Insect pathogens and entomoparasitic nematodes IOBC-WPRS Bulletin Vol. 90, 2013 pp. 359-363

Impact of Entomophaga maimaiga on gypsy moth populations in

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

Abstract: The entomopathogenic fungus Entomophaga maimaiga Humber, Shimazu and Soper (Entomophtorales) (Entomophtoraceae) was introduced into three populations of gypsy moth (Lymantria dispar L., Lepidoptera: Erebidae) in Bulgaria in 1999. After the first strong epizootics in 2005, the species was introduced in six outbreak populations of gypsy moth in different regions of the country from 2008 to 2011. Due to the resulting fungal epizootics, the calamities of the pest in Bulgaria were totally suppressed. The pathogen increased its impact by a natural range extension and it is now present in nearly all regions of the country in which L. dispar occurs.

Key words : gypsy moth, Entomophaga maimaiga , Bulgaria, biological control

Introduction

The gypsy moth (Lymantria dispar L., Lepidoptera: Erebidae) periodically causes severe damage in deciduous forests in several Central and Eastern European countries, as well as in the USA where it was introduced in the end of the 19 th century. In Bulgaria, oak stands of different age were infested over long periods of time (Georgiev et al ., 2007) . Repeated defoliations and decrease growth cause a physiological weakening of the host plants, thereby increasing their susceptibility to infestations of wood borers and plant pathogenic fungi. To reduce the pest density and control gypsy moth populations, broad spectrum chemical insecticides and the bacterial pathogen Bacillus thuringiensis var. kurstaki (Btk ) were used. Due to a lack of host specificity, these methods affect aquatic organisms and many other species within the order Lepidoptera, and thus reduce biodiversity in forest ecosystems (Miller, 1990). The entomopathogenic fungus Entomophaga maimaiga Humber, Shimazu & Soper (Entomophthorales: Entomophthoraceae) was described as a host specific pathogen of L. dispar from Japan (Soper et al. , 1988). It was introduced into the USA in the beginning of the 20 th century. Since then, it successfully reduced gypsy moth density in several states. In 1999, E. maimaiga was successfully introduced into Bulgaria from the USA (Pilarska et al. , 2000). Thereafter it caused epizootics and mortality in four outbreak populations of gypsy moth, located 30-70 km from the introduction sites (Pilarska et al. , 2006). In this paper we present results of recent introductions of E. maimaiga in gypsy moth populations in Bulgaria and on the impact of the fungus on the pest.

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Material and methods

From 2008 to 2011, six introductions of E. maimaiga were performed in outbreak populations of L. dispar in oak forests in different parts of the country (Table 1). Two of the introductions were conducted during the spring, four in the fall. Before use, the inoculum was stored in the soil for not less than 9 months under natural conditions.

Table 1: Main characteristics of studied areas, L. dispar density and origin of E. maimaiga

Locality State Forest Geographical Altitude, Tree Density Date of Origin of (Hunting) coordinates m a.s.l. species a of L. introduction E. maimaiga Enterprise dispar b Sadievo 42º31.783'N; 151 Q.r. 83 28.03.2008 Bulgaria 026º08.901'E Assenovo G. Oryahovitsa 43º17.695'N; 401 Q.c. 78 18.11.2009 USA 026º04.051'E Slavyanovo Popovo 43º17.090'N; 345 Q.c. 89 18.11.2009 USA 026º08.834'E Ruets 43º12.119'N; 312 Q.c.; 76 18.11.2010 Bulgaria 026º37.950'E C.b. Dalgach Targovishte 43º12.966'N; 193 Q.ru.; 86 18.11.2010 Bulgaria 026º42.478'E T.p. Solnik S. 42º54.268'N; 202 Q.f.; 183 05.04.2011 USA 027º44.296'E Q.c. a – C.b. – Carpinus betulus L.; Q.c. – Quercus cerris L.; Q.f. – Quercus frainetto Ten.; Q.r. – Quercus robur L.; Q.ru. – Quercus rubra L.; T.p. – Tilia platyphyllos Scop. b – egg masses per 100 trees

For the release of inoculum, within each experimental plot of 100 x 100 m, five circular sites were established – one central and four circles 50 m from the center to the magnetic north, south, east and west. Each circle contained at least five trees. The fungal introductions were conducted by mixing crushed infected larvae containing resting spores of E. maimaiga with soil, and dispersing the mixture around the base of 5 to 10 trees. The base of the tree was watered with 4-5 liters of water to achieve adequate humidity. To monitor larval density, in each study site burlap bands were placed on 25 oak trees (including the treated trees) at a height of 1.3 m from the ground. Larvae of L. dispar were collected from the burlap bands 2-3 times per month from early May to late July and transported to the laboratory, where they were reared on fresh oak foliage in plastic boxes. The foliage was changed daily, dead gypsy moth larvae were placed in Petri dishes with moisturized filter paper at 20 °C 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 number of egg masses on 100 trees in each study site was counted in the fall.

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Results and discussion

Impact of E. maimaiga on L. dispar in Bulgaria The introduction in Sadievo locality was conducted with dead gypsy moth larvae collected during the epizootic in 2005 in the village of Kremen. After the introduction, in the same year, E. maimaiga infected and killed 87.5% of the fifth and sixth instar L. dispar larvae. The reduction of egg masses was 96.4% and no egg masses were recorded two years later. Introductions of E. maimaiga in Assenovo and Slavyanovo area were conducted with an inoculum from the USA. As a result of the introduction epizootics occurred in both experimental sites in 2010. Mortality of young larvae reached 44-55%, whereas mortality of the late instar larvae was 95-98%; no defoliations were observed in the stands. The reduction of the egg masses was 55.1-81.8%, and 100% a year later. Interestingly, in 2010, gypsy moth epizootics caused by E. maimaiga were recorded not only in Assenovo and Slavyanovo, but also in many other areas in the adjacent forests of SFE and State Hunting Enterprise (SHE) Popovo. Introductions of E. maimaiga in Ruets and Dalgach in the region of SFE Targovishte were conducted with a mixed inoculum from the epizootic near Slavyanovo (collected in the summer 2010) and Sofia (collected in 2005 near Kremen, stored in soil substrate for about 5 years). In May and June 2011 frequent and heavy rainfall occurred in the region of Targovishte. We suppose that this favoured the establishment of E. maimaiga and resulted in an epizootic that killed almost all middle and late instar larvae of the pest. Defoliation in the experimental sites was not observed and no egg masses at all of L. dispar were recorded. Furthermore, conidial infections were registered in larvae in many areas near Targovishte. We believe that this caused the rapid suppression of the outbreak in the oak forests of Northeastern Bulgaria, where the strongest gypsy moth infestations in Bulgaria had been reported in the past. For the introduction in Solnik, inoculum from the USA was released in April 2011. In the late spring of 2011, 80.4% mortality of the late instar gypsy moth larvae was registered in the release site. The reduction of gypsy moth egg masses was 77.6% in 2011 and 86.3% in 2012. In 2011, on the Black Sea coast in the region of SHE Nessebar (30-50 km from Solnik) strong defoliation of oak forests by L. dispar was registered. In 2012, however, gypsy moth in this region as well as all over central Black Sea coast was suppressed by the pathogen. As seen in other areas, adjacent to release plots (e. g. Popovo, see above), we believe that this demonstrates the self-disseminating capacity of the fungus. It is well known that E. maimaiga can spread more than 100 km in one season (Elkinton et al ., 1991).

Infestations of Bulgarian forests by L. dispar Figure 1 presents data of the forest area infested by different gradations of L. dispar in a period of 60 years. Before the first introduction of E. maimaiga in Bulgaria in 1999, 492 to 1,028 thousand ha of forests were affected by the pest each decade, and annual defoliation reached from 150,000 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.

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1200

1023 1028 985 1000

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625 600 492

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Forests area infested by L. dispar (x 1,000 ha) 1,000(x dispar L.by infested area Forests 200 71 23 0 1953-1960 1961-1969 1970-1977 1978-1988 1989-2000 2001-2009 2010-2013

Figure 1: Forest area infested by L. dispar in Bulgaria during the period 1953-2013

From 2010 to 2013, gypsy moth is undergoing another outbreak, but only 71,000 ha of forest were 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. After the introduction of E. maimaiga in Bulgaria, 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. 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.

Acknowledgements

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

References

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