ISSN 0147
6874, 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
a
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 potential of soil actinomycetes for the biological con

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 [10]. 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 [8].
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 [2]. Thus, the
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 [1]. For this purpose, bacteria from the col

lections of the Department of Soil Biology (Faculty of

73
74 DEGTYAREVA et al.
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 [7].
For the selection of potential antagonists, actino

mycetes were grown on the medium with soybean
flour recommended for the search of active producers
[15]. For the initial characterization of the biological
activity of the isolated strains, the traditional method
of agar blocks was used [6].
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
MOSCOW UNIVERSITY SOIL SCIENCE BULLETIN
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 [3]. 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)
Vol. 64 No. 2 2009
 SOIL ACTINOMYCETES AS POTENTIAL BIOFUNGICIDES
1 µm
Fig. 1. Spiral spore chains of the Streptomyces hygroscopi

cus 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 [9].
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

MOSCOW UNIVERSITY SOIL SCIENCE BULLETIN
75
Factors
B
AB
A
AC
BC
C
0 10 20 30 40
Effect
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.
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
Vol. 64 No. 2 2009
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
50
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

20
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, %

CONCLUSIONS
100 The possibility for the biocontrol of the develop

ment of Fusarium phytopathogenic fungi via applica

80
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
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