Diversity of Entomopathogenic Microorganisms in Pest Populations of Bialowieza Forest Forest Stands

issn 1392-3714

Diversity of entomopathogenic microorganisms in pest populations of Bialowieza forest forest stands

Ludmila Prischepa, Natalia Мikulskaya, Мarina Gerasimovich RUC “Institute of plant protection”, Mira, 2, Priluki, Belarus; e-mail: [email protected] Danuta Sоsnowska Institute of plant protection – National Research Institute, ul. Wladyslawa Wengorka 20, 60-318 Poznan, Poland; e-mail: [email protected] Stanislaw Bałazy Institute for Agricultural and Forest Environment, ul. Bukowska, 19, 60-809, Poznan; e-mail: [email protected]

Annotation On the territory of Bialowieza forest the itinerary inspections on the reveal and collection of diseased and dead insects were carried out. On the whole, 29 dis- tricts with different type of vegetation and 81 samples were selected. Microscopic spore fungi and bacteria were present in the litter, grass stand, surface soil layer and under tree bark. In all, on Belarusian and Polish territories of Bialowieza forest 9 sections with different type of vegetation have been inspected, 147 samples have been selected. In different ecotopes of Bialowieza forest 36 species of entomopathogenic fungi, 7 isolates of entomopathogenic nematodes of Steinernema genus and 4 strains of the bacteria Bacillus thuringiensis have been isolated. By a percentage correlation, the fungi share has made 76.5 %, nematodes – 14.8 % and bacteria – 8.5 %. Key words: entomopathogenic microorganisms, diversity, Bacillus thuringiensis, Steinernema, Bialowieza forest.

INTRODUCTION The national park “Belovezhskaya Pushcha” is the oldest forest massive in Europe and located on the territory of Belarus and Poland. Forest maintenance in a relatively natural condition, flora and fauna richness, ecosystem diversity, position in a system of natural complexes

72 LUDMILA PRISCHEPA, NATALIA МIKULSKAYA, МARINA GERASIMOVICH, DANUTA SОSNOWSKA, STANISLAW BAŁAZY of Europe determine Bialowieza forest’s importance as an object of researches in the sphere of biology, ecology and biological diversity. Mixed and broad-leaved forests, the unusual combination of plants representatives of south and north, seaside and continental climate, meadows and water systems are kept there in their natural initial state. Bialowieza forest’s flora is characterized by a large number of plant species – Abies sp., Quercus spp., Picea abies, Carpinus betulus, Fraxinus sp. – which are annually damaged by pests (Priroda..., 2011). Under natural conditions insect diseases agents behave as authentic Arthropoda pest number regulators, the mechanism of their action is developed by centuries in the process of parasite-host relations evolu- tion (Воронцов, 1978). In separate years when favorable conditions are created for certain species, the phytophage mass reproduction outbreaks appear (Berriman, 1990; Воронцов, 1984). In Bialowieza forest there were the outbreaks of mass reproduction of green oak roller moth (Tortix viridana L.) (1908, 1909, 1957) and eight-toothed engraver (Ips typographus L.) (1903-1907, 1992, 1994- 1997), which were accompanied by pest number decrease at the cost of pathogenic microorganisms epizooties. Reveal of infected individuals in Belovezhskaya Pushcha insect populations is exclusively impor- tant for the evaluation of microorganisms role in the dynamics natural populations from the point of biological diversity. The aims of work on studying the entomopathogenic microorganisms and nematodes in phytophage populations were carried out in the National Park together with the Institute of Plant Protection – National Research Institute (Poland) in 2004-2005.

МАTERIALS AND METHODS The diseased and dead insects were collected in different botanical sections (mixed forests, groves, and pine forest) of “Bialowieza forest”. Dead insects were collected three times a year: spring, summer and autumn. A choice of sections for supervision was done considering forest pathological supervision data. On the whole, 29 compartments with different type of vegetation and 81 samples were selected. Microscopic

73 BOTANIKOS SODO RAŠTAI spore fungi and bacteria were present in the litter, grass stand, surface soil layer and under tree bark. In all, on Belarusian and Polish territories of Bialowieza forest 9 sections with different type of vegetation have been inspected, 147 samples have been selected. By supervision the tree trunks, undergrowth, soil surface in the range of crown projection was inspected. The pests inhabiting the upper soil layers and litter were collected on registration 0.5×0.5 m plots. In the course of researches the dead insects and the insects with the vivid disease signs were collected. The collected pathological material was inserted into Florinsky test-tube putting one exemplar, sometimes with a substrate proportion. The isolation of entomopathogens from soil was accomplished according to G. Zimmerman method (1986). Determination of specific be- longing was done according to manuals (Evlakhova, 1964; Koval, 1974).

RESULTS AND DISCUSSION Based on the analysis of forest pathological monitoring and the results of own inspections the most injurious Bialowieza forest’s phy- topathogens were revealed which are divided into 3 groups: conifer needles – biting, leaf-biting, trunked. The most massive species from conifer needles pests with two ge- nerations per one year is pine sawfly Diprion( pini L.). Also nun moth (Lymantria monacha L.), pine moth (Dendrolimus pini L.), pine beau- ty (Panolis flammea Schiff.), pine winter moth (Bupulus piniarius L.), pine false webworm (Acantholyda erythrocephala L.), star leaf-rolling sawfly Acantholyda( stellata Chr.), European pine sawfly Neodiprion( sertifer Geoffr.). Among leaf-biting pests Lymantria dispar L., Malacosoma neus- tria L.), Euproctis chrysorrhoea L., Dasychira pudibunda L., Tortrix viridana L., Apocheima hispidaria Sch., Phalera bucephala L.), Operophtera brumata L., Erannis defoliaria C., Notodonta anceps Geoze. are im- portant. Trunked pests – Blastophagus piniperda L., Blastophagus minor Hart., Monochamus galloprovincialis Oliv., Phaenops cyanea F., Scolytus ratzeburgi Jans., Blastophagus piniperda L., Ips typographus L.,

74 LUDMILA PRISCHEPA, NATALIA МIKULSKAYA, МARINA GERASIMOVICH, DANUTA SОSNOWSKA, STANISLAW BAŁAZY

I. sexdentatus Boern., I. stenographus Duft., I. duplicatus Sahlb., Pityogenes chalcographus L. and Blastophagus minor Hart. Among the above-mentioned phytophages the prevalent group of spruce trunk pests was a spring one presented by Ips typographus (the first generati- on), I. sextentatus, I. stenographus, I. duplicatus, Pityogenes chalcographus, Polygraphus polygraphus. Summer group was less numerous and was represented by eight-toothed engraver (the second and sister ge- nerations, different long-horned beetles, spruce weevils and horntails. A significant weakness of spruce forest stand, entomophages ab- sence during mass bark beetle reproduction and other factors pro- moted mass phytophage appearance. So, eight-toothed engraver number fluctuated from 2 thousand to 16-20 million individuals and on separate areas there were up to 32 million individuals per 1 ha. In 2003 spring leaf-biting pests’ reproduction was noticed in oak and birch plantations. Маss reproduction was marked for a moth complex and green oak roller moth. The itinerary inspections on the reveal and collection of diseased and dead insects were done using the common methods on Belarusian and Polish forest’s territories. On Belarusian and Polish territories of Bialowieza forest 29 sections with different type of vegetation have been inspected with different type of vegetation. Only 8 from 29 of the inspected section on the Belarusian territory of forest from the collected pathological material the entomopathogenic microorganisms and nematodes have been isolated. 30 isolates have been isolated, from which by the percentage correlation the fungi share has made 30.0 %, crystal-bearing bacteria – 46.6 %, nematodes – 23.4 %. Studying the insect localization places from which the entomopathogenic microorganisms have been isolated has shown that microscopic spore fungi and bacteria were present in a litter, grass stand, surface soil layer and under tree bark. On Polish territory the advantage in revealing the entomopathogenic complexes localization was given to the plots with broadleaf forest where lime, oak, maple, hornbeam, elm and ash were prevalent. By inspection, the vivid white spots (the entomopathogenic fungi mycelium and core- mium) were recorded. It is determined that among the entomopathogenic fungi two species (often accompanied by Paecilomyces tenuipes) were prevalent: P. far ino su s and Beauveria bassiana. The infection of

75 BOTANIKOS SODO RAŠTAI wintering insect stages has made 20–30 %. In the Polish part of forest the pathogens presence was determined, mainly, fungi on beetles and spiders found in forest biocoenosises under the dead tree bark, in forest grass stand or on tree leaves (Table 1).

Таble 1. Entomopathogenic microorganism species diversity on the Polish territory of Bialowieza forest 1 lentelė. Entomopatogeninių mikroorganizmų rūšių įvairovė Belovežo girioje Lenkijos teritorijoje Fungus name ARTHROPODA (place of inhabitance, other commentaries, cоelected in: (S) – studied plot, (B) – behind the studied plot) Chytridiomycetes Ips amitimes, I. duplicatus, I. typographus – adult beetles in Chytridiopsis their galleries (B) typographi Weiser Entomophthorales Diptera, Hemiptera, Lepidoptera, rare other order insects – Batkoa apiculata different species imago, as a rule, in a forest grass stand and (Thaxt.) Humber small forests (S) (B) Conidiobolus sp. Tipulidae, Limoniidae – imago; in a forest grass stand from the wet side(S) (B) Entomophaga Tenthredinidae (Dolerus sp. larvae) in the middle of forest tenthredinis (Fres.) meadows – rare (B) Batko E. tipulae (Fres.) Tipulidae, Limoniidae – imago; in a moistured forest soil (S) (B) Humber Entomophthora Anthomyidae – on imago, epizooties, rare single exemplars on muscae (Cohn) other flies (S) (B) Fres. E. israelensis Itonididae – in a dense undergrowth on separate host Ben-Ze’ev samples (B) E. planchoniana Aphids – mainly in undergrowth and in the middle of forest Cornu meadows (S) (B) Erynia ovispora Calliphoridae, Scatophagidae and perhaps other indeterminate (Nowak.) Remaud. flies in boggy forests and club grasses (S) (B) & Hennebert E. variabilis Small adult Nematocerous flies, more often Psychodidae, in (Thaxt.) Remaud. & brooks and ditches, simply on water surface (S) (B) Hennebert Eryniopsis Tipula and Nephrotoma species on single imago in moistured caroliniana (Thaxt.) places (B) Humber

76 LUDMILA PRISCHEPA, NATALIA МIKULSKAYA, МARINA GERASIMOVICH, DANUTA SОSNOWSKA, STANISLAW BAŁAZY

Furia Americana Calliphoridae imago in a wooden undergrowth, only separate (Thaxt.) Humber fragments (B) Neozygites fresenii As it is above-mentioned, from July to September (B) (Nowak.) Remaud. & S. Keller Pandora dipterigena Common for Nematocer in a forest litter and swamps (S) (B) (Thaxter) Humber P. echinospora Lauxaniidae and Dryomyzidae imago; only on single (Thaxter) Humber fragments in a forest litter, later on in summer and autumn (B) P. myrmecophaga Formica polyctena, on working ants in Belovezhskaya (Turiani Wuest) pushcha’s nests – only single samples, though they are Humber constantly met (B) P. lip ai Bałazy Rhagonycha lignosa – imago in a dense undergrowth of a mixed forest in June (S) P. mu s c iv ora As a rule, on different imago of flies from the end of May and (Schroeter) Bałazy up to autumn, excluding dry periods (S) (B) P. neoaphidis Aphid species, on field margins are rare and only single host (Remaud.) Humber species in a forest from June to autumn (B)(S) Zoophthora Mirid bugs (Discyphus or Stenodema sp.) on Stachys silvatica – aprophorae in one plot (B) (Rostrup) Z. autumnalis Dryomyzidae – imago; starting from September to November Bałazy (S) (B) Z. crassitunicata Malthodes spp. – imago; including May – June, rare in a litter Keller of broadleaf forests (B) Z. ichneumonis Ichneumonidae – imago; on leaves in a dense undergrowth; Bałazy from the end of May-September, local, even during dry period (S) (B) Z. nematoceris In a forest litter and undergrowth, in spring are constantly Bałazy met, in summer and autumn – single (S) Z. psyllae Bałazy Trioza urticae – not always, close to July (B) Z. radicans Operophthera brumata – caterpillars; in May – of June in (Brefeld) Batko broadleaf forests (B) Hyphomycetes Insects, seldom spiders, are constantly met (S) (B) Beauveria bassiana (Bals.) Vuill. Beauveria cf. Auchenorrhyncha – selects small species, prefers grass stand, bassiana later in summer and autumn, often epizooties are met (S) Gibellula leiopus Spiders (Juvenile forms and imago) in meadows, seldom in (Vuill. ex Maubl.) grass stand (S) (B) Mains

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G. pulchra Cavara Spiders (as it is above-mentioned) are found in moistured meadows, seldom in a forest litter (S) Hirsutella sp. Dendrolaelaps sp. (Acari) – single exemplars in bark beetle (brownorum or galleries on common spruce (B) nodulosa) Lecanicillium spp. On different insects and mites, sometimes on spiders in a litter, on plant leaves – species are determined (B) Paecilomyces Different insects, mainly, Lepidoptera, Hymenoptera and farinosus Brown et Diptera, seldom single spiders; in a forest litter are found Smith abundant in meadows in single exemplars, in a rainy period often epizooties are observed (S) (B) P. suff u ltu s (Petch) Bibio sp. – larvae in flood meadows litter Samson P. tenuipes (Petch) Lepidoptera caterpillars and pupae; in fallen leaves (S) (B) Samson Sesquicillium Predatory beetles larvae in a litter of fallen leaves, not in high candelabrum number in autumn (S) (B) (Bonorden) W. Gams

On Alnus leaves the insects infected with Paecylomyces suffultus and mummified by fungusSesguicllium candelabrum, spiders infected with Gibellula leiopus and Gibellula pulchara were found. In autumn entomopathogenic fungi collection in a forest litter of mixed and alder forests has made from 17 to 37 insects, mycosis infected. In all the isolated fungi dominated Paecilomyces farinosus (63 %) and Beauveria bassiana (21 %). The subdominant species was Paecilomyces tenuipes –10 %, at the same time the other entomopathogenic fungi species present in collections have made 7 %. In autumn in spider collections dominated fungi from Gibella and Beauveria bassiana. Arthropoda infected with entomopathogenic fungi, were found on herbaceous plant leaves, under tree bark, in the upper layer of alder trees. The insects with the vivid infection signs, mummified were collected. As a rule, first of all, there were insects infected with entomoph- torous fungi, detected in May and the beginning of June. At this time the appearance of Entomophaga tipulae, Erynia ovispora, Zoophthora nematoceris on flies imago andZoophthora radicans on winter moth caterpillars was marked (Operophthera brumata).

78 LUDMILA PRISCHEPA, NATALIA МIKULSKAYA, МARINA GERASIMOVICH, DANUTA SОSNOWSKA, STANISLAW BAŁAZY

The infected insects were registered and after June 10, 2009, were met till the end of the month. As a rule, at that time Pandora mus- civora were met on dipterous (Brachycera) and Zoophthora lipai on beetles Rhagomycha lignose, also Entomophthora muscae on beetles (Anthomyidae) and Pandora neoaphidis on some aphid species. The biological diversity of species appearing in summer depends on the rainfall amount. The entomopathogenic fungi number is significantly decreased if there is no rainfall in the course of the week and the weather is sunny. The only species not responding to draught was Zoophthora ichneumonis infecting Ichneumonidae caterpillars, was present in biocoenosis the whole vegetational season. From September to late autumn there were Zoophthora autumnalis, present on dipterous Dryomyzidae, sometimes it could be isolated even at the beginning of November. In present researches there were no insect epizooties. The species not registered ear- lier in forest were: Entomophthora israelensis, rare infecting Itonidiae; Beauveria cf. bassiana infecting Auchenorrhyncha; Paecilomyces suffultus – Bibionidae, and P. tenuipes – Arthropoda. It was determined that Geotrupes silvaticus infected 17 % of beetles were infected by Entomopoxvirus which were described for the first time in beetle populations by J. Lipa, J. Bartkowski (1971). Considering the fact that the life cycle of the majority of insects damaging forest crops is connected with soil their infection can take place in a forest litter. During the researches fungi growth was marked on a forest litter. The specific identification carried out by Polish specialists has shown that fungi belong to Paecilomyces fu- mosoroseus. The fungi penetration into organism of host insects can take practi- cally through any parts of the body, however, there is an opinion that a main way of entomopathogenic fungi entering is through insect cu- ticle. More often we find in samples the infected bark beetles’ imago. The deadIps typographus imago infected with green dying off (silkworm) fungus has been determined in 12 % of the phytophage popu- lation. The pest beetles’ death was noticed directly in galleries on the damaged spruce bark. More often the beetles were damaged by fungi Metarhizium anisopliae and Beauveria bassiana.

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CONCLUSIONS 1. On Belarusian and Polish territories in different ecotopes of Bialowieza forest 36 species of entomopathogenic fungi (76.5 %), 7 isolates of entomopathogenic nematodes (14.8 %) of Steinernema genus and Bacillus thuringiensis strain bacteria (8.5 %) have been isolated. 2. There were isolated the species which have not been registered in Bialowieza forest before: Entomophthora israelensis, rarely infecting dipterous (Itonidiae), Beauveria cf. bassiana infecting cicads (Auchenorrhyncha), Paecilomyces suffultus – from Bibionidae and P. tenuipes from Lepidoptera pupae. 3. From dying off (silkworm) fungi there were two prevalent species: Paecilomyces farinosus (63 %) and Beauveria bassiana (21 %), the subdominant species was Paecilomyces tenuipes (10 %). The infection of Ips typographus imago by Metarhizium anisoplia, Beauveria bassiana and Chytridiopsis typographi was noticed in galleries on the infected spruce bark.

REFERENCES BERRIMAN, A. 1990. Forest protection against pest insects. Моskva: Аgropromizdat, 287 p. LIPA, J. J.; BARTKOWSKI, J. 1971. A newly discovered poxlike virus disease of dung beetles, Geotrupes silvaticus (Coleoptera: Scarabaeidae). Journal of invertebrate pathology, 20: 218–219. Belovezhskaya Pushcha. Nature of Belarus. Prieiga per internetą: http:// belarusnature.sotela.html. [Žiūrėta 2011-01-01] ZIMMERMANN, G. 1986. “Galleria bait method” for detection of entomopathogenic fungi in soil. Zeitschrift für Angewandte Entomologie, 2: 213–215. ВОРОНЦОВ, А. И. 1978. Патология леса. Москва: Лесная промышленность, 272 с. ВОРОНЦОВ, А. И. 1984. Биологическая защита леса. Москва: Лесная про- мышленность, 264 с. ЕВЛАХОВА, А. А. 1964. Метод распознавания болезней насекомых. Москва: Колос, 252 с. КОВАЛЬ, Е. З. 1974. Определитель энтомофильных грибов СССР. Киев: Наукова Думка, 260 с.

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ENTOMOPATOGENINIŲ ORGANIZMŲ ĮVAIROVĖ BELOVEŽO GIRIOS KENKĖJŲ POPULIACIJOSE

Santrauka Tyrimo metu Belovežo girioje buvo renkami sergantys ir žuvę vabzdžiai. 29 kvartaluose buvo surinkta 81 mėginys. Tirti mikroskopiniai sporiniai grybai ir bakterijos, esantys miško paklotėje, ant žolių, paviršiniame dirvožemio sluoksnyje ir po medžių žieve. Baltarusijos ir Lenkijos teritorijose, Belovežo girioje, iš viso buvo atrinkti 9 kvartalai, išsiskiriantys didele augalijos įvairove; juose surinkti 147 mėginiai. Iš viso tyrimo metu buvo identifikuoti 36 rūšių entomopatogeniniai grybai, 7 rūšių entomopatogeniniai nematodai, priklausantys Steinernema genčiai, ir 4 rūšių bakterijos Bacillus thuringiensis. Tyrimo metu išskirti grybai sudarė 76,5 % mėginių, nematodai – 14,8 %, bakterijos – 8,5 %. Reikšminiai žodžiai: entomopatogeniniai mikroorganizmai, biologinė įvairovė, Bacillus thuringiensis, Steinernema, Belovežo giria.