The European corn borer Ostrinia nubilalis (Hubner, 1796) (Lepidoptera: Pyralidae) is a major pest of corn (Zea mays Linnaeus, 1753) in Serbia. Regular monitoring of O. nubilalis in the region of Kikinda (province of Vojvodina, Serbia) demonstrated a high percentage of parasitized eggs of this pest. Preliminary identification of the egg parasitoids based on morphological traits revealed that they belong to the genus Trichogramma Westwood, 1833 (Hymenoptera, Trichogrammatidae). The objective of this study was to identify the autochthonous Trichogramma species present in the region of Kikinda by using sequences of ribosomal DNA. Specimens of Trichogramma were reared from parasitized eggs of O. nubilalis collected at different localities in the region of Kikinda. The ITS2 gene was sequenced and subjected to BLAST searches of GenBank NCBI (National Center for Biotechnology Information). The majority of collected specimens were identified as Trichogramma brassicae Bezdenko, 1968, and a single specimen as T. evanescens Westwood, 1833. Intraspecific variation of T. brassicae was assessed by amplifying the mitochondrial cytochrome oxidase I (COI) gene. Wasps were also tested for the presence of the endosymbiotic bacteria Wolbachia Hertig, 1936 using Wolbachia-specific 16S and wsp primers as a preliminary test for evidence of thelytoky. Understanding of the diversity and biology of autochthonous Trichogramma species is a key step in the potential commercial implementation of these egg parasitoid wasps in the protection of corn fields in Serbia. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. TR31084 and Grant no. III43007]

The current study extends the faunistic survey of Trichogramma species parasitizing ECB to all agricultural growing regions in Serbia. Specimens of Trichogramma were reared from parasitized egg masses of ECB collected from field-grown corn and pepper crops. The number of egg masses parasitized varied by location. Using sequences of mitochondrial cytochrome oxidase I (COI) gene, we examined intra and interspecific variation in Trichogramma parasitizing the ECB egg masses. Seventy specimens were successfully sequenced of which, 65 were identified as T. brassicae. The remaining five were identified as T. evanescens, and this second species was only collected from pepper.

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Background The European corn borer (ECB), Ostrinia nubilalis (Hübner, 1796) (Lepidoptera: Crambidae), is the major pest of maize (Zea mays Linnaeus, 1753) in Serbia. One potential method for managing this pest is the augmentative release of naturally occuring egg parasitoids of the genus Trichogramma. The first step in this process is accurately identifying the naturally occuring species and estimating their natural distribution and abundance. Molecular identification, based on differences in DNA sequences, has commonly been employed for the identification of Trichogramma species. A simple, quick, and accurate molecular assay is urgently required for the identification of two common Trichogramma species, associated with ECB in Serbia: T. brassicae Bezdenko, 1968 and Trichogramma evanescens Westwood, 1833. Such an assay will facilitate an expansive survey of resident populations of Trichogramma associated with ECB across agricultural growing regions of Vojvodina province. Results A species-specific multiplex PCR assay for the 2 species was developed and validated that assay using a sample of 79 parasitoid wasps reared from ECB egg masses collected from sample sites across Vojvodina province. Trichogramma brassicae was confirmed as the dominant egg parasitoid of ECB in this region, accounting for 77 of the 79 wasps (97.47%). The remaining 2 were confirmed as T. evanescens. Trichogramma brassicae was detected at all 12 sample sites, while T. evanescens was detected at only 2 plots, Mokrin and Nakovo. Conclusions The species-specific multiplex PCR assay presented herein can provide the basis of a quick, cheap, and reliable means for identifying the species of Trichogramma that parasitize ECB egg masses in Serbia. Two currently documented species, T. brassicae and T. evanescens, are readily diagnosed by the size of the PCR product they produce in the assay. Any additional species are expected to not produce a band of a diagnostic size. Such species would subsequently be identified by sequencing, which may also allow them to be promptly incorporated into a revised assay.

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