ISSN 0026-2617, Microbiology, 2008, Vol. 77, No. 1, pp. 90–98. © Pleiades Publishing, Ltd., 2008. Original Russian Text © A.V. Kurakov, R.B. Lavrent’ev, T.Yu. Nechitailo, P.N. Golyshin, D.G. Zvyagintsev, 2008, published in Mikrobiologiya, 2008, Vol. 77, No. 1, pp. 103–112. EXPERIMENTAL ARTICLES Diversity of Facultatively Anaerobic Microscopic Mycelial Fungi in Soils A. V. Kurakova,1, R. B. Lavrent’evb, T. Yu. Nechitailoc, P. N. Golyshinc, and D. G. Zvyagintsevb a International Biotechnology Center, Moscow State University, Moscow, 119992 Russia b Department of Soil Biology, Faculty of Soil Science, Moscow State University, Moscow, 119992 Russia c National Biotechnology Center, Mascheroder Weg 1, 38124 Braunschweig, Germany Received March 26, 2007 Abstract—The numbers of microscopic fungi isolated from soil samples after anaerobic incubation varied from tens to several hundreds of CFU per one gram of soil; a total of 30 species was found. This group is com- posed primarily of mitotic fungi of the ascomycete affinity belonging to the orders Hypocreales (Fusarium solani, F. oxysporum, Fusarium sp., Clonostachys grammicospora, C. rosea, Acremonium sp., Gliocladium penicilloides, Trichoderma aureoviride, T. harzianum, T. polysporum, T. viride, T. koningii, Lecanicillum leca- nii, and Tolypocladium inflatum) and Eurotiales (Aspergillus terreus, A. niger, and Paecilomyces lilacimus), as well as to the phylum Zygomycota, to the order Mucorales (Actinomucor elegans, Absidia glauca, Mucor cir- cinelloides, M. hiemalis, M. racemosus, Mucor sp., Rhizopus oryzae, Zygorrhynchus moelleri, Z. heterogamus, and Umbelopsis isabellina) and the order Mortierellales (Mortierella sp.). As much as 10–30% of the total amount of fungal mycelium remains viable for a long time (one month) under anaerobic conditions. Keywords: facultatively anaerobic fungi, diversity of soil fungi, growth rate. DOI: 10.1134/S002626170801013X Soil is a habitat where reductive conditions are often MATERIALS AND METHODS present; in bog and floodplain soils, they may predom- The samples of the upper horizons of zonal soils inate for a long period [1]. Bacteria are traditionally were investigated (soddy podzolic, grey forest, and considered the only organisms which remain active in dark grey forest soils, leached, and ordinary cher- soils under conditions of limited oxygen supply. At the nozems), as well as the samples of peat soils taken from same time, it is known that both yeast and mycelial an ombrotrophic peat bog and a minerotrophic fen, of microscopic fungi have the capacity for fermentation. soddy-alluvial and alluvial-meadow floodland soil, They can therefore remain active under conditions of saline and alkali-saline soil, compost, mosses in various limited oxygen supply or in the absence of oxygen and degrees of decomposition, the intestinal contents and the presence of easy-to-access sugars [2–6]. fresh excrement (coprolites) of the earthworms Lum- bricus terrestris, Aporrectodea caliginosa and the Fungal biomass is often predominant in soils; there- muckworm Eisenia foetida. fore, they need to survive at low partial pressure of oxy- gen or under anaerobic conditions. In most cases, To estimate the mycelial biomass pool in the soils mycelial micromycetes have been considered aerobic with long-term reduction regime, we used samples of organisms. The data on the species diversity of faculta- typical soddy gley, peat podzolic gley, and oligotrophic peat soils taken from an ombrotrophic bog under a tive anaerobic fungi, their abundance, and occurrence sparse pine canopy (Central Forest State Nature Bio- in various soils are extremely scarce. There have been sphere Reserve, Tver oblast). The peat horizons T1 several reports on the isolation of microscopic fungi (10–20 cm), T2 (20–40 cm), Tn (40–60 cm) are charac- (Fusarium solani and Trichoderma harzianum) from terized by a high level of Sphagnum decomposition, soil, peat, and organic matter samples under anaerobic low pH (3.5–4.2), low ash content (2.4–6.0%), and an conditions [7–9]. absorption capacity of 80–90 mg-equiv/100 g of soil. The aim of the present work was to assess the abun- The effect of long-term anaerobiosis on the total and dance and diversity of microscopic fungi isolated from viable fungal mycelium was studied in fresh samples of various soils under anaerobic conditions. peat oligotrophic soils from the Central Forest State Nature Biosphere Reserve and samples of soddy pod- 1 Corresponding author; e-mail: [email protected] zolic reclaimed soil taken from a potato plot at the Soil 90 DIVERSITY OF FACULTATIVELY ANAEROBIC MICROSCOPIC MYCELIAL FUNGI 91 Station of the Moscow State University. The samples or, in rare cases, it was detected in trace quantities at the were taken from the Ap horizon (0–10 cm) with humus maximum sensitivity of the chromatograph. Methylene and total nitrogen concentrations of 2.7 and 0.24 %, blue (Eh of the half-transition point is +60 mV at pH 7) respectively. The pH of the soil was 6.7; the hydrolytic was used as an indicator of the oxygen content. The acidity, 2.2 mg-equiv/100 g of soil; the degree of base indicator was colored blue, while the oxygen partial saturation, 92%. pressure was higher than 0.05 atm [13]. Anaerobic incubation was carried out in penicillin Ten cell culture flasks were prepared for each type flasks. In the case of soddy podzolic soil, the samples of soil and inoculated under anaerobic conditions; (3.0 g) were preliminarily passed through a 2-mm under aerobic conditions, 10–20 petri dishes were inoc- sieve; then sterile water was added so that the soil ulated. humidity reached 100% of the maximum water capac- The cultures of fungi were isolated from fresh soil ity, and the flasks were sealed with rubber stoppers and samples by inoculating fine-grained soil on a solid tin caps. The air in the flasks was replaced with argon. nutrient medium under anaerobic conditions. To isolate The mycelial biomass content was determined by the fungal cultures, we used a glucose–peptone fluorescence microscopy in the samples before and medium of the following composition (g/l): KH2PO4, after incubation for 5–6 weeks under anaerobic condi- 1.0; KCl, 0.5; MgSO4, 0.5; FeSO4 · 7H2O, 0.01; tions. Calcofluor was used as a dye [10]. To assess the (NH4)2SO4, 2.0; agar, 15.0; peptone, 5.0; glucose, 5.0; length of the viable mycelium, the water–soil suspen- and yeast extract, 0.5. The medium was supplemented sion was supplemented with FDA (fluorescein diace- with a microelements solution (1.0 ml/l) containing the tate) (1 ml per 100 ml of the suspension) [11]. following (mg/l): FeCl2 · 7H2O, 200; ZnCl2 · 7H2O, 10; We used a modified Hungate technique [12] to MgCl2 · 4H2O, 3.0; H3BO4, 30; CoCl2 · 6H2O, 2.0; determine the numbers of colony-forming units (CFU), CuCl2 · 2H2O, 1.0; Na2MoO4, 3.0; NiCl2 · 6H2O, 2.0; as well as to isolate pure fungal cultures under anaero- and EDTA, 500. In addition, the medium was supple- bic conditions. mented with a vitamin solution (1.0 ml/l) containing (mg/l): 4-aminobenzoic acid, 25; D-biotin, 100; nico- During the preliminary experiments, in was estab- tinic acid, 25; calcium pantothenate, 25; pyridoxine lished that inoculation of soil samples into liquid glu- hydrochloride, 25; folic acid, 10; riboflavin, 25; Ç12, cose-containing mineral medium with peptone, vita- 0.5; and lipoic acid, 25. To inhibit bacterial growth in mins, and microelements, and subsequent incubation soil inocula, antibiotics (streptomycin and chloram- under anaerobic condition resulted in the predominance phenicol) were added to the molten cooled sterile of only one species of microscopic fungi. Thus, this medium in a concentration of 200 mg/l of medium. The approach hampers both the study of fungal diversity sterile vitamin, antibiotic, and microelement solutions (since it complicates the detection of minor species) were added after autoclaving. Difco Bacto agar was and the CFU numbers of facultatively anaerobic fungi used; other components were manufactured by Sigma. in soil. Therefore, in our further experiments, we used only solid agarized media placed in flat glass cell cul- Some soils (soddy podzolic, leached chernozem, ture flasks sealed with rubber stoppers. etc.) were also inoculated on wort agar and Czapek’s medium. Agarized medium placed in small cell culture flasks was incubated for 4–12 days at 24–26°ë; antibiotics To determine the cell numbers of facultatively were introduced in concentrations (200 mg/l) higher anaerobic fungi in soil samples, the media were inocu- than those usually used for inoculation. The air in the lated with the 1 : 100, 1 : 50, and 1 : 10 dilutions of the cell culture flasks was replaced with nitrogen. Oxygen water–soil suspension. The inoculum (1.0 ml) was was removed from nitrogen by passing through a Hun- spread evenly over the agar surface by gentle rocking of gate column packed with heated copper shavings. The the flask. For the isolation of pure cultures, inoculation solution of mineral salts was boiled in order to remove of the media with diluted suspensions was less produc- dissolved oxygen and cooled in a crystallizer with cold tive than that with fine-grained soil samples. Inocula- water. The solution was gassed with nitrogen prelimi- tion with dilutions higher than 1 : 100 or 1 : 50 yielded narily passed through the column packed with heated few colonies, whereas the probability of contamination copper shavings. After cooling, the solution was sup- of the obtained culture with the soil particles attached plemented with organic compounds, vitamins, and to the mycelium increased when lesser dilutions (1 : 10) microelements. The medium was poured into the glass were applied. When using fine-grained soil as inocu- cell culture flasks with weighed portions of agar (30 g/l) lum, the colonies spread from the soil particles to the surface of clean medium; their isolation as pure cul- under anaerobic conditions (under N2 flow).