Biological control of gypsy moth (Lymantria dispar) by the entomopathogenic fungus Entomophaga maimaiga in Bulgaria in 2021

A biological control programme against gypsy moth (Lymantria dispar) populations was carried out in 2021 via the inoculative release of the entomopathogenic fungus Entomophaga maimaiga on the territory of four State Forest Enterprises: Municipal Enterprise (ME) ‘Management of Municipal Forests, Agriculture and Forestry’, Nessebar; State Game Enterprises (SGE) Nessebar and Balchik; State Forestry (SF) Vidin. The pathogen was released during the period 15-26.03.2021 in 34 localities five in ME Nessebar, eight in SGE Nessebar, ten in SGE Balchik and eleven in SF Vidin. The average number of gypsy moth population density in the locations of introduction was relatively high, ranging between 0.4-15.9 egg masses/tree in the area of SGE Balchik and 11.9-65.0 egg masses/tree in the area of ME Nessebar. The average mortality of young gypsy moth caterpillars (first-third instar) due to E. maimaiga varied between 2.6% (SGE Balchik) and 13.0% (SF Vidin), and of caterpillars in later fourth-sixth instar between 20.7% (SF Vidin) and 52.4% (ME Nessebar). The lowest overall mortality of the gypsy moth caterpillars due to E. maimaiga was in the region of SGE Balchik (26.1%), followed by SF Vidin (33.7%), SGE Nessebar (48.5%) and ME Nessebar (55.9%). As a result of the release, the severe gypsy moth outbreaks in the region of Nessebar were significantly suppressed. There is a high number of E. maimaiga resting spores remaining in the surface layers of the soil in the other two areas (Vidin and Balchik). They have the potential to suppress L. dispar attacks in years to follow. Silva Balcanica 22(3): 17-27 (2021) doi: 10.3897/silvabalcanica.22.e78600 Copyright Georgi Georgiev. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. RESEARCH ARTICLE 18    Georgi Georgiev et al. / Silva Balcanica 22(3): 17–27 (2021)


Introduction
Gypsy moth (Lymantria dispar L., Lepidoptera: Erebidae) is the most important defoliator in the deciduous forests of Europe, Asia and North Africa. Soon after its accidental introduction into North America in 1869, the gypsy moth started its spread as an alien invasive species, causing severe defoliation and shade trees due to the absence of natural enemies (Hajek, 2007). In Bulgaria, during severe outbreaks, L. dispar defoliates 110-370 thousand ha of forest deciduous stands .
The entomopathogenic fungus Entomophaga maimaiga Humber, Shimazu and Soper (Entomophthorales: Entomophthoraceae) was established as a species-specific pathogen of Lymantria dispar japonensis Motschulsky in Japan, where it regularly causes epizootics. Some introductions of the pathogen into the United States were carried-out in 1910-1911 and 1985-1986. In 1989 the pathogen was identified in gypsy moth epizootic in seven north-eastern states (Andreadis, Weseloh, 1990). Since that time, E. maimaiga has expanded its range by natural spread and artificial introductions in new localities of L. dispar (Solter, Hajek, 2009). Inoculative releases of E. maimaiga accelerate the spread of the pathogen and help to reduce the impact of initial and future outbreaks of the pest (Hajek et al., 2021).
In 1999, E. maimaiga was successfully introduced into two localities of L. dispar in Bulgaria (Pilarska et al., 2000). Biological material with cadavers infected by the fungus were sent from the USA. Several years after the introductions, the first severe fungal epizootics were registered in forest stands located at long distances from the places of introductions (Pilarska et al., 2006). During the period 2008-2010, all outbreaks of the gypsy moth in the country were suppressed by E. maimaiga releases in over ten localities of the pest . Since then, the pathogen has expanded its range by natural spread, suppressing the pest's calamity in the Northwestern Bulgaria (Georgiev et al., 2014a). In 2013-2014, a new release of E. maimaiga was performed in the area of SFE Kirkovo where severe mortality of gypsy moth population was achieved (Georgiev et al., 2014b).
This study presents the results of an applied biological control programme by E. maimaiga release to suppress the gypsy moth outbreaks in four locations in Bulgaria.

Attacks of the gypsy moth (Lymantria dispar) in 2021
In 2021, the attacks of the gypsy moth (Lymantria dispar) on the territory of four State Forest Enterprises: Municipal Enterprise (ME) 'Management of Municipal Forests, Agriculture and Forestry' Nessebar, State Game Enterprises (SGE) Nessebar and Balchik, State Forestry (SF) Vidin, covered a total of 6810.9 ha of deciduous forests (Table  1). Of these, 5832.0 ha were severely infested and intended for biological control by release of Entomophaga maimaiga. The gypsy moth attacks on the territory of ME Nessebar, SGE Nessebar and SF Vidin were in natural oak forests, and on the territory of SGE Balchik -both in natural deciduous stands (the Rusalka Resort) and in field protection belts of Quercus cerris.

Release of Entomophaga maimaiga
The fungal pathogen was introduced in the period 15-26.03.2021 (Table 1) in 34 sample plots: five in ME Nessebar, eight in SGE Nessebar, ten in SGE Balchik (Eastern Bulgaria) and eleven in SF Vidin (Northwestern Bulgaria) ( Table 2).
The biological material used for inoculations included caterpillar cadavers infected by E. maimaiga, collected from the tree trunks during the epizootic in 2014 in the territory of SF Kirkovo. The biological material was stored in the region of Sofia in wooden crates, filled with soil to a depth of 20-30 cm.
In 2021, the inoculum (soil mixed with infected cadavers) was released around the base of the five stems in all sample plots, by the methodology described in a previous study .

Assessment of the effect of the biological control
The effect of E. maimaiga on the rate of mortality in gypsy moth populations was reported by: assessment of the average number of egg masses/tree during the spring and autumn seasons of 2021; collecting gypsy moth caterpillars and bringing them to laboratory where microscopic analyses of dead caterpillars were conducted; field examination for the presence of dead caterpillars on tree trunks.
The assessment of L. dispar egg masses in the spring (May 2021) was performed during the release of E. maimaiga in the sample plots. The number of egg masses of the new generation of gypsy moth was recorded in the autumn, at the end of September and in October, in 20 locations of the fungus release: ME Nessebar (3 places); SGE Nessebar (4); SGE Balchik (7) and SF Vidin (6).

Analysis of biological material for E. maimaiga infection
Microscopic analyses of dead caterpillars were performed at the Laboratory of phytopathology in Forest Research Institute, Sofia, with a Zeiss NU-2 light microscope at 125× magnification. Identification of the E. maimaiga infection was conducted by the presence of mycelium, conidia or resting spores (azygospores) of the pathogen.

Average density of Lymantria dispar at the locations of E. maimaiga releases
The population density of Lymantria dispar assessed at the locations of releases was considerably higher, with maximum value of 143 egg masses in SGE Nessebar (Table  1). The average number of egg masses varied between 11.9 and 65.0 per tree in the area of ME Nessebar (Fig. 1A), 18.3-64.7 -in SGE Nessebar (Fig. 1B), 0.4-15.9 -in SGE Balchik (Fig. 1C) and 0-29.0 in SF Vidin (Fig. 1D).

Mortality of Lymantria dispar caterpillars in the sample plots
A total of 1695 gypsy moth caterpillars collected from ten sample plots (Table 3), were analysed in laboratory conditions. The average mortality due to the infection with the pathogen E. maimaiga in young larvae (first-third instar) was assessed with the highest degree in the sample plots of SF Vidin (13.0%) and the lowest -in SGE Balchik (2.6%) ( Table 3). In the first collection, the average mortality of caterpillars in fourth to sixth instars, ranged between 20.7% (SF Vidin) and 52.4% (ME Nessebar) ( Table 3). The total average mortality of the gypsy moth populations due to the infection with the pathogen E. maimaiga was the lowest in the field protection forest belts in the area of SGE Balchik (26.1%). The highest pathogen efficacy was observed in the region of ME Nessebar (55.9%), followed by SGE Nessebar (48.5%) and SF Vidin (33.7%) ( Table 3).
The lack of rainfalls in May 2021 (scarce rainfall occurred only in the region of Vidin), reflected on the low mortality rates of gypsy moth young larvae in the sample plots in the Eastern Bulgaria. Completely defoliated trees (due to the high population density of L. dispar) were observed in these areas. In June, the successful infection of the larvae with E. maimaiga positively correlated with the large amounts of rainfall that occurred during the month. Field observations in the second half of June showed that there were plenty of dead third-forth instar larvae on the ground and in shrubs under tree crowns (Fig. 2B). Analysis of the biological material showed that the cadavers of gypsy moths contained resting spores of E. maimaiga -evidence that the mortality was due to the pathogen. At the same period, the first dead caterpillars (in the fourth-fifth instars) were registered on the tree trunks, with the typical symptoms of E. maimaiga infection (Fig. 2C). Mortality of L. dispar caterpillars from E. maimaiga was established at the end of June and the beginning of July, in the region of Nessebar. The heavy rainfalls led to the removing of the larvae from the trunks and their accumulation in the base of the trees (Fig. 2D). In the region of Vidin and Balchik, the presence of gypsy moth dead larvae on the tree trunks was registered, but no development of massive epizootic was observed.

Density of the new generation of Lymantria dispar
Only single egg masses of the gypsy moth's new generation were found during the observation in the autumn, in the region of Nessebar. The density was extremely low -0 to 0.04 egg masses/tree in the forest stands of ME Nessebar (Fig. 3A) and from 0.01 to 0.30 egg masses/tree in SGE Nessebar (Fig. 3B).
In the area of SGE Balchik, the density of the gypsy moth in October, in three sample plots, was lower than in March, and in the other four plots -it was higher (Fig.  3C). The population density of the new generation of the pest (1.4-10.7 egg masses/ tree) was preserved approximately at the levels of the previous generation (2.7-15.9 egg masses/tree). In the region of SF Vidin, the population densities of the new generations were significantly lower in four localities, and an insignificant increase in population density in the new generation of the pest was reported in two plots (Fig. 3D).

Discussion
After the introduction in Bulgaria, the fungus E. maimaiga has constantly expanded its range in the East European countries. In 2011, the pathogen was registered in two localities of the gypsy moth in Serbia (Tabaković-Tošić et al., 2012) and two localities in the European part of Turkey . In 2012, it was found in one locality in northern Greece and three localities in Northern Macedonia . In the same year, mortality of L. dispar was observed with symptoms typical for the E. maimaiga infection in three localities in southern Romania (Netoiu et al., 2016). In 2013, E. maimaiga was found in nine localities in Croatia (Hrašovec et al., 2013), five localities in Bosnia and Herzegovina (Milotić et al., 2015), seven localities in Hungary (Csóka et al., 2014) and two localities in Slovakia (Zubrik et al., 2014). In the following years, the range of the pathogen expanded in the countries in which it has established its presence (Hrašovec et al., 2013;Netoiu et al., 2016;Zubrik et al., 2016). In 2019, an epizootic of E. maimaiga was found in two localities of the gypsy moth in Austria (Hoch et al., 2019) and five localities in the Czech Republic (Holuša et al., 2020).
Site-specific factors (occurrence of rainfalls and high atmospheric humidity in May-June) also affect the prevalence of the fungus E. maimaiga and its efficiency to suppress the gypsy moth outbreaks (Georgiev et al., , 2020. In the current experiments, the release of E. maimaiga in the region of Nessebar was successful, although the lack of precipitation in the spring did not allow the development of an early epizootic, and as a result, by mid-June, caterpillars of L. dispar completely defoliated the infested forest stands. However, heavy rainfalls in June favoured the development of the pathogen, which suppressed the outbreak of the pest. The results in the area of SFE Vidin and Balchik were worse, but the high number of E. maimaiga resting spores still remaining in the surface layers of the soil in these areas has the potential to suppress L. dispar attacks in years to follow. In this regard, it is appropriate not to conduct additional pest control, but to rely on the pathogen to cause epizootic under more favourable conditions in 2022. Such results were registered in 2012 on the Central Black Sea coast, after the release of E. maimaiga in 2011 in the area of SGE Staro Oryahovo . Until the introduction of E. maimaiga in Bulgaria, the attacks of L. dispar during its outbreaks amounted to 492-1028 thousand ha, and after the introduction, they decreased to 23-90 thousand ha (Georgiev et al., 2020).
In conclusion, it should be emphasized that the use of E. maimaiga for biological control of the gypsy moth populations, under favourable conditions, contributes to reducing costs, preserving biodiversity and improving the recreational qualities of forest ecosystems. However, the effectiveness of the pathogen strongly depends on the specific environmental conditions -that is why it should only be used as a part of an integrated programme to control the pest.