Parasitoid inventarisation of European corn borer ( Ostrinia nubilalis Hübner , 1796 ) and options for its biological control in Slovenia

European corn borer (Ostrinia nubilalis) (ECB) is an important maize pest in central and northern Europe. Presently it is controlled by insecticides or biological agents such as Trichogramma brassicae in several European countries, excluding Slovenia, where the pest’s pressure is highly variable and no appropriate mechanization is available. Lessening the dependence on chemical pesticides is an integral part of the European Union’s agenda for agriculture. Mass release of Trichogramma spp. could be seen as a promising alternative for ECB control in countries with a highly fluctuating ECB pressure and no mechanization for insecticide applications. However, no records of naturally occurring hymenopteran parasitoids of ECB exist in Slovenia. To address this important under-researched topic and provide the expert basis for potential introduction of ECB egg parasitoids in Slovene maize production, a systematic inventarisation programme of ECB parasitoids was launched in 2010. Additionally, ECB flight was monitored in 2011 and 2012 at two locations in Slovenia: Jablje and Rakičan. In both locations two ECB generations were observed. ECB was fist observed at the end of May in Rakičan. During the five years of the systematic survey we discovered two ECB parasitoid species. ECB egg masses were parasitized by Trichogramma brassicae, whereas ECB pupae were parasitized by Tycherus nigridens, with 6 or 7 % parasitation rate, respectively. T. nigridens represents a new taxon report for Slovenia. We conclude that there is a strong need for undertaking systematic surveys of natural enemies of agricultural pests.


INTRODUCTION
Corn borers represent an important biotic stressor for maize (Zea mays L.) crops in Europe (Meissle et al., 2010).European corn borer (Ostrinia nubilalis (Hübner 1796)) (ECB) is the most important maize pest in central and northern Europe, while pink stem borer (Sesamia nonagrioides (Lef., 1827) is predominant in warmer areas of southern Europe (Velasco et al., 2002;Malvar et al., 2004;Meissle et al., 2010).ECB mostly causes damage by larvae that enter into the maize stalk after hatching and feed on the stalk pith.Yield is affected by ECB tunnelling which interferes with vascular system and increases the risk of stalk lodging and breakage (Gomboc et al., 1999).In addition, corn borer damage can affect plant health by vectoring Fusarium moniliforme J. Sheld.and facilitating fungal infections (Sobek and Munkvold, 1999).The yield loss produced by corn borer attack of the ear is sometimes less important than yield reduction associated to stalk tunnelling, as this kind of damage has been described as an important factor for favouring high levels of fumonisins in maize kernels (Sobek and Munkvold, 1999;Butrón et al., 2009).
Foliar insecticide applications in maize production are used in most European regions (i.e.Spain, Hungary, Poland, Germany, Italy, France and Denmark).The most commonly used active ingredients are pyrethroids and organophosphates (Meissle et al., 2010).However, Pons and Albajes's (2002) results illustrate how broad spectrum insecticides can have undesirable effects regarding ECB control: although treating maize seeds with imidacloprid reduced the incidence of cutworms (Agrotis segetum Denis & Schiffermüller, 1775), wireworms (Agriotes lineatus Linnaeus, 1767), pink stem borer and leafhoppers (Zyginidia scutellaris Herrich-Schäffer, 1838), treated plots were attacked significantly more by ECB.In Slovenia however, chemical measures in corn crops are not undertaken against ECB, partly due to highly fluctuating ECB pressure, partly due to nonexistent spraying mechanization.It is important to mention that ECB in Slovenia occurs also in vegetables like tomato, peppers and others, where the pest can be controlled with insecticides (Carlevaris et al., 2003).
Lessening the dependence on chemical pesticides is an integral part of the European Union's (EU) agenda for agriculture (European Parliament, 2009).The objective of this Directive is to reduce dependence on, as well as the risks and adverse impacts of, pesticide use on human health and the environment, and a key element to reach this goal is to promote the implementation of Integrated Pest Management (IPM), which has become compulsory in the EU in 2014.In sync with this directive and the previously mentioned reasons mass release of Trichogramma spp. is a promising alternative for ECB control in countries with a highly fluctuating ECB pressure and no insecticide applications.In Europe, the parasitoid wasps are already released mainly against ECB on about 150 000 ha per year with the largest area in France (Meissle et al., 2010).Encouraging results of biological control of ECB also exist from Asia (Zhang et al., 2010) and North America (Hoffmann et al., 2002;Hoffmann et al., 2006).An important ECB parasitoid, Trichogramma brassicae Bezdenko, 1968, was only recently discovered in Slovenia by Bohinc et al., (2015).
Despite encouraging results of biological control of ECB, records of naturally occurring hymenopteran parasitoids of ECB in Slovenia are limited.To address this important under-researched topic and provide the expert basis for potential introduction of ECB egg parasitoids in Slovene maize production, a systematic inventarisation programme of ECB parasitoids was launched in 2010.In this paper we report the first findings.

ECB flight monitoring
In the sampled areas (Jablje, Rakičan) ECB flight was monitored with a fluorescent light trap (Grote Lichtval zonder lamp, Entomologie-speciaalzaak, Vermandel, Hulst, Nederlands) from May to September in the years 2011-2012.One trap per location was used.

Scouting for ECB egg masses and pupae in the field
Our search for natural enemies of ECB was focused on egg and pupal parasitoids.Search for ECB egg parasitoids was performed by scouting for ECB egg masses on the abaxial side of maize leaves.Scoutings were carried out during the oviposition period of the second ECB flight.All potentially parasitized ECB egg masses were collected and transferred to the entomological laboratory of Agricultural Institute of Slovenia for observation of parasitoid emergence.Pupal parasitoids were searched for in plants with evident ECB damage (stalk tunnelling and breakage, frass on leaves, holes in leaves).ECB damaged plants were dissected to collect ECB pupae, which were also transferred to the entomological laboratory of Agricultural Institute of Slovenia for observation of pupal parasitoid emergence.

2.4
Observation of ECB egg masses and pupae for parasitoid emergence ECB egg masses on 10 cm 2 pieces of maize leaves and ECB pupae were put into individual 50 mL centrifuge tubes.The tubes were kept at room temperature for observation until ECB larva/imago or parasitoid emerged.

Parasitoid classification
The morphological classification of the ECB egg mass parasitoids was based on Cônsoli et al. (2010), Ferriere andKerrich (1958), Goulet and Huber (1993) to family and genus level.For species identification, Pintureau (2011) was used.Identification of Trichogramma species relies very heavily on examination of the male genitalia and to a lesser extent the male antennae.For this reason a slide-mounting technique modified from Platner et al. (1999) was necessary to allow the examination of male genitalia.Therefore, freshly emerged individuals of the egg parasitoid were killed and incubated in 90 % lactic acid for three days in order to allow their clearing.Then specimens were transferred, dorsum-up, to a small drop of Hoyer's medium on a slide and carefully covered with a small round coverslip.During this process the male genital capsule was gently pressed out of the gaster.After drying, the slides were examined at 100 x and 400 x magnification under the light microscope and morphological characters were determined for species identification according to Pintureau (2011).The morphological classification of pupal parasitoids was based on Goulet and Huber (1993) and a detailed description by Smith (1932).

ECB flight and parasitation levels
The ECB pressure varied between northeast Slovenia (Rakičan) and central Slovenia (Jablje).Higher catches were recorded in Rakičan.ECB first occurred in Rakičan at the end of May, early June.In Jablje, ECB was first observed in mid-June.In both localities two ECB flights were observed, although the two-flight trend was more evident in Rakičan.The first ECB flight peaked from 17-20 June, and the second from 15-20 July.In 2012 in Jablje the first ECB flight was not very distinct.ECB flight waned in August (Figure 1).Taxon T. nigridens had not been reported previously in Slovenia according to our knowledge.

Trichogramma brassicae Bezdenko, 1968
The parasitized ECB egg masses were typically discovered on the abaxial side of 3 rd or 4 th maize leaf from stem base up.ECB eggs usually formed aggregates of 20-35 eggs and were located as a single cluster near the central leaf vein.Some parasitized egg masses were completely parasitized, whereas some only partially.After transfer to the lab, minute wasps emerged in 3-5 days from the uniformly black ECB egg masses (Figures 1 and 2).The wasps were minute, ca.0.9 mm long, yellow or yellow and black / brown with bright red eyes, short antennae, compact bodies and severely reduced wing venation.Based on morphological characteristics described in Cônsoli et al. (2010), Ferriere andKerrich (1958), Goulet and Huber (1993), they were classified to the genus Trichogramma.The genus Trichogramma contains more than 200 described species (Pinto, 2006).Mostly they are generalists parasitizing lepidopteran, dipteran, neuropteran and coleopteran egg masses (Querino et al., 2010).
They are commonly used as an alternative measure to control major agronomical pests via inundative releases (Kölliker-Ott et al., 2004).For more detailed classification to the species level, Pintureau (2011) was used.Based on morphological characteristics of male genitalia the emerged Trichogramma wasps were classified as Trichogramma brassicae (Figure 3).The hymenopteran parasitoid Trichogramma brassicae Bezdenko (Hymenoptera: Trichogrammatidae) was introduced into Western Europe to control ECB more than 40 years ago (Babendreier, 2003b).The introduced strain originates from Moldavia.

Tycherus nigridens (Wesmael, 1845)
The pupae sampled for pupal parasitoid inventarisation were collected from maize plants evidently infested by ECB, exhibiting breakage, holes and / or frass.Most often, the pupae were discovered near the stem base.They were carefully removed from the plants using a Swiss army knife and tweezers.Upon transfer to the lab, the pupae were observed on a daily basis for ECB or parasitoid emergence.When an ECB emerged it was discarded.The emerged parasitoids were classified according to Goulet and Huber (1993) and Smith (1932) to Tycherus nigridens.T. nigridens (Wesmael, 1845) (Hymenoptera: Ichneumonidae; synonym: Tycherus planifrons Wesmael, 1845, Phaeogenes nigridens Wesmael) is an internal solitary parasite which attacks the pupal stage of ECB (Smith, 1932).It prefers 1-and 2-day old pupae, which it perforates with its ovipositor and lays the eggs free in the body cavity of the host (Baker et al., 1949).The earliest definite reference to this species in literature appeared in 1844, when Wesmael first described it as Phaeogenes nigridens.In addition to the corn borer the only other host from which Tycherus nigridens has been reared is Tortrix pronuboena Hübner (Smith, 1932).The adult is distinguished from the other parasites of ECB by its robust appearance and its rusty red-brown abdomen with black terminal portion.The larva has at least four, maybe five, distinct instars.The adults have long life spans; some females were kept alive at low temperature for 10 months (Smith, 1932).It overwinters as an adult female (Baker et al., 1949).
Originally described from Belgium, this species was initially reported from Sweden, Germany, Spain and France (Smith, 1932).Its present day areal includes most of the EU countries, with the exception of the following countries for which no data exists: Albania, Belarus, Bosnia and Herzegovina, Bulgaria, Croatia, Greece, Iceland, Ireland, Kosovo, Latvia, Lithuania, Macedonia, Moldova, Montenegro, Portugal, Serbia, Slovakia, Ukraine and Vojvodina (Fauna Europea, http://www.faunaeur.org/,accessed on June 4 th , 2015).

DISCUSSION
During the five years of the systematic survey we discovered two ECB parasitoid species.ECB egg masses were parasitized by Trichogramma brassicae, already reported in Slovenia by Bohinc et al. (2015), whereas ECB pupae were parasitized by Tycherus nigridens (new finding for Slovenia), with 6 or 7 % parasitation rate, respectively.More effort was invested in searching for egg mass parasitoids (> 3000 plants examined).Thus, to be sure of the exactness of T. nigridens's observed natural parasitation rate of 7 %, we would need to sample a much greater number of pupae.
Trichogramma occurs in all vegetated terrestrial habitats that have been sampled in all six biogeographic regions: Palearctic, Oriental, Nearctic, Neotropical, Afrotropical and Australasian (Querino et al., 2010).T. brassicae was isolated from several host species (Pintureau, 2011).In Slovenia it was first discovered on August 18, 2014, on Mamestra brassicae (Linnaeus, 1758) eggs (Bohinc et al., 2015).However, due to their small size, species of Trichogramma can potentially be transported by wind and intentionally or otherwise, by man from one country to another in a short period of time.Because of this it is often difficult to determine individual species' natural range of distribution (Querino et al., 2010).
Attempts at classical biological control of ECB employing Tycherus nigridens release were performed already in 1924 in the USA when a single colony was released in Massachusetts.Attempts intensified by releasing more numerous parasitoids imported from Europe and Japan, totalling more than 50.000T. nigridens between years 1926-1933 (Baker et al., 1949).In their report Baker et al. (1949) conclude that the biocontrol program was successful as entomologists were able to recover T. nigridens in localities where it was not released in the previous three years, but that more knowledge on the biology of the parasitoid would be needed in order to improve the storage conditions of the adults enabling better synchronization of the releases so the parasitoid would establish broader.Smith et al. (1932) reported that T. nigridens was the most effective ECB parasite in Europe with the maximum parasitism rate of 17.5 % recorded in 1930 in the fields around Padova in northern Italy.Factors that may limit its effectiveness as an effective biocontrol agent are the small number of eggs produced by each female; the mortality of some ovarian eggs, especially among hibernating females; the rather long oogenetic period; and the generally unsuitable oviposition conditions in the spring (Smith et al., 1932).Two related Tycherus species are reported in literature as pupal parasitoids of agricultural or forestry pests: cherry bark tortrix, Enarmonia formosana (Scopoli, 1763), an invasive orchard pest in northwest US, is parasitized by Tycherus vagus (Berthoumieu, 1899) (Jenner et al., 2004), whereas Cydia strobilella (Linnaeus, 1758), a Holarctic pest of spruce cones, is parasitized by Tycherus fuscibucca (Berthoumieu, 1901) (Brockerhoff andKenis, 1996).T. nigridens represents a new taxon report for Slovenia.However, this taxon has been previously reported from many EU countries, including some of Slovenia's neighbouring countries like Austria, Italy and Hungary, but not Croatia.This fact reveals the problem of reliability of such taxon distribution information, as the real, biological distribution is probably underestimated due to insufficient funding allocated to natural enemy inventarisation studies, especially in countries with a lower gross domestic product per capita.In line with the present Slovenian legislature (Official Gazette of the RS, 83/12, 36/10 40/14, 62/07 and 45/06), potential introduction of commercially reared natural enemies and their release can only be done after obtaining concrete evidence on the autochthonous presence of these species as the pests' natural enemies.Thus our study clearly illustrates the need for undertaking systematic surveys of natural enemies of agricultural pests, so that concrete evidence for their autochthonous presence can be confirmed and their introduction possible.
Specific risks exist when releasing large number of Lepidopteran egg parasitoids.Laboratory experiments demonstrated that T. brassicae can attack many non-target butterflies including endangered species (Babendreier et al., 2003a).However, non-target butterflies were very seldom attacked under natural conditions in Switzerland (Babendreier et al., 2003b).Besides having adverse effects on non-target organisms, inundatively released non-indigenous Trichogramma may compete with locally occurring Trichogramma.Therefore preference should always be given to indigenous strains or species, when developing a new IPM program based on Trichogramma (Herz et al., 2007).
Despite the mentioned risks, one should consider the benefits of parasitoid-based biological control strategies.The released parasitoids do not harm autochthonous population of generalist predators and other beneficial arthropods (Chapman et al., 2009;Zhang et al., 2010); the application has no negative effect on farmers' health; the environment, especially the ground-and surface water is not burdened by pesticides and their breakdown or transformation products (Zhang et al., 2010), and the food or feed is free from insecticide residues.

Figure 1 :
Figure 1: ECB flight in Jablje and Rakičan in 2011 2012.Green arrows show approximate times of ECB egg mass and pupae sampling.G1 or G2 -first or second ECB flight.During the five years of the survey over 3000 maize plants were inspected for the presence of second generation ECB egg masses.Altogether 84 ECB egg masses were discovered.Five of these 84 egg masses were parasitized, resulting in a percentage parasitism of nearly 6 %.Approximately 150 ECB-damaged plants were dissected to obtain potentially parasitized second generation ECB pupae.Approximately 30 pupae were transferred to the laboratory.Two of them were harbouring parasitoids (percentage parasitism: nearly 7 %).The egg masses were parasitized by Trichogramma brassicae Bezdenko, 1968, whereas the two parasitized pupae were parasitized by Tycherus nigridens Wesmael, 1845.Taxon T. nigridens had not been reported previously in Slovenia according to our knowledge.

Figure 3 :
Figure 3: Left picture: Trichogramma brassicae female (left) and male (right).Right picture: microscopic preparation of a Trichogramma brassicae male.Scale bar represents 0.1 mm on the left and 0.2 mm on the right picture.Photos: J. Razinger.