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Asiatic Journal of Biotechnology Resources Asiatic J. Biotech. Res. 2010; 01: 39-44 N.DEV AND Published online at http://www.pacificjournals.com/ajobr Copyright © 2010 by Pacific Publishers

A.Y. DAWANDE

Vol I Short Communication

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 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. Thus study revealed effectivty of Trichoderma spp. and P. fluorescens and their potential against plant pathogens. Copyright © 2010 by Pacific Publishers

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- [email protected]

[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 &

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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]

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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 4­15 m 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

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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, 4diacetylphloroglucinol (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.

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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 coculturing test organism on malt extract agar plate on similar culture plate and growth of Trichoderma spp. examined for 3rd - 7th day. Similarly cocultured 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.

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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

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