Identifikacija parazitoida kukuruznog plamenca, Ostrinia nubilalis (Hübner, 1796), roda Trichogramma, u Vojvodini

Abstract

Kukuruz (Zea mays Linnaeus, 1753) predstavlja ekonomski najznačajniju ratarsku kulturu u Srbiji, dok kukuruzni plamenac Ostrinia nubilalis Hübner, 1796 (Lepidoptera: Crambidae) predstavlja najznačajniju štetočinu ove kulture. Redovnim monitoringom populacija kukuruznog plamenca na teritoriji opštine Kikinda (Vojvodina, Srbija) registrovan je visok procenat parazitiranih jajnih legala ove štetočine. Preliminarna identifikacija, sprovedena na osnovu morfoloških karakteristika eklodiranih jedinki, pokazala je da su parazitirana jajna legla kukuruznog plamenca posledica aktivnosti jajnih parazitoida roda Trichogramma Westwood, 1833 (Hymenoptera: Trichogrammatidae). Imajući u vidu da je fauna osica roda Trichogramma u Srbiji nedovoljno istražena, cilj ovog rada je bio je da se izvrši identifikacija autohntonih vrsta roda Trichogramma, parazitoida kukuruznog plamenca na području Vojvodine, kao i utvrđivanje genetičke varijabilnosti u populacijama prisutnih vrsta. Osim toga jedan od ciljeva je bio da se pouzdanim molekularnim metodama u lokalnim populacijama Trichogramma utvrdi prisustvo endosimbiontske bakterije roda Wolbachia Hertig & Wolbach 1924 za koju je poznato da kod Trichogramma spp. favorizuje telitoknu partenogenezu. Monitoring kukuruznog plamenca, koji je sproveden sakupljanjem adulta pomoću svetlosnih klopki na dnevnoj bazi u toku sezone 2016. i 2017. godine na dva odabrana lokaliteta opštine Kikinda, pokazao je da ova štetočina produkuje tri generacije u toku sezone, od kojih je druga generacija, koja se pojavljuje u toku leta najbrojnija i ostvaruje maksimum krajem jula i početkom avgusta. Parazitirana jajna legla su nalažena u periodima koji su odgovarali letu, odnosno ovipoziciji druge i treće generacije kukuruznog plamenca. U 2016. godini, parazitirana jajna legla plamenca uzorkovana su na pet lokalitetana teritoriji opštine Kikinda (Vojvodina, Srbija): Kinđa, Stankić, Banatska Topola, Nakovo i Banatsko Veliko Selo. U zavisnosti od lokaliteta, prosečna stopa parazitiranosti jaja kukuruznog plamenca se kretala u granicama od 51,52% do 84,62%, a u proseku je za istraživano područje iznosila 62,5%. Nakon morfološke identifikacije eklodiranih odraslih jedinki do nivoa roda, osice Trichogramma sp. su bile podvrgnute molekularnoj identifikaciji do nivoa vrste, sekvenciranjem ITS2 regiona ribozomske DNK. Rezultati sekvenciranja ITS2 segmenta poređeni su uz pomoć BLAST programa sa poznatim sekvencama u onlajn genbanci National Center for Biotechnology Information. Većina identifikovanih jedinki iz uzoraka prikupljenih u 2016. godini je pripadala vrsti Trichogramma brassicae Bezdenko, 1968, dok je samo jedna jedinka identifikovana kao Trichogramma evanescens Westwood, 1833. Utvrđivanje intraspecijske varijacije uzoraka identifikovanih kao T. brassicae sprovedeno amplifikacijom segmenta mitohonrijalne DNK za citohrom c oksidazu I (COI gen) pokazalo je prisustvo 4 haplotipa T. brassicae i jednog haplotipa T. evanescens u lokalnim populacijama. Testiranje uzoraka osica na endosimbiotsku bakteriju Wolbachia Hertig, 1936 korišćenjem prajmera specifičnih za ovu bakteriju (16S i wsp) pokazalo je prisustvo ove bakterije u populacijama T. brassicae i njenom dominantnom haplotipu (TbSERB-1). Dizajniranjem specifičnih prajmera za amplifikaciju ITS2 segmenta kod T. brassicae i T. evanescens, sprovedena je Multipleks PCR metoda koja je omogućila identifikaciju ove dve vrste roda Trichogramma bez sprovođenja sekvenciranja. U 2017. godini uzorci su prikupljeni sa 12 lokaliteta u Vojvodini: Mokrin, Iđoš, Bašaid, Nakovo, Banatska Topola, Stankić, Njegoševo, Despotovo, Čoka, Senta, Vrbas i Gakovo. Prosečna stopa parazitiranosti jajnih legala kukuruznog plamenca se, u zavisnosti od lokaliteta, kretala u rasponu od 15% do 62,5%, odnosno u proseku za celokupno istraživano područje iznosila je 38,84%. Uzorci odgajenih osica su podvrgnuti su Multipleks PCR metodi, pri čemu je prisustvo T. brassicae utvrđeno na svim lokalitetima, a T. evanescens samo u uzorcima iz Mokrina i Nakova. Istraživanja u toku 2016. i 2017 godine su pokazala da T. brassicae predstavlja dominantnu vrstu osica parazitoida jajnih legala kukuruznog plamenca na istraživanom području Vojvodine. Razumevanje diverziteta, biologije i ponašanja autohtonih Trichogramma vrsta ključan je korak u potencijalnoj upotrebi ovih parazitoida u biološkoj zaštiti kukuruza i drugih poljoprivrednih kultura u Srbiji.

Maize (Zea mays Linnaeus, 1753) is the most important cereal crop in Serbia, while the European corn borer (ECB) Ostrinia nubilalis (Hübner, 1796) (Lepidoptera: Crambidae) is the major pest of this crop. Regular monitoring of ECB in the region of Kikinda (the province of Vojvodina, Serbia) demonstrated a high percentage of parasitized egg clasters of this pest. Preliminary identification of the egg parasitoids based on morphological traits of emerged adults revealed that they belong to the genus Trichogramma Westwood, 1833 (Hymenoptera, Trichogrammatidae). Considering that the fauna of Trichogramma wasps has not yet been enough explored in Serbia, the objective of this study was to identify the autochthonous species of Trichogramma parasitoids of ECB present in the region of Vojvodina, as well as determining the genetic variability of populations of present species. In addition, one of the objectives of this study was to confirm the presence of endosymbiotic bacteria of the genus Wolbachia Hertig and Wolbach 1924 in native populations of Trichogramma by reliable molecular methods, since it is known that these bacteria induce thelytokous parthenogenesis in Trichogramma species. Monitoring of ECB adults conducted by light traps on daily basis during the seasons of 2016 and 2017 at two localities in the region of Kikinda demonstrated that this pest produces three generations per year. The second generation of the ECB, which occurs during the summer was the most abundant and peaked in late July and early August. Parasitized egg clusters were found during the period of oviposition of the second and third generation of ECB. In 2016, parasitized egg clusters were collected at five localities in the region of Kikinda: Kindja, Stankic, Banatska Topola, Nakovo and Banatsko Veliko Selo. Depending on the locality, the average parasitism rate was ranging between 51.52% and 84.62%, while the average parasitism rate for the region was 62.5%. After morphological identification of emerged adults to the genus level, Trichogramma sp. wasps were molecularly identified to the species level by sequencing the ITS2 gene of ribosomal DNA. The ITS2 gene was sequenced and subjected to BLAST searches of the GenBank (NCBI – National Center for Biotechnology Information). The majority of specimens collected in 2016 were identified as Trichogramma brassicae Bezdenko, 1968 and a single specimen as T. evanecens Westwood, 1833. Intraspecific variation of samples of wasps was assessed by amplifying the mitochondrial cytochrome oxidase I (COI) gene. The results showed the presence of 4 haplotypes of T. brassicae in local populations, and a single haplotype of T. evanecens. Wasps were also tested for the presence of the endosymbiotic bacteria Wolbachia using Wolbachia-specific 16S and wsp primers and the results demonstrated the presence of the bacteria in the population of T. brassicae in its dominant haplotype (TbSERB-1). Specific primers for the amplification of ITS2 gene of T. brassicae and T. evanescens were designed. Using those primers, MultiplexPCR was conducted for identification of the two species without sequencing. In 2017, samples were collected from 12 localities in Vojvodina province: Mokrin, Iđoš, Bašaid, Nakovo, Banatska Topola, Stankić, Njegoševo, Despotovo, Čoka, Senta, Vrbas i Gakovo. Depending on the locality the parasitism rate was between 15% and 62.5%. The average parasitism rate for the entire studied area was 38.84%. Samples of wasps reared from egg clusters sampled in 2017 were subjected to the Multiplex PCR method. T. brassicae was determined in samples from all of the localities, while T. evanescens was identified only in samples from Mokrin and Nakovo. The research conducted in Vojvodina during 2016 and 2017 demonstrated that T. brassicae represents the dominant parasitoid species of ECB egg clusters. Understanding of the diversity, biology and behavior of autochthonous Trichogramma species is a key step in the potential commercial implementation of these egg parasitoid wasps in biological pest control in Serbia.