Entomophaga Maimaiga (Entomophthorales: Entomophthoraceae) in Balkan Peninsula – an Overview

Silva Balcanica, 17(1)/2016

Entomophaga maimaiga (Entomophthorales: Entomophthoraceae) in Balkan Peninsula – an overview

Daniela Pilarska1, 3, Georgi Georgiev2, Vassil Golemansky3, Plamen Pilarski4, Plamen Mirchev2, Margarita Georgieva2, Mara Tabaković-Tošić5, Milcho Todorov3, Danail Takov3, Milan Pernek6, Boris Hrasovec7, Marno Milotic7, Mirza Dautabasic8, Osman Mujezinovic8, Steria Naceski9, Irena Papazova-Anakieva9, Maria Matova2, Panagiotis Vafeidis10 1New Bulgarian University – Sofia 2Forest Research Institute – Sofia Bulgarian Academy of Sciences 3Institute of Biodiversity and Ecosystem Research – Sofia Bulgarian Academy of Sciences 4Institute of Plant Physiology and Genetics – Sofia Bulgarian Academy of Sciences 5Institute of Forestry – Belgrade 6Croatian Forest Research Institute – Jastrebarsko 7University of Zagreb 8University of Sarajevo – Sarajevo 9St. St. Cyril and Methodius University – Skopje 10Forest Department of Xanthi

Abstract

Through a fifteen-year period, by the continuous monitoring to establish the distribution of Entomophaga maimaiga in Bulgaria, it is clear that the fungus already occurs in many localities among 22 State Forest and/or Hunting Enterprises where it was introduced or has spread by natural means. Consequently, as a result of the introduction of E. maimaiga in Bulgaria, almost no insecticides were used over the past 15 years and more than 10 million BGN (5 million Euros) were saved. E. maimaiga epizootics occurred in the neighbouring countries in 2005, 2010 and 2011, intensifying the spread further, so by the end of 2013 it expanded its range throughout the Balkan Peninsula. The fungus is now present, and to our best knowledge, it was established in Serbia, FYRMacedonia, Greece, European part of Turkey, Croatia and Bosnia and Herzegovina. It has clearly shown a dramatic impact on gypsy moth populations, possessing a capability to cease gypsy moth outbreaks and maintain pest population density at low levels under favourable climatic conditions. Key words: entomopathogen, Lymantria dispar, introduction, biological control

Introduction

The fungus Entomophaga maimaiga Humber, Shimazu and Soper 1988 is an entomopathogen of gypsy moth, Lymantria dispar (Linnaeus, 1758) – one of the most harmful defoliators of broadleaf forests in Europe, Asia and North America. It was

31 originally discovered and described from a gypsy moth population in Japan (Koyama, 1954) where it periodically caused epizootics. E. maimaiga occurs as L. dispar pathogen in other parts of Asia Pacific (Nielsen et al., 2005). The fungus was introduced in North America in several locations near Boston, Massachusetts in 1910-1911 (Speare, Colley, 1912). However, E. maimaiga was not reported until 1989 when it caused epizootics in seven North-eastern States (Hajek et al., 1990). According to Dwyer et al. (1998) it has been estimated that in 1991 (despite the relatively dry spring) the pathogen spread >100 km/yr. E. maimaiga could potentially be transported over longer distances by humans, especially as resting spores in soil (Hajek et al., 1995; Kereselidze et al., 2011). In 1992, the fungus spread throughout the large gypsy moth distribution range of the north east, including 12 States (Hajek et al., 1996). Since then, E. maimaiga is considered as the most important natural enemy of this pest responsible for decreasing its density (Tobin, Hajek, 2012). Nowadays, some States do not apply gypsy moth management programs any more (Kereselidze et al., 2011). E. maimaiga is effective not only at high, but also at low gypsy moth density and can cause severe larval mortality (Hajek, 2007). It is a highly host-specific pathogen and possesses great potential as a control agent of gypsy moth populations. It has been shown that azygospores of E. maimaiga can persist in soil up to 12 years and in this way can assure long term persistence when the host is not available (Hajek et al., 2010). A detailed review about biology, pathology, host specificity and epizootiology of E. maimaiga was published by Hajek (1999). In order to improve the natural biological control of gypsy moth in Bulgaria, an introduction of the pathogen was conducted in 1999 in Karlovo region (Central Bulgaria) (Pilarska et al., 2000). In 1999 and 2000, two more introductions were conducted in other regions of Bulgaria and in 2005, the first epizootics caused by E. maimaiga occurred in four sites – Elovitsa (Montana region), Skravena (Botevgrad region), Spahievo (Haskovo region) and Kremen (Kardzhali region), located 30-70 km from the introduction sites (Pilarska et al., 2006). Since 2011, E. maimaiga has invaded several gypsy moth populations in Bulgaria (Georgiev et al., 2011). Because of this reason, it was suggested that the fungus should be expected to occur in other Balkan countries. The aim of this paper is to summarize the data of E. maimaiga introductions and its latest distribution in Bulgaria, as well as its presence and subsequent spread in the Balkan Peninsula countries.

Material and methods

The first successful introduction of E. maimaiga was conducted in the region of Karlovo (Central Bulgaria) into a medium density gypsy moth population (660 egg masses/ha) (Pilarska et al., 2000). Gypsy moth carcasses containing E. maimaiga resting spores and the procedure for introduction were obtained from Dr. Hajek via the European Biological Control Laboratory (Pilarska et al., 2006). Twenty dried carcasses

32 were crushed into fine dust and released on the forest floor layer within 10 cm distance from the base of five trees (central tree and four nearest trees in N, E, S and W direction). Following spores release, 4 L of water was sprinkled around the treated trees in order to improve conditions for resting spores germination. The area was watered weekly until the observed gypsy moth larvae reached the fourth instar. Burlap bands were placed on ten nearest trees where the azygospores were released (including the released trees itself) in order to collect sufficient numbers of locally developed gypsy moth larvae with fungus-induced infection. The bands were attached 1.3 m above ground. To facilitate collections of larvae, vertical cuts were made in both layers of each burlap band. Collections started when the gypsy moth larvae reached in the late third and early fourth instar. The collected larvae were transported to the laboratory and reared on oak foliage at 20-25°C. They were observed daily for mortality. When larvae died, the carcasses were placed at 20°C in a humid chamber (sterile Petri dishes with wet filter paper) for 3-4 days. Each carcass was individually dissected and presence of conidia or resting spores was detected by observation under light microscope (Pilarska et al., 2000). In the period 2000-2014, 17 introductions were carried out according with the above-described method. The used inoculum was obtained both from sites with epizootics and U.S. (Table 1). The resting spores for introductions were maintained in cotton bags buried 1-3 cm deep in the soil at natural conditions in order to preserve their viability (Hajek et al., 2001). Most of the introductions were conducted within the frame of a project financed by the National Science Fund of Bulgaria in the period 2009-2012. In the period 2011-2014, monitoring studies were conducted to detect the presence and prevalence of E. maimaiga in Bulgaria, Serbia, Turkey, FYRMacedonia, Greece, Croatia and Bosnia and Herzegovina. During the monitoring the collected gypsy moth larvae were processed as described previously. Dead larvae collected from the field were analyzed microscopically for the presence of E. maimaiga or other pathogens causing mortality.

Results and Discussion Introductions, distribution and impact of E. maimaiga in Bulgaria The results of conducted introductions within the period 1999-2014 showed that E. maimaiga was successfully introduced and caused severe mortality of gypsy moth (between 80.4 and 100%) in most of the released sites (Table 1). The first strong epizootics were observed in 2005 in different areas of Northern and Southern Bulgaria (Pilarska et al., 2006). In subsequent years new epizootics near Sadievo (2009), Slavyanovo, Assenovo (2010), Ruets, Dalgach (2011), Solnik (2012), Kirkovo (2014) (Fig. 1) occurred and suppressed several strong outbreaks of the pest (Georgiev et al., 2013; Mirchev et al., 2013; Georgiev et al., 2014). Through a fifteen-year period, by the continuous monitoring to establish the distribution of E. maimaiga in Bulgaria, it is clear that the fungus occurs and has already

33 Table 1. Conducted introductions of E. maimaiga in Bulgaria and mortality rates of the host

Geographic coordinates Alti- Mortality Year of Locality tude, Origin of inoculum caused by introduction m a.s.l. N E E. maimaiga

Gorni 375 1999 42o33.150’ 24 o54.032’ U.S. 10.0% Domlyan Gabrovnitsa 481 2000 43o05.331’ 23 o27.626’ U.S. 8.1% Izbeglii 237 2001 42o02.043’ 25o02.038’ U.S. No data Stryama 182 2005 42o13.710’ 24o51.659’ Bulgaria (Spahievo) No data Sadievo 151 2008 42o31.783’ 26 o08.901’ Bulgaria (Kremen) 87.5% Assenovo 401 2009 43o17.695’ 26 o04.051’ U.S. 95% Slavyanovo 345 2009 43o17.090’ 26 o08.834’ U.S. 98% Sofia 641 2009 42o37.862’ 23o21.217’ U.S. 100% Dalgach 193 2010 43o12.966’ 26 o42.478’ Bulgaria (Slavianovo) 100% Ruets 312 2010 43o12.119’ 26 o37.950’ Bulgaria (Slavianovo) 100% Solnik 202 2011 42o54.268’ 27 o44.296’ Bulgaria (Dalgach) 80.4% Chakalarovo 663 2013, 2014 41o16.468’ 25o17.574’ Bulgaria (Solnik) 100% Chakalarovo 559 2013 41o16.401’ 25o18.414’ Bulgaria (Solnik) 100% Dolno 618 2013 41o16.068’ 25o16.601’ Bulgaria (Solnik) 100% Kapinovo Strizhba 701 2013, 2014 41o17.445’ 25o25.480’ Bulgaria (Solnik) 100% Strizhba 591 2014 41o17.707’ 25o26.526’ Bulgaria (Solnik) 100% Strizhba 690 2014 41o18.188’ 25o25.746’ Bulgaria (Solnik) 100%

Tihomir 538 2014 41o18.723’ 25o28.304’ Bulgaria (Solnik) 100% been established in many localities among 22 State Forest and/or Hunting Enterprises where it was introduced or has spread by natural means (Fig. 2). Until the first E. maimaiga introduction in Bulgaria, between 492 and 1028 thousand ha of forests were attacked by each gradation of L. dispar. Since 2000, the infestations have been reduced to 23-90 thousand ha which is only 2-18% of the outbreaks values before the introduction (Fig. 3). Over the past 15 years, almost no insecticides have been used, unlike previous periods when large-scale control with bacterial insecticides was applied. As a result of E. maimaiga introduction in Bulgaria, more than 10 million BGN (5 million Euros) were saved. The studies on non-target phyllophagous insects conducted in 2009-2011 confirmed the host specificity of the fungus. A total of 1499 larvae belonging to 38 insect species from 10 families of Lepidoptera and two sawfly species (Hymenoptera: Tenthredinidae), all collected from the sites where E. maimaiga occurred, were checked on the presence of the fungus. The microscopic analyses indicated no evidence of E. maimaiga life stages in any of observed larvae (Georgieva et al., 2014). The presence and

34 Fig. 1. Epizootic of E. maimaiga (Kirkovo, 2014) activity of E. maimaiga has been very important for conservation of biodiversity in oak ecosystems in Bulgaria, which harbour many lepidopteran species including species with high conservation value. In Bulgaria, some of the epizootics (2005, 2010 and 2011) occurred near the border with Serbia, FYRMacedonia, Greece and Turkey. Considering the fact that the actively ejected air born conidia are distributed by wind, which facilitates rapid spread of this fungus (Hajek et al., 1999), it was expected that the fungus will occur in neighbouring countries of Bulgaria (Georgiev et al., 2011). Occurrence of E. maimaiga in Serbia In the spring of 2011 in two localities in Central Serbia – Borački Gaj (Belgrade region) and Monastery forests in Bogovađa (Valjevo region), where severe gypsy moth outbreaks were predicted (based on the egg masses/ha), no considerable defoliation in the oak forests was observed and in some studied plots high mortality of the older instar larvae was detected. Microscopic investigations revealed presence of conidia and resting spores of E. maimaiga in carcasses collected in both sites. In the field, the mortality caused by E. maimaiga of older instar gypsy moth larvae in Borački Gaj was 85% and in Monastery forests – 92.4%. Thanks to the fungus, the outbreaks were suppressed and the number of the pest was reduced from 5000 and 3200 egg masses/ha to almost

35 Fig. 2. Distribution of Entomophaga maimaiga on Balkan Peninsula negligible levels, 15 and 0 in Borački Gaj and Bogovađa, respectively (Tabaković-Tošić et al., 2012). In 2012, a new epizootic caused by the pathogen was observed in the region of Avala hill near Belgrade. In 2013 and 2014 the fungus was detected in over than 100 localities situated in 13 regions of Serbia (Fig. 2). Mortality rate in the field was very high – between 86.7 (in Kraljevo) and 98.0% (in Kruševac and Leskovac) respectively (Tabaković-Tošić, 2014). Thus E. maimaiga acted as a powerful reducer of the gypsy moth population density and prevented severe outbreaks of the pest in Serbia. Occurrence of E. maimaiga in Turkey In 2011, in order to establish whether E. maimaiga is present in the European part of Turkey, a field collection of gypsy moth larvae from Strandzha Mountain (Vize and Çatalca districts) was organised (Georgiev et al., 2012). The larvae were collected from seven localities with oak forests (Kiyiköy, Kizilagaç, Kömürköy, Sergen, Vize, Yaliköy and Celepköy), all close to the Bulgarian border. Despite the low density of the gypsy moth larvae, the fungus was found in two localities – Vize and Yaliköy (Fig. 2). Occurrence of E. maimaiga in Greece As one of the epizootics occurred in 2005 in the region of Kremen (Southern Bulgaria) near the border with Greece, it was supposed that the fungus has penetrated into the neighbouring country. This was the aim of conducted in 2012 collections of gypsy moth larvae in oak stands from three sites in Greece (Kidaris, Thermes and Meses Thermes). The microscopic analyses of collected and reared in the laboratory larvae

36 Fig. 3. Forest area infested by L. dispar in different gradations of the pest in Bulgaria during the period 1953-2015 (columns with dark pattern – before the introduction of Entomophaga maimaiga; columns with paler shading – after the introduction) showed presence of conidia and resting spores of E. maimaiga only in Kidaris located 7 km from the border with Bulgaria (Fig. 2). It was established that almost 79% of larval mortality was caused by the fungus. Analyses did not indicate the presence of other fungal species or microsporidia (Georgieva et al., 2013). Occurrence of E. maimaiga in FYRMacedonia In 2012, collections of gypsy moth larvae were conducted in three localities in FYRMacedonia – Toplica, Belovodica and Krushevo that are 130-170 km from the nearest known E. maimaiga occurrence in Southwestern Bulgaria (Fig. 2). The results of investigation confirmed the assumption that the fungus has expended its range throughout FYROM. It was established in all three localities, causing larval mortality of 12.3%, 16.3% and 8.3% consequently (Georgieva et al., 2013). Occurrence of E. maimaiga in Croatia and Bosnia and Herzegovina The establishment of E. maimaiga in countries on the eastern Croatian borders focused the attention of Croatian researchers on the possible pathogen appearance during the latest gypsy moth outbreak. In early summer of 2012 Croatian foresters reported on high incidence of dying gypsy moth larvae in the easternmost part of Croatia (Hrašovec et al., 2013). In spring of 2013, gypsy moth outbreak area increased and large areas were aerially sprayed with Bacillus thuringiensis kurstaki based formulations. Field surveys confirmed areas with unusually dramatic mortality of older larval instars. Consequently, dead gypsy moth larvae were collected from 10 localities situated in continental Croatia. In nine out of ten collected dead cadavers, either conidia or resting spores of E. maimaiga were confirmed. It is reasonable to assume that E. maimaiga appeared at least one year

37 earlier (in 2012) than officially declared and published (Hrašovec et al., 2013) but this cannot be proved now, in spite of the fact that the location of its possible presence is near to the far eastern border with Serbia where the fungus has been already reported earlier. In 2014 E. maimaiga presence was confirmed further to the west, in the middle part of Croatia, in forests around the city of Sisak. Positive E. maimaiga findings in 2013 and 2014 included both the floodplain oak forests as well as the colline beech belt in the neighbouring area. The collapse of gypsy moth population in 2014 coincided with high infestations of E. maimaiga together with nuclear polyhedrosis virus (NPV). It may be worth noting that, in spite the focused screening, no presence of E. maimaiga was recorded within the Croatian part of the Mediterranean region in the past two years. As recent research confirmed, this part of the country is inhabited by a population of a different gypsy moth haplotypes (Lacković et al., 2015). It will be interesting to see how suitable for the E. maimaiga establishment and pathogenicity this area will prove to be, both from thee climatic standpoint as well as its host. In Bosnia and Herzegovina the fungus was found in the north-east part of the country in summer 2013 at five close situated localities which were selected based on the vicinity to the Croatian sites where the fungus has been already detected and on L. dispar higher density (continuing outbreak) (Milotić et al., 2015).

Conclusions

E. maimaiga has expanded its range since the first successful introduction in Europe. It was established in most of the Balkan countries as an efficient pathogen of gypsy moth that can be characterized as a very promising biological agent, capable of breaking down gypsy moth outbreaks and maintaining pest population density at low levels under favourable conditions. The pathogen has also spread outside the Balkan Peninsula in the country of Eastern and Central Europe. In 2005, the species was established in Georgia (Kereselidze et al., 2011). In 2013 it was found in Hungary (Csóka et al., 2014), Slovakia (Zubrik et al., 2014) and Romania (unpublished). The confirmed presence of E. maimaiga in the border zones of the above mentioned countries implies that the pathogen most likely has penetrated further into the European continent. E. maimaiga can be regarded as a good alternative to the use of chemical and bacterial insecticides for pest control, which is of great importance for the quality of human environment and biodiversity conservation in forest ecosystems.

Acknowledgments: The idea for the introduction of E. maimaiga in Bulgaria was initiated by emeritus scientist, Dr. Michael McManus, US Department of Agriculture. We are especially indebted to Dr. Ann Hajek as well as to Prof. Dr. Leellen Solter and Prof. Andreas Linde for their support in this work. The research was supported by USDA/FAS/RSED 58=3148-7-013 with the Institute of Zoology (now Institute of Biodiversity and Ecosystem Research), Bulgarian Academy of Sciences, National Science Fund of Bulgaria, Project D0-02-282/2008, Ministry of Science of the Republic of Serbia, Project 31070, Croatian Ministry of Science and Higher education and Deutscher Academischer Austauch Dienst (DAAD).

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