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, 2009. Original Russian Text © E.A. Degtyareva, K.A. Vinogradova, A.V. Aleksandrova, V.A. Filonenko, P.A. Kozhevin, 2009, published in Vestnik Moskovskogo Universiteta. Pochvovedenie, 2009, No. 2, pp. 22–26.
Soil Actinomycetes as Potential Biofungicides
E. A. Degtyarevaa, K. A. Vinogradovab, A. V. Aleksandrovab, V. A. Filonenkoc, and P. A. Kozhevina
Department of Soil Biology, Faculty of Soil Science, Moscow State University, Moscow, 119899 Russia b Department of Microbiology, Biological Faculty, Moscow State University, 119899 Russia c EM Cooperation JSC, Moscow, Russia
Received April 18, 2008
Abstract—The biological control of fungal diseases in plants is considered an efficient and environmentally friendly alternative or supplement to fungicides. Soil antagonistic streptomycetes are particularly suitable for the biological control; they proved to be highly efficient in reducing the incidence of fungal pathogens. Strep tomycetes isolated from the podzolic soils were evaluated for the biosuppression of fungal populations. Sev enteen strains of streptomycetes (out of the total 279 isolates) were found to be strongly antagonistic to fungal pathogens in vitro and were selected for further experiments in situ. The full protection of plants against Fusarium spp. was obtained with the Streptomyces hygroscopicus strain K49. Key words: biological control, biofungicides, Streptomyces hygroscopicus, Fusarium spp. DOI: 10.3103/S0147687409020045
INTRODUCTION The control of phytopathogenic fungi that are responsible for various plant diseases remains a topical problem; its solution will make it possible to reduce yield losses considerably. The extensive use of chemi cals (fungicides) cannot be considered an optimum solution, because it increases the risk of the chemical pollution of the environment and agricultural produc tion. Therefore, an interest to biofungicides is grow ing. Biofungicides are microbial populations isolated from the natural environment that are capable to inhibit and destroy undesirable phytopathogens. Par ticularly, their careful application is especially impor tant in ecological farming aimed at obtaining high quality food products. Commercial biopreparations on the basis of different microorganisms [13, 14] are proposed; among them, only some preparations include populations of actinomycetes–antagonists, such as Alirin C, Mycostop, and Actinovate. Soil actinomycetes are suppliers of many antibiotic substances with antifungal activity. They also produce different exoenzymes, including chitinases that utilize cell walls of fungi . The capacity of actinomycetes– producers to form antibiotics and enzymes is displayed not only under laboratory and artificially controlled conditions but also in natural soils in situ . Ecological niches of soil fungi and actinomycetes are partly overlapped due to specific features of their life forms (mycelial growth, spore formation). Numerous data of direct microscopy in soil microbi ology, including microbial patterns (landscapes) on fouling glasses, attest to the active development of act inomycetes on dying fungal hyphae . Thus, the
potential of soil actinomycetes for the biological con trol of phytopathogenic fungi is beyond question. Recently, a significant role of actinomycetes in the development of the local and total resistance of plants to pathogenic fungi (chitinase and peroxidase activity of roots, etc.) was shown. Therefore, soil actino mycetes are of great interest as a source of efficient biofungicides. Our work was aimed at finding actinomycetes– antagonists of phytopathogenic fungi and studying prospects for their application as biofungicides. OBJECTS AND METHODS A collection of actinomycetes isolated previously from podzolic soils sampled at the Zvenigorod Exper imental Station of the Biological Faculty of Moscow State University and from a podzolic soil under a col ony of the Clitocybe geotropa agaricoid basidiomycete were analyzed [3, 4]. The samples were collected in different years and seasons on the plots exposed to for est fire, under a restoring forest, on a clear felled area, and under an undisturbed forest. Phytopathogenic fungi from the collection of the Department of Mycology and Algology (Biological Faculty, Moscow State University), populations iso lated from diseased plants (cucurbit, cucumber, potato, and tomato), and saprotrophic native fungi from the studied soil samples were used as test organ isms. In the work with biofungicides, it is important to estimate their action not only on the target population of pathogenic organisms but also on the entire native microflora . For this purpose, bacteria from the col lections of the Department of Soil Biology (Faculty of
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Soil Science) and the Department of Microbiology (Biological Faculty) were also used as test objects. Thus, the list of test objected included the following populations: (a) phytopathogenic fungi (Fusarium aquaeduct uum (Rabenh et Radhlk) Lagerh, F. heterosporum Nees:Fries, F. merismoides Corda, F. oxysporum Schlechtendahl: Fries, F. poae (Peck) Wollenweber, F. solani (Martius) Saccardo, Macrosporium solani (Ell et Mart), Phoma exigua Desmazieres, Verticillium luteoalbum (Link) Subramanian, and V. nubilum Pethybridge; (b) native saprotrophic fungi, the composition of which varied in dependence on the soil sample; (c) bacteria (soil and laboratory tests) Staphylococ cus aureus 209, Bacillus magatherium, B. subtilis, Micrococcus luteus 2665, M. roseus, Rhodococcus erythropolis 283, Rh. luteus 371, Arthrobacter globifor mis 281, Cellulomonas sp., Escherichia coli C600, Pseudomonas fluorescens var. lemonniere, Micrococcus + Spirillum mixed culture, and Aquaspirillum sp.; and (d) yeasts Asaccharomyces serevisidae 230 and Can dida guilliermondii 217. Actinomycetes were isolated using a traditional method of inoculation of soil suspension on the dense casein–glycerol medium (CGM) with nystatin . For the selection of potential antagonists, actino mycetes were grown on the medium with soybean flour recommended for the search of active producers . For the initial characterization of the biological activity of the isolated strains, the traditional method of agar blocks was used . Actinomycetes inhibiting three and more popula tions of phytopathogenic fungi were considered to have a wide spectrum of activity. The degree of activity was judged from the size of the zone, in which phyto pathogens were inhibited. When it was less than 5 mm, the antagonists were considered to be weakly active. The taxonomic belonging of actinomycetes was determined according to a set of commonly used mor phological–cultural and chemotaxonomic character istics [5, 9, 11, 12]. Polyphase taxonomic studies with the use of a set of phenotypic characteristics and molecular genetic methods (nucleotide sequence of the 16S gene of RNA) were performed to identify the streptomycetes at the species level. The surface of streptomycetes' spores was examined under transmis sion and scanning electron microscopes (Jeol 100 CX). The possibility of using the selected strepto mycetes as biofungicides was verified in model exper iments with wheat and watercress plants infected with phytopathogenic fungi Fusarium culmorum and F. oxysporum. The Trichoderma citrinoviride native fungal strain was used for the additional control. The tests were performed under laboratory conditions and in a phytotron. The laboratory experiments were performed with watercress. Suspensions of spores of 5 day culture of
F. oxysporum fungus (6 × 106 spores/g) and 7 day cul ture of streptomycetes (1 × 108 spores/g) were inocu lated into nonsterile and partly sterile (sterilization by microwave radiation, 800 J/g) samples from soddy podzolic soils in Petri dishes, and watercress was immediately sown. After 14 days, the germination capacity (%) and the hydrolytic activity of the micro bial community (using the fluorescein diacetate (FDA) hydrolysis method) were determined. Since the FDA hydrolysis is accomplished by active cells with a complex of enzymes, its rate may be an index of the active microbial biomass. In this soil, fungi predomi nated in the biomass and possessed a strong enzyme complex. Thus, it could be supposed that the rate of FDA hydrolysis depended on the activity of soil fungi. An independent verification of the biofungicide potential of the selected antagonists was performed with wheat plants on a phytotron at the Research Institute of Phytopathology (Golitsyno, Moscow oblast). Cultures of aggressive phytopathogenic fungi F. oxysporum and F. culmorum were used as phyto pathogens. The following characteristics were ana lyzed: the germination capacity of wheat seeds (%), the length of wheat roots at the tillering phase, and the number of plants with diseased roots. RESULTS AND DISCUSSION The collection of isolated soil actinomycetes mainly consisted of representatives of the Streptomyces genus, probably, due to the procedure of their count ing on the CGM medium favorable for them and their abundance in the investigated soils. Among 279 iso lates, those with the pronounced antifungal activity towards phytopathogenic fungi, including Fusarium aquaeductuum (Rabenh et Radhlk) Lagerh, F. het erosporum Nees: Fries, F. merismoides Corda, F. oxysporum Schlechtendahl:Fries, F. poae (Peck) Wollenweber, F. solani (Martius) Saccardo, Macrospo rium solani (Ell et Mart), Phoma exigua Desmazieres, Verticillium luteoalbum (Link) Subramanian, and V. nubilum Pethybridge, were selected. The effect of the native streptomycetes (their spe cies composition depended on the sample) on the aboriginal fungi was much higher than their effect on the phytopathogenic fungi atypical of the given habi tats. Approximately 14% of the streptomycetes iso lated from the samples of different zones within the colony of the agaricoid Clitocybe geotropa fungus inhibited the growth of native fungi. Among them, no more than 8% inhibited the growth of phytopatho genic fungi to some extent . The strong antagonists with a wide spectrum of activity inhibiting the growth of 2–3 species of the Fusarium and Verticillium luteoal bum fungi amounted to less than 3% of the total amount of the active isolates. The portion of streptomycetes inhibiting the growth of phytopathogenic in the samples isolated from the strongly podzolic soil (Zvenigorod Station)
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SOIL ACTINOMYCETES AS POTENTIAL BIOFUNGICIDES Factors B AB A AC BC C 0 1 µm 10 20 30 40 Effect
Fig. 1. Spiral spore chains of the Streptomyces hygroscopi cus population.
Fig. 2. Relative significance of the factors affecting the plant growth on a Pareto diagram: factor A is the soil microbial system, factor B is the Streptomyces hygroscopi cus population, and factor C is the Trichoderma cirinovir ide population.
was significantly higher: approximately one third of the analyzed isolates had the inhibiting effect on phy topathogenic fungi. Among them, populations with a narrow spectrum of antifungal effect (inhibition of the growth of 1–3 test fungi) predominated. The total number of identified strains of actinomycetes was 279; among them, 35 strains were with a wide spectrum of action, including 22 strains with the strong biofungi cidal action. For experiments on the biological control, 17 pop ulations of streptomycetes acting in vitro as potential biofungicides toward phytopathogenic fungi of the Fusarium genus (F. heterosporum, F. merismoides, F. oxysporum, and F. solani), Verticillium luteoalbum, and Phoma exigua) were selected. They differed in the spectra of their action: some of them inhibited only one of the studied phytopathogenic fungi, and some inhibited them all. On the basis of data on the mor phological characteristics of their cultures, the strep tomycetes were subdivided into several phenoclusters according to . As a result, Streptomyces sp. K 182 was attributed to phenocluster 10 of category 3 (Str. bambergiensis Wall hausser, Nesemann, Prave and Steigler 1966, 734AL); Streptomyces sp. K193, to phenocluster 8 of category 2 (Str. chattanoogensis Burns and Holtman 1959, 398AL); Streptomyces sp. 36, to phenocluster 8 of cate gory 2(Str. xanthocidicus Asahi, Nagatsu and Suzuki 1966, 196AL); Streptomyces sp. A10, to phenocluster 3 of category 1 (Str. halstedii (Waksman and Curtis 1916) Waksman and Henrici 1948, 953AL); the latter strain could be attributed to the Str. griseolus (Waks man 1923) Waksman and Henrici 938AL species. One of the most active streptomycetes—Strepto myces sp. K49—was identified as Str. hygroscopicus (Jensen 1931) Waksman and Henrici 1948, 953AL. It belongs to the taxonomic group Str. violaceusniger clade, which includes species with wrinkled spore exines in spiral chains (Fig. 1). The species identifica
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tion of this strain within the given taxonomic group was confirmed with the help of the PCR method (according to the sequence of RNA gene 16s). The experiments showed that streptomycetes with the antifungal activity could also inhibit the growth of different bacteria, including typical soil inhabitants. For instance, strains of streptomycetes K49 and K193 had a wide spectrum of antimicrobial effects; they inhibited the growth of bacteria (Bacillus megathe rium, B. subtilis, Micrococcus luteus 2665, M. roseus, Rhodococcus erythropolis 283, Rh. luteus 371, Arthro bacter globiformis 281, and Cellulomonas sp. lau 1) and two species of yeasts (Saccharomyces serevisiae 230 and Candida guilliermondii 217). However, they did not affect the Escherichia coli C600 and Pseudomonas fluorescens var. lemmoniere bacteria, and the mixed Micrococcus + Spirillum. Aquaspirillum sp. culture. An active antagonist of fungi—strain K182—did not inhibit most of the investigated soil bacteria and yeasts, but inhibited the growth of Staphylococcus aureus, Bacillus megaterium, B. subtilis, and Micrococ cus luteus. The population of streptomycetes A10 with the high activity toward Fusarium did not inhibit the growth of all the bacteria tested, including those iso lated from the soils. The preliminary information showed that biofungicides could be applied not only to control the growth of phytopathogenic fungi but also to regulate the structure and activity of the microbial communities. A possibilities to control fusarial infections with the Streptomyces hygroscopicus population was assessed in model experiments using a computer system of their planning (STATGRAPHICS Plus for Windows). The laboratory test (in Petri dishes) with the soil and water cress seeds infected with Fusarium showed that the most important and statistically significant factors were as follows: the introduction of a streptomyces– antagonist, the state of the microbial community at
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76 Effect, % 60 50 40 30 20
Partial Without sterilization treatment Present Soil Absent Soil
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Fig. 3. Major effects of the three factors (the state of soil microbial community, the introduction of streptomycetes, and the introduction of Trichoderma) on seed germina tion.
FDA hydrolysis index 5.5 5.3 5.1 4.9 4.7 4.5
Partial Without sterilization treatment Present Soil Absent Soil Absent Stretomyces treatment Trichoderma citrinoviride
Fig. 4. Effect of the studied factors studied on the FDA hydrolysis index.
the moment of sowing, and the interaction between these two factors (Fig. 2). The main effects of individual factors are shown in Fig. 3. The introduction of the Streptomyces hygro scopicus antagonist essentially improved the condi tions for the development of plants. A positive factor was also the undisturbed microbial community in the soil, which had a greater diversity (“mature” system) than the community after the partial sterilization (“young” system). In this experiment, the Tricho derma citrinoviride population did not perform func tions peculiar to fungi of this genus, which could be related to the specific properties of this aboriginal pop ulation. As expected, in the nonsterile soil, the FDA hydrolysis index significantly exceeded that in the soil after its partial sterilization. The introduction of the Trichoderma cirinoviride fungi regularly increased this index. However, after the introduction of strepto mycetes, this index value drastically decreased, which attested to a strong inhibition of the complex of fungal populations. Probably, such a decrease in the index of the active biomass evidenced the efficient regulation according to the type of necrotrophic mycophagy (Fig. 4). The surface of the responses for the two key factors demonstrates the possibility to control fusarial infec tions via application of Streptomyces hygroscopicus (Fig. 5). Evidently, information on the state of the soil microbial system at the moment of plant seeding and the introduction of an antagonist is necessary for the reliable prediction and control of the efficiency of this measure. An independent verification with the use of wheat (infected with F. oxysporum and F. culmorum) con firmed the possibility of using Streptomyces hygroscopi cus as a biofungicide. The streptomycetes introduced into the soil with the phytopathogenic fungi reduced their negative effects on the plants: the length of roots increased by 20%, the number of plants with diseased roots decreased by 36%, and the germination index rose by 48%. CONCLUSIONS
Seed germination capacity, % 100 80 60 40 20 0 Microbial system young mature
0.1 billion spores/g Level of population introduction
The possibility for the biocontrol of the develop ment of Fusarium phytopathogenic fungi via applica tion of streptomycetes has been shown. This fact yields promise with respect to the potential application of the selected populations of streptomycetes as biofungi cides. REFERENCES
1. Aleksandrova, A.V., Velikanov, L.L., Sidorova, I.I., and Sizova, T.P., Effect of Fungus Trichoderma harzianum on Soil Micromycetes, Mikol. Fitopatol., 2000, vol. 34, no. 3.
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Fig. 5. The surface of plant response as related to the appli cation of streptomycetes introduction and the state of the soil microbial community.
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SOIL ACTINOMYCETES AS POTENTIAL BIOFUNGICIDES 2. Aristovskaya, T.V., Mikrobiologiya podzolistykh pochv (Microbiology of Podzolic Soils), Moscow, 1965. 3. Vinogradova, K.A., Aleksandrova, A.V., Sidorova, I.I., and Voronina, E.Yu., Distribution of Soil Actino mycetes in the Collar Colony of Clitocybe geotropa (Vull). Quel. and Their Antagonistic Activity with Respect to Micromycetes, Mikol. Fitopatol., 2008, vol. 42, no. 3. 4. Vinogradova, K.A., Movchan, D.D., Aleksandrova, A.V., and Kozhevin, P.A., Characterization of Interpopula tion Interactions of Indigenous Fungi and Actino mycetes in Podzolic Soil, Mikol. Fitopatol., vol. 41, p. 207. 5. Gauze, G.F., Preobrazhenskaya, T.P., Sveshnikova, M.A., Terekhova, L.P., and Maksimova, T.S., Opredelitel’ aktinomitsetov (Guide to Actinomycetes), Moscow, 1983. 6. Egorov, N.S., Osnovy ucheniya ob antibiotikakh (Basic Theory of Antibiotics), Moscow, 2004. 7. Zenova, G.M., Pochvennye aktinomitsety redkikh rodov (Rare Soil Actinomycetes), Moscow, 2000. 8. Kozhevin, P.A., Ecology of Soil Microorganisms, in Ekologiya mikroorganizmov (Ecology of Microorgan isms), Netrusov, A.A., Ed., Moscow, 2004.
9. Bergey’s Manual of Systematic Bacteriology, Will iams, S.T., Sharpe, M., and Holt, J.A., Eds., Balti more, 1989, vol. 4. 10. Goves, R.C., Semedo, L.T.A.S., Soares, R.M.A., Alvi ano, C.S., Linhares, L.F., and Coelho, R.R.R., Chiti nolytic Actinomycetes from a Brazilian Tropical Soil Active against Phytopathogenic Fungi, Word J. Micro biol. Biotechnol, 2000, vol. 16. 11. Goodfellow, M., Kumar, Y., Labeda, D.P. and Sembir ing, L., The Streptomyces violaceus niger clade: a Home for Streptomycetes with Rugose Ornamented Spores, Antonie van Leeuwenhoek, 2007, vol. 92, pp. 173–199. 12. Kampfer, P., The Family Streptomycetacea. Part 1: Taxonomy, in The Procariotes, Dworkin, M. et al., Eds., 3rd ed., New York, 2004. 13. McSpadden Gardener, B.B. and Fravel, D.R., Biologi cal Control of Plant Pathogens: Research, Commercial ization, and Application in the USA, in APS net Feature: Biological Control of Plant Pathogens, 2002. 14. Nourozian, J., Etebarian, H.R., and Khodakaramian, G., Biological Control of Fusarium graminearum on Wheat by Antagonistic Bacteria, Songklanakarin J. Sci. Tech nol., 2006, vol. 28. 15. Warren, H.B., Non Synthetic Media for Antibiotic Producing Actinomycetes, Antibiot. Chemother., 1955, vol. 5, no. 10.
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