WO2007110686A2 - A synergistic composition useful as bioinoculant - Google Patents

Abstract

The present invention relates to a synergistic composition useful as bioinoculant, wherein the said composition comprising at least one fungal isolate of Trichoderma harzianum with an accession number NRRL 30595, NRRL 30596, and NRRL 30597 and optionally carrier.

Description

'A SYNERGISTIC COMPOSITION USEFUL AS BIOINOCULANT'

Field of the invention:

The present invention relates to a synergistic composition useful as bioinoculant, wherein the said composition comprising at least one fungal isolate of Trϊchoderma harzianum with an accession number NRRL 30595, NRRL 30596, and NRRL 30597 and optionally carrier.

More particularly, it relates to a a synergistic composition useful as bioinoculant, wherein the said composition comprising at least one fungal isolate of Trichoderma harzianum with an accession number NRRL 30595, NRRL 30596, and NRRL 30597 and optionally carrier having phytopathogenic fungi controlling activity, abiotic stress tolerating capability, and/or to stimulate plant growth, and/or to stimulate phenol contents in plants, and/or to induce systemic resistance in plants to diseases caused by phytopathogenic organisms, highly efficient root colonization capacity and long shelf life.

Background and prior art of the invention:

At the current state of the art, the methods that are used to control phytopathogenic fungi involve the use of large amounts of chemicals, which make it possible to select isolates that are resistant to them, making them unsuitable for use in the future, in addition to the fact that they remain in the soil for long periods of time and may prove toxic to both humans and to other species of animals and plants. Because of this, the ecosystem may be irreversibly affected, which is always undesirable. One alternative, which people have attempted to apply more widely, is to use agents that exist in nature and are the natural enemies of the phytopathogen(s) to be controlled. Many organisms that can be used in this way have been described in the state of the art. However, the use of said control agents has been limited, primarily because, in the majority of the cases the level of control has not been comparable to that achieved with a very efficient chemical fungicide. Among the most successful agents for biological control of phytopathogenic fungi are those belonging to the genus Trichoderma.

Trichoderma species have been investigated as biological control agents for over 75 years [L. Hjeljord and A. Tronsmo, In: Trichoderma and Gliocladium. Eds. C. P. Kubicek and G. E. Harman, Taylor & Francis, Ltd., London, United Kingdom (1998) pp 135-151], but it is only recently that isolates have become commercially available. These organisms have been favored because they are able to control a wide variety of phytopathogenic fungi that are of great importance to agriculture. Trichoderma spp. can control a wide variety of pathogens and appear in more products than any other microbe (Anti-Fungus; Binab T; Supresivit; T-22G and T-22HB; Trichopel, Trichoject, Trichodowels, and Trichoseal; TY). Products containing Trichoderma spp. control species of Amillaria, Botrytis, Chondrostrenum, Colletotrichum, Fulvia, Fusarium, Monilia, Nectria, Phytophthora, Plasmopara, Psendoperonospora, Pythium, Rhizoctonia, Rhi∑opus, Sclerotinia sclerotiorum, Sclerotium rolfsii, Verticillium, and wood rot fungi. Despite the relative success with which these organisms have been employed, it still has not been possible to achieve the desired levels of disease control. Various techniques have been used in attempts to obtain organisms of this genus that have improved efficiency as biological control agents; the improvement techniques utilized include that of mutagenesis of both the physical and the chemical types, and protoplast fusion. Even though the production of isolates that have been improved by these techniques has been described, one of the most serious problems that has been encountered is that in these cases some of the organism's desirable characteristics may be affected since it is not possible to direct the changes toward a single type of characteristic, at least as far as is now known in the prior art. These techniques do not make it possible to modify these organisms selectively, nor do they guarantee that improved isolates will be obtained. Thus despite the existence and use of biocontrol agents in agriculture, there continues to be a need for development of new plant biocontrol agents. The present invention is directed to fulfilling this need. Localized and systemic induced resistance occurs in all or most plants in response to attack by pathogenic microorganisms, physical damage due to insects or other factors, treatment with various chemical inducers and the presence of non-pathogenic rhizobacteria [R. Harnmerschmidt et al., European Journal of Plant Pathology, Volume 107, pp. 1-6 (2001)]. What was probably the first clear demonstration of induced resistance by Tήchoderma was published in 1997 by Bigirimana et al. [J. Bigirimana et al, Med. Fac. Landbouww. Univ: Gent., Volume 62, pp. 1001-1007 (1997)]. They showed that treating soil with Trichoderma harzianum isolate T-39 made leaves of bean plants resistant to diseases that are caused by the fungal pathogens namely B. cinerea and Colletotrichum lindemuthianum, even though T-39 was present only on the roots and not on the foliage. The same group extended their findings from B. cinerea to other pathogens [De Meyer et al., European Journal of Plant Pathology, Volume 104, pp. 279-286 (1998)]. Three classes of compound that are produced by Trichoderma isolates and induce resistance in plants are now known. These are proteins with enzymatic or other functions, homologues of proteins encoded by the avirulence (Avr) genes, and oligosaccharides and other low-molecular- weight compounds that are released from fungal or plant cell walls by the activity of Trichoderma enzymes [G. E. Harman et al., Nature Reviews, Volume 2, pp. 43-56 (2004)].

Trichoderma isolates are known for their ability to colonize roots, but Trichoderma conidia have also been applied to fruit, flowers and foliage, and plant diseases can be controlled by their application to any of these sites [G. E. Harman et al., Plant Disease, Volume 84, pp.

377-393 (2000)]. Several studies have shown that root colonization by Trichoderma isolates results in increased levels of defence-related plant enzymes, including various peroxidases, chitinases, β-1, 3-glucanases, and the lipoxygenase-pathway hydroperoxide lyase. In cucumber, the addition of Trichoderma asperellum T-203 led to a transient increase in the production of phenylalanine ammonia lyase in both shoots and roots [I.

Yedidia et al., Applied and Environmental Microbiology, Volume 69, pp. 7343-7353

(2003)]. The rapid mass production of promising antagonists in the form of spores, mycelia or tures of both, has been achieved by liquid-fermentation technology: mass production of biomasses of T. hamatum, T. harzianum, and T. viride was reached by utilizing commercially available, inexpensive ingredients such as molasses, brewer's yeast, cottonseed flour, or corn-steeped liquor [G. C. Papavizas et al., Phytopathology, Volume 74, pp. 1171-1175 (1984)]. Other techniques have been employed to improve the delivery of the biocontrol agents. A lignite-stillage (a by-product of sorghum fermentation) carrier system was tested for applying a T. harzianum preparation to the soil [Jones et al., Phytopatholgy, Volume 74, pp. 1167-1170 (1984)]. Encapsulation of the biocontrol agent in an alginate-clay matrix, using Pyrax as the clay material, improved yield and propagule viability over time [D. Fravel et al., Phytopathology, Volume 75, pp. 774-777 (1985)]. Pelletized formulations of wheat bran or kaolin clay in an alginate gel containing conidia, chlamydospores or fermentex biomass of several Trichoderma isolates revealed increased viability of stored pellets, and the number of CFUs formed after adding these pellets to the soil was comparable to that formed from freshly prepared pellets [J. A. Lewis and G. C. Papavizas, Plant Pathology, Volume 34, pp. 571-577 (1985)]. These growth media and delivery systems for formulations of biocontrol fungi show promise because they are able to introduce high levels (10⁶ tolO¹⁰ CFU/g) of fungi into soils not steamed, fumigated, or treated with other biocides. To enhance biocontrol eficacy, appropriate introduction of the antagonist into the microenvironment appears to be crucial: formulations have been applied to seedlings prior to planting [A. Sivan et al., Plant Disease, Volume 71, pp. 587- 592 (1987)] or to seeds in furrows [R. Lifshitz et al., Phytopathology, Volume 76, pp. 720- 725 (1986)]. Economic considerations have forced biotechnologists to improve the application techniques: seed-coating, a technique involving minimal amounts of inoculum was developed by Harman et al. [G. E. Harman et al., Phytopathology, Volume 70, pp. 1167-1172 (1980)]. Conidia of T. hamatun applied as a methocel slurry to pea and radish seeds protected seeds and seedlings against Pythium spp. and Rizoctonia solani almost as effectively as a chemical fungicide seed treatment. The population of T. hamatum increased 100-fold in soil planted with the treated seeds, whereas that of the pathogenic fungi (R. solani and Pythium spp.) was reduced, indicating the antagonist's establishment in the soil. Replanting these soils once or even twice with untreated seeds yielded lower disease incidence than soils originally planted with untreated seeds, indicating the long- term effect of the treatment. Adding chitin or R. solani cell walls as a food source to the seed coating increased T. hamatum's ability to protect the seeds, as well as the population density of Trichoderma in the soil (G. E. Harman et al., Phytopathology, Volume 70, pp. 1167-1172, (1980)]. The different aspects of biological control of plant diseases by Trichoderma are further reviewed by G. E. Harman et al., Nature Reviews, Volume 2, pp. 43-56, (2004).

As living organisms, biological control agents are dependent upon favorable environmental conditions for growth and antagonistic activity. Soils of the tropics and subtropics during summer season are subjected to high-temperature stress, which may have detrimental effects on the introduced Trichoderma isolates. Temperature can affect Trichoderma isolates persistence in inoculants during shipment or in storage, can influence survival in soil, and can limit both its phytopathogenic fungi controlling activity and ability to promote plant growth in the tropics and subtropics. In tropical soils temperature goes up to 55⁰C [S. Kulkarni and C. S. Nautiyal, Current Microbiology, Volume 40, pp. 221-226 (2000)]. Thus it will be prudent to develop approaches to solving production problems of tropical crops for Trichoderma isolates which are effective at high temperature in order to optimize production potentials, tolerance to abiotic stresses, ecological adaptability, and disease control strategies to ensure sustainability. Unfortunately, unsatisfactory or inconsistent disease control by Trichoderma isolates has frequently been reported, and insufficient knowledge of the environmental factors involved in biological control has been acknowledged [L. G. Hjeljord et al., Biological Control, Volume 19, pp. 149-160 (2000)]. Trichoderma harzianum has been used successfully as a biological control agent against several soil-borne plant pathogens. Asexual spores of this fungus, phyaloconidia [D. E. Eveleigh, Trichoderma, In - Biology of Industrial Microorganisms, Eds. A. L Demain, NA Solomon, The Benjamin/Cummings Publ Company Inc, California, (1985) pp, 487-509] have been frequently applied as propagules in biological control programmes [E. Agosin et al, World Jornal of Microbiology & Biotechnology, Volume 13, pp. 225-232 (1997)]. Many T. har∑ianum isolates are considered best adapted to warm temperatures, with optimal temperatures in the range of 15 to 35⁰C [K. H. Domsch et al., In "Compendium of Soil Fungi" Eds. K. H. Domsch, W. Gams, and T. H. Anderson, Academic Press, London (1980) pp. 794-809]. Lieckfeldt et al., Applied and Environmental Microbiology, Volume 65, pp. 2418-2428 (1999) have reported T. viride isolates of type II having the the ability to grow at 35°C. Temperature-tolerant microbes are likely to be found in environments affected by high temperature [S. Kulkarni and C. S. Nautiyal, Current Microbiology, Volume 40, pp. 221-226 (2000)]. We opted for three environments to screen for our temperature-tolerant Trichoderma isolates viz., soil used for Jhum cultivation, solarised and soils and exposed to high temperatures from desert. Jhum is a special kind of agricultural practice of the indigenous people of the north-eastern hill region of India [Ramakrishnan et al., Indian National Science Academy, New Delhi, Diamond Jubilee Publication, pp. 84 (1994)]. This method is also known as Sweden or slash and burn cultivation. Jhuming comprises cutting and burning of forest trees, clearing a small space and then sowing a variety of seeds. The livelihood and culture of the tribal people in the region depend on Jhum cultivation to a great extent. Soil solarisation was pioneered in Israel [Katan et al., Phytopathology, Volume 66, pp. 683-688 (1976)]. The technique involves levelling the soil with minimal soil compaction before thorough wetting, which increases the thermal sensitivity of the soil microfora and fauna as well as increasing heat transfer or conduction in the soil. The soil is then covered with thin clear polyethylene sheeting during the hottest months of the year. Increases in soil temperature can then eliminate or at least reduce soilborne pathogen inoculum as well as insects, nematodes and weed seeds [W. Otieno et al., Crop Protection, Volume 22, pp. 325-331, (2003)]. The tropical desert of Asia extends to India through Rajasthan and Gujarat where it is called the Thar. The climate of the desert region is characterized by extremes of temperatures ranging from even below freezing point in winters to as high as 52 °C in summers. In addition to scarce water and high temperatures, desert soils, typically rocky with a high content of alkali and salt [S. Kulkarni and C. S. Nautiyal, Current Microbiology, Volume 40, pp. 221- 226 (2000)]. Thus soils of the 3 environments were affected by the high temperature.

While work on Trichoderma isolates has been conducted in past there is no clear indication that heretofore that any detailed study has been conducted to demonstrate synergistic composition of high temperature tolerant Trichoderma isolates showing phytopathogenic fungi controlling activity, abiotic stress tolerating capability, stimulating plant growth and phenol contents in plants, ability to induce systemic resistance in plants to diseases caused by phytopathogenic organisms, highly efficient root colonization capacity and long shelf life, further a method of producing said composition thereof, and in addition, a method of isolating said high temperature tolerant Trichoderma isolates. Neverthless, high temperature tolerant Trichoderma isolates showing phytopathogenic fungi controlling activity and ability to promte growth, if one were discovered, could find immediate application, e.g., in soils affected by phytopathogens and high temperature in a desired improvement in crop development.

Tropical plant pathogens such as soil-borne pathogenic fungi are well recognized agricultural problems causing extensive damage, including damping off (Pythium spp.), collor rot (Sclerotium rolfsiϊ), root and stem rot (Rhizoctonia solanϊ), wilt {Fusarium spp.), white rot (Sclerotinia sclerotiorum) to various commercially important crops. For example, these fungi have been found to cause extensive damage to several plants such as chickpea, maize, sunflower, mustard, cauliflower, soybean, gladiolus, teak, chrysanthemum etc. and thus cause serious problems to the agriculture, floriculture, horticulture, and forestry industries. In the past the major approach in controlling these pathogens has been through the use of chemical pesticides. However, due to important economic and ecologic considerations, their use has been disfavored and alternative approaches are sought. Control of many pathogenic fungi through the use of antagonistic microorganisms has been demonstrated for several species of Trichoderma. While it has been found that different species or isolates within a species of Trichoderma may be differentially antagonistic to different pathogenic fungi, Trichoderma viride and Trichoderma harzianum have been shown to be generally effective as biocontrol agents. Some of the possible advantages associated with the biocontrol of pathogenic fungi through the application of Trichoderma as compared to the use of chemical pesticides include an improvement in food safety, a reduction of pollution in the environment, and a decreased incidence of occupational disease to workers in the industry.

Usefulness of Trichoderma is greatly limited in certain situations due to their intolerance to high temperatures. Because many plant pathogens, such as Sclerotium rolfsii causing collor rot, Rhi∑octonia solani causing root and stem rot, Sclerotinia sclerotiorum causing white rot, Fusarium spp. causing wilt and Pythium spp. causing damping off in chickpea, maize, sunflower, mustard, soybean, gladiolus, tomato etc. and several other economical important crops are most destructive in temperate soils exposed to high temperatures, the inability of these Trichoderma species to grow and function in these soils leaves the plants without protection at the time of greatest need. It would be desirable, therefore, to obtain a microorganism which is capable to withstand high temperature to function as biocontrol agents in tropical soils.

Such a high temperature tolerant biocontrol agent would be especially useful in tropical regions. By way of example, snow mold, a disease caused by low temperature pathogenic fungi, is the major cause of crop failures of, such as chickpea, maize, sunflower, mustard, soybean, tomato, gladiolus etc. in tropics. There is a need, therefore, in the art for a biocontrol agent that is effective against chickpea wilt complex, Fusarium corm rot and yellows of gladiolus, collor rot of betelvine, charcoal rot of soybean, Fusaium wilt of chrysanthemum and several other economically important plant diseases caused by pathogenic fungi at high temperature. Consequently, there exists a need in the art for a biocontrol agent that is effective against a wide range of pathogenic fungi existing at moderate as well as high temperature.

This invention relates to a synergistic composition comprising Trichoderma harzianum isolates of accession Nos. NRRL 30595, NRRL 30596, and NRRL 30597 which acts individually or in all possible combinations as phytopathogenic fungi controlling activity, abiotic stress tolerating capability, stimulating plant growth and phenol contents in plants, ability to induce systemic resistance in plants to diseases caused by phytopathogenic organisms, highly efficient root colonization capacity and long shelf life, and a method of producing the composition.

Objects of the invention:

The main object of the present invention is to provide a synergistic composition useful as bioinoculant, wherein the said composition comprising at least one fungal isolate of Trichoderma harzianum with an accession number NRRL 30595, NRRL 30596, and NRRL 30597 and optionally carrier.

Another object of the present invention is to provide a synergistic composition comprising Trichoderma isolates which is useful individually or in all possible combinations in controlling phytopathogenic fungi and stimulating plant growth and phenol contents in plants, ability to induce systemic resistance in plants to diseases caused by phytopathogenic organisms, highly efficient root colonization capacity and long shelf life, and a method of producing the composition.

Further in another object of the present invention is to provide a novel isolates of Trichoderma harzianum that have the ability to promote plant growth under field conditions. Yet another object of the present invention is to develop a method for the control of the transmission and spread of plant disease by applying an agriculturally effective amount of high temperature tolerant Trichoderma to a plant to be protected from a fungal pathogen, by applying an agriculturally effective amount of the Trichoderma isolate to a plant.

Still another object of the present invention is to develop a synergistic formulation comprising the 3 isolates with accession Nos. NRRL 30595, NRRL 30596, and NRRL 30597 phytopathogenic fungi controlling activity, abiotic stress tolerating capability, to stimulate plant growth, to stimulate phenol contents in plants, to induce systemic resistance in plants to diseases caused by phytopathogenic organisms, highly efficient root colonization capacity and long shelf life.

Summary of the invention:

The present invention relates to a synergistic composition useful as bioinoculant, said composition comprising Trichoderma harzianum isolates of accession Nos. NRRL 30595, NRRL 30596, and NRRL 30597 individually or in all possible combinations showing phytopathogenic fungi controlling activity, abiotic stress tolerating capability, and/or to stimulate plant growth, and/or to stimulate phenol contents in plants, and/or to induce systemic resistance in plants to diseases caused by phytopathogenic organisms, highly efficient root colonization capacity and long shelf life, further a method of producing said composition thereof, and in addition, a method of isolating said Trichoderma isolates. The isolates are useful in a method of imparting to plants protection against plant pathogens and promote plant growth by applying them to plants, plant seeds, or soil surrounding plants under conditions effective to impart disease protection and plant growth of the plants or plants produced from the plant seeds. Detailed description of the invention:

Accordingly the present invention provides a synergistic composition useful as bioinoculant, wherein the said composition comprising at least one fungal isolate of Trichoderma harzianum with an accession number NRRL 30595, NRRL 30596, and NRRL 30597 and optionally carrier.

In an embodiment of the present invention, the Trichoderma harzianum with accession number NRRL 30595 have the following characteristics:

(a) Morphological Character (i) Colour surface Dark: green

(ii) Colony mycelium: Compact

(iii) Phialide disposition: Whorl of 3

(iv) Phialide size (μm): 4.5 x 2.9

(v) Conidia shape: Subglobose (vi) Conidia size (μm): 1.9 x 1.5

(vii) Conidia colour: Green

(viii) Chlamydospores: Abundant

(b) Biochemical character

(i) Mycelial dry weight (mg) at Temperature ( °C)40-50: 140.6 to 48.6 (ii) Mycelial dry weight (mg) in NaCl cone. (%) 0.0 to 10.00: 205 to 351

(iii) Mycelial dry weight (mg) in H⁺ ion cone. at pH 3 to 11 : 107 to 305 (iv) Mycelial dry weight (mg) in poly Ethyl Glycol (PEG) 10% to 50: 247 to 78

In another embodiment of the present invention, the Trichoderma harzianum with accession number NRRL 30596 have the following characteristics: (a) Morphological Character

(i) Colour surface Dark: Glaucous to dark green (ii) Colony mycelium: Compact (Hi) Phialide disposition: Whorl of 2-3 (iv) Phialide size (μm): 5.7 x 3.5 (v) Phialide shape : Lageniform (vi) Conidia shape: Subglobose (vii) Conidia size (μm): 2.9 x 1.8

(viii) Conidia colour: Light green (ix) Chlamydospores: Fairly abundant (b) Biochemical character

( i ) Mycelial dry weight (mg)at Temperature ( °C)40-50: 120.9 to 39.6 ( ii ) Mycelial dry weight (mg) in NaCl cone. (%)0.0 to 10.00: 180 to 428

( i i i ) Mycelial dry weight (mg) in H ion cone. at pH 3 to 11 : 69 to 210 ( i v ) Mycelial dry weight (mg) in poly Ethyl Glycol (PEG) 10% to 50: 264 to 94

Further, in another embodiment of the present invention, the Trichoderma harzianum with accession number NRRL 30597 have the following characteristics:

(a) Morphological Character (i) Colour surface: Green

(ii) Colony mycelium: Slightly effuse (iii) Phialide disposition: Whorl of 3

(iv) Phialide size (μm): 3.9 x 2.5

(v) Phialide shape : Ampulliform

(vi) Conidia shape: Ovoid

(vi) Conidia size (μm): 3.2 x 2.5 (vii) Conidia colour: green

(viii) Chlamydospores: abundant

(b) Biochemical character

(i) Mycelial dry weight (mg) at Temperature ( °C)40-50: 151.7 to 50.5 (ii) Mycelial dry weight (mg) in NaCl cone. (%)0.0 to 10.00: 300 to 338 (iii) Mycelial dry weight (mg) in H⁺ ion conc.at pH 3 to 11 : 128 to 275

(iv) Mycelial dry weight (mg) in poly Ethyl Glycol (PEG) 10% to 50: 229 to 74

Yet in another embodiment of the present invention, the said carriers are selected from a group consisting of powdered sorghum grain, maize meal, maize cob, compost, rice husk, rice bran, wheat bran, cow dung, talc, a mixture of fermented sugar factory sulphitation press mud and distillery spent wash, and sugar factory carbonation press mud.

Still another embodiment of the present invention, the said composition is prepared by mixing the fungal isolates of Trichoderma harzianum with an accession number NRRL 30595, NRRL 30596, and NRRL 30597 in equal proportion.

Still in another embodiment of the present invention, the concentration of said fungal isolates used is in therange of 7-9 cfu/g of carrier and preferably 7-8 cfu/g of carrier.

Still in another embodiment of the present invention, the concentration of said fungal isolates used is 6-8 cfu/g of carrier and preferably 7-8 cfu/g of carrier.

Still in another embodiment of the present invention, the said composition has the ability to control phytopathogenic fungi.

Still in another embodiment of the present invention, the said composition has the ability to promote plant growth.

Still in another embodiment of the present invention, the said composition has the ability to tolerate abiotic stresses.

Still in another embodiment of the present invention, the said composition has the ability to stimulate phenol contents in plants. Still in another embodiment of the present invention, the said composition has the ability to induce systemic resistance in plants.

Still in another embodiment of the present invention, the said composition is efficient to colonize plant roots.

A Still in another embodiment of the present invention, the said composition has the ability of long shelf life.

The present invention relates to the Trichoderma isolates selected by the above process have the ability to control phytopathogenic fungi, promote plant growth, tolerance for abiotic stresses, to stimulate phenol contents in plants, induce systemic resistance in plants to diseases caused by phytopathogenic organisms, highly efficient root colonization capacity and long shelf life.

The present invention relates to 3 Trichoderma isolates have the taxonomic characteristics listed in Table 1 as compared to prior art isolate of Trichoderma harzianum ATCC No. PTA 3701.

Table 1 Comparison of biochemical and physical characteristics of Trichoderma harzianum NRRL 30595, Trichoderma harzianum NRRL 30596, and Trichoderma harzianum NRRL 30597 (invention), and Trichoderma harzianum ATCC No. PTA 3701 (descriptive).

NRRL 30595 NRRL 30596 NRRL 30597 ATCC NoT Invention Invention Invention PTA 3701

Descriptive*

Morphological Character Colour surface Dark green Glaucous to Green Dark green dark green

Colony mycelium Compact Compact Slightly effuse Compact Phialide disposition Whorl of 3 Whorl of 2-3. Whorl of 3 Whorl of 2-3 Phialide size (μm) 4.5 x 2.9 5.7x3.5 3.9x2.5 5.5x3.4

Phialide shape Ampulliform Lageniform Ampulliform Ampulliform

Conidia shape Subglobose Subglobose Ovoid Subglobose

Conidia size (μm) 1.9x1.5 2.9x1.8 3.2x2.5 2.5x2.0

Conidia colour Green Light green Green Green

Chlamydospores Abundant Fairly Abundant Abundant abundant

Biochemical character

Mycelial dry weight (mg) at

Temperature ( ^UC)

40 140.6 120.9 151.7 49.6

45 110.9 87.6 112.0 12.0

50 48.6 39.6 50.5 -

In NaCl cone. (%)

0.0 205 180 300 187

5.0 406 340 228 292

10.00 351 .428 338 127

In H⁺ ion cone. pH3 107 69 128 150 pH7 240 310 152 190 pH l l 305 210 275 95

Poly Ethyl Glycol (PEG) %

10 247 • 264 229 108

30 129 172 121 79

50 78 94 74 0.0

* J. Bisset, Canadian Journal of Botany, Volume 69, pp. 2357-2372 (1991)

The 3 isolates Trichoderma harzianum NRRL 30595, Trichoderma harzianum NRRL

30596, and Trichoderma harzianum NRRL 30597 isolated from 3 environments exposed to high temeperature viz., soil used for Jhum cultivation, solarised and desert soils respectively, selected by the method of screening as described above have the ability to control phytopathogenic fungi and stimulate plant growth. Trichoderma harzianum NRRL

30595, Trichoderma harzianum NRRL 30596, and Trichoderma harzianum NRRL 30597 have been deposited under the Budapest treaty on September 30, 2002 into ARS Patent culture collection, United States Department of Agriculture, 1815 North University Street,

Peoria, Illinois 61604, U.S.A.

In addition to the other properties noted above, the unexpected and surprising attributes of these specific Trichoderma harzianum isolates include the following characteristics. All the three isolates have been isolated from the 3 environments exposed to high temeperature viz., soil used for Jhum cultivation, solarised and desert soils. In pure culture the isolates inhibit the growth of many pathogenic fungi of plants. The isolates are capable of colonizing plant roots. These isolates reduce the plant disease in soil both under greenhouse and field conditions. The isolates of the present invention are capable of promoting plant growth of plants in soil both under greenhouse and field conditions.

The subject Trichoderma harzianum isolates also has tolerance to abiotic stresses like 10% salt (NaCl)₃ 3-11 pH, and 5O⁰C temperature. It is within the compass of the invention to isolate any type of fungal isolates having the ability to control phytopathogenic fungi, abiotic stress tolerating capability, stimulating plant growth and phenol contents in plants, ability to induce systemic resistance in plants to diseases caused by phytopathogenic organisms, highly efficient root colonization capacity and long shelf life, however, Trϊchoderma are the fungi of choice because (1) they can easily be isolated, cultured and identified; (2) being a naturally occurring isolate or isolate does not require genetic engineering to be effective; (3) being nutritionally versatile are able to utilize large number of organic substrates, including root exudates; (4) being suppressive to one or more pathogenic fungi; (5) having a stage in its life cycle that is resistant to harsh environmental conditions; (6) being tolerant to abiotic stresses (high salt, high pH, and high temperature); (7) being able to enhance phenol contents in plants; (8) ability to enhance induce systemic resistance in plants; (9) have highly efficient root colonization ability to colonise plants; (10) enhancing the yield of the host plant(s) under field conditions; and (11) being relatively easy to develop for commercial purposes.

Another aspect of the invention is directed to a method of controlling plant diseases and promoting plant growth of plants in soil both under greenhouse and field conditions. The 3 Trichoderma isolates designated Trichoderma harzianum NRRL 30595, Trichoderma harzianum NRRL 30596, and Trichoderma harzianum NRRL 30597 are especially preferred in this process. An inoculant of the subject isolate is μsed such that colonization is in the range of about Log 6-10 colony forming units/gram (cfu/g) root occurs and preferably Log 6-8 cfu/g. A mixture of the 3 isolates (consortium) designated Trichoderma harzianum NRRL 30595, Trichoderma harzianum NRRL 30596, and Trichoderma harzianum NRRL 30597 in the ratio of 1 :1:1, consisting of about Log 8-10 cfu/g each and preferably Log 7-8 cfu/g each is especially preferred in this process. The inoculum can be applied directly to the seeds or plants, can be present in the soil before planting or can be distributed, e.g., by spreading, dusting or the like, over the crop or soil top or in soil furrow where the crop has been planted. Seeds can be treated by coating with a composition containing the subject Trichoderma by dipping in a liquid containing these Trichoderma, by spraying with the liquid, or other method known in the art for applying Trichoderma to seeds.

According to yet another aspect of the invention cultures of Trichoderma har∑ianum NRRL 30595, Trichoderma harzianum NRRL 30596, and Trichoderma harzianum NRRL 30597 may be grown individually in molasses diluted with water in the ratio of 1 : 1 to 1 :10. However dilution of 1:5 is especially preferred in this process. The Trichoderma isolates may be used individually, or as a consortium in the ratio of 1 : 1 : 1 , consisting of about Log 6-10 cfu/ml each and preferably Log 8-9 cfu/ml each is especially preferred in this process. The consortium thus obtained may further be diluted with water in the ratio of 1:10 to 1 : 10000. However dilution of 1 : 100 is especially preferred in this process.

Growing the Trichoderma in molasses makes the process economically very viable. Trichoderma grown in such manner may further be used to treat seeds by coating with a composition containing the subject Trichoderma by dipping in a liquid containing these Trichoderma, by spraying with the liquid or other method known in the art for applying Trichoderma to seeds.

According to a further aspect the invention, the process of the invention may be used with any kind of fungi or other microorganisms capable of surviving under abiotic stress conditions e.g., tolerance to salt, pH, and temperature. Of particular interest are fungi, which have biocidal properties, e.g., biofungicidal, pesticidal, and other properties; promote plant growth, under abiotic stress conditions, e.g., high salt, high pH, and high temperature that are capable of living in the soil in the presence of the plants.

The carriers that may be used to disperse the subject isolates would include all those commonly used for inoculating crops and would include carriers such as powdered sorghum grain, fermented press mud, grain, maize meal, maize cob, compost, rice husk, rice bran, wheat bran, cow dung and talc. The fungi in such compositions are at a level of about Log 6-10 cfu/g carrier. Carriers such as talc or fermented press mud are especially preferred in this process. The fungi are grown in broth to the necessary amount, and then mixed with the carrier at the desired inoculum, followed by curing of the mixture by well- known methods.

According to this embodiment of the invention the optimum carrier may vary depending on the fungi used. Any of the above compositions, liquids, powders, talc, fermented press mud, sorghum grain, maize meal, maize cob, compost, rice husk, rice bran, wheat bran, cowdung and the like may have nutrients included therein or appropriate carrier medium such as water, oils or solid bases such as powders, peat, soil, vermiculite, charcoal, fermented, press mud and any other carrier agents. However, as demonstrated by the examples below, sorghum grain, maize meal, maize cob, compost, rice husk, rice bran, wheat bran, cowdung, talc, fermented press mud are preferred.

Further aspect of this invention relates to a process whereby the synergistic composition thus produced of the present invention may be used in any manner known in the art for example, including pretreatment of soil or seeds or pregerminated plant roots alone or in combination with other chemicals which is harmless to the growth and survival of fungi for example plant growth promoting compounds, pesticides, fertilizers, nematicides, herbicides with or without for example lime pelleting to limit the severity of the effect of these materials. However, as demonstrated by the example below, compatible pesticides are preferred.

The following examples are given by way of illustration of the present invention and should not be construed to limit the scope of present invention. EXAMPLE 1

Collection/Isolation of novel high temperature tolerant Trichoderma isolates having biological ability against phytopathogenic fungi

The temperature-tolerant Trichoderma isolates were selected by first screening 100

Trichoderma isolates each from soils from 3 environments exposed to high temeperature viz., soil used for Jhum cultivation, solarised and desert soils. One gram soil from each sample was suspended in 9 ml. sterile saline and diluted upto 1000 fold. 100 μl of soil suspension was spreaded on the Petri dishes containing Trichoderma selective media {Trichoderma selective media (g/L) MgSO₄, 0.20; K₂HPO₄, 0.90; KCl, 0.15; NH₄NO₃, 1.0; Glucose, 4.0; Penta Chloro Nitro Benzene (PCNB), 0.50 and Agar, 18.0) from each dilution. Plates were incubated at 25⁰C ± 2, as described by Papavizas [G. C. Papavizas, Phytopathology, Volume 72, pp. 121-125 (1982)]. After 24-48 hr. individual colonies were picked and maintained on Potato dextrose agar (PD A media (g/L) Potatoes, infusion from 300.0 g, Dextrose, 20.0 g and Agar, 18.0) slants for further study. Trichoderma isolates were grown in test tube for 7 days in SP3 media (SP-3 Media (g/L) Glucose, 50.0; Yeast extract, 1.0; Peptone, 1.0; KH₂PO₄, 1.5; (NH₄)₂SO₄, 1.5 and MgS O₄.7H₂O, 1.0). Test tubes having fully grown cultures were incubated at B. Braun make reciprocal water bath Ceromat WR at 50⁰C, 25 μl of culture was spotted on PDA plates at lhr interval. Plates were incubated at 25 ± 2⁰C to monitor the growth. Survival of selected Trichoderma isolates were monitored up to 10 days when grown at 50⁰C in 150 ml Erlenmayer flask containing 50 ml PDB (PDB media (g/L) Potatoes, infusion from 300.0 g and Dextrose, 20.0 g) inoculated with about 10⁷ colony forming units of Trichoderma isolates. Twenty- nine Trichoderma isolates out of the 300 isolates demonstrating survival at 50⁰C up to 6 days were thus selected for further characterization high temperature tolerant Trichoderma isolates. On the contrary, reference isolates Trichoderma isolate ATCC No.PTA-3701 survived for only for 1 day. . Twenty-nine Trichoderma isolates thus selcted were further screened for their effectiveness in controlling the growth of important phytopathogens viz., Fusarium oxysporium, Rhizoctonea solani, Sclerotium rolfsii, Sclerotinia sclerotiorum, Pythium aphanidermatum and Phytophthora nicotianae causing several diseases, under in vitro conditions. Twenty nine isolates of Trichoderma spp. were evaluated as antagonists under in vitro conditions against the Fusarium oxysporium, Rhizoctonea solani, Sclerotium rolfsii, Sclerotinia sclerotiorum, Pythium aphanidermatum and Phytophthora nicotianae by using dual culture plate inoculation technique [D.. J. Morton and W. H. Stroube, Phytopathology, Volume 45, pp. 417-420 (1955)]. In vitro comparisons consisted of removing 5 -mm diameter mycelial disks from the edge of expanding colonies of antagonist and pathogen, grown on Potato Dextrose Agar medium in Petri dishes. The paired isolates of Trichoderma spp. and phytopathogens were placed on opposite sides of 90 mm Petri plates containing 25 ml. of PDA medium. Paired cultures were incubated in BOD incubator at 25±2°C for observations. The plates were also used to record the type of colony interaction i.e. hyperparasitism or antagonism. Paired cultures were scored for degree of antagonism on modified scale of classes 1-5 [D. K. Bell et al., Phytopathology, Volume 72(4), pp. 379-382 (1982)]; Class A = Trichoderma completely overgrew the pathogen and covered the entire medium surface, Class B = Trichoderma overgrew at least two- thirds of the medium surface, Class C = Trichoderma and the pathogen each colonized approximately one half of the medium surface ( more than one third and less than two thirds), Class D = the pathogen colonized at least two thirds of the medium surface and appeared to withstand encroachment by Trichoderma and Class E= the pathogen completely overgrew the Trichoderma and occupied the entire medium surface. Percentage inhibition of sclerotia formation was also determined. Fusarium oxysporium, Rhizoctonea solani, Sclerotium rolfsii, Sclerotinia sclerotiorum, Pythium aphanidermatum and Phytophthora nicotianae held its growth till it comes in contact with the leading edge of the different Trichoderma isolates. Twenty-nine Trichoderma isolates isolated in the previous step are screened to select for their effectiveness in controlling the growth of important phytopathogens by using dual culture plate inoculation technique [D. J. Morton and W. H. Stroube, Phytopathology, Volume 45, pp. 417-420 (1955)]. In vitro comparisons consisted of removing 5-mm diameter mycelial disks from the edge of expanding colonies of antagonist and pathogen, grown on Potato Dextrose Agar medium in Petri dishes. The paired isolates of Trichoderma spp. and phytopathogens were placed on opposite sides of 90 mm Petri plates containing 25 ml. of PDA medium. Paired cultures were incubated in BOD incubator at 25±2°C for observations. The plates were also used to record the type of colony interaction i.e. hyperparasitism or antagonism. Thirteen Trichoderma isolates were selected as inhibiting mycelial growth of Fusarium oxysporium, Rhizoctonia solani, Sclerotium rolfsii, Sclerotinia sclerotiorum, Pythium aphanidermatum and Phytophthora nicotianae by at least 50% as compared to growth of Fusarium oxysporium, Rhizoctonia solani, Sclerotium rolfsii, Sclerotinia sclerotiorum, Pythium aphanidermatum and Phytophthora nicotianae under control conditions.

Thirteen Trichoderma isolates thus selected at a particular concentration promote plant growth under greenhouse conditions as described earlier [C. S. Nautiyal, Current Microbiology, Volume 34, pp. 12-17 (1997)]. The 13 Trichoderma isolates thus selected were further subjected to abiotic stress tolerance by first screened for their ability to grow in PDB containing 10% salt (NaCl; pH 7 and 25⁰C temperature), on a New Brunswick Scientific, USA, Innova Model 4230 refrigerated incubator shaker at 185 rpm. The finally isolates tolerant to 10% salt were grown at 11 pH (10% NaCl and 25⁰C temperature). Viable cells were counted by removing samples at various times in the presence or absence of stress, as indicated. Serial dilutions of each sample were spotted (25 μl) onto PDA plates, and incubated at 25⁰C in triplicate as described earlier [C. S. Nautiyal et al., FEMS Microbiology Letters, Volume 182, pp. 291-296 (2000)]. Viable cells were counted after 7-10 days. The 3 Trichoderma isolates tolerant to abiotic stresses (salt, pH, and temperature) selected as above were were designated as accession Nos. Trichoderma harzianum NRRL 30595, Trichoderma harzianum NRRL 30596, and Trichoderma harzianum NRRL 30597.

EXAMPLE 2

Effect of salt (NaCl) and pH on the growth of NRRL 30595, NRRL 30596, and NRRL 30597

As shown in Table 2 in comparison to without salt Trichoderma isolates NRRL 30595, NRRL 30596, and NRRL 30597 grew better in the presence of 5 and 10% salt concentration. Trichoderma isolates gives 47.25% less biomass at 10% salt and 56.14% more biomass in the presence of 5% salt. Results in Table 2 demonstrate that these isolates are salt loving and grow better in the presence of salt. Trichoderma isolates NRRL 30595, NRRL 30596, and NRRL 30597 grew best at high pH of 11, whereas for all other isolates the maximum growth was observed at pH 7 (Table 2).

Table 2 Effect of different Salt (NaCl) concentration and pH on the growth of selected Trichoderma isolates.

Isolates Mycelia I dry weight (mg)

InNaCl cone. (%) In H⁺ ion cone.

0.0 5.0 10.0 pH 3 pH 7 pH l l

NRRL 30595 205 406 351 107 240 305

NRRL 30596 180 340 428 69 310 210

NRRL 30597 300 228 338 128 152 275

ATCC No. 187 292 127 150 190 95

PTA 3701 EXAMPLE 3

Effect of NRRL 30595, NRRL 30596, and NRRL 30597 individually and in consortium showing pliytopathogenic fungi controlling and plant growth abilites.

Effect of NRRL 30595, NRRL 30596, and NRRL 30597 individually and in consortium was studied for ability to control the chickpea wilt disease and plant growth. As shown in Table 3 consortium of three Trichoderma isolates NRRL 30595, NRRL 30596, and NRRL 30597 was used to control the chickpea wilt disease. Results as presented in Table 3, reveal that % increase in plant height was 42.27 more in inoculated plants, over un-inoculated control. The change in the fresh weight and dry weight was also very significant when compared with un-inoculated control. The increase in fresh weight and dry weight observed was 55.48 and 48.15 respectively over the un-inoculated control. Increase in yield and weight of dry seeds observed was 49.17 and 197.05% more in the plants treated with consortium when compared with un-inoculated control plants. At the same time mortality of treated plants was only 7% in comparison to 41% for untreated plants (Table 3).

Table 3 Effectiveness of individual isolates of Trichoderma and their cpnsortium on chickpea wilt disease complex and growth parameters at CSAU A&T, Kanpur.

Treatments Mortality Plant Numbers Fresh Dry Dry. Number Yield (%) Height of wt. of wt.of wt. of of (kg

(cm) flowers shoot shoot seeds plants ha^"1)

& Pods plants^' plants^" plants^" (m^2)"' i i i

NRRL Ϊ95 5Ϊ6 4X9 ΪΪ03 349 248 36\0 795

30595

NRRL 25.9 48.2 47.5 94.6 30.7 21.0 34.2 648 30596

NRRL 24. 6 51 .8 48.9 108. 3 31 .8 23 .7 35 .5 754

30597

Consortium 7.9 56 .2 52.0 121. 9 42 .2 30 .6 36 .0 1182

Control 41. 8 39 .5 37.0 78.4 28 .5 20 .5 34 .0 398

Therefore consortium of NRRL 30595, NRRL 30596, and NRRL 30597 was used for further work as it demonstrated synergistic effect and thereby optimal protection against the phytopathogenic fungi and plant growth, compared with individual treatments.

EXAMPLE 4

Effect of NRRL 30595, NRRL 30596, and NRRL 30597 consortium showing phytopathogenic fungi controlling and plant growth abilites on different varieties of chickpea using seed and furrow treatments.

Table 4a and 4b demonstrates that the control of chickpea wilt on both the varieties of crop as in variety Radhey disease control was 78.2 and 74.4 when treatment of consortia was on seeds and furrow respectively (Table 4a), this disease control in the same treatments were 73.9 and 43. 9 when the grown crop was of variety H-208 (Table 4b). Rate of mortality observed was 78.2 and 74.4% less than un-inoculated plants in 2 types of seed and furrow treatments of variety Radhey and 73.9 and 43.9% respectively in seed and furrow treatments of variety H-208. Increase in yield was more significant in the chickpea variety of H-208, compared to radhey variety. Increase was 10.37 and 7.09 in Radhey variety and 44.58 and 43.80 in H-208 variety in the seed and furrow treatment respectively.

Table 4a Effectiveness of individual isolates of Trichoderma and their consortium on chickpea wilt disease complex and growth parameters at GBPUA&T, Pantnagar (Variety: Radhey). (Plot Size: 5 X I m²) Treatments Mortality Disease Dry wt. Yield Control of 100 (g/plof¹)* seeds (g)

Seed Treatment

NRRL 30595 29.6 44.9 20.9 524.8 NRRL 30596 33.9 36.8 20.5 514.6 NRRL 30597 27.7 48.5 21.7 533.1 Consortium 11.7 78.2 25.8 560.3

Furrow Application

NRRL 30595 32.8 38.9 20.5 519.4 NRRL 30596 37.3 30.5 20.5 510.5 NRRL 30597 29.0 45.9 20.9 524.9

Consortium (NRRL 30595 + 13.8 74.4 21.7 543.7 NRRL 30596 + NRRL 30597) Uninoculated Control 53.7 20.2 507.7

Plot size: 5 x 1 m

Table 4b Effectiveness of individual isolates of Trichoderma and their consortium on chickpea wilt disease complex and growth parameters at GBPUA&T, Pantnagar (Variety: H 208). (Plot Size: 5 X I m²)

Treatments Mortality Disease Dry wt. Yield Control of 100 (g/plof¹)* seeds

(g)

Seed Treatment NRRL 30595 25.6 46.2 14.2 547.2

NRRL 30596 29.9 37.1 14.7 513.7

NRRL 30597 23.7 50.2 15.2 578.8

Consortium (NRRL 30595 + 1 122..44 73.9 15.9 640.7

NRRL 30596 + NRRL 30597)

Furrow Application

NRRL 30595 39.5 20.2 12.8 526.8

NRRL 30596 37.3 21.5 12.6 501.6

NRRL 30597 31.4 33.8 12.8 565.9

Consortium (NRRL 30595 + 2 266..77 43.9 13.6 637.5

NRRL 30596 + NRRL 30597)

Uninoculated Control 47.5 12.6 443.3

* Plot size: 5 x 1 m

As shown in Table 4a and 4b consortium is able to control chickpea wilt on both the varieties of crop as well as when applied as seeds and furrow treatments.

EXAMPLE 5

Effect of NRRL 30595, NRRL 30596, and NRRL 30597 consortium showing phytopathogenic fungi control on cauliflower.

Table 5 demonstrates that the control of Sclerotinia stalk rot of cauliflower caused by Sclerotinia sclerotiorum. Rate of mortality observed was in the range of 7.5 to 18.5% in comparison to un-inoculated control where the 33.4% mortality was recorded. Disease control was in the range of 44.7 to 77.5% compared with un-inoculated control. Table 5 Effect of different isolates of Tήchoderma and its consortium to control the Sclerotinia stalk rot of cauliflower caused by Scleortinia sclerotiorum.

Treatments Mortality (%) Disease Control (%)

NRRL 30595 12.6 62.2

NRRL 30596 18.5 44.7

NRRL 30597 15.9 52.4

Consortium 7.5 77.5

Control 33.4

As shown in Table 5 the application of consortium totally suppressed the severity of sclerotinia rot of cauliflower caused by Sclerotinia sclerotiorum

EXAMPLE 6

Effect of NRRL 30595, NRRL 30596, and NRRL 30597 consortium showing phytopatho genie fungi control on teak {Tectona grandis) seedlings

Table 6 demonstrates the control of collor rot of teak {Tectona grandis) seedling caused by

Rhi∑octonia solani. Rate of mortality observed was in the range of 58.7 to 87.3% less than un-inoculated. Disease control was in the range of 29.2 to 69.2% compared with un- inoculated control.

Table 6 Effect of different isolates of Trichoderma and its consortium to control the collor rot of teak {Tectona grandis) seedling caused by Rhizoctonia solani

Treatments Mortality (%) Disease Control (%) NRRL 30595 26.5 35.8

NRRL 30596 29.2 29.2

NRRL 30597 23.4 43.4

Consortium 12.7 69.2

Control 41.3

Results in Table 6 clearly demonstrates that the seed treatment of consortium resulted in most consistent and effective suppression of collor rot of teak (Tectona grandis) seedling caused by Rhizoctonia solani

EXAMPLE 7

Effect of NRRL 30595, NRRL 30596, and NRRL 30597 consortium showing phytopathogenic fungi control on tomato seedling Table 7 shows the control of damping off of tomato seedling caused by Pythium aphanidermatum. Disease control was in the range of 59.5 to 78.9 % compared with un- inoculated control

Table 7 Effect of different isolates of Trichoderma and its consortium to control the damping off of tomato seedling caused by Pythium aphanidermatum

Treatments Mortality (%) Disease Control (%)

NRRL 30595 29.5 60.5

NRRL 30596 30.2 59.5

NRRL 30597 27.4 63.3

Consortium 15.7 78.9

Control 74.7 Results in Table 7 clearly demonstrates that the seed treatment of consortium resulted in most consistent and effective suppression of damping off of tomato seedling caused by Pythium aphanidermatum

EXAMPLE 8

Effect of plants representing various agronomic, floriculture, vegetables to NRRL 30595, NRRL 30596 and NRRL 30597 consortium

Responses of plants representing various agronomic and floriculture to treatment, (as described in Example 3), NRRL 30595, NRRL 30596, and NRRL 30597 consortium are shown in Table 8a to 8e. Treated plants demonstrated more yield and flowered earlier and produce significantly more flowers than the untreated control.

Table 8a Effect of individual isolates of Trichoderma and their consortium on growth and yield parameters of maize

Treatments Average Average Average Average Test Average Yield

Plant No. of internodal No. of Wt. No. q/ha height leaves/ distance/ cobs/plant (Wt. seeds/

(cm) plant Plant (cm) of cob

100 seeds in g)

NRRL 212.5 14.3 16.4 1.8 28.2 587.3 36.4 30595 ^' NRRL . 209.6 14.2 16.2 1.6 27.4 562.6 30596 34.7 NRRL 226.5 14.3 16.7 1.8 28.9 592.2 38.9 30597

Consortium 246.9 14. 8 16.7 2 .0 31 .9 623.4 43.8

Control 174.4 13. 8 16.1 1 .6 25 .7 549.72 32.5

Table 8b Effect of individual isolates of Trichoderma and their consortium on growth and yield parameters of chickpea

Treatments No. of Average Average no. No. of Nodulation Yield plant Plant of branches flowers per plant* q/ha per m² height per plant per plant

(cm)

NRRL 30595 27.4 44.7 7.8 43.9 +++ 9.1

NRRL 30596 27.6 44.8 8.0 44.5 +++ 9.7

NRRL 30597 24.9 43.8 7.5 39.4 +++ 8.5

Consortium 28.2 45.5 8.2 47.8 ++++ 10.9

Control 22.5 42.5 7.4 34.5 ++ 8.2

* Note: ++++ = 40-50, +++ = 30-39, ++ = 20-29 and + = < 20 nodule per plant

Table 8c Effect of individual isolates of Trichoderma and their consortium on growth parameters of gladiolus.

NRRL NRRL NRRL Consortium Control

30595 30596 30597

Germination 92.6 92.8 94.8 98.6 83.7

(%)

No. of 1.6 1.6 1.7 1.8 1.5 sprouts/ corm

Plant height 64.9 65.4 67.4 71.6 59.3

(cm)

Days to 85.0 85.5 84.2 82.2 87.6 flower

Spike length 72.0 72.9 75.9 82.3 61.6

(cm)

No. of 13.4 14.7 15.9 17.8 11.6 flowers/ spike

Duration of 19.8 19.6 20.4 22.0 19.6 flowering

(days

Weight of 54.0 58.0 62.7 69.7 51.3 corms/

Plant (g)

No. of 14.9 15.4 16.8 19.6 12.5 cormels/ plant

Table 8d Effect of individual isolates of Trichoderma and their consortium on growth and yield parameters of sunflower

Treatments Plant No. of Days No. of No. Dry Yield

Height Leaves/Plant taken to flowering of weight (q

(cm) flower buds/plant grains of ha^"1) bud per 1000 formation head grains (DAS)** (g)

NRRL 30595 108.3 29.1 33 1 885.0 49.5 12.3

NRRL 30596 121.0 28.2 32 1 876.6 50.7 13.1

NRRL 30597 119.4 27.0 35 1 723.2 47.9 11.5

Consortium 129.6 29.1 32 1 994.5 51.5 15.2

Control 93.6 23.7 38 1 709.4 46.8 10.4

* Days after sowing

Table 8e Effect of individual isolates of Trichoderma and their consortium on growth and yield parameters of mustard

Treatments No of No. of Total No. of No. of Test Yield

Plants/ Branches siliquae/ seeds/siliqua seeds/plant weight (q/ha.) m² /plant Plant of

1000 seeds

(g)

NRRL 7A5 ΪS5 227.0 VL9 1347.9 92 Ϊ33 30595

NRRL 22.8 18.2 219.5 17.2 1321.3 9.2 13.0 30596

NRRL 22.5 18.1 216.6 16.2 1290.9 9.1 12.3 30597

Consortium 26.8 18.9 244.6 18.6 1483.4 9.7 14.7

Control 21.3 17.3 209.8 15.7 1229.7 8.9 11.8

The results in Table 8a-8e showed that the application of consortium treatment in various crops viz., maize, chickpea, gladiolus, sunflower and mustard leads to considerable improvement in various growth and yield characters as compared with their un-inoculated control. The results revealed that seed treatment with consortium performs significant improvement in plant productivity.

EXAMPLE 9

Phenol content in plants inoculated with NRRL 30595, NRRL 30596, and NRRL 30597 consortium.

Phenoilc contents of various plants in response to inoculation with Trichoderma isolates NRRL 30595, NRRL 30596, NRRL 30597 and its consortium is shown in Table 9. Results showed that the seed treatments with consortium of selected Trichoderma isolates led to an increase in total phenolic content of the seedling selected under investigation.

Table 9 Effect of individual isolates of Trichoderma and their consortium on phenolic content of various crops

Treatments Phenolics gallic acid equivalent (mg/g of tissue ) sowing after

ψ 2P Ψ 2P Ψ 2p 7^stday 21^st ψ 2F^~ day day day day day day day day day

Chickpea Sunflower Mustard Maize Gladiolus

NRRL 0.721 4.987 0.765 6.321 0.601 4.320 0.654 3.154 0.732 5.102

30595

NRRL 0.643 4.876 0.723 5.985 0.572 4.121 0.612 3.032 0.698 4.653

30596 NRRL 0.765 5.765 0.876 6.876 0.623 4.875 0.714 3.259 0.793 5.296

30597

Consortium 0.954 6.143 1.124 7.432 0.768 5.321 0.946 4.645 0.928 6.213

Control 0.579 3.754 0.654 4.124 0.456 3.764 0.529 2.984 0.632 4.021

Phenolics or phenolic acids are intermediates in phenylpropanoid metabolism, and they play many important roles in plant development during seed germination. The results in Table 9 clearly showed that the increase in total phenolics of seedlings in response to seed treatment with consortium of Trichocherma isolates corresponds to enhance seedling vigour, which may contribute to improved lignification and antioxidant response.

EXAMPLE 10

Induction of induced systemic resistance in plants inoculated with NRRL 30595, NRRL 30596, NRRL 30597 and their consortium

Table 10 shows the induction of systemic resistance in plants in response to inoculation with NRRL 30595, NRRL 30596, NRRL 30597 and its consortium. Treated plants demonstrated higher induced systemic resistance than the untreated control.

Table 10 Influence of individual isolates of Trichoderma and its consortium on biochemical activity of sunflower in responst to infection of Alternaria blight caused by Alteranria helianthi

Treatment Phenylalanine Polyhenol Peroxidase Total Protein Catalase ammonia lyase oxidase activity in phenol content activity

(PAL) activity activity in units (mg/g (mg/g (units/mg

(nmol trans units* (Δ A «₀ plant of protein) cinnamic acid (Δ A ₄₉₅ mm^'1 g^"1 tissue) plant min^"1 g^"1 fresh min^"1 g^'1 fresh tissue) weight fresh weight) weight)

NRRL 30595 146 18.8 113.8 . 37.5 28.0 89.5

NRRL 30596 129 21.6 113.4 34.9 28.6 95.4

NRRL 30597 139 20.5 114.3 33.1 24.9 84.0

Consortium 184 27.5 127.6 47.8 37.9 123.9

Control 126 14.8 87.8 27.3 21.8 78.9

* 1 unit = 0.001 Absorbance

The result in Table 10 showed that Trichoderma spp. which is avirulent plant symbionts elicits the increases in the activities of phenylalanine ammonia-lyase (PAL), polyphenol oxidase, peroxidase, total protein, catalse and total phenol which ultimately led to induce systemic acquired resistance against chickpea wilt complex and play an important role in systemic acquired resistance in sunflower plant against foliar patghogen.

EXAMPLE 11

Shelflife of Trichoderma isolates NRRL 30595, NRRL 30596, and NRRL 30597 individually and in a consortium, on powdered sorghum grain as carrier Determination of the survival of 3 Trichoderma isolates NRRL 30595, NRRL 30596, and NRRL 30597 individually and in a consortium, on powdered sorghum grain as carrier, over the period of 12 months, at ambient temperature (28⁰C) was accomplished according to following method. Dried seeds of sorghum was crushed to form a powder and passed through 3 mm wire mesh to give a fine powder of sorghum to be used as carrier to grow the 3 Trichoderma isolates NRRL 30595, NRRL 30596, and NRRL 30597 individually and in a consortium. The 3 Trichoderma isolates NRRL 30595, NRRL 30596, and NRRL 30597 were grown individually in liquid growth medium PDB. Cultures were grown in 2- liters flasks containing 1.5 litres of PDB medium and incubated for 7 days at 28⁰C on a New Brunswick Scientific, USA, Innova Model 4230 refrigerated incubator shaker at 120 rpm. After 7 days of growth 300 ml of the culture was added to 1 kg of sterile powdered sorghum grain containing autoclavable plastic bag, which yielded approximately 30% moisture of the product. Consortium of the 3 Trichoderma isolates NRRL 30595, NRRL 30596, and NRRL 30597 was prepared by mixing the 3 cultures of approximately Log 8.0 cfu/ml, in the ratio of 1 : 1 : 1. Incubating the sealed bags for 2 days at 3O⁰C did curing of the bioinoculant preparation. After curing, the sealed bags were stored at 28⁰C and aliquots were periodically removed for viability measurements (Table 11). Viability of the product was determined by standard serial dilution method on TSM plates.

Table 11 Shelflife of Trichoderma isolates NRRL 30595, NRRL 30596, and NRRL 30597 individually and in a consortium, on powdered sorghum grain as carrier

Log 10 cfu/g in powdered sorj *hum grain months after

0 4 8 12

NRRL 30595 8.5 8.2 7.4 5.8

NRRL 30596 8.2 8.1 7.3 5.8

NRRL 30597 8.0 8.4 7.6 5.9

Consortium 8.6 8.4 7.6 5.9

As shown in the Table 11, all the 3 Trichoderma isolates NRRL 30595, NRRL 30596, and NRRL 30597 individually and in a consortium demonstrated good survival rates, during long-term storage in powdered sorghum grain at 28⁰C. After twelve months of storage, approximately Log 6 cfu/g of powdered sorghum grain was present. These data indicate that powdered sorghum grain works as an excellent carrier material for the isolates to be later inoculated onto seeds, plants or soil, as no appreciable loss of cell viability was observed. EXAMPLE 12

Growth of Trichoderma isolates NRRL 30595, NRRL 30596, and NRRL 30597 individually and in a consortium, on sugarcane molasses followed by fermented powdered sorghum grain as carrier

In our endeavour to reduce the production cost of 3 Trichoderma isolates NRRL 30595, NRRL 30596, and NRRL 30597 was accomplished using sugarcane molasses easily available as cheap source of growth media. AU the 3 isolates grew well in 1/5 diluted solution of sugarcane molasses in comparison to 1/10 or undiluted solution of sugarcane molasses. Initial experiment was executed in 500-ml Erlenmeyer flask having variable volume i.e. 100, 150, 200 and 300 ml of 1/5 diluted solution (with water) of sugarcane molasses. Optimal growth was profound in the flask having 100 ml of 1/5 diluted solution of sugarcane molasses. In I Lt. capacity flask having 100, 450, 500 and 750 ml of 1/5 diluted solution of sugarcane molasses growth of the isolates was about was 2xl0⁷cfu/ml after 7 days at 28⁰C on a New Brunswick Scientific, USA, Innova Model 4230 refrigerated incubator shaker at 120 rpm. After 7 days of growth 300 ml of the culture was added to 1 kg of sterile fermented press mud containing autoclavable plastic bag, which yielded approximately 30% moisture of the product. Consortium of the 3 Trichoderma isolates NRRL 30595, NRRL 30596, and NRRL 30597 was prepared by mixing the 3 cultures of approximately Log 8.0 cfu/ml, in the ratio of 1 : 1 : 1. Incubating the sealed bags for 2 days at 3O⁰C did curing of the bioinoculant preparation. After curing, the sealed bags were stored at 28⁰C and aliquots were periodically removed for viability measurements (Table 12). Viability of the product was determined by standard serial dilution method on TSM plates.

Table 12 Shelflife of Trichoderma isolates NRRL 30595, NRRL 30596, and NRRL

30597 individually and in a consortium, on sugarcane molasses followed by powdered sorghum grain as carrier Log 10 cfu/g in press mud months after

0 4 8 12

NRRL 30595 8.4 8.0 7.1 6.6

NRRL 30596 8.7 8.2 7.2 6.8

NRRL 30597 8.5 8.0 7.2 6.5

Consortium 8.8 8.6 7.5 6.9

As shown in the Table 12, all the 3 Trichoderma isolates NRRL 30595, NRRL 30596, and NRRL 30597 individually grown in sugarcane molasses and in a consortium demonstrated good survival rates, during long-term storage in powdered sorghum grain at 28⁰C. After twelve months of storage, approximately Log 6-7 cfu/g of powdered sorghum grain was present. These data indicate that powdered sorghum grain works as an equally excellent carrier material Compared with Example 11 product) for the 3 Trichoderma isolates NRRL 30595, NRRL 30596, and NRRL 30597 pre-grown in sugarcane molasses, to be later inoculated onto seeds, plants or soil, as no appreciable loss of cell viability was observed.

EXAMPLE 13

Effect of different agri based substrates as carrier of consortium of NRRL 30595, NRRL 30596 and NRRL 30597 Trichoderma on growth and yield parameters of sunflower

In our endeavour to reduce the production cost of Trichoderma based inoculant the different agri based substrates were compared for suitable carrier for selected isolates of Trichoderma as described in Example 11. Thereafter their field efficacy was tested in term of overall growth and yield parameters of sunflower plant.

Table 13 Effect of different agri based substrates as carrier of consortium of selected isolates of Trichoderma on growth and yield parameters of sunflower Substrates Days taken to No. of No. of Dry weight Yield flower bud flowering grains per of 1000 (q ha^'1) formation (DAS) * * buds/plant head grains (g)

Press Mud 33 1 865.0 49.5 12.9

Compost 32 1 856.6 50.7 12.7

Maize cob 35 1 876.2 47.8 12.9

Maize meal 32 1 914.5 49.5 14.2

Rice husk 36 1 836.9 40.6 12.8

Saw dust 34 1 815.9 • 40.1 11.3

Slurry 32 1 910.9 51.6 14.5

Used tea 33 1 836.4 40.5 11.5 leaves

Wheat bran 32 1 902.9 50.2 12.0

Wheat bran- 34 1 812.3 38.6 11.1 saw dust

Control 39 1 706.4 38.8 10.2

As shown in the Table 13, consortium formulation of selected Trichoderma isolates on different agri based substrates are efficient to increae the plant growth along with increase in overall yield of sunflower plant in comparison to uninoculated control.

EXAMPLE 14

Growth of Trichoderma isolates NRRL 30595, NRRL 30596, and NRRL 30597 individually and in a consortium, on cow dung as carrier

In our endeavour to further reduce the production cost of 3 Trichoderma isolates NRRL 30595, NRRL 30596, and NRRL 30597 grown in sugarcane molasses as described in Example 12. Fresh cow dung was from Sahiwal cows after drying in sun for about 7 days in the form of about thin 10 mm dough. Dried sun dried dough was crushed to form a granular powder and passed through 3 mm wire mesh to give a fine powder of cow dung to be used as carrier to grow the 3 Trichoderma isolates NRRL 30595, NRRL 30596, and NRRL 30597 individually and in a consortium. All the 3 Trichoderma isolates NRRL 30595, NRRL 30596, and NRRL 30597 individually grown in sugarcane molasses and in a consortium as described in Example 11 for inoculating fine powder of cow dung to be used as carrier. Moisture level of 80% was found suitable for the growth of NRRL 30595, NRRL 30596, and NRRL 30597 in cow dung. However size of initial inoculum did not matters as after 10 days of incubation, viable count was in the range of about 1x10⁶ cfu/g and after 30 days of incubation viable count was 7x10⁸ cfu/gm although at 0 day initial count was about 3x10³. Results demonstrate potential of cow dung as a source of carrier to grow Trichoderma isolates (Table 14). Viability of the product was determined by standard serial dilution method on PDA plates.

Table 14 Shelflife of Trichoderma isolates NRRL 30595, NRRL 30596, and NRRL 30597 individually and in a consortium, on cowdung as carrier

Log 10 cfu/g in press mud months after

0 1 2 4 8 12

NRRL 3.4 8.1 7.9 7.5 7.0 6.3

30595

NRRL 3.4 8.6 8.4 8.1 7.1 6.4

30596

NRRL 3.2 8.3 8.3 8.1 6.9 6.3

30597

Consortium 3.3 8.8 8.7 8.4 7.3 6.5

As shown in the Table 14, all the 3 Trichoderma isolates NRRL 30595, NRRL 30596, and NRRL 30597 individually grown in sugarcane molasses and in a consortium demonstrated good survival rates, during long-term storage in powder of cow dung at 28⁰C. After twelve months of storage, approximately Log 6 cfu/g of fermented press mud was present. These data indicate that powder of cow dung works as an equally excellent carrier material Compared with Example 7 and 8 products) for the 3 Trichoderma isolates NRRL 30595, NRRL 30596, and NRRL 30597 pre-grown in sugarcane molasses, to be later inoculated onto seeds, plants or soil, as no appreciable loss of cell viability was observed.

EXAMPLE 15

Responses of chrysanthemum to NRRL 30595, NRRL 30596, and NRRL 30597 consortium prepared in cow dung.

Response of chrysanthemum to treatment of NRRL 30595, NRRL 30596, NRRL 30597 and their consortium are shown in Table 15. Treated plants demonstrated more yield and flowered earlier and produce significantly more flowers than the untreated control.

Table 15 Effect of individual isolates of Trichoderma and its consortium prepared on cow dung on chrysanthemum

Treatments Plant ht. No. of No. of No. of Fresh wt.

(cm) branches/plant sprouts/plant flowers of plant

/plant (g)

NRRL 30595 57.6 6.5 47.3 53.5 521.9

NRRL 30596 55.7. 6.4 44.8 50.2 503.2

NRRL 30597 58.4 7.1 49.7 59.4 543.6

Consortium 63.3 8.7 58.4 75.8 678.3

Control 50.2 6.3 34.8 45.5 435.7 As shown in Table 15 the application of consortium showed increased, number of branches/plant, no of spouts/plant and yield/plant compared to either individual isolates of Trichoderma or an uninoculated control.

EXAMPLE 16

Responses of soybean to NRRL 30595, NRRL 30596, and NRRL 30597 consortium prepared in powdered sorghum grain

Response of soybean to treatment with NRRL 30595, NRRL 30596, NRRL 30597 and their consortium are shown in Table 16. Treated plants demonstrated more yield than the untreated control.

Table 16 Effect of individual isolates of Trichoderma and its consortium prepared on powdered sorghum grain on soybean .

Treatments Germination Plant Height No. of Test Grain

(%) (cm) Pods/Plant wt. of yield

100 (kg/ha) seed

NRRL 30595 87.9 80.5 155.9 8.2 1078.5

NRRL 30596 90.4 83.4 165.4 8.5 1145.6

NRRL 30597 84.3 79.9 149.4 7.9 1021.6

Consortium 92.5 84.3 187.7 10.8 1276.0

Control 78.6 74.0 137.4 7.4 864.3

Hence, the results in Table 16 showed that consortium has the potential to significantly stimulate the plant growth which ultimately increases the total yield of soybean plant in comparison to un-inoculated control. EXAMPLE 17

Effect of NRRL 30595, NRRL 30596, and NRRL 30597 individual and consortium culture filtrates on the growth and development of pathogenic fungi The effect of culture filtrates on the growth and development of pathogenic fungi was studied as follows. The result in Table 17 shows the effect of 10% concentration of secondary metabolites on % inhibition radial growth of economically important soil borne pathogens. 60.9 to 81.5 % reductions in radial growth of pathogens were recorded.

Table 17 Effect of cell free culture filtrates of selected isolates of Trichoderma spp. on radial growth of phytopathogens.

Phytopathogens Concentration of metabolites (10%) inhibition of growth

(%) NRRL 30595 NRRL 30596 NRRL 30597

Sclerotium rolfsii 71.8 70.5 74.3

Fusarium 69.5 64.3 60.9 oxysporium

Rhizoctonia solani 70.3 73.9 62.1

Sclerotinia 75.5 80.8 70.3 scleortiorum

Pythium 72.8 71.9 72.8 aphanidermatum

Phytophthora 81.5 65.3 73.4 nicotianae • Data recorded after 7 day of inoculation Results as shown in Table 17 demonstrate the strong antifungal effect of secondary metabolites of selectd isolates of Trichoderma against the soil borne pathogens under in vitro test.

EXAMPLE 18

Comparison of NRRL 30595, NRRL 30596, and NRRL 30597 consortium and chemical fungicide inhibition of chickpea wilt disease, Collor rot of betelvine, Fusarium corm rot and yellows of gladiolus, Fusarium wilt of chrysanthemum and Charcoal rot of soybean Using the same protocol as described in Example 3 the effect of the consortium was compared to the commercially available fungicide namely Carbendazim. Results are shown in Table 18.

Table 18 Comparitive evalution of consortium with commercial available fungicides to control the diseases caused by fungal pathogens.

Mortality (%) Disease Control (%)

Chickpea wilt Complex

Consortium 12.5 73.6 Carbendazim (50% a.i) 19.7 58.5 Control 47.5

Collor rot of betelvine

Consortium 18.3 75.1 Carbendazim (50% a.i) 34.8 52.5 Control 73.4

Fusarium corm rot and yellows of gladiolus

Consortium 6.9 81.5 Carbendazim (50% a.i) 14.1 62.1 Control 37.3

Fusarium wilt of chrysanthemum

Consortium 3.8 84.4

Carbendazim (50% a.i) 10.7 56.3

Control 24.5

Charcoal rot of soybean

Consortium 4^5 7O

Carbendazim (50% a.i) 11.6 45.5

Control 21.3

Results in Table 18 showed that the application of consortium was more efficient and has pronounce effect to control the disease in comparison to selcted commercial fungicide selected as well as un-inoculated control.

It is understood that the foregoing detailed description is given merely by way of illustration and that modification and variations may be made therein without departing from the spirit and scope of the invention.

Advantages:

The main advantages of the present invention are:

1. The present synergistic composition is useful to control phytopathogenic fungi and also promote the plant growth. 2. The present composition has the ability to tolerate abiotic stresses and also stimulates phenol contents in plants.

3. The present composition has the ability to induce systemic resistance in plants.

4. The present composition colonizes plant roots and has the ability of long shelf life.

5. The present composition survives all the seasons of the plant.

Claims

WE CLAIM:

1. A synergistic composition useful as bioinoculant, wherein the said composition comprising at least one fungal isolate of Trichoderma harzianum with an accession number NRRL 30595, NRRL 30596, and NRRL 30597 and optionally carrier.

2. A synergistic composition as claimed in claim 1, wherein the Trichoderma harzianum with accession number NRRL 30595 have the following characteristics: b) Morphological Character

(ix) Colour surface Dark: green (x) Colony mycelium: Compact

(xi) Phialide disposition: Whorl of 3

(xii) Phialide size (μm): 4.5 x 2.9

(xiii) Conidia shape: Subglobose

(xiv) Conidia size (μm): 1.9 x 1.5 (xv) Conidia colour: Green

(xvi) Chlamydospores: Abundant c) Biochemical character

( i ) Mycelial dry weight (mg)at Temperature ( °C)40-50: 140.6 to 48.6 ( ii ) Mycelial dry weight (mg) in NaCl cone. (%)0.0 to 10.00: 205 to 351 ( iii ) Mycelial dry weight (mg) in H⁺ ion conc.at pH 3 to 11: 107 to 305

( iv) Mycelial dry weight (mg) in poly Ethyl Glycol (PEG) 10% to 50: 247 to 78

3. A synergistic composition as claimed in claim 1, wherein the Trichoderma harzianum with accession number NRRL 30596 have the following characteristics: a) Morphological Character

(x) Colour surface Dark: Glaucous to dark green (xi) Colony mycelium: Compact (xii) Phialide disposition: Whorl of 2-3 (xiii) Phialide size (μm): 5.7 x 3.5 (xiv) Phialide shape : Lageniform (xv) Conidia shape: Subglobose (xvi) Conidia size (μm): 2.9 x 1.8 (xvii) Conidia colour: Light green (xviii) Chlamydospores: Fairly abundant d) Biochemical character

( i ) Mycelial dry weight (mg)at Temperature ( °C)40-50: 120.9 to 39.6 ( i i ) Mycelial dry weight (mg) in NaCl cone. (%)0.0 to 10.00: 180 to 428 ( i i i ) Mycelial dry weight (mg) in H⁺ ion conc.at pH 3 to 11 : 69 to 210 ( i v ) Mycelial dry weight (mg) in poly Ethyl Glycol (PEG) 10% to 50: 264 to

94

4. A composition as claimed in claim 1, wherein the the Trichoderma harzianum with accession number NRRL 30597 have the following characteristics: a) Morphological Character (i) Colour surface: Green

(ii) Colony mycelium: Slightly effuse (iii) Phialide disposition: Whorl of 3 (iv) Phialide size (μm): 3.9 x 2.5 (v) Phialide shape : Ampulliform (vi) Conidia shape: Ovoid

(vi) Conidia size (μm): 3.2 x 2.5 (vii) Conidia colour: green (viii) Chlamydospores: abundant b) Biochemical character (i) Mycelial dry weight (mg) at Temperature ( °C)40-50: 151.7 to 50.5

(ii) Mycelial dry weight (mg) in NaCl cone. (%)0.0 to 10.00: 300 to 338 (iii) Mycelial dry weight (mg) in H⁺ ion conc.at pH 3 to 11 : 128 to 275 (iv) Mycelial dry weight (mg) in poly Ethyl Glycol (PEG) 10% to 50: 229 to 74

5. A synergistic composition as claimed in claim 1, wherein said carriers are selected from a group consisting of powdered sorghum grain, maize meal, maize cob, compost, rice husk, rice bran, wheat bran, cow dung, talc, a mixture of fermented sugar factory sulphitation press mud and distillery spent wash, and sugar factory carbonation press mud.

6. A synergistic composition as claimed in claim 1, wherein the said composition is prepared by mixing the fungal isolates of Trichoderma harzianum with an accession number NRRL 30595, NRRL 30596, and NRRL 30597 in equal proportion.

7. A synergistic composition as claimed in claim 6, wherein the concentration of said fungal isolates used is in therange of 7-9 cfu/g of carrier and preferably 7-8 cfu/g of carrier.

8. A composition as claimed in claim 1, wherein concentration of each fungal isolates is 6-8 cfu/g of carrier and preferably 7-8 cfu/g of carrier.

9. A synergistic composition as claimed in claim 1, wherein the said composition has the ability to control phytopathogenic fungi.

10. A synergistic composition as claimed in claim 1, wherein the said composition has the ability to promote plant growth.

11. A synergistic composition as claimed in claim 1 , wherein the said composition has the ability to tolerate abiotic stresses.

12. A synergistic composition as claimed in claim 1, wherein the said composition has the ability to stimulate phenol contents in plants.

13. A synergistic composition as claimed in claim 1, wherein the said composition has the ability to induce systemic resistance in plants.

14. A synergistic composition as claimed in claim 1, wherein the said composition is efficient to colonize plant roots.

15. A synergistic composition as claimed in claim 1, wherein the said composition has the ability of long shelf life.