Asiatic Journal of Biotechnology Resources
Short Communication
Asiatic J. Biotech. Res. 2010; 01: 39-44
39
Published online at http://www.pacificjournals.com/ajobr
Copyright © 2010 by Pacific Publishers
Abstract
Mycolytic enzymes produced
by antagonists are very
important in biocontrol
technology. Study has
revealed the antagonistic
property of Trichoderma spp.
and Pseudomonas fluorescens
against rhizoctonia i.e. a
plant pathogen, on co-
culturing Trichodema and
Rhizoctonia on MEA, while P.
fluorescens and Rhizoctonia on
pseudomonas isolation agar,
growth of only antagonists
i.e. Trichoderma and P.
fluorescens was observed in
respective media while
growth of Rhizoctonia was
suppressed. Thus study
revealed effectivty of
Trichoderma spp. and P.
fluorescens and their potential
against plant pathogens.
Copyright © 2010 by Pacific
Publishers
Copyright © 2010 by Pacific Publishers
N.DEV AND
A.Y. DAWANDE Vol I
Biocontrol of soil borne plant pathogen
Rhiozoctonia solani using Trichoderma spp. and
Pseudomonas fluorescens
Neha Dev and A.Y. Dawande*
Department of Microbiology, Kamla Nehru Mahavidylaya, Sakkardara Chowk,
Nagpur-09
*correspondence: Ashok Dawande, email- ashokdawande@gmail.com
[Received 8 May 2010]
Keywords
Biocontrol, Rhizoctonia solani, Trichoderma spp., Pseudomonas flurescens,
Pathogen
Introduction
Soil borne pathogens are complex not only in their behavioral pattern but
also in their biochemical constituents. Hence, it is not very easy to
control these pathogens. Understanding and dealing with soil borne
pathogens is a very difficult and challenging task. At present, with an
effective management of plant diseases & microbial contamination in
several agricultural commodities is generally achieved by the use of
synthetic fungicides. However, the incessant & indiscriminate application
of these chemical fungicides has caused health hazards in animals &
humans due to residual toxicity.
In recent years, large no of synthetic fungicides have been banned in the
western world because of their undesirable attributes such as high &
acute toxicity. In developing countries such as India, they are still being
used despite their harmful effect. Many pathogenic microorganisms have
developed resistance against chemical fungicides. This seriously hinders
the management of diseases of crops & agricultural plants. Considering
the deleterious effects of synthetic fungicides on life supporting systems,
there is an urgent need for alternative agents for the management of
pathogenic microorganisms. [5]
Intensified use of fungicides has resulted in the accumulation of toxic
compounds potentially hazardous to humans & environment also in the
buildup of resistance of pathogens. In order to tackle these national &
Asiatic J. Biotech. Res. 2010; 01: 39-44
40 N.DEV AND A.Y. DAWANDE Vol I

global problems, alternatives of chemical control are investigated by the

use of antagonistic microbes. [4]

Biological control means control of disease through some biological

agency that is any living microorganism. Biocontrol may be defined as
any condition or practice where by survival or activity of pathogen is
reduced through the agency of any other living organism with the result
that there is reduction in the incidence of disease caused by the pathogen.
Biocontrol of soil-borne plant pathogens affecting agricultural plants can
be controlled by the use of species of Trichoderma, Aspergillus, Trichothecium
and Epicoccum in India. There are some antagonistic bacteria like Bacillus
subtilis, Enterobacter aerogenes, Pseudomonas fluorescence, Streptomyces spp. and
actinomycetes in disease control. The specific need of India is of complete
disease control, which the biological agent seldom offers due to the
problems associated with distribution of pathogen propagates in soil.
There is a growing concern in recent years, both in developed and
developing countries, about the use of hazardous fungicides for
controlling plant diseases. Chemical pesticides have already been proven
to cause adverse environmental effects and result in health hazards to
human as well as other organisms including beneficial natural enemies.
So there is need to develop safer and environmentally feasible control
alternatives. Biological control, i.e., the use of biological processes to
lower inoculum density of the pathogen in order to reduce the disease
producing activities thereby reducing crop loss, is a potential
nonhazardous alternative. [3]

Biological control of soil-borne plant pathogens is a potential alternative

to the use of chemical pesticides, which have already been proved to be
harmful to the environment. Several strains of the fungus Trichoderma
have been isolated and found to be effective biocontrol agents of various
soil-borne plant pathogenic fungi under greenhouse and field conditions.
Different application approaches have been used including integration of
Trichoderma with reduced doses of chemical agents. Biochemical and
molecular biology studies carried out to explore the mechanisms involved
in biological control revealed that Trichoderma is a rather specific
mycoparasite. Lectins were found to be involved in the recognition
between Trichoderma and its host fungi, whereas chitinase is involved in
the degradation of the host cell wall. Genetic engineering techniques
were employed in order to increase the effectiveness, stability, and
biocontrol capacity of Trichoderma spp. as well as other biocontrol agents,
such as Pseudomonass spp. and Rhizobium. [2]

Copyright © 2010 by Pacific Publishers Asiatic J. Biotech. Res. 2010; 01: 39-44
41 N.DEV AND A.Y. DAWANDE Vol I

Plant pathogen

Rhizoctonia solani (teleomorph: Thanatephorus cucumeris) is a plant

pathogenic fungus with a wide host range and worldwide distribution. It
is one cause of the condition known as damping off, which is a cause of
death of seedlings in agriculture. It is also responsible for wire stem, a
disease of cabbage, cauliflower and related plants that is similar to
damping off but attacks older seedlings and produces a constricted, wiry
stem Rhizoctonia solani does not produce spores and is hence identified
only from mycelial characteristics. Its hyphal cells are multinucleate. It
produces white to deep brown mycelium when grown on artificial
medium. The hyphae are 4–15 μm wide and tend to branch at right
angles. A septum near each hyphal branch and a slight constriction at the
branch are diagnostic. R. solani is subdivided into ana-stomosis groups
(AG) based on hyphal fusion between compatible strains.

It forms club-shaped basidia with four apical sterigmata on which oval,

hyaline basidiospores are borne.[1] The fungus is attracted to the plant by
chemical stimulants released by actively growing plant cells or
decomposing plant residues. As the attraction process proceeds, the
fungal hypha will come in contact with the plant and become attached to
its external surface. After attachment, the fungus continues to grow on
the external surface of the plant and will causes disease by producing a
specialized infection structure that penetrates the plant cell and releases
nutrients for continued fungal growth and development.

The infection process is promoted by the production of many different

extracellular enzymes that degrade various components of plant cell walls
(e.g. cellulose, cutin and pectin). As the fungus kills the plant cells, the
hyphae continue to grow and colonize dead tissue, often forming
sclerotia. New inoculum is produced on or in host tissue, and a new cycle
is repeated when new substrates become available. [6]

Biocontrol agents

Trichoderma Spp.

Trichoderma species are frequently isolated from forest or agricultural soils

at all latitudes. Hypocrea species are most frequently found on bark or on
decorticated wood but many species grow on bracket fungi (e.g. H.
pulvinata), Exidia (H. sulphurea) or bird's nest fungi (H. latizonata) or
agarics (H. avellanea). Several strains of Trichoderma have been developed
as biocontrol agents against fungal diseases of plants. The various
mechanisms include antibiosis, parasitism, inducing host-plant resistance,
and competition. Most biocontrol agents are from the species

Copyright © 2010 by Pacific Publishers Asiatic J. Biotech. Res. 2010; 01: 39-44
42 N.DEV AND A.Y. DAWANDE Vol I

Trichoderma harzianum, Trichoderma viride and Trichoderma hamatum. The

biocontrol agent generally grows in its natural habitat on the root surface,
and so affects root disease in particular, but can also be effective against
foliar diseases.

Trichoderma, being a saprophyte adapted to thrive in diverse situations,

produces a wide array of enzymes. By selecting strains that produce a
particular kind of enzyme, and culturing these in suspension, industrial
quantities of enzyme can be produced. Trichoderma spp. is highly efficient
producers of many extracellular enzymes. They are used commercially for
production of cellulases and other enzymes that degrade complex
polysaccharides.

Pseudomonas fluorescens

Pseudomonas fluorescens has multiple flagella. It has an extremely versatile

metabolism, and can be found in the soil and in water. It is an obligate
aerobe but certain strains are capable of using nitrate instead of oxygen as
a final electron acceptor during cellular respiration. Certain Pseudomonas
fluorescens isolates produce of the secondary metabolite 2, 4-
diacetylphloroglucinol (2,4-DAPG), the compound found responsible for
antiphytopathegenic and biocontrol properties in these strains. The phl
gene cluster encodes factors for 2,4-DAPG biosynthesis, regulation,
export, and degradation.

Figure 1.a.b. Luxurient growth of Trichoderma sp. on MEA on 7th day of incubation. c.and d. antagonistic property of
Trichoderma spp. and P. fluorescens against Rhizoctonia sp. on 7th day of incubation, e. and f. Growth of P. fluorescens
and Rhizoctonia on Pseudomonas isolation agar, g. and h. Effect of growth of only antagonists P. fluorescens, while
growth of Rhizoctonia was suppressed.

Copyright © 2010 by Pacific Publishers Asiatic J. Biotech. Res. 2010; 01: 39-44
43 N.DEV AND A.Y. DAWANDE Vol I

Materials and Methods

Microbial Cultures

Pure culture of plant pathogenic fungi Rhizoctonai solani was procured

from Panjabrao Deshmukh Agriculture Institute, Nagpur. Pure culture of
antagonist fungi Trichoderma spp. was procured from Rajiv Gandhi
Biotechnology Centre, Nagpur, inoculated on Potato Dextrose Agar plate
(PDA) incubated at 28-30 oC for 7days and & subcultured after 15 days.
Whereas pure culture of antagonist bacteria Pseudomonas fluorescens was
procured from NCL, Pune, cultured on Pseudomonas isolation agar &
incubated at 37oC for 24 to 48 hrs, Subcultured after every 15 days.

Biocontrol activity test

Biocontrol of plant pathogen R. solani by Trichoderma spp. tested by co-

culturing test organism on malt extract agar plate on similar culture plate
and growth of Trichoderma spp. examined for 3rd - 7th day. Similarly co-
cultured antagonistic bacteria P. fluorescens on pseudomonas isolation agar
with R. solani results were observed for same time span.

Results and Discussion

It has been found that the diseases caused by soil borne plant pathogen
Rhizoctonia solani can be controlled by the antifungal activity of Trichoderma
spp. and P. fluorescens. These two antifungal agents produces wide variety
of enzymes such as beta 1,4 glucanase, beta 1,3 glucanase, chitanases etc,.
Thus present study revealed the antagonistic property of Trichoderma spp.
and P. fluorescens against Rhizoctonia sp. i.e. a plant pathogen. So on
coculturing Trichodema and Rhizoctonia on MEA, while P. fluorescens and
Rhizoctonia on Pseudomonas isolation agar, growth of only antagonists i.e.
Trichoderma and P. fluorescens was observed in respective media while
growth of Rhizoctonia was suppressed. Figure 1

References
1. Adams, Jr., G. C. (1988). Thanatephorus cucumeris (Rhizoctonia solani):
A species complex of wide host range, Adv. Plant Pathol. 6:535-552.

2. Chet, I. (1987). Trichoderma–-application, mode of action, and

potential as a biocontrol agent of soilborne plant pathogenic fungi,
Wiley & Sons, NewYork, N.Y., 137-160.

Copyright © 2010 by Pacific Publishers Asiatic J. Biotech. Res. 2010; 01: 39-44
44 N.DEV AND A.Y. DAWANDE Vol I

3. Chet, I. (1990). Biological Control of Soilborne Plant Pathogens,

Hornby, D., ed., CAB Intl., Wallingford, UK, 274-277.

4. Deacon J.W. (1991). Significance of ecology in the development of

biocontrol agents against soil-borne plant pathogens, Biocontrol. Sci Techn 1: 5–
20

5. Paterson, R. R. M., J. M. Lynch (2001) Biological control by

antagonists.

6. Wiese, M.V. (1987). Compendium of wheat diseases, American

Phytopathological Society, 124-135

Copyright © 2010 by Pacific Publishers Asiatic J. Biotech. Res. 2010; 01: 39-44