Naturally-Occurring Entomopathogenic Fungi on Three Bark Beetle Species ( Coleoptera : Curculionidae ) in Bulgaria

Slavimira A. Draganova1, Danail I. Takov2 and Danail D. Doychev3 1Plant Protection Institute, 35 Panajot Volov Str., 2230 Kostinbrod, Bulgaria (sdraganova19@gmail.com) 2Institute of Zoology, Bulgarian Academy of Sciences, 1 Tsar Osvoboditel Boulevard, 1000 Sofia, Bulgaria 3University of Forestry, 10 St. Kliment Ohridski Boulevard, 1756 Sofia, Bulgaria Received: December 3, 2009 Accepted: February 2, 2010


INTRODUCTION
Ips typographus (L.) and Ips acuminatus Gyll.are some of the most damaging bark beetles (Coleoptera: Curculionidae: Scolytinae) on living trees in Europe according to Gregoire and Evans (2004).
In Bulgaria, about 300 ha of spruce stands in the forest reserve Bistrishko branishte (Vitosha Mountain) were destroyed some years ago due to damages caused by Ips typographus (Georgiev, 2006).The activity of their various natural enemies -pathogens, parasitoids, parasites or predators suppressing their population density is of great importance.
While Beauveria bassiana (Bals.-Criv.)Vuillemin has been reported as a fungal pathogen frequently occurring in populations of bark beetles, the species Isaria fumosorosea Wize (major synonym Paecilomyces fumosoroseus (Wize) Brown and Smith), Isaria farinosa (Holmsk.)Fries (major synonym Paecilomyces farinosus (Holmsk.ex Gray) Brown and Smith), Verticillium lecanii (Zimm.)Viegas and Metarhizium anisopliae (Metsch.)Sorokin have been mentioned only in a few reports (Landa et al., 2001;Jankevica, 2004;Sosnowska et al., 2004).Wegensteiner (2004), reviewing records about pathogens of bark beetles, pointed out that there was insufficient knowledge of the activity of pathogens on bark beetles worldwide in spite of a presumed potential of pathogens in controlling bark beetle populations.According to him, a number of investigations have shown the occurrence of fungi in bark beetle field populations.Several fungal species were most frequently mentioned, including B. bassiana and Isaria sp.(synonyms Spicaria, Paecilomyces).
Biodiversity of entomopathogenic fungi on bark beetles in Bulgaria has been sporadically investigated.An isolate obtained from a dead adult of Ips sexdentatus Boer., collected from a natural environment, has been identified by Draganova et al. (2007) as B. bassiana.
The aim of this study was to find, identify and study the morphological characteristics of fungal entomopathogens occurring naturally in populations of three scolytid species -Ips sexdentatus, Ips typographus and Dryocoetes autographus (Ratz.).

MATERIAL AND METHODS
Insects were collected from three localities.The first stand was located at an altitude of 1400 m above sea level in a forest on Zlatnite Mostove (part of the northern range of Vitosha Mountain, West Bulgaria).Adults of Ips typographus were found under the bark of dead spruce trees (Picea abies L.) in 2006.
Adults of Ips sexdentatus were collected from the bark of dead Scots pine trees (Pinus sylvestris L.) in 2008 from a second stand that was located at an altitude of 1230 m above sea level on Maleshevska Mountain (South-West Bulgaria).
Adults of D. autographus were found in 2008 under the bark of fallen and dead spruce trees in the forest reserve of Bistrishko Branishte (Vitosha Mountain).The stand was at 1550 m above sea level.
Dead pest adults were examined for fungal pathogens in 2008 and 2009 at the Department of Biological and Integrated Pest Control (Plant Protection Institute).Cadavers of insects were placed in a moist chamber to allow fungal reproductive structures to develop.Fungal pathogens were isolated into pure cultures on SDAY (Sabouraud dextrose agar with yeast extract) and were identified according to Samson et al. (1988) and Humber (1997) based on morphological characteristics both on the host and in the culture.Morphological characteristics of the isolates were studied by phenotypic methods.Colonies growing for 14 days at 23 o C under daylight were checked on plates with PDA (potato dextrose agar) and SDAY.Conidia and conidiogenous cells were observed in smears and durable specimens with lactophenol and aniline blue using a transmission interference microscope BX60 DIC Olympus equipped with digital camera and Cell B image capture software with a calibrated Carl Zeiss micrometer.
Dead insects placed in a moist chamber were covered after three days by dense mycelium of the fungus -white in the cases of B. bassiana and B. brongniartii and yellowish white in the case of I. farinosa.Mycelium started growing out through the inter-segmental membrane of the host.
Conidiogenous cells of B. bassiana were densely clustered in whorls, hyaline, smooth and short.In some smears and durable specimens of all fungal isolates, it was noticed that conidiogenous cells were branched, which made clusters thicker.Conidiogenous cells had globose (isolates 433, 434, 503 and 559 B. bassiana) 1) were smaller in size than conidia of the species reported by Brady (1979a) and by Evlakhova (1974) as 2-3 x 2-2.5 μm and as 2-3 μm in diameter, respectively, but they were similar in size to conidia of the isolate 426 B. bassiana with dimensions 1.8-2.0μm in diameter (Draganova et al., 2007).
On SDAY, B. bassiana isolates formed round raised colonies with powdery surface, with pigmentation from white to cream, reverse of the colonies with pale cream pigmentation.Only the isolate 501 B. bassiana released pink pigment into the media until the 10 th day, but it paled and gradually disappeared and the redish reverse of the colonies became pink-tanned.On PDA the colonies were round, flat, like a hyaline film from radial growing mycelium, downy in the centre.The colonies of B. bassiana isolates grew slowly.After 14 days at 23 o C and exposure to daylight they were between 17.7 and 34.0 mm in diameter on PDA and between 18.3 and 28.7 mm on SDAY ( Using a gene-genealogical approach to investigate molecular phylogenetic diversity of Beauveria and several presumptively related Cordyceps species, Rehner and Buckley (2005) analyzed 86 isolates from diverse geographic origins, habitats and insect hosts.They reported B. brongniartii isolates (one of them originating from Belgium and with insect host belonging to Coleoptera, Rhizophagidae) with smaller dimensions of conidia (2.1-2.9 x 1.9-2.3μm).
The isolate marked 500, obtained from dead adults of Ips sexdentatus, was identified as Isaria farinosa.Its colonies on PDA were not very different from colonies of B. bassiana and B. brongniartii.They were round, flat, like a hyaline film from radial growing mycelium, downy in the centre.In our opinion, PDA was not suitable for this species.On SDAY, the colonies of the isolate 500 I. farinosa were round, raised, with fluffy surface, yellowish to orange pigmentation, the reverse of the colonies of yellowish orange.The size of the colonies reached 34 mm in diameter on PDA and 36 mm on SDAY after 14 days of cultivation at 23 o C. Conidiophores arise from dense basal felt mycelium of the colonies, making them seem granular.Conidiophores were septate, hyaline, bearing groups of swollen lateral cells and whorls of conidiogenous cells.Conidiogenous cells were flask shaped phialides, smooth-walled, with average dimensions 5.36 x 1.07 μm, with cylindrical base tapering into a distinctly thin neck.Conidia of the isolate were one-celled, hyaline, smooth, lemon-shaped, with average dimensions 1.88-2.14x 0.79-1.36μm, borne on the tip of the phialides, forming dry chains.
In conclusion, although the present study was preliminary, the results contribute to our knowledge of the diversity of entomopathogenic fungi on bark beetles in Bulgaria.
or flask-like base (501, 502 B. bassiana) terminating in narrow extended denticulate apex.The growing apex repeatedly formed conidium and re-grew just below the new conidium, giving a distinctly zig-zag appearance (rachis) which in some cases reached 12.86 μm (isolate 501 B. bassiana) or even up to 18.44 μm (isolate 502 B. bassiana).Conidia were one-celled, hyaline, thinwalled, hydrophobic, globose in shape, very similar in size in different isolates -from 1.16 x 1.02 μm (isolate 502 B. bassiana) to 1.87 x 1.60 μm (isolate 559 B. bassiana) (Table 1).Conidiogenous cells of the isolates of B. bassiana have been observed in cultures often to be aggregated into clavate synnemata.The synnemata of the isolate 501 B. bassiana reached 10 mm in length in older cultures with SDAY.
Conidia of B. bassiana isolates (Table

Table 2 )
. Isolate 435 B. brongniartii had morphological traits very similar to the morphology of B. bassiana isolates.Its colonies on PDA were round, flat, like a hyaline film from radial growing mycelium, downy in the centre.Isolate 435 B. brongniartii grew better on SDAY,