Maize (Zea mays Linnaeus, 1753) is the number one crop in Serbia. The largest areas of maize production are located in the northern province of Vojvodina, where it is grown on about 620,000 ha, with a total production of over 4 million tons (Latković et al., 2018). Although the production of maize is affected by several harmful insects, the most important maize pest in Serbia is the European corn borer (ECB), Ostrinia nubilalis (Hübner, 1796) (Lepidoptera, Crambidae) (Čamprag et al., 1983). Management techniques commonly used to reduce ECB infestations include changes to cultural practices, the planting of resistant cultivars, and the application of chemical insecticides (Maissle et al., 2010), the last being the most commonly employed approach. Chemical insecticides are not entirely effective against ECB because of the relatively short period the insect spends outside the plant prior to boring into it. Furthermore, 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).
One particular biological control method that has attracted a lot of attention is the augmentative release of naturally occurring species of the genus Trichogramma Westwood, 1833 (Hymentoptera: Trichogrammatidae) (Grushevaya, 2020). Trichogramma spp. are tiny (<1mm) wasps that parasitize the eggs of many insect species and are widely used in inundative and inoculative biological control programs against several pests in the EU (Golbaghi et al., 2020). Several species of Trichogramma have been identified as promising biological control agents of ECB, including T. brassicae and T. evanescens (Van Schelt and Ravensberg, 1991). Both species appear to be widespread across Europe and are reared in commercial facilities for release as biological control agents.
Serbia is one of the countries, at the southast border of Europe, that does not actively use Trichogramma species in its agricultural practices, despite evidence suggesting high activity of native populations of these insects (Ivezić et al., 2020). Not all Trichogramma species (or populations) perform equally well in terms of mass rearing or field dispersal and parasitism rates (Borba et al., 2005). Morphological identification of Trichogramma requires expert taxonomic knowledge, due to their minute size (<1 mm long) and a general lack of reliable characters (Stouthamer et al., 1999). Consequently, alternative identification methods have been developed. Sequences of the internal transcribed spacer 2 (ITS2) region of the nuclear ribosomal DNA are one accepted means of identifying Trichogramma species (Fahriye et al., 2009). An intial survey of the Kikinda region identified two species of Trichogramma (T. brassicae and T. evanescens) parasitizing ECB eggs, based on sequences of the ITS2 gene (Ivezić et al., 2018). A second, more geographically expansive study identified the same two species, again based on DNA sequences, but this time of the mitochondrial cytochrome oxidase subunit 1 (COI) gene (Ivezić et al., 2020). In both studies, T. brassicae was found to be far more abundant in maize than was T. evanescens.
The utility of ITS2 and COI sequences as diagnostic tools for Trichogramma (and many other taxa) is widely accepted, but obtaining a DNA sequence incurs significant costs (time and money). One way that can be achieved is through the development of species-specific PCR primers that can be run alone, or better still, in a multiplex reaction. Species-specific multiplex PCR incorporates several PCR primers that bind to the same DNA strand, each of which is specific to only one “target” species (Rugman-Jones et al., 2020). When combined in a single reaction with a single complementary PCR primer that is “universal” to all target species, the multiplex PCR results in the production of a PCR product of a unique size for each of the target species, allowing specimens to be identified directly following gel electrophoresis of the PCR product (Rugman-Jones et al., 2020).
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.