Biological Control 49 (2009) 207–213 Contents lists available at ScienceDirect Biological Control journal homepage: www.elsevier.com/locate/ybcon Internal transcribed spacer-2 restriction fragment length polymorphism (ITS-2-RFLP) tool to differentiate some exotic and indigenous trichogrammatid egg parasitoids in India G. Ashok Kumar a, Sushil K. Jalali a,*, T. Venkatesan a, Richard Stouthamer b, P. Niranjana a, Y. Lalitha a a b Insect Biotechnology Laboratory, Post Bag No. 2491, H.A. Farm Post, Bellary Road, Hebbal, Bangalore 560024, Karnataka, India Department of Entomology, University of California, Riverside, CA, USA a r t i c l e i n f o Article history: Received 27 March 2008 Accepted 6 February 2009 Available online 20 February 2009 Keywords: Molecular identification ITS-2-RFLP Trichogrammatids Restriction enzymes a b s t r a c t ITS-2-RFLP method was employed to distinguish 12 different trichogrammatids consisting of indigenous and exotic species such as Trichogrammatoidea armigera and Tr. bactrae, Trichogramma achaeae, T. chilonis, T. japonicum, T. embryophagum, T. pretiosum (Thelytokous Form—TF), T. brassicae, T. dendrolimi, T. evanescens and T. mwanzai. ITS-2 region was amplified; complete ITS-2 sequences of nine species were deposited in Genbank. The size of the amplified product ranged from 500 to 900 bp. Restriction enzyme digestion of ITS-2 region showed different banding profiles for these 12 species. Dichotomous keys using the size of the ITS-2 product and the restriction fragment length polymorphism for the enzymes (EcoRI, MseI, MvaI, and TaqI) allowed quick species identification of these trichogrammatids. Ó 2009 Elsevier Inc. All rights reserved. 1. Introduction The family Trichogrammatidae consists of about 650 species (Grissel and Schauff, 1990) and is the most widely used taxon of natural enemies in various biocontrol programs worldwide. Although about 210 species of Trichogramma are known to attack the eggs of various crop pests (Pinto, 1998), at least 12 species are used in biological control programs. With the increasing preference for sustainable agriculture and awareness about side effects from indiscriminate use of insecticides, biological control agents are attracting more attention. However, with so many species, ecotypes and strains available and very frequent exchange of culture material, correct identification is the first step for a successful biological control program. Morphological identification of trichogrammatids remains difficult because of their minute size (<1 mm long), is time consuming and requires specialized skills (Pinto and Stouthamer, 1994). Morphological identification of most species is based on subtle differences in male genitalia (Nagarkatti and Nagaraja, 1971, 1977). Many important species share similar genital structures, forcing workers to rely on less dependable characters that often are intra-specifically variable (Pinto et al., 1989). Given the economic importance of trichogrammatids in biological control programs, species identification must be quick, simple and widely applicable. Novel approaches that used DNA sequences of the internal transcribed spacer 2 (ITS-2) helped to solve this difficulty (Stouthamer * Corresponding author. Fax: +91 080 23411961. E-mail address: [email protected] (S.K. Jalali). 1049-9644/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2009.02.010 et al., 1999). Ribosomal RNA (rDNA) comprises of three genes 18S, 5.8S and 28S separated by internal transcribed spacers (ITS-1 and ITS-2). ITS regions evolve faster than the regions coding for the ribosomal RNA (Hoy, 1994). The ITS region is widely used in identification studies (Hillis and Dixon, 1991). Sequence and restriction analysis of ITS-2 region has been used to distinguish Trichogramma species collected from tomato fields in Portugal (Silva et al., 1999). In India, twenty six species of trichogrammatids have been recorded and among them Trichogrammatoidea armigera, Tr. bactrae, Trichogramma achaeae, T. chilonis and T. japonicum are widely distributed and used against lepidopteran pests in several crops (Singh and Jalali, 1994; Jalali et al., 2003). Elsewhere in the world T. evanescens, T. dendrolimi, T. pretiosum and T. brassicae are most frequently used in the field (Smith, 1996). Trichogramma galloi is now extensively used as biocontrol agent in sugar cane fields of Brazil (Parra and Zucchi, 2004). In the past decade, several exotic species have been introduced in India to target pests such as codling moth (Cydia pomonella Linnaeus), diamondback moth (Plutella xylostella Linnaeus), corn borer [Chilo partellus (Swinhoe)] and American bollworm (Helicoverpa armigera Hübner) (Jalali et al., 2003). With the introduction of these non-native trichogrammatid species in addition to the native ones, there is a need for a quick and reliable identification technique to identify these parasitoids. While the identification of males of these species could be done using morphological features, many of the specimens collected from the field are females that are not identifiable using morphology. In the present study we develop an identification method based on the ITS-2 sequences of the 12 most commonly used spe- 208 G. Ashok Kumar et al. / Biological Control 49 (2009) 207–213 Table 1 Trichogrammatids studied, analysis performed, identity and origin of the cultures. Line designate Studies Species Origin Collection year Tra1 1,2 India 1990 Trb1 Ta1 Tj1 Tem1 Tc1 Tp1 Tev1 Tm1 Tbra1 Td1 Tbrass1 1,2 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 Trichogrammatoidea armigera Trichogrammatoidea bactrae Trichogramma achaeae T. japonicum T. embryophagum T. chilonis T. pretiosum T. evanescens T. mwanzai T. pretiosum (TF) T. dendrolimi T. brassicae India India India Russia India USA France Kenya USA Germany USA 1990 1990 1990 1993 1990 1993 2004 2003 1993 1994 2004 1, ITS-2; 2, sequencing; 3, restriction analysis. cies in India: Trichogrammatoidea armigera Manjunath, Tr. bactrae Nagraja, Trichogramma achaeae Nagaraja and Nagarkatti, T. chilonis Ishii, T. japonicum Ashmead, T. embryophagum Hartig, T. pretiosum Riley, T. pretiosum Riley (Thelytokous form-TF), T. brassicae Bezdenko, T. dendrolimi Matsumura, T. evanescens Westwood and T. mwanzai Schulten and Feijen. 2. Materials and methods 2.1. Parasitoid culture We studied 12 isofemale lines identified by Dr. H. Nagaraja as Trichogrammatoidea armigera, and Tr. bactrae, Trichogramma Fig. 1. PCR amplified ITS-2 plus flanking regions of 5.8s and 28s rDNA from different trichogrammatids (Lanes: M, 50 bp DNA perfect ladder (Novagen); 1, Tr. armigera; 2, Tr. bactrae; 3, T. achaeae; 4, T. embryophagum; 5, T. japonicum; 6, T. chilonis; 7, T. dendrolimi; 8, T. evanescens; 9, T. mwanzai; 10, T. brassicae; 11, T. pretiosum (TF); 12, T. pretiosum). achaeae, T. chilonis, T. japonicum (all indigenous), T. embryophagum, T. pretiosum, T. pretiosum (TF) often mistakenly referred to as T. brasiliense Ashmead (Pinto, 1997), T. brassicae, T. dendrolimi, T. evanescens and T. mwanzai (all exotic). Voucher specimens are kept by Dr. H. Nagaraja in PDBC (Bangalore, India). All cultures were maintained on rice moth, Corcyra cephalonica (Stainton) eggs (killed by ultraviolet irradiation) at 26 ± 1 °C, RH 60 ± 5%, LD 14:10. The origin of each species studied is provided in Table 1. Fig. 2. (a) Multiple sequence alignment of ITS-2 region of group II Trichogramma species. (b) ITS-2 sequences of group III Trichogramma species (A dash indicates that the nucleotides are absent,  indicates for similar nucleotides). G. Ashok Kumar et al. / Biological Control 49 (2009) 207–213 209 Fig. 2 (continued) 2.2. Isolation of DNA 2.3. ITS-2 PCR DNA was extracted using chelating agent Chelex100 (5%) (Biorad). One or two wasp previously frozen at 80 °C were crushed with a micro pestle in 20 ll of chelex. The homogenate was incubated at 56 °C for 3 h followed by 10 min at 100 °C. The supernatant was stored at 20 °C until used. The PCR reaction was performed in 50 ll reaction volumes using Biorad icycler, 2 ll DNA template, 5 ll (10) Taq assay buffer, 1 ll dNTP’s (each in 10 mM concentration), 1 ll forward and reverse primers (10 picomoles/ll), 0.25 ll taq polymerase (1 U). The primers used to amplify the ITS-2 region were 210 G. Ashok Kumar et al. / Biological Control 49 (2009) 207–213 Fig. 2 (continued) 50 -TGTGAACTGCAGGACACATG-30 (forward) and 50 -GTCTTGCCTG CTCTGAG-30 (reverse) (Stouthamer et al., 1999). The thermal cycling condition for PCR consisted of 30 cycles (Den: 94 °C for 1 min, Ann: 55 °C for 1 min, Ext: 72 °C for 2 min, with an initial denaturation: 95 °C for 5 min and final extension at 72 °C for 10 min). PCR products were electrophoresed on 1.8% agarose gel (ACROS). Gels were stained using ethidium bromide. Molecular standards were run along with the samples for reference. 2.4. Sequencing The PCR products were gel eluted using Qiagen gel clean up kit and direct sequenced using an Applied Prism 310. The resulting sequences were blasted against sequences in GenBank to confirm that the sequence was indeed ITS-2. 2.5. Restriction enzyme analysis Restriction digestion of the PCR product was performed in a 20 ll volume (10 ll PCR product, 2 ll (10) reaction buffer, 1 ll reaction enzyme and 7 ll distilled water). The mixture was incubated overnight at appropriate temperatures as per manufacturer’s specifications. RFLP products were analyzed by electrophoresis in 1.8% agarose gel. 50 bp DNA perfect ladder (Novagen) was included to estimate the size of restriction fragment. Four restriction enzymes used were EcoRI, MseI, MvaI and TaqI (MBI Fermentos). EcoRI was common enzyme selected for all the species and MseI to differentiate between T. chilonis, T. dendrolimi, T. evansecens, T. mwanzai, T. brassicae and T. pretiosum. These restriction enzymes were selected using Bioedit (Hall, 1999) restriction fragment analysis, as these were indicative for trichogrammatids used in the study. Table 2 Restriction sizes following digestion of PCR amplified ITS-2 plus flanking regions of 5.8s and 28s rDNA of 12 trichogrammatids with various restriction enzymes. Species Accession No. Group I (800–850 bp) Tr. armigera Tr. bactrae ITS-2 size EU251072 EU251071 900 bp 800 bp T. achaeae T. embryophagum EU251070 DQ344044 ITS-2 size 627 bp 592 bp T. japonicum DQ471294 565 bp T. chilonis DQ220703 ITS-2 size 538 bp EcoRI 233 bp 305 bp MseI  MvaI 470 bp 68 bp T. dendrolimi DQ344045 515 bp T. evanescens DQ381280 546 bp T. mwanzai DQ381279 500 bp T. brassicae DQ314611 522 bp 263 bp 252 bp 268 bp 278 bp 243 bp 257 bp 300 bp 222 bp 447 bp 68 bp 478 bp 68 bp 432 bp 68 bp 454 bp 68 bp T. pretiosum (thelytokous form) DQ381281 525 bp  434 bp 87 bp 454 bp 92 bp 447 bp 53 bp 412 bp 32 bp 68 bp  T. pretiosum DQ525178 528 bp   Group II (570–620 bp) EcoRI  229 bp 363 bp  Group III (500–550 bp) Note:  denotes no site present. a Only bands larger than 100 bp are listed. 457 bp 68 bp 393 bp 68 bp 63 bp TaqIa 141 bp 129 bp 143 bp 144 bp 129 bp 305 bp 323 bp 138 bp 135 bp 129 bp 307 bp 309 bp G. Ashok Kumar et al. / Biological Control 49 (2009) 207–213 Fig. 3. PCR amplified ITS-2 region from different trichogrammatids belonging to Group II digested with EcoRI (lanes1–3), (Lanes: M, 50 bp DNA ladder; 1, T. achaeae; 2, T. embryophagum; 3, T. japonicum). 3. Results 3.1. ITS-2 PCR and sequencing Based on the size of the ITS-2 rDNA PCR products, base pairs varied from 500 to 900 bp in the 12 trichogrammatid species used in the study (Fig. 1). Based on this size variation, three groups could be distinguished: Group I included Tr. armigera and Tr. bactrae, in which the size of ITS-2 product varied from 800 to 900 bp; Group II included T. achaeae, T. embryophagum and T. japonicum in which Fig. 4. PCR amplified ITS-2 plus flanking regions of 5.8s and 28s rDNA from different trichogrammatids belonging to Group III digested with EcoRI (Lanes: M, 50 bp DNA ladder; 1, T. chilonis; 2, T. dendrolimi; 3, T. evanescens; 4, T. mwanzai; 5, T. brassicae; 6, T. pretiosum (TF); 7, T. pretiosum. 211 Fig. 5. PCR amplified ITS-2 plus flanking regions of 5.8s and 28s rDNA from different trichogrammatids belonging to Group III digested with MvaI (Lanes: M, 50 bp DNA ladder; 1, T. chilonis; 2, T. dendrolimi; 3, T. evanescens; 4, T. mwanzai; 5, T. brassicae; 6, T. pretiosum (TF); 7, T. pretiosum. the size of ITS-2 product varied from 570 to 630 bp and Group III included T. brassicae, T. chilonis, T. dendrolimi, T. evanescens, T. mwanzai, T. pretiosum, and T. pretiosum (TF), in which the size of ITS-2 PCR products varied from 500 to 560 bp. These groups could be easily recognized after gel electrophoresis. Complete ITS-2 sequences of different species (Fig. 2a and b) have been deposited with NCBI Genbank (Table 2). 3.2. Restriction enzyme analysis Restriction digestion with PCR product obtained from two wasp for each species (Table 2) gave reproducible profiles. EcoRI showed no sites for T. achaeae, T. japonicum, T. pretiosum (TF) and T. pretiosum (Figs. 3 and 4). MseI showed no sites for T. chilonis, T. mwanzai, T. pretiosum (TF) and T. pretiosum (Fig. 6). Trichogrammatoidea Fig. 6. PCR amplified ITS-2 plus flanking regions of 5.8s and 28s rDNA from different trichogrammatids belonging to Group III digested with MseI (Lanes: M, 50 bp DNA ladder; 1, T. chilonis; 2, T. dendrolimi; 3, T. evanescens; 4, T. mwanzai; 5, T. brassicae; 6, T. pretiosum (TF); 7, T. pretiosum). 212 G. Ashok Kumar et al. / Biological Control 49 (2009) 207–213 4. Discussion Fig. 7. PCR amplified ITS-2 plus flanking regions of 5.8s and 28s rDNA from different trichogrammatids belonging to Group III digested with TaqI (Lanes: M, 50 bp DNA ladder; 1, T. chilonis; 2, T. dendrolimi; 3, T. evanescens; 4, T. mwanzai; 5, T. brassicae; 6, T. pretiosum (TF); 7, T. pretiosum). Table 3 Dichotomous key to differentiate the 12 Trichogramma species based on PCR product of the ITS-2 region plus flanking regions of the 5.8S and 28S rDNA and restriction analysis with various restriction enzymes. 1 2 3 4 5 6 7 8 9 10 Size of the PCR product larger than 700 bp Size of the PCR product smaller than 700 bp Size of the PCR product 850 bp Size of the PCR product 800 bp Size of the PCR product larger than 560 bp Size of the PCR product smaller than 560 bp PCR product restricted with EcoRI results in two bands PCR product restricted with EcoRI results in a single band Size of PCR product larger than 600 bp Size of the PCR product 600 bp PCR product restricted with MseI results in a single band >500 bp PCR product restricted with MseI results at least one band <500 bp PCR product restricted with EcoRI results in two bands (240, 300 bp) PCR product restricted with EcoRI results in a single band PCR product restricted with EcoRI results in two bands clearly different in size PCR product restricted with EcoRI results in two bands very similar in size often only visible as a single band Size of the band 275 bp Size of the band 250 bp PCR product restricted with TaqI results in largest band of >300 bp PCR product restricted with TaqI in largest product of <150 bp 2 3 Tr. armigera Tr. bactrae 4 6 T. embryophagum 5 T. achaeae T. japonicum 7 8 T. chilonis T. pretiosum T. brassicae 9 T. evanescens 10 T. mwanzai T. dendrolimi The ITS-2-RFLP tool was able to differentiate the 12 Trichogramma species used in this study. Length variation was observed in the ITS-2 region and it clearly separated the two genera, i.e., Trichogramma and Trichogrammatoidea from each other. Such length variation in the ITS region has been reported before (Orrego and Agudelo-Silva, 1993; Sappal et al., 1995). Using the length variation and the RFLP patterns we were able to construct a dichotomous key (Table 3). Studies on ITS-2-RFLP analysis to identify Trichogramma species have been limited to particular geographical regions (Silva et al., 1999; Ciociola et al., 2001; Thompson et al., 2003) or particular crops over a wide area (Pinto et al., 2002). This is one of the first attempts to differentiate some indigenous and exotic species involved in various biological control programmes in India. The intra-specific variability in both total ITS-2 length and restriction fragments length appears to be low within the two genera studied, as was found earlier in the analysis of ITS-2 sequences of some North American species (Stouthamer et al., 1999). PCR–RFLP analysis is very simple, fast and reliable. PCR–RFLP tool has been used in detection and species identification of fungal pathogen and nematodes (Dendis et al., 2003; Roberta et al., 2003). The restriction pattern shown for the MvaI digestion of the arrhenotokous form shows that the ITS-2 for this species is polymorphic for the MvaI site, so within individuals of the arrhenotokous T. pretiosum two different ITS-2 families exist one with the restriction site and one without that site. This is visible in the gel because for the arrhenotokous population two bands are visible a larger band of the same size as the thelytokous form and a smaller band only found in the arrhenotokous form. Slight variation within individuals is commonly found in Trichogramma wasps, for instance Trichogramma kaykai has a similar polymorphism for an EcoRI site (Stouthamer et al., 1999). The differentiation using MvaI between the arrhenotokous and thelytokous form of T. pretiosum works for these particular populations that have been imported to India. However, other arrhenotokous populations of T. pretiosum originating from Brazil and some from Riverside, California also show the same pattern as the arrhenotokous form shown here (see for instance GenBank Accession Nos. AF082820, AY187261). These methods are intended to replace the traditional morphological methods when large number of individuals need to be identified that belong to a known group of trichogrammatids. For instance, for checking laboratory contamination or the fate of released species in the field. These DNA based methods are faster, because no mounting of the specimens is required, and allows identification of both females and males. Application of the DNA based methods should free up time for systematists to identify and describe new species. From the present study it can be concluded that rDNA markers and restriction enzyme patterns can be used for reliable differentiation between two genera and also between various indigenous and exotic species to be field evaluated in India. Acknowledgments armigera and Tr. bactrae belonging to genus Trichogrammatoidea could be easily differentiated after amplification of ITS-2 region. Restriction digestion with EcoRI enzyme enabled differentiation of 10 species, while restriction digestion with MseI enabled differentiation of six species. MvaI enzyme (Fig. 5) proved useful to differentiate T. pretiosum (TF) from T. pretiosum. Cleavage activity of TaqI (Fig. 7) enzyme aided in discrimination of T. dendrolimi and T. mwanzai. Based on restriction patterns, a dichotomous key was constructed for easy differentiation of these 12 trichogrammatids (Table 3). We thank Dr. R.J. Rabindra, Project Director, Project Directorate of Biological Control, Bangalore for his keen interest in the study and Ms. R. Rajeshwari for valuable technical assistance. References Ciociola Jr., A.C., Zucchi, R.A., Stouthamer, R., 2001. Molecular key to seven Brazilian species of Trichogramma (Hymenoptera: Trichogrammatidae) using sequences of the ITS-2 region and restriction analysis. Neotropical Entomology 30, 256– 262. Dendis, M., Horvath, R., Michalek, J., Ruzicka, F., Grijalva, M., Bartos, M., Bendik, J., 2003. PCR–RFLP detection and species identification of fungal pathogens in G. Ashok Kumar et al. / Biological Control 49 (2009) 207–213 patients with febrile neutropenia. Clinical microbiology and infection 9, 1191– 1201. Grissel, E.E., Schauff, M.E., 1990. A handbook of the families of Nearctic Chalcidoidea (Hymenoptera). Entomological Society of Washington 1, 85. Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41, 95–98. Hillis, D.M., Dixon, M.T., 1991. Ribosomal DNA: molecular evolution and phylogenetic inference. Quarterly Review of Biology 66, 411–416. Hoy, M.A., 1994. Insect Molecular Genetics: An Introduction to Principles and Applications. Academic Press, San Diego. 540pp. Jalali, S.K., Rabindra, R.J., Rao, N.S., Dasan, C.B., 2003. Mass production of trichogrammatids and chrysopids. Project Directorate of Biological Control, Bangalore 560024, Technical Bulletin No. 33, 16pp. Nagarkatti, S., Nagaraja, H., 1971. Redescription of some known species of Trichogramma showing the importance of male genitalia as a diagnostic character. Bulletin of Entomological Research 61, 13–31. Nagarkatti, S., Nagaraja, H., 1977. Biosystematics of Trichogramma and Trichogrammatoidea species. Annual Review of Entomology 22, 157–176. Orrego, C., Agudelo-Silva, F., 1993. Genetic variation in the parasitoid wasp Trichogramma (Hymenoptera: Trichogrammatidae) revealed by DNA amplification of a section of the nuclear ribosomal repeat. Florida Entomologist 76, 519–524. Parra, J.R.P., Zucchi, R.A., 2004. Trichogramma in Brazil: feasibility of use after twenty years of research. Neotropical Entomology 33, 261–271. Pinto, J.D., 1997. Trichogrammatoidea brasiliensis (Ashmead)—New combination for a species historically placed in Trichogramma. Proceedings of the Biological Society of Washington 99, 593–596. Pinto, J.D., 1998. Systematics of the North American species of Trichogramma (Hymenoptera: Trichogrammatidae). Memoirs of Entomological Society of Washington 22, 1–287. Pinto, J.D., Stouthamer, R., 1994. Systematics of the Trichogrammatidae with emphasis on Trichogramma. In: Wajnberg, E., Hassan, S.A. (Eds.), Biological Control with Egg Parasitoids. CAB International, Oxon, UK, pp. 1–36. 213 Pinto, J.D., Koopmanschap, A.B., Platner, G.R., Stouthamer, R., 2002. The North American Trichogramma (Hymenoptera: Trichogrammatidae) parasitizing certain Tortricidae (Lepidoptera) on apple and pear with ITS-2 DNA characterization and redescription of a new species. Biological Control 23, 134–142. Pinto, J.D., Velten, R.K., Platner, G.R., Oatman, E.R., 1989. Phenotypic plasticity and taxonomic characters in Trichogramma. Annals of the Entomological Society of America 85, 413–422. Roberta, L.C., Omar, S.C., Cristine, L.F.G.M., Carlos, G.T., Marcia, C.F.S., Renata, B., Rafel, M., Walter, S.L., Henrique, L.L., 2003. Molecular differentiation of Angiostrongylus castaricensis, A. cantonensis and A. vasorum by polymerase chain reaction–restriction fragment length polymorphism. Mem Inst Oswaldo Cruz Rio de Janeiro 98, 1039–1043. Sappal, N.P., Jeng, R.S., Hubbes, M., Liu, F., 1995. Restriction fragment length polymorphisms in polymerase chain reaction amplified ribosomal DNAs of three Trichogramma (Hymenoptera: Trichogrammatidae) species. Genome 38, 419–425. Silva, I.M.M.S., Honda, J., van Kan, F., Hu, S.J., Neto, L., Pintureau, B., Stouthamer, R., 1999. Molecular differentiation of five Trichogramma species occurring in Portugal. Biological Control 16, 177–184. Singh, S.P., Jalali, S.K., 1994. Trichogrammatids. Project Directorate of Biological Control, H.A. Farm Post, Bangalore 560024, Technical Bulletin No. 7, 95pp. Smith, S.M., 1996. Biological control with Trichogramma: Advances, successes, and potential of their use. Annual Review of Entomology 41, 375–406. Stouthamer, R., Hu, J., van Kan, F.J.P.M., Platner, G.R., Pinto, J.D., 1999. The utility of internal transcribed spacer 2 DNA sequences of the nuclear ribosomal gene for distinguishing sibling species of Trichogramma. BioControl 43, 421– 440. Thompson, L.J., Rundle, B.J., Carew, M.E., Hoffmann, A.A., 2003. Identification and characterization of Trichogramma species from south-eastern Australia using the internal transcribed spacer 2 (ITS-2) region of the ribosomal gene complex. Entomologia Experimentalis et Applicata 106, 235–240.