Disease Caused by Beaveria Bassiana (Bals.-Criv.) Vuill

Silva Balcanica, 13(1)/2012

DISEASE CAUSED BY BEAVERIA BASSIANA (BALS.-CRIV.) VUILL. ON NEWLY HATCHED LARVAE OF THAUMETOPOEA SOLITARIA FREYER 1838

Plamen Mirchev, Georgi Georgiev Forest Research Institute – Sofia Bulgarian Academy of Sciences Slavimira Draganova Plant Protection Institute – Kostinbrod

Abstract: Samples of 34 еgg clusters of Thaumetopoea solitaria were collected on April 13, 2011 in a region near the town of Ivaylovgrad (Eastern Rho- dope Mountains, Southern Bulgaria). It has been found 5.6% of newly hatched caterpillars dead of mycosis caused by the entomopathogenic fungus Beaveria bassiana. This is the first report of natural fungal infection concerned young larvae of pistachio bud moth and the first announcement for Bulgaria about T. solitaria as a new host of the fungal species B. bassiana.

Key words: Thaumetopoea solitaria, Beaveria bassiana, newly hatched caterpillars, the Eastern Rhodopes, Bulgaria

INTRODUCTION

Thaumetopoea solitaria Freyer (Lepidoptera: Thaumetopoeidae) was established for the first time in Bulgaria in 1906 (Buresh, 1915). The pest is dis- tributed in the Southern Bulgaria, in Eastern Rhodope Mountains and Kresna Gorge (Mirchev et al., 2004). The pistachio bud moth is not well studied species. There are reports only about it‘s distribution (Buresh, 1918; Buresh, Tu- leschkov, 1930; Drenowski, 1930) and some aspects of bioecology (Mirchev et al., 2006). Data about natural enemies which regulate population dencity of the pest are very scanty in the world entomological literature. Kugler (1979) and Halperin (1983, 1990) announced the occurrence of parasitoids (Tachinidae) on caterpillars and pupae. The last author reported about a mycosis caused by an entomopathogenic fungus to laboratory reared T. solitaria caterpillars as well. The current study is a part of a project for investigation of parasitoids and pathogens in Thaumetopoea pityocampa and T. solitaria inhabiting the same habitat.

61 MATERIALS AND METHODS

Samples of 34 еgg clusters of T. solitaria were collected on April 13, 2011 (the hatching period of the caterpillars) in protected area ‘Dupkata’ in a region near the town of Ivaylovgrad (Southern Bulgaria, Eastern Rhodopes Mountains). The site is karst area at 300 m altitude, covert with 50 years old forest of Pinus nigra Arn., mixed with deciduous xerophytic species including Pistacia terebin- thus L. In this habitat constantly occurring insect pest of European black pine is Thaumetopoea pityocampa, as in 2011 its density was high. The collected clusters were transported to the Entomology Laboratory at the Forest Research Institute, where they were put singly into a test tube with a cotton stopper, and stored at room temperature of 20-22 °C. Periodically is carried out taking into account: the hatching of caterpillars; emerged parasitoids; and diseased and dead caterpillars. The final analysis was made after the end of the emergence of the parasitoids. The scales were removed and the eggs were examined for presence of parasitoids emergence openings. Through cutting the whole unhatched eggs were cut and examining them using an eyeglass-microme- ter (40x magnification). Microbiological analyses of dead larvae with symptoms of mycosis were carried out at Plant Protection Institute. Fungal pathogens were isolated into pure cultures on SDAY (Sabouraud dextrose agar with yeast extract) and identi- fied according to morphological characteristics (Samson et al., 1988; Humber, 1997). Conidia and conidiogenous cells were observed in smears stained with methylen blue and in durable specimens with lactophenol and aniline blue, using a transmission interference microscope (BX60 DIC Olympus), equipped with a digital camera and a Cell B image capture software.

RESULTS AND DISCUSSION

Observations at collected egg clusters exhibited that newly hatched larvae of the pest were covered with a white mycelium with a lot of conidia aggregated to spherical white masses (Fig. 1). According to microbiological analysis mycosis to T. solitaria was caused by the anamorphic entomopathogenic fungus Beaveria bassiana (Balsamo-Crivelli) Vuillemin 1912 (Hypocreales: Cordycipitaceae). Iso- lates of the fungus formed white round powdery colonies with colourless reverse on SDAY plates. Conidiogenous cells were densely clustered with a flask-shaped basal part and a well-developed zig-zag rachis, with denticles on which conidia were formed. Conidia were single-celled, hyaline, thin-walled, hydrophobic, glo- bose to broadly ellipsoidal in shape, 1.17 x 0.81 μm (Fig. 2). Conidiogenous cells and conidia of B. bassiana were similar to those described by Brady (1979). Newly hatched larvae infected with B. bassiana remain on or near the egg cluster they not move and dispersal.

62 Fig. 1. Egg cluster and died newly Fig. 2. Conidiophores and conidia of Beau- hatched caterpillars of B. bassiana veria bassiana. Bar = 20 μm

Table 1 Viability of Thaumetopoea solitaria in the egg stage

Factors Number Rate (%) Caterpillars hatched 3653 86.4 Caterpillars died in eggs, of them 61 1.4 - caterpillars died in eggs with opening 17 - caterpillars died in eggs without opening 44 Undeveloped eggs with dried-up yolk 79 1.9 Eggs totally empty, without any remains 9 0.2 Eggs destroyed by predators 13 0.3 Impact of egg parasitoids 177 4.2 Newly hatched caterpillars died of Beaveria bassiana 236 5.6 Total number of eggs 4228 100.0 Total number of egg clusters 34 100.0 Egg clusters with caterpillars died of B. bassiana 2 5.9

The final analysis of 4228 eggs in 34 egg clusters revealed that dead caterpillars were found in 61 eggs (1.4%). Some of the caterpillars had made a hole in the egg horion before their death but didn‘t leave the egg, the other eggs with dead caterpillars were without a hole (Table 1). Symptoms of mycosis were not established in both cases. The reason for the death of the caterpillars in the eggs was unknown. The lack of mycosis could be explained with the rarely occurrence of fungal isolates with ovicide action (Maniana et al., 1991; Long et al., 1998; Gavin et al., 2009) which is due to lack of the proper lipase capable of lysing wax of the egg surface. As it is known fungal pathogens infect their hosts percutaneously. Mode of action of fungal infections includes production of extracellular enzymes

63 by entomopathogenic fungi lysing the host cuticula, and penetration through the arthropod integument with support of mechanical pressure of the growing tubes of the germinating conidia (St. Leger et al., 1986; Draganova, 1988; Gupta et al., 1992; St. Leger, 1995). As it could be seen at Table 1 the rate of the hatched caterpillars was rela- tively high - 86.4% of the total number of eggs. Mycoses and entomophagous could be mentioned as two groups with comparatively similar parts in regulation of the population density of T. solitaria at the stage as newly hatched caterpillars. Eggs parasitized or damaged by predators presented 4.5%. The rate of sterile unfecun- dated eggs was small (1.9%) as eggs totally empty, without any remains (0.2%). Such sterile and empty eggs were registered for another species from family Thaumetopoeidae - T. pityocampa which eggs from the second group often were near or under 1% until sterile eggs could reach to 20.6% (Tsankov et al., 1998). Disease caused by a microsporidian pathogen affected insects in larval stage could be the reason butterflys to lay sterile eggs as it has been established for Lymantria dispar L. by Goertz, Hoch (2008). According to Halperin (1983, 1990) larvae of T. solitaria from laboratory breedings were affected by the entomopathogenic fungus B. bassiana which killed 9% of the larvae which penetrated the soil for pupation. The author supposed that rate of larvae with mycosis should be higher under natural conditions. According to Weiser (1972) inoculum of B. bassiana propagules and es- pecially of conidia was with a great importance for provocation of mycoses in a concrete biotope as single spores were not enough to cause it. Entomopathogenic fungus B. bassiana is a cosmopolitan species with a large host range (Goettel et al., 1990; Butt et al., 2001) which includes T. pityocampa (Sevim et al., 2010; Dra- ganova et al., 2011) as well. Collected samples of еgg clusters of T. solitaria were from the place called ‘Dupkata’ which was in occupation of another species from family Thaumeto- poeidae - T. pityocampa. In Bulgaria B. bassiana was announced as entomopatho- gen occurred in natural populations of the pine processionary moth (Draganova et al., 2011). Significance of the pine processionary moth in support of infectious stock in the biotope called ‘Dupkata’ is determined by the population density of the pest. Nearly 30 years T. pityocampa has been settled in the pine forest and often with high population density.

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