Rapid molecular identification of Trichogramma (Hymenoptera: Trichogrammatidae) parasitizing the eggs of the European corn borer, Ostrinia nubilalis (Lepidoptera: Crambidae) in Serbia

Aleksandar Ivezić,Paul F Rugman-Jones,Branislav Trudić

doi:10.1186/s41938-021-00414-5

Abstract

BackgroundThe 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.ResultsA 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.ConclusionsThe 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.

Highlights

The European corn borer (ECB), Ostrinia nubilalis (Hübner, 1796) (Lepidoptera: Crambidae), is the major pest of maize (Zea mays Linnaeus, 1753) in Serbia
The aims of this study were (1) to develop a diagnostic species-specific multiplex PCR assay for T. brassicae and T. evanescens in Serbia and (2) to use that assay to survey resident populations of Trichogramma associated with ECB across agricultural growing regions of Vojvodina province
Criteria for the design of species-specific primers were as follows: (1) they should lie in stretches of the internal transcribed spacer 2 (ITS2) that display minimal levels of intraspecific variation at the 3′ end of the primer, (2) they should work in tandem with the “universal” forward primer ITS2 for (Stouthamer et al 1999) which is located in the highly conserved 5.8S adjacent to ITS2, (3) at least one nucleotide at the 3′ end of the primer must be unique to its target, (4) complementarity between primers should be minimal to avoid the production of primer dimers, (5) they should all work at similar annealing temperatures, and (6) they should produce species-specific amplicons that differ sufficiently in size to allow their diagnosis using standard agarose gel electrophoresis

Summary

Introduction

The European corn borer (ECB), Ostrinia nubilalis (Hübner, 1796) (Lepidoptera: Crambidae), is the major pest of maize (Zea mays Linnaeus, 1753) in Serbia. 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. There are general concerns about the effects of insecticides on beneficial insects and the wider environment, and specific concerns among the general public about insecticide residues on agricultural products (Phoofolo, 1997) Due to such growing awareness, biological control methods are becoming more favored and increasingly important (Ivezić et al, 2020)

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