Isolation and identification of a phytotoxic substance from Hyptis suaveolens

D.L.: VG 308-2014 ISBN: 978-84-8158-647-3 2 7th World Congress on Allelopathy Complex Interactions in a Changing Climate Vigo - Spain July 28 - August 1, 2014 Book of Proceedings 7th World Congress on Allelopathy Complex Interactions in a Changing Climate International Allelopathy Society Edited by: Manuel Reigosa Roger and Adela Sánchez‐Moreiras Congress held in Vigo, Spain July, 28 – August, 1, 2014 3 4 INDEX Welcome letter… _____________________________________________________________________________________________________________ 6 Scientific Committee … _________________________________________________________________ 9 Organizing Committee… ______________________________________________________________ 10 International Allelopathy Society Board… _________________________________________ 11 Invited lectures… ______________________________________________________________________ 12 Session 1. … ____________________________________________________________________________ 33 Methodology, new methods, evaluation of methods New techniques. Use of –omics in allelopathy Fate of allelochemicals and dynamics of phytotoxin production, release and decomposition Session 2… ______________________________________________________________________________ 59 Chemical aspects of allelopathy Searching new secondary metabolites Chemistry of allelopathy Session 3… ______________________________________________________________________________ 81 Ecological aspects of allelopathy Ecology of allelopathy Allelopathy and climate change Invasive plants: possible role of secondary metabolites Soil and rhizosphere ecology Session 4… _____________________________________________________________________________ 117 Allelopathy in different environments Fungi and lichen allelopathy Freshwater allelopathy Allelopathy in marine environments Plant‐microbe interactions Session 5… _____________________________________________________________________________ 133 Allelopathy in agriculture, horticulture and forestry Session 6… _____________________________________________________________________________ 157 Weed control using allelopathy 5 Session 7… ____________________________________________________________________________ 197 Physiological aspects of allelopathy Physiology of allelopathy Mode of action of allelochemicals Detoxification of allelochemicals Session 8… _____________________________________________________________________________ 221 Genetics of allelopathy and breeding for allelopathy Essential oils and volatiles Plant – other organisms interactions Abiotic stress, tolerance, infections, other uses of allelopathy Author index… _________________________________________________________________________ 252 6 Welcome letter July 27, 2014 International Allelopathy Society President Leslie A. Weston Research Professor Plant Biology Charles Sturt University, Australia Conference Chair Manuel Reigosa Roger Professor Plant Physiology University of Vigo, Spain Dear Conference Participants: Welcome to Vigo! We are pleased you could attend and participate in the 7th World Congress on Allelopathy (webs.uvigo.es/7wca_vigo) – Complex Interactions in a Changing Climate. Our meeting is being held at the Centro Social Novacaixagalicia, located at St. Policarpo Sanz, 24 in the centre of old town Vigo. This is a lovely and functional venue for our congress. During our meeting, we will also be sharing our venue with conferences on phytoremediation of soil and on organic agriculture and weed suppression, so you may be interested in attending and participating in these interesting meetings as well. We look forward to a stimulating and informative meeting with internationally recognized keynote presenters describing plant interactions with insects, microbes, marine organisms and other plants. Our speakers will also focus on the current state of the science of chemical ecology in the areas of genomics, proteomics, and metabolomics. The International Allelopathy Society has been in existence for nearly 20 years and due to burgeoning interest in the field, has expanded to include over 1000 past or current members participating in allelopathy research world‐wide. The field of allelopathy includes plant/plant, plant/insect and plant/microbial interactions, among others. It is an exciting area that has spawned innovative research in above and below ground interactions and the chemistry of bioactive natural plant products and signalling molecules, leading to better management systems for weeds and pests in agroecosystems, vegetation management in natural settings, novel compound analysis and discovery, as well as their mode of action in complex ecosystems, including terrestrial and marine systems. Recent findings have also led to a better understanding of the mechanisms that aquatic and terrestrial plants and organisms utilize to invade new territories, colonize productive and non‐ productive soils, and defend themselves against a variety of pests. The International Allelopathy Society would like to thank all participating societies and sponsors for their generous support and interest in hosting this meeting. 7 These include the City of Vigo, the Xunta of Galicia, the University of Vigo, the Spanish Society of Plant Physiologists among others. We would also like to express our gratitude to the team at the University of Vigo, University of Santiago de Compostela, University of Coruña and University of Coimbra, Portugal for all their hard work in organizing and developing this meeting, as led by Prof. Manuel F. “Pachi” Reigosa Roger, chair, and vice‐chair and secretary, Dr. Adela Sanchez Moreiras, both of U. of Vigo. The IAS would also like to thank its executive committee for their contributions to planning and organization of this meeting, and financial contributions to support this meeting. Finally, the IAS would like to thank the scientific committees associated with planning this meeting and supporting conferences and the committee associated with selection of the latest IAS research and paper award winners in 2014. We know you will enjoy your time in Vigo and take advantage of associated tours of the palace, downtown historic Vigo and the Cies Islands. We also offer post‐ conference tours of local points of geologic, historical and touristic interest. Again, we thank the City of Vigo and associated travel agencies for their assistance in organizing these tours for conference participants, as well as the organizing committee for this meeting in providing these options. Students attending this meeting will also benefit from local cost‐effective lodging and reduced registration rates. We look forward to meeting our students and spending time in discussion of their research during this meeting. The society hosts an international congress every 3 years to draw together the international community to present noteworthy scientific research and ensure research collaboration. This year we have participants attending from over 33 nations. Please consider hosting one of our meetings in the future, in your country of origin! We welcome applications for consideration as sites of future world congresses and will entertain applications for new meeting sites at our business meeting of the IAS, held on Thursday, where conference award winners will be presented and business of the society discussed. Please attend if at all possible as we welcome your input into your society. Finally, we are pleased to announce and present the release of the first issue of the new journal affiliated with the IAS society and led by Editor Prof. Manuel F. Reigosa Roger and editorial staff in association with the University of Vigo Press. This new publication is entitled Journal of Allelochemical Interactions and is a result of hard work and planning efforts of those affiliated with the society who see a need for a high quality publication presenting research information on allelochemicals, their mode of action, their roles in chemical signalling processes and ecology, evolutionary biology and as natural products. This new journal offers 8 opportunities for publication of your research in a high quality publication, with leading experts in associated fields as contributors and editors. It will be published initially two times per year, and when fully developed, up to 4‐6 times per year, on demand. Careful development of this journal will ensure its success and achievement of a high impact factor over time, and potential future circulation by commercial publishing bodies. We hope you will consider publishing the work presented at this meeting in the Journal of Allelochemical Interactions and supporting its future development by serving as a reviewer. Enjoy your stay in lovely Vigo and surrounding Galicia. With our best wishes, Leslie A. Weston, President International Allelopathy Society [email protected] Manuel Reigosa‐ Roger Conference and Program Chair International Allelopathy Society [email protected] 9 International Scientific Committee Chair: Manuel Reigosa, Spain Vice‐Chair: Adela Sánchez‐Moreiras, Spain Committee members Adele Muscolo, Italy Ana Luisa Anaya, México Anne Osbourn, UK Azim Mallik, Canada Catherine Fernández, France Chang‐Hung Chou, Taiwan, China David Gealy, USA David Wardle, Sweden Francisco Macías, Spain François Pellissier, France Helena Freitas, Portugal Ibrahim S. Alsaadawi, Iraq Inderjit, India Ines Fritz, Austria Inge Fomsgaard, Denmark Jamal R. Qasem, Jordan Jeffrey Weidehnhamer, USA Jim Pratley, Australia Jing‐quan Yu, China José María Molinillo, Spain Juan Carlos Galindo, Spain Leslie Weston, Australia Luis González, Spain Margot Schulz, Germany Maria Rosa Abenavoli, Italy Marina Della Greca, Italy Min An, Australia Prasanta Bhowmik, USA Ragan M. Callaway, USA Regina G. Belz, Germany Ren‐sen Zeng, China Scott Baerson, USA Shaolin Peng, China Shibu Jose, USA Shi‐ming Luo, China Stephen Duke, USA Wen.xiong Lin, China Yoshiharu Fujii, Japan Zahid Ata Cheema, Pakistan Zhi‐qiang Pan, USA 10 Organizing Committee Organizing Chair: Manuel J. Reigosa‐Roger, Univ. of Vigo [email protected] Organizing Vice‐Chair & Secretary: Adela Sánchez Moreiras, Univ. of Vigo [email protected] Committee members: Emilio Carral Vilariño, University of Santiago de Compostela Federico Pomar, University of Coruña Helena Freitas, University of Coimbra Luis González Rodríguez, University of Vigo Nuria Pedrol Bonjoch, University of Vigo Carlos Souto Otero, University of Vigo François Pellissier, Université de Savoie Cristina Silvar Pereiro, University of Coruña Rubén Retuerto Franco, University of Santiago de Compostela Ana Martínez‐Peñalver Mas, University of Vigo Elisa Graña Martínez, University of Vigo Carla Díaz Tielas, University of Vigo Aitana Costas Gil, University of Vigo David López González, University of Vigo 11 IAS BOARD ‐ IAS Officers (2012‐2014) President Leslie Weston Charles Sturt University, Australia [email protected], [email protected] Program Vice President Manuel Reigosa University of Vigo, Spain [email protected] Secretary Catherine Fernandez Institut Méditerranéen d'Ecologie et de Paléoécologie (IMEP UMR CNRS 6116) Aix‐Marseille University – Campus St Charles, Case 4, France catherine.fernandez@univ‐provence.fr President‐Elect Ren‐sen Zeng South China Agricultural Uinversity, Guangzhou, 510642, China [email protected] Treasurer David Gealy Dale Bumpers National Rice Research Center, USDA‐ARS, Stuttgart, AR 72160 USA [email protected] Past President Steve Duke USDA‐ARS‐NPURU. Stoneville, MS, USA [email protected] Editor Azim Mallik Canada [email protected] 12 INVITED SPEAKERS Robert Verpoorte, Netherlands Aldo Barreiro, Portugal Francisco Macías, Spain Consuelo de Moraes Daniel Montesinos, Portugal Charlotte Poschenrieder, Spain Inge Fomsgaard, Denmark Antonio Fiorentino, Italy 13 14 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Invited Lecture 1. THE 4 DIMENSIONS OF PLANT SECONDARY METABOLISM. THE ROLE OF METABOLOMICS R. Verpoorte Natural Products Laboratory, Institute of Biology Leiden, Leiden University, PO Box 9505, 2300RA Leiden, The Netherlands, Email: [email protected] ABSTRACT Life Sciences are going through a rapid change. Since molecular biology started its advance some 30 years ago, it had a major landmark in obtaining the full sequence of the human genome, followed by that of various other organisms. We are now reaching the phase that the 1000 $ full sequencing of an organism becomes reality. It is almost cheaper to sequence again than to save the full sequence of an organism. At the same time it becomes clear that having a sequence does not help much to really understand a living organism. The high expectations for drug development, for example, have shown to be over optimistic, as so far no novel drugs have resulted from this knowledge. In fact a genome is like a blueprint, and a blueprint has only two dimensions, and not the four of life: 3 of space and 1 of time. Using these blueprints the research is now going to a more holistic approach: systems biology. That means in an integrated approach study organisms at all levels of phenotype, metabolome, proteome, transcriptome and genome. The importance of a systemic approach can be illustrated by the fact that plants can be considered to be super organisms in the sense that they are dependent on the collaboration of the plant with all kind of microorganisms, e.g. in the rhizosphere, but also endophytes in the plant itself. That means many new opportunities for natural products research. Plant interactions with their environment, health effects of our food, traditional medicine, biosynthesis, metabolic engineering are examples of areas where society expects us to translate basic research into novel products and concepts to the benefit of all of us. We all have many new opportunities but also many challenges. We have tools like metabolomics, genetic engineering, and phytochemistry to better understand plants but we need to closely collaborate with other disciplines to have an as complete view as possible of the systems we are studying. Keywords: metabolomics, secondary metabolism 15 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Invited Lecture 2. ALLELOPATHY DRIVES SPECIES COEXISTENCE 1,2 2 Aldo Barreiro1 and Vitor Vasconcelos2 CIIMAR, Interdisciplinary Center of Marine and Environmental Research. Porto, Rua dos Bragas 289, 4050-123 Porto, Portugal. e-mail: [email protected] Faculty of Sciences, Porto University, Rua do Campo Alegre, 4069 – 007 Porto, Portugal. email: [email protected] ABSTRACT Hutchinson's Paradox of the plankton states that phytoplankton diversity is paradoxical, since many coexisting species share few resources, contradicting the competitive exclusion principle. Ecologists began then searching mechanisms that could explain this paradox. Allelopathy was shown, in theory, to promote coexistence. Lotka-Volterra competition models with allelopathy predict one single stable coexistence equilibrium. Other models incorporating mechanisms (delays, remineralization, allelochemical uptake, adaptation...) show certain areas of parameter space predicting coexistence. However, no experimental demonstration exists for these model predictions. Our aim was to demonstrate experimentally that allelopathy is a mechanism promoting coexistence. We surveyed different phytoplankton species regarding competition for nitrate and allelopathy. We chose a couple showing a trade – off between allelopathy and competition for nitrate, so that the worst competitor for nitrate was the allelopathic species. Then, we parametrized a simple competition model for nitrate with an allelopathic interaction in continuous culture (hereafter chemostat) for those species. By model analysis, we chose a bifurcation parameter that could be manipulated in further long – term competition experiments in chemostats, whose results would be used to select an improved model among several candidates. Our selected species were the chlorophyte Ankistrodesmus falcatus and the allelopathic cyanobacteria Oscillatoria sp. The bifurcation parameter was the initial proportion of them. Our experimental results matched general predictions from several allelopathy models: A. falcatus dominance, coexistence (either stable or oscillating) and Oscillatoria sp. dominance. The best fitting models were versions of our simple nitrate-allelopathy-chemostat model, with features allowing oscillating coexistence (delay through allelochemical uptake or adaptation). Keywords: Allelopathy, Phytoplankton population dynamics, Competitive exclusion, coexistence, Cyanobacteria, Paradox of the plankton, mechanistic population model, model fitting, model selection. Tel: +351 22 340 18 00 ; fax: + 351 22 339 06 08 THIS INVITED LECTURE IS PART OF THE CONGRESS “TOXIC ALGAE. MARINE ALLELOPATHY” AND WILL BE FOLLOWED IN A JOINT SESSION INCLUDING THE 7TH WORLD CONGRESS ON ALLELOPATHY AND THE CITED CONGRESS 16 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Invited Lecture 3. INS AND OUTS OF ELEMENTAL DEFENSE IN PLANTS Charlotte Poschenrieder1*, Berta Gallego1, Soledad Martos1, Mercè Llugany1, Roser Tolrà1, Catalina Cabot2 and Juan Barceló1 1 Lab. Fisiologia Vegetal, Facultad Biociencias, Universidad Autónoma de Barcelona, Spain; e-mail: [email protected] 2 Depto. Biología, Universidad de las Islas Baleares, Spain; e-mail: [email protected]: ABSTRACT All organisms, including plants, have to maintain a strict ion homeostasis for the perfect performance of their cellular functions and their growth and developmental processes. Complex mechanisms have evolved not only for achieving adequate tissue concentrations of essential nutrients, despite the occasional low availability in the environment, but also to manage ion excess. However, the ability to maintain ion homeostasis by no means implies that all plant species require equal mineral concentrations. In fact quite large differences among species in the leaf concentrations of both essential and non-essential elements can be observed even when growing in the same substrate. Better nutrient uptake efficiency can lead to nutrient depletion and may contribute to outcompeting less efficient neighbors in the fight for essential nutrients. Contrastingly, high tolerance to excess foliar concentration of essential or non-essential trace elements can lead to enrichment of surface soil, due to decomposition of the elemental-rich litter, acting as an alellopathic mechanism for less tolerant species (1). In soils with high availability of metals (e.g. Zn, Cd, Ni, Mn, Al), or non-metallic trace elements (e.g. As, Se), most adapted species try to restrict the uptake and to exclude excess ions at least from their photosynthetic tissues. Some species, however, have evolved a different strategy (hyper)accumulating large, potentially toxic, concentrations of these elements in their shoots. Table 1 gives a few examples of trace element concentrations in shoots of some hyperaccumulating species along with an indicative range for non-hyperaccumulators, taking Brassica napus and Agrostis sp. or other grasses as references for less efficient and highly efficient shoot excluders, respectively. Huge differences in leaf concentrations between hyperaccumulators and non-hyperaccumulators are evident. Hyperaccumulation behavior can be found in different species from distant taxonomic groups, indicating that the mechanisms responsible for hyperaccumulation may have evolved separately at several sites and different times. This fact brings about the question concerning the possible evolutionary advantages of trace element hyper-accumulation in plants. Among the different hypothesis forecasted up-to–date, the elemental defense hypothesis has received the most support (2). According to this, hyper-accumulation helps the plant to better defend itself against herbivores, pathogenic microorganisms, and even competing plant species. Although other scenarios are possible, such as amelioration of abiotic stress factors due to elemental hyperaccumulation. Currently the mechanisms behind the protective effects against biotic stress are under intense investigation and it is being elucidated that different modes of action can lead to defense by elemental accumulation (3). 17 Table 1: Examples of elemental concentrations in leaves of excluders and hyperaccumulating species (recompiled from multiple sources) Elem Species Conc. (mg ent kg-1) Zn Agrostis sp 25-75a Brassica napus 25 -70a Noccaea 20000b caerulescens 4500b Viola calaminaria Ni Brassica napus 20 -170c Alyssum bertolonii 13400b Berkheya codii 11600b Mn Agrostis sp. 50-100a Brassica napus 30 -150a Virotia neurophylla 31200b Cd Agrostis sp. 0.6 - 3.3c Brassica napus 0.4 - 7.0c Noccaea praecox 6000b Al Agrostis sp 20-140d Brassica napus 160-240d Commelina sinensis >1000e (tea) Se grasses 0.4-13c Brassica napus 0.4 -470c Biscutella laevigata 12000b a sufficient range; b on soil with high concentration; c on contol (left) or polluted substrate (right); d under high (left) or low (right) pH; e soil pH ≤ 5.5; 18 The accumulated elements can be directly toxic to the herbivore, pathogen, or competing neighbor plant. Indirect mechanisms include either or both elementalinduced activation of specific defense genes (i.e. the trace elements are acting as elicitors of defense signaling pathways) and alterations in the metabolism producing organic substances that can deter leafconsuming herbivores, hamper tissue spreading of microorganisms, or act as alellopathic substances delivered from decaying leaves (Figure 1). The investigation of indirect mechanisms of the elemental defense has stimulated further research into the close interactions between mineral nutrition and plants’ tolerance to biotic stress. In fact, both the observation that trace element levels below the hyperaccumulator level can get toxic to generalist herbivores (4) and the relevance of essential trace elements such as Fe, Zn, Cu, or B in several defense reactions, including reactive oxygen species, (Fe and Cu), protein synthesis (Zn), and cell wall stability (B) extended the elementaldefense research even to species with con-centrations below the hyper-accumulation level. To illustrate the complex interactions between elemental hyper-accumulation and defense, and to highlight the difficulty to distinguish between direct and indirect mechanisms, two case studies with Noccaea from our lab will be discussed: I) Interaction between snails and Zn or Cd hyper-accumulation and II) Interaction between pathogenic fungi and Zn or Cd hyper-accumulation. Moreover, in line with the specific topic of this congress, we will briefly consider current knowledge on direct elemental allelopathy, as well as the still poorly explored possibility of elemental-induced metabolic changes that may alter organic alellopathy. Case study I revealed that multiple factors are conditioning herbivorism by the common garden snail, Helix aspersa on Noccaea species. Snails do not seem to have a taste for Zn and glucosinolates largely determine leaf consumption in N. caerulescens under Zn-hyperaccumulation (5). Moreover, hyperaccumulation of either Zn or Cd alters the glucosinolate profile (6). Leaf Cd hyper-accumulation had a clear inhibitory effect on leaf consumption by snails in N. praecox, while consumption was stimulated by total leaf sugar concentration (Llugany et al., unpublished). Likewise, multiple factors determine the interactions between pathogenic fungi and metal hyperaccumulation. Under low metal supply hyper-accumulating species use to be highly sensitive to fungal infection. Cadmium protected N. praecox against infection by the biotrophic Erysiphe by direct toxicity and/or fungal induced jasmonate signaling (7). In non-hyperaccumulating Arabidopsis thaliana Cd, close to the toxicity threshold, also potentiates JA signaling and plant defense against necrotrophic Botrytis cinerea (8). Zinc hyper-accumulation protects N. caerulescens against infection by necrotrophic Alternaria. Only low Zn leaves but not high Zn leaves responded to infection with enhanced transcription of defense genes, especially of the SA signaling pathway. This seems to support a direct protection by Zn. However, these high Zn leaves responded to fungal attack with a strong increase of glucosinolate levels (Gallego et al., unpublished). So, further research is required to distinguish between inhibition by glucosinolates and direct Zn effects. Elemental allelopathy has been suggested for several heavy metals, especially Ni and Zn. However field studies to proof this are scarce. Convincing data are available for Se in stands of Biscutella laevigata (9). Even less information of the influence of elemental (hyper)accumulation on the production of organic allelopathic substance are available. In this context, phenolics, especially flavonoids, deserve special attention (10) because of both their ability to complex metal ions and to act as signaling molecules in plant-microbe and plant-plant interactions. References: (1) Chemoecol. 8:1-7 (1997) (2) Oecologia 98:373-384 (1994) (3) Trends Plant Sci.11: 288-295(2006) (4) J. Chem Ecol. 39: 666-674 (2013) (5) New Phytol 165: 763-772 (2004) (6) Plant Soil 288:333-341 (2006) (7) Plant Cell Rep 32: 1243-1249 (2013) (8) Planta 237: 337-349 (2013) (9) New Phytol 191:120-131 (2011) (10) Biol. Fert. Soils 48:123-149 (2012). Acknowledgements: Supported by Spanish MICINN project BFU2010-14873. Keywords: elemental defense, metal hyperaccumulation, herbivorism, fungal infection, allelopathy… TEL: + 34 93 5812163 ; FAX: +34 935811321 19 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Invited Lecture 4. HOW TO SELECT THE RIGHT EXTRACTION METHOD FOR ALLELOCHEMICAL STUDIES Nuria Chinchilla, Rosa M. Varela, Ascensión Torres, José M. G. Molinillo and Francisco A. Macías* Grupo de Alelopatía, Departamento de Química Orgánica, Facultad de Ciencias, Instituto de Biomoléculas (INBIO), Universidad de Cádiz, Campus de Excelencia Internacional Agroalimentario (ceiA3), C/República Saharaui, s/n, 11510 Puerto Real, Cádiz, Spain; e-mail: [email protected] ABSTRACT In the process of isolating, identifying, characterizing their structures, and determining the bioactivity of allelochemicals, proper extraction techniques are paramount. In ecological studies, the most appropriate methodology involves mimicking natural conditions by using water as a solvent and then re-extracting with organic solvents, or other procedures. This way presents some important drawbacks due to the complex mixtures obtained, the low amount of metabolites isolated, the formation of artefacts, and easy microbial degradation. For that reason, direct extraction with organic solvents has been widely used because this method yields less complex extracts and significantly higher yields, which is particularly useful when allelochemicals are localized on an specific organ, such as trichomes. In these cases, some corrections have been proposed to ensure that the original concentrations of the metabolites in the plant and the ecological role of the isolated compounds can be determined. Several advanced extraction techniques can be applied to yield bioactive extracts and these include ultrasound-assisted extraction, supercritical fluid extraction and pressurized liquid extraction. Ultrasound extraction is a very common extraction technique for the recovery of active components, mainly due to the mild extraction conditions applied. Cavitation is the ultrasound mechanical effect that enables greater penetration of solvent into the sample. Additionally, these methods allow the recovery of compounds in shorter times and at lower temperatures. This avoids the destruction of active molecules due to high extraction temperatures. In the same way, supercritical fluid extraction technology, with or without the help of co-solvent, has been applied to the extraction of bioactive compounds. Throughout the extraction process, it is important to determine the most active fractions through a variety of bioassays and continue separation only of the active fractions in order to optimize the isolation of biologically active compounds. The bioassay selected in this study was the etiolated wheat coleoptile bioassay, which is both rapid (24 h) and sensitive. Furthermore, this bioassay can be considered as an initial assessment of phytotoxicity in which undifferentiated tissue cells are used. Keywords: Extraction methodologies, allelochemicals, bioguided extraction. Tel: +34 956012770; fax: +34 956016193 20 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Invited Lecture 5. WHY NOT ALL ADAPTATIONS ARE ADAPTIVE: INVASIVE AND NON-INVASIVE SPECIES SHOW SIMILAR TRAIT SHIFTS AMONG REGIONS. 1 Daniel Montesinos 1*, Yedra García2 Centro de Ecologia Funcional, Universidade de Coimbra, Portugal Tel: (+351) 239 855 238 (ext. 139); [email protected] 2 CIDE (CSIC-UV-UV), Valencia, Spain; [email protected] ABSTRACT Differences in morphological or ecological traits expressed by exotic species between their native and non-native ranges are often interpreted as evidence for adaptation to new conditions in the non-native ranges. In turn, this adaptation is often hypothesized to contribute to the successful invasion of these species. There is good evidence for rapid evolution by many exotic invasives, but the extent to which these evolutionary changes actually drive invasiveness is unclear. One approach to resolving the relationship between adaptive responses and successful invasion is to compare traits between populations from the native and non-native ranges for both exotic invaders and congeners that are exotic but not invasive. We collected seeds from several populations from both the native and non-native ranges of three closely related congeneric species of Centaurea, all of which are sympatric in the same native and non-native ranges in Europe and North America. Of these, C. solstitialis is highly invasive whereas C. calcitrapa and C. sulphurea are not. We grew plants in a common garden experiment and compared a suite of morphological traits that are commonly tested in the literature in the context of invasion. For all three species, plants from non-native populations showed similar shifts in key traits that have been identified in other studies as important putative adaptive responses to post-introduction invasion. For example, for all three species plants from populations in non-native ranges were (i) larger and (ii) produced seeds that germinated at higher rates. In fact, the non-invasive C. calcitrapa showed the strongest trait shift between ranges. Centaurea solstitialis was the only species for which plants from the non-native range increased allocation to defensive spines, and allocated proportionally less resources to reproduction, patterns contrary to what would be predicted by theory and other empirical studies to enhance invasion. Our results suggest caution when interpreting the commonly observed increase in size and reproductive capacity as factors that cause exotics to become invaders. Figure 1. Total plant biomass (g; mean ± SE) (A); relative growth rate of rosette diameter (mm mm-1 day-1; mean ± SE) (B) for each species from each region. Different letters indicate statistically significant differences (P ≤ 0.05) 21 Keywords: invasive species, trait-shifts, adaptation, speciation, allopatry, neoallopatry 22 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Invited Lecture 6. ALLELOPATHIC AND HEALTH-PROTECTIVE SMALL MOLECULES: SIMILAR STRUCTURES, MECHANISMS AND RESEARCH APPROACHES. 1 Inge S. Fomsgaard Department of Agroecology, Aarhus Universitet, Forsøgsvej 1, DK-4200 Slagelse, Denmark; e-mail: [email protected] ABSTRACT The biological activity of allelochemicals often is not limited to the allelopathic effects. Many of such compounds - originating from plants’ secondary metabolic pathways – can exert biological effects in the body of animals and humans, when they consume plant-based food and feed. A number of biologically active hydroxamic acids, lactams and benzoxazolinones that originate from the same biosynthetic pathway are often grouped as benzoxazinoids (BX). Selected examples of benzoxazinoids are shown in Figure 1. Lactams and hydroxamic acids generally are found as glucosides in plant tissues. Figure 1. Typical benzoxazinoids Benzoxazolin-2-one was the first benzoxazinoid to be discovered in rye seedlings (Virtanen et al, 1957). Subsequently other benzoxazinoids were discovered and the biosynthetic pathway was characterized in maize (Frey et al, 2010). The discovery of benzoxazinoids in grasses (cereals) made these compounds the probably most investigated compounds in allelopathy from the 50’ies until now as for instance in the project www.fateallchem.dk. Concurrently - starting in the 80’ies with Otsuka et al (1988) - other groups of scientists investigated the pharmacological properties of benzoxazinoids; - mostly isolated from dicotyledonous medicinal plants of the Acanthaceae, Ranunculaceae and Lamiaceae families. Immune-system stimulating and anti-cancer properties were reported, - most of them on basis of in-vitro studies (reviewed by Adhikari et al, in prep). Mutagenic properties of benzoxazinoids were also reported (Hashimoto et al, 1979). Numerous papers throughout the last 50 years that dealt with the allelopathic effects of benzoxazinoids in cereals stated that “these compounds are not 23 present in the mature grains”. However in 2009, our group identified and quantified 8 benzoxazinoid compounds in mature rye grains; - and we showed that upon consecutive watering and drying of wheat grains, these grains could produce benzoxazinoids at a concentration level similar to the level in dry rye grains but with differences in the pattern. These discoveries led to a patent application “Use of benzoxazinoid-containing cereal grain products for healthimproving purposes” (Fomsgaard et al, 2009) and initiated two research projects - www.breadandbreakfast.nu and www.ryeproc.dk - that focus on the possible exploitation of the effect of whole grain benzoxazinoids on the immune system and suppressive effects on prostate cancer. Research into fate and effects of bioactive compounds in the mammal body can benefit substantially form results obtained in agricultural and environmental research and vice versa, as illustrated in the following example: Absorption, distribution, metabolisation and excretion (ADME) of bioactive compounds in the mammal body includes Phase 1 metabolisation processes, which are oxidation, reduction and hydrolysis processes. However, recent results showed that metabolites from benzoxazinoids that are known to be formed through microbial transformations in soil should also be searched for when elucidating metabolisation in the mammal body. 2-aminophenoxazinone is a well-known microbial metabolite from BOA and HBOA (Gents et al, 2005; Zikmundova et al, 2002a). This compound was searched for in our recent ADME studies in rats and pigs and it was found in bile, urine and feces (Adhikari et al, 2012a, 2012b). Phenylacetamides (N-(2hydroxyphenyl)acetamide) and N-(2-hydroxy-5-nitrophenyl)acetamide)) as microbial metabolites (by endophytic fungus) of HBOA were reported by Zikmundova et al as well (2002a, 2002b). These compounds were recently identified as metabolites in rye consuming individuals (Beckmann et al, 2013; Hanhineva et al, 2014) and it was suggested that they were not only formed as colonic microbial metabolites but as ract, potentially by endogenous enzymatic activity in the endothelium of the small intestine. Additional recent research results will be presented in the symposium that explain the importance of sharing knowledge among research fields when investigating fate and effects of bioactive compounds. Adhikari, K.; Laursen, B.; Laerke, H.N.; Fomsgaard, I.S. Bioactive benzoxazinoids in rye bread are absorbed and metabolized in pigs. Journal of Agricultural and Food Chemistry 2012a, 60, 2497-506. Adhikari, K.B.; Laerke, H.N.; Mortensen, A.G.; Fomsgaard, I.S. Plasma and urine concentrations of bioactive dietary benzoxazinoids and their glucuronidated conjugates in rats fed a rye bread-based diet . Journal of Agricultural and Food Chemistry 2012b, 60, 11518 – 11524. Beckmann, M.; Lloyd, A.J.; Haldar, S.; Seal, C.; Brandt, K.; Draper, J. Hydroxylated phenylacetamides derived from bioactive benzoxazinoids are bioavailable in humans after habitual consumption of whole grain sourdough rye bread. Mol. Nutr. Food Res. 2013, 57, 1859-1873. 24 Fomsgaard, I.S.; Mortensen, A.G.; Holm, P.B.; Gregersen, P. PCT patent application PA 84245 "Use of benzoxazinoids-containing cereal grain products for health-improving purposes". 2008, submitted March 19, 2008; publicly available September 19, 2009. Frey, M.; Schullehner, K.; Dick, R.; Fiesselmann, A.; Gierl, A. Benzoxazinoid biosynthesis, a model for evolution of secondary metabolic pathways in plants: Evolution of Metabolic Diversity. Phytochemistry 2010, 70, 1645-1651. Gents, M.B.; Nielsen, S.T.; Mortensen, A.G.; Christophersen, C.; Fomsgaard, I.S. Transformation products of 2-benzoxazolinone (BOA) in soil. Chemosphere 2005, 61, 74-84. Hashimoto, Y.; Shudo, K.; Okamoto, T.; Nagao, M.; Takahashi, Y.; Sugimura, T. Mutagenicities of 4-hydroxy-1,4-benzoxazinones naturally occurring in maize plants. Mutation Research 1979, 66, 191-195. Hanhineva, K.; Keski-Rahkonen, P.; Lappi, J.; Katina, K.; Pekkinen, J.; Savolainen, O.; Timonen, O.; Paananen, J.; Mykk+ñnen, H.; Poutanen, K. The Postprandial Plasma Rye Fingerprint Includes Benzoxazinoid-Derived Phenylacetamide Sulfates. The Journal of Nutrition 2014, 144, 1016-1022. Otsuka, H.; Hirai, Y.; Nagao, T.; Yamasaki, K. Anti-Inflammatory Activity of Benzoxazinoids from Roots of Coix lachryma-jobi var. Ma-yuen. Journal of Natural Products 1988, 51, 74-79. Virtanen, A.; Hietala, P.; Wahlroos, O. Antimicrobial substances in cereals and fodder plants. Arch. Biochem. Biophys. 1957, 69, 486-500. Zikmundova, M.; Drandarov, K.; Bigler, L.; Hesse, M.; Werner, C. Biotransformation of 2-benzoxazolinone (BOA) and 2-hydroxy-1,4bewnzoxazin-3-one (HBOA) by endophytic fungi isolated from Aphelandra tetragona. Appl. Environ. Microbiol. 2002a, 68, 4863-4870. Zikmundova, M.; Drandarov, K.; Hesse, M.; Werner, C. Hydroxylated 2-amino3H-phenoxazin-3-one derivatives as products of 2-hydroxy-1,4-benzoxazin-3one (HBOA). Biotransformation by Chaetosphaeria sp. an endophytic fungus from Aphelandra tetragona. Zeitschr. Naturforsch. 2002b, 57c, 660-665. Keywords: allelopathy, benzoxazinoids Tel: +45 87158212; fax: +45 87156082 25 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Invited Lecture 7. CHEMICAL SIGNALING IN INTERACTIONS AMONG PLANTS, INSECTS, AND OTHER ORGANISMS 1 2 Consuelo de Moraes 1* Mark Mescher 2 Department of Environmental Systems Science, ETH Zürich; [email protected] Department of Environmental Systems Science, ETH Zürich; [email protected] ABSTRACT Olfactory cues mediate diverse interactions among plants and other organisms. Over the past two decades a key focus of chemical ecology has been on elucidating the role of constitutive and induced plant volatiles in conveying information to insect herbivores and their natural enemies1. It is now well established that volatile blends can carry complex information about the identity and status of emitting plants, and that insects and other organisms perceive and respond to such cues in sophisticated and sometimes surprising ways2-4. A great deal of research has documented the role of volatiles in tri-trophic interactions and explored implications for the control of herbivorous pests in agricultural ecosystems, and recent work is extending these insights in a variety of exciting directions. These include increasing attention to population-level and evolutionary processes occurring in natural communities, to the role of volatile chemistry in mediating interactions in complex environments in which plants simultaneously interact with a diverse community of other organisms (including microbial symbionts and pathogens as well as insects), and on the perception of olfactory cues by plants themselves. In this talk, I will describe these developments and discuss recent work from our program in each of these areas. Specifically, I will discuss recent studies exploring (i) the effects of inbreeding and genotypic variation within plant populations on community level plant-insect interactions5-8, (ii) the influence of pathogen-induced changes in host plant odors on interactions with vector and non-vector insects9-13, and (iii) plant responses to olfactory cues from other plants and from insects14-16. 26 Figure: Plant volatiles mediate diverse ecological interactions. The inset circles show various consumers of volatile cues, including (clockwise from top): (1) Insect pollinators; (2) Foraging herbivores (e.g., ovipositing moths); (3) Actively feeding herbivores (e.g., moth larvae); (4) Parasitic plants (discussed later in the text); (5) Below-ground herbivores (which respond to volatiles emitted from plant roots; e.g., beetle grubs); (6) Natural enemies of root-feeding herbivores (e.g., entomopathogenic nematodes); (7) Neighboring plants (or undamaged tissues of the emitting plant); (8) Insect disease vectors (e.g., aphids); (9) Natural enemies of foliar herbivores (e.g., parasitoid wasps). References: 1. Unsicker, S.B., Kunert, G., Gershenzon, J., 2009. Protective perfumes: the role of vegetative volatiles in plant defense against herbivores. Current Opinion of Plant Biology 12, 479-485. 2. De Moraes, CM & MC Mescher. 2004. Biochemical crypsis in the avoidance of natural enemies by an insect herbivore. Proceedings of the National Academy of Sciences 101: 8993-8997. 3. De Moraes, CM, MC Mescher & JH Tumlinson. 2001. Caterpillar-induced nocturnal plant volatiles repel conspecific females. Nature 410:577-580. 4. De Moraes, CM, WJ Lewis, PW Paré and JH Tumlinson. 1998. Herbivore infested plants selectively attract parasitoids. Nature 393:570-574. 5. Kariyat RR, KE Mauck, CM De Moraes, AG Stephenson & MC Mescher. 2012. Inbreeding alters volatile signalling phenotypes and influences tri-trophic interactions in horsenettle. Ecology Letters 15: 301–309. 6. Kariyat, RR, KE Mauck, CM Balogh, AG Stephenson, MC Mescher, & CM De Moraes. 2013. Inbreeding in horsenettle (Solanum carolinense) alters nighttime volatile emissions that guide oviposition by Manduca sexta moths. Proceedings of the Royal Society B 20130020. http://dx.doi.org/10.1098/rspb.2013.0020 7. Kariyat, RR, SR Scalon, RP Moraski, AG Stephenson, MC Mescher, & CM De Moraes. 2014. Plant inbreeding and prior herbivory influence the attraction 27 of caterpillars (Manduca sexta) to odors of the host plant Solanum carolinense (Solanaceae). American Journal of Botany, 101 (2): 376-380 8. Kariyat, RR, CM Balogh, RP Moraski, CM De Moraes, MC Mescher, & AG Stephenson. 2013. Constitutive and herbivore-induced structural defenses are compromised by inbreeding in Solanum carolinense L. American Journal of Botany, 100 (6): 464-472. 9. Mauck, KE, CM De Moraes & MC Mescher. 2010. Deceptive chemical signals induced by a plant virus attract insect vectors to inferior hosts. Proceedings of the National Academy of Sciences 107(8):3600-3605. 10. Shapiro LR, CM De Moraes, AG Stephenson & MC Mescher. 2012. Pathogen effects on vegetative and floral odors mediate vector attraction and host exposure in a complex pathosystem. Ecology Letters. 15:1430–1438. 11. Mauck, KE, CM De Moraes & MC Mescher. 2014. Biochemical and physiological mechanisms underlying effects of Cucumber mosaic virus on host-plant traits that mediate transmission by aphid vectors. Plant, Cell & Environment. doi: 10.1111/pce.12249 12. Mauck, KE, NA Bosque-Pérez, SD Eigenbrode, CM De Moraes & MC Mescher. 2012. Transmission mechanisms shape pathogen effects on host-vector interactions: evidence from plant viruses . Functional Ecology 26:1162-1175. 13. Shapiro LR, I Seidi-Adams, KE Mauck, CM De Moraes, A Stephenson & MC Mescher. 2014. Dynamics of short- and long-term association between a bacterial plant pathogen and its arthropod vector. Scientific Reports 4 (4155) doi:10.1038/srep0415 14. Runyon, JB, MC Mescher & CM De Moraes. 2006. Volatile chemical cues guide host location and selection by parasitic plants. Science 313: 1964-1967. 15. Frost, CJ, HM Appel, JE Carlson, CM De Moraes, MC Mescher & JC Schultz. 2007. Within-plant signaling via volatiles overcomes vascular constraints on systemic signaling and primes responses against herbivores. Ecology Letters 10:490-498 16. Helms, AM, CM De Moraes, JF Tooker & MC Mescher. 2013. Exposure of Solidago altissima plants to volatile emissions of an insect antagonist (Eurosta solidaginis) deters subsequent herbivory. Proceedings of the National Academy of Sciences. 110: 199-204 Keywords: Biocommunication, plant-insect interactions, plant-pathogen interactions TEL: + 41 44 632 39 20. 28 interactions, plant-plant 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Invited Lecture 8. NMR-BASED METABOLOMICS IN ALLELOPATHY: A USEFUL TOOL IN THE SEARCH FOR PLANT GROWTH REGUTATORS IN PLANTS OF MEDITERRANEAN VEGETATION Monica Scognamiglio, Brigida D’Abrosca, Assunta Esposito, Antonio Fiorentino* Department of Environmental Biological and Pharmaceutical Sciences and Technologies, Second University of Naples – via Vivaldi 43, 81100 Caserta, Italy. email: [email protected] ABSTRACT The study of allelopathic interactions between plants requires a systemic approach in which the input from several fields, such as botany, plant physiology, ecology, biochemistry, agriculture and natural products chemistry can be used for a better comprehension of the inherent physiological and metabolic processes. A valuable approach to study these interactions is systems biology, and among omics sciences, which constitute this platform, metabolomics could be very powerful. Despite its potential, it has been seldom applied to allelopathy studies. Here, we report a new NMR-based metabolomic approach useful to discover phytotoxic components in plant extracts. The method has been standardized with known allelochemicals. Then, crude extracts of Mediterranean plants were tested, in hydroponic system, against four target species. The analyses of both donor and target plants were carried out by NMR-based metabolomics. Furthermore, soil analysis of study site has been carried out by HPLC or NMR methods. Plants of Mediterranean vegetation have been studied in the search for plant growth inhibitors to be used as potential herbicides. Using the metabolomic approach, (a) the metabolome of donor plants and its seasonal variations have been evaluated; (b) the phytotoxic metabolites of selected plants have been identified; (c) the effects of phytotoxic extracts have been assessed; (d) the metabolome of target species has been characterized; (e) the biochemical changes induced in the receiving plants have been analyzed; (f) experiments using pure allelochemicals on selected target species allowed their mode of action to be hypothesized. Moreover, in field experiments allowed to demonstrate the release of allelochemicals in the soil. It is well-known that Mediterranean macchia ecosystems are characterized by a high level of spatial variability and structural complexity, mainly depending on environmental conditions and disturbance history. Consequently, Mediterranean plant species evolved and activated allelopathic mechanisms as life strategies to grow and survive in this environment. In order to study the allelopathic interactions in this area, an NMR-based metabolomics approach has been standardized. In order to study the existing chemodiversity, sixteen Mediterranean plants have been monthly collected for two years and analyzed for their metabolite variation during this period. The analyses revealed a high richness in secondary metabolites, depending on the 29 species and different classes of metabolites have been identified in the extracts. The metabolic profiling was strongly dependent by the seasonality and changed not only in concentration but also for the presence of different functional groups that periodically interchanged in the molecules present in the extracts. Concerning the intraspecific diversity, the attention was raised to metabolism seasonality, with the highlighting of peculiar trends, especially for phenolic compounds. These metabolic variation could be linked to the phoenological stages or induced by environmental stresses, such as allelopathy, competition, herbivores, drought, temperature, etc, to which plants are exposed in Mediterranean ecosystems. The observed changes could have an impact on the understanding of the roles of the secondary metabolites in the chemical interactions between Mediterranean plants and could contribute to explain the dynamic equilibrium existing among these species in the field. Preliminary phytotoxicity studies of the plant extracts evidenced that three species, Bellis sylvestris, Pistacia lentiscus and Melilotus neapolitana, induced different responses in the receiving plants. Figure 1. Experimental design The effects of these donor plants have been assessed on two crops, Triticum durum and Lactuca sativa, and two wild species, Aegilops geniculata and Avena fatua (Figure 1). The metabolome analysis of the target species furnished useful information on the biochemical changes induced by the toxics in treated plants compared to the control (Table 1). As the extracts produced different effects on the target species, a differentiated mode of action of the potential allelochemicals could be hypothesized. In order to classify the main metabolites identified in the extracts as allelochemicals, soil samples of the study site have been collected in field, 30 extracted in a Soxhlet apparatus and analyzed by NMR or HPLC. As their presence in the environment was detected, in order to have more information on the putative mode of action, specific experiments were carried out on selected target species in hydroponic system using the pure allelochemicals. Based on our observations, B. sylvestris and M. neapolitana were selected as good candidates for further studies. This choice was made upon consideration of their higher activity against wild species compared to the effects observed on the crops species, and based on the different biochemical changes induced in the target plants, suggesting a differentiated mode of action. M. neapolita ++ na - + -- ++ + B. ++ sylvestris -- - + * ++++ P. lentiscus --- -- - T durum A. geniculata Avena fatua - M. neapolita ++++ na -- ++ - --- --- --- #* ++ -- - + ++++ -- --- --- #* + - - - ++ + -- --- --- #* ++ + - + + + + - + - - - - * + + - + + + + * + + + -- - ++ ++ + ++ + + + + + + ++ - - - * + ++ + + + -- * + - - - +++ - - - - - - - - - * + ++++ -- -- - - - +++ - - - - - - - - + + -- + +++**** ++ - - - - * ++++ - +++ + + - P. lentiscus -- - + - -- - - - *** + - M. neapolita + na ---- - - - - +++ ++++ +++ - +++ Apigenin - Aromatics + Valine ++ B. --- ---sylvestris -- Nautigenin - Apigenin-8-gly + Apigenin-6-gly -- Oblongaroside - Betaine -- Choline + P. lentiscus Sucrose + Glucose GABA +++ Threonine Asparagine -- Glutamine Alanine + B. ++++ sylvestris Glutamate Malate - Citrate cis-Aconitate Table 1. Quantitative analysis of the compounds of receiving plant metabolome influenced by donor plants. Each (+) stands for 1-25 % increase, (*) 100% increase, (#) 500 % increase, (-) 125 % decrease. - --- Keywords: Allelopathy, Mediterranean vegetation, Chemical interferences, NMR-based metabolomics, Tel: +39 0823 274576; fax: +39 0823 274571 31 32 Session 1. Methodology, new methods, evaluation of methods New techniques. Use of –omics in allelopathy Fate of allelochemicals and dynamics of phytotoxin production, release and decomposition 33 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 1. GRAVITROPISM: NEW MODE OF ACTION OF ALLELOCHEMICALS FOR THE PRACTICAL MANAGEMENT OF TWINING PLANTS Fujii Yoshiharu, Maninang John, Tamura Naoyuki, and Wasano Naoya Tokyo University of Agriculture and Technology, Tokyo, Japan; e-mail: [email protected] ABSTRACT Twining weeds, such as kudzu (Pueraria lobata), Japanese honeysuckle (Lonicera japonica) and burr cucumber (Sicyos angulatus) have serious problems in many countries. Documented cases worldwide prove their negative impact on the productivity of farmlands and in altering the landscape of natural habitats. Moreover, these weeds pose risks to safety and reduce efficiency of power supply in urban communities as they invade electrical grids. In response to the request of electric power companies and government agencies in Japan, our laboratory has started a project to screen allelochemicals for use in the effective management of twining weeds. We have adapted laboratory bioassays using lettuce or Arabidopsis to evaluate the effect of allelochemicals on gravitropism. Practical assays to prevent twining of vine were also developed using leguminous vine plants such as Phaseolus vulgaris. We have, thus far, tested 49 allelochemicals including amino acids, phenolics, flavonoids, alkaloids and others, and found several promising candidates. Cis-cinnamic acid and its derivatives that we have found as allelochemical from Spiraea sp., and hinokitiol and its derivatives which are known volatile allelochemicals from Aomori-hiba (Thujopsis dolabrata) were among those that impair gravitropic response. The underlying mechanism of twining in plants is classified into two parts, gravitropism, and cell wall synthesis. Using transcriptome analysis, the mode of action of the candidate allelochemicals were evaluated. Cis-cinnamic acid for one appear to influence the expression of auxin-responsive gene families in Arabidopsis. Auxins are putative mediators of gravitropism in plants. Our results implicate that exogenous application of cis-cinnamic acid may effectively manage twining weeds by impairing their auxin-mediated gravitropic response. At present, we endeavor to develop derivatives by organic synthesis, and conduct field test for the development of practical management measures for twining weeds. Keywords: gravitropism, twining weed, allelochemicals, cis-cinnamic acid, hinokitiol Tel: + 81-42-367-5625; fax: +81-42-367-5625 34 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 2. SPATIAL AND TEMPORAL DYNAMICS OF ROOT EXUDATION: HOW IMPORTANT IS HETEROGENEITY IN ALLELOPATHIC INTERACTIONS? Jeffrey D. Weidenhamer1*, Brian K. Mohney1, Nader Shihada1 and Maduka Rupasinghe2 1 2 Department of Chemistry, Geology and Physics, Ashland University, Ashland, OH 44805 USA; e-mail: [email protected]; [email protected], [email protected] Department of Mathematics and Computer Science, Ashland University, Ashland, OH 44805 USA; e-mail: [email protected] ABSTRACT The understanding of allelopathy has been hindered by the lack of methods available to monitor the dynamics of allelochemicals in the soil. Previous work has demonstrated the feasibility of using polydimethylsiloxane (PDMS) microtubing (silicone tubing microextraction, or STME) to construct sampling devices to monitor the release of lipophilic allelochemicals in soil. The objective of this study was to use such sampling devices to intensively monitor thiophene fluxes beneath marigolds over several weeks to gain insight into the magnitude of temporal and spatial heterogeneity of these fluxes. Marigolds were grown in rhizoboxes (20.5 x 20.5 x 3.0 cm) with 16 individual STME samplers per box. Thiophene sampling and HPLC analysis began 45 days after planting,. At the end of the study, roots around each sampler were analyzed by HPLC. Results confirmed the tremendous spatial and temporal heterogeneity in thiophene production seen in our previous studies; STME probes show that thiophene concentrations increase over time, however, there is heterogeneity in various sampling ports.The reasons for this variability are unclear, but are being investigated. While the importance of such heterogeneity, or “patchiness”, in the root zone has been recognized for soil nutrients, the potential importance in allelopathic interactions has seldom been considered. Our results demonstrate that STME can be used as tool to probe the dynamics of allelochemicals in the root zone. Keywords: Allelopathy, diffusive sampling, PDMS, rhizosphere, root exudation, soil analysis, spatial heterogeneity, temporal heterogeneity, Tagetes patula. Tel: +01 419 289 5281 35 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 3. OPTIMIZED ANALYTICAL TECHNIQUES FOR EXTRACTION AND SEPARATION OF BIOACTIVE COMPOUNDS FROM DIVERSE PLANT TYPES Suparna Mandal Biswas1* and Nabanita Chakraborty2 1 Agricultural and Ecological Research Unit; Indian Statistical Institute 203, B.T. Road, Kolkata 700108, INDIA; E-mail: [email protected] or [email protected] 2 Agricultural and Ecological Research Unit; Indian Statistical Institute 203, B.T. Road, Kolkata 700108, INDIA; e-mail: [email protected] ABSTRACT Bioactive products from plants provide unlimited opportunities for pharmaceuticals and natural agrochemicals development due to availability of chemical diversity. Successful identification and development of natural product from plants necessitates a standard and integrated approach to screen compounds which further aids in determination of dose-response relationship. Here three different plant types namely; herbs (Piperomia pellucida with fibrous root and Cleome viscosa with tap root), shrubs (Piper chaba) and tree (Artocarpus lakoocha) are considered. In C. viscosa, allelochems are collected from Root Exudate Trapping System made of Buchner funnel and conical flask and in P. pellucida, a self-designed horizontal tube like glass ware with stopper and funnel at either ends has been developed. In case of P. chaba and A. lakoocha, bioactive compounds are extracted from stem and shedded leaves respectively. The basic sequential steps include grinding, homogenization, vacuum filtration followed by liquid-liquid extraction in which allelochemicals are fractioned into two major phases, viz. ethyl acetate layer and aqueous layer. In order to evaluate their chemical composition, the obtained extracts were further purified into a single pure compound by repetitive running through column and thin layer chromatography and finally subjected spectral analyses. Four major compounds are recovered from P. chaba and A. lakoocha with remarkable bioactivity. Lactam nonanic acid with allelopathic and antimicrobial properties has identified from Cleome viscosa and research continues for Piperomia pellucida. These new technologies will extend and enhance the usefulness of plants as renewable resources of valuable chemicals. Keywords: Bioactive compound, root exudates trapping system, Liquid-liquid extraction, Cleome viscosa, Piperomia pellucida, Piper chaba, Artocarpus lakoocha. Tel: +91(033) 2575 3225; fax: +91(033)25753049 36 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 4. A NEW APPROACH AND TECHNOLOGY ALLOW SEPARATION OF ALLELOPATHY AND COMPETITION UNDER GLASSHOUSE CONDITIONS J.R. Qasem Department of Plant Protection, Faculty of Agriculture. University of Jordan, Amman, Jordan; e-mail: [email protected] ABSTRACT Ideas allow separation of allelopathy from competition in pots are introduced and discussed. A new technology can be developed and examined to allow separation of plant interference components. In this new method, square pots of different sizes may be used. Each pot is divided by a double layer of fine (100150 micrometer) metal mesh barrier fixed from the bottom up to the pot surface. The mesh divides each pot into two identical sections filled by the same amount of soil mixture. Seeds or seedlings of donor and receiver species are planted separately each in a pot section. Density of the donor plant may be varied as needed to concentrate or dilute root exudates consisting allelochemicals released into the soil. Nutrient solution may be added to both sections to eliminate any shortage in nutrient supply to donor plants at high density, and also to avoid possible deficiency of mineral nutrients in the sections planted by receiver species. At extended period of growth a barrier may be also inserted from the pot surface to prevent competition for light. In control treatment, both receiver and donor plants can be grown separately in similar divided pots and treated similarly as those grown together per same pot. This technique allows prevention of direct physical root interference of donor and receiver species and can precisely differentiate allelopathic from non-allelopathic species through chemicals exuded with root exudates. Failure of receiver species to perform normal growth or to develop with supply of nutrients may be considered as an indicator on high possible influence of root exudates including allelochemicals received from the neighbor donor species. All kind of comparisons and densities can be tested and under excessive or shortage supply of growth factors imposed solely on donor species. Allelochemicals released from roots, foliage parts or residues incorporated into the soil all can be examined and at different planting depth or plant/seed placement in the soil. The schematic of new technique is presented and and will be discussed for any further comments and improvement in the layout or its precision in separating allelopathy from competition Keywords: receiver. Allelopathy, competition, separation, a new technology, donor, 37 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 1. COMPARING METHODS TO DETECT PHYTOTOXIC EFFECTS OF Acacia dealbata: EVERYTHING IS NOT THE SAME Paula Lorenzo, Jonatan Reboredo, Jonatan Rodríguez, Narciso Aguilera, Luís González, Luís Múñoz, Susana Rodríguez‐Echeverría and Helena Freitas 1 2 Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, 3000-455 Coimbra, Portugal; [email protected] Departamento de Bioloxía Vexetal e Ciencia do Solo, Universidade de Vigo, E-36310 Vigo, Spain 3 Depto de Química Orgánica, Universidade de Vigo, E-36310 Vigo, Spain ABSTRACT There are several methods to detect phytotoxic effects of metabolites from plants. Generally, organic compounds are pre-dissolved in organic solvents and then in water to bioassay its activity on target plants. However, compounds solubility and pH in water after removing solvents are not totally controlled. Other bioassays are performed using DMSO and MES as a solvent and buffer medium to improve compounds solubility and stabilize pH respectively. The aim of this work was to compare different methods to assess phytotoxic effects of chemical compounds extracted from flowers of Acacia dealbata. Chemicals of fresh flowers were extracted in methanol for ten weeks and sequentially fractionated with hexane, ethyl acetate and water. The dry organic fractions were re-dissolved in its own solvents plus water and in DMSO buffered with MES. The phytotoxic effect of each fraction was bioassayed at 0, 5, 10, 25 and 50 ppm on the germination and seedling growth of Lactuca sativa. In general, the growth of L. sativa was significantly stimulated when acacia chemicals were pre-dissolved with DMSO. However, L. sativa growth was solvent, concentration and pH dependent when organic solvents were used. Plant germination was not affected in any case. Our results show that the phytotoxic effect of chemicals from A. dealbata flowers may be influenced by the dissolving procedure. DMSO- or organic solvent-based bioassays must be carefully selected depending on research interests. DMSO may be useful to find the full effect of isolated compounds and organic solvents may be used under an ecological approach. Keywords: DMSO (Dimethyl sulfoxide), MES (2-[N- morpholino]ethanesulfonic acid), Organic solvents, Phytochemicals TEL: +351.239.855.244; FAX: +351.239.855.211 38 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 2. ALLELOCHEMICALS FROM LEAVES OF Helianthus annuus WITH APPLICATION OF SUPERCRITICAL FLUID EXTRACTION Zohuir El Marsini, Ascensión Torres, Lourdes Casas‐Cardoso, Rosa Varela, Jose Mª G. Molinillo, Casimiro Mantell, Enrique J. Martínez de la Ossa and Francisco A. Macías1 Grupo de Alelopatía, Departamento de Química Orgánica, Facultad de Ciencias, Instituto de Biomoléculas (INBIO), Universidad de Cádiz, Campus de Excelencia Internacional Agroalimentario (ceiA3), C/República Saharaui, s/n, 11510 Puerto Real, Cádiz, Spain; e-mail: [email protected] ABSTRACT There are many papers published on the isolation of bioactive compounds from the leaves of sunflowers, but in most methods of extraction are used conventional solvents. In view of increasing environmental and health concern about the use of organic solvents in the extraction of natural products, there has been growing interest in using supercritical fluids. Supercritical fluid extraction (SFE) has been demonstrated to be a valuable alternative for it requires less solvent, has a short extraction time and is capable of extracting thermally labile compounds under mild conditions. In most of these studies carbon dioxide is used as the solvent because of its relatively low critical temperature (31.1 ◦C), non-toxicity, non-flammability, good solvent power, ease of removal from the product and low cost. The work described here involved the extraction of bioactive compounds from the fresh leaf extracts of sunflower (Helianthus annuus L) var. Arianna, with supercritical carbon dioxide and identification of the major compounds responsible for the bioactivity of these extracts. The extraction conditions are also discussed. The dried extract obtain in the second separator was fractionated to give seven fractions. Herein, we also report the isolation, structural elucidation, and bioactivity of three new sesquiterpene lactones: helivypolide K, helivypolide L and helieudesmanolide B, in addiction to 47 known compounds: two fatty acids; twelve diterpenes; five triterpenes; nine flavonoids; two heliannuols; four bisnorsesquiterpenes, and thirteen sesquiterpene lactones. Keywords: Sunflowers, Helianthus annuus, Arianna, supercritical carbone dioxide, sesquiterpene lactones, bioactivity Tel: +34 956012770; fax: +34 956016193 39 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 3. DIFFERENCES ON PROTEOMIC PATTERNS IN ALLELOCHEMICAL AND ALLELOCHEMICAL+SALT TREATED Arabidopsis PLANTS Adela M Sánchez Moreiras, Elisa Graña, Ana Martínez‐Peñalver, Carla Díaz‐ Tielas, Aitana Costas Gil, David López‐González, Manuel J Reigosa Faculty of Biology, University of Vigo, Campus Lagoas-Marcosende s/n E-36310 Vigo, Spain; e-mail: [email protected] ABSTRACT Different abiotic factors such as temperature, irradiance, salt, etc. can interact with the negative effects of allelochemical action. According to the stress hypothesis (which says that in special situations, when plants are under stress, allelopathy could become important;1,2) we previously tested the combined stress of BOA (2-3H-benzoxazolinone), a well-established allelochemical compound, and different salt (NaCl) concentrations. Our results demonstrated that the combined stress (BOA+salt) induced greater inhibition of plant growth, vigor, and viability than either treatment alone (BOA or salt). Salt treatment alone induced mechanisms of tolerance to salt stress, while the combined BOA+salt treatment increased salt sensitivity.3 To determine the changes at the proteomic patterns caused by BOA, salt and BOA+salt treatments on lettuce plants, two-dimensional electrophoresis was conducted, getting reproducible gels that confirmed the previously found protein content decrease and RubisCO degradation in BOA-treated plants and the different protein content on plant metabolism under the different applied treatments, which resulted in significant differences on protein profile for plants treated only with BOA or with BOA+salt. We propose the identification of the showed spots for validating the nature of these spots as ‘stress proteins’ and their role in the whole plants stress response induced after BOA or BOA+salt treatments. 1Reigosa MJ, Sánchez‐Moreiras A, González L (1999) Crit Rev Plant Sci 18: 577– 608. 2Reigosa MJ, Pedrol N, Sánchez‐Moreiras AM, González L (2002) In: Reigosa MJ, Pedrol N (Eds), Allelopathy: From Molecules to Ecosystems. Science Publishers. Inc., Enfield, NH: 231–256. 3Sánchez‐Moreiras AM, Pedrol N, González L, Reigosa MJ (2009) Plant Biol 11: 582–590. Keywords: BOA, salt, stress hypothesis, 2D-electrophoresis, lettuce 40 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 4. 2D-ELECTROPHORESIS IN BOA-TREATED Arabidopsis PLANTS Ana Martínez‐Peñalver, Elisa Graña, Carla Díaz‐Tielas, Adela M Sánchez‐ Moreiras, Manuel J Reigosa Faculty of Biology, University of Vigo, Campus Lagoas-Marcosende s/n, E-36310, Vigo, Spain; e-mail: [email protected] ABSTRACT 2-3(H)-Benzoxazolinone (BOA) is a secondary metabolite commonly present in the Poaceae, Acanthaceae, Ranunculaceae and Scrophulariaceae families1, 2 with a high phytotoxic capacity3 and a role of chemical defense against other species4. In previous studies5, measurements of chlorophyll a fluorescence, total protein content, malonyldialdehyde and in situ O2 - and H2O2 measurements, suggested that the primary effect of BOA could be the induction of early senescence, with oxidative stress as a secondary effect that appears later (1 to 2 days). The aim of this study was to determine the primary effect of BOA and corroborate it by 2D electrophoresis. For this reason, adult plants of Arabidopsis thaliana (3 weeks old) were treated with 1 mM BOA for 96 h. To determine the appropriate harvesting time, chlorophyll a fluorescence was measured with a Maxi-Imaging-PAM M Series Fluorometer from Walz at 1, 6, 12, 24, 36, 48 and 96 h after BOA watering and maximal PSII quantum yield (Fv/Fm), quantum yield of non-regulated energy dissipation (ϕNO), effective PSII quantum yield (ϕII), and the coefficient of non-photochemical quenching (ϕNPQ) were recorded to detect the first symptoms of damage at the plant metabolism. According to the fluorescence measurements, the plants were harvested 24 h after BOA treatment to obtain the proteomic profile. Proteomic measurements revealed both a quantitative and qualitative decrease of protein content, suggesting strong effects at the plant metabolism. Noteworthy is the strong and clear disappearance of RubisCO in treatment gels compared to the control. 1 Niemeyer HM (1988) Phytochemistry 27: 3349–3358. Pratt K, Kumar P, Chilton WS (1995) Syst Ecol 23: 781–785. 3 Batish DR, Singh HP, Setia N, Kaur S, Kohli RK (2006) Plant Physiol Biochem 44: 819-827. 4 Bravo HR, Copaja SV (2002) Ann Appl Biol 140: 129–132. 5 Sánchez-Moreiras AM, Martínez-Peñalver A, Reigosa MJ (2011) J Plant Physiol 168: 863-870. 2 Keywords: Benzoxazolin-2,3(H)-one, BOA, 2D Electrophoresis, Arabidopsis Tel: +34 986 818 714; fax: +34 986 812 556 41 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 5. METABOLOMICS AS A POWERFUL TOOL TO STUDY CHEMICAL INTERACTIONS AMONG PLANTS: A CASE STUDY OF MEDITERRANEAN MACCHIA VEGETATION Monica Scognamiglio*1, Brigida D’Abrosca1, Assunta Esposito1, Young Hae Choi2, Robert Verpoorte2 and Antonio Fiorentino1 1 2 Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples; via Vivaldi 43, 81100 Caserta, Italy e-mail: [email protected] Natural Products Laboratory, Institute of Biology Leiden, Leiden University, P.O. Box 9502, 2300 RA, Leiden, The Netherlands ABSTRACT Introduction: Metabolomics has shown to be a useful tool for studying plant responses to biotic and abiotic stresses and it seems a promising approach also for the study of allelopathic interactions. Such interactions play probably a central role in structuring ecosystems like the Mediterranean one, characterized by high plant diversity. Objective: To explore the potential of a metabolomic approach in allelopathy studies, focusing on chemical interactions among Mediterranean plants. Materials and Methods: Donor plant extracts were analyzed by NMR-based metabolomics and used for the bioassays on the receiving plant. The receiving plants were analyzed and compared with controls by NMR-based metabolomics using chemometrics. Results and discussion: The experimental design allowed the chemical analysis of donor plant extracts as well as the assessment of the effects of the potential allelochemicals on receiving plant metabolism. Among active plants, different groups could be observed. The activity was correlated to the donor plant metabolome (hence, putative allelochemicals or active “phytocomplexes” were identified). Concerning the receiving plants, the metabolic pathways affected by the allelochemicals were identified. Furthermore, the fate of allelochemicals in the receiving plants was studied and many of them seem to be taken up by the receiving plants. Conclusions: Although the full elucidation of allelopathic interactions requires further studies, the NMR based metabolomic approach proved to be a useful tool and gave important new insights in the interactions occurring in Mediterranean ecosystems. Keywords: Allelopathy, Mediterranean macchia, Metabolomics, NMR TEL: +390823274579 ; FAX: +390823274571 42 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 6. THE METABOLIC RESPONSE OF Arabidopsis TREATED WITH GLYPHOSATE AND FLAVONOIDS Hans Albert Pedersen 1 and Inge S. Fomsgaard 2* 1 2 Department of Agroecology, Aarhus University, Forsøgsvej 1, Flakkebjerg, DK-4200 Slagelse, Denmark; e-mail: [email protected] Department of Agroecology, Aarhus University, Forsøgsvej 1, Flakkebjerg, DK-4200 Slagelse, Denmark; e-mail: [email protected] ABSTRACT The growth of certain plants can be inhibited by isoflavonoids such as biochanin A. These compounds are produced by leguminous species such as clover that are used as cover crops and for weed suppression. It is therefore possible that isoflavonoids play a role in chemical weed suppression by legumes. To put such chemical weed suppression into context, three compounds were evaluated in a simple cultivation assay: biochanin A, an isoflavone, catechin, a flavanol, and glyphosate, a synthetic herbicide. Arabidopsis thaliana was used as the target species and was most susceptible to glyphosate (EC50 = 11µM) followed by catechin (EC50 = 275 µM). Biochanin A was not phytotoxic and a concentration of >3100 µM was needed to produce a small (15%) but significant decrease in biomass. Library-based GC-TOF metabolomics was used to characterize plant samples at various doses of the three compounds, resulted in the detection of about 70 metabolites and made it possible to follow the accumulation of the signature compounds shikimic acid, catechin, and biochanin A in the treated plants. Keywords: isoflavones, Arabidopsis, metabolomics, GC-TOF, biochanin A, catechin, glyphosate Tel: +45 87158178 / *+45 87158212; fax: +45 87156082 43 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 7. INTRASPECIFIC VARIABILITY IN ALLELOPATHY OF H. MANTEGAZZIANUM IS LINKED TO METABOLIC PROFILE 1 2 Kateřina Jandová1,2*, Petr Dostál2, Tomáš Cajthaml1,3, Zdeněk Kameník3 Faculty of Science, Institute for Environmental Studies, Charles University in Prague, Albertov 6, CZ-128 43 Prague 2, Czech Republic Institute of Botany, Academy of Sciences of the Czech Republic, CZ-252 43 Průhonice, Czech Republic 3 Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ-142 20 Prague 4, Czech Republic ABSTRACT According to the novel weapon hypothesis invasive species possess novel chemical compounds being allelopathic in new ranges and thus facilitating spreading 1. Allelopathy in exotic species is generally studied at the species level and relatively little is known about intraspecific variation in this trait. However, allelopathy is influenced by evolutionary history and environmental conditions and therefore quantifying variation and factors influencing this variation is key to a better understanding of plant interactions 2. Since, we observed variable allelopathy in well known invasive species Heracleum mantegazzianum, we suppose, there is considerable intraspecific variation in allelopathy and this variation is likely due to differences in concentration of allelochemical compounds. We addressed the intraspecific variability of allelopathy in 40 genotypes of H. mantegazzianum using bioassays of its root exudates in combination with UPLC-TOF-MS untargeted metabolomics. The genotypes were collected from separate maternal plants in several populations in separate areas. The partitioning of variation in allelopathic effects showed that the highest variance lies in variation among genotypes. The effect of root exudates was species-specific as there was no correlation between germination succes of our two assay species. The exudates stimulated germination of A. thaliana, however, they decreased germination success and root development of P. lanceolata. The responses of P. lanceolata were explained by the metabolic profile in orthogonal partial least squares analyses (OPLS) (P < 0.05). Biomarkers most associated with allelopathic effect were determined according to their position in S-plots. We subsequently inspected these features and proposed their tentative structure. References: (1) Callaway, R. M., and Aschehoug, E. T. (2000) Invasive Plants Versus Their New and Old Neighbors: A Mechanism for Exotic Invasion. Science 290, 521–3. (2) Inderjit, Wardle, D. a, Karban, R., and Callaway, R. M. (2011) The ecosystem and evolutionary contexts of allelopathy. Trends Ecol. Evol. 26, 655–62. Keywords: bioassay; giant hogweed; Heracleum mantegazzianum; invasive species; metabolomics; novel weapon hypothesis; root exudate; UPLC-TOF-MS TEL: +420 775670135 (Katerina Jandova) 44 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 8. DE NOVO CHARACTERIZATION OF THE Rehmannia glutinosa LIBOSCH LEAF TRANSCRIPTOME AND ANALYSIS OF THE GENES ASSOCIATED WITH ALLELOPATHIC AUTOTOXICITY 1 Zhong Yi Zhang1, Ming Jie Li2, Peng Fei Wang2, Feng Qing Wang2, Wen Xiong Lin1, Xin Jian Chen2* College of Crop Sciences, Fujian Agriculture and Forestry University, Fuzhou, China, 350002 2 College of Life Sciences, Henan Agricultural University, Zhengzhou, China, 450002 *Corresponding author E-mail: ZZY: [email protected]; [email protected] ABSTRACT The perennial herbaceous plant, Rehmannia glutinosa Libosch, is one of traditional Chinese medicines because of active extractives from its dried tuberous roots. However, R. glutinosa productivity and quality has been seriously affected by allelopathic autotoxicity (or replanting disease), which cannot be effectively prevented or controlled to date. Since there is very little known about the molecular mechanism of replanting disease, we aimed to investigate transcriptional changes in replanted R. glutinosa leaves and identify genes responding to the disease. Here, we constructed a cDNA library from total RNA isolated from the mixture of leaves of the first year planted (L1) and the second year replanted R. glutinosa (L2) at the tuberous root expansion stage. We generated ~37 million high-quality reads from the cDNA library using deep sequencing and obtained 94,544 distinct sequences by de novo assembly and gap-filling. From this set, a total of 54,490 transcripts containing a complete or partial encoding region were annotated in public protein databases. Based on this resource, we screened differentially expressed genes in the L1 and L2 libraries by digital gene expression (DGE) technique. Finally, a set of 1,954 genes may be in differential expression of L2. By bioinformatics and qRT-PCR, the 117 most strongly differentially expressed ones were considered to be prime candidates for responsible for replanting disease. Functional analysis of the candidates showed that ethylene signaling was exaggerated and the genes in key metabolism pathways were abnormally expressed in L2. The study provides an important resource for further investigating the cause of allelopathic autotoxicity and developing the methods to control or subtract its injury. Keywords: R. glutinosa, allelopathic autotoxicity, leaf transcriptome, deep sequencing, transcriptional changes This work was supported by grants from the National Natural Science Foundation of China (Nos. 81072983, 31271674 and 81274022) and China Postdoctoral Science Foundation (2013M541977 45 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 9. PHYTOTOXICITY OF MEDITERRANEAN PLANTS AND EVALUATION OF BIOCHEMICAL CHANGES INDUCED IN CROP AND WILD SPECIES BY NMRBASED METABOLOMICS Antonio Fiorentino*, Monica Scognamiglio, Nikolaos Tsafantakis, Assunta Esposito and Brigida D’Abrosca Department of Environmental, Biological and Pharmaceutical Sciences and Technologies; Second University of Naples; via Vivaldi 43, 81100 Caserta, Italy; e-mail: [email protected] ABSTRACT Introduction: Pests, invasive species and the overuse of synthetic pesticides for their control, represent a serious threat to the complex balance of ecosystems. Pesticides can be toxic to beneficial plants, insects and other organisms, thus damaging biodiversity and causing risks for human health. In this context, the search for new herbicide formulations based on natural products is gaining more and more importance. Objective: In this study, a new NMR-based metabolomic approach, useful to discover phytotoxic components in plant extracts, is reported. Materials and Methods: Crude plant extracts of Mediterranean plants were tested, in Petri dishes and in hydroponic system, against three target species. The analyses of both donor and target plants were carried out by NMR. Principal component analysis was performed with scaling based on Pareto method. Results and discussion: Five plants from Mediterranean vegetation were investigated for their ability to interfere with the growth and development of a crop (durum wheat) and two wild species (wild wheat and wild oat). Using an NMR-based metabolomic approach, the effects of phytotoxins on the target plants were evidenced by evaluating the induced biochemical changes. Moreover, the metabolic profiling of the plant extracts furnished important information useful to identify the metabolite or metabolites responsible for the phytotoxicity. Conclusions: The NMR-based metabolomic approach led to the identification of the bioactive metabolites present in the investigated plants. As the extracts produced different effects on the target species, a differentiated mode of action of the potential allelochemicals could be hypothesized. Keywords: NMR-based metabolomics, Phytotoxicity, Mediterranean plants, Triticum durum, Triticum ovatum, Avena fatua TEL: +390823274576 ; FAX: +390823274571 46 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 5. TRANSCRIPTOMIC ANALYSIS OF PLANT GROWTH-INHIBITORY ACTIVITY CAUSED BY A SPIRAEA THUNBERGII-DERIVED ALLELOCHEMICAL Naoya Wasano1 and Yoshiharu Fujii2 1 Graduate School of Agriculture, Tokyo University of Agriculture and Technology; e-mail: [email protected] 2 Graduate School of Agriculture, Tokyo University of Agriculture and Technology; e-mail: [email protected] ABSTRACT Thunberg’s meadowsweet (Spiraea thunbergii) is a popular garden shrub. While screening natural chemical alternatives to synthetic herbicides, Morita et al. (2005) found that leaf extract of S. thunbergii had high plant growth-inhibitory activity against lettuce. They identified the inhibitory compounds as ciscinnamoyl glucosides (Hiradate et al., 2005). The essential chemical structure responsible for the bioactivity of cis-cinnamoyl glucosides, cis-cinnamic acid (cis-CA), strongly inhibits the root growth of several plant species; however, its mode of action has not been characterized at the gene expression level. We conducted a time–course microarray analysis of gene expression in seedlings of Arabidopsis thaliana in response to 20 µM cis-CA. Comparison of the microarray profiles revealed a 10-fold upregulation of several auxinresponsive GRETCHEN HAGEN-3 (GH3) genes and LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKE (LBD) genes from 2 h to 6 h post-treatment. A qPCR analysis revealed that revealed that two early auxin-responsive gene families, the Aux/IAA family (IAA1, IAA5) and the GH3 family (GH3.1, GH3.2, GH3.3), and an LBD gene (LBD16) were markedly upregulated at 2 h after treatment in the roots, but not in the shoots, of Arabidopsis and remained highly expressed for 4 h. The influence of an exogenous application of cis-CA on the indole-3-acetic acid pathway strongly suggests that a root-targeted induction of auxin-responsive genes is involved in the cis-CA-mediated plant growth inhibition. Keywords: Spiraea thunbergii, DNA microarray, Gene Ontology analysis, ciscinammic acid, early auxin-responsive genes, Arabidopsis thaliana, auxin Tel: +81 42-367-5625; fax: +81 42-367-5625 47 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 6. A METABOLOMIC APPROACH TO STUDY COUMARIN MODE OF ACTION ON Arabidopsis thaliana SEEDLINGS Monica Scognamiglio1,*, Fabrizio Araniti2, Antonio Fiorentino1, and Maria Rosa Abenavoli2,* 1 Department of Environmental Biological and Pharmaceutical Sciences and Technologies, Second University of Naples; via Vivaldi 43, 81100 Caserta, Italy; e-mail: [email protected] 2 Department of Biotechnology for the Environmental Monitoring and Agri-Food, Mediterranean University of Reggio Calabria, Feo di Vito I-89124, Reggio Calabria, Italy; e-mail: [email protected] ABSTRACT Introduction: Among the most promising techniques adopted to identify allelochemicals and understand their mode of action, the metabolomic approach plays a pivotal role. Coumarin is an allelopathic compound released into the environment, that affects plant growth and development of many species. In particular, this metabolite interferes with root system, one of the main target of allelochemicals. Despite many information on its morphological and physiological effects, no evidences concerning coumarin effects on the metabolome of A. thaliana seedlings have been reported. Objective: In this study, NMR-based metabolomics was utilized to assess metabolic changes induced by coumarin on the model plant A. thaliana. Materials and Methods: In the present study A. thaliana seedlings grown in hydroponic culture, were treated with 100 µM coumarin. After treatment, the seedlings were collected (at different time points), frozen in liquid nitrogen and lyophilized. The analysis of target plants was carried out by NMR-based metabolomics. Results and discussion: NMR-based metabolomics analysis of A. thaliana treated with coumarin showed strong biochemical changes in treated plants compared to control. In particular, drastic variation in the aromatic component was observed, while less marked effects were detected for amino acids, organic acids and sugars. Conclusions: The NMR-based metabolomic approach was proved a powerful tool in studying the effects of allelochemicals at molecular level. Keywords: Coumarin, metabolome, allelopathy, mode of action, NMR-based metabolomics TEL: +390965 324077; FAX: +39965311092 48 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 10. MICROBIAL BIOTRANSFORMATION OF CATECHIN ENHANCES THE ALLELOPATHIC EFFECTS OF RHODODENDRON FORMOSANUM Chang‐Hung Chou and Chao‐Min Wang Research Center for Biodiversity, China Medical University, Taichung, Taiwan 404; email: [email protected] ABSTRACT Rhododendron formosanum, belonging to Ericaceae, exhibits a unique pattern of lacking understory species on the floor of the plants. The vegetation is distributed widely in the central mountain in Taiwan and the major allelopathic compound in the leaves has been identified as (-)-catechin, which is also as major allelochemical of an invasive spotted knapweed in North America. Soil microorganisms were thought to play an important role in the allelopathic mechanism of some dominant vegetations, yet, little evidence has proved the important mechanism. We present the clear evidence that microorganisms, such as Pseudomonas, Herbaspirillum, and Burkholderia, in the rhizosphere of R. formosanum play a major role in biotransformation of (-)-catechin, in particular the Pseudomonas catalized the conversion of catechin into protocatechuic acid in vitro. The phytotoxicity of (-)-catechin was siginificantly lower than that of protocatechuic acid (PCA). Thus, the PCA becomes an active allelopathic agent in the processes of allelopathic inhibition of understory species. We have quantitatively identified the concentrations of the metabolites, catechin taxifolin, protocatechuic acid and glycerol, in 72 hr by using LA-ESIMS/ MS method, and concluded that the process of allelopathic interaction involved ecological, biochemical, and microbiological analyses, demonstrating a multidisciplinary approach in understanding the successful invasion of new areas by alien plant. Keywords: invasive plants, catechin, allelopathy 49 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 11. PHYTOTOXIC DYNAMICS OF DECAYING LEAF LITTER OF Artemisia arborescens IN SOIL MICROCOSMS Araniti F., Zumbo A., Scalise A., Abenavoli M.R., Gelsomino A. Università degli Studi “Mediterranea” di Reggio Calabria, Feo di Vito I-89124, Reggio Calabria, Italy; [email protected] ABSTRACT Allelopathic effects of Artemisia arborescens L. have been extensively studied, but a little attention has been given to changes in the phytotoxic potential of leaf litter during the decomposition process in soil. To this aim, a laboratory-scale experiment was set up with microcosms filled with either a sterilized (by autoclaving) or not-sterilized clay loam agricultural soil. Finely ground A. arborescens leaf litter was added at three levels of amendment (0, 1 and 10% w/w) and incubated in the dark at 25°C for a 60-day period. Soil samples were taken after 0, 15, 30, 45 and 60 days from the beginning of the trial, extracted with sterile water and assayed on seed germination and root growth of lettuce. Interestingly, the phytotoxic activity was found to be higher in the non-sterilized than in the sterilized soil over the first 30 days at 10% amendment level. However, at later stages (45 and 60-day sampling) the phytotoxic potential remained almost unchanged in sterilized soil, whereas it became negligible in non-sterilized soil. Soil basal respiration strongly increased with the increasing amount of added leaf litter, although no differences were found between sterilized and non-sterilized soils. Conversely, microbial biomass C was added by both amendment and soil sterilization. These findings suggest that the potential phytotoxicity of soil incorporated leaf litter from A. arborescens during the decomposition process could be affected by microbial community. Keywords: Artemisia arborescens, allelopathy, microbial biomass, litter decomposition, basal respiration Tel: +39 0965 324077; Fax: +39 0965 311092 50 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 12. FATE OF 2-BENZOXAZOLINONE IN SOIL: IDENTIFICATION OF HITHERTO UNKNOWN METABOLITES Stine Krogh Steffensen 1 and Inge S. Fomsgaard 2* 1 2 Department of Agroecology, Aarhus University, Forsøgsvej 1, DK-4200 Slagelse, Denmark; e-mail: [email protected] Department of Agroecology, Aarhus University, Forsøgsvej 1, DK-4200 Slagelse, Denmark; e-mail: [email protected] ABSTRACT Rye is a common crop in Denmark, both in food production and as a cover crop during winter. When used as cover crop, the immature plants are plowed under in the spring before sowing. This contributes to soil fitness by adding nutrients but also through the allelochemical effects of the benzoxazinoids, the main allelochemical of rye. To understand these effects it is important to investigate the degradation of these compounds in the soil. The main benzoxazinoid in rye is glycosylated DIBOA. When the plant is damaged, the highly reactive and unstable DIBOA is released to the surroundings. In an aqueous environment DIBOA will spontaneously transform into BOA within a few hours, hence we decided to investigate the degradation products from BOA in soil taken directly from the field. The aim was to identify new metabolites and confirm the formation of previously described metabolites. The experimental setup contained a 140-day period with sampling every second day in the beginning, falling to every second week at the end of the period. A variety of HPLC coupled mass spectrometers were used for initial identification, accurate mass determination and final identification and quantification. In conclusion, BOA is mainly transformed into 2-aminophenol and further on into APO and AAPO as previously seen. But catechol, HPAA and a hydroxyl analog to APO, HPO, were also identified as degradation products from BOA. These metabolites, however, are not stable for the duration of the experiment and are further transformed into a number of degradation products not fully identified. Keywords: rye, benzoxazinoids, BOA, APO, degradation, soil TEL: +45 87158139 / *+ 87158212 ; FAX: +45 51 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 7. ALLELOCHEMICALS FROM DECAYING LILAC (Syringa vulgaris L.) RESIDUES: PHYSIOLOGICAL AND BIOCHEMICAL ANALYSIS Pavliuchenko N.A.,1 Macias F.A.,2 Igartuburu J.M.3 1 2 M.M. Grishko National Botanical Garden, Department of Allelopathy, Timiryazevska 1, 01014 Kyiv, Ukraine; e-mail: [email protected] University of Cadiz, Faculty of Science, Department of Organic Chemistry, Republica Saharaui s/n, Apdo. 40, 11510 Puerto Real (Cadiz), Spain; e-mail: [email protected] 3 University of Cadiz, Faculty of Science, Department of Organic Chemistry, Republica Saharaui s/n, Apdo. 40, 11510 Puerto Real (Cadiz), Spain; e-mail: [email protected] ABSTRACT The lilac (Syringa vulgaris L.) is a very valuable ornamental, essential oilcontaining, medicinal plant. Collection of lilac of M.M. Grishko National Botanical Garden is of interest as genetic bank of rare cultivars. The aim of the research was carrying out of physiological and biochemical analysis of decay products from lilac cultivars residues. Lilac seedlings were grown for 15 months in grey forest soil mixed with residues (a mixture of roots, fallen flowers and leaves at 1:1:1, at 2% to soil weight) of several lilac cultivars under greenhouse conditions.Reduction in basic photosynthetic pigments content and simultaneous increase in allelopathic activity, proline and phenolics contents in leaves of lilac seedlings growing in soil with decaying residues were found, which indicated stress state of plants. As a result, inhibition of growth of lilac seedlings was observed. Decay products of lilac residues changed biochemical characteristics of soil. They reduced oxidation-reduction potential in soil, which can be explained by accumulation of mobile organic substances in soil. Total concentrations of phenolics in soil increased during decay of lilac residues. 4-hydroxybenzoic, 4-hydroxycinnamic, ferulic, 3-hydroxycinnamic, vanillic, syringic and 2-hydroxycinnamic acids were identified by HPLC in soil with decaying lilac cultivars residues. Content of phenolic acids depended on lilac cultivars characteristics, duration of decay of residues. Ferulic, 4hydroxycinnamic and 2-hydroxycinnamic acids were the dominant phenolic acids in soil with decaying lilac residues (13-38 % of the total spectrum). Increase in the phytotoxicity of soil with decaying lilac residues depended on accumulation of phenolic compounds. Keywords: Syringa vulgaris L., lilac cultivars residues, phenolic acids, phytotoxicity 1 Tel: + 38 (044) 285-54-53 ; fax: + 38 (044) 285-26-49 52 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 8. ALLELOPATHIC EFFECT OF CAFFEIC ACID DERIVATIVES FROM Bellis sylvestris: BIOACTIVITY, ORGAN LOCALIZATION AND RELEASE Monica Scognamiglio*, Brigida D’Abrosca, Vittorio Fiumano, Mimmo Golino, Assunta Esposito and Antonio Fiorentino 1 Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples; via Vivaldi 43, 81100 Caserta, Italy e-mail: [email protected] ABSTRACT Introduction: Plants of Mediterranean area are a rich source of bioactive secondary metabolites. Although the positive or negative effects on plant germination and growth of many metabolites isolated from these plants has been reported, further evidences are needed in order to assess their allelopathic potential. Objective: A study was undertaken with the aim to demonstrate the allelopathic potential of caffeic acid and seven of its derivatives, isolated from leaves of Bellis sylvestris Cyr. Materials and Methods: Caffeic acid derivatives were isolated from Bellis sylvestris leaves. The structures of the metabolites were elucidated on the basis of their spectroscopic features. Allelopathic effects were evaluated on seed germination and seedling growth of two coexisting Mediterranean species (Dactylis hispanica and Aegilops geniculata). Finally, the distribution of caffeic acid derivatives in plant organs, as well as their presence in soil community was investigated by HPLC. Results and discussion: Cluster analysis of biological data pointed out interesting relationships between the chemical structures of the compounds and their biological effects. From the analysis of the distribution of these compounds in plant organs and in the soil, it was shown that leaf decomposition could justify the presence of caffeic acid derivatives in the soil. Conclusions: The study evidenced an allelopathic role of caffeic acid derivatives on coexisting species growth. The detection of most of the metabolites in the soil supported the hypothesis of their contribution in modulating plant community composition. Keywords: Allelopathy, Bellis sylvestris, Caffeic acid derivatives, Soil analysis TEL: +390823274579 ; FAX: +390823274571 53 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 9. DYNAMICS OF PHENOLICS AND ENZYMATIC ACTIVITY IN SOIL VARY AMONG CROP GROWTH STAGE AND DURATION OF RESIDUES DECOMPOSITION Abdul Khaliq1*, Farhena Aslam1, Asif Tanveer1, Amar Matloob1 and Zahir Ahmad Zahir2 1 2 Department of Agronomy, University of Agriculture, Faisalabad-38040- Pakistan Institute of Soil and Environmental Sciences, University of Agriculture, Faisalabad 38040Pakistan 1; e-mail: [email protected]; 2; e-mail: [email protected]; 3; e-mail: [email protected]; 4; e-mail: [email protected]; 5; e-mail: [email protected] ABSTRACT Decomposition of crop herbage over time changes the pattern of release of allelopathic compounds in in the soil and modifies activities of soil microorganisms. Release of allelopathic compounds also varies among cultivars and developmental stages of crop. Bioassays were conducted to appraise the allelopathic compounds released during different decomposition periods and changes in soil enzymatic activities were evaluated. Herbage of four wheat cultivars (Faisalabad-2008, Lasani-2008, AARI-2011, Millat-2011), at different developmental stages (tillering, anthesis and maturity) was collected and dried under shade. Wheat herbage of respective cultivars and growth stage was soil incorporated at 8 g kg-1. Herbage was allowed to decompose and sampling was done at fortnightly interval. The experiment was replicated thrice in a completely randomized design under factorial arrangements. Herbage of different wheat cultivars and developmental stages had differential effect on soil enzymatic activities. Herbage collected at anthesis and maturity significantly changed the activities of soil enzymes and level of phenolic contents during decomposition. Phenolic contents were increased during initial period of decomposition but decreased with the passage of time. Herbage collected at anthesis and maturity exhibited higher phenolics than that collected at tillering stage. On the other hand, enzymatic activities (urease, dehydrogenase) increased with decomposition as compared to initial stage. Overall results showed that decomposed herbage of wheat cultivars significantly affected the allelopathic compounds and changed the activities of soil enzymes and such results were more pronounced for herbage collected at anthesis and maturity than tillering. Keywords: allelopathy, cultivar, phenolics, soil enzymatic activity, wheat growth stage TEL: +92 321 66 15 848 54 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 10. EFFECT OF PHENOLIC COMPOUNDS ON AMERICAN GINSENG SEEDLING GROWTH JIAO Xiao‐Lin 1 Bi XIao‐Bao 2 and GAO Wei‐Wei 3* 1 2 Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, China; e-mail: [email protected] Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, China; e-mail: [email protected]:[email protected] 3 Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, China; e-mail: [email protected] ABSTRACT The cultivation of American ginseng (Panax quinquefolium L.) is frequently hindered by problems during replanting. Our previous studies showed that nine phenolic compounds existed in American ginseng root and rhizosphere respectively inhibited the radicle growth of its own embryo on filter paper. Here, we investigated the combined actions of phenolic compounds on its seedling growth and potential physiological mechanism involved. The 3-yearold American ginseng seedlings used in the experiments were grown in nutrient culture, added by p-coumaric acid or nine phenolic compounds mixture (PCM), which included 60% p-coumaric acid (w/w) and other eight phenolic acids mimicked their content in the field. The results showed that either 0.012-0.3 mg/mL p-coumaric acid or 0.02-0.5 mg/mL PCM significantly delayed leaf expansion and inhibited the above-ground growth of American ginseng plant (P<0.05). The decreases of chlorophyll synthesis and photosynthesis were responsible for the reducing of biomass. The inhibition strength in a mixture of nine phenolics treatment was similar to that in individual p-coumaric acid treatment. Furthermore, the pot experiment was conducted to evaluate the effect of phenolic compounds in soil. After added 1 mg/g PCM in soil, the plant growth and PCM concentration in the rhizosphere were monitored through high performance liquid chromatography (HPLC). The results showed plant growth and biomass had no significant difference between PCM treatment and control. The nine phenolic compounds were degraded by 95%-100% after 30 days of treatment. The allelopathic effects of phenolic compounds were diminished due to a majority of them was degraded in soil. Keywords: Panax quinquefolium L., phenolic compounds, p-coumaric acid, allelopathic effect, biomass, photosynthesis, soil degradation Tel: +08601057833423; fax: +08601057833020 55 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 11. SORGOLEONE PRODUCTION WITH INCREASING CONCENTRATIONS OF WATER EXTRACT OF ROOTS OF Abutilon theophrasti Paulo Roberto Fidelis Giancotti1, Kumudini M. Meepagala2, Jason Martin2, Stephen O. Duke2, Pedro Luís da Costa Aguiar Alves1* 1 2 Department of Applied Biology, FCAV/UNESP—São Paulo State University, Jaboticabal SP, Brazil; e-mail: [email protected], *[email protected] USDA-ARS, Natural Products Utilization Research Unit, P.O. Box 8048, University, Mississippi 38677, United States ABSTRACT The aim of this study was to determine if the root extract of Abutilon theophrasti could stimulate the sorgoleone production in sorghum seedlings. Each plot consisted of a petri dish with 20 seeds of sorghum hybrid SX17 and filled with glasswool. The treatments were increasing concentrations of water extract of A. theophrasti (0.624 mg mL-1, 1.25 mg mL-1, 2.5 mg mL-1, 5 mg mL-1, 10 mg mL-1 and a control with just deionized water). A sorgoleone standard curve was built in order to determine the sorgoleone amount in each plot. After 7 days in chamber with controlled temperature and light, the Petri dishes were opened and its sorghum roots were separated in a beaker filled with dichloromethane. The material was sonicated in order to collect root exudate and the roots were placed in an oven to be dried. Trough HPLC, in each plot it was detected the peak correspondent with the sorgoleone standard in order to determine the sorgoleone amount present in the sorghum exudate. The root dry mass of sorghum, the amount of sorgoleone per plot and the concentration of sorgoleone per dried root weight did not differ between the treatments, according to Tukey’s test (p<0.05). However, the results of concentration of sorgoleone per dried root weight showed an increase trend as the concentration of water extract of A. theophrasti increased, corroborating with Dayan (2006). Keywords: sorghum, allelochemical, secondary metabolism Tel: + 55 16 98133 8529 56 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 12. ALLELOPATHIC POTENTIAL OF Bidens sulphurea (Cav.) Sch. Bip ROOT EXUDATE AT THREE PHENOLOGICAL STAGES ON TWO LETTUCE CULTIVARS Bruna Pires da Silva1* Mariluce Pascoina Nepomuceno2 Pedro Luís da Costa Aguiar Alves3 Paulo Roberto Fidelis Giancotti4 1,2,3,4 Departamento de Biologia Aplicada à Agropecuária, FCAV – UNESP, Jaboticabal, SP, Brazil. 1 2 3 e-mail: [email protected]; e-mail: [email protected]; e-mail: 4 [email protected], [email protected] ABSTRACT Bidens sulphurea (Cav.) Sch. Bip. has been showing a high invasive behavior on crops of agricultural interest, probably due its allelopathic potential. This feature of B. sulphurea makes it a potential resource of natural herbicides (allelochemicals). When isolated and characterized they can be used directly or used for the semi-synthesis of new compounds. The present study aimed to evaluate the allelopathic potential of B. sulphurea on lettuce by the method of release by root exudation ("in vivo" plant). In order to obtain the root exudates, B. sulphurea seedlings (20 days after germinated) were placed in inverted bottles of 2 L (with usable area of 90 mm diameter and 270 mm long), painted with silver paint and filled with washed sand and gravel. In the bottle cap it was installed a polyethylene tube (5 mm diameter and 200 mm long) for root exudates collection. The collection was performed at three stages of the studied plant: vegetative, flowering and senescence stage. The exudates collected were stored in refrigerator until be used in germination bioassays. For the bioassays, it was tested two cultivars of lettuce (Lactuca sativa): Veronica and Itapuã Super. The osmolality and pH of each exudate was measured. We evaluated the percentage of germination, length and dry weight of roots and shoots. With statistical analysis, we concluded that the exudates collected during flowering and senescence promoted a significant reduction in all parameters evaluated in the two cultivars of L. sativa. Keywords: allelopathy, allelochemicals, natural herbicides. Tel: +55 16 3209 2620; fax: +55 16 3209 2620 57 58 Session 2. Chemical aspects of allelopathy Searching new secondary metabolites Chemistry of allelopathy 59 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 13. IDENTIFICATION OF THE ALLELOCHEMICALS IN SOILS OF ALLELOPATHIC RICE Jia‐Yu Li, Qi Zhang, Zhi‐Hua Lin, Zhen‐Ming Yu, Hai‐Bin He* Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, and Agroecological Institute/School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, 350002, P.R. China; e-mail: [email protected] ABSTRACT Allelochemicals is the critical topic in allelopathy research because it is the cause of plant allelopathy phenomena. In order to investigate the potent allelopathic substances in rice allelopathy, a weed inhibit ring of an allelopathic rice PI312777 and a nonallelopathic rice Lemont has been established. The PI312777 had an effective inhibition on barnyardgrass which planted 12cm distance from the rice, showing 53.0%, 50.0%, and 74.4%, respectively of inhibitory rates on root length, plant height and dry weight (aboveground) of barnyardgrass. Further, the soils in 12cm distance from the rice were extracted by water and 70% aqueous ethanol. The allelopathic activity of extracts was studied by Petri dishes used lettuce as target. And the soils before and after extracted were also bioassay by Agar-Soil-Sandwich Method (ASSM) used barnyardgrass as target. By ASSM, the inhibitory rates of the no-extracted soils on root length and plant height of barnyardgrass was 51.03% and 18.89% respectively. However, they were 12.76% and 5.75% respectively after soils were extracted by water, and 27.57% and 5.46% respectively used aqueous ethanol extracted soils. The extracts bioassays showed that the inhibitory rate of water extracts and aqueous ethanol extracts on root growth of lettuce was 42.6% and 21.4%, respectively. These results implied that the potential allelochemicals in the soils of allelopathic rice would be water-soluble components. Keywords: Extract Rice (Oryza sativa. L), Allelopathy, allelochemicals, Paddy soil, Tel: +86-591-83789352; fax: +86-591-83769440 60 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 14. PHYTOTOXICITY OF Cynara cardunculus ALLELOCHEMICALS ON WEEDS Carlos R. Cumbrera1, Paula Novaes1, Rosa M. Varela1, José M. G. Molinillo1 and Francisco A. Macías1*. 1 Grupo de Alelopatía, Departamento de Química Orgánica, Facultad de Ciencias, Universidad de Cádiz, Campus de Excelencia Internacional Agroalimentario, ceiA3, Institute of Biomolecules (INBIO). C/ República Saharaui s/n, Apdo. 40, 11510-Puerto Real (Cádiz), Spain. (*Phone: +34 956012770; fax: +34 956016193; email address: [email protected]). ABSTRACT Cynara cardunculus L. is a plant native from Iberian Peninsula and the European Atlantic coast and invasive in American environments. It allelochemicals could be used as natural origin herbicides to inhibit the development of important weeds as barnyardgrass (Echinochloa crus-galli L.) and brachiaria (Urochloa decumbens (Stapf) R.D. Webster). Leaves of artichoke were defatted with hexane at room temperature. The residual vegetal material was extracted with dichloromethane (DCM), ethyl acetate (EtOAc), acetone (acetone) and methanol (MeOH). These four extracts were assayed on etiolated wheat coleoptiles. The ethyl acetate extract had the higher inhibitory activity and therefore, was proved on the germination and growth of standard target species (STS: lettuce, watercress, tomato and onion) and weeds (barnyardgrass and brachiaria). Ethyl acetate extract was very active on root length in both STS and weeds (≥80% at 0.8 mg mL-1), and therefore, was chromatographic fractionated. The spectroscopic studies of the most active fractions showed that major compounds were sesquiterpene lactones with guayanus skeleton: aguerin B, grosheimin, 8α-acetoxialuzanin C, deshydromelitensin, cynaropicrin and 11,13-dihydroxi-8-desoxigrosheimin. Aguerin B, grousheimin and cynaropicrin were very active on etiolated wheat coleoptile, STS and weed growth. The compounds elevated phytotoxicity can explain the bioactivity of the extract and it fractions. Their strong phytotoxicity on important weeds as brachiaria and barnyardgrass shows their strong potential to be used as natural origin herbicides. Keywords: Allelopathy, Aguerin B, Cynaropicrin, Grousheimin, gayanolide sesquiterpene lactones. 61 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 15. STRUCTURAL ELUCIDATION AND ALLELOPATHIC EVIDENCES OF OLEANANE SAPONINS FROM Bellis sylvestris CYR. Monica Scognamiglio, Brigida D’Abrosca, Valeria Severino, Vittorio Fiumano, Nikolaos Tsafantakis, Angela Chambery, Assunta Esposito and Antonio Fiorentino* Department of Environmental, Biological and Pharmaceutical Sciences and Technologies; Second University of Naples; via Vivaldi 43, 81100 Caserta, Italy; e-mail: [email protected] ABSTRACT Objective: In this study, the isolation, structural elucidation and phytotoxicity of six new oleanane saponins from Bellis sylvestris, is reported. Materials and Methods: The structures of saponins were established by the extensive use of 2D-NMR experiments, including COSY, TOCSY, NOESY, HSQC, HMBC, CIGAR, H2BC, and HSQC–TOCSY, along with Q-TOF HRMS2 analysis. Their phytotoxic effects was assayed against Aegilops geniculata Roth., a coexisting test species at three different concentrations: 1 mM, 1 µM and 1 nM. The metabolite profile of the field soil was obtained by NMR analysis. Results and discussion: Six oleanane saponins were isolated for the first time from leaves of the southern daisy. All of the compounds are constituted by bayogenin as aglycone, and characterized by the presence of an oligosaccharide moiety, consisting of two to four sugar unities esterified at the C-28 carboxyl carbon. One of the isolated compounds is a bisdesmoside containing an additional sugar moiety at the C-3 carbon. The phytotoxic activity has been evaluated revealing that all the compounds, at the highest concentrations, showed strong phytotoxicity against the leaf development. Furthermore, an agravitropic response was observed for saponin treated roots. The NMR profile of the methanolic extract of field soil revealed that these compounds are released into the environment. Conclusions: This work demonstrates the inhibiting activity of B. sylvestris saponins against shoot development of the coexisting herbaceous species A. geniculata. The effects on root gravitropism could be related to their interference with auxin metabolism. Keywords: Bellis sylvestris, Saponins, Besylvosides, Spectroscopic analysis, Phytotoxicity TEL: +390823274576; FAX: +390823274571 62 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 16. ISOLATION AND IDENTIFICATION OF A PHYTOTOXIC SUBSTANCE FROM Hyptis suaveolens A K M Mominul Islam a * Osamu Ohno b, Kiyotake Suenaga b and Hisashi Kato‐ Noguchi a a Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, 2393 Ikenobe, Miki, Kagawa 761–0795, Japan b Department of Chemistry, Faculty of Science and Technology, Keio University, 3–14–1 Hiyoshi, Kohoku, Yokohama 223–8522, Japan *Corresponding author: [email protected] ABSTRACT Hyptis suaveolens is an herbaceous weed belonging to Lamiaceae family and grows in clumps. It has been reported that the growth of other plant species near the clumps are quite restricted. Earlier reports also confirmed that H. suaveolens plant extracts and its residues have phytotoxic properties. However, to date no phytotoxic substance has been reported from this plant. Therefore, the present study was undertaken to investigate the presence of phytotoxic substances in H. suaveolens. The aqueous methanol extract of this plant inhibited significantly the seedling growth of garden cress, lettuce, alfalfa, rapeseed, Italian ryegrass, crabgrass, barnyard grass and timothy at concentrations greater than 10 mg dry weight equivalent extract mL-1. These results indicate that the aqueous methanol extract of H. suaveolens is phytotoxic, and thus contains phytotoxic substances. The extract was then purified by several chromatographic runs. A phytotoxic substance was characterized by high-resolution ESI-MS, 1H-, 13C- NMR and specific rotation. The isolated compound inhibited the shoot and root growth of garden cress, Italian ryegrass and barnyard grass, and shoots of lettuce at concentrations greater than 30 µM. Inhibitory activity of the substance was increased with the increase of concentration. The concentration required for 50% shoot and root growth inhibitions (I50) of the test plant species were ranged from 100 – 236 µM and 76 – 1155 µM, respectively. Therefore, the substance is phytotoxic and may be responsible for the phytotoxic activity of H. suaveolens extract. Keywords: Phytotoxicity, Lamiaceae, invasive plant, growth inhibitor TEL: +81-090-2896-1982; FAX: +81-878913086 63 As.cgoo7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 17. ISOLATION AND CHARACTERIZATION OF A NEW BIOACTIVE ALLELOCHEMICAL FROM Tephrosia purpurea LINN. R.N.Yadava1* and Prakrati Yadav2 1 Department of Chemistry,Dr.H.S.Gour Central University, Sagar (M.P); e-mail: [email protected] 2 Department of Chemistry,Dr.H.S.Gour Central University, Sagar (M.P); e-mail: [email protected] ABSTRACT Tephrosia purpurea Linn. (Family Leguminosae), is commonly known as “Sarphonka” in Hindi and found throughout in India. This plant is specifically recommended as a blood purifier and anthalmintic for children. Its roots are bitter and used in the treatment of dyspepsia and also used as a fish poison. The whole plant is used to cure tumors, ulcers, leprosy and inflammatory conditions such as rheumatism, asthma and bronchitis. Its aerial part and roots are used in hepatic ailments, pain and inflammation. It is also used as an antidiabetic, antipyretic, anticancer and antiulcer agent in addition to its usefulness in treatment of diseases related to oxidative stress and free radical activity. It is beneficial for liver spleen and kidney disorder. The present paper deals with the isolation and structural elucidation of a new flavone glycoside [m. p 247-248 °C], m.f. C33H40O20, [M]+ 746 (FABMS) from the methanolic extract of the stems of this plant alongwith two new compounds Apigenin and Kaempferol. A new compound was characterized as 5, 7, 3′, 4′tetrahydroxy-8-methoxyflavone-5-O-α-L-rhamnopyranosyl (1→6)O-β-Dglucopyranosyl(1→3)O-β-D-xylopyranoside by various chemical degradations, color reactions and spectroscopic techniques. This new compound exhibited higher radical scavenging activity in the 1,1-diphenyl-2-pycrylhydrazyl (DPPH) assay system. Keywords: Tephrosia purpurea Linn., Leguminosae, flavone Antioxidant activity. Tel: +91,94256-3568 64 glycoside, 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 18. PHYTOTOXIC POTENTIAL OF ROSMARINIC ACID ON Arabidopsis SEEDLINGS Aitana Costas‐Gil, David López‐González, Carla Díaz‐Tielas, Manuel J Reigosa, Adela Sánchez‐Moreiras Department of Plant Biology and Soil Science Faculty of Biology. University of Vigo Campus Lagoas-Marcosende s/n E-36310, Vigo (Spain); e-mail: [email protected] ABSTRACT Rosmarinic acid is a natural phenolic compound commonly found in plants of the families Boraginaceae and Lamiaceae, and in aquatic monocots of the families Zosteraceae and Potamogetonaceae.1 Although the antibacterial, antiinflammatory, antimutagenic, fungicide, bactericide and antioxidant activity of rosmarinic has been demonstrated,2 its phytotoxic activity has been poorly investigated. The phytotoxicity of this compound was tested on Arabidopsis thaliana (L.) Col0 and Lactuca sativa (L.) cv. Great Lakes seedlings with concentrations of 0, 50, 100, 200, 400, 800 and 1200 µM. In general, the results showed a higher inhibitory effect on Arabidopsis than on Lactuca growth. Rosmarinic treatment caused a strong effect on the root growth of Arabidopsis with a low IC50 of 175 µM. The root structure, thickness and presence of root hairs in Arabidopsis roots were studied under a magnifier. Rosmarinic-treated roots showed a strong tissue disorganization, which resulted in necrotic areas. Ultra-structural analysis (by electron microscopy; TEM) of rosmarinic-treated roots showed higher bigger intercellular spaces and increased secretion of Golgi complexes, which could be the consequence of detoxification processes. As well, rosmarinic treatment resulted in an increased number of vacuoles and broken and condensed mitochondria, which could be related to energy deficit conditions. Measurements of mitochondrial membrane potential with JC-1 fluorochrome (confocal microscopy) confirmed the energy deficit signals. The necrotic areas, confirmed by Trypan Blue staining, together with the decrease of mitochondrial membrane potential suggest that cell death processes are present in rosmarinic treated-roots.3 The strong root growth inhibition, the changes at cellular level and the cell death processes confirmed the strong phytotoxicity of rosmarinic acid, a promising candidate for weed management. 1 Petersen M, Simmonds MSJ (2003) Phytochemistry 62: 121–125. Bais HP, Park S, Weir TL, Callaway R, Vivanco JM (2004) Trends Plant Sci 9: 26–32. 3 Díaz-Tielas C, Graña E, Reigosa MJ, Sánchez-Moreiras AM (2012) Plant Signal Behav 7: 1274–1276. 2 Keywords: rosmarinic acid, Arabidopsis thaliana, phytotoxicity, energy deficit, cell death Tel: +34986812616; fax: +34986812556 65 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 13. BIOASSAY-GUIDED FRACTIONATION AND ISOLATION OF A NATURAL INHIBITOR FROM Raphanus sativus L ON THE GROWTH OF WEED SPECIES. Emy L. Ishii‐Iwamoto1*, Gislaine C. Mantovanelli1, Letycia L.R. Fiorucci2 Mauro C. Barbosa1, Fernanda L. Kagami1, Márcio S. Mito1, Jéssica D. Almeida1, Darlon I. Bernardi2, Rubem S.Oliveira Jr3, Jamil Constantin3 and Debora C. Baldoqui2 1 Department of Biochemistry, University of Maringá, Paraná, Brazil;* [email protected] 2 3 Department of Chemistry, University of Maringá, Paraná, Brazil; Department of Agronomy, University of Maringá, Paraná, Brazil. ABSTRACT Mulches of Raphanus sativus L. used in no-till systems affect negatively the germination and establishment of weed seeds. This species are rich in compounds like polyphenols, glucosinolates and saponins, but the identity of phytochemicals involved in the weed suppression is unknown. In the present work, a bioassay-guided identification of phytotoxic compounds of R. sativus on the weed species Euphorbia heterophylla, Ipomoea grandifolia and Bidens pilosa was conducted. The aerial parts of the R. sativus were extracted with solvents of varying polarity (hexane, dichloromethane, ethyl acetate and butanol). The weed seeds were incubated in the presence of the partitioned fractions (250 – 1000 µg/mL) in a growth chamber, at 25 or 30°C, and at variable time intervals. The dichloromethane fraction was the most active among the partitioned fractions. The ethyl acetate fraction was less active and the other ones did not exert significant effects. The growth of primary roots was the most sensitive variable. The calculated IC50 for primary root and stem growth inhibition of I. grandifolia was 153.8 ± 4.9 µg/mL and 430.0 ± 83.4 µg/mL, respectively. The dichloromethane fraction was submitted to successive chromatographic columns to afford dehydrovomifoliol, which was identified by comparison of their spectroscopic data with those reported in the literature. Dehydrovomifoliol, that was isolated for the first time in aerial parts of R. sativus, is structurally related to abscisic acid. An interference on the balance between phytohormones is a possible mechanism for herbicidal action that deserves to be investigated in future works. Keywords: Raphanus spectroscopy. sativus, weeds, TEL: +55-44-30114712; FAX: +55-44-30114896 66 dehydrovomifoliol, mulches, 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 14. IDENTIFICATION OF ALLELOCHEMICALS IN GARLIC STALK DECOMPOSITION 1 Xu Han1, 2, Zhihui Cheng1,*, Huanwen Meng1 College of Horticulture, Northwest A & F University, Yangling, Shaanxi, 712100, China. 2 Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences, Danzhou, Hainan, 571737, China. Xu Han: E-mail:[email protected]; Zhihui Cheng: E-mail: [email protected]; Huanwen Meng: E-mail: [email protected] ABSTRACT Garlic (Allium sativum L.) stalk is a byproduct of garlic production that is normally thought of as waste but is now considered to be a useful biological resource. It is necessary to utilize this resource efficiently and reasonably to reduce environmental pollution and achieve sustainable agricultural development. The allelochemicals in garlic stalk decomposition were identified by the method of GC-MS in this research, and allelophaty of the allelochemicals was verified by bioassay using lettuce as the test plant. Garlic stalk resolved or created new allelochemicals during it decomposing. Caffeine, N-à-(tertButoxycarbonyl)-L-Valine and diflunisal were detected from petroleum ether, ethyl acetate and chloroform extractions of garlic stalk decomposition, respectively. The petroleum ether and ethyl acetate extractions had the favorable allelopathy on lettuce bioassay, but the chloroform extractions had the inhibitive allelopathy. The results of bioassays of exogenous chemical reference substance showed that higher concentration of caffeine, N-à-(tertButoxycarbonyl) -L-Valine and diflunisal had the inhibitive allelopathy on lettuce bioassay. The results were the similar as the results of different organic reagent extractions and the general rule of allelopathy. It is concluded that the three substances were verified the effective allelochemicals of garlic stalk decomposition. Keywords: allelochemicals; garlic stalk decomposition; identification Tel: +86-29-87091332; Fax: +86-29-87082613 67 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 15. PHYTOTOXIC POTENTIAL OF Artemisia arborescens: BIOGUIDED FRACTIONATION ASSAY AND PHYTOCHEMICAL CHARACTERIZATION OF LITTER 1 Araniti F.1, Gullì T.1, Marelli M.2, Statti G.2, Gelsomino A.1, Abenavoli M.R.1 Università degli Studi “Mediterranea” di Reggio Calabria Feo di Vito I-89124, Reggio Calabria, Italy; [email protected] 2 Università della Calabria, via Pietro Bucci, I-87036 Rende (CS) ABSTRACT Artemisia arborescens L. (Asteraceae family) is a pioneer defoliant Mediterranean shrub characterized by high growth rate and abundant production of leaf litter. Under natural conditions, the decaying of leaf litter at the soil surface could release biologically active compounds representing an important ecological strategy for new area colonization. In the present study, the leaf litter of A. arborescens has been investigated for its potential phytotoxicity through a bioguided-fractionation assay and characterized by using HTPLC and GC-MS. After collection, leaf litter was extracted through an hydroalchoolic maceration (methanol:water, 9:1) and then sequentially fractionated using three solvents with increasing polarity: hexane, chloroform and ethyl acetate. The methanolic extract and each fraction were assayed in vitro on both seed germination and root growth of lettuce (Lactuca sativa L.) and two weeds (Amaranthus retroflexus L. and Cynodon dactylon L. ) that generally colonized the same plant habitat of A. arborescens. Estimates of ED50 parameters were obtained by fitting data into non linear regression model. Germination and root growth of lettuce were strongly inhibited by all the fractions showing the following hierarchy of phytotoxicity for both physiological processes: chloroform ≥ ethyl acetate > hexane. Moreover, phytochemical characterization pointed out an high presence of terpenes, fatty acids, lignans, flavonoids and phenolic acids. These results make A. arborescens litter a source of active phytotoxic compounds possibly responsible of its high colonization ability. Keywords Artemisia arborescens, leaf litter, phytotoxicity, phytochemical characterization Tel: +39 0965 324077; Fax: +39 0965 311092 68 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 16. PHYTOTOXIC POTENTIAL OF EXTRACTS OF Ocotea pulchella NEES ET MART 1 Lafayette P. Candido 1, Rosa M. Varela1, Ascensión Torres1, José M. G. Molinillo1, Sonia C. J. Gualtieri, Francisco A. Macías1* Grupo de Alelopatía, Departamento de Química Orgánica, Facultad de Ciencias, Instituto de Biomoléculas (INBIO), Universidad de Cádiz, Campus de Excelencia Internacional Agroalimentario (ceiA3), C/República Saharaui, s/n, 11510 Puerto Real, Cádiz, Spain; e-mail: [email protected] 2 Departamento de Botanica, Universidade Federal de São Carlos, Rodovia Washington Luís, km 235,São Carlos – São Paulo, CEP 13565-905, Brazil ABSTRACT Few studies have been described the chemical profile or evaluated the biological activity of Brazilian Savannah (cerrado) species. The genus Ocotea has aroused greater interest of Brazilian phytochemicals due to their wide distribution in national territory. This work had the objective study phytochemical and biological activity of extracts leaves of Ocotea pulchella. Extracts of leaves, stem and roots were obtained with different solvents (dichloromethane, ethyl acetate, acetone, methanol), and evaluated in bioassays wheat coleoptile and seed germination. The leaf extracts EtOAc at three concentrations, showing that at 0,8 mg.mL1 inhibited significantly the wheat coleoptiles growth. This extract was tested on germination and seedling growth of model species (STS) (lettuce, cress, tomato and onion) and weeds (Echinochloa cruss-galli and Brachiaria sp.).The results showed that ethyl acetate leaf extract of O. pulchella was active on STS. The most affected parameter was root growth with inhibitions higher than 40%, being tomato the most sensitive species with inhibitions higher than 60% on root and shoot growth. Regarding the weed Brachiaria sp., a remarkable stimulation of germination (60%) was observed. Until now, compounds isolated from the most active fractions are 1β,6α-dihydroxy-eudesm-4(5)ene, (-)-loliolide and spathulenol. Keywords: Ocotea pulchella, Standar target species (STS), Brachiaria. Phone: + 34956012770; fax: +34956016193 69 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 17. PHYTOTOXIC ACTIVITY OF LEAF, STEM AND ROOT EXTRACTS OF Tithonia diversifolia ON GERMINATION AND SEEDLING GROWTH Maria A.F.M. Miranda1, Rosa M. Varela1, Ascensión Torres1, José M. G. Molinillo1, Sonia C. J. Gualtieri2, Francisco A. Macías1* 1 Grupo de Alelopatía, Departamento de Química Orgánica, Facultad de Ciencias, Instituto de Biomoléculas (INBIO), Universidad de Cádiz, Campus de Excelencia Internacional Agroalimentario (ceiA3), C/República Saharaui, s/n, 11510 Puerto Real, Cádiz, Spain; e-mail: [email protected] 2 Departamento de Botanica, Universidade Federal de São Carlos, Rodovia Washington Luís, km 235,São Carlos – São Paulo, CEP 13565-905, Brazil ABSTRACT Tithonia diversifolia (Hemsl.) A. Gray (Mexican sunflower) is a perennial plant of the family Asteraciae, native to Mexico and Central America (TONGMA et al., 1999). This plant grows rapidly and it’s a dominant plant. This suggests a strong competitive ability and possible allelopathic activity on growth of other plants under natural conditions. The present study was conducted to determine the phytotoxic activity of T. diversifolia extracts. For that, were prepared extracts of leaves, stems and roots, using a polar solvent (Methanol) and a medium polarity solvent (Ethyl acetate) in with were carried out under wheat coleoptile and seedlings growing. We used target plants (lettuce, sugar-beet, tomato and onion) and weeds (barnyard grass and brachiaria). For the wheat coleoptiles and seedlings growing bioassays the extracts were tested at concentrations of 0.8, 0.4 and 0.2 mg mL-1. The results showed that Ethyl acetate extracts of leaves, stems and roots inhibited significantly the wheat coleoptiles growth over control, whereas the leaf extract at 0.8 and 0.4 mg mL-1 had similar inhibitory effects to the herbicide Logran. In the seedlings growth bioassay, the results showed that Ethyl acetate extracts of leaves had a high activity on the roots both as target plants to weeds, approximately 90% of inhibition at 0.8 mg ml-1). Until now, from this extract Tagitinin C, Tagitinin A, 1β-methoxydiversifolin have been isolated. The high phytotoxic activity of this extracts may be responsible for its dominance and may thus affect the development process of native species, ensuring their establishment in the environment. Keywords: Phytotoxicity, alellopathy, plant extracts Phone: + 34956012770; fax: +34956016193 70 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 18. TRANS-CINNAMALDEHYDE, A PHENOLIC COMPOUND WITH PHYTOTOXIC ACTIVITY David López‐González, Aitana Costas‐Gil, Elisa Graña, Carla Díaz‐Tielas, Ana Martínez‐Peñalver, Manuel J Reigosa, Adela M Sánchez‐Moreiras Faculty of Biology University of Vigo, Campus Lagoas-Marcosende s/n, E-36310, Vigo, Spain; email: [email protected] ABSTRACT Trans-cinnamaldehyde is a phenolic compound that is naturally synthesized as secondary metabolite in species of the family Lauraceae, like Cinnamomun osmophloeum and C. zeylanicum.1 It is used as flavouring in foods and has demonstrated cytotoxic, fungicide and antimicrobial activities,2 but its phytotoxic activity was not deeply investigated. Thereby, this work was aimed to establish the phytotoxic potential of this compound on the model species Arabidopsis thaliana (L.) Col-0 by treating Arabidopsis seedlings with different trans-cinnamaldehyde concentrations (from 0 to 1250 µM) to establish its phytotoxic potential. Arabidopsis root growth was highly affected by trans-cinnamaldehyde treatment showing a so low IC50 concentration as 46 µM, while the germination process was completely inhibited with 800 µM. Trans-cinnamaldehyde-treated roots showed increased number of root hairs, loosening of root dominance and a high proliferation with a batch of several roots, which made difficult to distinguish the main and the secondary roots. This high proliferation of roots could be due to an alteration in the auxins/cytokinins balance. Structural analysis by light and transmission electron microscopy of 5 and 10 days-old trans-cinnamaldehydetreated Arabidopsis roots showed important disturbances compared to the control, as abnormal nucleus shape, fragmented chromatin and increased number of mitochondria, which could be related to programmed cell death processes. Auxin quantification by ELISA, measurements of mitochondrial membrane potential with JC-1 and cell death staining with Trypan Blue were done in order to better know the effects and the mode of action of this secondary metabolite on plant metabolism. The strong phytotoxicity of trans-cinnamaldehyde on Arabidopsis suggests the potential of this molecule for weed management. 1 Fang S-H, Rao YK, Tzeng Y-M (2004) Int J Appl Sci Eng 2: 136-147. 2 Gill AO, Holley RA (2004) Appl Environ Mic 71 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 19. SCOPOLETIN, A NATURAL COMPOUND WITH PHYTOTOXIC EFFECT Aitana Costas‐Gil, Sabela Longueira, Luz Cabeiras, Elisa Graña, Manuel J Reigosa, Adela Sánchez‐Moreiras. Department of Plant Biology and Soil Science Faculty of Biology. University of Vigo Campus Lagoas-Marcosende s/n E-36310, Vigo (Spain); e-mail: [email protected] ABSTRACT Scopoletin is a coumarin found in roots of plants of the genus Scopolia and members of the family Solanaceae.1 This natural compound has antiinflamatory, antiproliferative, antioxidant and bactericide activities,1-3 but its phytotoxic effect has been poorly investigated. Therefore, in this study, scopoletin phytotoxicity has been evaluated on model species Arabidopsis thaliana. The results showed a high inhibitory effect in root growth with an IC50 value of 100 µM. Under magnifier, Arabidopsis treated-roots showed an increased number of secondary roots with a necrotic appearance of the main root. This result could be explained by a possible hormonal unbalance due to the action of this coumarin. Therefore, effects on hormone balance (auxin and ethylene) in 14 days old A. thaliana seedlings were evaluated. Ultra-structural analysis by electron microscopy (TEM) in Arabidopsis roots treated with the IC50 of scopoletin, showed detoxification signs, such as increased activity of Golgi apparatus, increased exocytosis, and an increment of cell wall deposits. Alteration of cell division was also detected, evidenced by alterations in the division patterns, the presence of binucleated cells and condensed chromatin. This suggests a possible microtubule alteration, which was confirmed by immunofluorescence. Moreover, Trypan Blue staining was used to evaluate the presence of cell death. The effects at cellular level and the strong root growth inhibition in Arabidopsis seedlings confirmed the phytotoxic activity potential of scopoletin, although deepest study will help to elucidate the mode of action of this compound. 1 Rollinger JM, Hornick A, Langer T, Stuppner H, Prast H (2004) J Med Chem 47: 6248–6254. 2 Kayser O, Kolodziej H (1997) Planta Med 63: 508–510. 3 Shaw C-Y, Chen C-H, Hsu C-C, Chen C-C, Tsai Y-C (2003) Phytother Res 17: 823– 825. Keywords: scopoletin, phytotoxicity, Arabidopsis thaliana, microtubules, cell death, mode of action, Tel: +34986812616; fax: +34986812556 72 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 20 PHYTOTOXIC ACTIVITY OF 2-AMINOPHENOXAZIN-3-ONE (APO) ON Arabidopsis thaliana SEEDLINGS Ana Martínez‐Peñalver, Elisa Graña, Paula Baltasar, Carla Díaz‐Tielas, Adela M Sánchez‐Moreiras, Manuel J Reigosa Faculty of Biology, University of Vigo, Campus Lagoas-Marcosende s/n, E-36310, Vigo, Spain; e-mail: [email protected] ABSTRACT APO (2-Aminophenoxazin-3-one) is a compound belonging to the group of benzoxazolinones, specifically produced by the degradation of the benzoxazolinone BOA1, a secondary metabolite mainly found in the families Poaceae, Acanthaceae, Ranunculaceae and Scrophulaceae. Previous works2 have suggested a strong phytotoxic potential for this compound, although no deeper studies have been still done. Therefore, the objective of this work is to study the phytotoxic potential of APO on Arabidopsis thaliana (L.) Col-0 seedlings and to give light on its effects on plant metabolism. A dose-response curve of the root growth and a study of its root structure was carried out with a structural and ultra-structural analysis by magnifying glass, light and TEM microscope. The dose-response curve was done with 0, 0.39, 0.781, 1.562, 3.125, 6.25, 12.5 and 25 µM APO, obtaining Inhibitory Concentrations for root growth as low as IC50 of 7.25 µM and IC80 of 25.06 µM for Arabidopsis root length, strongly down when compared to the IC50 of BOA2 (619.1 µM). Furthermore, the APO-treated roots showed important alterations as increased number of lateral roots and a strong density of roots hairs. The ultra-structural analysis showed several differences compared to the control both at structural and cellular level, being more evident at 7 than 14 days of treatment. Treated roots showed irregular walls (thickening and zig-zag signals), slack and broken up amyloplasts, electrodense deposits in cell corners, numerous secretion points and depositions in the cell walls, fragmented chromatin, distorted nuclei (possible microtubules alteration) and highly vesiculated Golgi complex, probably due to detoxification processes. The strong root growth inhibition and the ultra-structural alterations in Arabidopsis seedlings confirmed the high phytotoxicity of this secondary metabolite. 1 Macías FA, Marín D, Oliveros-Bastidas A, Marín D, Castellano D, Simonet AM, Molinillo JMG (2005) J Agric Food Chem 53: 554-561. 2 Macías FA, Marín D, Oliveros-Bastidas A, Simonet AM, Molinillo JMG (2007) In: Fujii Y. & Hiradate S. (eds.) Allelopathy. New concepts and methodology. Science publishers. Enfield. USA. pp. 91-108. 3 Reigosa MJ, Malvido-Pazos E (2007) J Chem Ecol 33(7): 1456–1466. Keywords: 2-Aminophenoxazin-3-one, APO, phytotoxicity, Arabidopsis, mode of action Tel: +34 986 818 714; fax: +34 986 812 556 73 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 19. IDENTIFICATION AND LOCALIZATION OF ISOHEXENYLNAPHTHAZARINS IN PRIMARY AND SECONDARY ROOTS OF PATERSON’S CURSE (Echium plantagineum) Xiaocheng Zhu1, Brigette Ryan1, Dmitry V. Sokolov1, Geoff M. Gurr2 and Leslie A. Weston*1 1 Charles Sturt University, Graham Centre for Agricultural Innovation, Life Sciences Building, Wagga Wagga NSW 2678 Australia; [email protected] 2 Charles Sturt University, School of Agriculture and Wine Science, Orange NSW 2800 Australia; [email protected] ABSTRACT Isohexenylnaphthazarins, also referred to as naphthoquinones (NQs), are lipophilic red pigments derived from shikonin/alkannin. They have been identified in the roots of several plants of the Boragineaceae family. This chiral pair has been widely studied as potent pharmaceuticals with associated antimicrobial, anti-inflammatory and anticancer activities. We have first identified isohexenylnaphthazarins in the invasive species Echium plantagineum L., now widespread across Australia and native to the Mediterranean. Their roles in plant defense including herbivory, allelopathy and plant invasion are now under investigation. Little is known about the biosynthesis and localization of these compounds in E. plantagineum roots. We identified various NQs in ethanolic extracts of root periderm, which exhibited significant activity in plant growth inhibition of a common local weed, annual ryegrass (Lolium rigidum Lam.). Using bright field and confocal fluorescence imaging, we examined fresh root and shoot tissues for the presence of NQs, and found them to be localized in highest concentration in root periderm tissues. In the periderm, they were found in cytoplasmic vesicles and appear to be deposited mainly in the plant cell wall or in intracellular space between the cell wall and plasmalemma. NQs inherently exhibit unique autofluorescence at specific wavelengths of light. Spectral imaging was performed to assist in localization by comparison to known NQ standards such as shikonin. Similarly coloured red pigments appear to be associated with cells below tubercles (trichomes with red bases) on leaf and stem surfaces. The tubercle structure and its associated chemistry is currently under investigation. Keywords: naphthoquinones, Boraginaceae, shikonin, light and confocal microscopy, spectral imaging, absorbance, fluorescence Tel: +61 (2)69334689 ; fax: +61 (2)69332429 74 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 20. STUDIES TOWARDS SYNTHESIS OF ACTIVE GUAIANOLIDE-TYPE ALLELOCHEMICALS Alejandro Santana1, José M. G. Molinillo1, Frank R. Fronczek2, Rosa M. Varela1, Francisco A. Macías1* 1 Grupo de Alelopatía, Departamento de Química Orgánica, Facultad de Ciencias, Instituto de Biomoléculas (INBIO), Universidad de Cádiz, Campus de Excelencia Internacional Agroalimentario (ceiA3), C/República Saharaui, s/n, 11510 Puerto Real, Cádiz, Spain; e-mail: [email protected] 2 Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803, U.S.A. ABSTRACT Sesquiterpene lactones (SLs) have been reported to possess anti-inflammatory, antiprotozoal, cytotoxic and phytotoxic activity, being particularly abundant in plants of the family Compositae. Among various classes of allelochemicals screened for weed suppressing ability, sesquiterpene lactones form an important group because of their wide spectrum of biological activities and structure-activity relationship From the endemic Cape Verdean Artemisia gorgonum, seco-guaianolide 1 was isolated, with good antimalarial activity. In a previous work, we described the total synthesis of this compound and tested it on the wheat coleoptile bioassay, as well as on STS phytotoxicity bioassay, showing high activity levels. Retrosynthetic analysis scheme of seco-guaianolide 4-5. Considering the promising results obtained, these natural products can serve as excellent leads for herbicides. More so if we consider that many of the biological activities reported for the SLs are attributed to covalent bonding to the α,β-unsaturated carbonyl groups. Our interest is focused on developing a series of derivatives with the α-methylene-γ-lactone being really interesting as a Michael addition fashion. Total synthesis and phytotoxic activity will be presented showing an interesting approach for achieve the desired products and good levels of activity obtained as expected. Keywords: secoguaianolides, sesquiterpene lactones, allelophaty, phytotoxicity Tel: +34 956012769; fax: +34 956016193 75 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 21. SPECIOSIN G AND P FROM Hexagonia speciosa: SYNTHESIS AND BIOACTIVITIES Nuria Chinchilla, Guillermo A. Guerrero‐Vásquez, José M. G. Molinillo and Francisco A. Macías* Grupo de Alelopatía, Departamento de Química Orgánica, Facultad de Ciencias, Instituto de Biomoléculas (INBIO), Universidad de Cádiz, Campus de Excelencia Internacional Agroalimentario (ceiA3), C/República Saharaui, s/n, 11510 Puerto Real, Cádiz, Spain; e-mail: [email protected] ABSTRACT Speciosins have been isolated for the first time in 2009 from the chinese fungus basiodiomycete Hexagonia speciosa. Their structures, which consist in oxygenated cyclohexanoids, possess a wide range of bioactivities. We have a particular interest in speciosin G (1) and P (2) because are structurally similar to siccayne (3) isolated from deuteromycete Helniinthosporium siccans in 1968. Reports made of siccayne showed that it is a moderately active antibiotic and inhibits mitochondrial respiration in Saccharomyces cerevisiae. With this background, in this work has raised the first total synthesis of these compounds. They have been prepared via Sonogashira coupling from readily available starting materials. If speciosins P and G could offer properties similar to siccayne would do speciosins as an important future target in the development of drugs and ecoherbicides. These facts provided sufficient reason to develop an efficient synthetic route to access them. In addition, our group has proposed a preliminary study on etiolated wheat coleoptiles to demonstrate the preliminary bioactivities in all the intermediates generated in the process of total synthesis and some derivatives. Keywords: Speciosins, synthesis, bioassay, activities. Tel: +34 956012769; fax: +34 956016193 76 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 21. FUROCOUMARINS: ALLELOCHEMICALS OF HERACLEUM SOSNOWSKYI MANDEN SEEDS Mishyna Maryia1, Laman Nikolai2, Prokhorov Valery2 and Fujii Yoshiharu1* 1 Tokyo University of Agriculture and Technology, Tokyo, Japan; e-mail: [email protected] 2 Institute of Experimental Botany, Minsk, Belarus ABSTRACT Endemic to the Caucasus mountains, Heracleum sosnowskyi Manden (Apiaceae) is now widely distributed in Baltic countries, Belarus and Russia as invasive weed. The present research was conducted to examine whether allelopathy mediated by the seeds is involved in the invasiveness of Heracleum sosnowskyi. Heracleum plants are known to contain high amounts of furocoumarins. Our research focused on the identification and quantification of furocoumarins in Heracleum seeds and its growth inhibitory properties. Seeds of Heracleum sosnowskyi were collected in August-September 2012 from different locations in Belarus and western Russia. Cold stratification treatment (90 days, 2-3°C) was applied to facilitate germination. Stratified and non-stratified seed extracts were used for lettuce growth inhibitory assay. Primary screening revealed high inhibitory property of the seed coat be it stratified (EC50=1.4 g/L) or not (EC50=1.8 g/L). HPLC analysis confirmed that furocoumarins are highly concentrated in the seed coat compared to other parts. Angelicin was found to be the predominant compound in the seed coat at a concentration 11.8-26.3 mg/g FW. While in the endosperm, the compound was found at 2.7-11.1 mg/g FW. Cold stratification treatment decreased the concentration of furocoumarins in the seed coat. Angelicin content in seed coat declined 2.1-5.4 folds compared to that of non-stratified seeds concomitant with a weakened growth inhibitory property. Results may suggest the role of angelicin in the overall phytotoxity of Heracleum. We hereby show that furocoumarins in seeds of the invasive Heracleum sosnowksyi plants impart allelopathic effect on the growth of lettuce seedlings. Keywords: Heracleum sosnowskyi, allelochemicals, furocoumarins, angelicin Tel: +81-42-367-5625; fax: +81-42-367-5625 77 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 22. THEORETICAL STUDY OF THE ALLELOCHEMICAL DEHYDROCOSTUSLACTONE IN CYCLODEXTRINS INCLUSION COMPLEXES. 1 Antonio Cala1, José A. A. Saura2, Jesús Ayuso, José M. G. Molinillo1 and Francisco A. Macías1,* Grupo de Alelopatía, Departamento de Química Orgánica, Facultad de Ciencias, Instituto de Biomoléculas (INBIO), Universidad de Cádiz, Campus de Excelencia Internacional Agroalimentario (ceiA3), C/República Saharaui, s/n, 11510 Puerto Real, Cádiz, Spain; e-mail: [email protected] 2 Departamento de Química Física ABSTRACT In a previous communication, we presented the study of the synthesis and evaluation of phytotoxic activity of inclusion complexes of the allelochemical dehydrocostuslactone (DHC) in natural cyclodextrins, where the advantages of the encapsulation of this compound were reported. In this report, we describe at the molecular level the main parameters that control the molecular transport with DHC in cyclodextrins. Computational studies have been performed at different levels: molecular mechanics (MM+, Amber), semiempirical (AM1, PM3), and because of the size of the cyclodextrins and the nature of the complexes, at ab initio level, HF (6-311G*) and DFT (B3LYP, 6-31G*) have been performed. Better results were obtained with molecular mechanics and semiempirical calculations, so a deep study with MP2 about the molecular mechanisms of equilibrium complex formation and release of DHC with cyclodextrins is undergoing. Keywords: dehydrocostuslactone; theoretical studies; Tel: +34 956012770; fax: +34 956016193 78 cyclodextrins; inclusion complexes; 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 23. SYNERGISTIC INTERACTIONS OF PHENOLIC COMPUNDS FROM CITRUS SINENSIS FRUIT PEEL EXTRACT Benito Fernández, Ascensión Torres, Jose L. G. Galindo, and Francisco A. Macías* Grupo de Alelopatía, Departamento de Química Orgánica, Facultad de Ciencias, Instituto de Biomoléculas (INBIO), Universidad de Cádiz, Campus de Excelencia Internacional Agroalimentario (ceiA3), C/República Saharaui, s/n, 11510 Puerto Real, Cádiz, Spain; e-mail: [email protected] ABSTRACT Synergistic interactions are of vital importance in phytomedicines, to explain difficulties in always isolating a single active compound, and explain the efficacy of apparently low doses of active constituents in plant products. This concept, that a whole or partially purified extract of a plant offers advantages over a single isolate compound, also intensifies in general the philosophy of bioactivity plants. It is likewise common practice in the study of plant extracts, or other raw materials as byproducts or waste from the food industry, assigning a chemical compound responsible for the activity that presents these extracts. This approach can lead to inconclusive results, since during the purification process activity in successive fractions and finally pure compounds may decay and even disappear. Through bioactivity-guided isolation is possible to observe changes in the activity of extract, fractions and purified compounds, being able to suspect the presence of interactions between chemicals. A first approach is through the study of the interaction of binary mixtures, from compounds that have been isolated in different fractions with different profiles of activity, can provide information of how these compounds may be interacting in such fractions. The present study has been made bioactivity-guided isolation of extracts obtained from waste of the citrus industry, which have insecticidal and fungicidal activity. Through bioactivity-guided isolation using bioassay of coleoptile etiolated wheat, extracts, fractions and purified compounds have interesting profiles of activities, noting that some extracts and fractions have effect levels with little variation with decreasing dose, a fact that you can suspect the presence of interactions between compounds. For possible interactions between compounds, study has been made of binary mixtures of polymethoxyflavones, major compounds present in most of the fractions. It has been used for bioassay coleoptile etiolated wheat and isobole method, representing qualitatively with isobolograms and quantitatively through the fractional inhibitory concentration (FIC). Keywords: citrus waste, poymethoxyflavones, isobolograms, synergism, antagonism and additive action, bioactivity. Tel: +34 956012770; fax: +34 956016193 79 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 24. SPIROSTANE SAPONINS FROM Furcraea hexapetala. PHYTOTOXICITY ON STS Juan M. Calle1, Andy J. Pérez2, Ana M. Simonet1, José O. Guerra3 and Francisco A. Macías1*. 1 Grupo de Alelopatía, Departamento de Química Orgánica, Facultad de Ciencias, Universidad de Cádiz, Campus de Excelencia Internacional Agroalimentario, ceiA3, Institute of Biomolecules (INBIO). C/ República Saharaui s/n, Apdo. 40, 11510-Puerto Real (Cádiz), Spain. [email protected] 2 Department of Biochemistry, Institute of Soil Science and Plant Cultivation, State Research Institute, ul. Czartoryskich 8, 24-100, Puławy, Poland. 3 Departamento de Licenciatura en Química, Facultad de Química y Farmacia, Universidad Central “Marta Abreu” de Las Villas, Carretera a Camajuaní km 5.5, 54830 Santa Clara, Cuba. ABSTRACT The Agavaceae family includes more than 480 species that occur natively in America. The major natural products isolated in those plants are steroidal saponins, to which various biological properties such as haemolytic, molluscicide, anti-inflammatory, antifungal, anti-bacterial, antiplatelet aggregation and cytotoxic have been attributed. However, few studies have reported phytotoxicity for such metabolites. Furcraea hexapetala is an Agavaceae plant present in Cuba without previous phytochemistry studies. A guided bioassay fractionation of their leaves allowed the isolation of fifteen spirostanol saponins, four of which have not been described previously. The structures of new compounds were determined by comprehensive spectroscopic analysis, including one- and two-dimensional NMR techniques, mass spectrometry and chemical methods. The phytotoxicity of the isolated compounds on the standard target specie Lactuca sativa L. was evaluated. Keywords: Allelopathy, spirostane saponins, Furcraea hexapetala, Lactuca sativa Phone: +34 956012770; fax: +34 956016193. 80 Session 3. Ecological aspects of allelopathy Ecology of allelopathy Allelopathy and climate change Invasive plants: possible role of secondary metabolites Soil and rhizosphere ecology 81 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 22. Examination of Rhizo- and Endosphere Microbiome Composition as Affected by Developmental Stage and Allelochemical Status in S. bicolor Scott R. Baerson1, Zhiqiang Pan1, Colin R. Jackson2, Megan A. Rúa2, Jason D. Hoeksema2 1 United States Department of Agriculture, Agricultural Research Service, Natural Products Utilization Research Unit, P.O. Box 8048, University, MS 38677, USA. 2 Department of Biology, University of Mississippi, University, MS 38677, USA. ABSTRACT The thousands of diverse microbial taxa closely-associated with plant root systems, collectively referred to as 'microbiomes', play a critical role in determining overall plant vigor. Additionally, recent studies have indicated that the chemical constituents of plant root exudates play a significant role in shaping the composition of these microbial communities. One of the predominant root exudate components found in Sorghum spp. is the lipid benzoquinone sorgoleone (5-[(8’Z,11’Z)-8’,11’,14’- pentadecatrienyl]resorcinol), which likely accounts for much of the allelopathic properties associated with this genus. While numerous studies have indicated a major role for sorgoleone in plant-plant allelopathic interactions, several reports have also documented significant antimicrobial activity for this compound. To further explore the potential influence of sorgoleone rhizosecretion on microbiomes, the compositions of both rhizosphere and endophytic communities of transgenic sorghum events engineered for loss of sorgoleone production were compared with both wild-type and null-segregant control plants at two different developmental stages. DNA samples prepared from rhizosphere soil fractions as well as surface-sterilized root tissues were used as templates in PCR reactions with primers targeting conserved fungal and bacterial rDNA gene sequences, and the resulting amplicon libraries were subjected to pyrosequence analysis. Comparisons of microbial community structures in flowering as well as post-flowering S. bicolor (genotype Tx430) individuals, as well as insights obtained concerning the potential effects of sorgoleone on these communities will be presented. Keywords: Sorgoleone, microbiomes, root exudates 82 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 23. ALLELOPATHIC RESEARCH IN MÉXICO: FROM PLANTS TO ENDOPHYTIC FUNGI Ana Luisa Anaya1*, Blanca E. Hernández, Rocío Cruz‐Ortega, Martha L. Macías‐Rubalcaba2, and María C. González3 1 Instituto de Ecología, Universidad Autónoma de México (UNAM); e-mail: [email protected] 2 Instituto de Química, UNAM; e-mail: [email protected] 3 Instituto de Biología, UNAM; e-mail: [email protected] ABSTRACT We look for the defense role of the diversity of secondary metabolites of Mexican biodiversity that could avoid the emergence of resistance in pathogens and weeds. We look for promising allelopathics that could rehabilitate pesticides, antibiotics or chemo therapeutics. We performed allelopathic studies in humid and dry natural tropical ecosystems and in different temperate agroecosystems. In tropical forests of Yucatan we found allelopathic plants as Stauranthus perforatus (Rutaceae) which diversity of compounds has selected bioactivity: inhibited root growth of weeds, and kill corn weevil. Selective bioactivity of tropical trees allelopathics, inhibited the root growth of weeds but stimulated that of others, and also stimulated arbuscular mycorrhizae and Rhizobium in the roots. We follow our allelopathic research looking now in endophytic fungi (EF) of selected allelopathic plants from Yucatan tropical forests. We discovered new genus and species of EF in the leaves of some of these trees. We isolated naphthoquinone spiroketals, including three new compounds and palmarumycin from a novel EF Edenia gomez-pompae. Spiroketals 1–3 displayed significant growth inhibition against phytopathogenic fungi (FF). A new species of Muscodor, M. yucatanensis, produced a mixture of volatile organic compounds (VOCs) with allelochemical effects against other EF, FF, fungoids, and plants. VOCs were lethal to many of the test species, but they have not autotoxic effects. VOCs inhibited also root elongation in some weeds. We discussed the relevance of allelopathy studies in the knowledge of many biological and ecological processes, and the benefits that we can obtain from this basic knowledge. Keywords: Allelopathics, tropical forests, selective bioactivity, biodiversity, chemodiversity. Tel: + 52 (55) 5622 9032; fax: +52 (55) 5622 1976 83 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 24. ROOT EXUDATION OF LIPOPHILIC NAPHTHOQUINONES BY PATERSON’S CURSE: A CLUE TO THEIR ECOLOGICAL ROLE? Jeffrey D. Weidenhamer1,2*, Xiaocheng Zhu1, Dominik Skoneczny1, James Mwendwa, Paul A. Weston1 and Leslie A. Weston1 1 E.H. Graham Centre, Charles Sturt University, Wagga Wagga, NSW 2678 Australia Department of Chemistry, Geology and Physics, Ashland University, Ashland, OH 44805 USA; e-mail: [email protected]; [email protected]; [email protected]; 2 [email protected]:[email protected]; [email protected]; [email protected]. ABSTRACT Roots of Paterson’s curse (Echium plantagineum) produce an array of coloured and colourless naphtho- and anthraquinones in their roots. Under certain environmental conditions, young roots produce large quantities of naphthoquinones in the outer layers of root periderm, and root hairs of seedlings exude droplets of dark red naphthoquinones. Polydimethylsiloxane (PDMS) microtubing is a useful tool to measure lipophilic allelochemicals in soil, and has been used in several forms to probe the release of these compounds directly from Paterson’s curse roots, both in and out of soil growth media. Droplets of exudate have been collected directly from root hairs, and solid phase root zone extraction probes (constructed from silicone tubing mounted on a stainless steel wire core) placed in soil around Paterson’s curse plants have also been used to measure the release of these compounds from roots. Further metabolic profiling studies using LC-MS QToF (HPLC coupled to time of flight mass spectrometry) are underway to establish the specific composition of the root exudates and quantities of compounds being released. Our results demonstrate polydimethylsiloxane in various forms is a useful tool to monitor the release of nonpolar to moderately polar allelochemicals into soil. These naphthoquinones show potent antimicrobial, fungitoxic and phytotoxic activity due to their impact on electron transport and cellular respiration processes. Measurement of the dynamics of their release into the soil environment will be valuable in assessing the ecological role of these compounds. Keywords: Allelopathy, diffusive sampling, Echium plantagineum, PDMS, rhizosphere, root exudation, soil analysis. Tel: +01 419 289 5281 84 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 8. Ecological aspects of allelopathy Oral 25. PLANT AUTOTOXICITY AND ITS ALLEVIATION BY PLANT DIVERSITY 1 Rensen Zeng1,2, Ruilong Wang2, Xiaoting Liang1,2 Yuanyuan Song1,2, Azim Mallik3, Shiming Luo2 College of Crop Science, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, P.R. China. E-mail: [email protected], [email protected] 2 Institute of Tropical and Subtropical Ecology, South China Agricultural University, Guangzhou 510642, P.R. China 3 Department of Biology, Lakehead University, Thunder Bay, Ontario, Canada P7B 5E1 ABSTRACT Autotoxicity refers to inhibition of plant growth and productivity by toxic chemical substances released from the same plant species. Autotoxicity is a common phenomenon leading to continuous cropping obstacle in agriculture and regeneration failure in forestry. In this study three generations of alfalfa were planted in sand culture to evaluate the dynamic effects of alfalfa autotoxins on the growth of sequential generations of alfalfa and the influence of plant diversity and activated carbon application on alfalfa autotoxicity. Continuous planting of alfalfa resulted in lower survival rate and biomass. However, AC addition in culture sand improved alfalfa growth. Mixture culture with Bidens pilosa L. and Celosia argentea L. in previous generation significantly improved alfalfa growth. AC addition and mixture culture increased the chlorophyll content of alfalfa, but they reduced total polyphenol content and malondialdehyde (MDA). HPLC analysis of sand extracts revealed that alfalfa monoculture led to chemical accumulation in sands, and AC addition and mixture culture diluted the concentration of most compounds. Mixture culture of different cultivars also improved alfalfa growth. Similar phenomenon was also observed in pepper plants (Capsicum annuum L.). Our results suggest that the autotoxicity can be alleviated by increasing species and genetic diversity. In nature autotoxicity may be an important mechanism of biodiversity maintenance. Autotoxicity leads to poor growth of pure stand of a plant and creates opportunity for coexistence with other species. Keywords: Autotoxicity, allelopathy, plant diversity, alfalfa Tel: + 86-591-8372-2796; fax: +86-591-8378-9483 85 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 26. THE ALLELOPATHIC MECHANISM OF ALSTONIA SCHOLARIS Chang‐Hung Chou, Hsiao‐Ting Chen and Chao Min‐Wang Research Center for Biodiversity, China Medical University, Taichung, Taiwan 404; email: [email protected] ABSTRACT Alstonia scholaris, belonging to the family of Apocynaceae is widely distributed throughout tropical regions of Africa and Asia. A. scholaris, which is commonly called blackboard tree or devil tree, is a tropical evergreen native to south and southeast Asia. Underneath A. scholaris it is often lacking understory species. Similar phenomenon has been found in many plantation areas. The senior author has observed the allelopathic pattern for, at least, 10 years. We have carried out experiments in field, pot experiments, laboratory assays and found that the clear evidences of allelopathic inhibition of A. scholaris. The floor of A. scholaris showed significantly low relative weed growth as compared to its adjacent grassland control site. However, at distances of 1 m and 3 m from the Alstonia forest, the relative weed growth increased three to ten-fold, respectively. The relative coverage of dominant species, Bidens pilosa, was 0% on the floor of the A. scholaris floor, but increased substantially at distances of 1 to 3 m away from the Alstonia forest. In comparison with the control soil and soil sampled from outside the Alstonia, the leaves, litter and soil from Alstonia inhibited growth of B. pilosa. The aqueous and methanolic extracts of A. scholaris leaves and litter inhibited radicle growth of tested plants. The responsible allelopathic compounds from Alstonia leaves were identified as pentacyclic triterpenoids, namely ursolic acid, betulinic acid and oleanolic acid through ‘H and 13C-MNR and LC-MS/MS spectroscopy analyses. Further analysis concluded that the ursolic acid was present in forest soil at a concentration of 3, 095 ug/g, i.e. exceeding the 1C50. In the field, ursolic acid accumulated abundantly in the soil of A. scholaris forests, and suppressed weed growth during summer and winter. It is concluded that A. scholaris pentacyclic triterpenoids influence the growth of neighboring weeds by inhibiting seed germination, radicle growth, and functioning of photosystem II. Ref.: J. Chem. Ecol. (2014) 40 : 90-98. 86 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 27. HOW MUCH DIFFERENT TYPE OF PLANT INTERFERENCES IMPACT COMPETITIVE AND NEIGHBOR-DEFENSIVE BEHAVIORS? 1 Yogan Monnier1,2, Christiane Gallet3, Anne Bousquet‐Mélou1, Bernard Prévosto4, Catherine Fernandez1 Institut Méditerranéen de Biodiversité et d’Ecologie (IMBE) Aix-Marseille Université, CNRS, IRD, Univ. Avignon, Campus St Charles Case 4, 13331 Marseille Cedex 03, France. [email protected] 2 BotAnique et bioinforMatique de l'Architecture des Plantes (AMAP) Cirad-Cnrs-Inra-Ird-Université Montpellier 2 37/PS1, Bd de la Lironde, 34 398 Montpellier cedex 5, France 3 Laboratoire d’Ecologie Alpine (LECA) UMR 5553 Université de Savoie Savoie Technolac F-73376 Le Bourget-du-Lac, France 4 Institut national de recherche en sciences et technologies pour l’environnement et l’agriculture, 3275 route de Cézanne - CS 40061, 13182 Aix-en-Provence Cedex 5, France ABSTRACT In contrast to plant-animal interactions, a poor conceptual framework remains to sustain the effectiveness of secondary metabolites implication in mediating plant-plant interactions and plant community dynamic. In this way, we addressed here questions about the role of allelopathy as a driver of forest dynamic through differential sensitivity of succeeding species, and hypothesize that plants adopt specific behavior to cope with different types of plantinterferences. In a two-year nursery multifactorial experiment, we studied overall phenotypic responses (OPR) of two Mediterranean forest species, a pioneer (Pinus halepensis) and a late-successional (Quercus pubescens), to different plant-plant interferences (chemical interferences by leachates, constraint by physical neighboring, and both combined). We studied the competitive and the neighbor-defensive responses of saplings through assessing the whole-plant growth and targeted ecometabolomic approach at leaf-level. We confirmed that allelopathy alone impacted differentially growth of both species. But contrary to what expected, Quercus showed a greater sensitivity to allelopathic treatment, increasing with time, while Pinus showed weak autotoxic effects. At the leaflevel, the induction of secondary-metabolites varied specifically to different types of plant interferences, highlighting the interest to improve our knowledge of neighbor-defensive behaviors. Finally, our results highlight how understanding OPR of successionally distinct species to diverse type of plant interferences helps provide insights about the role of plant secondary metabolism in dynamic of non-invaded plant community. Keywords: Forest regeneration dynamic; Pinus halepensis; Quercus pubescens; Allelopathy; Competition; Secondary metabolism; Ecometabolomic Tel: +33 (0) 4 13 55 12 22 87 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 28. ALLELOPATHY AS A DRIVING FORCE OF BIODIVERSITY CHANGE Luo Shiming*, Zeng Rensen, Liang Xiaoting, Liu Yinghu 1 Institute of Tropical and Subtropical Ecology, South China Agricultural University, Guanzhou, China 510642; e-mail: [email protected] ABSTRACT Relationship of allelopathy and biodiversity can be divided into three phases. In a short term, the dosage to show inhibition effect for donor plant releasing allelochemicals is usually higher than for receptor plants which affected by the allelochemicals. It has advantage for the donor plant to compete for resources such as nutrition, water and space and it is a reward for donor plant after spending the energy and material to make those chemicals. This allelopathy phenomenon exists in many indigenous species and invasive species as well. Wedelia trilobata in South China is one example. In this short term effect, allelopathy helps individual plant species to compete, to survive, to establish and to expand. The result of this stage usually ends up in communities with one dominant species with no or only few other plant species. In practice, this knowledge will help us to design intercropping system, to find suitable cover crop, to select weed resistance variety and to identify weed control chemicals. However, allelochemicals accumulated in field or in plantation for long enough duration will sooner or later reach the autotoxicity level of that donor plant species. Then allelopathy effect enters to the second phase. New species which can tolerate those allelochemicals and their accumulation levels enter the community. Biodiversity in the community increases. The increase of biodiversity also helps to reduce the autotoxicity of the donor species in the community because of the density effect. The individuals of the donor plant species will be forced to disperse into new habitat. If they can survive, it also helps other plant communities to increase diversity. In practice, this knowledge is important for the design of crop rotation system and perennial pasture system. Continue cropping of monoculture alfalfa (A) was compared with mix cropping with Bidens pilosa and Celosia argentea L. (M3). Result showed that mix cropping with other species could reduce the allelopathy effect and even have stimulated effect. The plant biomass of alfalfa in the third crop of M3-M3-A rotation was 106.9% to 165.6% more than in the third crop of A-A-A. The fifth crop of alfalfa was reduced by 30.6% to 31.63% in continue mono-alfalfa system (A). It was reduce to only 20.37% in a continue mix-alfalfa system (MA). Long term continue allelochemical pressure will show a third stage effect of allelopathy. Allelopathy becomes an evolution force. The donor species which adapts new habitat or develop higher resistance to its own allelochemicals will develop into new ecotypes. Plant community which accumulated enough allelochemical will force the pioneer plant species to yield their ways to more tolerate succession species and finally climax species. The biodiversity in a community reaches its top stage. In reforestation practice, the selection of tree species according to their position in local succession stage and the goal of reforestation are important. Research results from Peng Shaolin and Chen Baoming show that abscisic acid (ABA) as an allelochemical is an important driving force for forest succession and also important factor to prevent the invasion of alien species. Our modeling result by using Penna Cellular Automata Model shows that a more diversified community in terms of inhibition thresholds caused by allelopathy can help the species in a plant community to withstand the invasion. Keywords: allelopathy, biodiversity, cropping system, alfalfa 88 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 29. Anthemis sp.: THE PHENOMENON AND EXPERIMENTATION J.R. Qasem Department of Plant Protection, Faculty of Agriculture University of Jordan, Amman, Jordan E-mail: [email protected] ABSTRACT Field observations revealed that Anthemis sp. tends to grow in pure colonies excluding all other vegetations in its vicinity. The weed inhibited and totally excluded wheat, barley and the wild barley “Hordeum bulbosum” in the field. These observations encouraged conduct of several laboratory and glasshouse experiments to examine possible allelopathic activity of this common weed. High concentrations of fresh foliage extracts inhibited seed germination and growth of wheat and barley seedlings grown in Petri-dishes. Roots were more inhibited than shoots and for both crops. In contrast, shoot leachates stimulated growth of both crops at all concentrations and the effect was concentrationdependent. Barley appeared more tolerant to extract harmful effects but better responsive to leachates stimulatory action. Incorporation of chopped fresh shoots in potted soil increased growth of both crop species and growth increased with residue rate. Shoot fresh and dry weights and plants heights were all increased with Anthemis shoot residues incorporated in the soil. Decayed residues of Anthemis stimulated growth of both crops especially at high rates left in the soil. Results indicate that Anthemis colonies in nature and exclusion of other species from within and in its surroundings are not necessarily due to allelopathy mechanism but rather to other mechanisms of plant interference including competition. This however, necessitates the development of a new technology enable separation of allelopathy from competition or other mechanisms of plant interference. Keywords: Anthemis sp., allelopathy, interference, wheat, barley, field colonies 89 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 25. POPULATION DYNAMIC OF PHLEUM SUBULATUM IN A PLANT COMMUNITY OF MEDITERRANEAN COASTAL VEGETATION: IS ALLELOPATHY A KEY COMPONENT? Assunta Esposito*, Antonio Croce, Brigida D’Abrosca, Vittoria Graziani, Antonio Fiorentino and Monica Scognamiglio 1 Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples; via Vivaldi 43, 81100 Caserta, Italy e-mail: [email protected] ABSTRACT Introduction: The knowledge of biodiversity and of its regulatory mechanisms is a fundamental issue in the understanding of plant community composition and dynamics. Allelopathy has been shown to be one of the most important mechanisms regulating plant diversity and succession through several biological traits (e.g. seed germination, seedling growth, site quality). The modulation of these mechanisms can affect intra and interspecific plant interactions and thus dynamical vegetation processes. Objective: This study aims to investigate the effects of allopathic factors of several coexisting species on the germination and growth of Phleum subulatum, a characteristic steno-Mediterranean therophyte of sandy coastal dune herbaceous community. Materials and Methods: Plant samples of six coexisting species and P. subulatum seeds were collected in the field. To investigate on the potential allelopathic effects of donor plants, the aqueous crude plant extracts, extracts obtained from the aerobic plant decomposition experiments (collected at different times), and metabolites were assayed. Data on the germination and growth were analyzed by univariate and multivariate statistical approaches. Results and discussion: P. subulatum responses varied in relation to tested species, plant organ (leaf, root), time of decomposition and metabolite content. The most active species were those of late successional stage suggesting that colonizing species may be disadvantaged by organic residues that accumulate in the soil. Conclusions: The results support the hypothesis that late-successional competitive exclusion may be determined by the release of allelopathic factors present in the litter of the coexisting species. Keywords: Biodiversity Mediterranean plants, Phleum subulatum, Population dynamic, TEL: +390823274544 ; FAX: +390823274571 90 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 30. SPHAGNUM PHENOLICS GOVERN ALLELOCHEMICAL INTERACTIONS IN PEATLAND UNDER CLIMATE CHANGE Geneviève Chiapusio 1*, Jassey Vincent1,2, ML Toussaint1, F Bellvert3, G Comte3, and Philippe Binet1 1 2 Chrono-Environnement, Université de Franche-Comté UMR UFC/CNRS 6249, Montbéliard 25211 cedex, France; [email protected]; [email protected]; [email protected] School of Architecture, Civil and Environmental Engineering (ENAC), Ecole Polytechnique Fédérale de Lausanne EPFL, Ecological Systems Laboratory (ECOS), Swiss Federal Institute for Forest, Snow and Landscape Research (WSL), Station 2, 1015 Lausanne, Switzerland; [email protected] 3 Ecologie Microbienne USC INRA UMR 5557 CNRS-Université de Lyon, 69622 Villeurbanne Cedex, France; [email protected] ; [email protected] ABSTRACT Peatland ecosystems represent a crucial terrestrial sink of carbon by containing one-third of the earth soil carbon. Nevertheless, warming is likely to alter aboveground and belowground processes which regulate soil organic dynamic. Sphagnum genus is of particular interest because of its fundamental ecological role in peatlands, forming dense carpets that slowly decompose into peat, sequestering carbon. Sphagnum species are characterized by a specific morphology (living and decaying layers) and physiology (limited secondary metabolism) but remain largely unknown. Since 2008, our original research focuses on Sphagnum allelochemical interactions in a french peatland where an experimental moderate climate forcing (1 up to 3°C) was created by Open Top Chambers over vegetation. Objectives are to understand 1) the production, degradation and allelopathic effect of living Sphagnum polyphenols and 2) how warming affects Sphagnum allelochemical interactions and the peatland functionning. Results show that water-soluble phenolics are well produced by living Sphagnum fallax (around 1 mg g-1 DW) and that Sphagnum-peroxidases constituted their main oxidative system (10-3 U/g DW). Phenolics play a crucial role in the microdistribution of associated Sphagnum micro-organisms (specially the top predators) but also on surrounding Andromeda mycorrhizal symbiosises. Phenolics represent then a main ecological factor as chemical and physical ones. Under climate change, Sphagnum decreased its phenolic concentrations (by around -1.5 times) and so greatly impact phenolic-microbial interactions. To conclude, for the first time, sphagnum phenolics were found to be of great interest for the functioning of peatlands under climate change especially on aboveground and belowground processes. Keywords: bryophyte, sphagnum, phenolics, phenoloxydases, mycorrhizal fungi, microorganisms, peatland, climate change *Tel: +33 3 81 99 46 94; fax: +33 3 81 81 99 46 61 91 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 31. GLOBAL WARMING AND DOMINANCE OF ALGAL BLOOMS A S Ahluwalia Department of Botany, Panjab University, Chandigarh-160014, India E mail: [email protected] ABSTRACT Since temperature is one of the effective abiotic factor guiding the growth and establishment of planktons, it is worthwhile to undertake/review such a study for aquatic bloom forming algae/cyanobacteria. Competitive advantage of one alga/cyanobacterium over other co-occurring species in a water body determines the formation and persistence of water blooms. This advantage is guided by a variety of biotic and abiotic factors including allelopathy which in turn determines the invasion success of an organism. Most of the cyanobacteria perform better at higher temperature and hence appearance of frequent and intense water blooms is expected with changing global climate resulting in global warming. Frequent and intense algal/cyanobacterial blooms usually result in ecological and economic loss. Observations are in progress in a natural water body concerning dominant organism and succession of phytoplanktons and possible role of various factors in guiding their behavior of dominance throughout the year/ season, through field and laboratory experiments. Keywords: Algal bloom, climate change, temperature 92 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 26. EFFECT OF LITTER SECONDARY METABOLITES ON DIVERSITY, ABUNDANCE AND ACTIVITY OF DECOMPOSERS IN A MEDITERRANEAN FOREST Virginie Baldy, Mathieu Santonja, Alexandra Popova, Marine Bouyssou, Magali Proffit, Catherine Fernandez Institut Méditerranéen de Biodiversité et d’Ecologie (IMBE) Aix-Marseille Université, CNRS, IRD, Univ. Avignon, Campus St Charles Case 4, 13331 Marseille Cedex 03, France. [email protected] ABSTRACT Mediterranean region is considered as a hot spot of biodiversity and is also supposed to be seriously threatened by climate changes. These changes can lead to a shift in plant communities with important feedbacks for ecosystem functioning. Within this, leaf litter decomposition is a key process controlled by leaf chemistry determining diversity and activity of decomposers (dominated by collembola). However, the way that leaf chemistry can affect these decomposers is still insufficiently understood. In order to explore this relationship, three complementary experimentations were carried out to test at different scales the potential impact of four different types of litter (Quercus pubescens, Acer monspessulanum, Cotinus coggygria and Pinus halepensis) on diversity, abundance and activity of soil decomposers: (i) in vitro ecotoxicological tests on Folsomia candida (ubiquitous species of collembola); (ii) mesocosms experiment that mimic effects of natural litter leachates on soil biodiversity; (iii) in situ litter decomposition experiment to check if laboratory results were consistent with field observations. In laboratory, aqueous extracts of C. coggygria showed inhibitory effects on survival and reproduction of F. candida and on collembola abundance over the mesocosms experiment. During the in situ experiment, C. coggygria litter exhibited the lowest abundance and diversity on collembola contrary to the three other plant species. P. halepensis and Q. pubescens showed the highest diversity with a strong increase of some collembola species associated to these litters. These results suggest that litter chemistry can drive decomposers communities and then highlight the key role of secondary metabolites in plant-decomposers interactions. Keywords: Mediterranean forest, litter decomposition, diversity-functioning relationship, secondary metabolites, soil biodiversity, collembola TEL: +33 (0) 4 13 55 12 18 93 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 32. METABOLOMIC PROFILING OF SECONDARY PRODUCTS IN Echium plantagineum AND Echium vulgare, TWO EXOTIC INVADERS IN AUSTRALIA. Dominik Skoneczny1, Alexandra G.‐Duran2, Aitana Costas Gil3, Paul A. Weston4, Ascension Torres2, Francisco A. Macías2, Manuel Reigosa Roger3, Geoff M. Gurr5 and Leslie A. Weston4* 1 2 Charles Sturt University, School of Agricultural and Wine Sciences, Life Sciences Building Wagga Wagga, NSW 2678, Australia; [email protected] Grupo de Alelopatía, Departamento de Química Orgánica, Facultad de Ciencias, Instituto de Biomoléculas (INBIO), Universidad de Cádiz, Campus de Excelencia Internacional Agroalimentario (ceiA3), C/República Saharaui, s/n, 11510 Puerto Real, Cádiz, Spain; [email protected] 3 University of Vigo, Department of Plant Biology and Soil Sciences, Vigo, Spain; [email protected] 4 Charles Sturt University, Graham Centre, Life Sciences Building, Wagga Wagga, NSW 2678, Australia; [email protected] 5 Charles Sturt University, School of Agricultural and Wine Sciences, Orange, NSW 2800, Australia; [email protected] ABSTRACT Australian populations of Paterson’s curse (Echium plantagineum L., Boraginaceae) were investigated with respect to accumulation of selected secondary plant defense compounds. Key families of bioactive compounds evaluated in the study included isohexenylnaphthazarins (IHNs) (shikonin and related compounds) which occur as red pigments in the outer layer of root periderm and pyrrolizidine alkaloids (PAs) which are mainly accumulated as their N-oxides in foliage. Ethanolic extracts of root periderm and methanolic leaf extracts were subjected to solvent and solid phase extraction and analyzed qualitatively and quantitatively by LC/MS Q-ToF using ESI. Additional isolation of IHNs employed TLC followed by HPLC, and NMR for structural confirmation. A chemical library of key secondary products was created using Mass Profiler Professional Software (Agilent). Composite root and shoot extracts were separately evaluated from 14 geographically distinct populations across NSW Australia to evaluate intra-populational differences in secondary product accumulation. Extracts of the related species E. vulgare, a less widespread Australian invader, were also evaluated. In addition, extracts of plants grown under uniform conditions in the glasshouse were studied so the role of environment and genotype in accumulation of IHNs and PAs could be estimated. Interestingly, there were some significant similarities and differences in metabolomic profiles of shoots and roots in both species. Identifying the patterns of abundance of the most common constituents in root and shoot extracts of Echium spp. and expanding this study to include native European genotypes should provide critical information needed to generate a broader understanding of plant invasion mechanism(s). Keywords: plant invasions, E. plantagineum, E. vulgare, pyrrolizidine alkaloids, isohexenylnaphthazarins, mass spectrometry. Tel: +61 (2)69334689; fax: +61 (2)69332429 94 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 33. THE PHYTOTOXICITY CAPACITY OF VARIOUS INVASIVE PLANTS Ioana Cristina Marinas 1*, Mihaela Dinu 2, Viorel Robert Ancuceanu 2, Marilena Viorica Hovanet 2, Eliza Oprea 3 and Veronica Lazar 1 1 2 3 University of Bucharest, 1-3 Portocalilor Way, Bucharest, Romania; e-mail: [email protected] Faculty of Pharmacy, Traian Vuia street, no. 6; e-mail: [email protected]; University of Bucharest, Faculty of Chemistry, 4-12 Regina Elisabeta, Bucharest, Romania; email: [email protected]; ABSTRACT Allelopathy interactions between different plant species in natural ecosystems and agroecosystems represent a form of competition which involves inhibition of seeds germination or seedlings. The phytotoxicity testing was intended to demonstrate a specific trait of invasive plants, namely their ability to remove native species. For this purpose, we determined the effect of extracts obtained from Robinia pseudoacacia, Amaranthus retroflexus, Helianthus tuberosus, Phytolacca americana, Fallopia japonica and Aillanthus altissima on root elongation and the mitotic film by Triticum assay. The method consists in determining the maximum dilution of the extracts studied which, depending on the duration of action, influences root elongation and the caryokinetic film. Plant material on which the test was carried out consisted of embryonic roots of wheat. Microscopic examination allows assessing the changes induced by extracts studied in the mitotic film on radicular tips of Triticum vulgare. Chromatic material is put into evidence by acetic orcein staining. The occurrence of significant mitotic abnormalities may be due to interaction of polyphenolic compounds with proteins involved in mitosis, clastogenic effects or other perturbations leading to the harming of chromosomes. The extracts obtained from reproductive organs seem to show the highest level of phytotoxicity, thus demonstrating one of the specific features of invasive plants, namely their ability to remove native species or plants of agricultural interest. Keywords: compounds invasive plants, phytotoxicity, Triticum test, polyphenolic Tel: 0040757257980 95 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 34. Melinis minutiflora (POACEAE) PHYTOTOXIC STUDY Paula Novaes 1, Rosa M. Varela1, José M. G. Molinillo1, Francisco A. Macías1* 1 Grupo de Alelopatía, Departamento de Química Orgánica, Facultad de Ciencias, Universidad de Cádiz, Campus de Excelencia Internacional Agroalimentario, ceiA3, Institute of Biomolecules (INBIO). C/ República Saharaui s/n, Apdo. 40, 11510-Puerto Real (Cádiz), Spain. (*Phone: +34 956012770; fax: +34 956016193; email: [email protected]). ABSTRACT Melinis minutiflora (P. Beauv.) is a species native from Africa that is invasive in natural and agricultural areas of Brazil. We believe that allelochemicals should be present in their leaves and they could act in the establishment of these plants in the invaded environments. A factorial design optimization program was performed to determinate the conditions that would result in higher phytotoxic activities. The better conditions were: ultra-sound probe extraction, using mixture of ethyl acetate/acetone (90:10%), for 25 minutes. The extract was divided in two phases: chlorophyll present and absent parts. The chlorophyll absent part strongly inhibited wheat etiolated coleoptile growth and moderately affected the development of standard target species (lettuce, watercress, tomato and onion) and important weeds (Echinochloa crus-galli and Urochloa decumbens). It intermediate fractions showed elevated phytotoxic activity. The continuum fractionation and purification of these fractions showed that they are very complex, presenting high diversity of minority compounds, with reduced masses, that made impossible their identification. Many of these substances were fatty acids. Among the compounds that could be identified were: sinensetin, 1,3dihydroxipropan-2-ilo palmitate, estigmasta-4,7,22-trien-3-ol, 6, 10, 14trimetilpentadecan-2-ona, palmitic acid and fucosterol, all of them with reduced masses. We concluded that the phytotoxic activity of M. minutiflora is probably due to the synergism of many minority compounds. Keywords: Alien plants, allelopathy, weeds 96 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 35. Diplotaxis erucoides: AN ALLELOPATHIC INVASIVE SPECIES IN DISTURBED ARID LAND WITH POTENTIAL ANTIMICROBIAL ACTIVITIES. Mohammed H. Abu‐Dieyeh1*, H. AL‐Abeid2 and Wafaa Abu Taleb2 1 * Department of Biological and Environmental Sciences, College of Arts and Sciences, Qatar University, P.O. Box: 2713, Doha – Qatar, Email: [email protected] 2 Department of Biology and Biotechnology, The Hashemite University, P.O. Box 15049, Zarqa 13115 Jordan. ABSTRACT Disturbances of natural habitats due to human activities are often considered as a precursor for weed invasion. Allelopathic interactions have been proposed as a strategy for the success of some invasive plants. In stressed conditions allelopathy may be the factor that guidelines the outcome of competition to the benefit of the allelopathic species. An agricultural disturbance in arid land in Zarqa- Jordan was accompanied by weed invasion. Diplotaxis erucoides is a weed predominate the agricultural fields during January to June. A three year field work indicated a year after year gradual and significant increase of D. erucoides accompanied with a significant decrease in densities of other weeds. Laboratory experiments showed that foliage and root aqueous extracts of D. erucoides caused significant decrease in seed germination of Salsola baryosma as well as Eruca sativa Sonchus oleraceus, Tragopogon coelesyriacus, Launea nudicaulis. Soil amended with full strength or 1:1 diluted solutions of leaf leachates of D. erucoides decreased the shoot growth of E. sativa seedlings. On the other side, ethanol and hexane foliage extract of D. erucoides exerted a potential antimicrobial effect on certain bacteria: Methicillin resistant (MRSA) and Methicillin sensitive (MSSA) Staphylococcus aureus, Klebsiella sp., Escherichia coli, Shigella sp., Pseudomonas aeruginosa; and certain fungi: Candida albicans, C. glabrata, C. tropicalis, Fusarium oxysporum, F. chlamydosporum, F. moniliforme, Aspergillus terreus, Penicillium italicum and Cladosporium spheasperium. Results from laboratory and field experiments indicate that D. erucoides may use allelopathic interactions to shift the balance of competition to its favour and consequently to predominate in the disturbed arid land. Keywords: Allelopathy, weed invasion, Diplotaxis erucoides, plant interactions, arid land, antimicrobial. TEL: + 974-44036571; FAX: + 974-44034531 97 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 36. INDUCTION OF CHEMICAL DEFENCES IN THE INVASIVE WEED PATERSON’S CURSE (Echium plantagineum) BY A GENERALIST HERBIVORE INSECT AND A SPECIALIST BIOLOGICAL CONTROL AGENT 1 Saiful Z Jamil1, Paul Weston2, Dominik Skoneczny2, Leslie A. Weston2 Geoff M. Gurr*1 Graham Centre, Charles Sturt University, Orange, NSW 2800, Australia; * Presenting author [email protected] 2 Graham Centre, Charles Sturt University, Wagga Wagga, NSW 2678, Australia ABSTRACT Paterson’s curse is native to the Mediterranean region, where it is not considered a weed, but is an invasive weed of national significance in Australia and responsible for large economic losses to agriculture. The plant is known to produce a range of secondary compounds including naphthoquinones and pyrrolizidine alkaloids but the biological roles of these are not well known. This study aims to identify the significance of plant chemistry in the success of Paterson’s curse as an invasive species. Plants were exposed to two herbivore species with a chewing mode of feeding: a generalist herbivore (Helicoverpa punctigera caterpillars) and a specialist biological control agent (Mogulones larvatus adults). Analysis via LC-QTOF mass spectrometry revealed that damage by the two herbivores both resulted in marked changes in the secondary chemistry of roots and foliage of test plants. Significantly, however, the two herbivores triggered strongly divergent changes in plant chemistry, indicating that the nature of the plant’s response differed when challenged by a co-adapted specialist versus a newly-associated generalist. Results will be discussed in relation to the success of Paterson’s curse as an invasive species in Australia where the native, H. punctigera, is ubiquitous across the plant’s invaded range yet establishment of the introduced biological control agent, M. larvatus, is sporadic. Keywords: invasive species, biological control, induced defences, herbivory, mass spectrometry, naphthoquinones, pyrrolizidine alkaloids TEL: +61 (2)63657551; FAX: +61 (2)63657278 98 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 27. LITTER DECOMPOSITION REGULATES NATIVE AND EXOTIC TREE SPECIES EFFECT ON UNDERSTORY PLANT SPECIES IN RIVERINE AREAS 1 2 Pérez‐Corona, M.E.1; De las Heras, P. and Vázquez de Aldana, B.R.2* Departamento de Ecología. Universidad Complutense de Madrid. 28040. Spain.; email: [email protected]; [email protected] Instituto de Recursos Naturales y Agrobiología. IRNASA-CSIC. 37008. Salamanca. Spain; email: [email protected] ABSTRACT Introduction: Invasion of plant exotic species has become a global concern. It is often quoted a relevant role of allelopathic substances from invasive species as a novel weapon that could affect the presence and growth of native plant, microbial or animal species. Litter decomposition may regulate timing and chemical characteristics of allelopathic substances from the dead leaves to the soil. Objective: To determine the effects of litter from invasive and native riparian species at different decomposition phases on growth of two natives understory plant species. Materials and Methods: A greenhouse pot experiment was carried out by adding litter from invasive (Ailanthus altissima, Robinia pseudoacacia and Ulmus pumilla) and native (Populus alba, Populus nigra, Ulmus minor) riparian tree species, at four decomposition phases (0, 1, 2 and 3 months) on pots with seeds of the target species (Trifolium pratense and Dactylis glomerata). Discussion: All treatments reduced root and shoot growth of T. pratense. The effect of decomposition phase was similar for aboveground and belowground biomass but showed different pattern according to tree species: A.altissima and P.alba produced lower biomass inhibition when litter was more decomposed. On the contrary, P.nigra and U.minor litter produced greater growth reduction when litter was less decomposed. The inhibition of D. glomerata growth was lower and it was mainly due to time decomposition but not to litter species. No overall effect of native or invasive species was found. Plant understory specific response was found. Keywords: Germination, growth parameters, biomass allocation, allelopathy, decomposition TEL: +3413945079; FAX: +3413945081 99 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 28. THE INFLUENCE OF ALLELOPATHY ON SEED GERMINATION OF Galinsoga ciliata Dong‐Sheng Tang1*, Yang Fu1, Rui‐xiang Li1, Yi‐qing Guo1 1 College of Plant Protection, Yunnan Agricultural University, Kunming, China; [email protected] ABSTRACT Galinsoga ciliata was an exotic species introduced into Yunnan province of China in 1920’s. The weed is an agricultural pest infesting summer corn and soybean fields and causing serious economic loss. The allelopathy on seed germination of G. ciliata has not been documented, and this gap in knowledge has limited analysis the causes of it to be a dominant weed in upland field. To investigate the response to weed secretion substances from different sources on seed germination of G. ciliata, laboratory experiments were conducted to examine the role of roots exudates, leaves water leaching matters, water extraction of shoots, seeds of G. ciliata on seed germination of its self. Four serious weeds of Rhizoma fagopyri, Vicia villosa, Digitaria sanguinalis, Echinochloa crus-galli, Chenopodium serotinum, Nicandra physaloides, and Ageratina adenophora on seed germination of G. ciliata were surveyed either. Leaves leaching matters showed a slight inhibitory effect on seed germination. Germination rate of G. ciliata seeds incubated near its root was gradually decreased as the density of seedlings was improved. The allelopathic role of homogenate of fresh shoots on seed germination was far lower than that of water extraction of dry powder of shoots. Germination rate of seeds in high density was higher than in low density. Roots exudates of R. fagopyri, V. villosa, N. physaloides, A. adenophora showed significant allelopathic effect on seed germination; while there was no any bioactive effect of root exudates of D. sanguinalis, E. crus-galli, C. serotinum. These attributes could give some explanations for the phenomenon of observed in field. Keywords: allelopathy, substance, root exudates seed germination, Tel: 86+153-3172-7676; fax: 86-871-65228581 100 Galinsoga ciliata, secretion 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 29. INVASIVE PLANTS: A PRIME THREAT TO NATIVE BIODIVERSITY K.N. Dhumal Department of Botany, University of Pune, Pune – 411007, M.S., India E-mail: [email protected] ABSTRACT Allelopathy is an important ecological mechanism influencing phytodiversity as well as their dominance, succession and encroachment. The invasion of exotic plants in forests of Western Ghats by Ageratina, Ageratum, Eupatorium, Lantana etc. is considered as the prime threat to the native phytodiversity. To elucidate this secret of invasion, dominance and substitution by some dominant forest weeds forming pure stands in the forest of Mahabaleshwar were investigated to focus their ecophysiological, biochemical and enzymological aspects. At the same time, the allelochemicals existing in them were envisaged and characterized. Therefore ecological survey was conducted in forest of Mahabaleshwar, followed by their ecophysiological analysis. The allelochemicals were characterized by HPTLC. The results of present investigation revealed that dominant invasive plants had negative influence on associated natives. The leaf, bark and fruit extracts showed presence of twelve different types of allelochemicals/ ecochemicals such as terpenoids, flavonoids, glycosides and bitter essential oils. Similarly invasive weeds had higher photosynthetic efficiency and tolerance to biotic stress. It was concluded that the invasive plant species were responsible to reduce the forest phytodiversity. Keywords: weeds Allelopathy, allelochemicals, forest of Mahabaleshwar, invasive TEL: +09604393523 101 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 30. PHENOLICS IN TWO Alternanthera SPECIES RESIDUES AFFECT THE GERMINATION AND EARLY SEEDLING GROWTH OF RICE (Oryza sativa L.) Asif Tanveer1 Azhar Mehmood2 Department of Agronomy, University of Agriculture, Faisalabad , Pakistan; 1 [email protected] 2 [email protected] ABSTRACT Alternanthera species are invasive aquatic/semi aquatic weeds posing a serious threat to agro-biodiversity in several countries of the world. The present study was conducted to assess the phytotoxic effects of Alternanthera philoxeroides and A. sessilis residues on emergence and early seedling growth traits of rice (Oryza sativa L.). Soil was infested with 4% Alternanthera species residues separately and allowed to decay for 0, 15 and 30 days. Rice emergence was significantly decreased but increase in mean emergence time and time to 50% emergence was observed in soils modified with Alternanthera species residues compared with seed sown in unmodified soils. Rice emergence was reduced to the tune of 50-67% and 52-75% by A. sessilis and A. philoxeroides, respectively. A significant reduction in root, shoot length, their biomass and reduction in rice seedling growth traits of rice was also noted with Alternanthera species infested soil. The phenolic compounds identified were Quercitin, Chlorogenic acid, P- Coumeric acid, Trans-4-hydroxy3-methoxy, Cinamic acid, Caffeic acid, Syringic acid, Sinapic acid, Vanillic acid, 4-hydroxy3-methoxy benzoic acid. The total phenolics increased with increasing residues decay time in both species thereby showing their direct interaction with emergence and seedling traits of rice. Keywords: Allelopathy, Alternanthera species, rice, germination, seedling growth, decomposition periods, phenolics Tel: +923457734324 102 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 37. PATHOGENIC PLANT-MICROBE RHIZOSPHERE INTERACTION UNDER Rehmannia glutinosa CONSECUTIVE MONOCULTURE REGIME Linkun Wu 1,2 and Wenxiong Lin 1,2* 1 2 College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, P.R. China; e-mail: [email protected]; * e-mail: [email protected] Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, P.R. China ABSTRACT Under consecutive monoculture regime the biomass and quality of Rehmannia glutinosa, an important Chinese medicinal plant suffers significant decline. The objective was to evaluate the response of soil bacterial and fungal communities to consecutive monoculture using Terminal Restriction Fragment Length Polymorphism (T-RFLP) technique and assess the positive and negative effects of root exudates on the key microbes associated with consecutive monoculture problems. Most of diversity and evenness indices of bacterial community were significantly lower in two-year monoculture soil than in newly-planted soil. But fungal community showed the opposite trend. Two-year consecutive monoculture led to significant reduction of populations assigned to genus Pseudomonas. However, Fusarium oxysporum or Fusarium sp. belonging to Ascomycota was significantly higher in two-year monoculture soil than in newlyplanted soil. Real-time PCR assay confirmed the reduction in abundance of Pseudomonas sp. and an increase in F. oxysporum in two-year monoculture soil. Furthermore, the relative abundance of Pseudomonas sp. with the antagonistic activity against F. oxysporum was greatly lower in two-year monoculture soil than in newly-planted soil. We aslo found that phenolic compounds mixture in an ratio as detected in the soil could greatly promote the growth of pathogenic F. oxysporum, but inhibit the beneficial antagonistic bacteria. The isolated host-specific F. oxysporum caused wilt disease on the tissue culture seedlings of R. glutinosa. This study demonstrated that consecutive monoculture resulted in the alteration of rhizospheric microbial composition with fewer microorganisms providing beneficial functions and more microorganisms with pathogenicity, which had a negative impact on R. glutinosa growth and development. Keywords: Consecutive monoculture Belowground diversity, Root exudation problems, Microbial ecology, Tel: +8613809511917; fax: +86059183769440 103 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 38. Abutilon theophrasti´s ROOT ASSOCIATED MICROORGANISMS SUPPORT HYDROXYLATION-DEPENDENT BENZOXAZOLINONE DETOXIFICATION AND DEGRADATION Margot Schulz 1*, Sevda Haghi kia 1, Dieter Sicker2, Christian Paetz3, Bernd Schneider3, Letizia Mondani4, Cristina Ganimede4, Vincenzo Tabaglio4, and Adriano Marocco4 1 IMBIO Institute Molecular Biotechnology of Plants, University of Bonn, Karlrobert Kreiten Str. 13, 53115 Bonn, Germany; e-mail:[email protected] 2 Institute for Organic Chemistry, University of Leipzig, Johannisallee 29, 04103 Leipzig, Germany; e-mail:[email protected] Max Planck Institute for Chemical Ecology, Hans-Knöll-Straße 8, 07745 Jena, Germany; e-mail: [email protected] 4 Institute of Agronomy, Plant Physiology and Field Crops, Università Cattolica del Sacro Cuore, 29122 Piacenza, Italy [email protected] ABSTRACT Abutilon theophrasti is one of the most problematic weeds worldwide. Rye mulches, rich in benzoxazinoids, could not suppress Abutilon (Herbiseeds) due to its avoidance of high BOA/BOA detoxification product accumulation and phytotoxic 2-aminophenoxazinone production. Since Abutilon theophrasti seed coats harbour a variety of fungi and bacteria, a participation of those organisms in BOA degradation was assumed. The avoidance of high detoxification product/BOA accumulation in the roots was correlated with the presence of the Zygomycete Actinomucor elegans, which is known to colonize Abutilon seed coats. The microorganism(s) produced plant growth promoting compounds, whereby tryptophan excretion was stimulated by BOA. In Fenton reactions with BOA, tryptophan accelerates the destruction of the allelochemical via hydroxylated BOA molecules, when used in defined ratios. Destruction of BOA via hydroxylated molecules presents a new pathway for BOA degradation without accumulation of 2-aminophenoxazinone. During this process tryptophan is also degraded. Applications of high concentrations of tryptophan led, however, to a higher, channelled synthesis of BOA-6-O-glucoside in the roots, probably to slow down oxygen consuming Fenton reactions. From all possible hydroxylated BOA molecules, BOA-6-OH is the best substrate for Oglucosylation. Incubations in presence of the mono-oxygenase effector 2bromo-4´-nitroacetophenone revealed that those enzymes are mainly responsible for BOA-6-OH synthesis and only a minor part results from Fenton reactions. The study elucidates the important role of plant-microorganism associations in the survival of allelopathic attacks. Keywords: tryptophan Abutilon theophrasti, Actinomucor elegans, BOA degradation, Tel: + 49-(0)228-732151; fax: + 49-(0)228-732151 104 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 39. SCREENING OF ALLELOCHEMICALS FROM ACHYRANTHES BIDENTATA RHIZOSPHERE WHICH REMITTING CONTINUOUS MONOCULTURE PROBLEMS IN Rehmannia glutinosa Juan Li1*, Jiafang Du1, Hui Qi1, Junyan Shen1, Fengqing Wang1, Xinjian Chen1, Zhongyi Zhang2 1. . Agronomy College, Henan Agricultural University, Zhengzhou 450002, China, E-mail: [email protected] 2 Institute of Agroecology, Fujian Agriculture and Forestry University, Fuzhou 350002, China ABSTRACT ︐ Continuous monoculture problem in medicinal plant culture has become a core problem that hinders the sustainable development of Chinese medicinal materials production. Producing practice and previews research indicated that Achyranthes bidentata has a positive allelopathy and Rhemannia glutinosaAchyranthes bidentata rotation can effectively remit continuous monoculture disease of Rehmannia glutinosa. The experiments carried out with pots, and Rehmannia cultivars “wen-85-5”, the tested material, was replanted in rhizosphere soil of previous Rehmannia. Effective fractions were determined by adding different fractions of water extraction of rhizosphere soil of Achyranthes bidentata into different pots and the result showed that the most effective fraction was n-butyl alcohol extract, followed by the extraction of petroleum ether and ethyl acetate. The survival rate of replanting Rehmannia glutinosa treated with N-butyl alcohol extract approached 80% while the survival rate of control was less than 10%. Treated by different concentration of extracts of Nbutyl alcohol and petroleum ether increased root activity and decreased plasma membrane permeability of replanting Rehmannia glutinosa. HPLC-MS are employed to analyze the chemical constituents of N-butyl alcohol extract fractions, and determine target compounds by comparing effective fraction chromatograph of water extraction from rhizosphere soil of Achyranthes bidentata with that of the same fraction from blank soil. By using the information of the mass spectral library, the molecular ion peak, fragment ions and their fragmentation patterns, the structure of target compounds was inferred and the allelochemical in effective fraction could be phytosterones and triterpenoid saponins. This research explore the material of positive allelopathy of continuous cropping Achyranthes bidentata, and illustrate the mechanism of allelochemical from Achyranthes bidentata rhizosphere resolving continuous monoculture problems in Rehmannia glutinosa, hence, contributes to provide theory basis and technical support to further solve the continuous monoculture problems in the production of Chinese medicinal material. Keywords: Achyranthes bidentata, Rehmannia glutinosa, Allelopathy, continuous monoculture problems, positive allelopathy of continuous monoculture Tel: + 86-371-63558122; fax: +86-371-63558126 105 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 40. THE INFLUENCE OF WINTER RAPE RESIDUES DECOMPOSED IN THE SOIL ON Sinapis arvensis L. GERMINATION Zita Kriaučiūnienė1, Rimantas Velička1, 2, Aušra Marcinkevičienė1, 2, Rita Čepulienė 2, Rita Pupalienė 1, 2, Robertas Kosteckas 1, Sigitas Čekanauskas 1 1 2 Experimental Station, Aleksandras Stulginskis University, Rapsų str. 7, LT-53363 Noreikiškės, Kaunas distr., Lithuania; e-mail: [email protected] Institute of Agroecosystems and Soil Science, Faculty of Agronomy, Aleksandras Stulginskis University, Studentų str. 11, LT-53361, Akademija, Kaunas dist., Lithuania; e-mail: [email protected] ABSTRACT Allelopathic effect of Brassicaceae plants on weeds germination is an important factor in agriculture. Model field experiment and laboratory tests were carried out in 2011–2014 in order to evaluate the allelopathic effects of aqueous extracts of winter oilseed rape residues after harvesting and decomposed in the soil for a different duration (3, 7, 14, 19 and 26 months) on Sinapis arvensis L. emergence. Various concentrations: 1:6250 (the ratio of rape residues and water), 1:1250, 1:250, 1:50 and 1:10 of aqueous extracts of winter rape residues were evaluated for phytotoxicity. It was estimated, that the total amount of phenolic compounds in winter rape threshing remains, stubble and roots residues after 3 months of decomposition in the soil significantly decreased from 22.0 to 35.2 %. Contrarily in the root residues after 7, 19 and 26 months of decomposition it significantly increased from 10.0 to 14.0 %. Germination of S. arvensis L. seeds was inhibited by all concentrations of aqueous extracts of all of winter rape residues after harvest, 3, 7, 14 and 19 months of decomposition in the soil, except extracts of higher concentrations (1:10, 1:50 and 1:250) of root residues after harvest, 1:6250 concentration after 14 months decomposition and that of threshing remains and stubble of 1:10 concentration after 19 months decomposition. Germination of this weed seeds was significantly stimulated by aqueous extracts at concentration of 1:10 and 1:50 of winter rape roots, also – 1:50, 1:1250 and 1:6250 concentrations of aqueous extracts of winter rape stubble after 26 months decomposition. Keywords: winter oilseed rape, residues, decomposition, aqueous extracts, weeds, emergence Tel/fax: +370 37 753 377 106 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 41. EFFECT AND MECHANISM OF INTERCROPPING GARLIC OR GREEN GARLIC ON ALLEVIATING THE CONTINUOUS CROPPING OBSTACLES OF CUCUMBER GROWN IN PLASTIC TUNNEL Cheng, Zhihui1; Xiao, Xuemei2, and Meng, Huanwen3 College of Horticulture, Northwest A&F University 1 e-mail: [email protected] 2 e-mail: [email protected] 3 e-mail: [email protected] ABSTRACT Continuous cropping obstacle is prevalent for cucumber in China. Garlic is one of the important allelopathic crops and green garlic is another product form, sowing bulbs and cut harvest sprouted seedlings. A three-year study was conducted during 2009 to 2012 to investigate the effect and mechanism of intercropping on alleviating continuous cropping obstacles of cucumber in plastic tunnel. The results showed that the intercropped garlic significant stimulated the growth of plant, number of female flowers of cucumber. Green garlic showed the same effect except the first intercropping year as compared to monoculture cucumber (control). Moreover, this effect on cucumber appeared stronger at co-growth stage of autumn cultivation than spring cultivation. The nutritional quality of cucumber fruit was improved by intercropping and became better with the increase of intercropping year. Intercropping significantly increased cucumber yield and the best effect appeared in the second year. Intercropping garlic or green garlic significantly increased numbers of soil bacteria and actinomycetes, while decreased fungi. Activities of soil catalase, invertase, urease, and alkaline phosphatase were encouraged under intercropping. The effect on soil biological feature got stronger with intercropping year increasing. Intercropping increased the harvest of garlic bulbs ripened 30－40 days earlier than the outdoor garlic or the harvest of green garlic, which significantly increased the yearly net output of the plastic tunnel. These results suggest that intercropping garlic or green garlic can improve soil microbal structure and alleviate continuous cropping obstacle of cucumber, and both are efficient and sustainable intercropping patterns. Keywords: intercropping cucumber, alleviate continuous cropping obstacle, garlic, Tel: +15129183300; fax: +86 29 87082613 107 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 42. PLANT ALLELOPATHY TYPES AND THEIR APPLICATION IN AGRICULTURE Wenxiong Lin1,2 Fujian Provincial Key Laboratory of Agroecological Processing and Safety Monit oring (School of Life Sciences, Fujian Agriculture and Forestry University), Fuzh ou 35002, People's Republic of China Key Laboratory of Crop Ecology and Molecular Physiology. Fujian Agriculture and Forestry University, Fujian Province University, Fuzhou 35002, People's Republic of China ABSTRACT As the in-depth study of rhizosphere ecology, the researches on plant allelopathy gradually focus on rhizospheric biological processes. The cross-talk between plants and microbes is the key factor for plant allelopathy. Plant allelopathy consists of kinds of action modes, such as allelopathic inhibition (amensalism), allelopathic autotoxicity, and allelopathic autostimulation, etc. Increasing studies showed that plant allelopathy was the resultant from the comprehensive effect of plant-microbe interactions mediated by root exudates. Our researches showed that the phenolic compounds released by the roots of allelopathic rice could greatly promote the growth of host-specific myxobacteria in rhizosphere soil. Together with the phenolics, these myxobacteria are able to inhibit the germination and growth of target barnyard grass. Allelopathic amensalism has been widely applied in weed control, and great achievements have been made in the practice. Allelopathic autotoxicity, also known as consecutive monoculture problems, was shown as serious soil sickness in plants, which often results in decreased biomass and quality of plant under consecutive monoculture. Consecutively monocultured medicinal herbs often suffered from serious soil disease, consequently declined the yield and its quality. Recent researches have displayed that the ecological effects of soil microbial functional disorder mediated by root exudates were the main reasons for the consecutive monoculture problems. These findings suggested that the restoration and improvement of disease-conducive soil environment was crucial to overcome the problems. However, allelopathic autostimulation (positive allelopathy) was the case that the monocultured plants grew and harvests well, showing higher biomass and its quality with the increasing years of monoculture. Although the mechanism for the allelopathic autostimulation was not deeply understood, it was suggested that the ecological phenomena mainly resulted from the improvement of soil microbial community structure and their positive interactions with plant root systems, and hence increased soil fertility and nutrient supply ability, enforced root resistance to disease, consequently resulted in enhanced plant yield and quality. Achyranthes bidentata Blume was found to be a typical medicinal plant with positive allelopathy in consecutive monoculture, and had been used as a rotation/intercropping crop with other medicinal plants sensitive to consecutive cropping to keep sustainable production of medicinal herbs. In conclusion, various positive and negative plant-plant interactions among intra- or interspecific plants such as amensalism, autotoxicity and autostimulation, are all the results of the comprehensive effect of the rhizospheric talk between plants and microbes. The deep understanding of plant-soil-microbe interactions mediated by root exudates has important meaning for elucidating the functions of rhizosphere microecology and providing practice guidelines in agriculture production. Keywords: Allelopathy, autotoxicity, medicinal plant, rice, rhizospheric biology, soil microbes TEL: + 86-591-83737535; FAX: +86-591-83769440 108 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 31. ALLELOPATHIC INFLUENCE OF PEPPER-GARLIC INTERCROPPING SYSTEMS ON SOIL BIOLOGY AND NUTRIENT VARIATION UNDER PLASTIC TUNNEL ENVIRONMENT Muhammad Azam Khan1,2, Cheng Zhihui2, Abdul Rehman Khan2, Shah Jahan Rana2, Bushra Ghazanfar2 1 Department of Horticulture, PMAS-Arid Agriculture University, Rawalpindi, 46300, Pakistan 2 College of Horticulture, Northwest A&F University, Yangling, Shaanxi 712100, PR China *Corresponding email: [email protected] ABSTRACT The current study was carried out to investigate soil biological and nutrient property of garlic and green garlic intercropping systems with pepper in plastic tunnel for overcoming the nutrient alleviation and other soil poverty obstacles due to continuous monocropping. Higher concentration of soil bacteria and actinomycetes was observed during both the years of experiment after one month of garlic intercropping as compared to the control; however, concentration of fungi showed decreasing trend. Soil protective enzyme activity (invertase, urease, and alkaline phosphatase) showed dynamic changes after the intercropping of garlic. The principal soil nutrients (NPK) also showed higher levels in pepper-garlic intercropped treatments as compared to the control especially with green garlic (cv. G064). It is therefore concluded that the intercropping of pepper with garlic enhances the soil fertility by changing nutrient levels, enzymatic activity and soil microbes. Overall, it provides a convenient organic farming model for vegetable growers across the globe to accomplish the dire need of new era. Keywords: Intercropping systems, Pepper, garlic, allelopathy, soil microbial activity, soil enzymes, soil nutrition Tel: 00923006884470; fax: 0092519290160 109 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 32. RESIDUAL ALLELOPATHY OF PARTHENIUM: CONSEQUENCES FOR EMERGENCE, SEEDLING GROWTH OF SOME WINTER CROPS, WEEDS AND SELECTED SOIL PROPERTIES Abdul Khaliq1*, Farhena Aslam1, Ibrahim Alsaadawi2, Amar Matloob1 and Asif Tanveer1 1 Department of Agronomy, University of Agriculture, Faisalabad-38040- Pakistan 2 Department of Biology, College of Science, Baghdad University, Baghdad, Iraq 1 2 3 e-mail: [email protected]; e-mail: [email protected]; e-mail: 4 5 [email protected]; e-mail: [email protected]; e-mail: [email protected] ABSTRACT Allelopathic effect of residue incorporation and rhizosphere-infested soil of invasive weed parthenium were studied on emergence and seedling growth of winter crops (wheat, canola) and weed species (wild oat, canary grass) in two separate pot studies. Parthenium whole plant residue was incorporated at 6 and 8 g kg-1 soil five days before sowing of test species. Pots without residue incorporation were maintained as control. In another study, parthenium infested-rihzosphere soil collected from depth (15 and 22.5 cm) and collar distance (15 and 22.5 cm) was used as growing medium for test species. Parthenium-free soil from the same field was used as control. Parthenium residue amendment as well its rhizosphere-soil inhibited emergence and suppressed seedling growth of test species. Residue incorporation inhibited emergence of canola, wild oat and canary grass by 11-20%, 20-28% and 2026%, respectively. Seedling biomass was reduced by 41-48% (wheat), 53-61% (canola), 31-45% wild oat and 30-45% (canary grass) by residue incorporation. Soil phenolics, electrical conductivity, organic carbon and nitrogen contents were increased over unamended soil while pH showed a decline. Phenolics increased considerably with increasing rate of parthenium residue incorporation. Degree of inhibition of emergence and seedling growth of test species strongly corresponded to phenolic contents. Soil from a rhizospheric depth of 15 cm and collar distance of 15 cm inhibited the emergence (15%) and reduced seedling growth (40%) in all test species. Test species manifested reduced chlorophyll and increased phenolic contents in response to parthenium- residue incorporation and infested-rhizosphere soil. Highest chlorophyll inhibition coupled with enhanced tissue phenolic contents was forced by residue incorporation (8 g kg-1 soil) and rhizosphere soil (15 cm soil depth and 15 cm collar distance) in all test species. Canola was more susceptible than wheat as was canary grass than wild oat. Keywords: allelopathy, chlorophyll, rhizosphere soil, winter crop and weeds TEL: +92 321 66 15 848 110 emergence attributes, phenolics, 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 33. ROOT SENSITIVITY TO DETECT PHYSICAL OBSTACLE AND SOURCE OF RESOURCES 1* 2 3 Shamima Sultana1*, Saira Hussain2 and Md. Asaduzzaman3 College of Development Alternative, Dhaka-1207, Bangladesh, Email: [email protected] School of Biomedical Science, Charles Sturt University, Wagga Wagga, NSW, Australia, School of Agricultural and Wine Sciences, Charles Sturt University, Wagga Wagga, NSW, Australia ABSTRACT In crop rhizosphere setting, crop roots-resources are much complicated and are much debated. However evidence suggests that root are able to detect their required nutrients in rhizosphere soil media. The preference of crop choice response to physicals obstacle for hunting nutrients yet not clear. By using Equal Compartment Agar Method (Wu et al. 2000), we tested the hypothesis that plant extent their root growth to reach into resource sources by avoiding any physical barrier. We found that fourteen days old wheat’s (cv. Jazz) root detached from their siblings root, avoided closely (3 cm) situated physical obstacle and aggressively reached into resource sources (228 cm; P<0.05), while same species seedlings roots were less active (167 cm; P<0.05) when physical obstacle was far away (5 cm). However roots tendency was always avoid their siblings and physical barrier and moved to resources lines. The results suggest that the observed behavior of self discrimination is regulated by sensitivity of root’s tip and plant physiological coordination. Keywords: Root, rhizosphere and resources 111 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 43. DO WHEAT ALLELOCHEMICALS CONTRIBUTE TO FHB RESISTANCE AND MYCOTOXIN ACCUMULATION? Thomas Etzerodt 1, Bente B. Laursen 2, Lise N. Jørgensen 3, Helene Kristjansen 4, Sidsel Kirkegaard 5, Jette Lilholt 6 and Inge S. Fomsgaard 7* Department of Agroecology, Aarhus Universitet, Forsøgsvej 1, DK-4200 Slagelse, Denmark; 1 2 3 e-mail: [email protected]; e-mail: [email protected]; e-mail: 4 5 [email protected]; e-mail: [email protected]; e-mail: 6 7 [email protected]; e-mail: [email protected]; e-mail: [email protected] ABSTRACT Fusarium head blight (FHB) is a severe disease in wheat caused by mainly F. graminearum and F. culmorum causing yield losses and grain accumulation of the trichothecene mycotoxins DON and NIV. Susceptibility to Fusarium and mycotoxin accumulation in grain is cultivar dependent, e.g. Sumai-3 cultivar exhibiting high FHB resistance and low accumulation of DON and NIV in grain. We wish to utilize wheat allelochemicals for their ability to inhibit trichothecene biosynthesis in order to reduce the use of fungicides. In a field trial in Flakkebjerg, Denmark, 18 wheat cultivars with different susceptibility to FHB were inoculated naturally (from Fusarium inoculated maize debris) and artificially at flowering stage (by spray inoculation with a spore suspension). Wheat heads were harvested at 5 time points (BBCH59 to mature kernels) and analyzed for DON and NIV and 40 wheat allelochemicals (carotenoids, phenolic acids, benzoxazinoids and flavonoids). Analysis was done using liquid chromatography coupled to DAD and MS including high resolution TOF-MS with sensitivity as low as 3.125 ng/ml. The data matrix is evaluated using multivariate statistics to assess correlations between mycotoxin and allelochemical concentrations. In future studies, selected candidates from the 40 allelochemicals will be tested in in vitro setups with liquid Fusarium cultures and TRI protein inhibition assays in a research stay at the University of Nagoya, Japan. Keywords: Fusarium, mycotoxins, secondary metabolites, chromatography, mass spectrometry, multivariate statistics Tel: +45 87158139 / *+45 87158212; fax: +45 87156082 112 liquid 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 34. INVESTIGATION OF ENDOPHYTIC FUNGI ASSOCIATED TO Abutilon theophrasti ROOT 1 Cristina Ganimede1*, Adriano Marocco1, Margot Schulz2 and Vincenzo Tabaglio1 Institute of Agronomy, Genetics and Field Crops, Università Cattolica del Sacro Cuore, Via Emilia Parmense 84, 29122 Piacenza, Italy; e-mail: [email protected] 2 IMBIO Institute, University of Bonn, Karlrobert-Kreiten-Str. 13, 53115 Bonn, Germany ABSTRACT Introduction Velvetleaf is one of the most aggressive weeds for some important crops, such as maize and soybean. Rye is an allelopathic cover crop who produces benzoxazinoids. These allelochemicals can control some weeds, but not velvetleaf. Previous studies have shown the biochemical mechanisms by which velvetleaf may avoids the accumulation of allelochemicals, being able to detoxify them. Velvetleaf can host fungal endophytes which can help plants’ survival under stressed ecological conditions. Objectives Velvetleaf seeds have some microorganisms associated to the external seed coat. Our purpose is to investigate if there are, also, endophytic fungi that could help to survive in presence of benzoxazinoids. Materials and methods Abutilon seeds collected from 3 sites (Meleti, Pralboino and Gambara) in Northern Italy were tested to verify the presence of endophytic fungi. Seeds were surface sterilized with ethanol (80%), hypochlorite (2%) and then washed with distilled water. Seeds were incubated at 25° C. 3 and 6 days after emission, roots were transferred into PDA medium. Results and Discussion Interesting results were observed for the roots portions cut at 6 days after germination: Alternaria alternata was isolated in 27% of samples from Meleti and Fusarium spp. in the 36% of the samples. For the seeds collected from Pralboino there was 42% presence of Fusarium spp. The seeds from Gambara were not affected by root associated fungi. The isolation of fungi from 6 days old roots suggest a presence of endophytic host that migrate from the seed to the young root. Alternaria alternata and Fusarium spp. are frequently associated to Abutilon seeds and can show an endophytic behavior. Keywords: Abutilon theophrasti Medicus, velvetleaf, endophytic fungi, weed control, allelopatic cover crops. Tel: +39 0523-599202; fax: +39 0523-599222. 113 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 35. ISOLATION AND CHARACTERIZATION OF PHYTOTOXINS PRODUCED BY Phoma sp. № 19 Ekaterina V. Poluektova1, Alexander O. Berestetskiy2 All-Russian Institute of Plant protection, Saint-Petersburg, Russia, 196608; 1 e-mail: [email protected] 2 e-mail: [email protected] ABSTRACT The fungal genus Phoma is known for its diverse spectrum of secondary metabolites including phytotoxins. These substances often play an important role in the interactions of the fungi with other organisms, especially, with plants. Our bioassays had shown that extracts of Phoma sp. № 19, an endophyte of Cirsium arvense, are phytotoxic. The aim of this work was to isolate phytotoxins of this fungus and to characterize them. Fungus was grown on both liquid and solid media (YMG and pearl barley, respectively). Phytotoxins were isolated from the extracts of culture filtrate and mycelium of fungus using preparative column and thin layer chromatography. Phytotoxic activity was determined using leaf disk-puncture bioassay. Selectivity of phytotoxins was observed on plants of different families. Antimicrobial activity against a number of bacteria and fungi was studied by a paper disc technique. Activity against ciliates was determined on Paramecium caudatum. Structures of compounds were elucidated by 1D- and 2D-NMR-, UVspectroscopy, mass-spectrometry techniques. Two phytotoxins, identified as curvulin and phaeosphaeride A (PhA), were isolated from Phoma sp. culture filtrate and mycelium, respectively. PhA caused necrotic lesions on punctured leaves of C. arvense and Elytrigia repens at the minimal concentration 250 µg/ml and 125 µg/ml, respectively; curvulin was less phytotoxic. Both phytotoxins were not selective. PhA was shown to be weakly toxic against ciliates, while curvulin was nontoxic. Antimicrobial activity of both compounds was not observed. Phytotoxic activity of PhA was observed for the first time. PhA was reported earlier as an inhibitor of STAT3 signaling in human cells. The analogue target in plants seems is not known yet. It supports that PhA has potential for development as a herbicide agent or for mode–of–action studies. Keywords: Phoma sp., phytotoxin, curvulin, phaeosphaeride A 114 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 36. ALLELOPATHIC EFFECT OF ROOT EXUDATES FROM DIFFERENT RESISTANT EPPLANTS ON Verticillium dahliae Chen Z.X.1 and Zhou B.L.2* 1 Department of Environment & Resourses Science, Liupanshui Normal University, Liupanshui City, 553004, Guizhou Province, China; e-mail:[email protected] 2 College of Horticulture, Shenyang Agricultural University, Shenyang, 110866, China; email:[email protected] ABSTRACT Verticillium wilt is a destructive disease in eggplant that limits produciton worldwide, which is mainly caused by infection with Verticillium dahliae through the root surface to the vascular system. The plant resistance mechanisms of eggplant against verticillium wilt are related to physiological and biochemical conditions and biochemical compounds released in root exudates. To determine the relationship between root exudates and resistance to verticillium wilt in eggplant, three eggplant cultivars (resistant, tolerant and susceptible) with different levels of resistance to this disease were inoculated with Verticillium dahliae. The root exudates of eggplant cultivars were collected 14 days after inoculation, using the root soaking method. The extracts were analyzed by gas chromatography-mass spectrometry (GC-MS). The results showed that chemical substances of root exudates differed among the different eggplant cultivars, which was related to both the cultivar and inoculation status. Upon Verticillium dahliae infection, the amount and variety of ketone, ester and phenol increased in the susceptible type, and phenolic acid was only detected in this treatment group, while the contents of phenol and quinones increased in the resistant cultivar but decreased in the tolerant and susceptible types. The chemicals dibutyl phthalate, 2,4-bis(1,1-dimethylethyl) phenol and 2,6-bis(1,1dimethylethyl) were the main components identified in all treatment groups, which might be the allelopathic chemicals in root exudates. To detect the potential allelochemicals from those substances,we used the most abundant component, phenol, 2,4-bis(1,1-dimethylethyl) and 2,6-bis(1,1-dimethylethyl) as simulatory substances to determine their effects on germination and growth of eggplant, mycelium growth of Verticillium dahliae and incidence of verticillium wilt. The results showed that at low concentration (0.05 mmol·L-1), they promoted the growth of eggplant, inhibited the growth and development of Verticillium dahliae and decreased the incidence of verticillium wilt, but at higher concentrations they exhibited an opposite trend. So the concentration of phenol 2,4-bis(1,1-dimethylethyl) and 2,6-bis(1,1-dimethylethyl) in soil were closely related to development of Verticillium dahliae and incidence of verticillium wilt. Keywords: allelopathy, root exudates, eggplant, Verticillium dahliae, TEL: +86-13804013306; FAX: +86-024-88487143 115 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 37. PHYTOCHEMISTRY AND JOINT ACTION STUDIES OF ANTARCTIC LICHEN (Umbilicaria antarctica) Jose L. G. Galindo1, Ascensión Torres1, Benito Fernández1, Juan C. G. Galindo1, Joane Romagni2 and Francisco A. Macías1,* 1 Grupo de Alelopatía, Departamento de Química Orgánica, Facultad de Ciencias, Instituto de Biomoléculas (INBIO), Universidad de Cádiz, Campus de Excelencia Internacional Agroalimentario (ceiA3), C/República Saharaui, s/n, 11510 Puerto Real, Cádiz, Spain; e-mail: [email protected] 2 Department of Science, University of St. Thomas, Houston, TX, USA ABSTRACT Lichens are stable self-supporting mutualism between an exhabitant fungus (mycobiont) and inhabitant (photobiont: alga or cyanobacterium). According to recent estimations, lichens comprise about 18.500 species, 427 of them present in Antarctica. Lichens had been used in traditional medicine in a large variety of treatments as in cardiac or respiratory problems, or urinary and skin sickness. Their chemical composition and their biologic activities had been studied for pharmacologic effects on tumor cells, virus, bacteria and other living organism as herbivore, plants, fungi and other microorganism. Therefore, their ecological function should be relevant and need to be deeply study. Nevertheless, the pharmaceutical industries pay no attention to these fact due the difficulty for prepare axenic cultures and fast growth conditions and use it as source of natural products, but these investigations gather importance because the discovered compounds and their bioactivity behavior can be used as reference and target compounds by the chemical industries. The joint action of these compounds can be determine the better form of use of them and explain the rule of lichens in its ecosystem. The Dose-Response Curve and isobolograms methodology, based on the IC50 generated by curves has been largely recommended to demonstrate synergism, antagonism or additive situation. In this study, we carry out several coleoptiles bioassay testing the influence of pure compounds and different binary mixtures of them, and determine the interaction in the activity of the compounds. This results were confirmed with the comparative activity levels obtained for the original fractions versus the theoretical activity obtained from the combination activity of the compounds applying the joint detected. An important clue of this investigation was the use of the coleoptile bioassay as a new tool for the determination of synergistic interactions. This tool has, sufficient statistical support for discrimination or determining the relationship between activity value and dosses as well as proportions of components in binary or more complex mixtures. Keywords: Antarctic lichen, Umbilicaria antarctica, poliols, isobolograms, synergism, antagonism and additive action, bioactivity Tel: +34 956012770; fax: +34 956016193 116 Session 4. Allelopathy in environments Fungi and lichen allelopathy Freshwater allelopathy Allelopathy in marine environments Plant-microbe interactions different 117 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 44. FILAMENTOUS GREEN ALGAE PRODUCE POLY-UNSATURED FATTY ACIDS AS ALLELOCHEMICALS Joey Allen 1, Loïc Ten‐Hage 2 and Joséphine Leflaive 3 Laboratoire Ecologie Fonctionnelle et Environnement, UMR 5245 CNRS-INP-UPS, Université de Toulouse, 118 route de Narbonne, 31062 Toulouse, France; 1 [email protected] 2 3 [email protected] [email protected] ABSTRACT Phototrophic biofilms are complex assemblages of phototrophic and heterotrophic micro-organisms, where short distances between cells lead to intense competition. Our hypothesis is that allelopathy is implied in competition between benthic algae in biofilms. Particularly, filamentous species might produce allelochemicals to avoid epiphytes competitors. We used agar diffusion assay to test for allelopathic effect of freshwater filamentous algal species. Methanol and acetone extracts of algal biomass were tested for inhibitory effects on a common epiphytic diatom (Nitzschia palea). UPLC and HPLC were used to separate different fractions of the extracts. Once the allelochemicals are identified, we measured their concentration in the culture medium and the biomass of five filamentous green algae (Uronema conferviculum, Ulothrix sp., Stigeoclonium sp. and two strains of Oedogonium sp.), a diatom (Nitzschia palea) and a filamentous Xantophycea (Tribonema sp). U. conferviculum had the most important allelopathic activity. We found that fractions of the biomass extracts with inhibitory activity contained free fatty acids (stearic, palmitic, myristic, linoleic and alpha-linolenic acids). These fatty acids were also detected in culture medium of U. conferviculum. Standards of linoleic and alpha-linolenic acids have both inhibitory activity at 250 µg/L against various benthic strains, the others were less active. The allelopathic function of polyunsatured fatty acids is already known in planktonic habitats, these results confirm that they are also produced by benthic microalgae as allelochemicals. Keywords: Freshwater allelopathy, biofilm, fatty acid, benthic algae Tel: +33 5 61 55 89 52; fax: + 33 5 61 55 89 01 118 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 45. ALLELOPATHIC INHIBITION OF DIATOM ADHESION BY FILAMENTOUS GREEN ALGAE Joey Allen 1 and Joséphine Leflaive 2 Laboratoire Ecologie Fonctionnelle et Environnement, UMR 5245 CNRS-INP-UPS, Université de Toulouse, 118 route de Narbonne, 31062 Toulouse, France; 1 [email protected] 2 [email protected] ABSTRACT Filamentous green algae compete for space, light and nutrients with many other benthic phototrophic microorganisms such as epiphytic diatoms. Allelopathic inhibition of other algae fixation could be a way to avoid competitors in biofilms. Filamentous green algae were tested for effect on the adhesion ability of benthic diatoms (Nitzschia palea, Fistulifera saprophila). We tested the effect of 1) co-cultivation of diatoms and filamentous green algae, 2) filamentous green algae culture filtrate and biomass extract and 3) two poly-unsaturated fatty acids (linoleic acid and alpha-linolenic acid) identified in the culture medium and biomass of some filamentous green algae. The adhesion was measured as the density of diatom attached to a microscope cover glass after 4h or 24h of incubation. The adhesion of N. palea was strongly inhibited by the presence of Uronema conferviculum in the culture media. The filtrate of a dense culture of U. conferviculum decreased by 50% the number of adherent diatom. Biomass extracts also have strong effects on diatoms adhesion. However the two polyunsaturated fatty acids (linoleic acid and alpha-linolenic acid) tested did not have any effect on diatom adhesion even in high concentration (1000 µg.L-1). We conclude that filamentous green algae produce and release in the culture medium an allelopathic compound that acts as a cell adhesion inhibitor. The inhibition of adhesion was not an effect of linoleic acid or alpha-linolenic acid produced by these algae. Keywords: Freshwater allelopathy, biofilm, biofouling, benthic algae Tel: +33 5 61 55 89 52; fax: + 33 5 61 55 89 01 119 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 46. INHIBITION OF TOXIC AND NON-TOXIC STRAINS OF THE CYANOBACTERIUM MICROCYSTIS AERUGINOSA BY ALLELOCHEMICAL TANNIC ACID Algirdas Švanys1*, Falk Eigemann2, Paškauskas Ričardas3, Hans‐Peter Grossart4 and Sabine Hilt5 1 Marine Science and Technology Centre, Klaipėda University, H. Manto 84, 92294 Klaipėda, Lithuania & Leibniz-Institute of Freshwater Ecology & Inland Fisheries, Müggelseedamm 301, 12587 Berlin, Germany; email: [email protected] 2 Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Müggelseedamm 301, 12587 Berlin, Germany; email: [email protected] 3 Marine Science and Technology Centre, Klaipėda University, H. Manto 84, 92294 Klaipėda, Lithuania & Institute of Botany of Nature Research Center, Žaliųjų ežerų g. 49, 08406 Vilnius, Lithuania; email: [email protected] 4 Leibniz-Institute for Freshwater Ecology and Inland Fisheries, Department of Limnology of Stratified Lakes, Alte Fischerhütte 2, 16775 Stechlin, Germany & Potsdam University, Institute of Biochemistry and Biology, Maulbeerallee 10, 14469 Potsdam, Germany; email: [email protected] 5 Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Müggelseedamm 301, 12587 Berlin, Germany; email: [email protected] ABSTRACT Harmful cyanobacterial blooms dominated by microcystins-producing and hence toxic Microcystis aeruginosa are increasing in freshwater ecosystems worldwide. The impact of several environmental factors (e.g. nutrients, light and temperature) on toxicity potentials of M. aeruginosa has been studied intensively, however, how allelopathic substances exuded by aquatic plants may affect toxic and non-toxic M. aeruginosa developments is still an open question. We investigated effects of the polyphenolic allelochemical tannic acid (TA) on 7 toxic and 6 non-toxic (single) strains of M. aeruginosa of different origin under laboratory conditions. Concentrations of chlorophyll a and maximum quantum yield of photosystem II (photosynthetic yield) were measured after 3 days of exposure to TA (initial concentrations 5, 10 and 20 mg L-1) under constant light (200±15 µE m-2 s-1) and temperature (22±1 °C). Both chlorophyll a concentrations and photosynthetic yield were significantly more diminished for non-toxic relative to toxic strains. In contrast, origin of strains or their initial growth rate did not affect their sensitivity towards TA. Our results indicate that the presence of allelopathically active aquatic plants exuding polyphenols can favour toxic M. aeruginosa developments and thus potentially alter M. aeruginosa toxicity potential in freshwater ecosystems. Keywords: allelopathy, Microcystis aeruginosa, tannic acid Tel: + 370 46 398843; fax: + 370 46 39 8845 120 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 47 PHYTOTOXIC EFFECTS OF MICROALGA Scenedesmus quadricauda CULTURED UNDER DIFFERENT CONDITIONS ON LETTUCE Araniti F., Bargagli A., Gennari M., Abenavoli M.R. Università degli Studi “Mediterranea” di Reggio Calabria Feo di Vito I-89124, Reggio Calabria, Italy; [email protected] ABSTRACT The search of novel natural products from plants, fungi or microbes with potential application in weed and pest management has become attractive in the recent years. In contrast, compared to these organisms, few information on biologically active compounds from microalgae by using as herbicidal templates have been reported. In the present study, the phytotoxic effects of Scenedesmus quadricauda Meyen (SQ), cultured under two nutritive conditions, were investigated. Microalgae were grown in BG11 aerated medium without or with sodium bicarbonates at 2g/L concentration (BC2), and then placed in a growth chamber at room temperature with 12/12 h light (3500 lux)/dark photoperiod, for 21 days. After this period, microalgal biomass was collected through filtration and dried. The production of biomass and the total carbohydrate content were higher in SQ-BC2 (30% and 35%, respectively) compared to SQ-BG11. The dry biomass was then extracted by a hydroalcoholic solution (methanol:water, 9:1), and assayed in vitro on seed germination and root growth of lettuce (Lactuca sativa L.). The data were fitted through non linear regression model to obtain the ED50 for both physiological processes. The ED50 values on lettuce root growth, treated with two different extracts, were not statistically different (1.76 vs 1.66 mg/ml). Conversely, the SQ-BG11 extract was more phytotoxic than SQ-BC2, on seed germination, showing ED50 values by 1.81 and 5.1 mg/ml, respectively. These results provide the first evidence that microalga Scenedesmus quadricauda produced phytotoxic compounds and the growth condition could affect their composition and phytotoxicity. Keywords: microalgae, allelopathy, bioassay, phytotoxicity Tel: +39 0965 324077; Fax: +39 0965 311092 121 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 38. ENVIRONMENTAL EFFECTS ON ALLELOPATHIC GROWTH INHIBITION OF A CYANOBACTERIUM (Microcystis aeruginosa) BY A SUBMERGED MACROPHYTE (Myriophyllum spicatum) AND A CLUE TO UNDERSTAND ITS MECHANISMS Satoshi Nakai 1*, Tetsuji Okuda 2, and Wataru Nishijima 2 1 Grad. School Eng., Hiroshima Univ., Higashi-Hiroshima 739-8527, Japan; Email: [email protected] 2 Environ Res Manag Centr, Hiroshima Univ, Higashi-hiroshima 739-8513, Japan; 2 3 Email: [email protected]; email: [email protected] ABSTRACT Investigated here is an effect of temperature and light intensity on the growth inhibition of the cyanobacterium Microcystis aeruginosa and its photosynthesis reduction by eight allelochemicals consisting of five polyphenols and three fatty acids released from Myriophyllum spicatum. By the addition of eight allelochemicals, the maximal PSII quantum yield of M. aeruginosa and its growth were suppressed. An analysis of the relationship between normalized specific growth rate and normalized maximal quantum yield showed that the reduction in photosystem II was the major mechanism in the growth inhibition of M. aeruginosa. The growth inhibition effect of the eight allelochemicals was strengthened or weakened along with the change of temperature and light intensity under the experimental conditions: 20 °C―30 °C and 25 µmol m-2 s–1 75 µmol m-2 s–1. The lower temperature and light intensity resulted in the stronger growth inhibition of M. aeruginosa: the reduction of specific growth rate of M. aeruginosa at 20 °C was 1.9 times greater than that at 30 °C, and the 1.5 times difference was observed in the reduction of specific growth rate between 25 µmol m-2 s–1 and 75 µmol m-2 s–1. This paper provides the evidence that estimation of anti-cyanobacterial allelopathic effect of macrophyte in the aquatic environment should consider its strengthening or weakening along with the changes of water temperature and light intensity. Keywords: allelopathy; cyanobacterium; fatty acid; photosystem II quantum yield; polyphenol Tel: +81-82-424-7621; fax: +81-82-424-7622 122 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 48. FEEDING OF Fragilidium cf. duplocampanaeforme AND F. subglobosum ON FOUR Dinophysis SPECIES: PREY SPECIFICITY, LOCAL ADAPTATION AND FATE OF TOXINS F. Rodríguez1*, P. Riobó2, P. Rial1, B. Reguera1, J. M. Franco2 1* Instituto Español de Oceanografia (IEO), Centro Oceanográfico de Vigo (UA Fitoplancton Tóxico CSIC-IEO), Subida a Radio Faro 50-52, Cabo Estay, Canido, 36390 Vigo, Spain. 2 Instituto de Investigaciones Marinas, CSIC (UA Fitoplancton Tóxico CSIC-IEO), Eduardo Cabello, 6, 36208 Vigo, Spain ABSTRACT We studied the feeding behaviour of 2 Fragilidium species — F. cf. duplocampanaeforme (strains VGO1120 and VGO692 from NW Iberia and the Mediterranean Sea, respectively), and F. subglobosum (IO97-01, W Iberia) — on Dinophysis acuminata, D. acuta, D. caudata and D. tripos from NW Iberia. Only the Atlantic strain of F. cf. duplocampanaeforme fed upon D. acuminata (2.52 ± 0.48 cells Fragilidium−1 d−1) and D. caudata (0.58 ± 0.32 cells Fragilidium−1 d−1); furthermore, this strain fed weakly on D. tripos (< 0.10 cells Fragilidium−1 d−1) and not at all on D. acuta. Neither the Mediterranean F. duplocampanaeforme nor F. subglobosum ingested Dinophysis. Deleterious effects caused by Fragilidium, namely cell immobilization or even death, were observed in some cases (D. acuminata and D. caudata). The Atlantic F. cf. duplocampanaeforme that had previously fed on D. acuminata and D. caudata showed low intracellular toxin levels at the end of the exponential growth phase (LC-MS analyses). However, extracellular toxin levels were higher in the Dinophysis cultures exposed to the Atlantic F. cf. duplocampanaeforme than in those without Fragilidium (the contrary being true for the toxin amount per cell). We concluded that (i) Fragilidium cf. duplocampanaeforme feeds differentially on certain Dinophysis species, (ii) inter-specific and intraspecific differences exist in Fragilidium-Dinophysis predator-prey interactions, (iii) the amount of extracellular Dinophysis toxins is enhanced by the presence of Fragilidium, and (iv) decreased final yields and higher frequencies of small cells are obtained in Fragilidium mixotrophic cultures. Keywords: Dinophysis, Fragilidium, prey specificity, okadaic acid, DSP toxins transfer, mixotrophy Tel: 986 49 21 11; fax: 986 49 86 26 123 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 49. ALLELOPATHIC EFFECTS OF ANIMAL HORMONES ON GROWTH OF MARINE PHOTOSYNTHETIC BACTERIA (PSB) AND DEGRADATION OF PESTICIDES 1 2 Lansheng Li1, Le Li1 and Xiuhai Wang2* College of Environmental Science and Engineering, Ocean University of China, 5 Yushan Road, Qingdao, P.R.China; e-mail: [email protected]; [email protected] Key Laboratory of Marine Environment and Ecology of MOE, Ocean University of China, 238 Songling Road, Qingdao, P.R.China; e-mail: [email protected] ABSTRACT In this study, allelopathic effects of animal hormones on proliferation of marine photosynthetic bacteria (PSB) were evaluated. In such a simple, fast and economical way, PSB population was enhanced to a high enough level in quite a short period of time to achieve degradation of pesticides in water. Study results showed that with an appropriate addition of animal hormones, growth of PSB was promoted significantly. The optimal level of both prednisone (cblconin) and dexamethasone were 0.5 mg/L, with a growth promotion rate of PSB varying between 13% and 15%. PSB prospered under organophosphorus pesticide (omethoate) concentration less than 1000 mg/L; otherwise, with the increase of omethoate concentration, their growth was inhibited accordingly. PSB used omethoate as their sole sustaining source of C and N elements; therefore, they can be used to degrade omethoate in a certain concentration range. For omethoate at a concentration of 500 mg/L, its degradation rate reached nearly 50% in 24 hours, and ranged from 70% to 80% in 4 days. Keywords: allelopathic effect, marine photosynthetic bacteria (PSB), proliferation, animal hormone, prednisone (cblconin), dexamethasone, degradation of pesticide, organophosphorus pesticide, omethoate Tel: +86-13325001885; Fax: +86-532-82883085 124 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 39. ALLELOPATHIC EFFECTS IN DINOFLAGELLATES: A CASE STUDY OF THE BENTHIC GENUS Ostreopsis AND TWO NON-TOXIC SPECIES FROM THE GENERA Prorocentrum AND Coolia. 1 G. Portela M.1, Riobó P.2, Franco J.M.2, Rodríguez F.1 Instituto Español de Oceanografia (IEO), Centro Oceanográfico de Vigo (UA Microalgas Nocivas CSIC-IEO), Subida a Radio Faro 50-52, Cabo Estay, Canido, 36390 Vigo, Spain. email: [email protected] 2 Instituto de Investigaciones Marinas, CSIC (UA Microalgas Nocivas CSIC-IEO), Eduardo Cabello, 6, 36208 Vigo, Spain. ABSTRACT The genus Ostreopsis includes several toxic species that can develop blooms in benthic ecosystems, with potential harmful consequences to the human health and marine invertebrates. Despite of this, little is known about the allelopathic effects caused by these organisms in other co-occurring microalgae. The aim of this study was to study these effects in mixed cultures of Ostreopsis and two benthic genera of dinoflagellates (Coolia and Prorocentrum), when sharing the same environment, under controlled conditions. The potential allelopathic effects were studied in two non-toxic species (Coolia monotis and Prorocentrum elegans) exposed separately to the clarified medium and cells of two strains of Ostreopsis. For this purpose, growth rates of each species, toxin profiles and toxin content per cell were determined by LC-HRMS in mixed cultures. The obtained results are discussed in terms of the interespecific differences observed in toxin contents for both Ostreopsis strains and the inhibitory effects in both Coolia and Prorocentrum cultures. Keywords: Ostreopsis, allelopathy, ovatoxins, PLTXs, interespecific effects, inhibitory effects Tel: (+34) 986 49 21 11; fax: (+34) 986 49 86 26 125 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 50. Novel phytoalexins from Brassicales: synthesis, biosynthesis and biological activity M. Soledade C. Pedras*, Huy To and Estifanos Yaya Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, SK, Canada; e-mail: [email protected]; e-mail: [email protected]; e-mail: [email protected]; e-mail: [email protected] ABSTRACT Crucifer plants respond to stress employing metabolic pathways that involve the biosyntheses of numerous and structurally diverse natural products. Phytoalexins are natural products involved in induced plant defence pathways against plant pathogens and various other stresses. Importantly, in general the chemodiversity of phytoalexins correlates with the biodiversity of their sources. For example, within crucifers, cultivated Brassica species produce the phytoalexins brassinin, cyclobrassinin, brassilexin, spirobrassinin, and rutalexin (5), whereas wild species such as Arabidopsis thaliana and Thellungiella salsuginea produce camalexin and wasalexins, respectively. We have investigated additional cultivated and wild cruciferous species native of various habitats for production of phytoalexins. The chemical structures of novel phytoalexins produced by various crucifers, including nasturlexins, novel non-indolyl phytoalexins, and tenualexin together with their chemical syntheses and biosynthetic pathways will be reported. De novo incorporations of deuterated compounds and amino acids into pertinent phytoalexins and chemical analysis by LC-MS-DAD will be described. Results from these experiments showed incorporation of deuteria into nasturlexins, indicating for the first time that an additional phytoalexin biosynthetic pathway operates in parallel to the tryptophan derived pathway in the same plant species. In concluding, the production of glucosinolates that are biosynthetic precursors of some cruciferous phytoalexins and proposed biosynthetic pathways will be discussed. Knowledge of new phytoalexins and their biosynthetic pathways will facilitate the identification of plant defence genes and the potential breeding of plants producing more potent antifungal metabolites. Keywords: Brassicales, crucifer, nasturlexin, phytoalexin, tenualexin 126 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 51. DOES BIOLOGICAL DENITRIFICATION INHIBITION BY PLANT SECONDARY METABOLITES EXIST? Clément Bardon1,2*, Florence Piola2, Haichar Feth el Zahar1, Thomas Pommier 1,3, Floriant Bellvert1,4, Sara Puijalon2, Guillaume Meiffren1,4 ,Noelline Tsafack2 and Franck Poly1,4 1 Université de Lyon, UMR 5557 Laboratoire d’Ecologie Microbienne, Université Lyon 1, Villeurbanne, France; *Corresponding author: [email protected] 2 Université de Lyon, UMR 5023 Laboratoire d’Ecologie des Hydrosystèmes Naturels et Anthropisés, Université Lyon 1, Villeurbanne, France. 3 INRA, UMR 5557 Laboratoire d’Ecologie Microbienne, Université Lyon 1, Villeurbanne, France; 4 CNRS, UMR 5557 Laboratoire d’Ecologie Microbienne, Université Lyon 1, Villeurbanne, France; ABSTRACT Studies on plant-driven microbial functioning and selection by secondary metabolites mostly focused on the effect of plant on the nitrogen (N) cycle but discarded biological denitrification inhibition (BDI), which was never demonstrated. The invasive complex species Fallopia sp. was shown to reduce denitrification (Dassonville et al 2011), a major way of N loss by gaseous emission, in soil. We investigated the BDI by secondary metabolites from Fallopia on a panel of cultivated strains and on a complex microbial community in soil by comparing effects of root extracts from Fallopia on metabolic activity and respiratory activity, in aerobic condition and anaerobic condition (Denitrification). The analysis of root extracts shows the presence of families of compounds recognized for their biological activities. In denitrifying bacteria denitrification inhibition (95%) by root extracts from Fallopia sp. is higher than respiration inhibition (50%). Anaerobic metabolic activity was used to investigate dose-response effects and molecules involved by extract fractionation. It appeared that anaerobic metabolic reduction is correlated to the amounts of some compounds of the extracts. In soil, extracts reduced the Denitrification Enzyme Activity / Substrate Induced Respiration ratio without affecting denitrifier gene abundances / total bacteria gene abundances ratio. Our findings show that secondary metabolites from Fallopia sp. inhibit denitrification probably by targeting the respiratory process of denitrifying bacteria. This is a new insight in plant-soil interactions and furthers our understanding on plant ability to shape microbial soil functioning by the release of secondary metabolites. Keywords: denitrification, Fallopia, secondary metabolites, denitrifying bacteria, biological inhibition Tel: +33472431478; fax: +33472431223 127 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 52. MOLECULAR AND METABOLIC ASPECTS OF PLANT-MICROBE INTERACTIONS DURING ORGANIC NITROGEN ACQUISITION IN THE RHIZOSPHERE Weronika Graj1, Jim Rasmussen2, Mogens Nicolaisen1, Inge S. Fomsgaard1* 1 2 Department of Agroecology, Aarhus University, Forsøgsvej 1, DK-4200 Slagelse, Denmark; e-mail: [email protected] Department of Agroecology, Aarhus University, Blichers Alle 20, DK-8830 Tjele, Denmark; email: [email protected] 3 Department of Agroecology, Aarhus University, Forsøgsvej 1, DK-4200 Slagelse, Denmark; email: [email protected] 4 Department of Agroecology, Aarhus University, Forsøgsvej 1, DK-4200 Slagelse, Denmark; email:[email protected] ABSTRACT It has been proven that besides inorganic nitrogen (N) plants from different ecosystems can assimilate organic N. However, knowledge about its contribution to the total N budget in leguminous plants is very limited. Therefore questions about the importance of organic N in legume nutrition remain to be addressed. In order to reveal legume potential for organic N acquisition we have to understand interactions between plants and microbes regulated by allelochemicals during competition for N sources. To show the organic N dynamics in rhizosphere I will quantify amino acid assimilation by a legume plant (Trifolium repens) and microorganisms over a short time scale. Moreover, to define the molecular and metabolic basis underlying plant-microbe interactions during N competition, chemical compounds mediating their relationships will be analyzed. Established legume seedlings growing in a split-root rhizobox system will be treated with dual-labelled 13C,15N asparagine through the injection into soil. I expect that collected results will help me to answer the following questions: how do microorganisms modulate plant organic N assimilation and conversely, how do plants contribute to the structure and activity of microbial community? How does this relationship influence plant productivity and N dynamics in the rhizosphere? The integration of metagenomic and metabolomic approaches will undoubtedly help to depict how plants use organic N in the context of plant nutrition. Keywords: plant-microbe nitrogen cycling interactions, nutrient Tel: +45 87158139 / *+45 87158212; fax: +45 87156082 128 acquisition, rhizosphere, 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 53. TRANSCRIPTOMICS OF Pseudomonas sp. ISOLATED FROM DIFFERENT WATER REGIMES Humaira Yasmin1, Neil Wilson, Asghari Bano, Rosalind Deaker, Ivan R. Kennedy Department of Plant Sciences. Quaid I Azam University, Islamabad, Pakistan 1 [email protected] ABSTRACT Pseudomonas sp. is a gram negative bacteria with many favourable plant growth promoting capabilities. It is also an ideal candidate to improve drought tolerance in plants in water stress habitat. Here we report the first transcriptomic analysis of Pseudomonas sp. strains isolates from rhizospheres of maize and rice grown at varying moisture regimes during desiccation. Filtered Pseudomonas sp. cells incubated under dessicated (10% RH) as well as hydrated (85% RH) condition were analysed for cell viability and transcriptomic study of dessication resistant genes. Cell viability of all Pseudomonas sp. decreased significantly under dessicating (10% RH) condition as compared to that of hydrating (85% RH) conditions in a dessicating tolerance assay. Trehalose synthase (tre), sigma factor (rpoS), 2, 3 butanediol (adh), acetion dehydrogenase (acoA), alginate (algF), flagellar protein (flgG), flagellar motor switch protein (fliM) and alginate regulatory protein (mucA) were detected in all Pseudomonas strains. RT-PCR showed significant increase in relative expression of trehalose synthase (tre), sigma factor (rpoS), alginate regulatory gene (mucA) and flagellar motor switch protein gene (fliM) in desiccated (10% RH) condition. Marked up-regulation in relative expression of trehalose synthase (tre), sigma factor (rpoS), alginate regulatory gene (mucA) and flagellar motor switch protein (fliM) gene in response to induced desiccation (10% RH) of Pseudomonas sp. cells in relation to their water stressed habitat (from rhizosphere of rice grown in raised bed condition) was observed. It is inferred from the study that trehalose and exopolysaccharides production plays a prominent role in promoting desiccation tolerance in Pseudomonas strains isolated from water scarce environment (semi-arid and raised bed). These desiccation specific responses represent the best candidates for mechanism of desiccation resistance in Pseudomonas sp. strains in relation to their host and habitat Keywords: allelopathy, Pseudomonas, desiccation resistance genes, trehalose synthase (tre), sigma factor (rpoS), 2, 3 butanediol (adh), acetion dehydrogenase (acoA), alginate (algF), flagellar protein (flgG), flagellar motor switch protein (fliM), alginate regulatory protein (mucA). Tel: +923325570471 129 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 54. IN VITRO ANTIFUNGAL AND ANTIMICROBIAL ACTIVITY OF Eucalyptus camaldulensis ESSENTIAL OIL AGAINST PATHOGENIC MICROORGANISM Mehani Mouna1, Salhi Nesrin2, Valeria Terzi3 and Ladjel Segni1 1 2 Laboratory of Process Engineering, University of Kasdi Merbah, Ouargla, Algeria; e-mail: [email protected] Laboratory for Bioressources Saharan preservation and enhancement, University of kasdi Merbah, Ouargla, Algeria 3 Instituto Sperimentale per la cerealicoltura, Fiorenzuola, Italy ABSTRACT Introduction: The vegetable world is the origin of a large number of drugs. Recently, researchers have estimated that there are about 400,000 species of plants in the world, about a quarter or a third has been used by companies for medicinal purposes. The humans have used the plants for thousands of years to treat various ailments and in several countries in the developing world a large part of the population relies on traditional doctors and their collections of medicinal plants for treatment. Essential oils have many therapeutic properties. In herbal medicine, they are used for their antiseptic properties against infectious diseases of fungal origin, against dermatophytes, those of bacterial origin. Objectives: The object of our study was to determine the antimicrobial effect of essential oils of Eucalyptus camaldulensis plants on some pathogenic bacteria and two fungal strains (Fusarium graminearum and Fusarium sporotrichioides), it is a medicinal plant used in traditional therapy. Materials and Methods: The test adopted is based on the diffusion method on solid medium (antimicrobial), this method allows determining the sensitivity or resistance vis-à-vis micro-organism extract studied. Discussion: Our study reveals that the essential oil of Eucalyptus camaldulensis plants has a different effect on the resistance of germs. Proteus, Staphylococcus, Escherichia coli strains are moderately susceptible. By contrary, Pseudomonas aeruginosa is a strain that represents a low sensitivity. In addition, the technique of direct contact is contacting the essential oils and micro-organisms, and observing the growth of the latter. The essential oil of Eucalyptus camaldulensis had a significant inhibitory activity against the fungus (Fusarium sporotrichioides and Fusarium graminearum) diameters, speed and antifungal index mycelium growth are reduced every time you increase the concentration of essential oil to the non-germination of the disc affect the CMI 50 µl/ 20 ml of PDA ). Keywords: Essential oil, plant, bacteria, champigion, antibiogram Tel: +213790034016 130 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 55. ALLELOPATHIC INTERACTIONS IN THE COMMUNITIES OF HALOPHYTE VEGETATION OF CRIMEA Simagina N.O. V.I. Vernadsky Taurida National University, [email protected] ABSTRACT Natural halophyte vegetation has been used in methods of phytomelioration for recultivation of the secondary salted grounds of Crimea (Ukraine). Complex research including phytocenotic, hystochemical, biochemical methods allows to investigate allelopathic activity of halophytes. It was established that perennial species of halophyte vegetation of Crimea Artemisia santonica L., Halocnemum strobilaceum (Pall.) M.Bieb., Limonium gmelinii (Willd.) O. Kuntze possess high allelopathic activity, which dynamics changes during the vegetative period, and in ontogenesis as a whole. The allelopathic potential of annual species of halophyte Saliconia europaea L., Suaeda prostrata Pall. is caused by activity of excretions of germinated seeds, and also water-soluble excretions of virginive and generative individuals. Morphometric parameters (height, weight, diameter of stem, degree of branching) of plants - acceptors Salicornia europaea L., Suaeda prostrata Pall., Petrosimonia oppositifolia (Pall.) Litv. increase on a gradient of remotness from plants-donors of allelopathic substances A. santonica, H. strobilaceum, L. gmelinii. The reproductive effort of annuals S. europaea, S. prostrata, P. oppositifolia grows at increasing distance from the basis of stem of perennial halophytes. The ryzosperous ground in phytogenous field of A. santonica, H. strobilaceum, L. gmelinii shows high phytotoxic activity which increases in this list. At action of the allelopathic factor plant-acceptor S. europaea has essential changes at the histological level: intensive lignification and suberinization of conducting and mechanical tissues, significant accumulation of pectinaceous substances, phenolic connections. It was revealed that allelopathic activity of excretion of halophytes is caused by the presence of amines, alkaloids, phenolic substances (phenolcarbonic acids, tannins, flavonoids). Keywords: allelopathy, halophyte Tel:+(380)999848796 131 132 Session 5. Allelopathy horticulture and forestry in agriculture, 133 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 56. DOES LAMIACEAE PLANTS POSSESS ALLELOPATHIC SUBSTANCES: A PLAUSIBLE ANSWER FROM Leucas aspera AKM Mominul Islama* Osamu Ohnob, Kiyotake Suenagab and Hisashi Kato‐ Noguchia a Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, 2393 Ikenobe, Miki, Kagawa 761–0795, Japan *Corresponding author: [email protected] b Department of Chemistry, Faculty of Science and Technology, Keio University, 3–14–1 Hiyoshi, Kohoku, Yokohama 223–8522, Japan ABSTRACT Plants of Lamiaceae family attracted the attention of many researchers in pharmacological interest because of their toxic potential and medicinal properties. However, information about the allelopathic activities and allelopathic substances of that family are quite negligible. Therefore, current research was conducted to investigate the allelopathic properties of five Lamiaceae medicinal plants: Leucas aspera, Leonurus sibiricus, Ocimum tenuiflorum, Mentha sylvestris and Hyptis suaveolens against eight test species. The results revealed that the extracts of those plants are allelopathic, and thus contain allelopathic substances. Their inhibitory activities on test species were in the order of L. aspera> H. suaveolens> M. sylvestris> L. sibiricus> O. tenuiflorum. The greatest inhibitory activity of L. aspera motivated us to further work with this plant extract to isolate and identify the allelopathic substances responsible for its inhibitory activity. The extract of L. aspera was then purified by several chromatographic runs. Final purification was achieved by reversedphase HPLC to give an equilibrium (or inseparable) 3:2 mixture of two labdane type diterpenes, compounds 1 and 2. These two compounds were characterized by high-resolution ESI-MS, 1H-, 13C- NMR and optical rotation. The mixture of these two compounds significantly inhibited the growth of all the test species. The concentration required for 50% growth inhibition (I50) of the test species ranged from 31−180 µM. These results suggest that the mixture of compounds 1 and 2 is responsible for the growth inhibitory activity of L. aspera, and may play an important role in the allelopathic properties of this plant. Keywords: inhibitor Allelopathy, Lamiaceae, medicinal plant, diterpene, plant growth TEL: +81-090-2896-1982; FAX: +81-878913086 134 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 57. THE INDUCED EFFECTS OF BARNYARDGRASS (Echinochloa crus-galli L.) ON RICE ALLELOPATHY Hai‐Bin He*, Zhen‐Ming Yu, Jia‐Yu Li, Qi Zhang, Zhi‐Hua Lin Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, and Agroecological Institute/School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, 350002, P.R. China; e-mail: [email protected] ABSTRACT In paddy field, barnyardgrass always grows together with rice. We presume that there are chemical crosstalk between rice and barnyardgrass, and rice allelopathy enhanced under barnyardgrass stress is contributed partly to the chemicals in barnyardgrass exudates. To attest this hypothesis, the allelopathic rice PI312777 and non allelopathic rice Lemont were used as inductive targets. Inductive agents, which extracted from the barnyardgrass tissues and concentrated from the culture solutions of barnyardgrass, were added to the culture solutions of two target rice accessions. The bioassay of water extracts from rice leaves on barnyardgrass were evaluated the inductive effect. The results showed that, (1) treated by tissues extracts, the inhibitory rate of rice leaves extracts on root length, plant height and dry weight of barnyardgrass has increased by 17.0%, 10.8% and 12.0% respectively in PI312777, compared to its untreated control, and 11.2%, 9.1% and 7.7% respectively in Lemont, compared to its untreated control; (2) treated by concentrated culture solution, the inhibitory rate has increased by 21.2%, 16.8% and 17.9% in PI312777, and 13.8%, 13.1% and 15.7% respectively in Lemont. The most suitable dosage was 15mL concentrated solution for 5d, in which the inhibitory rate has increased by 19.8%, 18.97%, 23.0% in PI312777, and 19.2%, 19.9%, 20.5% respectively in Lemont. The most suitable time was 2d in 15mL concentrated solution. The inhibitory rate reached to 74.6%, 69.5%, 75.0% in PI312777, and 29.7%, 32.0%, 30.7% in Lemont. These results indicated that increase of rice allelopathy by induction would be a novel approach. Keywords: Rice (Oryza sativa L), allelopathy, barnyardgrass (Echinochloa crus-galli L.), induction TEL: +86-591-83789352; FAX: +86-591-83769440 135 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 58. ALLELOPATHY IN CROPPING SYSTEMS Muhammad Farooq*1, Zahid Ata Cheema2 and Sardar Alam Cheema3 Allelopathy Lab, Department of Agronomy, University of Agriculture, Faisalabad-38040, Pakistan E-mail: 1 [email protected]; 2 [email protected]; 3 [email protected] ABSTRACT Crops grown in short rotations and monocultures may experience growth suppression and yield reduction than the crops grown in longer rotations. Although the allelopathic effects may be stimulatory or inhibitory, suppressive effects of allelopathic, plants both in monocultures and multiple cropping systems, have been observed. Crop rotations are practiced to reduce the ill effects of monocultures, but the subsequent crop may be affected by the allelochemicals released by the preceding one. For instance, when grown in rotation with tobacco, stand establishment and growth of maize were improved whereas mungbean stand and growth were suppressed. When grown in rotation mungbean, stand establishment and growth of soybean were suppressed; however, stand establishment and growth of maize were improved. Long term trials of aerobic rice also indicated yield decline over time. This yield decline has been attributed to biotic factors such as buildup of plant pathogens, and allelochemicals in the rhizosphere. Therefore, allelopathic nature of the crops must be considered while making the crop rotations. Keywords: Crop rotation, cropping system, growth Tel: +92(41)9201098; Fax: +92(41)9200605 136 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 40. PHYTOCHEMICAL CHARACTERISTICS OF TWO ALLELOPATHIC RICE VARIETIES CULTIVATED IN ARGENTINA Hugo D. Chludil1* and Silvia R. Leicach2 Chemistry of Biomolecules, Department of Applied Biology and Food, School of Agronomy, University of Buenos Aires. Avda.San Martın 4453,CABA C1417DSE, Bs.As., Argentina. e-mail1: [email protected] 2 e-mail : [email protected] ABSTRACT El Paso 144 and Bluebonnet 50, two out six rice cultivars most frequently used in Argentinean Mesopotamia have previously shown significant inhibitory effects on barnyardgrass (Echinochloa crus-galli L.). Phytochemical analysis of their roots and those from an inactive variety (Supremo 13) showed significant differences among their chemical compositions. Presence of momilactones B and A, was only detected in similar amounts in both allelopathic varieties. Significant differences were found among the three varieties when comparing relative amounts of non polar and polar constituents. Linear hydrocarbons were main non-polar constituents for Supreme 13 (non- allelopathic) cyclohexane extract with heneicosane, docosane, tricosane, and eicosane as major components. Hexadecanoic acid methyl ester was the most abundant compound for Bluebonnet 50 and a higher amount of oxygenated derivatives was found in El Paso 144, which showed palmitic acid as cyclohexane extract major component, being the only one that also contained octadecanal, 6.10.14 trimethyl-2- pentadecanone, and 8-heptylpentadecane in significant amounts. Both allelopathic cultivars contained around three times the relative amount of oxygenated non-polar derivatives compared to Supremo 13. On the other hand, Bluebonnet 50 methanol extract showed much higher concentrations of phenolic derivatives; quercetin glucoside and caffeic, vanillic, syringic, p-coumaric, ferulic, and chlorogenic acids were significantly more abundant in this variety compared to the other two. Presence of higher amounts of oxygenated non-polar compounds in El Paso 144 and phenolic derivatives in Bluebonnet 50 may contribute to the negative effects of these varieties on barnyardgrass seeds germination and development. Keywords: rice allelopathy, barnyardgrass, oxygenated non-polar compounds, phenolics Tel: +541145248001; fax: +541145148737 137 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 41. ALLELOPATHIC EFFECTS OF SENECIO GRISEBACHII ON TWO WHEAT CULTIVARS. Chludil, H.D.1*; Yaber Grass, M.A.2; Del Fueyo, P.A.3; Leicach, S.R.4 1,2,4 Chemistry of Biomolecules, Department of Applied Biology and Food, e-mail1: [email protected] 2 e-mail : [email protected] 4 e-mail : [email protected] 3 Laboratory of Seed Biology 3 e-mail : [email protected] School of Agronomy, University of Buenos Aires. Avda. San Martın 4453,CABA, Buenos Aires C1417DSE, Argentina. ABSTRACT Direct seeding and widespread use of glyphosate have favored an increase of weeds population density, particularly including species such as Senecio grisebachii with low susceptibility to the herbicide, most of which have been shown to deleteriously affect wheat, soybean, and corn production. This weed produces mainly pyrrolizidine alkaloids and phenolics, and also terpenic derivatives, in concentrations that depend on soil quality, affecting the intensity of its allelopathic effects on crops. A pot trial was performed to obtain S. grisebachii samples grown under N and P deficit (deteriorated soil, D), and also in pristine soil (non-deteriorated, ND). Aerial tissues from S. grisebachii samples were submitted to phytochemical analysis. Plant material was analyzed by UV-Vis spectrophotometry to determine the presence of phenol derivatives and by GC and GC- MS to determine pyrrolizidine alkaloid profile. Their allelopathic effects on wheat germination were determined for two crop cultivars (Buck 75 and Baguette 11). Soils with low P and N caused an increase of total alkaloid content in S. grisebachii aerial parts, also modifying the senecionine / senecifiline ratio. Aqueous extracts of weed aerial tissues from samples grown under N and P deficit significantly affected wheat cultivars germination, delaying Buck 75 germination and inhibiting that of Baguette 11. Allelopathic effect of this weed on wheat might be associated in part to the presence of pyrrolizidine alkaloids in its residues that could affect in different ways wheat cultivars under direct seeding. Keywords: Senecio grisebachii, wheat cultivars, pyrrolizidine alkaloids, phenolics. Tel: +541145248001; fax: +541145148737 138 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 42. VARIETAL RESPONSE OF CHICKPEA (Cicer arietinum L.) TOWARDS THE ALLELOPATHY OF DIFFERENT WEEDS 1 Rahamdad Khan1 and Ijaz Ahmad Khan 2* Department of Weed Science,The University of Agriculture, Peshawar- Pakistan 1; e-mail:[email protected] 2 Department of Weed Science,The University of Agriculture, Peshawar- Pakistan 2;e-mail:[email protected] ABSTRACT In a laboratory trail three chickpea varieties viz, Karak-I, Karak-III and Shenghar were tested against the phytotoxicity of four weed species: Parthenium hysterophorus L., Phragmites australis (Cav) Trin., Datura alba L., Cyperus rotundus L. and Convolvulus arvensis L. in January, 2013. The weed extracts were prepared @ 120 g/liter (w/v) after shade dry. The results indicated highly significant inhibitory effect of all the tested weed species on chickpea varieties. Results also revealed that the chickpea variety “Karak-III” was more susceptible to the phytotoxcity of the tested weed extracts. Among the extract C. arvensis proved much toxic in term of inhibition of germination by giving only 43.33% germination in comparison with control where, 97.50% germination recorded. On the other hand, the effect of P. australis extract found a little stimulator by speeding the seed germination in all varieties and giving a low (2.21) Mean germination time (MGT) value. From the current results it can be concluded that the infestation of C. arvensis can pollute the soil by accumulating toxic chemicals that leads to the germination failure and growth suppression in chickpea. Therefore, the prevention and removal of C. arvensis in the chickpea growing areas could be recommended. In addition P. australis must be tested against chickpea weeds because, chickpea varieties with stand against its phytotoxcity, so it can be popularized as bioherbicide in chickpea if it gave promising results in controlling chickpea weeds. Keywords: Allelopathy, chickpea, germination, parthenium, weeds Tel: +923329238965 139 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 59. ALLEVIATION OF CONSECUTIVE MONOCULTURE PROBLEMS OF Rehmannia glutinosa BY APPLICATION OF SPENT MUSHROOM SUBSTRATE Ming‐jie LI1,2, Xuan‐zhen LI1, Feng‐qing WANG1, Rui‐hong RU1, Fa‐jie FENG2, Bao ZHANG2, Zhong‐yi ZHANG2,1 ∗ 1 2 College of Life Sciences, Henan Agricultural University, Zhengzhou, China, 450002 College of Crop Sciences, Fujian Agriculture and Forestry University, Fuzhou, China, 350002 ABSTRACT Cultivation of Rehmannia glutinosa Libosch, a traditional Chinese medicine herb, can cause the accumulation of phenolic acids in the soil and it is supposed to be the reason of the so called “consecutive monoculture problems”. In our study, phenolic acids (hydroxybenzoic acid, vanillic acid, eugenol, vanillin and ferulic acid) were degraded by the extracts of all the tested spent mushroom substrate (SMS) and the maximal total degradation rate was 75.3%, contributed by extracts of SMS of Pleurotus eryngii Quel. The pot experiment indicated that hydroxybenzoic acid and vanillin in soil were also degraded effectively by SMS of Pleurotus eryngii Quel. At the same time, the fresh and dry weight and total catalpol concentration of tuberous root of Rehmannia glutinosa increased to 2.70, 3.66 and 2.25 times by application of SMS, respectively. The application of SMS also enhanced ecophysiology index of plant, such as crown width, leaves number, leaf length, leaf width and height near to the normal levels. The increase of bacteria, fungi and actinomycetes number in rhizosphere soil were observed after the application of SMS by microbial counts. These results indicated that application of SMS alleviated the consecutive monoculture problems of Rehmannia glutinosa Libosch in some extent, and its possible mechanism was in one hand, phenolic acids were degraded, and in another hand, it supplied the additional nutrition and stimulated the microbial activities. Keywords: Spent mushroom substrate, Rehmannia glutinosa Libosch, phenolic acids, consecutive monoculture problems 140 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 60. PHYTOTOXICITY OF CRUDE ALKALOID FRACTION FROM Rhazya stricta Nazif Ullah1, Sehar Amjad2, Ihsan‐Ul‐haq3, Shibu Jose4 and Bushra Mirza2* 1 Department of Biotechnology, Abdul Wali Khan University Mardan, Pakistan: Email: [email protected] 2 Department of Biochemistry Quaid-I-Azam University, Islamabad, Pakistan: Email: [email protected] 2*Department of Biochemistry Quaid-I-Azam University, Islamabad, Pakistan: Email: [email protected] 3 Department of Pharmacy Quaid-I-Azam University, Islamabad, Pakistan: Email: [email protected] 4 The Centre for Agroforestry University of Missouri, Columbia, USA: Email: [email protected] ABSTRACT Rhazya stricta, an important medicinal plant in Pakistan, belonging to the family Apocynaceae is well known for its alkaloids and allelopathic potential. Evidence from the literature suggests that alkaloids negatively affect growth and development of plants. The present research focuses on the hypothesis that alkaloids of R. stricta are responsible for the allelopathic potential of this plant. For this purpose aerial parts and roots of R. stricta were subjected to fractionation followed by phytotoxicity assay. Four fractions (aqueous fraction, chlorofrom fraction, ammonical-chloroform fraction and alkaloid fraction) each from the aerial part and root extracts were prepared using acid-base partitioning methodology, with the aim to optimize the method for isolation of crude alkaloids from R. stricta. Phytotoxic behaviour of these extracts/fractions was then studied using both monocot (Triticum aestivum) and dicot (Brasica napus) seeds as standard target species. The alkaloid mixtures both from the root and aerial part inhibited seedling growth up to 100% at higher concentration of 10,000 ppm which decreased with decrease in concentration. Although aqueous fractions also showed promising phytotoxic activity, the phytotoxic chemicals were found to be at higher concentrations in the alkaloid fraction than in the aqueous since it showed greater reduction in seedling growth. Although additional experimental evidence would be necessary, these results demonstrated high phytotoxic potential of the crude alkaloid fractions of R. stricta. Chromatographic analysis was also done to validate the fractionation method and optimization of alkaloids extraction. The current findings confirm the allelopathic nature of the alkaloids found in R. stricta. Keywords: Alkaloid, phytotoxicity, Rhazia stricta Tel: +92-333-9718064 141 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 61. BIOSOURCE OF AGROCHEMICALS FROM Tamarindus indica, L. – A COMPREHENSIVE STUDY FROM EXPERIMENTAL AND STATISTICAL PERSPECTIVES: Nabanita Chakraborty1 and Suparna Mandal Biswas2* 1 Agricultural and Ecological Research Unit, Indian Statistical Institute 203, B.T. Road, Kolkata 700108, INDIA; e-mail: [email protected] 2 Agricultural and Ecological Research Unit, Indian Statistical Institute 203, B.T. Road, Kolkata 700108, INDIA; e-mail: [email protected] ABSTRACT Tamarindus indica L., a common economic tree under the family Fabaceae, has been a well known as a source of herbal medicine from ages. The tree sheds leaves continuously in all seasons and the fact which grabs my attention is that vegetation under the tree is very sparse. Isolation and identification of bioactive compounds from Tamarind leaves is a potential approach for developing plant based agrochemicals for eco-friendly agricultural practices. Three major fractions have been isolated by liquid-liquid extraction process. In order to evaluate their chemical composition, the obtained extracts were further purified into a single pure compound by repetitive running through column chromatography and thin layer chromatography and finally the purified compounds were subjected spectral analyses (MS, 1HNMR, 13CNMR and FTIR). Methyl 4, 4-dimethyl-tetra-decanoate (mol.wt.270), caffeic acid derivative (mol.wt.390), and Methyl-2,3,4-trihydroxyhexanoate (mol.wt.178), have been identified from Hexane, ethyl acetate and methanol fraction respectively. All the compounds showed strong antimicrobial activity against several bacterial and fungal species. Concentration-dependent inhibitory and stimulatory bioassay on rice seeds was revealed by different fractions of tamarind. The comparative studies of cause and effect relationship of different fraction were also statistically signified. In conclusion, the use of natural product based agrochemicals verses environmental toxicity for the control of pests and diseases in agriculture will provide for a safe and better natural resource management by reducing dependence on synthetic chemicals. Keywords: Tamarindus indica, bioactive compounds, agrochemicals, methyl 4, 4-dimethyl-tetra-decanoate, caffeic acid derivative, methyl-2,3,4trihydroxyhexanoate. Tel: (+91) (033) 2575 3225, Fax: (+91) (033)25753049 142 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 62. ALLELOPATHIC EFFECTS OF AQUEOUS EXTRACTS OF SUNFLOWER ON WHEAT (Triticum aestivum L.) AND MAIZE (Zea mays L.) Abdul Majeed1*, Zubeda Chaudhry1 and Zahir Muhammad2 1 2 Department of Botany, Hazara University Mansehra, Khyber Pakhtunkhwa, Pakistan; e-mail: [email protected] Department of Botany, University of Peshawar, Peshawar, Khyber Pakhtunkhwa, Pakistan; email: [email protected] ABSTRACT Sunflower is a potent allelopathic plant which possesses important allelochemicals with known allelopathic activity on other plants. This aim of study was to evaluate the effects of fresh and air dried aqueous extracts of root, shoot and leaves of sunflower on wheat (Triticum aestivum L.) and maize (Zea mays L.) in laboratory experiments. Results showed significantly inhibitory effects of aqueous extracts on germination, growth and dry weight of seedling of the target plants. In wheat seedlings significant germination inhibition (15.21 %), increased mean germination time (57.76%), reduced plumule and radical growth (21.66 and 28.44 %) and low seedling dry weight (31.05 %) were recorded for leaf dry aqueous extracts when compared to control. Germination percentage of maize seedling was inhibited by leaf dry aqueous extracts by 7.81 %, germination index by 16.51 %, mean germination time increased by 25.53 %, decreased plumule and radical lengths by 29.00 and 36.12 % respectively, and lowered maize seedling dry weight 34.02 %. In both experiments, dry aqueous extracts (DAE) were more phytotoxic than fresh aqueous extracts (FAE). Similarly, inhibitory effects of aqueous extracts of different parts of sunflower were recorded in the order leaf > shoot > root for both the tested plants. The study suggests growth inhibitory effects of sunflower on wheat and maize in lab bioassay. Keywords: Allelopathy, competition, phytochemicals, secondary metabolites TEL: + 92 997 530732 ; FAX: + 92 997 530046 143 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 63. ALLELOTOXICITY OF OUDNEYA AFRICANA R. BR. AQUEOUS LEACHATE ON GERMINATION EFFICIENCY OF BROMUS TECTORUM L. AND TRITICUM AESTIVUM L. Salhi Nasrine1,Salama M. El‐Darier2 and Halilat M.El‐Taher3 1- Université Kasdi Merbah Ouargla Laboratoire de Bio-ressources sahariennes: préservation et valorisation, Faculté des Sciences de la Nature et de la Vie et des Sciences de la Terre et de l’Univers Ouargla 30 000 Algérie ([email protected]) ([email protected]) 2- Departments of Botany and Microbiology, Faculty of Science, University of Alexandria, Alexandria, Egypt (E mail:[email protected]) 3- Laboratoire de Bio-ressources sahariennes : préservation et valorisation, Faculté des Sciences de la Nature et de la Vie et des Sciences de la Terre et de l’Univers Ouargla 30 000 Algérie. ABSTRACT In agroecosystams, some weeds interfere with crop plants through allelochemicals, which reduce the crop growth and crop development and ultimately may reduce also directly weeds yield. The present study was conducted to investigate the possible allelopathic effect of Oudneya africana (donor species) on Bromus tectorum (weed species) and Triticum aestivum (cv. Sahel1; crop species) through germination bioassay experiment. Bromus tectorum is a winter annual grass that grows in winter wheat and other crops and in both disturbed and undisturbed grasslands. The effect of O. africana aqueous leachate (OAAL) on germination and seedling growth of B. tectorum and T. aestivum was investigated. High concentrations of the aqueous leachate significantly reduced the germination rate of B. tectorum. Similarly, coleoptile (CL) and radicle (RL) lengths of B. tectorum seeds were significantly inhibited. To go through with this, seedling growth of the weed species was also affected. On the other hand, the germination parameters of wheat seeds were slightly affected by applying different OAAL concentration levels compared to B. tectorum. In conclusion, allelochemicals extracted from the donor species caused a significant reduction in germination and growth parameters of B. tectorum > T. aestivum. Keywords: Aqueous germination rate. 144 leachate, Oudneya africana, Bromus tectorum, 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 64. USING ALLELOPATHY EFFECTS OF FIELD BINDWEED (Convolvulus arvensis L.) FOR IMPROVING GERMINATION RATE AND GROWTH OF WHEAT IN ORGANIC FARMING Pold V.1, Friedel J.K.1, Fritz I.2, and Gollner G.1 1 University of Natural Resources and Life Sciences, Vienna Department of Sustainable Agricultural Systems, Division of Organic Farming Gregor Mendel Straße 3,1180 Vienna: [email protected] 2 Department for Agrobiotechnology, IFA-Tulln Institute for Environmental Biotechnology: [email protected] ABSTRACT Wheat (Triticum aestivum ssp. vulgare L.) is the most important cereal cultivated in Austria and plays an important role in organic farming. Generally spoken, the yield in organic farming is lower than in conventional systems, most probably because chemical fertilizers are not allowed. Field bindweed (Convolvulus arvensis L.) can be found all over the world, especially in springsown crops. This weed is known to have an allelopathic effect on different field crops because of released phenolic acids, especially from the shoots. Phenolic acids can stimulate the germination and can increase the growth rate of receiver plants, however, only at low concentrations. In our study, extracts with distilled water and with ethanol were produced from dried field bindweed, both in rates 1:10 based on plant dry mass and solvent. Phenolic acids from the shoots of the weed should be dissolved by the procedure. Winter wheat was “seed primed” for 24 hours in different concentrations of both extracts. Afterwards the seeds were dried to the original weight and germination tests were done in plastic boxes. After seven days, length of shoots and roots and dry weights of the wheat plants were measured. Additionally, the germination rate (n=100, 4 replications) was observed. The study concluded with recommendations about the solvent to be used and about the most beneficial concentration in prospect of an upcoming field test. Possibly, such an extract could be used as an effective growth stimulator for organically cultivated winter wheat in the future. Keywords: Allelopathy, field bindweed, Triticum aestivum, ethanol extract, water extract Tel.: +43660/1425558 145 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 65. ALLELOPATHIC SORGAAB-GROWTH STIMULATOR FOR STRESSED MAIZE 1 Nazimah Maqbool1*, A. Wahid1* Department of Botany, University of Agriculture, Faisalabad-PAKISTAN [email protected], [email protected] ABSTRACT Sorgaab, a water extract of sorghum leaves, was applied to probe the modulation in growth and physiological attributes of maize (Zea mays L.) under drought in lab and greenhouse studies. Sorgaab soaked seeds (10 dilutions from 0 mL L-1 to 2mL L) were germinated to assess for drought tolerance at germination and seedling growth stages. More concentrated sorgaab solutions (2 mL L-1) were damaging, while lower concentration in the range of 0.75 to 1.5 mL L-1 improved the root and shoot growth, of maize under drought. Applied drought stress decreased chlorophyll b greater than chlorophyll a, leading to an increase in the chlorophyll a:b ratio. Application of sorgaab also improved the internal CO2 assimilation, which increased the net photosynthesis and water use efficiency under drought. Soluble phenolics and anthocyanins were also increased in plant receiving sorgaab under drought than control. Although greater concentrations of phenolic acids inhibit the uptake of ions, but its lower concentrations stimulated the uptake of K+, Ca2+, NO3, PO4. In conclusion, although high concentrations of sorgaab reduced the growth of plants, it may alleviate the adverse effects of drought, if applied at low concentration. Dilute concentrations of sorgaab can be utilized as a natural source for improving drought resistance in maize both at germination and later growth stages. Keywords: Sorgaab, growth stimulator, drought Tel: +923414292152 146 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 43. THE INFLUENCE OF BOA (2- Benzoxazolinone) ON THE RESPONSE OF CUCUMBER SEEDLINGS TO HERBICIDE BASTA Anna Demczuk*, Elżbieta Sacała, Edward Grzyś Department of Plant Nutrition, Wrocław University of Environmental and Life Sciences, Grunwaldzka 53, 50-357 Wrocław, Poland *e-mail: [email protected] ABSTRACT BOA (2- benzoxazolinone), a type of hydroxamic acid - an allelochemical present in cereals, is being viewed as a potential candidate for the development of natural herbicides. Although extensive research on the mode of action of BOA has been conducted, there is little information on its interactions with herbicides. Basta (also known as glufosinate or phosphinothricin) is a commonly used herbicide in both agricultural and non-agricultural systems and relatively nontoxic to the environment. The aim of the study was to determine the combined action of BOA (0,5 mM) and Basta (0,011 mM glufosinate) on cucumber seedlings grown in hydroponic cultures. Chemicals were applied to a nutrient solution. After 7 days of cultivation the growth parameters were evaluated (length, fresh and dry weight of roots and shoots), lipid peroxidadation and concentrations of photosynthetic pigments and total protein. The combined action of BOA and Basta caused a significant decrease in growth of cucumber seedlings (there was 2-fold decrease in growth than when the chemicals were added separately) and a slight decrease (10%) in the total protein content in shoots. BOA and Basta caused 22% and 12% increase respectivealy in the chlorophyll concentration in cotyledons compared to the control plants, while the combined action of BOA and Basta resulted in 27% decrease. The combined action of BOA and Basta on lipid peroxidadation in seedlings was similar to that recorded when the chemicals were applied separately. Keywords: glufosinate, 2-benzoxazolinone, cucumber growth, total protein, lipid peroxidadation Tel: +48713205605; fax: + 48713205605 147 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 44. THE COMBINED ACTION OF ALLELOCHEMICALS AND THE SULFONYLUREA HERBICIDE ON MAIZE SEEDLINGS Anna Demczuk*, Edward Grzyś, Elżbieta Sacała Department of Plant Nutrition, Wrocław University of Environmental and Life Sciences, Grunwaldzka 53, 50-357 Wrocław, Poland *e-mail: [email protected] ABSTRACT Crotonic acid [(E)-2-butenoic acid] – an allelopathic substance isolated from edible carrot´s seeds and BOA (2- benzoxazolinone), a type of hydroxamic acid - an allelochemical present in cereals, are being viewed as a potential candidates for the development of natural herbicides. Although extensive research on the mode of action of these allelochemicals has been conducted, there is little or no information on their influence on the response of plants to synthetic herbicides. Titus (a.i. – active ingredient rimsulfuron) is sulfonylurea herbicide that inhibits the activity of acetolactate synthase (ALS) a key enzyme in the biosynthesis of the branched- chain amino acids. It is a commonly used herbicide in agricultural systems and relatively nontoxic to the environment. The aim of the study was to determine the combined action of BOA (1 mM) and Titus (100 nM a.i.) and the combined action of crotonic acid (0.5 mM) and Titus (100 nM a.i.) on maize seedlings grown in hydroponic cultures. Chemicals were applied to a nutrient solution. After 7 days of cultivation the growth parameters were evaluated (length, fresh and dry weight of roots and shoots), ALS activity and concentrations of photosynthetic pigments. Maize seedlings were more sensitive to the influence of the Titus when applied in combination with BOA than when the herbicide was applied alone. Maize seedlings exhibited less sensitivity to the impact of the Titus herbicide when applied in the presence of crotonic acid. Keywords: allelochemicals, ALS activity, 2-benzoxazolinone, crotonic acid, combined action, herbicide, growth, maize seedlings Tel: + 48713205605; fax: + 48713205605 148 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 66. INFLUENCE OF LEAF LEACHATE OF Delonix regia (Boj Ex Hook) Raf. ON GERMINATION AND SEEDLING GROWTH OF MUNG BEAN K. B. Pawar1and Rawal A.V.2 1 Department of Botany, Shivaji University, Kolhapur-416 004 (MS) India [email protected] 2 Department of Botany, Shivaji University, Kolhapur-416 004 (MS) India ABSTRACT Delonix regia (Boj Ex Hook) Raf is a common ornamental plant. It is also grown as shade tree in farms and along the lines of street. There is frequent shedding of dry and senescent leaves of D. regia. Mung bean (Vigna radiata (L.) R. Wilczek) is an important legume crop commonly cultivated in Maharashtra state of India. Influence of leaf leachate (prepared in 1:5 proportions, high concentration) and diluted leaf leachate (1:50 proportion, low concentration) on seed germination and seedling growth of V. radiata (variety 1089) was studied. Seed germination and seedling growth with respect to root length, shoot length and fresh weight were reduced due to leaf leachate at both low and high concentration in petriplate bioassay. In soil bioassay root length, shoot length and fresh weight were inhibited due to leaf leachate at high concentration and at low concentration there is slight reduction in root length and increase in shoot length and fresh weight. The leaf leachate is having appreciable amount of total polyphenols. Analysis of leaf leachate has been continued for the detection of phytochemicals responsible for such alterations. Keywords: Delonix regia (Boj Ex Hook) Raf, leaf leachate, seed germination, seedling growth, Vigna radiata (L.) R. Wilczek Tel: + 09422324078 149 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 67. EVALUATION OF DIFFERENT CONCENTRATIONS OF REDROOT PIGWEED’S (Amaranthus retroflexus L.) LEACHATE EFFECTS ON GERMINATION & GROWTH OF WHEAT Rouhollah Motafakkerazad1*, Seyed Yahya Salehi‐lisar1 and Hamide Bakhshayeshan‐Agdam1 1 Department of Plant Sciences, Faculty of Natural Sciences, University of Tabriz, Tabriz, Iran; *e-mail: [email protected] ABSTRACT Allelopathic effects of different concentrations of redroot pigweed’s leachate on germination and seedling growth of wheat (Triticum auestivum L.) was evaluated in this study. This experiment was conducted as factorial based on completely randomized design with three replications under lab conditions. Plant organs were mixed and dried in lab conditions; then it was powdered using a mixer. The redroot pigweed’s leachate was prepared as 10% (W/V) of powder after shacking for 24 h. Experiment was done by using four concentrations of leachate; For this purpose leachate was diluted to different concentrations (7.5, 5 and 2.5%) after filtration. Double distilled water was considered as control. Treatment of wheat seeds with 5 ml of leachate was done on filter papers in petri dishes. After that, some parameters including germination percentage, seedling, plumule and radicle length, seedlings fresh and dry weight, relative growth and seedling survival rates were evaluated. Based on results it was shown that treatment of wheat seeds with different concentrations of leachate can significantly reduce germination and subsequent growth of seedlings. Inhibitory effects of redroot pigweed’s leachate were elevated by increasing of leachate concentrations. However, leachate effect on different parameters was not similar. The allelopathic effects of redwood pigweed on germination percentage and seedling survival rate of wheat were lower than other studied parameters. Finally this study revealed that 5% redroot pigweed’s leachate is the suitable choice in order to make visible allelopathic effects on wheat plants on filter paper. Keywords: allelopathy, redroot pigweed, leachate concentration, wheat Tel: +98 914 403 9626; fax: +98 411 335 6027 150 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 45. ALLELOPATHIC STUDY OF Polygonum aviculare, Tribulus terrestris AND Setaria sp. ON WHEAT GERMINATION AND GROWTH 1 Rouhollah Motafakkerazad1*, Seyed Yahya Salehi‐lisar1 and Elham Rakhshaie1 Department of Plant Sciences, Faculty of Natural Sciences, University of Tabriz, Tabriz, Iran; *e-mail: [email protected] ABSTRACT Allelopathy is defined as the interactions between plants by their metabolites, which are called ''allelochemicals''. These compounds belong to the plant secondary metabolites. Wheat is the most important crop and usually weeds reduce crops yield in the world by allelopathic effects. Polygonum aviculare, Tribulus terrestris, and Setaria sp. are widespread weeds in crops fields in Iran. Therefore, this study was conducted to investigate the allelopathic effects of those weeds on germination and seedlings growth of wheat. The experiment was carried out based on completely randomized design with three replications under lab conditions. Experimental factors were three weed species and leachate concentration in four levels (control, 2.5%, 5%, 7.5% and 10%). Results indicated that percentage of seed germination and seedling growth of wheat considerably decreased by increasing concentrations of T. terrestris leachate. While, low concentrations of P. aviculare and Setaria sp. leachates stimulated many growth parameters, higher concentrations significantly decreased wheat germination and growth. Generally, based on the obtained results it can be suggested that among studied weed species, T. terrestris has higher allelopathic potential against wheat. Keywords: allelopathy, T. terrestris, P. aviculare, Setaria sp., wheat Tel: +98 914 403 9626; fax: +98 411 335 6027 151 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 68. INVESTIGATION ON ALLELOPATHIC EFFECTS OF REDROOT PIGWEED (Amaranthus retroflexus L.) ON GERMINATION AND GROWTH OF WHEAT AND CUCUMBER UNDER DIFFERENT IRRIGATION REGIME Seyed Yahya Salehi‐lisar1*, Rouhollah Motafakkerazad1 and Mehri Arianfar1 1 Department of Plant Sciences, Faculty of Natural Sciences, University of Tabriz, Tabriz, Iran; *e-mail: [email protected] ABSTRACT Allelopathy is defined as direct or indirect effects of plants on each other by production and release of allelochemicals into the environment. Different environmental conditions can influence allelopathic potential of plants. Hence, the aim of this study is the investigation of allelopathic effects of redroot pigweed on germination and seedling growth of wheat (Triticum auestivum L.) and cucumber (Cucumis sativus L.) plants under different irrigation condition. Redroot pigweed is widespread weed in Iran with well-known allelopathic effects. Wheat and cucumber seeds were cultivated in soils containing 5% leachate (W/V) of redroot pigweed and irrigated at 100% and 70% field capacity. The results showed that allelopathic effects of redroot pigweed on wheat were higher than those effects on cucumber. The inhibitory effects of leachate in soils with lower water content were higher than those effects in soils with high moisture. It can be due to an increase of allelochemicals concentrations with decrease of soil water content. In both plants, germination was more sensitive than seedling growth towards redroot pigweed leachate. It was shown wide varieties of results depending on species of plants and content of soil water. In general, these findings revealed that allelopathic effects of redroot pigweed on crops were under control of environmental conditions and it can be regarded to management of weed in crop fields. Keywords: allelopathy, redroot pigweed, wheat, cucumber, irrigation Tel: +98 914 900 3272; fax: +98 411335 60 27 152 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 69. ALLELOPATHIC EFFECTS OF REDROOT PIGWEED (Amaranthus retroflexus L.) ON GERMINATION & GROWTH OF CUCUMBER, ALFALFA, COMMON BEAN AND WHEAT PLANTS Seyed Yahya Salehi‐lisar1*, Rouhollah Motafakkerazad1 and Hamide Bakhshayeshan‐Agdam1 1 Department of Plant Sciences, Faculty of Natural Sciences, University of Tabriz, Tabriz, Iran; *e-mail: [email protected] ABSTRACT Allelopathy is the interaction between plants by synthesis and releasing of certain secondary metabolites into the environment. Allelopathic effects of weeds can lead to considerable yield lose of crops. In order to the study of allelopathic effects of redroot pigweed on germination and growth of four important crop species, an experiment was conducted as factorial based on completely randomized design with three replications under lab conditions. Experimental factors in this research were 1) crop species including cucumber (Cucumis sativus L.), alfalfa (Medicago sativa L.), common bean (Phaseulus vulgaris L.) and wheat (Triticum aestivum L.) and 2) different concentrations of redroot pigweed leachate (5% and 10%). Double distilled water was considered as control. Measured parameters include germination percentage, germination rate, seedling, plumule and radicle length, seedlings fresh and dry weight, relative growth and seedling survival rate. Analysis of variance showed that despite of different sensitivity of studied species, the germination and subsequent growth of seedlings significantly affected by different concentrations of redroot pigweed leachate. According to the obtained results, wheat and common bean were the most resistant species, cucumbers was resistance species at low concentration but sensitive at high concentration, and alfalfa was the most sensitive species against redroot pigweed leachate. Therefore, among the four studied crop species cultivation of wheat and common bean plants in the regions with redroot pigweed’s invasion was affordable, and avoidance of alfalfa cultivation in these regions is essential because this species is quite sensitive to redroot pigweed. Keywords: allelopathy, redroot pigweed, resistance, crop Tel: +98 914 900 3272; fax: +98 411335 60 27 153 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 70. ALLELOPATHY OF Urochloa ruziziensis (R. GEMAN & EVRARD) CRINS IN SOYBEAN Mariluce Pascoina Nepomuceno1* Rosa Maria Varela Montoya,2 José M. G. Molinillo3 Francisco Antonio Macias4 Pedro Luís da Costa Aguiar Alves5 1,5 2,3,4 Departamento de Biologia Aplicada à Agropecuária, FCAV – UNESP, Jaboticabal, SP, Brazil Grupo de Alelopatia de Cádiz, Dept. of Organic Chemistry, Faculty of Sciences, Universiy of Cadiz – Spain 1* e-mail: [email protected]; 5 e-mail: [email protected] ABSTRACT The cover crop’s decomposition can produce and/or release allelopathic compounds that affect the growing of weeds and can harm crops of economic importance. Forage grasses from the genus of Urochloa have allelopathic inhibitory activity and its aboveground is the major source of active chemical substances on seed germination and growing of different weed species. Through extraction, isolation and identification of chemical substances from dried aboveground of U. ruziziensis, this study has the aim of evaluate the allelopathic effect of its major compound on genetically modified soybean (Glycine max L.). U. ruziziensis plants at early flowering were desiccated with 1.44 kg of glyphosate ha-1 and sampled 10 days after germinated. There was also a control without herbicide. It was prepared hexane, dichloromethane (DCM) and methanol (MeOH) extracts (in this sequence) from dried and ground U. ruziziensis aboveground. Through cytotoxicity bioassays with DCM and MeOH extract, it was verified that MeOH extract was more harmful than DCM one, regardless glyphosate application. Using liquid column chromatography techniques, with thin layer of high and average performance, it was fractionated the MeOH extract with water and MeOH as solvents. The fractions MeOH/water 2:2 (v/v) and MeOH/water 3:1 (v/v) were the most harmful, once again regardless glyphosate application; and the fraction MeOH/water 2:2 (v/v) was the most phytotoxic to soybean seedlings. In these fractions, a major compound, the steroidal saponin protodioscin, was isolated as a major compound. The protodioscin showed phytotoxicity on soybean seedlings, with the inhibitory concentrations (IC50) of 97, 160 and 186 µM for root dry mass, root length and aboveground length, respectively. Keywords: allelochemicals, Brachiaria ruziziensis, saponin steroidal, ID50. Tel: +55 16 3209 2620; fax: +55 16 3209 2620 154 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 71. ALLELOPATHY; A BRIEF REVIEW Javed Kamal Department of Plant Sciences, Quaid-i-Azam university, Islamabad. E-mail: [email protected] ABSTRACT Allelopathy is a well known area of active research in ecology. However, its importance in agro-ecology is still underappreciated. This review sets out to address this situation and introduce this new and developing field to a wider research audience and to stimulate new research in it. The review starts with an introduction, followed by discussions of allelochemicals, The role of allelopathy in crop production, Allelopathy related problems in crop production, and Suggestions for future research. It also describes broader research into allelopathy in agriculture and the biosciences, and literature resources on the subject. We hope that it will encourage more scientists to initiate research into this exciting new field. Keywords: allelopathy, allelochemicals biological control. Tel: 86+153-3172-7676; fax: 86-871-65228581 155 156 Session 6. Weed control using allelopathy 157 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 72. The use of allelopathic plant species to manage weed species of the Kurdistan region of Iraq Hadi M. Aliki1, Matthew A. Back2, John P.H. Reade3 * * Harper Adams University, Newport, Shropshire, TF10 8NB, UK 1 [email protected] 2 [email protected] [email protected] 3 ABSTRACT Weeds are one of the main causes of losses to farmers’ incomes in Kurdistan and are recognised for hindering crop growth and decreasing production as they compete for nutrients, moisture, light and space. Using allelopathy to control weeds is a novel approach which may reduce environmental impact and improve sustainable agriculture. Previously water extracts from Brassica napus (L.) containing allelochemicals such as caffeic, ferulic, chlorogenic vanillic acids, thiocyanates and isothiocyanates have been shown to reduce germination and the growth of weed species. In this study an in-vitro experiment was carried out to investigate the effect of Brassica napus (L.) extracts from different plant parts on the seed germination and seedling growth of Phalaris minor (Retz.), Convolvulus arvensis (L.) and Sorghum halepense (L.) Water extracts were obtained by soaking 100g fresh tissue in 1 litre distilled water for 24 hours. Ten ml of extract solution from each plant part were added to each petri dish and distilled water was used as a control. Seed germination, shoot and root length, shoot and root weight of all weed species were inhibited significantly (P<0.001) by all treatments compared to the control. Flower water extracts had the greatest effect causing a 100%, 93.9% and 92.9% inhibition in the germination of Phalaris minor (Retz.), Convolvulus arvensis (L.) and Sorghum halepense (L.) respectively. Also, exposure to flower and stem extracts caused a reduction in the shoot length and weight of Phalaris minor (Retz.), Convolvulus arvensis (L.) and Sorghum halepense (L.). Keywords: Allelopathy, Brassica napus L., water extract, weed germination, Phalaris minor (Retz.), Convolvulus arvensis (L.), Sorghum halepense (L.). Tel: 00447407239434 158 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 73. HERBICIDE POTENTIAL OF SESQUITERPENE LACTONE AND FLAVONOID ON GERMINATION AND ROOT LENGTH IN Arabidopsis thaliana M. Iftikhar Hussain1 and Manuel J. Reigosa2 12 Department of Plant Biology and Soil Science, University of Vigo, Campus Lagoas-Marcosende, 36310-Vigo, Spain 1; [email protected], 2; [email protected] ABSTRACT Natural product-based research strategies can provide industry led discovery of herbicide lead compounds that are environmentally safe and easily degradable. Phytotoxic effect of natural product, artemisinin (sesquiterpene lactones) and rutin (flavonoids) on germination, seedling growth and root length of Arabidopsis thaliana were tested under controlled bioassays. Seeds of A. thaliana L. ecotype Columbia (Col-0) were placed in 24-well plates (1.25-cmdiam) having Whatman no.1 paper disks watered with 200 µl of the test solution (artemisinin or rutin) or controls. Plates were placed in growth chamber with day/night temperatures of 22 and 20 ºC, respectively. Germination was assessed with a magnifying glass and data was used to calculate germination indices according to Chiapusio et al. (1997) and vigor index as reported by Orchard (1977). For root length (RL), sterilized seeds of A. thaliana L. (Heyn.) ecotype Columbia (Col- 0) were placed in square petri plates with a semisolid media and allelochemical (5 mL/dish) containing 0.5× Murashige and Skoog salts, 1× Gamborg’s B5 vitamins, 1% sucrose (w/v), and 2% Gelrite® (w/v) adjusted to pH 6. The square dishes were then placed vertically in a thermostatically controlled chamber and RL was measured 15 d after treatment. Inhibitory effect of phytochemicals on RL of Arabidopsis was calculated by using the best-fit equation based on the coefficient of determination (r2). Total germination % age was reduced in A. thaliana after treatment with artemisinin and maximum reduction was observed at 160 and 80 µM. Rutin (100, 250, 500, 750, 1000 µM) decreased germination % age but 1000 µM proved to be most deleterious. Artemisinin inhibited the speed of germination (S) of A. thaliana at all concentrations. Rutin-delayed the A. thaliana “S” at all the concentrations tested and maximum inhibition was recorded at 1000 µM. The effect of artemisinin and rutin on radicle length (RL) of A. thaliana was concentration (conc.) dependent that gradually decreased after rutin treatment. Seedling vigour index (SVI) of A. thaliana was decreased following treatment with both allelochemicals at all concentrations. References Chiapusio G, Sánchez AM, Reigosa MJ, Gonzalez L, Pellisier F. 1997. Do germination indices adequately reflect allelochemical effects on the germination process? J Chem Ecol 23: 2445- 53 Orchard T. 1977. Estimating the parameters of plant seedling emergence. Seed Sci Tech 5: 6169. Keywords: Natural products, germination bioassays, stress Tel: +34986812616, Fax: +34986812556 159 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 74. INTEGRATION OF REDUCED RATE OF HERBICIDE WITH ALLELOPATHIC SORGHUM RESIDUES FOR WEED MANAGEMENT IN BROAD BEAN Ibrahim S. Alsaadawi1, Nabil R. Lahmod2 and Resan K. Shatty3 1 Department of Agronomy, College of Agriculture, Waset University, Waset, Iraq 2 3 Department of Biology, College of Science, Baghdad University, Baghdad, Iraq Department of Agronomy, College of Agriculture, Waset University, Waset, Iraq *Email: [email protected] ABSTRACT A two-year field trial was conducted to explore the response of weeds and broad bean (Vicia faba) to different rates (3.5, 5.3 and 7.6 t ha-1) of sorghum residues cv Rabeh alone or in combination with 50% (1.2 L ha-1) of the label rate of treflan (trifluralin) herbicide. Weedy check, weed free and label rate of treflan treatments were included for comparison. Plots treated with 50% of label rate of treflan and amended with sorghum residues recorded least weed density and dry biomass and this suppression was much greater than the residue treatments and reduced herbicide when used alone. Application of 50% dose of herbicide in plots amended with sorghum residue resulted in similar yield advantage as was noticed with 100% herbicide dose. Integration of sorghum residues with lower herbicide dose can furnish adequate weed suppression without compromising yield as a feasible and environmentally sound approach in broad bean fields. Chromatographic analysis of residue-infested field soil indicated the presence of several phytotoxic compounds of phenolic nature. Periodic data revealed that maximum suppression in weed density and dry weight synchronized with peak values of phytotoxins observed 4 weeks after incorporation of m residues Keywords: Allelopathy, sorghum residues, herbicide, broad bean. 160 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 75. ALLELOPATHIC AND WEEDICIDAL EFFECT OF Emblica officinalis GAERTN.ON Tephrosia purpurea L. AND Cassia occidentalis L. M. Jayakumar1* M. Eyini2 and Mary Sajitha Jeev3 1 2 Thiruvalluvar University, Serkkadu, Vellore - 632 115, Tamil Nadu, India 1; e-mail: [email protected] Research Centre in Botany, Thiagarajar College (Autonomous), Madurai – 625 009, Tamil Nadu, India 2; e-mail: [email protected] 3 Research Department of Botany, VHNSN College (Autonomous), Virudhunagar– 626 001, Tamil Nadu, India ABSTRACT The allelopathic and herbicidal potential of tree species Emblica officinalis leaf aqueous extract on weeds viz. Tephrosia purpurea and Cassia occidentails were studied. Four different concentration of aqueous leaf extract were used for the experiment. The inhibition of seed germination and bioassay of the weeds decreased with the increasing concentration of the aqueous extract of Emblica officinalis leaves. Among the weeds, seeds of Tepherosia purpurea were sensitive to the aqueous leaf extract of Emblica officinalis. Seed germination of Cassia occidentails was affected less than the seeds of Tephrosia purpurea. In pre-emergence, seed germination of both the weed seedlings were inhibited above 50% of the control values even at the highest concentration of the extract used. The shoot length, root length, number of compound leaves, leaf area, biomass, and mortality were sensitive to the higher concentration (20%) only. In the pre-emergence treatment, both weeds were affected. In post emergence treatment the seedlings of weeds were treated with aqueous leaf extract of Embalica officinalis. Post emergence treatment inhibited the growth of the weed seedlings considerably only at 20% extract concentration. The inhibitory effect of the extract on shoot length, root length, number of compound leaves, leaf area, biomass, and mortality, was concentration dependent. In post emergence treatment the decrease of all growth parameters in Tephrosia purpurea was affected more than the Cassia occidentalis seedlings. From the aqueous leaf extract of tree species Emblica officinalis the phenolic acids viz., Gallic, protocatechuic, chlorogenic, vanillic, caffeic, syringic, p-coumaric, ferulic, mcoumaric, o-coumaric, ellagic and trans-cinnamic acids were identified by HPLC technique. Keywords: Emblica officinali, Tepherosia purpurea, Cassia occidentails, phenolic acid, allelochemicals, HPLC Tel: +914162274746; fax: +914162274748 161 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 76. EVALUATION OF CANOLA (Brassica napus) ALLELOPATHY: FROM LABORATORY TO FIELD Md. Asaduzzaman1,3*, David J Luckett2,3, Min An3,4, James E Pratley1,3, and Deirdre Lemerle1,3 1 School of Agricultural and Wine Sciences, Faculty of Science, Charles Sturt University, Wagga Wagga, NSW 2650, Australia *e-mail: [email protected] 2 3 NSW Department of Primary Industries, Wagga Wagga, NSW 2650, Australia Graham Centre for Agricultural Innovation (an alliance between Charles Sturt University and NSW Department of Primary Industries), Wagga Wagga, NSW 2650, Australia 4 Faculty of Science, Charles Sturt University, Wagga Wagga, NSW 2650, Australia ABSTRACT Canola (Brassica napus L) is a major oilseed crop widely grown in Australia. The concern about herbicide-resistance weeds is encouraging to include nonchemical weed management tactics such as allelopathy. The impact of crop allelopathy on weeds can demonstrate under controlled conditions, but field studies are required to confirm commercial impact of this phenomenon It is difficult to prove such phenomena in field studies but laboratory to field linking can provide an overall assessment of the allelopathic effects crop species. This study was conducted to validate laboratory allelopathic outcomes of several canola genotypes under field condition. Results from laboratory studies using the equal compartment agar method on annual ryegrass (Lolium rigidum), was linked with suppression of Shepherd’s purse (Capsella bursa-pastoris), Indian hedge mustard (Sisymbrium orientale), barley grass (Hordeum leporinum) and annual ryegrass in the field using the ecological parameter of Simson’s diversity index (%SDI). A strong correlation co-efficient of 77** was observed between laboratory and field suppression outcomes. The allelopathic genotypes in the laboratory bioassay, such as Av-opal, Sardi603, Rivette and Atr-beacon, also performed well in the field whereas cv. Barossa and X-06-6-3725 consistently poorly performed in field condition as well. It is recognized that the specificity of allelopathy only results in effects from certain genotypes against a specific weed. These results do not imply that allelopathic potential in canola will negate the necessity to apply chemical herbicides but rather that allelopathy can be a valuable component in an integrated weed management program. Keywords: Canola, allelopathy and genotypes Tel: + 61-0402095316 162 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 77. WEED MANAGEMENT IN MAIZE THROUGH ALLELOPATHIC WATER EXTRACTS Shahzad M.A. Basra Department of Crop Physiology, University of Agriculture, Faisalabad [email protected] ABSTRACT Modern agriculture is productivity oriented and depends largely on synthetic inputs (herbicides) to tackle weeds. However, non-judicious use of herbicides could cause environmental, health and herbicides resistance problems. Therefore, a focus has been given since last two decades on the use of plant derived materials as alternative to herbicides for weed control. Allelopathy is an eco-friendly and organic weed management approach which may be used as a tool in controlling weeds. In this study, allelopathic water extracts of sorghum, sunflower, brassica and mulberry were applied at 18 L ha-1 alone or in combination with half dose of Primextra Gold as pre-emergence spray, for weed management in maize. Horse purslane (Trianthema portulacastrum), purple nutsedge (Cyperus rotundis), digera (Digera arvensis) and goose grass (Eleucine indica) were the dominating weeds in the experimental field while other weeds were less in number. All the weed control methods significantly reduced total weed density and dry weight recorded at 40 and 60 days after sowing (DAS). Hand weeding employed at 15 and 30 DAS reduced the total weed density and total weed dry weight by 75 and 67.17%, 80.07 and 79.66% recorded at 40 and 60 DAS, respectively than weedy check. Sorghum + sunflower + brassica + mulberry water extract each applied at 18 L ha-1 combined with half dose of Primextra Gold as pre-emergence spray provided 68.71 and 65.51% reduction in total weed density and 73.77 and 71.88% reduction in total weed dry weight as compared to control recorded at 40 and 60 DAS, respectively. Sorghum + sunflower + brassica + mulberry water extract each at 18 L ha-1 combined with half dose of Primextra Gold applied as preemergence spray gave significantly higher cob length, number of grain rows per cob and 1000-grain weight over control (weedy check) but statistically at par with full dose of herbicide. Allelopathic plant water extracts tank mixed with half dose of Primextra Gold gave maximum grain yield, which was 45.07% higher than weedy check (vs 31.47% increase over control from label dose of herbicide). In conclusion, allelopathic crop water extracts reduced the dry weight of weeds by 63%. Application of sorghum, sunflower brassica and mulberry water extract in combination with half dose of Primextra Gold 720 SC (360 g a.i. ha-1) provided as affective weed control as was achieved from standard herbicide dose. However yield increase (over control) was higher than the application of standard herbicide dose. This study suggests that allelopathy offers an attractive and environmental friendly method of weed control. Keywords: Allelopathy, maize, water extracts, weed control Tel: +923336519675 163 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 78. USE OF ALLELOPATHIC PLANTS AS LIVING MULCH IN APPLE ORCHARDS Mine Ruşen¹ Ayşe Işik² Adnan Doğan¹ Barış Albayrak¹ Ahmet B.Tinmaz¹ Hasbi Yilmaz¹ Hüseyin Özpinar3 Ahmet Uludağ4* 1 Atatürk Horticulture Research Institute; e-mail: [email protected] 2 Batı Akdeniz Agricultural Research Institute; 3 Ege Agricultural Research Institute; 4* Canakkale Onsekiz Mart University, Faculty of Agriculture; e-mail: [email protected] ABSTRACT Using non chemical methods in farming has become a necessity for economy, health and environment. Living mulches with plants with allelopathic effects are among choices. A three-year-field study with Festuca rubra L., Trifolium repens L., Trifolium subterraneum L. and Agrostis tenuis Sibth., which their allelopathic effects have been documented in the literature, was conducted in an apple orchard from 2010. No measurement was taken in 2010 to let mulches established well. The main weeds in the site were Sonchus oleraceus L., Cynodon dactylon (L) Pers., Convolvulus arvensis L., Cyperus rotundus L., Anagallis arvensis L., Galium aparina L., Sinapis arvensis L., Sorghum halepense (L) Pers., and Papaver rhoeas L. The number of weeds and percent weed cover were reduced by all applications comparing to weedy control. The highest weed control (94%) was obtained from current farmer application for weed control, which is combination of tillage and herbicide. The ratio of weed control by living mulches changed between 73% by T. subterraneum to 60% by A. tenuis. The apple yield was not statistically different in 2011 but 2012. T. subterraneum gave the best weed control and apple yield among living mulches and comparable with current farmer application. Measured soil properties did not affected by mulches. Keywords: Festuca rubra L., Trifolium repens L., Trifolium subterraneum L., Agrostis tenuis Sibth., living mulch, apple orchard, weed control Tel: +905375781211 164 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 46. POSSIBLE USES OF ALLELOPATHY IN WEED CONTROL IN TURKEY Ahmet Uludag1* İlhan Uremis2 Mine Rusen3 Nihat Tursun4 1 *Canakkale Onsekiz Mart University, Faculty of Agriculture; e-mail: [email protected] 2 Mustafa Kemal University, Faculty of Agriculture; 3 Atatürk Horticulture Research Institute; 4 Ismet Inonu University, Faculty of Agriculture; ABSTRACT Weeds are one of the main obstacles in farming systems. In the most agricultural systems, herbicides are the main tool to solve weed problems in spite of problems bearing from excess use of chemicals. On the other hand, organic farmers rely on mechanical techniques which include tillage that can make soil open to erosion if it becomes only technique to be used. Allelopathic relations have been used to establish proper farming systems, control directly weeds and other pests, and develop chemicals less harmful to environment. There have been researches on alleopathy with many crops, trees, shrubs and weeds under both laboratory and field conditions to determine their allelopathic potential and its use for weed control, and the role of allelopathy in weed interference on crop production in Turkey since 1980’s. However, it is not easy to say that results of these studies have had a chance to be implemented. In this paper, allelopathy studies in Turkey will be reviewed, how they will be integrated with agricultural systems will be discussed. Furthermore, research and implementation needs elaborated. Keywords: farming systems, weed control, Turkey, research TEL: + 905375781211 165 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 47. ALLELOPATHIC POTENTIAL OF CANOLA AND WHEAT TO CONTROL WEEDS IN SOYBEAN (Glycine max) A. Dadkhah1, J. Alboghbeish2 and B. Babainezhad2 1 Ferdowsi University of Mashhad, I.R. of Iran; e-mail: [email protected] 2 M.Sc. Students of Agronomy, Ferdowsi University of Mashhad, I.R. of Iran ABSTRACT A filed experiment was done to develop management practices to reduce the use of herbicides, in the arid and semi-arid agricultural ecosystems of north east of Iran. Five treatments including I: chopped residues of canola (Brasica vulgaris), II: chopped residues of wheat (Triticum aestivum) both were separately incorporated to 25 cm depth soil, 20 days before sowing, III: shoot aqueous extract of canola, IV: shoot aqueous extract of wheat which were separately sprayed at post emergence stage and V: without any residues and spraying as control. The weed control treatments reduced the total weed cover, weed density and biomass of weed. The reduction in weed density with canola and wheat residues incorporation were up to 57.3 and 61.2% respectively, at 40 days after sowing and 52.1% and 63.2%, respectively, at 90 days after sowing, compared to control. However, post emergence spraying of shoot aqueous extract of canola and wheat, suppressed weed density up to 45.3 and 36.6% at 40 days after sowing and 48.8% and 48.7% at 90 days after sowing respectively, compared to control. Weed control treatments reduced weed cover (%), weed biomass and weeds stem length. Application of canola and wheat residues increased soybean yield by 57% and 69% respectively, compared to control while post emergence application of shoot aqueous extract of canola and wheat increased soybean yield by 38% and 30% respectively. Incorporation of canola and wheat residues in soil reduced weed cover (%) by 63.7% and 62.5% respectively, while spraying of shoot water extract of canola and wheat suppressed weed cover (%) by 39.6 and 40.4 respectively at 90 days after sowing. Keywords: Allelopathy, bioherbicide, Brassica oleracea, plant residues, Triticum aestivum. TEL: +989155090727; FAX: +985856353663 166 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 79. WEED CONTROL IN ONION (Allium cepa, L.) USING ALLELOPATHY OF SAWDUST, RICE STRAW, CLOVER WEED OR COGONGRASS Hussein F. Abouziena1* and S.M.A. Radwan2,3 1 Botany Department, National Research Center, Dokki, Cairo, Egypt, 12622; e-mail: [email protected] 2 3 Agricultural Microbiology Department, National Research Center, Dokki, Cairo, Egypt Soil Science Department, College of Food and Agricultural Sciences, King Saud University, P.O. Box 2460 Riyadh 11451, Saudi Arabia; e-mail: [email protected] *Corresponding author: [email protected] ABSTRACT Onion is poor competitive with weeds, especially at the early stage of growth, so relatively weed-free conditions are required for successful production. A great attention has been given recently to use allelopathy having a definite beneficial role in weed control and crop production. Owing to acute shortage of hand labours and to avoidance herbicides uses, the present study was conducted using the organic mulches to overcome the problem of weed infestation in onion fields. However, the response of weeds and crop plants to allelopathy may vary according to plant species, plant parts and thickness of mulch used. A field experiments were conducted to examine the effect of four organic mulches included sawdust (SD), rice straw (RS), bur-clover weed (CW) or cogongrass (CG) in comparison with hand hoeing (HH) and butralin with one hoeing (BUH) on growth, bulb nutrient concentration, yield, and quality of onion (Allium cepa, L) plants and associated weeds. The results indicated that mulching decreased weed density to different extents and Lolium multiflorum weed was less affected by mulch treatments compared to the broadleaved weeds. Application of SD, RS, CW, CG, HH and BUH, decreased significantly the total weeds dry weight by 42, 51, 62, 63, 92 and 98%, respectively. Weed competition caused a significant decrease of plant dry weight (43-56%), bulb diameter (44%) and marketable yields (66%). Among mulches, CW gave the highest marketable yield with insignificant difference with HH, although CW efficacy control was much more modest (up to 62%) compared to HH (98%). Keywords: mulch, onion, sawdust, rice straw, burclover weed, cogongrass, mineral TEL: +201006273653; FAX: +2023370931 167 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 80. INHIBITORY EFFECT OF Nicotiana tabacum L. ON Sinapis arvensis L. Masoumeh Younesabadi1, Leila Habibian2, Yousef Nikpey3, Abdoltaher Mirzaali4, Behnam Daftari5 1,2,5 Address: Postal Code: 49156-77555, Plant Protection Research Department, Agricultural Research Centre, Beheshti Ave., Gorgan, Golestan, Iran 3,4 Address: Azad University, Shahid Kalantari Blvd., Daneshjoo st., Gorgan, Iran e-mail:[email protected], [email protected], [email protected], [email protected], [email protected] ABSTRACT In recent years using of natural compounds in weed control has been increased as a result of disadvantages of herbicides usage in agriculture. This experiment was carried out as a factorial design with three replications to examine the inhibitory effect of tobacco (Nicotiana tabacum L.) water extracts on Sinapis arvensis growth and germination in 2014. The main factors were water extracts that prepared from tobacco leaves with two grades including high (completely yellow leaves) and low quality (completely green leaves) leaves and with two different extraction methods including extraction after incubation in water bath110°c for 30 minutes (WB) and extraction after three days incubation in darkness at 20°c (Cheema's method, 2005). Distilled water was considered as control treatment. Sub factors were different doses of mentioned extracts including: 5%, 10%, 15%, 20%, 25%, 50%, 75% and 100% (1:10 w/v). The results showed that there was significant difference between main factors with respect to rootlet and stem let length and germination of S. arvensis. Regardless of leaf grades, inhibitory effect of extracts that prepared with WB method was more than Cheema's extraction method. Length of rootlet and stem let and germination percentage were decreased 76.89, 71.95 and 80.0 % by yellow leaf water extract and 75.51, 78.65 and 76.0% by green leaf water extract prepared with WB method respectively. With increasing extracts concentrations, inhibitory effects were increased. Sensitivity to tobacco extracts was in this order: rootlet length>stem let length>germination. Based on the result of this experiment, tobacco extracts may be useful as alternative materials of biological weed control. However, for demonstrating their herbicidal potential, complementary studies are needed. Key Words: Allelopathy, tobacco, water extract, weed control, wild mustard Tel: +98-171-3354871(office), +98-171-3358471(home), +98-9113707175(mobile); fax: +98-171-3359813 168 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 81. ISOLATION AND IDENTIFICATION OF STEROIDAL SAPONINS FROM STRAWS OF Brachiaria ruziziensis SHOWING INHIBITORY ACTIVITY ON THE GROWTH OF WEED SPECIES Mauro C. Barbosa1*, Adriano A. Silva2, Márcio S. Mito1, Gislaine C. Mantovanelli1, Fernanda L. Kagami1, Aneliz B. A. Alencar1, Maria H. Sarragiotto2 and Emy L. Ishii‐Iwamoto1 1 Department of Biochemistry, University of Maringá, Paraná, Brazil; *[email protected] 2 Department of Chemistry, University of Maringá, Paraná, Brazil; ABSTRACT The cover straws of Brachiaria species reduce the emergence of weeds in the field. However, the potential mechanisms—particularly the allelochemicals involved—remain unclear. The aim of the present study was to identify the active compounds of Brachiaria ruziziensis and confirm their allelopathic activity on weed species. The bioguided fractionation of the most active fraction obtained from the partition of MeOH crude extract of B. ruziziensis straws afforded the butanolic subfraction (BF-III). Analysis of 1H and 13C NMR data demonstrated the presence of the steroidal saponins protodioscin and protoneodioscin. The effects of protodioscin on germination and initial growth of Euphorbia heterophylla, Ipomoea grandifolia, Conyza bonariensis and Bidens pilosa were measured in gerboxes placed in germination chamber adjusted for specific photoperiod and temperature for each species. The seeds were grown in the absence (control) or presence of protodioscin, at concentrations of 250, 500 and 1000 µg ml-1, for 120 hours. For all species the primary root growth was the most sensitive parameter. At 1000 µg ml-1, the primary root growth of E. heterophylla, I. grandifolia and B. pilosa was inhibited by 81%, 82% and 70%, respectively. Protodioscin also inhibited the germination of C. bonariensis, a troublesome weed resistant to glyphosate in soybean plantations. At 1000 µg ml-1 a nearly complete inhibition of germination was found. Comparisons between the activities of protodioscin and two structurally related compounds, dioscin and diosgenin on Bidens pilosa suggested that the chemical moiety βD-glycopyranosyl-22-hydroxyfurost-5-ene-3 in the protodioscin structure is essential for its herbicidal effect. Keywords: Brachiaria, cover plants, weeds, saponin, protodioscin Tel: +55-44-30114712; fax: +55-44-30114896 169 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 82. HERBICIDAL POTENTIAL OF DRYLAND PLANTS ON GROWTH AND TUBER SPROUTINGIN PURPLE NUTSEDGE (Cyperus rotundus L.) 1 Javaid Iqbal 1,3, Syeda Tahreem Zahra2, Antonio DiTommaso3, Muhammad Ahmad2 and Asghari Bano2 College of Agriculture, Dera Ghazi Khan 32200 subcampus University of Agriculture, Faisalabad Pakistan 2 3 Quaid e Azam University Islamabad Pakistan College of Agriculture and Life Sciences, Cornell University, Ithaca, NY USA 14853. Email:[email protected] ABSTRACT Allelopathy can serve as an effective alternative weed control strategy. Dryland plants are considered to have higher herbicidal potential since salinity, water stress, and overgrazing are features of this ecosystem favoring the release of allelochemicals. Moreover, the relatively low activity of soil microflora and reduced leaching in this dryland ecosystem results in greater availability of allelochemicals in the soil. Based on previous findings, the ability of a suite of dryland plants to suppress growth and tuber spouting in the noxious weed purple nutsedge (Cyperus rotundus L.) was investigated. The plant species evaluated were Fagonia indica, Aerva javanica, Calotropis procera, Rhazya stricta and Withania coagulans. In a greenhouse experiment, 5 sprouted and 5 non-sprouted tubers of nutsedge were planted in pots containing 250g fieldcollected soil and aqueous extracts of the test plants were irrigated in pots at five concentrations (0, 25, 50, 75 and 100%; original extract was concentrated 20 times and was considered as 100% concentrated and further concentrations were made according to treatments). Extracts of all test plants significantly inhibited nutsedge tuber sprouting and growth. A non-significant interaction was observed between the timing of sprouting initiation and mean sprouting time (MST), while a significant interaction was observed between sprouting index (SI) and final sprouting percentage. Maximum reductions in SI and final sprouting percentage were recorded with Rhazya stricta extracts but all test plants showed similar effects on timing of sprouting initiation and mean sprouting time. Extracts of Rhazya stricta showed maximum suppressive potential of nutsedge density, root and shoot length, root and shoot fresh and dry weight. Overall, the least effective suppression of purple nutsedge was observed for extracts of Fagonia indica. Calotropis procera extracts resulted in the lowest reductions in nutsedge root length of all test plants. The 100% and 75% concentrations provided complete suppression of nutsedge. For all test plants, the 25% extract concentration was least effective in suppressing nutsedge and in some cases results were similar to the water-only control treatment. Our findings suggest that several dryland plant species with strong allelochemical properties have the potential to substantially reduce the deleterious impact of purple nutsedge in dryland cropping systems and warrant further study. Keywords: Allelopathy, dry land plants, water extract, purple nutsedge Tel: +1-607-279-4162 170 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 83. SYNTHESIS OF NOVEL PHTHALIMIDE-LACTONE DERIVATIVES AS PROMISING AGROCHEMICALS Kala Ghooray, Antonio Cala, José M. G. Molinillo, Juan C. G. Galindo and Francisco A. Macías* Grupo de Alelopatía, Departamento de Química Orgánica, Facultad de Ciencias, Instituto de Biomoléculas (INBIO), Universidad de Cádiz, Campus de Excelencia Internacional Agroalimentario (ceiA3), C/República Saharaui, s/n, 11510 Puerto Real, Cádiz, Spain; e-mail: [email protected] ABSTRACT Strigolactones have long been known to act as germination stimulants of Orobanche species, parasitic plants that afflict sunflower populations. Several analogues of strigolactones have been synthesized in an effort to control these plagues. Our research group has modified rings A and B in the strigolactones, leading to the synthesis of new strigolactones, guainestrigolactones, and pthalimide lactones. Thus, we have found that 5-phthalimide-3-methyl-2(5H)furanone acts as a germination stimulant of Orobanche ramosa, but not of Orobanche cumana. In order to improve its bioactivity, we have proposed the synthesis of novel phthalimide-lactone derivatives. Thus far, seven have been synthesized via a nucleophilic substitution of 5-bromo-3-methyl-2(5H) furanone with pthalimide salt derivatives. The yield of the pthalimide lactone originally synthesized has been improved, and new derivatives containing amino-, nitro-, chloro-, and naptho- functional groups have also been synthesized with acceptable yields. Bioactivity data of the novel phthalimide-lactone derivatives will be presented. Keywords: phthalimide-lactone, strigolactone, Orobanche spp. Tel: +34 956012770; fax: +34 956016193 171 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 84. PHYTOTOXIC EVALUATION OF NAPHTHOQUINONE ANALOGUES Guillermo A. Guerrero‐Vásquez, Nuria Chinchilla, Rosa M. Varela, José M. G. Molinillo, and Francisco A. Macías* Grupo de Alelopatía, Departamento de Química Orgánica, Facultad de Ciencias, Instituto de Biomoléculas (INBIO), Universidad de Cádiz, Campus de Excelencia Internacional Agroalimentario (ceiA3), C/República Saharaui, s/n, 11510 Puerto Real, Cádiz, Spain; e-mail: [email protected] ABSTRACT Natural naphthoquinones are compounds with a broad spectrum of biological activities against microbes, trypanosomes, viruses, and cancer cells, and it has been demonstrated that some quinones can inhibit enzymes that triggers apoptosis Regarding their allelopathic activity, they were early described as phytotoxic agents, although currently the number of these compounds that have shown activity is comparatively low. Well-known examples of allelochemicals are quinones juglone, sorgoleone and its related compounds. A set of 29 quinones have been prepared with several different chemical substituents including hydroxy, halogen, methoxy, and acetate as functional groups. In a first step the etiolated wheat coleoptiles bioassay was used to check for their biological activities. Most of assayed compounds have showed high activity levels. We have selected 12 showing the highest activity to be evaluated on a subsequent STS seedling bioassay. These selected compounds have different electron attracting substituents, mainly located on the quinone ring. The most active compounds in STS have been tested on weeds Echinochloa crus-galli and Urochloa decumbens. The most phytotoxic compounds are proposed as models for the development of new herbicides based on natural products. Keywords: Quinones, allelopathy, phytotoxic compounds Phone: +34 956012770; fax: +34 956016193 172 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 85. NAPHTHOQUINONES AS LEAD COMPOUNDS FOR THE DEVELOPMENT OF NEW AGROCHEMICALS Alexandra G. Durán, Nuria Chinchilla, José M. G. Molinillo, Juan C.G. Galindo, Francisco A. Macías * Grupo de Alelopatía, Departamento de Química Orgánica, Facultad de Ciencias, Instituto de Biomoléculas (INBIO), Universidad de Cádiz, Campus de Excelencia Internacional Agroalimentario (ceiA3), C/República Saharaui, s/n, 11510 Puerto Real, Cádiz, Spain; e-mail: [email protected] ABSTRACT The intensive use of synthetic herbicides over the last 50 years has caused several problems, among them, the environmental impact and the phenomenon of resistance. This loss of efficiency is due mainly to they present the same mode of action that herbicides discovered before 1985. Because of this, we need to find new agrochemicals with different modes of action, new chemical structures and more environment friendly. Naphthoquinones are natural pigments with two carbonyl groups at positions 1,4 and less frequently in positions 1,2 or 1,3 of the naphthalene ring. Owing to the wide range of biological activities that these structures possess such us anti-inflammatory, antifungal, antiviral and anti-carcinogenic effects, they are proposed as lead compounds for the search of new natural herbicides. Physico-chemical properties of two natural naphthoquinones (juglone and lawsone) have been modified in order to achieve this purpose (logP, number of rotatable bonds, solubility, etc), through the introduction of esters and ethers of varying chain length to optimize the biological activity levels and obtain improved compounds. All products were evaluated in a first bioassay of general activity (etiolated wheat coleoptiles bioassay) in which the effect of compounds over vegetable undifferentiated tissues are measured and after this screening, the most active compounds were evaluated on STS phytotoxicity bioassay. Keywords: naphthoquinones, phytotoxicity, bioassay, herbicide Tel: +34 956012770; Fax: +34 956016193 173 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 86. ALLELOPATHIC EFFECT OF RICE BRAN ON ORGANIC RICE PRODUCTION 1 2 P. Gnanasoundari 1 and E. Somasundaram 2* Department of Agronomy, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India; e-mail: [email protected] Department of Agronomy, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India; e-mail: [email protected] ABSTRACT Allelopathic weed control encourages weed suppression rather than elimination. This is done by the growth suppression of one plant species by another due to the release of toxic compounds. Rice bran application just after transplanting has been increasingly practiced as a herbicide-substitute for organic rice production. Field experiment was conducted at Tamil Nadu Agricultural University, Coimbatore during Rabi 2012-13 (October-February) to identify the ideal weed management technique in organic rice production. The experiment was laid out in randomized block design with three replications. The treatments comprised of fourteen different weed management practices viz., Paired row planting of rice with daincha as intercrop and incorporation on 35 DAT, Azolla as dual crop & manual incorporation on 20 DAT and 40 DAT, Azolla as dual crop and rotary weeder incorporation on 20 DAT and 40 DAT, Azolla as dual crop and cono weeder incorporation on 20 DAT and 40 DAT, Rotary weeder four times on 10, 20, 30 and 40 DAT, Cono weeder four times on 10, 20, 30 and 40 DAT, Rice hull solution (50 %) spray on 3 DAT and hand weeding on 35 DAT, Rice hull solution (50 %) spray on 15 DAT and hand weeding on 35 DAT, Sunflower dried stalk solution (1:10 w/v) spray on 3 DAT and hand weeding on 35 DAT, Sunflower dried stalk solution (1:10 w/v) spray on 15 DAT and hand weeding on 35 DAT, Rice straw @ 3 t ha-1 on 3 DAT and hand weeding on 35 DAT, Rice bran @ 2 t ha-1 on 3 DAT and hand weeding on 35 DAT, Hand weeding on 15 DAT and 35 DAT and Unweeded control. Rice byproducts meal was also found to be quite suppressive to annual weed growth. The application of rice bran significantly decreased the total weed density (15.00 No. m2) and weed dry weight (3.38g m2)as compared with weedy control. The results revealed that application of rice bran @ 2 t ha-1 on 3 DAT followed by a hand weeding on 35 DAT kept the weed density and dry weight below the economic threshold level and also increased the growth and yield of organic rice production. Keywords: Organic rice, Weed control, Rice bran, Growth, Yield Tel: +04222667413; fax: +04226611416 174 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 87. DYNAMIC OF RELEASE OF ALLELOCHEMICALS FROM Eucalyptus globulus LEAVES INCORPORATED INTO THE SOIL AND THEIR POTENTIAL PHYTOTOXICITY FOR WEED CONTROL Carolina G. Puig 1, Paula B. Andrade 2, Patrícia Valentão 2, Manuel J. Reigosa 1 and Nuria Pedrol1 1 2 Department of Plant Biology and Soil Science, laboratory of Plant Ecophysiology, University of Vigo. Campus Lagoas-Marcosende, 36310-Vigo, Spain; e-mail: [email protected] REQUIMTE/laboratory of Pharmacognosy, Department of Chemistry, University of Porto, 4050-313 Porto, Portugal. ABSTRACT In order to minimize the reliance of chemical herbicides for a sustainable agriculture, alternatives strategies based on natural products, such as the use of allelopathic plants as cover crops or applied to the soil as green manure for weed control, have been reported. It is well known the allelopathic nature of Eucalyptus globulus Labill., its herbicidal activity and its chemical composition, especially of the essential oil. But studies on the use of eucalyptus leaves incorporated into the soil as green manure for weed control in sustainable agriculture are very recent in literature (Puig et al. 2013). Based on the temporal phytotoxic effects of eucalyptus leaves observed on the germination and growth of Amaranthus retroflexus L. and Echinocloa crus-galli (L.) P. Beauv., highperformance liquid chromatography coupled with diode array detection was performed to identify and quantify the phenolic composition of eucalyptus leaves incorporated into the soil throughout time. Phytochemical analysis showed the presence of polyphenols such as flavonoids (hyperoside and rutin) and phenolic acids (chlorogenic acid and ellagic acid) in eucalyptus leaves. The phenolic quantification of leaf litter along time showed a continuous reduction of each compound concentration, according to the phytotoxic effects observed on weeds. The continuous release of allelochemical compounds from leaves may be partially responsible for the observed phytotoxic activity, which could explain the durability of the bioherbicide effect of E. globulus green manure. Reference: Carolina G. Puig, Lorena Álvarez-Iglesias, Manuel J. Reigosa, Nuria Pedrol (2013). Eucalyptus globulus leaves incorporated as green manure for weed control in maize. Weed Science 61, 154-161. Keywords: Tasmanian blue-gum, phenolic compounds, phytotoxicity, weed control, sustainable agriculture. Tel: +34986812616; fax: +34986812556 175 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 48. EVALUATION THE ALLELOPATHIC POTENTIAL OF WATER EXTRACT OF COMMON VETCH AND RYE 1 Negin Norouzi1 Gholam R. Muhammadi1 and Iraj Nosratti1 Dep. of Agronomy and plant breeding, Paradise of Agricultural and Natural Resources, Razi University, Kermanshah, Iran; e-mail: [email protected] ABSTRACT Allelopathy is a biological process including interactions between two plants through the production of chemical compounds (allelochemicals), which can diminish weed problems. Common Vetch and rye have strong allelopathic effects on many weed species. Therefore in order to assess the weed control potential of rye and common vetch, this experiment was conducted in the campus of paradise of Agriculture and Natural, Razi University, Kermanshah, Iran, in 2012. Allelopathic effects of water extract of common vetch, Rye and common vetch + rye on redroot pigweed, lambsquarter, and common cocklebur which are the main weeds of maize and maize were evaluated in pots. The extract was sprayed on plants. The results of this experiment showed that water extract of rye, common vetch and rye + common vetch reduced germination and growth of the weeds but didn’t strong effect on maize. The water extract of rye controlled redroot pigweed and common cocklebur better than other treatments and water extract of common vetch+rye better controlled lambsquarter. Keywords: allelochemicals, biological process, weed control Tel: 988314294165 176 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 49. THE ALLELOPATHIC EFFECT OF SOME CROPS ON JOHNSONGRASS SEED GERMINATION 1 Yasin Norouzi1, Gholam R. Muhammadi1 and Iraj Nosratti1 Dep. of Agronomy and plant breeding, Paradise of Agricultural and Natural Resources, Razi University, Kermanshah, Iran; e-mail: [email protected] ABSTRACT Allelopathy has a great impact on the weed biology and management practices therefore outdoor pot experiments were conducted at the campus of paradise of Agriculture and Natural, Razi University, Kermanshah, Iran, during the summer of 2013, to evaluate the effect of soil on activity of allelopathic crops on emergence and early growth of johnsongrass. Treatments were included of using grinded powder of alfalfa, sorghum and tobacco subterranean and shoot on emergence and early growth of weed species johnsongrass, barnyardgrass and redroot pigweed. Results of this study showed that presence of microorganisms in soil decreased the inhibitory effect of allelopathic plant materials on weed growth. Alfalfa had significantly higher growth reduction than all other used allelopathic plants followed by tobacco and sorghum. Lowering pH resulted in higher allelopathic activity of all plant materials. Keywords: Alfalfa, barnyardgrass, johnsongrass, redroot pigweed, sorghum, tobacco Tel: 988314294165 177 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 88. WHEAT (Triticum aestivum L.) ALLELOPATHY FOR MANAGEMENT OF LITTLESEED CANARYGRASS (Phalaris minor Retz.) Muhammad Saleem Kashif1, Zahid Ata Cheema1 and Muhammad Farooq1 1 Allelopathy Lab, Department of Agronomy, University of Agriculture, Faisalabad, Pakistan. Corresponding author’s email: [email protected] ABSTRACT Organic weed management is the need of current agricultural systems due to ecological toxicities of synthetic chemicals. Allelopathy can serve the purpose productively due to its ability to suppress the weeds under field conditions through the production of secondary metabolites by certain plants. A field experiment was conducted to investigate the allelopathic effects of 3 wheat cultivars (Shafaq-06, Faisalabad-08 and Sehar-06) against different density levels (0, 100, 200 and 300 plants m-2) of littleseed canarygrass. Wheat cultivar Shafaq-06 suppresses littleseed canarygrass more efficiently than other cultivars due to its ability to produce relatively higher amount of total soluble phenolics. Grain yield and all other growth and yield related parameters was also found maximum in Shafaq-06 at all density levels of littleseed canarygrass, whereas cultivar Sehar-06 showed relatively poor allelopathic potential against littleseed canarygrass with comparatively lower growth indices and less yield. In conclusion, Shafaq-06 is highly allelopathic wheat cultivar against littleseed canarygrass. Keywords: Allelopathy, total soluble phenolics, wheat, littleseed canarygrass TEL: +92-321-8872511 178 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 50. ALLELOPATHIC POTENTIAL OF Bidens sulphurea (CAV.) SCH. BIP. João Paulo Ferreira Cabreira1 Mariluce Pascoina Nepomuceno2* Pedro de Figueiredo Rocha Barbosa Martins3 Bruna Pires da Silva4 Pedro Luís da Costa Aguiar Alves5 1,2,3,4,5 Departamento de Biologia Aplicada à Agropecuária, FCAV – UNESP, Jaboticabal, SP, Brazil. 1 e-mail: [email protected]; 2* e-mail: [email protected]; 3 e-mail: [email protected]; [email protected] ; 5 e-mail: [email protected] ABSTRACT The natural effects of allelochemicals on other species vary, and they go from stimulation and inhibition of growth of the receiver plants to inhibition of seedlings germination. The search for alternatives for weed control besides synthetic herbicides has increased once weeds have getting resistance to those herbicides. This research was carried out in order to evaluate the allelopathic potential of water extract of young leaves of Bidens sulphurea (Cav.) Sch. Bip. (Asteraceae) on germination and development of lettuce (Lactuca sativa L.), pigweed (Amaranthus viridis L.) and Guinea grass (Panicum maximum Jacq). Fresh young leaves of B. sulphurea were collected (100 g) and extracted by blender with deionized water (300 mL). The extract was filtrated and then diluted to the concentrations of 2.5, 5, 10, 15, 20 and 25%. With 4 replicates, it was spread 6 mL of the dilutions in Petri dishes (9 cm diameter) with filter paper and 25 seeds of target plants. The dishes were identified, sealed and wrapped with aluminum foil. Then they were kept in a germination chamber at 25 oC for suitable periods for each target plant. After the growing period, the plants were frozen for at least 24 h and the parameters evaluated: germination percentage, root and aboveground length and dry mass. The water extract affected significantly the growing of the species (causing reduction), especially regarding roots. It affected also the allocation of dry mass in high concentrations. The water extract of B. sulphurea showed allelopathic potential in the evaluated species and promoted inhibition mostly on P. maximum and A. viridis. Keywords: allelochemicals, Cosmos sulphureus, seedling inhibition. Tel: +55 16 3209 2620; fax: +55 16 3209 2620 179 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 51. ENHANCING ALLELOCHEMICALS EFFICIENCY FOR WEED CONTROL IN FIELD Muhammad Jamil1 and Zahid Ata2* 1 Department of Biosciences, COMSATS Institute of Information Technology, Islamabad, Pakistan; e-mail: [email protected] 2 Department of Agronomy, University of Agriculture, Faisalabad, Pakistan; e-mail: [email protected] ABSTRACT Allelopathic suppression of weeds is receiving greater attention as a possible alternative for weed management. A number of plants possess potent allelopathic activity that had already been confirmed by a number of ways like bioassays with aqueous or various solvent extracts and residues, fractionation and identification of causative allelochemicals. Even though many possible explanations have been given for the weed suppressing effect of allelochemicals, but the exact allelochemicals and their mechanism by which they work to suppress weed spp. is lacking. The allelochemicals responsible for weed suppression can be quantified with liquid chromatography mass spectrometer (LC/MS). After screening and quantification, the highly producer of allelochemicals can be identified and introduced in the field for effective weed suppression. Furthermore possible ways to enhance efficiency of allelochemicals can be studied to reduce weed attack in the field. The development of a allelochemicals analysis based screening method, identification of highly allelochemicals producing spp. as well as enhancement of their efficiency could be useful idea to reduce weed attack in the field and may also helpful to decrease the use of pesticides and lessen environmental deterioration. Keywords: allelopathy, weed control 180 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 89. ALLELOPATHIC ACTIVITY AND CHEMICAL COMPOSITION OF RAPESEED EXTRACTS Abusteit, E.O 1 and Ibrahim R.A 2* 1 Agronomy Dept., Fac., of Agric., Cairo Univer., Egypt. e-mail: [email protected] 2 Agronomy Dept., Fac., of Agric., Cairo Univer., Egypt. e-mail: [email protected] ABSTRACT Wheat (Triticum aestivum L.) is the first food crop in Egypt. Its production is by heavy use of herbicides, which resulted in herbicide-resistant weeds, with disturbed ecological balance and human health there has been an increasing interest in weed control strategies with natural compounds released by plants Rapeseed is contains allelochemical compounds, These compounds affect the germination and growth of other crops. This research aims to study the inhibitory effect of rapeseed extracts on one of the main weeds in wheat yield in Egypt, wild oats (Avena fatua L). Aerial parts of rapeseed plants were extracted with methanol, chloroform and water after that 4 concentrations (0, 1%,10%,100%) were prepared. Then, 30 ml of each concentration were added to Petri dishes, which contained 25 seeds of wild oats. The experiment was conducted as randomized with 3 replications at university of Opole, Poland. Results showed that after one week the methanolic extract had great inhibitory effect on germination percentage, as well as seedling length and fresh weight The 100% extract concentration, compared to other concentrations had a high inhibitory effect on germination indices analysis methanolic extract with HPLC. Keywords: Allelopathy, weed control, rapeseed extracts, HPLC, wild oats Tel: +02357177355; Fax: +0235684565 181 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 ORAL 90. TWO ALLELOPATHIC SUBSTANCES FROM AQUEOUS METHANOL EXTRACT OF SOIL UNDER JAPANESE RED PINE FOREST Fukiko Kimura 1*, Osamu Ohno 2 and Hisashi Kato‐Noguchi 1 1 2 Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Miki, Kagawa, Japan, 761-0795 1; e-mail: [email protected] Department of chemistry, Faculty of Science and Technology, Keio University ABSTRACT The vegetation under Japanese red pine (Pinus densiflora Sieb. et Zucc.) forests is sparse. This phenomenon was considered to be due to allelopathy of the pine. Based on the literature, allelopathic substances are probably released and accumulated into the soil from pine needles and litter. These allelopathic substances in the soil may cause the growth inhibition of neighboring plants. Therefore, allelopathic substances in the soil are important for the elucidation of allelopathy. However, the information of active substances in soil is limited. In this study, inhibitory substances in the soil under the pine forests have been isolated and identified. The soil extract showed the concentration-dependent inhibition on the growth of 7 test plants, which suggest that the soil may contain growth inhibitory substances. The soil extract was purified by silica gel column, Sephadex LH-20 column, C18 cartridge and HPLC, and two growth inhibitory substances were isolated. These substances were characterized by spectral data as 7oxodehydroabietic acid and methyl 15-hydroxy-7-oxo-dehydroabietate. The concentrations required for 50 % inhibition of 7-oxodehydroabietic acid on the hypocotyls and roots of cress were 0.17 and 0.18 mM, respectively. Those of methyl 15-hydroxy-7-oxo-dehydroabietate were 9.9 and 1.0 mM, respectively. The results suggest that these two substances may play an important role in the allelopathy of Japanese red pine and contribute to form its sparse forest floor. Keywords: allelopathy, Pinus densiflora Sieb. et Zucc., soil, aqueous methanol extract 182 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 91. ALLELOCHEMICALS IN AQUEOUS VOLATILE FRACTIONS FROM SELECTED FOUR Eucalyptus SPECIES Hanwen Wu1, Jinbiao Zhang2, Min An3, Rex Stanton3 1 Graham Centre for Agricultural Innovation (a collaborative alliance between Charles Sturt University and Industry & Investment NSW), Wagga Wagga Agricultural Institute, Wagga Wagga, NSW 2678, Australia 2 Analytical Center, and School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China 3 Graham Centre for Agricultural Innovation (a collaborative alliance between Charles Sturt University and Industry & Investment NSW), Pugsley Place, Wagga Wagga, NSW 2678, Australia [email protected] ABSTRACT The rapid development of herbicide resistance in weeds posses significant threats to agricultural sustainability. There has been an increasing demand for alternative weed control. There is limited understory vegetation within the dripline of certain Eucalyptus species in fields. The aqueous volatile fractions (AVFs) from four selected Eucalyptus species have been previously confirmed to have stronger inhibitory effects on germination and seedling growth of the perennial weed, silverleaf nightshade (Solanum elaeagnifolium Cav.). The AVFs were the water soluble volatile fractions produced together with the essential oils (water insoluble fractions) during the steam distillation process. The allelochemicals in AVFs was further investigated in this work. Solid phase microextraction (SPME) was used to extract compounds from AVFs, and then the analytes extracted were determined by gas chromatograph–mass spectrometry (GC-MS). The GC-MS profile of each AVF was different from each other, indicating that their composition varied between species. The identification analysis revealed that there were 32 components in the AVF from E. spathulata leaves. The compounds found in the AVFs of E. salubris, E. brockwayii and E. dundasii were 29, 35 and 34, respectively. The quantification analysis showed that 1.8-cineole was the most abundant component in all AVFs, ranging from 37.15 to 80.1%. Finally, the relationship between the composition of AVFs and the chemical structure of components were also discussed. Keywords: Allelopathy, Eucalyptus, elaeagnifolium Cav), weeds. silverleaf nightshade (Solanum Tel: +61 2 69381602; fax: +61 2 69381861 183 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 92. ALLELOPATHIC EFFECTS OF ESSENTIAL OIL FROM FOUR EUCALYPTUS SPECIES ON PRAIRIE GROUND CHERRY (Physalis viscosa L.) Jinbiao Zhang1*, Hanwen Wu 2, Min An3, Rex Stanton3 1 2 Analytical Center, and School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China; e-mail: [email protected] Graham Centre for Agricultural Innovation (a collaborative alliance between Charles Sturt University and Industry & Investment NSW), Wagga Wagga Agricultural Institute, Wagga Wagga, NSW 2678, Australia 3 Graham Centre for Agricultural Innovation (a collaborative alliance between Charles Sturt University and Industry & Investment NSW), Pugsley Place, Wagga Wagga, NSW 2678, Australia ABSTRACT There is limited understory vegetation within the dripline of certain Eucalyptus species in fields. The allelopathic effects of the essential oils from eucalyptus may be one of the reasons. The aim of this paper was to assess the allelopathic effects of essential oils from four selected eucalyptus species on prairie ground cherry (Physalis viscosa L. PGC), a highly invasive weed originated from South America. The study was carried out under both laboratory and greenhouse conditions. Our results showed that the germination and seedling growth of PGC were inhibited by these eucalyptus oils. The inhibition was dose dependent and varied among these four eucalyptus species. E. salubris oil demonstrated the strongest bioactivity with 99.2% in germination inhibition and more than 98% in growth inhibition when 30 ml of oil was used. The greenhouse study showed that the essential oils would cause damage on the plants. As observed in the bioassay in lab, there were differences in injury rating among these eucalypts oils. The injury rating of essential oils on plants increased with the increase of treatment time and oil concentrations used. E. salubris oil showed the highest bioactivity on PGC in greenhouse experiment and the dead injury occurred just one day after treatment when 40% of oil concentration was used. Our results provide the possibility for the alternative weed control. Keywords: Allelopathy, essential oil, Eucalyptus, prairie ground cherry, weeds. Tel: +86 0591 83789371; fax: +86 0591 83789371 184 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 93. ALLELOPATHY IN Shorea robusta FORESTS Ombir Singh* Silviculture Division, Forest Research Institute. Dehradun, Uttarakhand, India – 248006, India. * E. Mail: [email protected] ABSTRACT Sal (Shorea robusta) forests in India have been a source of perennial supply of timber, fuel and various other products since time immemorial. These forests occupy ecologically and economically vital position but large-scale mortality in moist as well as in dry sal forests coupled with absence of regeneration, recalcitrant seeds are posing great threat to these forests. The factors responsible to this state of affair are many e.g. recurrence of drought, deficient rainfall, excessive biotic pressure, illicit and irregular felling, and forest fires etc. To understand the process of natural regeneration failure in these forests, allelopathic interactions of one under-storey species (Ardisia solanacea) and two invasive species (Ageratina adenophora & Lantana camara) with the keystone species (Shorea robusta) were studied along with regeneration surveys in sal areas. The foliage leachates (0, 5%, 10%, 15%, 20%) of these species were investigated on the seed germination, root and seedling growth of S. robusta in laboratory as well as in nursery. The leachate concentrations have no effect on seed germination but affected the root growth, root hairs; causes yellowing of leaves and weakening of seedlings subsequently. Regeneration survey in these forests showed that there is no sal regeneration in areas having either Ageratina adenophora or Ardisia solanacea, and less regeneration with Lantana camara but interestingly good regeneration in areas infested with Ageratum conyzoides. These findings may explain the problems of seedling establishment in sal forests infested either with invasive or under-storey associates. By knowing which trees are particularly susceptible and which plants are most likely to produce toxic effects, these allelopathic problems can be avoided or dealt with by site preparation and weed control in plantations and assisted natural regeneration in these forests. Keywords: Shorea robusta, regeneration, under-storey, allelopathy Tel:+911352224469; Fax: +911352756865 185 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 94. ALLELOPATHY OF Cistus ladanifer ON THE REGENERATION OF Quercus ilex Juan Carlos Alias Gallego, Teresa Sosa Díaz, Cristina Valares Masa and Natividad Chaves Lobón* 1 Department Ecology, Faculty of Science, University of Extremadura, Av. De Elvas S/N, 06080 Badajoz, Spain; e-mail: [email protected] ABSTRACT Mediterranean forests are currently dominated by evergreen oak species, the holm oak Quercus ilex L. being the commonest in the Iberian Peninsula. The long-term persistence of these ecosystems critically depends on the regeneration of the tree layer. Different authors suggest a facilitative effect of shrubs on seedling survival due to nurse effects associated with shade and protection from herbivores. Nonetheless, some shrubs species provide clear evidence of allelopathic effect. Allelochemicals derived from living, dead and decomposed tissues of the shrubs can interfere with tree regeneration directly by affecting seed germination and seedling growth. The objective of our study are to determine if Cistus ladanifer affects Quecus ilex regeneration (inhibition or facilitation). Seed germination and growth of Quecus ilex were studied. 200 acorns were sown in plastic pots that have been filled with vermiculite. Seeds were placed at 1-2 cm depth and then 100 acords were covered with litter of Cistus ladanifer (treatment A) and 100 were covered with litter (the exudate having been previously extracted with methanol) of Cistus ladanifer (control). More than 80% of the acorns germinated during the experiment in the control and significant differences were found among treatments, treatments A: 53% germinated. Seedlings in vermiculite with litter of Cistus ladanifer grew considerably less than those growing in the control. We can conclude that C. ladanifer played a strong role in inhibiting establishment and growth of Quercus ilex and allelopathy is an interaction which could partially explain the difficulties in natural regeneration of Quercus ilex. Keywords: Cistus ladanifer, Quercus ilex, litter, allelophaty Tel: +34924289300 (86929) 186 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 52. ALLELOPATHY OF Cotinus coggygria IN MEDITERRANEAN OAK FORESTS Jordane Gavinet1,2, Catherine Fernandez1, Mathieu Santonja1, Samuel Peano1, Theodore Tchong1, Anne Bousquet‐Melou1 1 2 Institut Méditerranéen de Biodiversité et d’Ecologie (IMBE) Aix-Marseille Université, CNRS, IRD, Univ. Avignon, Campus St Charles Case 4, 13331 Marseille Cedex 03, France. E-mail: [email protected] Institut national de recherche en sciences et technologies pour l’environnement et l’agriculture, 3275 route de Cézanne - CS 40061, 13182 Aix-en-Provence Cedex 5 ABSTRACT The pubescent oak forest is an important ecosystem in northern Mediterranean basin showing a lack of natural regeneration. Cotinus coggygria, a dominant shrub in the understorey of these forests, is particularly rich in secondary metabolites (phenolics and terpenoids). In this study, we tested the influence of Cotinus allelochemicals on: (i) germination and growth of six herbaceous species and the role of microorganisms in these interactions through in vitro bioassays, (ii) regeneration of pubescent oak and mycorrhizal association in mesocosms. Finally, (iii) we checked if the results obtained in laboratory were consistent with observations made in the field. The herbaceous species were inhibited or insensitive (germination and growth) to Cotinus extracts while microorganisms were stimulated (biomass and respiration). Generally, microorganisms altered the expression of allelochemicals. Young pubescent oaks showed a decrease in their root biomass in presence of litter or extracts whereas older oaks were not affected. The high mycorrhization rate of older oaks, with a dominance of contact mycorrhizae known for their ability to degrade polyphenols, could explain this result. These data will be confronted to the results obtained in the field. Keywords: Allelopathy, biodiversity, bioassay, soil microorganisms, Pubescent oak forest, Cotinus coggygria, phenolic compounds, mycorrhizae TEL: +33 (0) 4 13 55 12 22 187 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 95. WHAT IS THE IMPACT OF THE VARIOUS MODES OF RELEASE ON ALLELOPATHIC ACTIVITY OF Pinus halepensis? Mathieu Santonja1, Anne Bousquet‐Melou1, Elisabetta D’Addio2, Catherine Fernandez1 1 Institut Méditerranéen de Biodiversité et d’Ecologie (IMBE) Aix-Marseille Université, CNRS, IRD, Univ. Avignon, Campus St Charles Case 4, 13331 Marseille Cedex 03, France. E-mail: [email protected] 2 Seconda Università degli Studi di Napoli, Viale Beneduce 10, 81100 Caserta. Italy ABSTRACT Pinus halepensis, known to synthesize a wide range of secondary metabolites, afforest abandoned agricultural land that present high species richness in north part of Mediterranean basin. Previous studies have shown that green needles leachates of P. halepensis strongly affect various natural species. However, this species is also recognized to be a strong terpene emitter and this mode of secondary metabolites release is not usually studied in allelopathic processes. The objective of this study is to improve our knowledge on allelopathic expression of Pinus halepensis secondary metabolites by testing all mode of release: volatilization of leaves, litter and roots, litter decomposition, leachates of green leaves and roots exudates. Germination and growth of Lettuce were tested in laboratory according the different mode of release (simulated by macerates or volatiles) and the presence or not of soil microorganisms (filter paper or soil). These two parameters were significantly altered in dose-dependent ways. Litter decomposition and green leaves leachates exhibited inhibitory effects. Root exudates were the only mode of release that presented positive effects on seedling growth. Volatized compounds from leaves, litter or roots also affected negatively Lettuce but in a lesser extent. Allelopathic effect was reduced on natural soil, particularly with leachates comparing to volatized compounds. This highlights the key role of microorganisms in chemical plant-plant interactions. The allelopathic activity of P. halepensis varies according to the different modes of release proving the complexity of chemical interaction between plants. Keywords: Allelopathy, Pinus halepensis, soil microorganisms, bioassays, biogenic volatile organic compound (BVOC), terpenoids, phenolics TEL: +33 (0) 4 13 55 12 22 188 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 96. THE ROLE OF ALLELOPATHY IN EARLY STAGES OF POST FORESTFIRE SECONDARY PLANT SUCCESSION Hassan A. M. Mezori*, Salih T. Wali** * Biology Department, Faculty of Science, ** Faculty of Agriculture and Forestry, University of Duhok, Duhok, Kurdistan Region, Iraq . Email: [email protected] ABSTRACT Forests play an important role in regulation of climatic conditions, decreasing of air pollution, recycling of nutrient, and improving the soil properties. Fires remove allelochemicals from the environment, which could play an important role in plant succession, directly through its effect on invader plants seed bank or indirectly by its effect on the nitrogen cycle. The aim of this study is to test the hypothesis which argued that allelopathy plays a crucial role in the early stages of post forest fire succession. The study was carried out in forest area burned in (Ashawa) within Duhok, to investigate (i) the forest community composition, (ii) the seed bank of plant species post forest fire and (iii) the effect of aqueous extracts, residues and residues of Quercus infectoria decomposing in the soil for different periods, directly through its effect on germination and growth of pioneer species, or indirectly through its effect on nitrification. The results indicated that the relative dominance, density and abundance of Q. infectoria was more than all other trees, the seed bank revealed that there were two groups of plants, the first one (10) species appeared in burned and unburned lands, while the second group (5) species was appeared in the burned land only. The density, abundance, biomass and diversity of plant species in burned land were increased. The aqueous extract and residues of Q. infectoria significantly inhibited the seed germination and growth of Medicago polymorpha, residues decomposition for different periods was persisted for 14 weeks. Residues of Q. infectoria highly inhibited nitrification rate; the inhibition persisted over 36 days.The forest fire caused appearance of some pioneer plants which change the community structure of the area. Keywords: allelopathy,community structure, forest fire, seed bank, nitrification, pioneer plant Tel: +964 750 457 96 41 189 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 97. ALLELOPATHIC INFLUENCE OF Carpinus betulus ON UNDERSTOREY VEGETATION Lamine Bensaddek*, Deborah Closset‐Kopp and Guillaume Decocq Unité de recherche « Ecologie et Dynamique des Systèmes Antropisés » (FRE 3498 CNRS – UPJV), Université de Picardie Jules Verne, 1, rue des Louvels, F-80037 Amiens Cedex 1; [email protected] ABSTRACT In forest ecosystems, trees interact with understorey species through negative (e.g. soil water and nutrient depletion, shading) or positive (e.g. nitrogen fixation) effects on these species' environmental conditions. Interference between neighboring plants through the release of allelochemicals into the environment has been widely reported. Hornbeam (Carpinus betulus L., Betulaceae) is a shade tolerant tree species, which is widely distributed on deep fertile soils with wide adaptability to varied environmental conditions. It is widely distributed in north - west and central Europe where it has long been promoted as coppice wood. As a result, many deciduous forests of the temperate area now consist of oak-hornbeam forests, inherited from the coppice-with-standards silviculture. Many field observations have emphasized the fact that very poor vegetation is seen under the canopy of Carpinus betulus as compared to adjoining forest areas. Among potential causes for this, competitive (i.e. depletion of one or more limiting resources such as light, nutrients and water), as well as allelopathic (i.e. effect of allelochemicals) interference may be hypothesized. The allelopathic effects of hornbeam leaf extracts on understorey plant species (Poa trivialis and Urtica dioica) were investigated following different experimental conditions. Seed germination tests and leaf surface analyses were carried out in order to evaluate the studied extracts influence. In this study our first results tend to highlight an allelopathic effect. Keywords: Carpinus betulus, understorey vegetation, allelopathy, seed germination test Tel:+ 33 322825421 190 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 98. CAN THE ATLANTIC SHRUBLAND CONTROL THE INVASION OF AILANTHUS ALTISSIMA (MILL.) SWINGLE? ALLELOPATHIC EVIDENCES 1 Tonio López‐Nogueira 1, María Pardo‐Muras1, Lorena Álvarez‐Iglesias1, Carlos Cavaleiro2, and Nuria Pedrol1 Department of Plant Biology and Soil Science, Faculty of Biology, University of Vigo, 36310-Vigo, Spain. e-mail: [email protected] 2 Department of Pharmacognosy, Faculty of Pharmacy/CEF, University of Coimbra, 3000Coimbra, Portugal ABSTRACT The loss of biodiversity is the main problem caused by plant invasions in terrestrial ecosystems. One example is Ailanthus altissima that invades some areas of Galicia (NW Spain) being one of the main invasive tree species across Europe and other continents, except Antarctica. Usually, research in biological invasions deals on the invasive competition effects against the native plants. On the contrary, we focus on the effects of the native dominant species against the invasive species. Our field observations suggest that the mature native shrubland is able to stop the invasion of A. altissima, acting as a natural barrier. Both dominant species of the Atlantic shrubland: Ulex europaeus and Cytisus scoparius have a great competitive ability; in fact, they are top invasive plant species in other parts of the world. Under the hypothesis that competition by passive interference (allelopathy) may underlie the resistance to invasion, we tested the phytotoxicity of volatiles emitted by U. europaeus and C. scoparius on the in vitro early growth of A. altissima, by using the method of hermetic chambers (Barney et al. 2005). Moreover, for the first time in literature, we determined the composition of the volatile extracts of both shrub species by GC-MS. The volatile compounds emitted by the native shrub species produced a significant phytotoxic effect on A. altissima, by inhibiting drastically seedling growth. The volatile profile of U. europaeus and C. scoparius revealed the presence of some allelochemicals previously described as phytotoxic in other species. From our results, we conclude that Allelopathy can, at least in part, underlie the competitive ability of the Atlantic shrubland against the invasion of A. altissima by seed setting. Barney JN, Hay AG, Weston LA (2005) Isolation and characterization of alllelopathic volatiles from mugwort (Artemisa vulgaris). Journal of Chemical Ecology 31: 247-265. Keywords: native species, invasive species, phytotoxicity, volatile compounds, Ulex europaeus, Cytisus scoparius. 191 th 7 World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 53. RESISTANCE OF THE NATIVE SHRUB Cytisus scoparius (L) Link TO NON-POLAR EXTRACTS OF THE INVASIVE Acacia dealbata Link Pablo Souza‐Alonso1*, Carolina G. Puig1, Luís González1 1 Plant Biology and Soil Science Department, University of Vigo, 36310 Vigo, Spain; [email protected]; [email protected]; [email protected]. ABSTRACT Acacia dealbata Link is an aggressive invader belonging to Acacia genus that currently threatens Mediterranean-type ecosystems worldwide. Allelochemicals have been suggested as drivers of oxidative stress in various target species. Natural allelochemical concentration responsible for this interaction is not known. Therefore, we tested the possible oxidative stress and the antioxidant response, which were caused on the native species Cytisus scoparius (L) Link by A. dealbata. For this, we used natural concentrations previously quantified in soils. We extracted chemical compounds from aerial parts, roots and soil of A. dealbata plants using the non-polar solvents dichloromethane (DCM) and acetone. At the end of the treatment period, total germination, germination indices (S, AS and CRG), radicle and shoot length, seedling biomass, soluble protein content, hydrogen peroxide (H2O2) content, malondialdehyde (MDA) content, and the antioxidant enzymes superoxide dismutase (SOD) and peroxidase (POX) were measured. The results revealed that total germination, S, and AS indices and early growth were slightly enhanced. Soluble protein content was significantly increased for almost all A. dealbata extracts, but the rise in H2O2 level was strictly found in the DCM fraction. Acetone soil extracts significantly enhanced POX activity and flowers extracted with DCM enhanced SOD activity. There was no evidence for significant accumulation of MDA neither in acetone nor DCM extracts. At natural concentrations A. dealbata extracts did not cause severe oxidative stress on hard-coat species as C. scoparius. Therefore, our findings suggested that oxidative stress is not a powerful tool negatively affecting the early growth of the pioneer species C. scoparius. Keywords: Allelopathy, oxidative stress, extracts, enzymatic activities, germination, non-polar solvents Tel: +34 986 812594; fax: +34 986 812555 192 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 99. Mentha piperita VOLATILES AND ALLELOPATHIC MONOTERPENES AS GROWTH MODIFIERS IN Brassica oleracea CULTURE SYSTEMS Andreas Ulbrich1*, Margot Schulz2, Hannah Kahle1, and Philip Krämer1 1 Department of Horticultural Production, University of Applied Science, Am Krümpel 31 49090 Osnabrück, Germany; [email protected] 2 IMBIO Institute Molecular Biotechnology of Plants, University of Bonn, Karlrobert Kreiten Str. 13, 53115 Bonn, Germany; e-mail:[email protected] ABSTRACT White cabbage (Brassica oleracea convar. capitata var. alba cv LENNOX F1) were grown with two Mentha x piperita varieties, “Englische Minze cv Micham” and “Multimentha” in randomly designed arrangements in greenhouses during spring (set 1) and autumn (set 2). The single plots were exposed to one or four Mentha plants of the same variety, each with three main shoots of 7 nodes. Controls were cultured in another greenhouse without Mentha plants. In the first set, Brassica plants exposed to Mentha volatiles exhibited significantly higher specific leaf weight, dry weight percentage and number of plants with a second leaf 21 days after sawing. Except for leaves of the 4th node, which were significantly longer, leaf shape from other nodes was not affected. In the second set (autumn), specific leaf weight was significantly higher, the number of plants with a second leaf at stage 2 was significantly higher when exposed to 4 Multimentha plants, but there was only a tendency of higher dry weight percentage. Using single monoterpenes, menthol or menthone, only effects on the leaf phenotype were found. In summary, the results indicate that stimulatory effects strongly depend on the developmental stage of the leaves. When applied at sensitive stages, Mentha volatiles can enhance the productivity and increase the quality and quantity of the biomass. Keywords: Terpenoids, Mentha volatiles, Brassica oleracea, growth modifier. Tel: + 49-(0)541 969-5116; fax: + 49 541 969 – 5170 193 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 100 EFFICACY OF LEAF EXTRACT OF Moringa oleifera AS BIO-STIMULANT ON TOMATO Shahzad. M.A. Basra1 and Carol J. Lovatt 2* 1 2 Department of Crop Physiology, University of Agriculture, Faisalabad; [email protected] Botany and Plant Sciences, University of California, Riverside; [email protected] ABSTRACT Moringa oleifera leaf extract (MLE), rich in growth enhancing compounds was investigated to analyze the plant growth stimulants and its effect on improving tomato yield and quality. In first phase, moringa plants were grown in walk-ingrowth chamber and MLE was analyzed to identify and quantify PGRs in itself from. MLE was found rich cytokinins, (cis) Zeatin riboside was found in a fair quantity while very low or bound forms of other PGRs. Moringa leaf extract was also rich in total phenolics and enzymetic antioxidants (SOD,CAT, POD). Later, MLE (30 times diluted pure extract) was applied exogenously (root and foliar) in comparison with BAP, adenosine and zeatin (100 ppm each) to tomato plants (Super sweet-100, hybrid). Exogenous applications of natural and synthetic plant growth stimulants significantly affected the yield and quality of tomato leaves and fruits. Maximum flowers, fruits and total yield were recorded when tomato plants were treated with Zeatin (root) followed by MLE (root) and BAP (foliar). MLE (foliar) was ranked second in total yield and yield per plant. Total chlorophyll and chlorophyll a contents were maximally improved by MLE (root) followed by zeatin (root) application while chlorophyll b and β-carotene contents were not significantly affected by exogenous applications. Maximum arginine contents and total antioxidant activity in moringa leaves were recorded in MLE (foliar) treated plants while proline contents were maximally improved by MLE (root) application in comparison with all other treatments. Good quality indicators of fruits like sugar, lycopene contents and total proteins were maximally found by MLE (foliar) and zeatin (root) applications, respectively while total phenolic contents in tomato fruits were not significantly affected by any treatment but total antioxidant activity of tomato fruits was significantly improved by zeatin (foliar) application. The present study suggests MLE is a good alternate of synthetic PGRs in improving the yield and quality of tomato fruits and leaves when used 30 times diluted solution. Keywords: Moringa oleifera, leaf extract, zeatin, TEL: +92 333 6519675 194 th 7 World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 101. VOLATILE ORGANIC COMPOUNDS (VOCS) of Acacia dealbata Link INTERFERING WITH PLANT GROWTH Pablo Souza‐Alonso1*, Luís González1, Carlos Cavaleiro2 1 Plant Biology and Soil Science Department, University of Vigo, 36310 Vigo, Spain; [email protected]; [email protected]. 2 Faculdade de Farmácia e Centro de Neurociências e Biologia Celular de Coimbra, Universidade de Coimbra, 3000-295, Coimbra, Portugal; [email protected] ABSTRACT Acacia dealbata Link is a widespread invader in Mediterranean type ecosystems and traits promoting its invasiveness are currently under research. Due to the dense atmosphere below its canopy we hypothesized that volatile organic compounds (VOCs) released from flowers, leaves, litter or a mixture of treatments could affect seeds and seedlings underneath acacia canopy. We tested the possible effect on germination, early growth and oxidative stress, on the native species Trifolium subterraneum, Lolium multiflorum, Medicago sativa and also on A. dealbata seeds by A. dealbata VOCs. We reported that VOCs from flowers significantly reduced germination in L. multiflorum and A. dealbata and also S and AS indices. Moreover, VOCs from flowers decreased root length, stem length, above and belowground biomass for all species studied. VOCs from flowers and mixture also increased significantly malondialdehyde (MDA) content in T. subterraneum and L. multiflorum. However, antioxidant enzymatic activities showed an irregular trend. In T. subterraneum, peroxidase activity was significantly enhanced in flowers and leaves whereas superoxide dismutase activity decreased after the exposure to VOCs from flowers. On the contrary, VOCs released from leaves increased the activity of superoxide dismutase in L. multiflorum. GC-MS analyses showed that the volatile fraction from flowers presented the lowest diversity with four compounds namely heptadecadiene, n-nonadecane, n-tricosane, and octadecene, representing the 62% of its isolate. Nevertheless, 11 compounds were exclusively found in the volatile fraction from flowers. Here, we present evidence that the VOCs released from A. dealbata flowers strongly inhibited germination and seedling growth of selected species. As far as we know, this is the first evidence of phytotoxicity induced by VOCs in invasive species belonging to Acacia genus. Keywords: Volatile organic compounds, plant invasion, essential oils, phytotoxicity, seed germination, oxidative stress Tel: +34 986 812594; fax: +34 986 812555 195 196 Session 7. Physiological aspects of allelopathy Physiology of allelopathy Mode of action of allelochemicals Detoxification of allelochemicals 197 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 102. CALCIUM SIGNAL SYSTEM INVOLVES IN THE FORMATION OF ALLELOPATHIC AUTOTOXICITY IN THE ROOTS OF Rehmannia glutinosa L. Xin Jian Chen1, Ming Jie Li1, Peng Fei Wang1, Feng Qing Wang1, Wen Xiong Lin2, Zhong Yi Zhang2,1 1. College of Life Sciences, Henan Agricultural University, Zhengzhou, China, 450002 2. College of Crop Sciences, Fujian Agriculture and Forestry University, Fuzhou, China, 350002 [email protected] ABSTRACT Rehmannia glutinosa L, a traditional Chinese medicine herb, is unable to grow normally in a soil where the same species has recently been cultivated. The biological basis of this so called allelopathic autotoxicity or replanting disease is unknown. To elucidate the injured reason happened in the roots of replanting plants, we performed on three level experiments as follows. Firstly, based on the suppression subtractive libraries, transcriptome sequencing, digital differentially expressed analysis and qPCR technique, we found that calcium signal system was abnormally expressed in root of replanting plant. In the roots of replanted Rehmannia glutinosa L, several Ca2+ signaling-related genes (such as those encoding a calcineurin B-like calcium binding protein, a calmodulinbinding protein and a calcium ion binding protein) were all strongly upregulated. In contrast, the gene encoding a Ca2+ antiporter/cation exchanger, which is presumed to regulate the Ca2+ concentration in the cell, was sharply down-regulated. Secondly, using calcium signal blocking agents such as TPC, CNGC, GLR and Annexin, which are commonly used in therapy of human being’s disease, both states of injured by allelochemicals and expression of t the abnormal genes related to Ca2+ signal were improved or compromised in the roots of replants plants. And finally, the distribution of Ca2+ ion was observed by fluorescence microscope and transmission electron microscope and results showed that in root cytoplasmof replanting plants Ca2+ concentration was dramatically higher than that in normally grew plants. Our experiment strongly indicated that Ca2+ signal plays an important part in different allelochemical stresses in Rehmannia glutinosa L. Keywords: Rehmannia glutinosa L, allelopathic autotoxicity, calcium signal system 198 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 103. CHLOROPHYLL PIGMENTS, PHOTOSYNTHETIC PERFORMANCE AND REACTIVE OXYGEN SPECIES GENERATION DURING ARTEMISININ-INDUCED STRESS IN Arabidopsis thaliana M. Iftikhar Hussain1 and Manuel J. Reigosa2 1,2 Department of Plant Biology and Soil Science, University of Vigo, Campus Lagoas-Marcosende, 36310-Vigo, Spain 1; [email protected], 2; [email protected] ABSTRACT Plant-derived secondary compounds have great potential in the development of environmentally safe herbicides with novel molecular sites of action. Artemisinin, a potent antimalarial drug, is phytotoxic to many crops and weeds. We reported here the effects of artemisinin on stress markers, including fluorescence parameters, photosystem II photochemistry, photon energy dissipation, lipid peroxidation and reactive oxygen species generation in Arabidopsis thaliana. Arabidopsis ecotype Columbia (Col-0) seedlings were grown in perlite and watered with 50% Hoagland nutrient solution. Adult plants of Arabidopsis were treated with artemisinin (at 0, 40, 80, 160 µM) after every second day (1, 3, 5, 7) up till one week and fluorescence measurements were performed by using a Maxi Imaging PAM Chlorophyll Fluorescence System. At each measuring time, the plants were kept in darkness for 5 min and successively illuminated with a saturating pulse of intensity 2700 µmol m−2 −1 for the measurement of Fm (maximum fluorescence of dark-adapted leaves), and all other fluorescence parameters; ΦII (effective photochemical quantum yield), ΦNPQ (quantum yield of light-induced nonfluorescent nonphotochemical fluxes), ΦNO (quantum yield of all fluxes other than ΦNPQ and ΦII) as defined by Bilger and Bjorkman (1990) and Demmig-Adams et al. (1995). Artemisinin, in the range 40-160 µM, decreased the fresh biomass, chl a, b, but increased carotenoid contents. The Fv/Fm level decreased significantly following treatment with artemisinin (80 µM) than control from day 1 up till day 7. Artemisinin (40 µM) reduced photosynthetic yield (ΦII) during all 7 days and reduction was more severe from day 4 to 7. The electron transport rate in Arabidopsis was significantly affected following exposure to artemisinin at 80 and 160 µM. The ΦNPQ and NPQ were significantly lower than control. These results confirmed that excitation energy fluxes of NPQ can be used as biomarkers for rapid phytotoxicity assessment. Artemisinin stress caused higher lipid peroxidation and root oxidizability of Arabidopsis than control. Demmigs-Adams B, Adams III WW. 1992. Ann Rev Plant Physiol Plant Mol Biol 43:599-626. Bilger W, Björkman O. 1990. Photosyn Res 25: 173–185. Keywords: Arabidopsis, reactive oxygen species, photosynthesis, root oxidizability, secondary metabolites TEL: +34986812616, FAX: +34986812556 199 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 54. ALLELOPATHIC POTENTIAL OF Eucalyptus rostrata LEAF RESIDUE ON SOME METABOLIC ACTIVITIES OF Zea mays L. Gabr, M. A.1*; Hegab, M. M.2; Al‐Wakeel, S. A. M.1 and Hamed, B. A.2 1 Department of Botany, Faculty of Science, Cairo University, Giza, Egypt 2 Department of Botany, Faculty of Science, Beni-Suef University, Beni Suef, Egypt. *(e-mail: [email protected] - Mobile: +2 01113468691) ABSTRACT A greenhouse pot experiment was conducted to investigate the possible allelopathic effect of Eucalyptus rostrata leaf residue on some metabolic activities associated with growth of 10, 20 and 30-day-old corn plants. The lower level of Eucalyptus treatment (0.5 %, w/w) induced a stimulatory effect on the growth of shoot and root of corn plants. The positive effect was more obvious in shoot than in root growth. Inversely, high levels of incorporated Eucalyptus leaf residue (1 and 2 %, w/w) reduced all growth parameters of both shoot and root relative to the respective control, and this reduction was concentration dependent. The total phenolic aglycone content of mature E. rostrata leaf was 16.89 mg g-1 dry weight present as water-soluble secondary metabolites. Qualitative and quantitative HPLC analysis of E. rostrata leaves revealed the presence of nine phenolic compounds. The p-coumaric acid was the most dominant compound, while catechol and chlorogenic acid were detected in lesser amounts. A trace amount of salicylic acid, vanillin, coumarin, as well as caffeic, cinnamic and ferulic acids were also detected. The total phenolic content of corn shoot increased with the lower concentration of E. residue and decreased with higher treatments. The lowest level of Eucalyptus residue (0.5%, w/w) increased the contents of photosynthetic pigments, particularly chlorophyll “a” content, which was correlated with increase in the total carbohydrate production. Similarly, the total nitrogen and total phosphorus, as well as nucleic acids were markedly higher than the control treatment. Negative pattern of changes in the contents of photosynthetic pigments, carbohydrate, nitrogen, phosphorus, phenolic compounds and nucleic acids, was observed in corn plant treated with high rates of Eucalyptus leaf residue (1 and 2%, w/w) as compared with their respective controls. These negative effects were concentration dependent. Keywords: Phenolic compounds, growth, pigments and metabolic activities 200 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 104. EFFECT OF TWO ETHYLENE INHIBITORS ON THE GROWTH OF REPLANTING Rehmannia glutinosa Juan LI1*, Junyan Shen1, Jiafang DU1, Hui QI1, Fengqing Wang1, Jun wang, Xinjian CHEN1, Zhongyi ZHANG2 1. Agronomy College, Henan Agricultural University, Zhengzhou 450002, China E-mail: [email protected] 2. Institute of Agroecology, Fujian Agriculture and Forestry University, Fuzhou 350002, China ABSTRACT Continuous monoculture problem in medicinal plant culture has become a core problem that hinders the sustainable development of Chinese medicinal materials production. Information of differential expression gene in replanting Rehmannia glutinosa and their functional annotation of differentially expressed genes shows that continuous monoculture has a profound effect on expression of the genes in R. glutinosa and that signal transduction of allelochemicals may be related to the calcium signal and ethylene production. Some of our previous research indicates that calcium signal system is involved in allelochemical perceptive system of continuous cropping R. glutinosa. In order to confirm that ethylene production is also involved in the allelochemical perceptive system, successive cropping of R. glutinosa was treated with different levels of ethylene the inhibiting agents 1-MCP and AOAA in the whole growing period. The results show that, compared with the control plants, R. glutinosa treated with ethylene inhibitors have comparatively higher survival rate, biomass, root activity, SOD activity and lower relative conductivity and MDA content. It indicates that ethylene inhibiting agents such as 1-MCP and AOAA can reduce the replanting disease of R. glutinosa by conducted interference of the ethylene biosynthesis process of it. The ethylene inhibitors can affect the plant perception of allelopathic chemicals and block the abnormal expression of specific genes, thus increasing the resistance of replanting R. glutinosa. Keywords: Rehmannia glutinosa, replanting diseases, allelopathy, 1-MCP, AOAA Tel: + 86-371-63558122; fax: +86-371-63558126 201 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 105. GENERATION OF SUPEROXIDE ANION AND CHANGES IN ACTIVITY OF SUPEROXIDE DISMUTASE UNDER JUGLONE INFLUENCE IN SOME PLANTS OF CEREAL AGROSYSTEM Leszczynski Bogumil, Kozak Agnieszka, and Sytykiewicz Hubert Department of Biochemistry and Molecular Biology, University of Life Science and Humanities, Prusa 12, Siedlce, Poland, e-mail: [email protected] ABSTRACT Juglone is a Juglandaceae allelochemical that stimulates generation of free oxygen radicals including superoxide anion (O2•−) and promotes oxidative burst within plant tissues. Such molecular modifications result in inhibition of seed germination and seedling growth of various plant species. The purpose of the study was to determine the level of superoxide anion and the activity of superoxide dismutase under juglone influence in seeds of some cereals and accompanied weeds. Experiments were carried out on seeds of the following monocots: spring wheat (Triticum aestivum L.), spring triticale (×Triticosecale Wittm. ex A.Camus), barley (Hordeum vulgare L.) and oat (Avena sativa L.), as well as seeds of dicotyledonous weeds: corn poppy (Papaver rhoeas L.), common corn cockle (Agrostemma githago L.) and cornflower (Centaurea cyanus L.). The seeds were treated with juglone at concentrations ranged from 0,001 to 10 mM. Then the measurements were taken in suitable intervals of time while the first eight days of the experiment. Level of superoxide anion within the seeds was determined using the method of Chaitanya and Naithani (1994), instead activity of the superoxide dismutase was measured by the method of Elstner and Heupel (1976). Juglone at the tested concentrations caused an increase in generation of O2•− within seeds of the tested monocots, and generally the highest increase was found at 10 mM juglone treatment. The lowest level of O2•− occurred in kernels of triticale, followed by the highest activity of superoxide dismutase. On the most occasions, juglone caused an increase in activity of the antioxidative enzyme in kernels of the cereals. Seeds of the dicotyledous weeds generated approximately two times more O2•− than the cereal kernels. Among tested weeds the highest level of the superoxide anion was found within seeds of common corn cockle treated with 0,001 mM juglone. Generally, the tested seeds of dicotyledous weeds were characterized by much lower activity of the superoxide dismutase, with exception of cornflower. In addition, juglone induced expression of genes encoding detoxifying enzymes, involved in neutralization of the free oxygen radicals within plant tissues. Summing up, dicotyledonous weeds of cereal agrosystems were more sensitive to juglone than the cereals. The studied monocots were also much better adapted to neutralize the superoxide anion by the superoxide dismutase than the monitored weeds. Keywords: juglone, SOD 202 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 106. BENZOXAZOLIN-2(3H)-ONE (BOA) INHIBITS SOYBEAN GROWTH AND ALTERS THE LIGNIN MONOMER COMPOSITION Ângela Valderrama Parizotto, Jennifer Munik Bevilaqua, Gisele Adriana Bubna, Osvaldo Ferrarese‐Filho and Maria de Lourdes Lucio Ferrarese* Laboratory of Plant Biochemistry, University of Maringá, Av. Colombo, 5790, 87030-900, Maringá, PR, Brazil; e-mail: [email protected] ABSTRACT Benzoxazolin-2-(3H)-one (BOA) has been investigated for phytotoxic effects in monocot and dicot weeds and crops. Multiple metabolic events in BOA toxicity have been suggested to explain its inhibitory effect on plant growth. However, no investigation has been carried out about the effects of BOA on soybean lignification. So, we evaluated the effects of BOA on seedlings growth, lignin content and its monomers p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) in roots, stems and leaves of soybean. To this end, 3-day-old seedlings were cultivated in nutrient solution (pH 6.0) in a growth room (25°C, 14-h light/10-h dark photoperiod, irradiance of 400 µmol m-2 s-1) for 10 d. After, seedlings were treated with or without 0.1 to 0.4 mM BOA for 5 d. Lignin content was evaluated by spectrophotometry while monomers were determined by HPLC. BOA decreased the lengths and fresh weights of roots and stems, and the fresh weights and areas of leaves. Reductions in seedling growth were accompanied by enhanced lignin content in all tissues. In roots, BOA increased the content of H, G and S monomers as well as the overall amount of lignin (H+G+S), but did not alter the S/G ratio. In stems and leaves, BOA increased the H, G, S and H+G+S contents while decreasing the S/G ratio. In brief, our results suggest that BOA-induced inhibition of soybean may be due to excessive production of monomers that increase the degree of polymerization of lignin, limit cell expansion, solidify the cell wall and restrict seedling growth. Keywords: allelochemical, benzoxazolinones, lignin monomers, plant growth Tel: +55 44 30114717; fax: +55 44 30114714 203 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 107. CHANGES OF PHYSIOLOGICAL PARAMETERS IN CONTINUOUSLY CROPPED EGGPLANT UNDER RELAY INTERCROPPING WITH GARLIC Mengyi Wang 1 and Zhihui Cheng 2* College of Horticulture, Northwest A&F University, Yangling, Shaanxi 712100, China 1 e-mail: [email protected] 2* e-mail: [email protected] ABSTRACT Continuous monocropping leads to continuous cropping obstacles, causing injury to plants. This study was conducted to determine whether relay intercropping with normal or green garlic protects eggplant from these injuries. The malonaldehyde (MDA) content and the activity of resistance-related enzymes, including superoxide dismutase (SOD), peroxidase (POD), polyphenol oxidase (PPO), and phenylalanine ammonia-lyase (PAL), were investigated in leaves from eggplants under plastic tunnel cultivation from the beginning of 2011 to the end of 2012. Three treatments were replicated thrice each (randomized block design): eggplant monoculture (CK), eggplant under relay intercropping with normal garlic (NG), and eggplant under relay intercropping with green garlic (GG). The overall MDA content first increased and then decreased in both years and was higher in 2012 than in 2011, but the MDA contents in the NG and GG treatments were often significantly lower than that in the CK treatment. The SOD and POD activities of the NG and GG plants were generally lower than those of the CK plants for both years. The trends for the levels of PPO activity were generally opposite those for POD because the two enzymes act synergistically and offset each other. The overall variation of PAL was similar to that of MDA, and there were no strong differences among the treatments. The studied enzymes work to maintain normal eggplant growth under relay intercropping systems. Eggplant relay intercropping with normal or green garlic is a beneficial cultivation practice, promoting a healthy eggplant crop and alleviating continuous cropping obstacles. Keywords: Eggplant; garlic; relay intercropping; malonaldehyde; resistancerelated enzyme Tel: + 86-29-87091332; fax: +86-29-87082613 204 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 108. PHYTOTOXICITY OF THE ESSENTIAL OIL COMPONENT CITRAL DETECTED BY CHLOROPHYLL A FLUORESCENCE IMAGING Elisa Graña, Carla Díaz‐Tielas, Adela M. Sánchez‐Moreiras and Manuel J. Reigosa Faculty of Biology, University of Vigo, Campus Lagoas-Marcosende s/n E-36310 Vigo, Spain; e-mail: [email protected] ABSTRACT Citral (3,7-dimethyl-2,6-octadienal) is a monoterpene commonly found as volatile component of essential oils in different aromatic plants1. Although it has a demonstrated phytotoxic activity on seedling metabolism2,3, nothing is known to date about its effects on adult plant metabolism. In order to establish its phytotoxic potential, adult Arabidopsis thaliana plants were treated for 21 days (spraying or watering) with 0, 300, 600 and 1200 µM citral concentrations. Chlorophyll a fluorescence was monitored every 2 days for the 21 days of treatment and post-harvest analyses were done on the citraltreated and untreated leaves at the end of the treatment. Clear morphological differences were observed among concentrations and among application ways. Results showed a decrease in effective PSII quantum yield for the two treatments, which was confirmed by an increase in φ(NO) (quantum yield of non-regulated energy dissipation). Spraying treatment affected also Fv/Fm (maximal PSII quantum yield) and electron transport rate (ETR) in a very significant way, suggesting the damage at the antenna complex. The results of photosynthetic activity, decrease of growth rate and pigment content and increase of total proteins suggests a general reduction of metabolism in citral watered-plants, while the results obtained for spraying could be suggesting more direct by-contact damage, likely causing alterations in the plant oxidative status4. In conclusion, citral appears as a promising plant growth regulator and a potential bioherbicide. 1: Djorjdevic D et al. (2008), Food Chem 106: 698-705. 2: Graña E et al. (2013) J Chem Ecol 39: 271-82. 3: Chaimovitsh D et al. (2010) Plant J 61: 399-408. 4: Graña E et al. (2013) Weed Sci 61: 469-81. Keywords: citral, weed control, chlorophyll a fluorescence phytotoxicity Tel: +34 986 818 714; fax: +34 986 812 556 205 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 55. EFFECT OF JUGLONE ON GENERATION OF HYDROGEN PEROXIDE AND CATALASE ACTIVITY IN SEEDS OF SOME PLANT SPECIES Kozak Agnieszka, Sytykiewicz Hubert, and Leszczynski Bogumil Department of Biochemistry and Molecular Biology, University of Life Science and Humanities, Prusa 12, Siedlce, Poland, e-mail: [email protected] ABSTRACT The first step of neutralization of the superoxide anion (free oxygen radical) is based on reaction catalyzed by superoxide dismutase. The antioxidative enzyme converts the superoxide anion to hydrogen peroxide (H2O2) that is also highly toxic to numerous plant species. In further metabolic pathway it’s degraded by catalase to water molecule and oxygen. The research presented here reports on level of hydrogen peroxide and activity of catalase in seeds of some plants of cereal agrosystems under juglone treatment. The experiments were carried out on seeds of monocots: spring wheat (Triticum aestivum L.), spring triticale (×Triticosecale Wittm. ex A.Camus), barley (Hordeum vulgare L.) and oat (Avena sativa L.), as well as seeds of dicotyledonous weeds: corn poppy (Papaver rhoeas L.), common corn cockle (Agrostemma githago L.) and cornflower (Centaurea cyanus L.). The seeds were treated with juglone at concentrations ranged from 0,001 to 10 mM, and measurements were taken in suitable intervals of time, after 1st, 2nd, 3rd and 8th day of the experiment. Hydrogen peroxide was determined by the method of Patterson at al. (1984) with slight modifications, instead catalase activity was measured following the method of Beers and Sizer (1952). Obtained results showed that kernels of the tested monocots generated much lower concentrations of H2O2 under the applied juglone doses, in comparison to seeds of the dicotyledonous weeds. Generally, higher concentration of the allochemical caused stronger induction of H2O2 generation. Among studied cereals, the highest catalase activity was observed for wheat kernels. On the most occasions the lowest levels of juglone inhibited activity of the enzyme. The catalase activity fluctuated in seeds of dicotyledonous weeds under juglone treatment. It was pretty clear in case of corn poppy and cornflower, instead within seeds of common corn cockle a strong induction of the enzyme was found. In conclusion, the juglone caused generation of hydrogen peroxide and modified activity of catalase within the studied seeds of monocots and dicotyledones. Results of the study showed that cereal monocots are less sensitive to the allelochemical than the dicotyledonous weeds. Thus it might be possible to control such weeds with juglone preparation in the future. Keywords: allelopathy, juglone, ROS 206 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 56. THE ALLELOCHEMICAL L-DOPA AFFECTS THE UPTAKE AND ASSIMILATION OF NITRATE AND AMMONIUM IN SOYBEAN Tiara da Silva Bortolo‐Coelho, Rogério Marchiosi, Joselaine Viganó and Osvaldo Ferrarese‐Filho* Laboratory of Plant Biochemistry, University of Maringá, Av. Colombo, 5790, 87030-900, Maringá, PR, Brazil; *e-mail: [email protected] ABSTRACT Velvetbean (Mucuna pruriens) is widely used in tropical regions as a soilimproving crop, a cover crop to control weeds, green manure, and a forage plant. Many secondary compounds are produced by velvetbean, but the nonprotein amino acid L-DOPA (L-3,4-dihydroxyphenylalanine) is its main allelochemical. Reduction of plant growth by L-DOPA has been reported in several plant species, but their effects on nitrogen metabolism in soybean (Glycine max L. Merrill) are poorly understood. In the current research, we investigated the effects of L-DOPA on the uptake of nitrate and ammonium, contents of nitrate, ammonium and amino acids in roots, and activity of nitrate reductase in leaves of soybean. For this, seeds were dark-germinated (at 25°C) on two sheets of moistened filter paper. Three-day-old seedlings were supported on adjustable acrylic plate and dipped into an 10×16 cm glass container filled with nutrient solution. The container was kept in a growth room at 25°C, with a light/dark photoperiod of 14/10 h and a photon flux density of 400 µmol m−2 s−1. After 10 and 12 days, L-DOPA (0.25 mmol L-1) was added to the nutrient solution. All parameters were evaluated on the 14th day of cultivation. In roots, the uptake of nitrate and ammonium and contents of nitrate and amino acids decreased whereas ammonium levels increased after L-DOPA treatments. In leaves, the nitrate reductase activity decreased after L-DOPA exposure. Taken together, the present findings suggest a strong influence of LDOPA on the uptake and assimilation of nitrate and ammonium in soybean seedlings. Keywords: nitrogen assimilation, ammonium uptake, soybean L-DOPA, nitrate reductase, nitrate and Tel: +55 44 30114717; fax: +55 44 30114714 207 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 57. EFFECTS OF HYDROMETHANOLIC EXTRACTS OF PEARL MILLET ON WILD POINSETTIA Hingrid A. da Silva, Rogério Marchiosi, Rita de Cássia Siqueira‐Soares, Graciene de Souza Bido, Maria de Lourdes L. Ferrarese and Osvaldo Ferrarese‐Filho* Laboratory of Plant Biochemistry, University of Maringá, Av. Colombo, 5790, 87030-900, Maringá, PR, Brazil; *e-mail: [email protected] ABSTRACT Wild poinsettia (Euphorbia heterophylla), a well-known weed distributed in tropical and subtropical areas of the world, has a high potential to reduce the productivity of many crops. Due to its herbicide resistance, an alternative management by using allelopathic plants has been recently adopted. Because pearl millet (Pennisetum glaucum) affects the growth of some plants species, we have evaluated its effects on wild poinsettia. For this, seeds of wild poinsettia were dark-germinated (at 25°C) on two sheets of moistened filter paper. Five-day-old seedlings were supported on adjustable acrylic plate and dipped into an 10×16 cm glass container filled with 250 mL of nutrient solution with or without 250 to 1000 mg L-1 hydromethanolic extracts of pearl millet´s shoots. The container was kept in a growth chamber (25 °C, 14-h light/10-h dark photoperiod, irradiance of 400 µmol m-2 s-1) for 15 d. At the end, the plant growth, oxidative stress (lipid peroxidation and soluble peroxidase activity), activities of enzymes (phenylalanine ammonia-lyase and cell wall-bound peroxidase), root cell viability and apex radicular scanning electron microscopy were evaluated. Overall, the results showed that hydromethanolic extracts 1) reduced root and stem growths, fresh and dry weights and leaves areas; 2) decreased activities of phenylalanine ammonia-lyase and cell wall-bound peroxidase and 3) reduced root elongation, damaged plant root and increased cell mortality. In brief, these results suggest that hydromethanolic extract of pearl millet has a high potential to reduce the wild poinsettia growth. Isolation and identification of allelochemicals in the hydromethanolic extract are in progress. Keywords: allelochemical, Euphorbia heterophylla, Pennisetum glaucum Tel: +55 44 30114717; fax: +55 44 30114714 208 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 58. ALLELOPATHIC POTENTIIAL OF Casuarina equisetifolia J R ON GERMINATION GROWTH OF GROUND NUT (Arachis hypogae Willd - VRI -2) Natarajan S Department of Plant Biology & Plant Biotechnology, Guru Nanak College, Velachery, Chennai- 42,Tamil Nadu, India Email: [email protected] ABSTRACT Allelopathy is the biochemical interaction (stimulatory and inhibitory) among plants. Allelochemicals refers mostly to the secondary metabolites produced by plants and are byproducts of primary metabolic process. They have an allelopathic effect on the growth and development of the same plant or neighboring plant. In the present investigation, the allelopathic effect of the root and leaf powder extract of Casuarina equisetifollia J. R. on the germination and growth of Arachis hypogaea Willd, were studied. The 50 grams powder of leaf and root (Casuarina) were soaked in 500ml of distilled water. After 24 hours, they were filtered with the help of Whatman No:1 filter paper and from this pure extract of 0, 5%, 10%, 25%, 50% and 100% of various extracts were prepared by using distilled water and used in the present study. The germination, growth studies were conducted with groundnut (VRI-2) and fresh weight, dry weight, bio chemical studies like chlorophyll, protein and reducing sugar content were analyzed. Allelopthic effect of leaf extract on ground nut are higher in 25% and 50% of leaf extract of Casuarina in all parameters. The effect of aqueous root extract of Casuarina on groundnut was higher in 5% concentration and stimulatory effect was observed in all parameters in lower concentration and inhibitory effect was observed in higher concentration. Moreover, leaf extract have stimulatory effect up to 50% concentration level and root extract show 5% level as stimulatory effect and higher concentration no germination was observed. Finally concluded that the lower concentration of leaves and root extract shows stimulatory effect and higher concentration shows inhibitory allelopathic effects. Keywords: Allelopathy, Allelochemicals, Casuarina equisetifollia, Arachis hypogaea, stimulatory and inhibitory Willd. TEL: +91 9444001541, +91 9840867232 209 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 59. PHYTOTOXIC ACTIVITY OF THE SECONDARY METABOLITE TRANSCHALCONE IN Arabidopsis thaliana ADULT PLANTS Carla Díaz‐Tielas, Elisa Graña, Ana Martínez‐Peñalver, Manuel J. Reigosa, Adela M. Sánchez‐Moreiras. Faculty of Biology, University of Vigo. Campus Lagoas-Marcosende, 36310, Vigo, Spain; e-mail: [email protected] ABSTRACT The flavonoid chalcone (1,3-diphenyl-2-propen-1-one) is known to show many biological activities as anti-bacterial, anti-malarial, anti-cancer, etc., and has been found to be strongly phytotoxic on Arabidopsis thaliana seedlings1. However, little is known up to now about the phytotoxic potential of chalcone on adult plant metabolism. Therefore, in this study, Arabidopsis adult plants were treated with chalcone (0, 100, 200 and 400 µM) either by watering or by spraying, and damage was measured by monitoring fluorescence quenching during 3 weeks every 2 days. At the end of the treatment, dry and fresh biomass, elemental analysis, pigment concentrations, total protein content, and lipid peroxidation were determined. Since the 11th day of treatment, both sprayed and watered plants showed significantly smaller φII values, decreased ETR, and greater φNPQ and φNO than in controls. Fv/Fm fluorescence images of sprayed plants showed, in addition, damages on the photosynthetic apparatus at the leaf margins, but not at the vascular bundles or the young leaves. Chalcone treatment increased pigment (chlorophylls a and b and carotenoids) concentrations, but was detrimental to the development of Arabidopsis plants, as confirmed by fresh and dry weight (20-40% of the control). The Ca2+ and Zn2+ levels of watered plants fell (~60-70% of the control), and also total protein content (~80%), suggesting an ion-selective effect on nutrient uptake by the roots, which lead to decreased protein production in watered plants. However, malonyldialdehyde content significantly increased in sprayed plants, indicating oxidative damage on treated plants. These results confirm the phytotoxic effects of trans-chalcone on Arabidopsis adult plants, and show differential mode of action depending on the administering way, spraying or watering. 1 Díaz-Tielas C, Graña E, Sotelo T, Reigosa MJ, Sánchez-Moreiras AM (2012) Plant Cell Environ 35: 1500-1517. Keywords: trans-chalcone, Arabidopsis thaliana, phytotoxicity, mode of action Tel: +34986812616; fax: +34986812556 210 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 60. THE STRONG PHYTOTOXIC EFFECT OF THE ALKALOID NORHARMANE ON Arabidopsis ADULT PLANTS Elisa Graña, David Ledo, David López‐González, Aitana Costas‐Gil, Manuel J. Reigosa and Adela M. Sánchez Moreiras Faculty of Biology, University of Vigo, Campus Lagoas-Marcosende s/n E-36310 Vigo, Spain; e-mail: [email protected] ABSTRACT Norharmane (9H-pyrido(3,4-b)indole) is an indol alkaloid commonly excreted as active exometabolite by some cyanobacteria as Nodularia harveyana.1 Its antibacterial, antifungal and algicide activities have been already described,2 showing a possible applicability as antifouling agent; but nothing is known up to now about its effects on plant metabolism. Therefore, the aim of this work was to determine the phytotoxic potential of norharmane on the model species Arabidopsis thaliana. A dose-response curve (from 0 to 1200 µM) was carried out in previous studies3 to establish the IC50 and IC80 concentrations for A. thaliana 10 days-old seedlings in 62 and 153 µM, respectively. Considering this strong effect, adult plants were treated with 153, 306 and 612 µM norharmane solutions for 21 days (spraying or watering), by everyday monitoring of chlorophyll a fluorescence with a Maxi Imaging-PAM (Walz). Although no strong changes were found on norharmane-sprayed plants, there was a strong reduction on PSII efficiency, electron transport rate (ETR) and PSII maximum efficiency (Fv/Fm) and an increase on the energy dissipated as fluorescence (φ(NO)) on norharmane-watered plants. These results were related to a highly significant reduction of the size of the rosettes. The post-harvest analysis of Arabidopsis adult leaves after 21 days norharmane treatment revealed decreased water and osmotic potentials, DW/FW ratio, total protein content, strong anthocyanin accumulation and alterations on photosynthetic pigments content. Although more studies need to be done to clearly state the mode of action of norharmane on Arabidopsis adult plants, these results suggest that norharmane could be a promising plant growth inhibitor to be used as bioherbicide. 1: Volk R-B, Furkert FH (2005) Microbiol Res 161: 180-6. 2: Volk R-B (2006) Microbiol Res 163: 307-13. 3: Graña et al. (2013) J Chem Ecol 39: 271-82. Keywords: norharmane, phytotoxicity, Arabidopsis thaliana, chlorophyll a fluorescence Tel: +34 986 818 714; fax: +34 986 812 556 211 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 109. MOMILACTONE HIGH-SENSITIVE AND LOW-SENSITIVE MUTANTS OF Arabidopsis Hisashi Kato‐Noguchi, Tomotaka Itaya and Yasuomi Tada Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Miki, Kagawa 761-0795, Japan; [email protected] ABSTRACT We found momilactone high-sensitive and low-sensitive mutants of Arabidopsis. Large field screening programs and laboratory experiments in many countries have indicated that rice is allelopathic and releases allelochemical(s) into its environment. A number of compounds have been identified as potential rice allelochemicals. However, the labdane-related diterpenoid momilactones were reported to be the most important rice allelochemicals, with momilactone B playing a particularly critical role. In addition, genetic studies have shown that selective removal of only the momilactones from the complex mixture found in rice root exudates significantly reduces allelopathy. However, there has been no information available for the mode of action of momilactone. Therefore, we investigated the action of momilactone on the growth inhibition. Two momilactone high-sensitive and two low-sensitive mutants of Arabidopsis were identified in 14,585 SALK T-DNA insertion lines. Those mutations were related to flavonoid biosynthetic pathway. Then, the expressions of the genes involved in flavonoid biosynthesis were determined by real-time PCR. The present results indicate that momilactone inhibits the growth of Arabidopsis due to its involvement in the gene expressions in the flavonoid biosynthesis. Keywords: Momilactone, mode of action, Arabidopsis, mutant Tel: + 81-87-891-3086; fax: +81-87-891-3086 212 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 110. ALLELOPATHIC POTENTIAL OF M-TYROSINE ON TOMATO (Solanum lycopersicum L.) ROOT GROWTH IS DUE TO MODIFICATION IN ROS/RNS METABOLISM Joanna Olechowicz, Anna Antosik, Olga Andrzejczak, Wojciech Borucki, Agnieszka Gniazdowska, Urszula Krasuska, Renata Bogatek Warsaw University of Life Sciences - SGGW, Nowoursynowska 159, 02-776 Warsaw, Poland, e-mail: [email protected] ABSTRACT Allelopathy is defined as a plant interference to the surrounded environment by secondary metabolites, which finally induce modification of crucial physiological and biochemical processes in cells of acceptor plants. Meta-tyrosine (m-tyr) is a non proteinogenic amino acid identified as allelochemical synthesized by chewing fescue (Festuca rubra L. ssp. commutata) and donkey-tail spurge (Euphorbia myrsinites). This compound is considered as phytotoxin which can be used as a natural herbicide involved in weeds management. Increase of mtyr content was observed during oxidative stress in animal cells as a result of non-enzymatic oxidation of phenylalanine. Therefore, m-tyr may be a marker of oxidative stress. The aim of this study was to investigate the mode of action of m-tyr during inhibition of tomato (Solanum lycopersicum L. cv Ożarowski Malinowy) seedling root growth. Tomato seedlings were exposed to m-tyr at concentration responsible for 50% inhibition of root growth (0.050 mM) and concentration at which root growth was completely arrested (0.250 mM), but was not lethal. It was demonstrated that mtyr induced oxidative stress manifested as accumulation of reactive oxygen species (ROS; H2O2 and O2•-) and alteration in action of cellular enzymatic antioxidant system (activity of catalase, superoxide dismutase and peroxidase). It was accompanied by modifications in production of reactive nitrogen species (RNS), mostly nitric oxide (NO). Moreover, m-tyr treatment led to typical deterioration of plasma membrane manifested as increased electrolyte leakage. It may be assumed that toxicity of m-tyr may be due to alteration of nitrooxidative status of the cells leading to structural disintegration. Keywords: m-tyrosine, nitric oxide, oxidative stress, ROS, root growth Tel: +48 22-593-25-30; fax: +48-22-593-25-21 213 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 111. NON-PROTEIN AMINO ACIDS AS INHIBITORS OF ROOT GROWTH OF TOMATO (Solanum lycopersicum L.) SEEDLINGS Olga Andrzejczak, Urszula Krasuska, Paweł Staszek, Anna Antosik, Joanna Olechowicz, Renata Bogatek, Agnieszka Gniazdowska Department of Plant Physiology, Warsaw University of Life Sciences - SGGW, Nowoursynowska 159, 02-776 Warsaw, Poland; e-mail: [email protected] ABSTRACT Phytotoxic properties of various plant metabolites determine their function in ecosystems. Many non-protein amino acids play critical roles within cell structure and physiology and have been associated with allelopathic or insecticidal properties. L-canavanine (Can; structural analog of arginine), 5hydroxy-tryptophan (5-HT; analog of tryptophan) and m-tyrosine (m-tyr; analog of phenylalanine) have been demonstrated to be produced in plants. In opposite, 5-methyl-tryptophan (5-MT; synthetic tryptophan analog) is commonly used as inhibitor of anthranilate synthase, the key enzyme of tryptophan biosynthetic pathway. Plant growth and development is under control of phytohormones. Auxin, synthesized from tryptophan is a phytohormone of the main regulatory role in root architecture and gravitropic response. Auxin signaling is influenced by nitric oxide (NO), a molecule which biosynthesis is arginine dependent, if catalysed by NOS-like enzyme. The aim of this work was to investigate impact of these non-protein amino acids on growth of roots of tomato (Solanum lycopersicum L.) seedlings. Tomato seedlings were treated with Can, 5-HT, 5-MT, m-tyr at various concentration (0.05 -0.5 mM) for 3 days. All tested amino acids exhibited inhibitory effect on growth of roots. Replacement of tested chemical by its analog not always led to recovery, suggesting its direct effect on cell structure/function. Inhibition of root growth was accompanied with disturbances in gravitropic response. It correlated well with modifications in auxin level, suggesting that non protein amino acids led to disorder of plant hormonal regulation. Keywords: non-protein amino acids, root growth, auxin, Tel: +48 22-593-25-30; fax: +48-22-593-25-21 214 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 112. CANAVANINE - PHYTOTOXIC NON-PROTEIN AMINO ACID ACTS AS INHIBITOR OF APPLE EMBRYO GERMINATION BY MODIFICATION OF ROS AND RNS PRODUCTION Krasuska U., Ciacka K., Staszek P., Bogatek R., Gniazdowska A. Department of Plant Physiology, Warsaw University of Life Sciences-SGGW, Nowoursynowska 159, 02-776 Warsaw, Poland; e-mail: [email protected] ABSTRACT L-canavanine (Can), a structural analog of L-arginine (Arg), is a non-protein amino acid synthesized in jack bean and highly toxic to bacteria, fungi, plants, insects, mammals. It is mostly recognized as a chemical active in plant defense against insects, but may also play role as inhibitor of plant growth. Apple seeds are characterized by deep dormancy and do not germinate even in favorable conditions. Seed dormancy and germination is under regulation of classical phytohormones (ABA, gibberellins), plant growth regulators (polyamines) and signaling molecules such as reactive oxygen (ROS) or nitrogen species (RNS). Arg acts as substrate for nitric oxide (NO) generation in oxidative NOS-like dependent biosynthetic pathway. It is also a precursor of polyamines. The purpose of our study was to investigate the impact of Can as a antimetabolite of Arg on dormancy status and germination of apple embryos. Embryos isolated from dormant seeds were imbibed in Arg or Can solution. Arg removed dormancy and stimulated germination leading to development of seedlings without morphological abnormalities. Can strengthened embryos dormancy, abolished germination and halted greening of cotyledons. Influence of Can was comparable to negative effect of NO scavenger on embryos germination and growth of seedlings, therefore we focused on RNS production. NO and ONOO- emission by embryonic axes was reduced by Can. It was accompanied by modification in ROS production. We assume that, toxic effect of Can is due to perturbation in oxidative/nitrosative status of cells, leading to inhibition of embryo transition from dormant status to germination. Keywords: canavanine, RNS, ROS, seed dormancy, seed germination Tel: +48 22-593-25-30 ; fax: +48-22-593-25-21 215 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 113. PAST, PRESENT AND FUTURE OF BENZOXAZOLIN-2(3H)-ONE PHYTOTOXIC STUDIES Adela M Sánchez Moreiras, Ana Martínez‐Peñalver, Iftikhar Hussain and Manuel J Reigosa Faculty of Biology, University of Vigo, Campus Lagoas-Marcosende s/n E-36310 Vigo, Spain; e-mail: [email protected] ABSTRACT Benzoxazolin-2(3H)-one (BOA) is a well-known secondary metabolite, commonly found in several Poaceae species and a few dicotyledonous species belonging to the Acanthaceae, Lamiaceae, Scrophulariaceae and Ranuculaceae families. This benzoxazinone is an important compound, which has been deeply studied for the last 20 years in order to establish its mode of action, detoxification pathways and weed control potential. After testing BOA phytotoxicity on different model species, different modes of action on seedling and adult plant metabolisms have been suggested for this compound. In this way, our group has demonstrated by transcriptomic studies the phytotoxic activity and the presence of detoxification processes on Arabidopsis seedlings after BOA treatment1, and has suggested that the primary phytotoxic action of BOA could be the induction of premature senescence, with oxidative stress as a secondary effect that sets in a day or two later2,3. The new omics studies and the analyses of BOA derivatives appear to be the new promising research lines in the study of this benzoxazinone. 1 Baerson SR, Sánchez-Moreiras AM, Pedrol-Bonjoch N, Schulz M, Kagan IA, Agarwal AK, Reigosa MJ, Duke SO (2005) J Biol Chem 280: 21867-21881. 2 Sánchez-Moreiras AM, Reigosa MJ (2005) J Chem Ecol 31: 2689-2703. 3 Sánchez-Moreiras AM, Martínez-Peñalver A, Reigosa MJ (2011) J Plant Physiol 168: 863-870. Keywords: benzoxazinone, mode of action, -omics, BOA derivatives TEL: +34 986 818 714; FAX: +34 986 812 256 216 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 61. MODE OF ACTION OF THE SECONDARY METABOLITE TRANSCHALCONE IN Arabidopsis thaliana SHOOTS: LATE AND EARLY EFFECTS Carla Díaz‐Tielas, Elisa Graña, Ana Martínez‐Peñalver, Aitana Costas‐Gil, Manuel J. Reigosa, Adela M. Sánchez‐Moreiras Faculty of Biology, University of Vigo. Campus Lagoas-Marcosende, 36310, Vigo, Spain. e-mail: [email protected] ABSTRACT The flavonoid chalcone (1,3-diphenyl-2-propen-1-one) is a plant secondary metabolite known to show many biological activities as anti-bacterial, antimalarial, anti-cancer, etc. Recently, trans-chalcone has been found to induce programmed cell death on Arabidopsis thaliana roots after 35 µM (IC50) treatment1. Due to the strong phytotoxicity detected on roots, more studies were carried out on A. thaliana aerial parts under IC50 chalcone concentration. A strong albinism of the shoots of Arabidopsis seedlings was detected after 14 days chalcone treatment. Electron microscopy revealed important alterations on chloroplasts, as thylakoids dilation and disintegration. Also, a strong reduction of chlorophylls a and b, zeaxanthin, violaxanthin, and β-carotene contents were detected by HPLC, suggesting the presence of incomplete or malformed photosynthetic apparatus. All these alterations, together with a pronounced H2O2 production resulted in a significant increase of death cells in the aerial parts, revealed by trypan blue staining. When the seedlings were grown in sodium-supplemented medium, the shoots showed less degreening and a significant increase of all photosynthetic pigments, suggesting a dysfunction of the membrane pumps. This was confirmed by strong membrane potential depolarization, which was detected already 30 min after chalcone treatment. Furthermore, PIN-3 Arabidopsis mutants revealed an early effect of chalcone stimulating PIN-3 expression in roots, results linked with auxin and ethylene imbalance, both in treated roots and shoots. These results confirm the strong phytotoxic activity of chalcone on Arabidopsis seedlings, and reveal an early effect of this compound on membrane potential and auxin transport. Keywords: allelopathy, substance, root exudates seed germination, Galinsoga ciliata, secretion Tel: 86+153-3172-7676; fax: 86-871-65228581 217 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 62. PHYTOTOXICITY OF CYANAMIDE ON TOMATO AND MAIZE ROOT TIP – DISTINCT SPECIES SIMILAR RESPONSES Dorota Soltys1,2 Anna Rudzińska‐Langwald3, Renata Bogatek2, Agnieszka Gniazdowska2 1 Plant Breeding and Acclimatization Institute-National Research Institute, Młochów Research Center; [email protected] 2 Department of Plant Physiology, Warsaw University of Life Sciences‐SGGW, Nowoursynowska 159, 02‐776 Warsaw, Poland 3 Department of Botany, Warsaw University of Life Sciences-SGGW, Nowoursynowska 159, 02776 Warsaw, Poland ABSTRACT Cyanamide (CA) is a natural compound produced by vetch species and black locust (Robinia pseudoacacia L.). It was found that CA may act as allelopathic agent. We have shown that CA inhibits plant growth, especially root growth. This effect may be a consequence of root tip disorder. Based on our previous experiments we have compared phytotoxic effects of CA on dicot – tomato (Solanum lycopersicum L.) and monocot – maize (Zea mays L.) root tip function. CA has inhibited post-germination growth of tomato and maize root after 3 days of culture. However, 50% inhibition of root growth has been reached at 1.2 mM CA for tomato and 3 mM for maize. In tomato, as well as in maize CA decreased number of dividing cells and induced modification in root tip organization due to earlier cell differentiation. Moreover, CA significantly reduced number of border cells and columella cells of root cap. Apart from more frequent vacuolization, CA did not induce any significant changes in the organelle structure of other root cells. This comparison shows that maize is less sensitive to CA treatment then tomato. Both species characterizes similar physiological response, however at various doses of CA. Keywords: allelopathy, vacuolization, cell differentiation, Tel: + 48 22 729 92 48; fax: +48 22 729 92 47 218 cyanamide, phytotoxicity, 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 114. DETOXIFICATION OF CRUCIFEROUS PHYTOALEXINS AND AROMATIC GLUCOSINOLATES BY PHYTOPATHOGENIC FUNGI M. Soledade C. Pedras,* Abbas Abdoli, and Sajjad Hossain Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, SK, Canada; e-mail: [email protected]; e-mail: [email protected]; [email protected]; [email protected] ABSTRACT Crucifers such as canola, mustard, and cauliflower respond to stress employing metabolic pathways that involve the biosynthesis of numerous and structurally diverse natural products, including phytoalexins and phytoanticipins. These plant metabolites participate in responses to stress caused by pathogens, metal salts, UV radiation, etc. Detoxification of phytoalexins and phytoanticipins by pathogens deprives the plant of useful chemical defenses, making them more susceptible to microbial diseases. The phytoalexins brassinin, cyclobrassinin and camalexin are important antimicrobial metabolites produced de novo by cruciferous plants. Glucosinolates are phytoanticipins derived from different amino acids including phenylalanine and tryptophan also produced by crucifers. To better understand the interaction of fungal pathogens with their host plants, the metabolism of the phytoalexins brassinin, cyclobrassinin and camalexin and indolyl and benzyl glucosinolates with the fungal pathogens Alternaria brassicicola, a selective fungal pathogen that infects stems, leaves and pods of Brassica species and Sclerotinia sclerotiorum, a generalist pathogen that infects above ground organs of many plant species, was analyzed. It was discovered that the phytoalexins were detoxified by both fungal species, while only A. brassicicola metabolized benzyl glucosinolate and indolyl glucosinolates were not transformed by any of the pathogens. This work demonstrated that A. brassicicola was able to detoxify camalexins through a pathway similar to that followed by the generalist pathogen Botrytis cinerea but different from S. sclerotiorum. Details of this work will be presented, pathways will be proposed and implications in plant disease resistance will be discussed. Keywords: Alternaria brassicicola, brassinin, camalexin, cyclobrassinin, phytoalexin 219 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 115. THE REDOX-NETWORK OF Zea mays BOA DETOXIFICATION: A ROOT – MICROBE COOPERATION Margot Schulz1*, Barbara Filary1, Dieter Sicker2, Lothar Hennig2, Tom Colby3, Anne Harzen3, Jürgen Schmidt3 and Diana Hofmann4 1 IMBIO Institute Molecular Biotechnology of Plants, University of Bonn, Karlrobert Kreiten Str. 13, 53115 Bonn, Germany; e-mail:[email protected] 2 Institute for Organic Chemistry, University of Leipzig, Johannisallee 29, 04103 Leipzig, Germany; e-mail: [email protected] 3 Max-Planck-Institut für Züchtungsforschung, Carl von Linné Weg 10, 50829 Köln, Germany e-mail: [email protected] 4 Forschungszentrum Jülich IBG-3, Wilhelm-Johnen-Straße, 52428 Jülich, Germany; e-mail: [email protected] ABSTRACT In Zea mays, (auto)toxification by the allelochemical benzoxazolinone is first avoided by glucosylation of BOA-6-OH and subsequently by glucoside carbamate and malonylglucoside carbamate synthesis. UGT BX9 is responsible for the BOA-6-OH glucosylation step, but its function in glucoside carbamate synthesis was unclear. We assumed that BOA is not the direct substrate for glucoside carbamate synthesis and that BX9, although involved in the process, is not directly responsible for the glucosylation step. The maize endophyte Fusarium verticillioides was isolated and incubated with BOA under suboptimal oxygen supply. Under these conditions, a BOA-like compound (BC) accumulated which is the assumed precursor of the fungal and plant detoxification products, oHPMA and glucoside carbamate. Apoplast-located endoglucanases, known to possess trans-glucosylation activity participate in glucoside carbamate synthesis from precursor compounds and have probably also a function in the release from cell wall fixed N-glucosylated benzoxazolinones in maize seedlings. The switch from BOA-6-O-glucoside to glucoside carbamate production is redox-sensitive, involves Fenton reactions, oxidative cleavage of BX9 to shut down BOA-6-O-glucosylation and the participation of several compartments. The glucoside carbamate detoxification pathway is complected with Fusarium verticillioides, since the endophyte can release BOA from glucoside carbamate. Therefore, detoxification products fixed in the cell wall can serve as an allelochemical depot. Our results are in agreement with the idea that pathogenic fungi can have a mutualistic lifestyle. Keywords: BOA-detoxification, Zea mays, endophyte Fusarium verticillioides Tel: + 49-(0)228-732151; fax: + 49-(0)228-732151 220 Session 8… Genetics of allelopathy and breeding for allelopathy Essential oils and volatiles Plant – other organisms interactions Abiotic stress, tolerance, infections, other uses of allelopathy 221 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 116. PRELIMINARY STUDIES ON GENES REGULATION IN RICE ALLELOPATHIC POTENTIAL 1 Jian Fu 1 Yiding Sun 1 Yiqing Guo 2* Yongliang Lu 3 Biotechnology & Genetic Germplasm Resources Institute, Yunnan Academy Of Agricultural Sciences, Yunnan Province of China 650223, e-mail: [email protected] 2 Yunnan Agricultural University, Kunming, Yunnan Province of China 650201 e-mail: [email protected] 3 China National Rice Research Institute, Fuyang, Zhejiang Province of China 311400, e-mail: [email protected] ABSTRACT Allelopathy is defined as process involving secondary metabolites produced by plant, algae, bacteria, and fungi that influence the growth and development of agricultural and biological systems. The process of allelopathy is regulated by many genes such as signaling, allelochemicals synthesized and metabolited by plant. Phenolics, terpenoids and steriods were repoted as the most important allelochemicals of rice varities. Identification of genes in regulation of these allelochemicals is necessary and theorical meaning to understand the mechanism of allelopathy in rice. With strong allelopathic potential to some species of weeds, PI312777 was known as a standard material for evaluating rice lines, and also as a good material to carry out the further researches on relative genes identification and expression. In this research, PI312777 and Lemont are used as material of allelopathic and non-allelopathic rice variety, and affected by artificial stress, and then RNA extracted. The techniques of gene chip and bio-informatics were used to analyze and select the relative genes in regulation of rice allelopathy according to reported types of allelochemicals. Preliminary results showed that there are around 8060 genes involved in response to the stress condition, in which there are 4970 and 3080 genes upregulated and downregulated respectively, and around 40 genes related to phenolics and steroids synthesis and metabolited. The findings suggested that phenolic acids and steroids are the most important chemicals in rice allelopathic potential. And the results provide a theorical basis for identify or use the genes and well understand the mechanism in rice allelopathy. Keywords: Rice, allelopathy to weed, genes related to allelochemicals Correspondent: Yiqing Guo. e-mail: [email protected], Tel: +86-871-65228581; Fax: +86-87165227145 Tel: +86-871-65111863; Fax:+86-871-65151112 (Jian Fu and Yiding Sun) 222 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 63. BIOSYNTHESIS AND GENE REGULATION OF BENZOXAZINOIDS DURING GRAIN DEVELOPMENT IN RYE Fariha Tanwir 1, Inge S. Fomsgaard 2 and Per L. Gregersen 3* 1 2 Department of Molecular Biology and Genetics, Aarhus University, Forsøgsvej 1, DK4200 Slagelse, Denmark; e-mail: [email protected] Department of Agroecology, Aarhus University, Forsøgsvej 1, DK-4200 Slagelse, Denmark; email: [email protected] 3 Department of Molecular Biology and Genetics, Aarhus University, Forsøgsvej 1, DK-4200 Slagelse, Denmark; e-mail: [email protected] ABSTRACT Benzoxazinoids is a group of plant secondary metabolites having allelopathic properties and found in grasses, e.g. maize, wheat, rye and wild barley, as well as in some dicots. Studies have also shown a possible role of these compounds in health promotion, i.e. in reducing cancer and obesity. These allelopathic and health-promoting properties of benzoxazinoids point to more detailed investigations of gene regulation and biosynthesis in relation to accumulation of the compounds during different stages of plant growth. Rye was chosen because it is an important crop used as green manure during young growth stages, and because rye grains also are a major part of food in Northern Europe. Secondly, rye grains have higher benzoxazinoid contents compared to wheat. For investigating the gene regulation and biosynthesis of these secondary metabolites, samples were collected from field-grown rye during grain development, starting from anthesis until grain filling and maturity. qRTPCR was performed to estimate the regulation of genes involved in benzoxazinoid biosynthesis in rye, whereas LCMSMS analysis was performed to quantify their chemical contents. Our results showed the accumulation of DIBOA-glc-hexose as a major form of di-glycosylated hydroxamic acid during grain filling. Biosynthesis and accumulation of HBOA and DIBOA was also under corresponding gene regulation. A detailed overview of benzoxazinoid accumulation and transformation under genetic control will be discussed. Keywords: benzoxazinoid hydroxamic acid biosynthesis, rye qRT-PCR, diglycosylated Tel: +45 87158212 / *+45 87158246; fax: +45 87156082 223 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 117. EXPLORING OPTIMIZATION OF WEED SUPPRESSION, YIELD, AND BIOTIC STRESS TOLERANCE IN AN ALLELOPATHIC X NON-ALLELOPATHIC RICE MAPPING POPULATION David Gealy1, Yulin Jia2, and Shannon R.M. Pinson3 1 Dale Bumpers National Rice Research Center, USDA, Agricultural Research Service, 2890 Hwy 130 East, Stuttgart, AR 72160, USA; [email protected]: 2 Dale Bumpers National Rice Research Center, USDA, Agricultural Research Service, 2890 Hwy 130 East, Stuttgart, AR 72160, USA; [email protected]: 3 Dale Bumpers National Rice Research Center, USDA, Agricultural Research Service, 2890 Hwy 130 East, Stuttgart, AR 72160, USA; [email protected]: ABSTRACT Indica rice cultivars such as PI 312777 can be highly productive as well as suppressive to barnyardgrass (Echinochloa crus-galli) in drill-seeded systems in the US. To understand the molecular basis of the important genes underlying crop productivity, weed suppression, and disease resistance, a recombinant inbred line (RIL) population was developed using single seed descent from a cross between ‘Katy’, a moderate-yielding, non-weed-suppressive tropical japonica, and PI 312777, a high-yielding allelopathic indica. A mapping population of 350 RILs (presently F6 generation) has been developed. Phenotypic traits are being evaluated for: rice emergence and growth rates; number, timing, and angle of tillers produced; leaf area; height and yield; root architecture, allelopathic activity, and weed suppression; and blast disease reactions. Greenhouse experiments are ongoing, and field and high-throughput growth chamber experiments will be conducted 2014-2016. Important genomic regions associated with the evaluated traits, will be established by associating phenotypic data and genotypic data generated using Genotyping by Sequencing or other appropriate genotyping methods. This mapping population, with the accompanying genetic and phenotypic information, will provide a foundation for association mapping, understanding the genetic basis of beneficial traits for yield, crop protection, and insights into optimizing yield traits with key components of weed-suppressive traits, and disease resistance. Keywords: mapping population, recombinant inbred line (RIL), allelopathy, weed suppression, rice blast disease, tillering, genotyping by sequencing (GBS), Oryza sativa, rice, Echinochloa crus-galli, barnyardgrass. Tel: + 870-672-9300; fax: + 870-673-7581 224 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 118. ROOT DISTRIBUTION AND INTERACTIONS BETWEEN ALLELOPATHIC RICE AND C4 GRASS WEED SPECIES AS DETERMINED BY 13C ISOTOPE DISCRIMINATION ANALYSIS David R. Gealy1, Karen A. Moldenhauer2, and Sara Duke3 1 Dale Bumpers National Rice Research Center, USDA, Agricultural Research Service, 2890 Hwy 130 East, Stuttgart, AR 72160, USA; [email protected]: 2 Dale Bumpers National Rice Research Center, USDA, Agricultural Research Service, 2890 Hwy 130 East, Stuttgart, AR 72160, USA; [email protected]: 3 Dale Bumpers National Rice Research Center, USDA, Agricultural Research Service, 2890 Hwy 130 East, Stuttgart, AR 72160, USA; [email protected]: ABSTRACT Cultivars which carry traits that enable them to suppress weeds could improve the economical management and sustainability of rice production. Interactions between roots of rice and weeds are thought to be modulated by the weedsuppressive activity of some rice cultivars, but these phenomena are difficult to measure and not well understood. Above-ground crop-weed interactions and root distribution of PI 312777 and Taichung Native 1 (TN-1) (rice cultivars known to release weed-suppressive allelochemicals from roots), breeding selections, and non-suppressive commercial cultivars, and the C4 grass weed species, sprangletop (Leptochloa fusca (L.) Kunth var. fascicularis (Lam.) N. Snow) and barnyardgrass (Echinochloa crus-galli (L.) Beauv.), were evaluated in field plots at Stuttgart, Arkansas, USA in a two-year study. The allelopathic cultivars and other weed-suppressive cultivars produced more tillers and suppressed both weed species to a greater extent than other cultivars. 13C isotope discrimination analysis of intermixed root samples showed that the allelopathic cultivars produced a greater fraction of their total root mass in the upper 5 cm of soil compared to the non-suppressive cultivars. These trends in root distribution were observed early in the season (pre-flood) as well as at crop maturity in both weed-free and weed-infested plots. Barnyardgrass reduced rice yield and root mass more than did sprangletop. Our results demonstrated that roots of the allelopathic cultivars used in these studies grew aggressively and proliferated near the soil surface. Rice plant types exhibiting such optimized root distribution and allelochemical exudation traits in combination with high tillering and yield might be useful for improved weed management and crop production in organic rice or other reduced-input systems. Keywords: allelopathy, barnyardgrass, Echinochloa crus-galli, sprangletop, Leptochloa fusca, root architecture, 13C isotope discrimination analysis, weed suppression, tillering Tel: + 870-672-9300; fax: + 870-673-7581 225 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 119. STUDIES ON ALLELOPATHIC POTENTIAL OF WILD RICE RESOURCES FROM YUNNAN CHINA Yiqing Guo * Yang Fu and Weiling Xu College of Plant Protection, Yunnan Agricultural University, Kunming, Yunnan Province of China, 650201 e-mail: [email protected] ABSTRACT Allelopathic interactions resulting from release of allelochemicals into the environment. H. Molish coined allelopathy in 1937 from allelon (of each other) and pathos (to suffer). Combining allelopathic potential characteristics in rice cultivars may be a way of reducing weed infestation and herbicide use in rice production. A wild kindred population of crop is containing the specialties of high yield, disease resistance and stress physiology resistance, and has wealth genes variety becoming an important source of new genes in modern rice breeding. This research focuses on selection and evaluation of allelopathic wild rice species against barnyardgrass in order to further facilitate research. Alleopathic potential in wild rice species, rufipogon, Officinalis, granulate, against Echinochloa crus-galli was evaluated by means of bioassay and method of HPLC allelopathic index, and PI312777, Longistaminata and Lemont were used as the positive and negative contrast. The results of bioassay and cluster analysis divided the experimental materials into 3 groups, in which PI312777 and Officinalis were allelopathic group, rufipogon, Longistaminata and granulate were just in the middle, and Lemont belonged to non-allelopathic group. And the results from HPLC analysis showed that the allelopathic potential was Officinalis > PI31277 > rufipogon > Longistaminata > granulate > Lemont, with the allelopathic potential index as 0.788, 0.582, 0.575, 0.525, 0.486 and 0.132 respectively. These results from different evaluating methods showed similar tendency and suggested that the three wild rice species with good allelopathic potential could be used in wide hybridization programs for weed suppressive variety development. Keywords: wild rice species, allelopathy to weed, evaluation Tel: +86-871-65228581; Fax: +86-871-65227145 226 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 120. A CHEMICAL BASIS FOR DIFFERENTIAL ALLELOPATHIC POTENTIAL OF ROOT EXUDATES OF Sorghum bicolor L. (Moench) CULTIVARS ON COMPANION WEEDS 1 Ibrahim S. Alsaadawi1, Tamara A. Al‐Khateeb2, Hameed A. Hadwan2 and Nabil R. Lahmood3 Department of Biology, College of Science, Baghdad University, Baghdad, Iraq 2 National Center for Organic Farming, Ministry of Agriculture, Baghdad, Iraq 3 Department of Agronomy, College of agriculture, Waset University, Waset, Iraq. E mail: [email protected] ABSTRACT Study was conducted to test if the variation in weed population and biomass between the stands of Enkath and Rabeh sorghum cultivars, which was observed in the field, was due to differences in their allelopathic potential. Field experiment revealed that Enkath cultivar significantly suppressed weed density and dry weight biomass over Rabeh cultivar by 34 and 29% after 35 DAS, and 22 and 24% after 65 DAS. Stair case experiment indicated that root exudates of Enkath cultivar showed more suppression to weeds than Rabeh giving additional evidence for the superiority of Enkath cultivar in its allelopathic weed suppression. Chemical analysis revealed that sorgoleone and several phenolic acids were present in higher concentrations in root exudates of Enkath compared to Rabeh. The results recommend screening more sorghum cultivars in order to offer a potential source of allelopathic germplasm that could be manipulated to enhance weed suppression in an effective and environmentally sustainable approach. Keywords: Allelopathy, sorghum cultivars, weeds, root exudates, phytotoxins 227 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 121. ALLELOCHEMICALS MODIFY Salvia officinalis EFFECTS´ ON NEIGHBORED Arabidopsis thaliana Margot Schulz 1, Andreas Ulbrich2*, Hannah Kahle2, Kristin Rotherm1 and Diana Hofmann3 1 Address 1; e-mail: 1IMBIO Institute Molecular Biotechnology of Plants, University of Bonn, Karlrobert Kreiten Str. 13, 53115 Bonn, Germany; e-mail:[email protected] 2 Department of Horticultural Production, University of Applied Science, Am Krümpel 31 49090 Osnabrück, Germany; [email protected] 3 Forschungszentrum Jülich IBG-3, Wilhelm-Johnen-Straße, 52428 Jülich, Germany; e-mail:[email protected] ABSTRACT Depending on the dosage, volatile compounds of aromatic plants can have beneficial or harmful effects on neighbored plants. Low concentrations of some monoterpenes can promote plant growth whereas higher ones inhibit the growth, affects the cytoskeleton, alter gene expressions, lead to permanent stomata opening and desiccation of receiver plants. In a recent study we investigated caffeine absorption of Salvia officinalis, previously used as an intercrop in coffee plantations. A possible influence of caffeine on monoterpene synthesis in sage was assumed. Therefore we investigated differential gene expression of the most important monoterpene synthases in Salvia officinalis, (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase by qPCR after caffeine application in comparison to control plants. Despite of the age-dependency of the gene activity in the leaves, the expression of the monoterpene synthase genes was generally down regulated after three and four days of caffeine treatment. This had effects of the flowering time of neighbored Arabidopsis thaliana plants. In comparison to the control Arabidopsis plants, Salvia exposed Arabidopsis showed a retarded flower development. The caffeine treatment abolished the inhibitory effect and, depending on the sage: Arabidopsis ratio, inflorescence development could occur even accelerated. Currently, the volatile composition after caffeine treatment is investigated. We assume that many allelochemicals can modify, probably temporarily, the volatile composition with different influences on neighbored plants. Keywords: Salvia officinalis, monoterpene synthases, differential gene expression, caffeine, Arabidopsis thaliana. Tel: + 49-(0)228-732151; fax: + 49-(0)228-732151 228 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 122. ALLELOPATHIC POTENTIAL OF VOCS FROM THE DESERT PLANT Seriphidium kaschgaricum Hua Shao and Chi Zhang Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi 830011, China; e-mail: [email protected] ABSTRACT The allelopathic potential of VOCs from a desert shrub - Seriphidium kaschgaricum was investigated. Seedling growth of amaranth (Amaranthus mangostanus) and ryegrass (Lolium perenne L.) cultivated in rhizosphere soil of S. kaschgaricum as well as in air tight boxes containing fresh stems and leaves of S. kaschgaricum was significantly affected, indicating that S. kaschgaricum can release biologically active VOCs to affect neighboring plants’ growth. The phytotoxic activities of essential oils from stems, leaves, flowers and whole plant of S. kaschgaricum were assayed against amaranth and ryegrass at 0.2, 0.5, 1.5, 3 and 5 µl/mL. These oils exhibited different phytotoxic activities on receiver plants, with the whole plant oil possessing the weakest activity. Remarkable reduction in root growth of receiver plants was observed when 0.5 µl/mL stem, leaf and flower oils were applied, but not the whole plant oil, possibly due to antagonistic effect among its ingredients. 1.5 µl/mL oils almost killed all seedlings, and 3 µl/mL oils completely inhibited seed germination of receiver plants. GC/MS analysis revealed that among the total 37 compounds identified in the oils, 19 were common, with eucalyptol (43.00%, 36.66%, 19.52%, and 38.68% in stem, leaf, flower and whole plant oils, respectively) and camphor (21.55%, 24.91%, 21.64%, and 23.35%, respectively) consistently being the principal ingredients in all oils. Eucalyptol and camphor have been reported to possess plant growth inhibitory activities, implying that they might be responsible for the oils’ toxicity. Further study is needed to investigate how these VOCs are released and function as effective allelochemicals. Keywords: allelopathy, dominant species, phytotoxicity, S. kaschgaricum, eucalyptol, camphor Tel: + 86-991-782-3131; Fax: +86-991-788-5320. 229 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 123. ALLELOPATHIC POTENTIAL OF Mentha suaveolens: VOLATILE BIOASSAY Carolina G. Puig*, Manuel J. Reigosa and Nuria Pedrol Department of Plant Biology and Soil Science, laboratory of Plant Ecophysiology, University of Vigo. Campus Lagoas-Marcosende, 36310-Vigo, Spain; *e-mail: [email protected] ABSTRACT Mentha is a genus of the Labiatae family, which is known to produce large quantities of volatile compounds. Essential oils have been studied for their chemical composition and biological activities. At the present work, the potential herbicidal activity of the volatile compounds released from M. suaveolens Ehrh. aerial parts was studied for the first time. Different amounts of freshly-harvested leaves and flowers were evaluated for herbicidal activity in a manner that only atmospheric contact was allowed between the test species and the mint tissues, following the methodology of Barney et al. (2005). The phytotoxic effects were tested on the germination and growth of Lactuca sativa L. and Agrostis stolonifera L. as dicot and monocot model target species, Zea mays L. as crop species, and Amaranthus retroflexus L. as one of its representative accompanying weeds. Results show that the volatile cocktail released from mint aerial tissues delayed the germination and reduced significantly the radicle length of lettuce. Although germination of weed species was not affected, growth was significantly inhibited by mint volatile cocktail. On the other hand, no effects on crop were observed. This selectivity and capability of controlling weeds without damaging the crop is a key factor when considering a potential herbicide for its use in the field. Our results indicate that M. suaveolens produce and release a cocktail of volatile compounds that could be used as natural herbicides for weed control. Barney JN, Hay AG, Weston LA (2005) Isolation and characterization of alllelopathic volatiles from mugwort (Artemisa vulgaris). Journal of Chemical Ecology 31: 247-265. Keywords: mint, volatile compounds, phytotoxicity, weed control Tel: +34986812616; fax: +34986812556 230 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 124. CHEMICAL DEFENSE OF Bellardia trixago (L.)All. IN JAPAN Masanori Morimoto, Miki Ishida and Kazuhiko Matsuda Kinki University School of Agriculture, 3327-204 Nakamachi Nara city, Nara, Japan 631-8585; e-mail: [email protected] ABSTRACT Recently, the alien hemiparasitic weed, Bellardia trixago (Orobanchaceae) invaded Japanese liver side. They possess glandular trichomes on the leaf surface. Notably, young leaf exudates are more abundant than from the other parts of this species. The exudates contain the labdane-type diterpene alcohol and its hemimalonate as major components and deter from herbivore feeding, but does not kill herbivores in the field. However, lepidopteran larva die by eating this plant species, suggesting that phytochemicals other than the trichome exudate possibly contribute to such toxicity. Here, we have investigated the phytochemicals underlying the lethal action against lepidopteran larvae. Additionally, this study has disclosed how these phytochemicals act for expansion in Japan. The exudate was prepared by rinsing the field collected plant materials with dichloromethane, and then the plant residue was extracted with methanol to obtain non-exudate phytochemicals. The labdane-type diterpenes and iridoid glucosides were purified from the rinsate and the methanol extract, respectively. Structures of these compounds were identified on spectroscopic data. The antifeedant and insecticidal activities of these compounds were evaluated on the common cutworms (Spodoptera litura). Also plant growth inhibitory activity was evaluated on lettuce and Italian ryegrass seedlings. The diterpenes had no significant biological activities against tested organisms, whereas iridoid glcosides showed an insecticidal activity and a moderate phytotoxicity. Thus the exudate may act as a physical defense against herbivores, while iridoid glucosides act as a toxicant against herbivores as a chemical defense. Keywords: Chemical defense, diterpene, iridoido, alien plant, weed Tel: +81-742-43-7162; fax: +81-742-43-1445 231 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 28. Plant‐insect interaction Oral 125. PLANT CHEMICAL DEFENSE PRIMED BY SILICON AND MYCORRHIZAL FUNGUS Rensen Zeng1,2, Yuanyuan Song1,2, Mao Ye1,2, Scott R. Baerson3, Zhiqiang Pan3, Kai Lu1, Shiming Luo2 1 2 College of Crop Science, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, P.R. China. E-mail: [email protected], [email protected] Institute of Tropical and Subtropical Ecology, South China Agricultural University, Guangzhou 510642, P.R. China 3 United States Department of Agriculture-Agricultural Research Service, Natural Products Utilization Research Unit, University, Mississippi 38677, USA ABSTRACT Plants are good at the production of an extensive array of secondary metabolites, which can be used for both allelopathy against their competing neighbors and chemical defense against herbivores and pathogens. Priming of plant defense refers to increased readiness of induced defense through the perception of indicative signal cues or the experience of previous enemy attack, which allows plant to induce more effective and rapid defense responses upon subsequent attack. We examined priming effects of silicon (Si) and arbuscular mycorrhizal fungus (AMF). Si pre-treatment increased rice resistance against caterpillar Cnaphalocrocis medinalis (rice leaffolder, LF). Upon LF attack, wildtype plants subjected to Si pre-treatment exhibited enhanced defense responses relative to untreated controls, including higher JA accumulation levels, increased levels of transcripts encoding defense marker genes, and elevated activities of peroxidase, polyphenol oxidase and trypsin protease inhibitor. Exogenous application of methyl jasmonate amplified Si-induced defense and enhanced Si deposition and Si cell expansion in rice leaves. Mycorrhizal inoculation negatively affected larval performance of caterpillar Helicoverpa arimigera on tomato (Solanum lycopersicum Mill.). Insect feeding on AMF pre-inoculated plants resulted in much stronger defense response induction of four defense-related genes LOXD, AOC, PI-I and PI-II in the leaves of tomato plants relative to non-inoculated plants. Our results suggest that both Si and mycorrhizal fungus could prime systemic defense responses in plants and the jasmonate pathway is involved in defense priming against herbivore insects. Appropriate manipulation of plant defense priming is a promising approach to reduce human dependence on synthetic chemicals. Keywords: Plant chemical defense, defense priming, silicon, mycorrhizal fungus, jasmonate pathway TEL: + 86-591-8372-2796 ; FAX: +86-591-8378-9483 232 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 126. WEAVER ANT FAECES: ELUCIDATION OF ALLELOCHEMICALS AND NUTRITIONAL EFFECTS Nanna Hjort Vidkjær 1, Inge S. Fomsgaard 2*, Bernd Wollenweber 3 and Karl‐ Martin Vagn Jensen 4 1 2 Department of Agroecology, Aarhus University, Forsøgsvej 1, DK-4200 Slagelse, Denmark; e-mail: [email protected] Department of Agroecology, Aarhus University, Forsøgsvej 1, DK-4200 Slagelse, Denmark; email: [email protected] 3 Department of Agroecology, Aarhus University, Forsøgsvej 1, DK-4200 Slagelse, Denmark; email: [email protected] 4 Department of Agroecology, Aarhus University, DK-Forsøgsvej 1, 4200 Slagelse, Denmark; email: [email protected] ABSTRACT Weaver ants (Oecophylla) have been used for centuries in pest control in e.g. mango and citrus orchards. They are carnivorous tropical ant species renowned for their ability to weave leaves into nests in the canopies of trees using silk from their larvae1. Like most ant species they live in high numbers and thus have a high impact on their environment. Weaver ants excrete high amounts of waste material (“ant manure” (AM)) as visible droplets on the leaves of their host tree. It has been hypothesized that AM contains compounds of nutritional value for the host tree and preliminary studies of its chemical composition have found that AM contains urea, a known foliar nutrient. In a laboratory plant experiment using GC-MS-based metabolomics, it has been demonstrated that plants hosting an Oecophylla smaragdina colony elicit metabolic responses similar to plants having received higher levels of nitrogen-containing nutrients compared to low-level control plants. This is evident by e.g. elevated levels of several amino acids in leaves from ant plants and elevated levels of carbohydrates in control plants. Furthermore, it has been shown that herbivorous pest insects are deterred from feeding on leaves collected from ant-hosting plants2 and it is hypothesized that allelochemicals deposited together with AM are responsible for this. In ongoing studies elucidation of allelochemicals will be performed and deterring effects investigated by e.g. gaschromatography coupled to electroantennography as well as behavioural assays. 1 2 Crozier, R.H. et al. (2010). Myrmecol. News, 13, 57-71. Offenberg, J. et al. (2004). Proc. R. Soc. Lond. [Biol.] 271, S433-S435. Keywords: Weaver ants, Oecophylla smaragdina, semiochemicals, metabolomics, pest control, ant-plant interactions, ant-pest insect interactions. Tel: +45 87158139 / *+45 87158212; fax: +45 87156082 233 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 127. VOLATILE INTERACTIONS BETWEEN UNDAMAGED PLANTS INDUCE RESPONSES IN THE PLANTS AFFECTING HERBIVORE INSECTS AND THEIR NATURAL ENEMIES Velemir Ninkovic Swedish University of Agricultural Sciences, Box 7044, SE-750 07 Uppsala, Sweden; e-mail: [email protected] ABSTRACT Herbivore damaged plants release volatile organic compounds (VOCs) that are involved in a wide range of interactions and play important roles in the coexistence between plants and organisms on other trophic levels. However, plants release VOCs even when they are not attacked or mechanically damaged, and these volatiles are available as signals for neighboring plants. Plant interactions by volatiles emitted from foliage between undamaged plants have not previously been considered an important factor for induced resistance to herbivores. The aim of our studies was to investigate whether volatile transfer between undamaged plants can contribute to the effects of intercropping on herbivores and their natural enemies. This idea was tested in a system consisting of potato (Solanum tuberosum L.) intercropped with onion (Allium cepa L.) or garlic (Allium sativum L.), and the green peach aphid Myzus persicae (Sulzer), which uses potato as a host plant and seven- spotted ladybird (Coccinella septempunctata L.) which is the most important predator of aphids on different crops. In the present study we have found that exposure to plant volatiles can result in differences in volatile emission in the exposed plant, which has not been shown previously. Exposing potato to VOCs from undamaged onion plants had a deterrent effect against host-seeking green peach aphid and attracting effect on ladybird. In a field experiment, migration of aphids into potato was significantly reduced by intercropping with onion. Our findings represent a novel bottom-up effect of plant co-existence on insect and provide new evidence of the role of chemically-mediated mechanisms. Keywords: plant-plant communication, terpenoids, (E) nerolidol, (3E, 7E)-4, 8, 12-trimethyl-1, 3, 7, 11-tridecatetraene (TMTT) aphids, Tel: +4618672541; fax: +4618672890 234 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 128. EFFECT OF LEAF LEACHATE OF Azadirachta indica AND Albizia lebbeck ON THE INCIDENCE OF INSECTS PESTS ON Azolla pinnata Quazi Taheruzzaman Department of Botany,Sarat Centenary College,Dhaniakhali,Hooghly, West Bengal,India. Email: [email protected] ABSTRACT Numerous studies have been carried out on the effect of leaf leachate on terrestrial plants but there are only a few on the effects of terrestrial leaf leachate on aquatic macrophytes.Azolla pinnata, a common aquatic fern has a paramount importance in agriculture as biofertilizer in South East Asia.But there are some constraints as regard to the year round culture of Azolla.Azolla is usually infected by insects and pests.The present study was conducted in order to evaluate the effect of leaf leachate of Azadirachta indica and Albizia lebbeck,the common trees of India,on the growth of Azolla pinnata.The study revealed that the leaf leachate of Azadirachta indica enhanced the growth of the fern by reducing insects pests throughout the seasons compared to less growth in Albizia lebbeck leaf leachate media.Evidently,the leaf leachate of Azadirachta indica may be used in sustainable development to control insects pests of Azolla. Keywords: Azolla pinnata, leaf leachate, Azadirachta indica, Albizia lebbeck, insects pests control Tel: +91 9434637587; fax: +91 03213 2557747 235 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 129. INFLUENCE OF THE LITTER CHEMICAL COMPOSITION ON Folsomia candida'S FITNESS Mathilde Chomel1, Annie Desrochers2, Marie Larchevêque3, Virginie Baldy1, 1 2 Institut Méditerranéen de Biodiversité et d’Ecologie (IMBE) Aix-Marseille Université, CNRS, IRD, Univ. Avignon, Campus St Charles Case 4, 13331 Marseille Cedex 03, France. [email protected] Institut de Recherche sur les forêts - Université du Québec en Abitibi-Temiscamingue, 341 rue principale Nord, Amos (Québec) J9T2L8, Canada 3 Institut de recherche en mines et en environnement - Université du Québec en AbitibiTemiscamingue, 341 rue principale Nord, Amos (Québec) J9T2L8, Canada ABSTRACT Springtails (Collembola) are among the groups of the most abundant arthropods on earth and present a huge biodiversity, with more than 7900 species described today. They play an essential role on soil processes, mainly by an indirect effect by stimulating microorganisms colonizing the litter. Even if the effects of soil conditions and the nature of organic matter on collembola are demonstrated, the precise mechanisms responsible of the structure of communities remain little studied. The question is to know to what extent springtails are influenced by the chemical quality of litter, especially through allelopathic mechanisms. To answer this question, we realized laboratory tests by following the standard ISO 11267. These tests consist to breed 10 individuals of Folsomia candida in mesocosms containing a standardized substratum to which we added separately poplar, spruce and herbaceous litter or a mixture of poplar and spruce litter. In order to separate chemical and physical effects of litter, other mesocosms were done by mixing litter leachates (at 5 % and 10 % concentrations) to the substratum. After 30 days of incubation, we determined reproduction and mortality rates of collembola. Results showed that leachates had a greater impact on the springtails compared to litter. In addition, there were between 94 and 100% of mortality in mesocosms that received leachates of poplar or mixed poplar/spruce, indicating a strong influence of the compounds contained in the poplar leaves on springtails. However there is a positive effect of litter or leachates of herbaceous on the rate of reproduction. Keywords: Folsomia candida, collembola, reproduction, mortality, allelopathy, ecotoxicological test. TEL: +33 (0) 4 13 55 12 33 236 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 130. BENZOXAZINOID ALLELOCHEMICALS ARE ABSORBED AND METABOLIZED IN MAMMALS Khem B. Adhikari 1, Bente B. Laursen 2, Helle N. Lærke 3, Heidi J. Schnoor 4, Marianne Witten 5, Lars K. Poulsen 6, Bettina M. Jensen 7 and Inge S. Fomsgaard 8* 1 2 Department of Agroecology, Aarhus University, Forsøgsvej 1, DK-4200 Slagelse, Denmark; email: [email protected] Department of Agroecology, Aarhus University, Forsøgsvej 1, DK-4200 Slagelse, Denmark; email: [email protected] 3 Department of Animal Science, Aarhus University, Blichers Alle 20, DK-8830 Tjele, Denmark; e-mail: [email protected] 4 Allergy Clinic, Copenhagen University Hospital, Niels Andersens Vej 65, DK-2900 Hellerup, 5 Denmark; e-mail: [email protected]; e-mail: [email protected]; 6 7 e-mail: [email protected]; e-mail: [email protected]; 8 Department of Agroecology, Aarhus University, Forsøgsvej 1, DK-4200 Slagelse, Denmark; email: [email protected] ABSTRACT Benzoxazinoids are a group of naturally occurring bioactive allelochemicals mostly found in cereal plants. In addition to their allelopathic effects, benzoxazinoids contain a range of health-protecting effects and pharmacological properties. The presence of these chemicals in mature cereal grains and bakery products was recently identified, making them interesting compounds from a nutritional point of view. Rye grain is the major source of dietary benzoxazinoids followed by wheat. The objective of these studies was to evaluate the uptake, distribution and metabolism of dietary benzoxazinoids in mammals. We fed a benzoxazinoid-containing rye bread-based diet to pigs (n=6), rats (n=6), and humans (n=19) and analyzed the content of benzoxazinoids and their potential metabolites in plasma, urine, bile and faeces by LC-MS/MS using electrospray ionization and multiple reaction monitoring mode. The most dominant dietary benzoxazinoid, 2-β-D-glucopyranosyloxy-4hydroxy-1,4-benzoxazin-3-one (DIBOA-Glc), was apparently reduced to 2-β-Dglucopyranosyloxy-1,4-benzoxazin-3-one (HBOA-Glc), the most dominant circulating benzoxazinoid in the plasma in all three experiments. Eight different benzoxazinoids and their derivatives including some glucuronide and sulfate conjugates were excreted through the urine, HBOA-Glc being the most dominant urinary benzoxazinoid. Interestingly, 2-amino-3H-phenoxazin-3-one, a potent antibiotic, was also detected in the bile and urine. These results indicate that benzoxazinoids could be one of the key ingredients contributing to the overall health-protecting effects of whole-grain consumption. More information: www.BREADANDBREAKFAST.nu Keywords: benzoxazinoids, bioavailability, wholegrain, absorption Tel: +45 87158178 / *+45 87158212; fax: +45 87156082 237 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 131. CHARACTERIZATION OF PHOSPHOLIPASE D FROM Chorispora bungeana CALLUS IN RESPONSE TO FREEZING STRESS Ning Yang1 and Sun Kun2 1 2 College of Life Sciences, Northwest Normal University,No. 967 Anning East Road, Lanzhou 730070, People’s Republic of China; [email protected] College of Life Sciences, Northwest Normal University,No. 967 Anning East Road, Lanzhou 730070, People’s Republic of China; [email protected] ABSTRACT The influence of freezing on phospholipase D (PLD) was studied in Chorispora bungeana Fisch. & C.A. Mey., which is a naturally cold-tolerant species. During the freezing treatment (-4 °C), PLD activities in both microsomal and mitochondrial membranes increased at day 3, remained at a high level at day 6 and then declined to a moderate level. The RT-PCR analyses showed that PLD activity partially corresponded to the CbPLD gene transcript level. The freezing treatment resulted in increases in the Km and Vmax for microsomal and mitochondrial PLD, respectively. Freezing injury, as measured by electrolyte leakage and malondialdehyde content, peaked at day 6 and then gradually decreased. Alleviation of freezing injury was related to a decreased content of membrane-associated Ca2+. We suggest that the specific mechanism of cold resistance of C. bungeana is linked with PLD. Keywords: calcium, electrolyte leakage, malondialdehyde, microsomal and mitochondrial membranes, RT-PCR. Tel: 13919280080; fax: 0086-0931-7971207 238 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 132. POTENTIAL OF ALLELOPATHIC WATER EXTRACTS IN IMPROVING THE DROUGHT RESISTANCE DURING REPRODUCTIVE AND GRAIN-FILLING PHASES OF WHEAT Ahmad Nawaz*1, Muhammad Nabeel2 and Muhammad Farooq3 Allelopathy Lab, Department of Agronomy, University of Agriculture, Faisalabad-38040, Pakistan, E-mail: 1 [email protected]; 2 [email protected]; 3 [email protected] ABSTRACT Drought is the most prevalent abiotic stress, which drastically reduces the crop yield. Allelopathic water extrcats of different crop have ability to cope with stress conditions due to presence of secondary metabolites. This study was aimed to evaluate the role of allelopathic water extracts in improving the drought resistance during reproductive and grain filling phases in wheat. The experiment was laid out in completely randomized design in factorial arrangement with three replications. Wheat cultivars Faisalabad-2008 and Maraj-2008 were used in this study. Pots were maintained at 70% water holding capacity (well-watered) till booting stage and then managed drought was applied at booting, anthesis and post-anthesis stages by maintaining 35% water holding capacity (drought stress). After one week of drought imposition at each reproductive stage, the allelopathic water extracts (of sorghum, sunflower, brassica and moringa), water were applied at each respective stages. Agromorphological, physiological and yield related parameters were measured following standard procedures. Performance of Mairaj-2008 was quite better in term of yield related traits than Fsd-2008 in drought stress conditions. Similarly, application of allelopathic water extracts improved the agronomic and yield related traits especially when applied at booting and anthesis stage. Application of sunflower water extract was quite better than other allelopathic water extracts in drought stress conditions. Severity of drought stress was more detrimental when applied at booting and anthesis stage and was less when applied at grain filling stage. In conclusion, growing of stay green varieties accomplished with allelopathic water extract application at reproductive stages may be used as an option to elevate the drought induced yield losses in bread wheat Keywords: Drought, water extracts TEL: +92(41)9201098; FAX: +92(41)92 239 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 133. PHYSIOLOGICAL AND ALLELOPATHIC RESPONSES OF FIELD GROWN LEMONGRASS TO SEASONAL CHANGES Aziz, A1 and A. Wahid2* 1 2 Botany Department University of Agriculture Faisalabad Pakistan; [email protected]: Botany Department University of Agriculture Faisalabad Pakistan; [email protected]: ABSTRACT Lemongrass (Cymbopogon citratus) is known for its very high antioxidants and nutritional value. A landrace of lemongrass was investigated to evaluate the seasonal changes in its metabolite profiles in the leaves of different ages i.e. penultimate (second fully expanded leaf from the top), middle (a leaf from the central position of a tiller) and bottom (a lowermost green leaf on a tiller).. Measurements were made for some primary and secondary metabolites while the essential oils profile of leaves was analyzed using GC-MS. The accumulation trends of osmolytes in lemongrass showed notable seasonal variation. The antioxidant enzymes minimized the effect of oxidative damage by scavenging H2O2 and MDA in extreme environmental conditions especially during summer. The secondary metabolites i.e. alkaloid, phenolic and flavonoid contents in lemongrass leaves were markedly increased with increasing temperature. Seasonal variation affected the nutritional profile of lemongrass significantly. Lemongrass oil was rich in neral, citral, geranial, allerthin, caryophyllene oxide. Therefore, the allelopathy of lemongrass should not be ignored when planted it with other crops due to its high profile of secondary metabolites. Keywords: Cymbopogon citratus, secondary metabolites, essential oil, seasonal effects Tel: +92 3144942918 240 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 134. ALLELOPATHIC POTENTIAL OF Medicago sativa L. IN ASSESSING THE QUALITY OF SALINE SOIL K. B. Pawar1, J.A. Hawale2 and M.V. Suryawanshi2 1 Department of Botany, Shivaji University, Kolhapur-416 004 (MS) India [email protected] 2 Department of Botany, Shivaji University, Kolhapur-416 004 (MS) India ABSTRACT In India in Sangli District of Maharashtra State hundred acres of land has become saline due to monoculture of sugarcane. Now a day’s land is not under cultivation of any crop plant. Alfalfa, Medicago sativa L. is a forage legume. Many workers have tried to evaluate salt tolerance of alfalfa. So an attempt has been to study allelopathic potential of M. sativa L. for assessing the quality of saline soil. We have selected some sites from Sangli District. We have collected soil samples from each site and started to germinate and grow alfalfa seeds in the soils under laboratory conditions. Seedling growth with respect to root length, shoot length and fresh weight was recorded after one month growth. After removing alfalfa seedlings from the soil, seeds of chickpea (Cicer arietinum L.) were sown. Seedling growth of chickpea was also recorded after one month. Good growth performance with respect to root length, shoot length and fresh weight was observed in both the crops. This study is continued to carry out analysis of crop plants and soil samples for the presence of phytochemicals as well as micro and macronutrients. Keywords: Medicago sativa L., monoculture, seedling growth, sugarcane, saline soil Tel: +09422324078 241 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 135. INFLUENCE OF ENHANCED UV-B (285-320 nm) RADIATION CHANGES ON THE MORPHOLOGY AND IN ALLELOCHEMICALS (SECONDARY METABOLITES) CONTENT IN Adhatota vasica M. Eyini1 P. Seetharaman2 P. Periyakaruppiah2 and M. Jayakumar3* 1 Research Centre in Botany, Thiagarajar College (Autonomous), Madurai – 625 009, Tamil Nadu, India 2; e-mail: [email protected] 2 Research Department of Botany, VHNSN College (Autonomous), Virudhunagar– 626 001, Tamil Nadu, India 1; e-mail: [email protected] 3 Thiruvalluvar University, Serkkadu, Vellore - 632 115, Tamil Nadu, India 3; e-mail: [email protected] ABSTRACT There are many reports on Ultraviolet-B (280-320 nm) induced changes in morphology of plants, plant growth, photosynthesis, chloroplast structure, function in higher plants. But very few papers have appeared on the UV-B influences on allelochemicals. In this study, the influence of enhanced UV-B radiation on the morphological changes and in the content of some allelochemicals in Adhatota vasica. Allelochemicals not only helps the plants to overcome extreme environmental conditions, but also acts as a defense tool to with stand UV-B stress. In UV-B exposed plants, shoot height leaf area, fresh weight, dry weight was decreased than the control plants in Adhatota vasica. The qualitative analysis of allelochemicals in IR spectrophotometer and HPLC in the presence of similar groups viz. protocatechuic acid, p-hydroxy benzoic, caffeic acid acid and dopamine. These results indicated that in Adhatota vasica in allelochemicals are enhanced by UV-B radiation. This will improve the quality of the plants for its medicinal values. Keywords: Ultraviolet-B, Adhatota vasica, allelochemicals, IR spectrum, HPLC Tel: +914162274746; fax: +914162274748 242 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 136. ALLELOPATHIC AND BIOCHEMICAL PROPERTIES OF SOIL OF C3, C4 PLANTS UNDER DROUGHT CONDITIONS Zaimenko N.V.,1 Pavliuchenko N.A.,2 Kharytonova I.P.3 M.M. Grishko National Botanical Garden, Department of Allelopathy, Timiryazevska 1, 01014 Kyiv, Ukraine; 1 e-mail: [email protected], 2 e-mail: [email protected], 3 e-mail: [email protected] ABSTRACT Allelochemical production is influenced by different environmental stress factors. Drought is one of the most important. The aim of the work was to study effect of soil drought on allelopathic activity and biochemical characteristics of soil of C3, C4 plants. Plants with C3 (cultivars of Brassica juncea L. Czern. and Raphanus sativus L. var. oleiformis Pers.) and C4 (cultivars of Amaranthus paniculatus × Amaranthus caudatus) pathways of carbon metabolism were grown under field conditions. Soil moisture content, allelopathic activity, oxidation-reduction potential, humus and phenolic substances contents, macro- and microelements composition were measured. The critical phases (soil moisture deficit) of development were flowering and fruitage for C4 plants, fruitage and the end of vegetation period for C3 plants. Reduction in growth-stimulating activity of soil of C3 plants under drought factor action was observed. Increase in the phytotoxicity of soil of C4 plants under drought conditions was found. Oxidation-reduction potential, content of phenolic substances in soil increased during soil moisture deficit. Amount of humus was invariable or lesser under drought. We found differences in the levels of macroand microelements ions in soil of C3 and C4 plants under water deficit. Drought caused increase in content of Mn in soil both C3 and C4 plants. Thus, allelopathic and biochemical characteristics of soil of C3 and C4 plants changed under drought factor action. Keywords: C3 and C4 plants, soil drought, humus, phenolic substances, macroand microelements, allelopathic activity, oxidation-reduction potential TEL: + 38 (044) 285-54-53 ; FAX: + 38 (044) 285-26-49 243 th 7 World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 137. INDUCTION OF HEAT TOLERANCE IN MAIZE (Zea mays) USING AQUEOUS EXTRACTS OF FOLIAR PARTS OF MORINGA (Moringa oleifera) THROUGH SOIL APPLICATION Asima Batool1 and Abdul Wahid2 1 Department of Botany, University of Agriculture Faisalabad, Pakistan 2 Department of Botany, University of Agriculture Faisalabad, Pakistan [email protected] [email protected] ABSTRACT Changing climate and increase in temperature is damaging to agricultural activities. Moringa (Moringa oleifera L.) is rich in minerals, ascorbates and phenolics, hormones like zeatin and cytokinin can be used to mitigate the effect of heat stress in maize. In this experiment, influence of aqueous extracts of moringa fresh leaves (3.3%), dry leaves (10%) and flower extract (10%) were soil applied on the plants of maize hybrids ICI-984 and SB-11 under normal and heat stress. Heat stress was imposed after the uniformity of stand establishment. Application of these extracts improved over all plant growth in both high and ambient temperature as compared to control. Maximum root fresh and dry weight was recorded with dry leaf extract. The osmoprotectants like free proline contents were higher in shoots under high temperature, while GB increased in both shoot and root with application of fresh leaves extract. However, total free amino acids and soluble sugars contents were improved in both shoot and root as a result of dry leaf extract. In conclusion, moringa extracts has potential to cope with abiotic stresses like heat stress and may be used as a potential growth promoter for crops. Keywords: moringa, proline, cytokinin 244 th 7 World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 138. SIGNAL REGULATION OF PROLINE METABOLISM IN CALLUS OF THE HALOPHYTE Nitraria tangutorum Bobr. GROWN UNDER SALINITY STRESS Yingli Yang • Fan Yang • Xiaoning Li • Ruxia Shi • Jin Lu School of Life Science, Northwest Normal University, Lanzhou 730070, Gansu, People’s Republic of China ABSTRACT The calli from desert halophyte Nitraria tangutorum Bobr. were used to investigate proline metabolism and its signal regulation under salinity stress. Enhanced concentrations of proline and hydrogen peroxide (H2O2) were observed in the calli exposed to salinity stress, and elevated level of calcium (Ca) was the early response to 75 mM NaCl treatment. Additionally, NaCl treatment induced significant elevation of ornithine-δ-aminotransferase (OAT) activity, but remarkable decreases in the activities of glutamyl kinase and proline dehydrogenase (PDH). H2O2 scavenger dimethylthiourea and pyruvate inhibited the accumulation of proline and the stimulation of OAT activity in salinity-stressed calli. Moreover, the utilization of Ca chelator EGTA and Ca channel blocker verapamil abolished the enhancement of proline level induced by 75 mM NaCl treatment for 3 days. These results suggest that in Nitraria tangutorum Bobr. calli the accumulation of proline is correlated to the increase of OAT activity and the decrease of PDH activity in response to salinity. Here it is also indicated that increased Ca signal during the early stage of NaCl treatment and the excitation of OAT activity, which may be the consequence of increasing H2O2 generation from salinity-stressed calli, is essential for proline accumulation in Nitraria tangutorum Bobr. calli under NaCl treatment. Keywords: Calcium, hydrogen peroxide, Nitraria tangutorum Bobr., proline, salinity TEL: 0086-0931-7971575 245 th 7 World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 64. TOXIC EFFECTS OF HEAVY METALS ON MAIZE: SPERIMIDINE PRIMING IS CURATIVE, AND POLYAMINES AND ANTIOXIDANTS PLAY A ROLE Momtaz M. Hegaba; Gaurav Zintab; Hamada Abd Elgawad ab a Department of Botany, University of Beni-Suef, Egypt. ([email protected]) b Department of Biology, University of Antwerp, Belgium 2610 Belgium ([email protected] - [email protected]) ABSTRACT Toxicity caused by heavy metals is a major threat to crop production worldwide. It restricts plant growth and results in significant yield losses. In our experiment, maize plants were imposed, separately, to the sub-lethal doses of four toxic metals viz., Cu, Ni, Zn and Cd for the duration of two and three weeks. Objectives of the study was to assess the impact of metal(s) toxicity on the whole gradient of maize plant, i.e., whole root, leaf no. 1-2, leaf 3-4. Also, the role of polyamines in stress mitigation was investigated. Sub-lethal concentration of each metal metal increased the concentrations of the applied metals within the plants in the following order: roots> leaf 1-2> leaf 3-4. Moreover, the accumulation of heavy metals was enhanced with longer stress duration. Consistently, roots growth was more inhibited than leaf 1-2 and leaf 34 was least stressed. Physiological analysis revealed decrease in photosynthesis and chlorophyll fluorescence in stressed plants. Seed pretreatment with 1 mM of sperimidine reduced the stress impact on plant growth. Moreover, it decreased the concentrations of accumulated metals within plants and induced the accumulation of total polyamines in roots and leaves. It also improved antioxidants system by induction of glutathione and glutathione reductase enzymes. Moreover activities of peroxidase, ascorbate perioxidase and catalase enzymes were induced. We concluded that sperimidine recovers maize plants from the impact of metal toxicity, somehow by lowering the accumulation of toxic metals within plants, as well as by further induction of antioxidant defence system. Keywords: heavy metals, maize, polyamines, sperimidine, photosynthetic reactions antioxidants. Corresponding author: Dr. Momtaz Mohamed Yehya Hegab - Tel.: 00201001992231 Fax: 0020822336381 246 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 139. Baccharis glutinosa L. (ASTERACEAE) AS A PROMISING ALLELOPHATIC PLANT TO CONTROL SOME PESTS OF ORGANIC TOMATO CROP Mariana Miranda1*, Guillermo Delgado2, Blanca E. Hernández‐Bautista1, Ricardo Reyes‐Chilpa2, Martha L. Macías‐Rubalcava2, and Ana Luisa Anaya3** 1 CIBA TLAX, Instituto Politécnico Nacional e Instituto de Ecología, Universidad Nacional Autónoma de México. 2 3 Instituto de Química. Universidad Nacional Autónoma de México. Instituto de Ecología, Univesidad Nacional Autónoma de México. Ciudad Universitaria, D.F. México. *[email protected]. **[email protected]. ABSTRACT Modern agriculture has contributed to the problem of environmental damage by the indiscriminate use of synthetic agrochemicals. This problem has given rise to an extensive effort to find more ecological controls of pests, trying to preserve environmental quality and to reach multiple agricultural and organic production to achieve a sustainable use of natural resources. Allelopathy could be one of the solutions to solve several agricultural problems. Farmers in Tlaxcala, Mexico want to growth organic tomato and asked us to find some natural controls for several of the main diseases and pests of this crop. We starting the search of several allelopathic plants to deal with some of these diseases and weeds of tomato crop using allelochemicals. Baccharis glutinosa (Asteraceae) was a promising plant with a strong in vitro allelopathic effect on some plants (Amaranthus sp.) and phytopathogenic fungi (Fusarium oxysporum and Phytophthora capsici). These two last species caused crown and root rot to the crop. Aqueous leachates of the dry aerial part (1%) significantly inhibited A. hypochondriacus root growth (100%), and mycelia growth of both phytopathogens (50%). Dichloromethane-methanol (CH2Cl2-CH3OH,1:1) extract at 200 µg ml-1 inhibited (40%) the mycelia growth of both. Biodirected fractionation of this organic extract produced some secondary and tertiary fractions with a strong inhibition effect on mycelia growth of those fungi (83 and 100%). Our results suggest that possibly, a mixture of similar allelopathic compounds of B. glutinosa could be a good precursor of a natural pesticide for an organic production of tomato crop. Keywords: Baccharis glutinosa, tomato, Amaranthus sp., Fusarium sp., Phytophthora sp. Tel: 0155 56229043; etx.29043 247 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 140. COATING MATERIALS FOR SUSTAINED RELEASE OF ALLELOCHEMICALS Ines Fritz and Stephan Strasser‐Altrogge Universität für Bodenkultur, IFA-Tulln, Konrad Lorenz Str. 20, A-3430 Tulln, Austria, eMail: [email protected]. ABSTRACT The increasing public rejection of synthetic pesticides and especially their adverse impact on the environment and on consumer’s health accentuate the need for biological or ecological pest management in present-day high-tech agriculture. Biopesticides need to comply with the same important prerequisites like conventional synthetic pesticides. Microencapsulation of active ingredients mediates site- and time-specific release at controlled rates. Fluidised bed coating is suitable for encapsulation of active ingredients to mediate sustained release. The release behaviour of coating materials of natural origin was investigated in order to obtain appropriate formulations for biologically active substances or microorganisms to be used as biopesticides. Waxes and resins are useful to prolong the release of coated ingredients, while their mechanism of performance is mostly based on slow biological degradation or erosion. The evaluation of sustained release systems was carried out by encapsulating the model substance aluminium sulphate in four different coatings of natural origin and further by investigating the sustained release kinetics of the encapsulated compound. The study revealed that carnauba wax and tall oil rosin mediate slower release patterns than shellac and soya oil coatings. Depending on the desired release kinetics all four natural coating substances are potential candidates to be used for encapsulating biopesticides, such as allelochemicals. Keywords: Sustained release, biopesticides, allelochemicals, biodegradation, microencapsulation TEL: +43 2272 66280 559; FAX: +43 2272 66280 503 DIF 248 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 141. ALLELOPATHIC INFLUENCE OF THE PERENNIAL HALOPHYTES ON ANATOMICAL STRUCTURE OF ANNUAL HALOPHYTES Simagina N.O. V.I. Vernadsky Taurida National University, [email protected] ABSTRACT The halophytes - a specialized group of plants, developed during evolution mechanisms of stability to specific аbiotic factors of ecotope. However, growth of halophyte vegetation is determined by аbiotic as well as biotic factors. Due to allelopathic influence in natural ecosystems (N-W Crimea, Ukraine) and model experiments there are significant changes on the histological level of plantsacceptors (p-ac). At annual halophytes Salicornia europaea L. (p-ac), grown in the zone of maximum allelopathic effects Halocnemum strobilaceum (Pall.) M.Bieb. and Artemisia santonica L.- donors of allelopathic substances, intense lignification of xylem vessel walls of the central cylinder (CC), sclerenchyma strands, idioblasts were detected. Area of lignification zones of CC S. europaea, growing in a phytogenous field A. santonica, is 1,6-1,8 times higher than in monocoenosis. It was established negative correlative relation between the degree of lignification CC zone along the remoteness gradient from A. santonica r=-0,51±0,10, H. strobilaceum r=-0,47±0,12 (P<0,05). Idioblasts in parenchyma of primary cortex showed positive reaction in the qualitative detection of lignin, suberin. In tissues S.europaea the number and size of idioblast in 1,9 times more at allelopathic influence of perennials than in monocoenosis. It was established the negative correlation between the distance from A.santonica and the amount of idioblasts r=0,73±0,09, and their size r=0,60±0,12 in tissues plant-acceptor. Comparative histochemical studies of annual halophytes, grown in monocenosis and in maximum allelopathic effects zone allowed to establish significant accumulation of pectins, phenolic compounds in tissues of S. europaea (p-ac). Detected changes on histological level provide realization of protective reaction of plant- acceptors on the action of allelopathic factor. ial candidates to be used for encapsulating biopesticides, such as allelochemicals. Keywords: allelopathy, halophytes Tel.:+(380)999848796 249 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 65. GLYPHOSATE AND AMPA IN SOIL AND SEEDS IN GLYPHOSATERESISTANT SOYBEAN Giani Mariza Barwald Bohm1; Léa Schneider2; César Valmor Rombaldi3 1 2 3 Federal Institute Sul-Rio-Grandense, IFSul-Pelotas Campus, Department of Environmental Management, RS-Brazil; zip code: 96015360, e-mail: [email protected]; Federal Institute Sul-Rio-Grandense, IFSul-Pelotas Campus, Department of Environmental Management, RS-Brazil; zip code: 96015360, Eliseu Maciel School of Agronomy, Federal University of Pelotas. Campus Universitario, s / n °, Capon Lion, RS, zip code 96010-900 ABSTRACT Genetically modified soybean [Glycine max (L.) Merr.] resistant to glyphosate (N-phosphono-methyl-glycine) herbicide (GR) has turned out to be one of the most highly used products of plant engineering. The impacts of the soybean, genetically transformed for glyphosate resistance, on the environment under Brazilian soil and climatic conditions are still unknown. For this purpose, two soybean genotypes, one genetically modified (GR BRS 243 RR) and a conventional (isogenic genotype) were grown under different weed control methods: hand weeding and glyphosate herbicide application at doses 960, 1920 e 3840 g ha-1. The study was performed the Agricultural Center of Palma, Federal University of Pelotas, in Rio Grande do Sul State, in a completely randomized design with four replications, with evaluations of glyphosate and aminomethylphosphonic acid (AMPA) residues in seeds and soil. The method described by Veiga et al. (2001) was used for detection and quantification of glyphosate and AMPA residues in soil end seed. All the analyses were performed in triplicate and results expressed as mean± standard derivation. Differences between means were first analyzed by ANOVA test and then least significant difference (LSD) test (p < 0.05). It was verified that the glyphosate use at recommended application doses for weed control in GR resulted the glyphosate residues in the seeds above permitted by Brazilian law. Also, AMPA residues were detected in the soil and the seeds. Keywords: glyphosate, AMPA, transgenic soybean Tel: +55 53 81153656; fax: +55 53 21231006 250 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Poster 66. GLYPHOSATE EFFECTS ON DIAZOTROPHIC BACTERIA 1 2 Giani Mariza Barwald Bohm1; César Valmor Rombaldi3 Federal Institute Sul-Rio-Grandense, IFSul-Pelotas Campus, Department of Environmental Management, RS-Brazil; zip code: 96015360, e-mail: [email protected]; Eliseu Maciel School of Agronomy, Federal University of Pelotas. Campus Universitario s/n°, Capon Lion, RS, zip code [email protected] ABSTRACT The technology of genetically modified plants (GR) is the result of innovation and strategic management in biotechnology and represents a new opportunity for the control of pests and diseases, among other possibilities. Despite the potential inherent in the biotechnology, much concern about the biosafety has been demonstrated by the population and segments of the scientific community. Regarding the cultivation of soybeans GR, environmental concerns include effects on non-target organisms. In this context, we developed this study to evaluate the effects of application of glyphosate, used in weed control for soybean crop, on the variability of strains of Rhizobium present in nodules in of plant, using the technical of restriction analysis of amplified ribosomal DNA (ARDRA). The experiment was conducted at the Agricultural Center of Palma, Federal University of Pelotas, Rio Grande do Sul, in the area of Alfissol dystrophic. Were Tested two soybean genotypes, one genetically modified (GR BRS 243 RR) and a conventional (isogenic genotype) were grown under different weed control methods: hand weeding, and glyphosate or imazethapyr herbicide application. It was found that use of glyphosate one or two applications at a dosage of 960 g ai ha-1, as well as an application of imazethapyr at a dosage of 100 g ai ha-1 BRS 243 RR, resulted in lower variability of strains of Rhizobium. Keywords: Rhizobium, ARDRA, transgenic soybean Tel: +55 53 81153656; fax: +55 53 21231006 251 7th World Congress on Allelopathy. Vigo, July 28 – August 1, 2014 Oral 140. Identification and comparative analysis of microRNAs and functional microbe associated with rice allelopathy on bardyardgrass Changxun Fang, Yingzhe Li, Xiaomei Zeng, Changliang Ye, Lihua Shen, Wenxiong Lin Fujian Provincial Key Laboratory of Agroecological Processing and Safety Monitoring (School of Life Sciences, Fujian Agric ulture and Forestry University), Fuzhou 35002, People's Republic of China 1. Key Laboratory of Crop Ecology and Molecular Physiology Fujian Agriculture and Forestry University , Fujian Province University, Fuzhou 35002, People's Republic of China （ ） ABSTRACT Rice allelopathy on weed had been reported for more than 20 years, and studies were well conducted in the last 10 years, however, the underlying mechanism has not been clearly elucidated. Regulation of gene expression is an initial response in plant interaction, and the miRNAs expression pattern on allelopathic rice PI312777 and its accompanied barndyardgrass (BYG) was investigated respectively by using high throughput sequencing technology. The results show that miRNAs relevant to plant hormone signal transduction, nucleotide excision repair, tropane, piperidine and pyridine alkaloid biosynthesis were enrich expressed on BYG, while only tropane, piperidine and pyridine alkaloid biosynthesis relative miRNAs were found enrichment on PI312777. Most of the enrich miRNAs from BYG that accompanied with PI312777 was enhanced up-regulated folds under elongation on coth cultured days, and it was peak at the 7 day. The reverse was true in the condition of BYG co-cultured with non-allelopathic rice Lemont, based on the qPCR detection of miRNAs dynamic expression under different co-cultured days. Increasing of BYG numbers accompanied with PI312777 also resulted in enhanced gene expression folds of enrich miRNAs on these BYG. Overexpression of PAL gene on PI312777 contributes to increase the miRNAs expression on the accompanied BYG, and silence of PAL gene on PI312777 leads to a reverse result. The target genes were presented a revere expression pattern in contrast to their relevant miRNAs. Moreover, increasing of BYG numbers with PI312777 resulted in decreased the IAA content but increased the apurinic/apyrimidinic sites (AP sites) in the root of BYG, and the BYG accompanied with PAL-OE transgenic lines had lowest IAA content and largest AP sites, but the reverse was true in the BYG accompanied with PAL-RNAi transgenic lines, however, no significant difference was found in the BYG co-cultured with non-allelopathic rice Lemont. In addition, BYG under phenolic acid allelochemicals treatment showed greatly enhancement on the gene expression level of the enrich miRNAs, which in turn to reduce the target gene expression. However, no significant change was found on the BYG treated by terpenoids. Further determination on microbe population numbers in the hydroponic solution showed that a special microbe named Myxococcus species were closely correlated with rice allelopathy potential. The number of Myxococcus was significantly increased following the enhancing BYG density accompanied with PI312777, and hydroponic system of PAL-OE co-cultured with BYG contained highest Myxococcus numbers while the mono-cultured of PAL-RNAi lines had lowest Myxococcus contains, however, hydroponic solution from Lemont had lower Myxococcus numbers than PI312777 that under same treatment, and no significant difference was found among different ratios of Lemont/BYG co-cultured system. Phenolic acids contributed to increased Myxococcus numbers in the hydroponic solution, and the number was higher than that of terpenoids used. BYG co-cultured in sterile hydroponic solution contained Myxococcus showed increased gene expression level on the enrich miRNAs, and the miRNAs expression level was further enhanced in the condition of Myxococcus with its metabolisms, which indicated the important role of Myxococcus on rice allelopathy. According to the research, it was clear that rice allelopathy specially suppress the IAA synthesis and DNA damage repair on the surrounding BYG, by promoting of the enhanced expression on the relevant miRNAs, which in turn to inhibit BYG growth and development, and Myxococcus species play a vital part in the process of rice allelopathy on BYG. Keywords: Allelopathy, allelochemicals miRNA, Myxococcus, rice Tel: +86-591-83737535; fax: +86-591-83769440 / 0 () )4)*)5*- $ % ! &1, 0 ! " # + ! () (%. ( )1- $ % 0 2 ! &! ' ()*)( + " # (, ,)! $ # 3 + % . &! AUTHOR INDEX Abd Elgawad, Hamada 246 Abdoli, Abbas 219 Abenavoli, Maria Rosa 48, 50, 60, 121 Abouziena, Hussein F. 167 Abu-Dieyeh, Mohammed H. 97 Abusteit, E.O. 181 Adhikari, Khem B. 237 Aguilera, Narciso 38 Ahluwalia, A.S. 92 Ahmad, Muhammad 170 Al-Abeid, H 97 Al-Khateeb, Tamara A. 227 Al-Wakeel, S.A.M. 200 Alam Cheema, Sardar 136 Albayrak, Barış 164 Alboghbeish, J. 166 Alencar, Aneliz B.A. 169 Alias Gallego, Juan Carlos 186 Aliki, Hadi M. 158 Allen, Joey 118, 119 Alsaadawi, Ibrahim 110, 160, 227 Álvarez-Iglesias, Lorena 191 Alves, Pedro Luís da Costa Aguiar 56, 57, 154, 179 Amjad, Sehar 141 An, Min 162, 183, 184 Anaya, Ana Luisa 83, 247 Ancuceanu, Viorel Robert 95 Andrade, Paula B. 175 Andrzejczak, Olga 213, 214 Antosik, Anna 213, 214 Araniti, Fabrizio 48, 50, 68, 121 Arianfar, Mehri 152 Asaduzzaman, Md. 111, 162 Aslam, Farhena 54, 110 Ata Cheema, Zahid 136, 178 Ata, Zahid 180 Ayuso, Jesús 78 Aziz, A. 240 Babainezhad, B. 166 Back, Matthew A. 158 Baerson, Scott R. 82, 232 Bakhshayeshan-Agdam, Hamide 150, 153 Baldoqui, Debora C. 66 Baldy, Virginie 93, 236 Baltasar, Paula 73 Bano, Asghari 129, 170 Barbosa, Mauro C. 66, 169 Barceló, Juan 17 Bardon, Clément 127 Bargagli, A. 121 Barreiro Aldo, 16 252 Basra, Shahzad M.A. 163, 194 Batool, Asima 244 Bellvert, F. 91 Bellvert, Floriant 127 Bensaddek, Lamine 190 Berestetskiy, Alexander O. 114 Bernardi, Darlon I. 66 Binet, Philippe 91 Bogatek, Renata 213, 214, 215, 218 Bohm, Giani Mariza Barwald 250, 251 Bortolo-Coelho, Tiara da Silva 207 Borucki, Wojciech 213 Bousquet-Mélou, Anne 87, 187, 188 Bouyssou, Marine 93 Bubna, Gisele Adriana 203 Cabeiras, Luz 72 Cabot, Catalina 17 Cabreira, João Paulo Ferreira 179 Cajthaml, Tomáš 44 Cala, Antonio 78, 171 Calle, Juan M. 80 Candido, Lafayette P. 69 Casas-Cardoso, Lourdes 39 Cavaleiro, Carlos 191, 195 Čekanauska, Sigitas 106 Čepulienė, Rita 106 Chakraborty, Nabanita 36, 142 Chambery, Angela 62 Chaudhry, Zubeda 143 Chaves Lobón, Natividad 186 Chen, Hsiao-Ting 86 Chen, Xin Jian 45, 105, 198, 201 Chen, Z. X. 115 Cheng, Zhihui 67, 204 Chiapusio, Geneviève 91 Chinchilla, Nuria 20, 76, 172, 173 Chludil, Hugo D. 137, 138 Choi, Young Hae 42 Chomel, Mathilde 236 Chou, Chang-Hung 49, 86 Ciacka, K. 215 Closset-Kopp, Deborah 190 Colby, Tom 220 Comte, G. 91 Constantin, Jamil 66 Costas Gil, Aitana 40, 65, 71, 72, 94, 211, 217 Croce, Antonio 90 Cruz-Ortega, Rocío 83 Cumbrera, Carlos R. 61 D.Almeida, Jéssica 66 D’Abrosca, Brigida 29, 42, 46, 53, 62, 90 D´Addio, Elisabetta 188 Da Silva, Hingrid A. 208 Dadkhah, A. 166 Daftari, Behnam 168 De las Heras, P. 99 De Moraes, Consuelo 26 De Souza Bido, Graciene 208 Deaker, Rosaling 129 Decocq, Guillaume 190 Del Fueyo, P.A. 138 Delgado, Guillermo 247 Demczuk, Anna 147, 148 Desrochers, Annie 236 Dhumal, K.N. 101 Díaz-Tielas, Carla 40, 41, 65, 71, 73, 205, 210, 217 Dinu, Mihaela 95 DiTommaso, Antonio 170 Doğan, Adnan 164 Dostál, Petr 44 Du, Jiafang 105, 201 Duke, Sara 225 Duke, Stephen O. 56 Durán, Alexandra F. 94, 173 Eigemann, Falk 120 El Marsini, Zohuir 39 el Zahar, Haichar Feth 127 El-Darier, Salama M. 144 El-Taher, Halilat M. 144 Esposito, Assunta 29, 42, 46, 53, 62, 90 Etzerodt, Thomas 112 Eyini, M. 161, 242 Farooq, Muhammad 136, 178, 239 Feng, Fa-jie 140 Fernández, Benito 79, 116 Fernandez, Catherine 87, 93, 187, 188 Ferrarese-Filho, Osvaldo 203, 207, 208 Ferrarese, Maria de Lourdes Lucio 203, 208 Fidelis Giancotti, Paulo Roberto 56, 57 Filary, Barbara 220 Fiorentino, Antonio 29, 42, 46, 48, 53, 62, 90 Fiorucci, Letycia L.R. 66 Fiumano, Vittorio 53, 62 Fomsgaard, Inge S. 23, 43, 51, 112, 128, 223, 233, 237 Franco, J.M. 123, 125 Freitas, Helena 38 Friedel, J.K. 145 Fritz, Ines 145, 248 Fronczek, Frank R. 75 Fu, Jian 222 Fu, Yang 100, 226 Gabr, M.A. 200 Galindo, José L.G. 79, 116 Galindo, Juan C. G. 116, 171, 173 Gallego, Berta 17 Gallet, Christiane 87 Ganimede, Cristina 104, 113 Ganiver, Jordane 187 García, Yedra 21 Gealy, David 224, 225 Gelsomino, A. 50, 68 Gennary, M. 121 Ghazanfar, Bushra 109 Ghooray, Kala 171 Gnanasoundari, P. 174 Gniazdowska, Agnieszka 213, 214, 215, 218 Golini, Mimmo 53 Gollner, G. 145 González, Luís 38, 192, 195 González, María C. 83 Graj, Weronika 128 Graña, Elisa 40, 41, 71, 72, 73, 205, 210, 211, 217 Graziani, Vittoria 90 Gregersen, Per L. 223 Grossart, Hans-Peter 120 Grzyś, Edward 147, 148 Gualtieri, Sonia C.J. 69, 70 Guerra, José O. 80 Guerrero-Vásquez, Guillermo A. 76, 172 Gullì, T. 68 Guo, Yi-qing 100, 222, 226 Gurr, Geoff M. 74, 94, 98 Habibian, Leila 168 Hadwan, Hameed A. 227 Haghi-kia, Sevda 104 Hamed, B.A. 200 Han, Xu 67 Harzen, Anne 220 Hawale, J.A. 241 He, Hai-Bin 60, 135 Hegab, Montaz M. 200, 246 Hennig, Lothar 220 Hernández-Bautista, Blanca E. 83, 247 Hilt, Sabine 120 Hoeksema, Jason D. 82 Hofmann, Diana 220, 228 Hossain, Sajjad 219 Hovanet, Marilena Viorica 95 Huanwen, Meng 107 Hussain, M. Iftikhar 159, 199, 216 Hussain, Saira 111 Ibrahim, R.A. 181 Igartuburu, J.M. 52 Ihsam-Ul-haq 141 Iqbal, Javaid 170 253 Ishida, Miki 231 Ishii-Iwamoto, Emy L. 66, 169 Işik, Ayşe 164 Islam, A.K.M. Mominul 63, 134 Itaya, Tomotaka 212 Jackson, Colin R. 82 Jamil, Muhammad 180 Jamil, Saiful Z. 98 Jandová, Kateřina 44 Jayakumar, M. 161, 242 Jeev, Mary Sajitha 161 Jensen Bettina M. 237 Jensen Karl-Martin Vagn 233 Jia, Yulin 224 John, Maninang 34 Jørgensen, Lise N. 112 Jose, Shibu 141 Kagami, Fernanda L. 66, 169 Kahle, Hannah 193, 228 Kamal, Javed 155 Kameník, Zdeněk 44 Kashif, Muhammad Saleen 178 Kato-Noguchi, Hisashi 63, 134, 182, 212 Kennedy, Ivan R. 129 Khaliq, Abdul 54, 110 Khan Rahamdad 139 Khan, Abdul Rehman 109 Khan, Ijaz Ahmad 139 Khan, Muhammad Azam 109 Kharytonova, I.P. 243 Kimura, Fukiko 182 Kirkegaard, Sidsel 112 Kosteckas, Robertas 106 Kozak, Agnieszka 202, 206 Krämer, Philip 193 Krasuska, Urszula 213, 214, 215 Kriaučiūnienė, Zita 106 Kristjansen, Helene112 Kun, Sun 238 Lærke, Helle N. 237 Lahmod, Nabil R. 160 Lahmood, Nabil R. 227 Larchevêque, Marie 236 Laursen, Bente B. 112 Laursen, Bente B. 237 Lazar, Veronica 95 Ledo, David 211 Leflaive, Joséphine118, 119 Leicach, Silvia R. 137, 138 Lemerle, Deirdre 162 Leszczynski, Bogumil 202, 206 Li, Jia-Yu 60, 135 Li, Juan 105, 201 Li, Lansheng 124 254 Li, Le 124 Li, Ming Jie 45, 140, 198 Li, Rui-xiang 100 Li, Xiaoning 245 Li, Xuan-zhen 140 Liang, Xiaoting 85, 88 Lilholt, Jette 112 Lin Wenxiong 103 Lin, Wen Xiong 45,108, 198 Lin, Zhi-Hua 60, 135 Llugany, Mercè 17 Longueira, Sabela 72 López-González, David 40, 65, 71, 211 López-Nogueira, Tonio 191 Lorenzo, Paula 38 Lovatt, Carol J. 194 Lu, Jin 245 Lu, Kai 232 Lu, Yongliang 222 Luckett, David J. 162 Luo, Shiming 85, 88, 232 Macías-Rubalcaba, Martha L. 83, 247 Macías, Francisco Antonio 20, 39, 52, 61, 69, 70, 75, 76, 78, 79, 80, 94, 96, 116, 154, 171, 172, 173 Majeed, Abdul 143 Mallik, Azim 85 Mandal Biswas, Suparna 36, 142 Mantell, Casimiro 39 Mantovanelli, Gislaine C. 66, 169 Maqbool, Nazimah 146 Marchiosi, Rogério 207, 208 Marcinkevičienė, Aušra 106 Marelli M. 68 Marinas, Ioana Cristina 95 Marocco, Adriano 104, 113 Martin, Jason 56 Martínez de la Ossa, Enrique J. 39 Martínez-Peñalver, Ana 40,41, 71, 73, 210, 216, 217 Martins, Pedro de Figueredo Rocha Barbosa 179 Martos, Soledad 17 Maryia, Mishyna 77 Matloob, Amar 54, 110 Matsuda, Kazuhiko 231 Meepagala , Kumudini M. 56 Mehmood, Azhar 102 Meiffren, Guillaume 127 Meng, Huanwen 67 Mescher, Mark 26 Mezori, Hassan A.M. 189 Min-Wang, Chao 49, 86 Miranda, Maria A.F.M. 70 Miranda, Mariana 247 Mirza, Bushra 141 Mirzaali, Abdoltaher 168 Mito, Márcio S. 66, 169 Mohney, Brian K. 35 Moldenhauer, Karen A. 225 Molinillo, José M.G. 20, 39, 61, 69, 70, 75, 76, 78, 96, 154, 171, 172, 173 Mondani, Letizia 104 Monnier, Yogan 87 Montesinos, Daniel 21 Morimoto, Masanori 231 Motafakkerazad, Rouhollah 150, 151, 152, 153 Mouna, Mehani 130 Muhammad, Zahir 143 Muhammadi, Gholam R. 176, 177 Munik Bevilaqua, Jennifer 203 Muñoz, Luis 38 Mwendwa, James 74, 84 Nabeel, Muhammad 239 Nakai, Satoshi 122 Naoya, Wasano 34, 47 Naoyuki, Tamura 34 Nasrine, Salhi 144 Natarajan, S 209 Nawaz, Ahmad 239 Nepomuceno, Mariluce Pascoina 57, 154, 179 Nesrin, Salhi 130 Nicolaisen, Mogens 128 Nikolai, Laman 77 Nikpey, Yousef 168 Ninkovic, Velemir 234 Nishijima, Wataru 122 Norouzi, Negin 176 Norouzi, Yasin 177 Nosratti, Iraj 176, 177 Novaes, Paula 61, 96 Ohno, Osamu 63, 134, 182 Okuda, Tetsuji 122 Olechowicz, Joanna 213, 214 Oliveira Jr., Rubem S. 66 Oprea, Eliza 95 Özpinar, Hüseyin 164 Paetz, Christian 104 Pan, Zhiqiang 82, 232 Pardo-Muras, María 191 Pavliuchenko N.A. 52, 243 Pawar, K.B. 149, 241 Peano, Samuel 187 Pedersen, Hans Albert 43 Pedras, M. Soledade C. 126, 219 Pedrol, Nuria 175, 191, 230 Pérez-Corona, M.E. 99 Pérez, Andy J. 80 Periyakaruppiah, P. 242 Pinson, Shannon R.M. 224 Piola, Florence 127 Pires da Silva, Bruna 57, 179 Pold, V. 145 Poluektova, Ekaterina 114 Poly, Franck 127 Pommier, Thomas 127 Popova, Alexandra 93 Portela, G. 125 Poschenrieder, Charlotte 17 Poulsen, Lars K. 237 Pratley, James E. 162 Prévosto, Bernard 97 Proffit, Magali 93 Puig, Carolina G. 175, 192, 230 Puijalon, Sara 127 Pupalienė, Rita 106 Qasem, Jamal R. 37, 89 Qi, Hui 105, 201 Radwan, S.M.A. 167 Rakhshaie, Elham 151 Rana, Shah Jahan 109 Rasmussen, Jim 128 Rawal, A.V. 149 Reade, John P.H. 158 Reboredo, Jonathan 38 Reguera, B. 123 Reigosa, Manuel J. 40, 41, 65, 71, 72, 73, 94, 159, 175, 199, 205, 210, 211, 216, 217, 230 Reyes-Chilpa, Ricardo 247 Rial, P. 123 Ričardas, Paškauskas 120 Riobó, P. 123, 125 Rodríguez-Echeverría, Susana 38 Rodríguez, F. 123 Rodríguez, Jonatan 38 Romagni, Joane 116 Rombaldi, César Valmor 250, 251 Rotherm, Kristin 228 Ru, Rui-hong 140 Rúa, Megan A. 82 Rudzińska-Langwald, Anna 218 Rupasinghe, Maduka 35 Ruşen, Mine164, 165 Ryan, Brigette 74 Sacała, Elżbieta 147, 148 Salehi-lisar, Seyed Yahya 150, 151, 152, 153 Sánchez Moreiras, Adela M 40, 41, 65, 71, 72, 73, 205, 210, 211, 216, 217 Santana, Alejandro 75 Santonja, Mathieu 93, 187, 188 Sarragiotto, Maria H. 169 255 Saura, José A.A. 78 Scalise, A. 50 Schmidt, Jürgen 220 Schneider, Bernd 104 Schneider, Léa 250 Schnoor, Heidi J. 237 Schulz, Margot 104, 113, 193, 220, 228 Scognamiglio, Monica 29, 42, 46, 48, 53, 62, 90 Seetharaman, P. 242 Segni, Ladjel 130 Severino, Valeria 62 Shao, Hua 229 Shatty, Resan K. 160 Shen, Junyan 105, 201 Shi, Ruxia 245 Shihada, Nader 35 Sicker, Dieter 104, 220 Silva, Adriano A. 169 Simagina, N.O. 131, 249 Simonet, Ana M. 80 Singh, Ombir 185 Siqueira-Soares, Rita de Cássia 208 Skoneczny, Dominik 74, 84, 94, 98 Sokolov, Dmitry V. 74 Soltys, Dorota 218 Somasundaram, E. 174 Song, Yuanyuan 85, 232 Sosa Díaz, Teresa 186 Souza-Alonso, Pablo 192, 195 Stanton, Rex 183, 184 Staszek, Pawel 214, 215 Statti, G. 68 Steffensen, Stine Krogh 51 Strasser-Altrogge, Stephan 248 Suenaga, Kiyotake 63, 134 Sultana, Shamima 111 Sun, Yiding 222 Suryawanshi, M.V. 241 Švanys, Algirdas 120 Sytykiewicz, Hubert 202, 206 Tabaglio, Vicenzo 104, 113 Tada, Yasuomi 212 Taheruzzaman, Quazi 235 Taleb, Wafaa Abu 97 Tang, Dong-Sheng 100 Tanveer, Asif 54, 102, 110 Tanwir, Fariha 223 Tchong, Theodore 187 Ten-Hage, Loïc118 Terzi, Valeria 130 Tinmaz, Ahmet B. 164 To, Huy 126 Tolrà, Roser 17 256 Torres, Ascensión 20, 39, 69, 70, 79, 94, 116 Toussaint, M.L. 91 Tsafack, Noelline 127 Tsafantakis, Nikolaos 46, 62 Tursun, Nihat 165 Ulbrich, Andreas 193, 228 Ullah, Nazif 141 Uludağ, Ahmet 164, 165 Uremis, Ílhan 165 Valares Masa, Cristina 186 Valderrama Parizotto, Ângela 203 Valentão, Patricia 175 Valery, Prokhorov 77 Varela, Rosa M 20, 39, 61, 69, 70, 75, 96, 154, 172 Vasconcelos Vitor 16 Vázquez de Aldana, B.R. 99 Velička, Rimantas 106 Verpoorte, Robert 15, 42 Vidkjær, Nanna Hjort 233 Viganó, Joselaine 207 Vincent, Jassey 91 Wahid, A. 146, 240 Wahid, Abdul 244 Wali, Salih T. 189 Wang, Chao-Min Wang, Feng Qing 45, 198 Wang, Fengqing 105, 201 Wang, Fenq-qing 140 Wang, Jun 201 Wang, Mengyi 204 Wang, Peng Fei 45, 198 Wang, Ruilong 85 Wang, Xiuhai 124 Wei-Wei, Gao 55 Weidenhamer, Jeffrey D. 35, 84 Weston, Leslie A. 74, 84, 94, 98 Weston, Paul A. 74, 84, 94, 98 Wilson, Neil 129 Witten, Marianne 237 Wollenweber, Bernd 233 Wu, Hanwen 183, 184 Wu, Linkun 103 Xiao-Bao, Bi 55 Xiao-Lin, Jiao 55 Xu Weiling 226 Xuemei, Xiao 107 Yaber Grass, M.A. 138 Yadav, Prakrati 64 Yadava, R.N. 64 Yang, Fan 245 Yang, Ning 238 Yang, Yingli 245 Yasmin, Humaira 129 Yaya, Estifanos 126 Ye, Mao 232 Yilmaz, Hasbi 164 Yinghu, Liu 88 Yoshiharu, Fujii 34, 47, 77 Younesabadi, Masoumeh 168 Yu, Zhen-Ming 60, 135 Zahir, Zahir Ahmad 54 Zahra, Syeda Tahreem 170 Zaimenko, N.V. 243 Zeng, Rensen 85, 88, 232 Zhang, Bao 140 Zhang, Chi 229 Zhang, Jinbiao 183, 184 Zhang, Qi 135 Zhang, Zhong Yi 45 Zhang, Zhong-yi 140, 198 Zhang, Zhongyi 105, 201 Zhihui, Cheng 107, 109 Zhou, B.L. 115 Zhu, Xiaocheng 74, 84 Zinta, Gaurav 246 Zumbo, A. 50 257