Impact of Entomophaga maimaiga on gypsy moth populations in

1 2 3,5 4 1 1 Plamen Mirchev , Andreas Linde ,, Daniela Pilarska , Plamen Pilarski , Margarita Georgieva , Georgi Georgiev

1Forest Research Institute, Bulgarian Academy of Sciences, , 132 St. Kliment Ohridski Blvd., Sofia 1756, Bulgaria; 2University of Applied Sciences Eberswalde, Alfred-Moeller-Str.1, 16225 Eberswalde, Germany; 3Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, 1, Tsar Osvoboditel, 1000 Sofia, Bulgaria; 4Institute of Plant Physiology and Genetics, Acad. G. Bonchev Str., Bldg, 21, 1113 Sofia, Bulgaria; 5Czech University of Life Science, Prague, Czech Republic

Introduction Results and discussion

The gypsy moth (Lymantria dispar L., Lepidoptera: Erebidae; Fig. 1 and 3) periodically causes severe Introduction in 2008. In 2008, E. maimaiga infected and destroyed 87.5% of the fifth and sixth instar damage in deciduous forests in several Central and Eastern European countries, as well as in the USA L. dispar larvae in Sadievo locality (Table 2 and Fig. 8). where it was introduced in the end of the 19th century. In Bulgaria, oak stands of different age were A late epizootic, however, led to a 60-80% defoliation of the stand by the pest. The reduction of new infested over long periods of time (Georgiev et al., 2007). Repeated defoliations decrease growth and cause generation number was 96.4% (Fig. 9). No egg masses of the pest were recorded in the experimental a physiological weakening of the host plants, increasing their susceptibility to infestations of wood borers site two years later. and pathogenic fungi. To reduce the pest density and control gypsy moth populations, broad Introductions in 2009. In the spring of 2010, an epizootic affecting mid- and late instar larvae of the spectrum insecticides and the bacterial pathogen Bacillus thuringiensis var. kurstaki (Btk) were used. Due gypsy moth occurred in Assenovo and Slavyanovo localities. Mortality of mid larvae reached 44.1- to a lack of host specificity, these methods affect aquatic organisms and many other species within the 54.1%, and of late stage larvae – 95.0-97.4% (Table 2). No defoliation was observed in the oak stands. order Lepidoptera, and thus reduce biodiversity in forest ecosystems (Miller, 1990). The reduction of the new generation was 55.1-81.8%, and was 100% one year later (Fig. 9). In 2010, gypsy moth epizootics occurred in many areas in the forests of SFE and State Hunting Enterprise (SHE) Popovo, and caused attenuation of pest outbreaks in the region. Introductions in 2010. Frequent and heavy rainfall occurred in May and June of 2011 in the region of and a strong E. maimaiga epizootic destroyed mid- and late instar L. dispar larvae in Ruets and Dalgach localities (Table 2). Defoliation in the experimental sites was not observed and no egg masses were recorded for the next L. dispar generations (Fig. 9). Conidial infections were recorded in many areas near Targovishte, which undoubtedly caused the rapid suppression of the outbreak in northeastern Bulgaria where most oak forests and strongest gypsy moth infestations in the country typically occur. E. maimaiga is known to spread more than 100 km in one season (Elkinton et al., 1991). Introduction in 2011. In the spring of 2011, 80.4% mortality of the late instar gypsy moth larvae was recorded in the experimental site at Solnik (Table 2 and Fig. 8). The reduction of L. dispar in the next Fig. 1: Late instar L. dispar Fig. 2: Spores of E. maimaiga Fig. 3: Male adult of L. dispar larva generation was 77.6%, and was 86.3% one year later (Fig. 9). Strong defoliation of oak forests of L. dispar was observed in 2011 on the Black Sea coast in the region of SHE Nessebar, approximately The entomopathogenic fungus Entomophaga maimaiga Humber, Shimazu & Soper (Entomophthorales: 30-50 km from Solnik. E. maimaiga was not introduced in the area, but because it was found in many Entomophthoraceae) was described as a host specific pathogen of L. dispar from Japan (Soper et al., 1988) areas along the coast and in Strandzha Mt. (unpublished data), it is possible that it was naturally and was introduced into the USA in the beginning of the 20th century. Since then, it successfully reduced dispersed to the region. The lack of rainfall from mid-May to mid-July could be the reason that no gypsy moth density in several states. In 1999, E. maimaiga was successfully introduced into Bulgaria from epizootic occurred. E. maimaiga is not dependent on L. dispar population density due to long survival the USA (Pilarska et al., 2000) and thereafter caused epizootics and mortality in four gypsy moth outbreak of resting spores in the soil but successful epizootics depend largely on the availability of rainfall populations, located 30-70 km from the introduction sites (Pilarska et al., 2006). during the period of larval development of the host. In 2012, the conditions for development of E. In this paper we present information on the results of recent introductions of E. maimaiga in gypsy moth maimaiga in the area were more favourable and the outbreaks that occurred in the central Black Sea populations in Bulgaria and on the impact of the fungus on the pest. coast area were suppressed by massive epizootics caused by the pathogen (Fig. 8).

Material and methods Table 2: Mortality of late instar L. dispar larvae 10 0 10 0 10 0 10 010 0 10 0 10 0 From 2008 to 2011, six introductions of E. maimaiga were performed in outbreak populations of L. dispar in oak caused by E. maimaiga 100 9 6 . 4 8 6 . 3 8 1. 8 forests in different parts of the country (Table 1; Fig. 4 and 5).Two introductions were conducted during spring, 80 77. 6 four in the fall. Before use, the inoculum was stored in soil for not less than 9 months under natural conditions. Locality Year Number of Mortality caused 60 55. 1 studied L. by % 1-st generation E of Greenwic h 23 24 25 26 27 28 dispar larvae E. maimaiga, % 40 2-nd generation Table 1 and Fig. 4: Location and main characteristics of R O M A N I A Sadievo 2008 128 87.5 44 20 studied areas, L. dispar density and origin of E. maimaiga 44 u Assenovo 2010 39 97.4 0 Locality State Forestry Altitude, Tree Density of Date of Origin of Slavyanovo 2010 40 95.0 Slavyanovo (Hunting m a.s.l. speciesa L. disparb introduction E. maimaiga Assenovo Dalgach Ruets 2011 8 100.0 N Enterprise) 43 Ruets Sadievo 151 Q.r 83 28.03.2008 Bulgaria Solnik 43 Dalgach 2011 53 100.0 Assenovo G. Oryahovitsa 401 Q.c 78 18.11.2009 USA Черно Solnik 2011 107 80.4 Slavyanovo Popovo 345 Q.c 89 18.11.2009 USA Sadievo море Fig. 9: Reduction of population density of Ruets Targovishte 312 Q.c. ;C.b. 76 18.11.2010 Bulgaria L. dispar after E. maimaiga introduction 42 Dalgach Targovishte 193 Q.ru.; T.p. 86 18.11.2010 Bulgaria 42 Solnik S. 202 Q.f.; Q.c 183 05.04.2011 USA

50 km Infestations of Bulgarian forests by L. dispar

A

M a – C.b. – Carpinus betulus L.; Q.c. – Quercus cerris L.; Q.f. – Quercus G R E E C E Figure 10 presents data of the forest area infested by different gradations of L. dispar in a period of 60 frainetto Ten.; Q.r. – Quercus robur L.; Q.ru. – Quercus rubra L.; T.p. – Tilia 23 24 25 26 27 28 years. Before the first introduction of E. maimaiga in Bulgaria annual defoliation reached from 150,000 platyphyllos Scop. b – egg masses per 100 trees to 370,000 ha. After the introduction of E. maimaiga, no large-scale pest calamities were observed and the gypsy moth s annual infestation area did not exceed 25,000 ha, only 2-5% of the infestation levels observed before the introduction. From 2010 to 2013, gypsy moth is undergoing another outbreak, but only 71,000 ha of forest are severely affected. By extrapolation we estimate that by the end of the current gradation (expected for 2017/18), the total forest area affected by gypsy moth will not exceed 150,000 ha, corresponding to only 15-30% of the infestation levels observed during gradations before the establishment of E. maimaiga. The decrease of L. dispar damages to the forest after a one-time introduction of E. maimaiga shows that the pathogen effectively reduces and regulates the pest density and chemical control of gypsy moth was used in very small areas only, dismissing the previous practice of large-scale use of

microbial and chemical insecticides.

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a 1200

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Fig. 5 and 6: Oak forest in Solnik / Bulgaria and eggs of L. dispar on bark Fig. 7.: L. dispar larvae Fig . 8 : L.dispar killed by E. maimaiga (Solnik) r

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625 Fig.10: Forest area infested by L. dispar L.

For the release of inoculum, within each experimental plot of 100 x 100 m, five circular sites were established –

y 600 b 492 in Bulgaria during the period 1953-2013

one central and four circles 50 m from the center to the magnetic north, south, east and west. Each circle d

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contained at least five trees. The fungal introductions were conducted by mixing crushed infected larvae, f 400

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containing E. maimaiga resting spores (Fig. 2), with soil and dispersing the mixture around the base of 5 to 10 a

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trees. Larvae of L. dispar were collected from the burlap bands (Fig. 7) 2-3 times per month from early May to late s t

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July and transported to the laboratory, where they were reared on fresh oak foliage in plastic boxes. The foliage re o

F 0 was changed daily, and dead gypsy moth larvae were placed in Petri dishes with moisturized filter paper at 20 C 1953-1960 1961-1969 1970-1977 1978-1988 1989-2000 2001-2009 2010-2013 for 5-7 days and then refrigerated at 5 C until microscopic evaluation. Each cadaver was dissected individually and observed under light microscope at 125x magnification for the presence of conidia or azygospores of E. maimaiga. To estimate the influence of the fungal infection on the density of the gypsy moth population, the Conclusions number of egg masses (Fig. 6) on 100 trees in each study site was counted in the fall. Е. maimaiga is expanding its range in the Balkan countries (Georgiev et al., 2012) and in the near future it is expected to spread into other areas of Southeast Europe. The high virulence and species specificity of E. maimaiga and its ability to reduce L. dispar density characterize the fungus as an effective, economical and environmentally safe biological control option for L. dispar. References

Elkinton, J.S., Hajek, A.E., Boettner, G.H. & Simons, E.E. 1991: Distribution and apparent spread of Entomophaga maimaiga (Zygomycetes: Entomophthorales) in gypsy moth (Lepidoptera: Lymantriidae) populations in North America. Environmental Entomology 20: 1601-1605. Georgiev, G., Mirchev, P., Pilarska, D., Golemansky, V., Pilarski, P., Tomovski, H. & Bochev, N. 2007: The gypsy moth will be neutralized. Gora, 5: 8-10 (In Bulgarian). Georgiev, G., Tabaković-Tošić, M., Pilarska, D., Mirchev, P., Georgieva, M., Petkov, P. & Pilarski, P. 2012: Distribution of Entomophaga maimaiga Humber, Shimazu and Soper (Entomophthorales: Entomophthoraceae) on Balkan Peninsula. In: International Scientific Conference „Forests in Future-Sustainable Use, Risks and Challenges“, Belgrade, ed. Rakonjac, L.: 619-622. Miller, J. C. 1990: Field Assessment of the effects of a microbial pest control agent on non-target Lepidoptera. American Entomologist, 36: 135-139. Pilarska, D., McManus, M., Hajek, A., Herard, F., Vega, F., Pilarski, P. & Markova, G. 2000: Introduction of the entomopathogenic fungus Entomophaga maimaiga Hum., Shim. & Sop. (Zygomycetes: Entomophtorales) to a Lymantria dispar (L.) (Lepidoptera: Lymantriidae) population in Bulgaria. Journal of Pest Science, 73: 125-126. Pilarska, D., McManus, M., Pilarski, P., Georgiev, G., Mirchev, P. & Linde A. 2006: Monitoring the establishment and prevalence of the fungal entomopathogen Entomophaga maimaiga in two Lymantria dispar L. populations in Bulgaria. Journal of Pest Science, 79: 63-67

Acknowledgements

The study was supported by National Science Fund of Bulgaria, Project DO-02/282/2008.