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ISSN 01476874, Moscow University Soil Science Bulletin, 2009, Vol. 64, No. 2, pp. 73–77. © Allerton Press, Inc., 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
Department of Microbiology, Biological Faculty, Moscow State University, 119899 Russia
c EMCooperation 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 potential of soil actinomycetes for the biological con
trol of phytopathogenic fungi is beyond question.
The control of phytopathogenic fungi that are Recently, a significant role of actinomycetes in the
responsible for various plant diseases remains a topical development of the local and total resistance of plants
problem; its solution will make it possible to reduce to pathogenic fungi (chitinase and peroxidase activity
yield losses considerably. The extensive use of chemi of roots, etc.) was shown. Therefore, soil actino
cals (fungicides) cannot be considered an optimum mycetes are of great interest as a source of efficient
solution, because it increases the risk of the chemical biofungicides.
pollution of the environment and agricultural produc
Our work was aimed at finding actinomycetes–
tion. Therefore, an interest to biofungicides is grow
antagonists of phytopathogenic fungi and studying
ing. Biofungicides are microbial populations isolated
prospects for their application as biofungicides.
from the natural environment that are capable to
inhibit and destroy undesirable phytopathogens. Par
ticularly, their careful application is especially impor OBJECTS AND METHODS
tant in ecological farming aimed at obtaining high
quality food products. Commercial biopreparations A collection of actinomycetes isolated previously
on the basis of different microorganisms [13, 14] are from podzolic soils sampled at the Zvenigorod Exper
proposed; among them, only some preparations imental Station of the Biological Faculty of Moscow
include populations of actinomycetes–antagonists, State University and from a podzolic soil under a col
such as AlirinC, Mycostop, and Actinovate. ony of the Clitocybe geotropa agaricoid basidiomycete
were analyzed [3, 4]. The samples were collected in
Soil actinomycetes are suppliers of many antibiotic different years and seasons on the plots exposed to for
substances with antifungal activity. They also produce est fire, under a restoring forest, on a clear felled area,
different exoenzymes, including chitinases that utilize and under an undisturbed forest.
cell walls of fungi [10]. The capacity of actinomycetes– Phytopathogenic fungi from the collection of the
producers to form antibiotics and enzymes is displayed Department of Mycology and Algology (Biological
not only under laboratory and artificially controlled Faculty, Moscow State University), populations iso
conditions but also in natural soils in situ [8]. lated from diseased plants (cucurbit, cucumber,
Ecological niches of soil fungi and actinomycetes potato, and tomato), and saprotrophic native fungi
are partly overlapped due to specific features of their from the studied soil samples were used as test organ
life forms (mycelial growth, spore formation). isms. In the work with biofungicides, it is important to
Numerous data of direct microscopy in soil microbi estimate their action not only on the target population
ology, including microbial patterns (landscapes) on of pathogenic organisms but also on the entire native
fouling glasses, attest to the active development of act microflora [1]. For this purpose, bacteria from the col
inomycetes on dying fungal hyphae [2]. Thus, the lections of the Department of Soil Biology (Faculty of

74 DEGTYAREVA et al.

Soil Science) and the Department of Microbiology F. oxysporum fungus (6 × 106 spores/g) and 7day cul
(Biological Faculty) were also used as test objects. ture of streptomycetes (1 × 108 spores/g) were inocu
Thus, the list of test objected included the following lated into nonsterile and partly sterile (sterilization by
populations: microwave radiation, 800 J/g) samples from soddy
(a) phytopathogenic fungi (Fusarium aquaeduct podzolic soils in Petri dishes, and watercress was
uum (Rabenh et Radhlk) Lagerh, F. heterosporum immediately sown. After 14 days, the germination
Nees:Fries, F. merismoides Corda, F. oxysporum capacity (%) and the hydrolytic activity of the micro
Schlechtendahl: Fries, F. poae (Peck) Wollenweber, bial community (using the fluorescein diacetate
F. solani (Martius) Saccardo, Macrosporium solani (FDA) hydrolysis method) were determined. Since the
(Ell et Mart), Phoma exigua Desmazieres, Verticillium FDA hydrolysis is accomplished by active cells with a
luteoalbum (Link) Subramanian, and V. nubilum complex of enzymes, its rate may be an index of the
Pethybridge; active microbial biomass. In this soil, fungi predomi
(b) native saprotrophic fungi, the composition of nated in the biomass and possessed a strong enzyme
which varied in dependence on the soil sample; complex. Thus, it could be supposed that the rate of
FDA hydrolysis depended on the activity of soil fungi.
(c) bacteria (soil and laboratory tests) Staphylococ
cus aureus 209, Bacillus magatherium, B. subtilis, An independent verification of the biofungicide
Micrococcus luteus 2665, M. roseus, Rhodococcus potential of the selected antagonists was performed
erythropolis 283, Rh. luteus 371, Arthrobacter globifor with wheat plants on a phytotron at the Research
mis 281, Cellulomonas sp., Escherichia coli C600, Institute of Phytopathology (Golitsyno, Moscow
Pseudomonas fluorescens var. lemonniere, Micrococcus + oblast). Cultures of aggressive phytopathogenic fungi
Spirillum mixed culture, and Aquaspirillum sp.; and F. oxysporum and F. culmorum were used as phyto
pathogens. The following characteristics were ana
(d) yeasts Asaccharomyces serevisidae 230 and Can lyzed: the germination capacity of wheat seeds (%),
dida guilliermondii 217. the length of wheat roots at the tillering phase, and the
Actinomycetes were isolated using a traditional number of plants with diseased roots.
method of inoculation of soil suspension on the dense
casein–glycerol medium (CGM) with nystatin [7].
For the selection of potential antagonists, actino RESULTS AND DISCUSSION
mycetes were grown on the medium with soybean The collection of isolated soil actinomycetes
flour recommended for the search of active producers mainly consisted of representatives of the Streptomyces
[15]. For the initial characterization of the biological genus, probably, due to the procedure of their count
activity of the isolated strains, the traditional method ing on the CGM medium favorable for them and their
of agar blocks was used [6]. abundance in the investigated soils. Among 279 iso
Actinomycetes inhibiting three and more popula lates, those with the pronounced antifungal activity
tions of phytopathogenic fungi were considered to towards phytopathogenic fungi, including Fusarium
have a wide spectrum of activity. The degree of activity aquaeductuum (Rabenh et Radhlk) Lagerh, F. het
was judged from the size of the zone, in which phyto erosporum Nees: Fries, F. merismoides Corda,
pathogens were inhibited. When it was less than 5 mm, F. oxysporum Schlechtendahl:Fries, F. poae (Peck)
the antagonists were considered to be weakly active. Wollenweber, F. solani (Martius) Saccardo, Macrospo
The taxonomic belonging of actinomycetes was rium solani (Ell et Mart), Phoma exigua Desmazieres,
determined according to a set of commonly used mor Verticillium luteoalbum (Link) Subramanian, and
phological–cultural and chemotaxonomic character V. nubilum Pethybridge, were selected.
istics [5, 9, 11, 12]. Polyphase taxonomic studies with The effect of the native streptomycetes (their spe
the use of a set of phenotypic characteristics and cies composition depended on the sample) on the
moleculargenetic methods (nucleotide sequence of aboriginal fungi was much higher than their effect on
the 16S gene of RNA) were performed to identify the the phytopathogenic fungi atypical of the given habi
streptomycetes at the species level. The surface of tats. Approximately 14% of the streptomycetes iso
streptomycetes' spores was examined under transmis lated from the samples of different zones within the
sion and scanning electron microscopes (Jeol 100 CX). colony of the agaricoid Clitocybe geotropa fungus
The possibility of using the selected strepto inhibited the growth of native fungi. Among them, no
mycetes as biofungicides was verified in model exper more than 8% inhibited the growth of phytopatho
iments with wheat and watercress plants infected genic fungi to some extent [3]. The strong antagonists
with phytopathogenic fungi Fusarium culmorum and with a wide spectrum of activity inhibiting the growth
F. oxysporum. The Trichoderma citrinoviride native of 2–3 species of the Fusarium and Verticillium luteoal
fungal strain was used for the additional control. The bum fungi amounted to less than 3% of the total
tests were performed under laboratory conditions and amount of the active isolates.
in a phytotron. The portion of streptomycetes inhibiting the
The laboratory experiments were performed with growth of phytopathogenic in the samples isolated
watercress. Suspensions of spores of 5day culture of from the strongly podzolic soil (Zvenigorod Station)


0 10 20 30 40
1 µm
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
Fig. 1. Spiral spore chains of the Streptomyces hygroscopi cus population, and factor C is the Trichoderma cirinovir
cus population. ide population.

was significantly higher: approximately onethird of tion of this strain within the given taxonomic group
the analyzed isolates had the inhibiting effect on phy was confirmed with the help of the PCR method
topathogenic fungi. Among them, populations with a (according to the sequence of RNA gene 16s).
narrow spectrum of antifungal effect (inhibition of the
growth of 1–3 test fungi) predominated. The total The experiments showed that streptomycetes with
number of identified strains of actinomycetes was 279; the antifungal activity could also inhibit the growth of
among them, 35 strains were with a wide spectrum of different bacteria, including typical soil inhabitants.
action, including 22 strains with the strong biofungi For instance, strains of streptomycetes K49 and K193
cidal action. had a wide spectrum of antimicrobial effects; they
inhibited the growth of bacteria (Bacillus megathe
For experiments on the biological control, 17 pop rium, B. subtilis, Micrococcus luteus 2665, M. roseus,
ulations of streptomycetes acting in vitro as potential
Rhodococcus erythropolis 283, Rh. luteus 371, Arthro
biofungicides toward phytopathogenic fungi of the
Fusarium genus (F. heterosporum, F. merismoides, bacter globiformis 281, and Cellulomonas sp. lau 1) and
F. oxysporum, and F. solani), Verticillium luteoalbum, two species of yeasts (Saccharomyces serevisiae 230
and Phoma exigua) were selected. They differed in the and Candida guilliermondii 217). However, they did
spectra of their action: some of them inhibited only not affect the Escherichia coli C600 and Pseudomonas
one of the studied phytopathogenic fungi, and some fluorescens var. lemmoniere bacteria, and the mixed
inhibited them all. On the basis of data on the mor Micrococcus + Spirillum. Aquaspirillum sp. culture. An
phological characteristics of their cultures, the strep active antagonist of fungi—strain K182—did not
tomycetes were subdivided into several phenoclusters inhibit most of the investigated soil bacteria and
according to [9]. yeasts, but inhibited the growth of Staphylococcus
aureus, Bacillus megaterium, B. subtilis, and Micrococ
As a result, Streptomyces sp. K 182 was attributed to cus luteus. The population of streptomycetes A10 with
phenocluster 10 of category 3 (Str. bambergiensis Wall the high activity toward Fusarium did not inhibit the
hausser, Nesemann, Prave and Steigler 1966, 734AL); growth of all the bacteria tested, including those iso
Streptomyces sp. K193, to phenocluster 8 of category 2 lated from the soils. The preliminary information
(Str. chattanoogensis Burns and Holtman 1959, showed that biofungicides could be applied not only to
398AL); Streptomyces sp. 36, to phenocluster 8 of cate control the growth of phytopathogenic fungi but also
gory 2(Str. xanthocidicus Asahi, Nagatsu and Suzuki
to regulate the structure and activity of the microbial
1966, 196AL); Streptomyces sp. A10, to phenocluster 3
of category 1 (Str. halstedii (Waksman and Curtis communities.
1916) Waksman and Henrici 1948, 953AL); the latter A possibilities to control fusarial infections with the
strain could be attributed to the Str. griseolus (Waks Streptomyces hygroscopicus population was assessed in
man 1923) Waksman and Henrici 938AL species. model experiments using a computer system of their
One of the most active streptomycetes—Strepto planning (STATGRAPHICS Plus for Windows). The
myces sp. K49—was identified as Str. hygroscopicus laboratory test (in Petri dishes) with the soil and water
(Jensen 1931) Waksman and Henrici 1948, 953AL. It cress seeds infected with Fusarium showed that the
belongs to the taxonomic group Str. violaceusniger most important and statistically significant factors
clade, which includes species with wrinkled spore were as follows: the introduction of a streptomyces–
exines in spiral chains (Fig. 1). The species identifica antagonist, the state of the microbial community at

76 DEGTYAREVA et al.

Effect, % the moment of sowing, and the interaction between
60 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
40 was also the undisturbed microbial community in the
soil, which had a greater diversity (“mature” system)
than the community after the partial sterilization
30 (“young” system). In this experiment, the Tricho
derma citrinoviride population did not perform func
tions peculiar to fungi of this genus, which could be
Partial Without Soil Absent Soil Absent
related to the specific properties of this aboriginal pop
sterilization treatment ulation.
Present Stretomyces Trichoderma As expected, in the nonsterile soil, the FDA
hygroscopicus citrinoviride hydrolysis index significantly exceeded that in the soil
after its partial sterilization. The introduction of the
Fig. 3. Major effects of the three factors (the state of soil
microbial community, the introduction of streptomycetes,
Trichoderma cirinoviride fungi regularly increased this
and the introduction of Trichoderma) on seed germina index. However, after the introduction of strepto
tion. 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
FDA hydrolysis index
5.5 according to the type of necrotrophic mycophagy
(Fig. 4).
5.3 The surface of the responses for the two key factors
demonstrates the possibility to control fusarial infec
5.1 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
4.9 the introduction of an antagonist is necessary for the
reliable prediction and control of the efficiency of this
4.7 measure.
An independent verification with the use of wheat
4.5 (infected with F. oxysporum and F. culmorum) con
Partial Without Soil Absent Soil Absent
sterilization treatment firmed the possibility of using Streptomyces hygroscopi
Present Stretomyces Trichoderma cus as a biofungicide. The streptomycetes introduced
treatment citrinoviride into the soil with the phytopathogenic fungi reduced
their negative effects on the plants: the length of roots
Fig. 4. Effect of the studied factors studied on the FDA increased by 20%, the number of plants with diseased
hydrolysis index.
roots decreased by 36%, and the germination index
rose by 48%.

Seed germination capacity, %
100 The possibility for the biocontrol of the develop
ment of Fusarium phytopathogenic fungi via applica
tion of streptomycetes has been shown. This fact yields
60 promise with respect to the potential application of the
40 selected populations of streptomycetes as biofungi
0.1 billion
20 spores/g cides.
0 Level of
Microbial system population
young mature REFERENCES
1. Aleksandrova, A.V., Velikanov, L.L., Sidorova, I.I., and
Fig. 5. The surface of plant response as related to the appli Sizova, T.P., Effect of Fungus Trichoderma harzianum
cation of streptomycetes introduction and the state of the on Soil Micromycetes, Mikol. Fitopatol., 2000, vol. 34,
soil microbial community. no. 3.


2. Aristovskaya, T.V., Mikrobiologiya podzolistykh pochv 9. Bergey’s Manual of Systematic Bacteriology, Will
(Microbiology of Podzolic Soils), Moscow, 1965. iams, S.T., Sharpe, M., and Holt, J.A., Eds., Balti
more, 1989, vol. 4.
3. Vinogradova, K.A., Aleksandrova, A.V., Sidorova, I.I.,
and Voronina, E.Yu., Distribution of Soil Actino 10. Goves, R.C., Semedo, L.T.A.S., Soares, R.M.A., Alvi
mycetes in the Collar Colony of Clitocybe geotropa ano, C.S., Linhares, L.F., and Coelho, R.R.R., Chiti
(Vull). Quel. and Their Antagonistic Activity with nolytic Actinomycetes from a Brazilian Tropical Soil
Respect to Micromycetes, Mikol. Fitopatol., 2008, Active against Phytopathogenic Fungi, Word J. Micro
vol. 42, no. 3. biol. Biotechnol, 2000, vol. 16.
11. Goodfellow, M., Kumar, Y., Labeda, D.P. and Sembir
4. Vinogradova, K.A., Movchan, D.D., Aleksandrova, A.V., ing, L., The Streptomyces violaceus niger clade: a Home
and Kozhevin, P.A., Characterization of Interpopula for Streptomycetes with Rugose Ornamented Spores,
tion Interactions of Indigenous Fungi and Actino Antonie van Leeuwenhoek, 2007, vol. 92, pp. 173–199.
mycetes in Podzolic Soil, Mikol. Fitopatol., vol. 41,
p. 207. 12. Kampfer, P., The Family Streptomycetacea. Part 1:
Taxonomy, in The Procariotes, Dworkin, M. et al., Eds.,
5. Gauze, G.F., Preobrazhenskaya, T.P., Sveshnikova, M.A., 3rd ed., New York, 2004.
Terekhova, L.P., and Maksimova, T.S., Opredelitel’ 13. McSpadden Gardener, B.B. and Fravel, D.R., Biologi
aktinomitsetov (Guide to Actinomycetes), Moscow, cal Control of Plant Pathogens: Research, Commercial
1983. ization, and Application in the USA, in APS net Feature:
6. Egorov, N.S., Osnovy ucheniya ob antibiotikakh (Basic Biological Control of Plant Pathogens, 2002.
Theory of Antibiotics), Moscow, 2004. 14. Nourozian, J., Etebarian, H.R., and Khodakaramian, G.,
Biological Control of Fusarium graminearum on Wheat
7. Zenova, G.M., Pochvennye aktinomitsety redkikh rodov by Antagonistic Bacteria, Songklanakarin J. Sci. Tech
(Rare Soil Actinomycetes), Moscow, 2000. nol., 2006, vol. 28.
8. Kozhevin, P.A., Ecology of Soil Microorganisms, in 15. Warren, H.B., NonSynthetic Media for Antibiotic
Ekologiya mikroorganizmov (Ecology of Microorgan Producing Actinomycetes, Antibiot. Chemother., 1955,
isms), Netrusov, A.A., Ed., Moscow, 2004. vol. 5, no. 10.