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SOIL ACTINOMYCETES AS POTENTIAL BIOFUNGICIDES

SOIL ACTINOMYCETES AS POTENTIAL BIOFUNGICIDES

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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. Streptomycetes isolated from the podzolic soils were evaluated for the biosuppression of fungal populations. Seventeen 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.
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. Streptomycetes isolated from the podzolic soils were evaluated for the biosuppression of fungal populations. Seventeen 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.

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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.
73
INTRODUCTIONThe control of phytopathogenic fungi that areresponsible for various plant diseases remains a topicalproblem; its solution will make it possible to reduce yield losses considerably. The extensive use of chemicals (fungicides) cannot be considered an optimumsolution, because it increases the risk of the chemicalpollution of the environment and agricultural production. Therefore, an interest to biofungicides is growing. Biofungicides are microbial populations isolatedfrom the natural environment that are capable toinhibit and destroy undesirable phytopathogens. Particularly, their careful application is especially important in ecological farming aimed at obtaining highquality food products. Commercial biopreparationson the basis of different microorganisms [13, 14] areproposed; among them, only some preparationsinclude populations of actinomycetes–antagonists,such as AlirinC, Mycostop, and Actinovate.Soil actinomycetes are suppliers of many antibioticsubstances with antifungal activity. They also producedifferent exoenzymes, including chitinases that utilizecell walls of fungi [10]. The capacity of actinomycetes–producers to form antibiotics and enzymes is displayednot only under laboratory and artificially controlledconditions but also in natural soils in situ [8].Ecological niches of soil fungi and actinomycetesare 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
) onfouling glasses, attest to the active development of actinomycetes on dying fungal hyphae [2]. Thus, thepotential of soil actinomycetes for the biological control of phytopathogenic fungi is beyond question.Recently, a significant role of actinomycetes in thedevelopment of the local and total resistance of plantsto 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 studyingprospects 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 MoscowState University and from a podzolic soil under a colony of the
Clitocybe geotropa
agaricoid basidiomycete were analyzed [3, 4]. The samples were collected indifferent 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 theDepartment of Mycology and Algology (BiologicalFaculty, Moscow State University), populations isolated from diseased plants (cucurbit, cucumber,potato, and tomato), and saprotrophic native fungifrom the studied soil samples were used as test organisms. In the work with biofungicides, it is important toestimate their action not only on the target populationof pathogenic organisms but also on the entire nativemicroflora [1]. For this purpose, bacteria from the collections of the Department of Soil Biology (Faculty of 
Soil Actinomycetes as Potential Biofungicides
E. A. Degtyareva
a
, K. A. Vinogradova
b
, A. V. Aleksandrova
b
, V. A. Filonenko
c
, and P. A. Kozhevin
a
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 fungalpathogens 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
 
74
MOSCOW UNIVERSITY SOIL SCIENCE BULLETIN Vol. 64 No. 2 2009
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 followingpopulations:(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,
Verticilliumluteoalbum
(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, Rhodococcuserythropolis
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 traditionalmethod of inoculation of soil suspension on the densecasein–glycerol medium (CGM) with nystatin [7].For the selection of potential antagonists, actinomycetes were grown on the medium with soybeanflour recommended for the search of active producers[15]. For the initial characterization of the biologicalactivity of the isolated strains, the traditional methodof agar blocks was used [6]. Actinomycetes inhibiting three and more populations of phytopathogenic fungi were considered tohave 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 wasdetermined according to a set of commonly used morphological–cultural and chemotaxonomic characteristics [5, 9, 11, 12]. Polyphase taxonomic studies withthe use of a set of phenotypic characteristics andmoleculargenetic methods (nucleotide sequence of the 16S gene of RNA) were performed to identify thestreptomycetes 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
nativefungal strain was used for the additional control. Thetests were performed under laboratory conditions andin a phytotron.The laboratory experiments were performed with watercress. Suspensions of spores of 5day culture of 
F. oxysporum
fungus (6
×
10
6
spores/g) and 7day culture of streptomycetes (1
×
10
8
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 wasimmediately sown. After 14 days, the germinationcapacity (%) and the hydrolytic activity of the micro bial community (using the fluorescein diacetate(FDA) hydrolysis method) were determined. Since theFDA hydrolysis is accomplished by active cells with acomplex of enzymes, its rate may be an index of theactive microbial biomass. In this soil, fungi predominated in the biomass and possessed a strong enzymecomplex. Thus, it could be supposed that the rate of FDA hydrolysis depended on the activity of soil fungi. An independent verification of the biofungicidepotential of the selected antagonists was performed with wheat plants on a phytotron at the ResearchInstitute of Phytopathology (Golitsyno, Moscowoblast). 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 thenumber of plants with diseased roots.RESULTS AND DISCUSSIONThe collection of isolated soil actinomycetesmainly 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
Fusariumaquaeductuum
(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 theaboriginal fungi was much higher than their effect onthe phytopathogenic fungi atypical of the given habitats. Approximately 14% of the streptomycetes isolated from the samples of different zones within thecolony of the agaricoid
Clitocybe geotropa
fungusinhibited the growth of native fungi. Among them, nomore than 8% inhibited the growth of phytopathogenic fungi to some extent [3]. The strong antagonists with a wide spectrum of activity inhibiting the growthof 2–3 species of the
Fusarium
and
Verticillium luteoalbum
fungi amounted to less than 3% of the totalamount of the active isolates.The portion of streptomycetes inhibiting thegrowth of phytopathogenic in the samples isolatedfrom the strongly podzolic soil (Zvenigorod Station)
 
MOSCOW UNIVERSITY SOIL SCIENCE BULLETIN Vol. 64 No. 2 2009
SOIL ACTINOMYCETES AS POTENTIAL BIOFUNGICIDES75
 was significantly higher: approximately onethird of the analyzed isolates had the inhibiting effect on phytopathogenic fungi. Among them, populations with anarrow spectrum of antifungal effect (inhibition of thegrowth of 1–3 test fungi) predominated. The totalnumber 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 thespectra of their action: some of them inhibited only one of the studied phytopathogenic fungi, and someinhibited them all. On the basis of data on the morphological characteristics of their cultures, the streptomycetes were subdivided into several phenoclustersaccording to [9]. As a result,
Streptomyces
sp. K 182 was attributed tophenocluster 10 of category 3 (
Str. bambergiensis
Wallhausser, Nesemann, Prave and Steigler 1966, 734
 AL 
);
Streptomyces
sp. K193, to phenocluster 8 of category 2(
Str. chattanoogensis
Burns and Holtman 1959,398
 AL 
);
Streptomyces
sp. 36, to phenocluster 8 of category 2(
Str. xanthocidicus
Asahi, Nagatsu and Suzuki1966, 196
 AL 
);
Streptomyces
sp. A10, to phenocluster 3of category 1 (
Str. halstedii 
(Waksman and Curtis1916) Waksman and Henrici 1948, 953
 AL 
); the latter strain could be attributed to the
Str. griseolus
(Waksman 1923) Waksman and Henrici 938
 AL 
species.One of the most active streptomycetes—
Streptomyces
sp. K49—was identified as
Str. hygroscopicus
(Jensen 1931) Waksman and Henrici 1948, 953
 AL 
. It belongs to the taxonomic group
Str. violaceusniger clade
, which includes species with wrinkled sporeexines in spiral chains (Fig. 1). The species identifica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 withthe antifungal activity could also inhibit the growth of different bacteria, including typical soil inhabitants.For instance, strains of streptomycetes K49 and K193had 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) andtwo species of yeasts (
Saccharomyces serevisiae
230and
Candida guilliermondii 
217). However, they didnot affect the
Escherichia coli 
C600 and
Pseudomonas fluorescens
var.
lemmoniere
bacteria, and the mixed
Micrococcus
+
Spirillum. Aquaspirillum
sp. culture. Anactive antagonist of fungi—strain K182—did notinhibit most of the investigated soil bacteria and yeasts, but inhibited the growth of 
Staphylococcusaureus, Bacillus megaterium, B. subtilis
, and
Micrococcus luteus
. The population of streptomycetes A10 withthe high activity toward
Fusarium
did not inhibit thegrowth of all the bacteria tested, including those isolated from the soils. The preliminary informationshowed that biofungicides could be applied not only tocontrol the growth of phytopathogenic fungi but alsoto regulate the structure and activity of the microbialcommunities. A possibilities to control fusarial infections with the
Streptomyces hygroscopicus
population was assessed inmodel experiments using a computer system of their planning (STATGRAPHICS Plus for Windows). Thelaboratory test (in Petri dishes) with the soil and watercress seeds infected with
Fusarium
showed that themost important and statistically significant factors were as follows: the introduction of a streptomyces–antagonist, the state of the microbial community at
1
µ
m
Fig. 1.
Spiral spore chains of the
Streptomyces hygroscopicus
population.
B AB A  ACBCCFactors403020100 Effect
Fig. 2.
Relative significance of the factors affecting theplant growth on a Pareto diagram: factor A is the soilmicrobial system, factor B is the
Streptomyces hygroscopicus
population, and factor C is the
Trichoderma cirinoviride
population.

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