CLIAMS:We Claim:

1. A formulation for biological control of soil borne pathogens, and pest, the formulation comprising any one or combination of Trichoderma viride and Bacillus subtilis strains mixed with a water soluble carrier.

2. The formulation as claimed in claim 1, wherein the water soluble carrier is dextrose.

3. The formulation as claimed in claim 1, wherein the Trichoderma viride strain includes dried spores thereof.

4. The formulation as claimed in claim 1, wherein the Bacillus subtilis strain includes lyophilized spores thereof.

5. A method for preparing formulation of Trichoderma viride for biological control of soil borne pathogens, and pest as claimed in claim 1, the method comprising:
preparing mother culture of Trichoderma viride strain for conidial mass production in liquid medium containing dextrose 10g, yeast extract 5g, K2HPO4 2g and MgSO4 1g per liter of water.
washing and sterilizing of substrate sorghum grains in 200 gauge polypropylene bags;
inoculating 72 hrs old mycelium broth of Trichoderma viride in polypropylene bags filled with sterilize sorghum grains followed by incubation for at least 5 days at room temperature;
separating spores from sorghum grain base substrate using spore harvesting liquid;
purifying spores of Trichoderma viride by centrifugation; and
drying the spores of Trichoderma viride at 38±10C for 48 hrs in hot air oven, followed by preparation of fine powder of spores for formulation with water soluble carrier.

6. A method for preparing formulation of Bacillus subtilis for biological control of soil borne pathogens, and pest as claimed in claim 1, the method comprising:
preparing mother culture of Bacillus subtilis in liquid medium containing nutrient broth;
inducing nutrient stress on Bacillus subtilis in a pilot scale fermenter for maximum spore formation followed by harvesting of cells/spore by centrifugation; and
freeze drying of Bacillus subtilis cells followed by viability testing for formulation development with water soluble carrier.
,TagSPECI:FIELD OF THE INVENTION

The present disclosure generally relates to formulation for biological control of soil borne pathogens and more particularly, to formulation for biological control of soil borne plant pathogens, pest and for promotion of plant growth and method for mass multiplication of bio control fungi Trichoderma viride and bacterium Bacillus subtilis.

BACKGROUND OF THE INVENTION

Soil borne plant pathogen significantly reduces yield and quality in various crops. Many diseases caused by soil borne plant pathogens are difficult to predict, detect and diagnose. To control soil borne plant pathogen is challenging because they often survive in soil for many years. For the management of soil borne plant pathogens synthetic chemicals are frequently use, which gives good control. But recently due to wide application of agrochemicals a serious impact was observed on environment of soil and water. Further, resistance is also developed in the pest and the pathogen is another serious problem.

Hence, biological control of plant pathogens is a crucial activity for eco friendly agriculture production. There are many synthetic agrochemicals have been used, but due to large amount of application of agrochemicals there are many adverse environmental and health problem have occurred. Many chemical pesticides are toxic for humans, wildlife and for many other species. In addition, some of these pesticides have been found in ground water as well.

There are number of carriers that are used for formulation development of bio control agents. Conventional carries for bio agents are talcum and liquids. However, there are some lacunas with exiting formulations, such as these formulations are not completely soluble in water, high pH range, less shelf life of product, problem of stability and problem of contamination. Further, talc based formulations shows scorching effect on grapes and the like. The major drawback of existing technology of bio pesticide production is that during storage and marketing, population of bio agents decreases rapidly and formulation fails to give expected results. It is aware that when such bio pesticide is applied in field, satisfactory management of target disease does not occur.

Accordingly, there exists a need to develop a formulation for biological control of soil borne pathogens which overcomes abovementioned drawbacks.

OBJECTS OF THE INVENTION

Object of the present invention is to provide a formulation for biological control of soil borne pathogens, which is suitable for soil drenching and foliar application.

Another object of the present invention is to provide a formulation for biological control of soil borne pathogens, which has long shelf life.

Yet object of the present invention is to provide a formulation for biological control of soil borne pathogens, which has water soluble carrier.

Yet another object of the present invention is to provide a formulation for biological control of soil borne pathogens, which is stable and effective.

Further object of the present invention is to provide a formulation for biological control of soil borne pathogens, which promotes plant growth.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides a formulation for biological control of soil borne pathogens, and pest. The formulation comprises any one or combination of Trichoderma viride and Bacillus subtilis strains mixed with a water soluble carrier (Dried spore of T.viride and lyophilized cells of B.subtilis 0.50g and 2g (Minimum) per Kg of dextrose).
Typically, the water soluble carrier is dextrose.

Typically, the Trichoderma viride strain includes dried spores thereof.

Typically, the Bacillus subtilis strain includes lyophilized spores thereof.

DETAILED DESCRIPTION OF THE INVENTION

The foregoing objects of the present invention are accomplished and the problems and shortcomings associated with the prior art, techniques and approaches are overcome by the present invention as described below in the preferred embodiment.

The present invention provides a formulation for biological control of soil borne plant pathogens a method of preparation thereof. Specifically, the formulation uses a completely water soluble carrier, for example dextrose, which makes formulation most suitable for soil drenching and foliar application. Further, the formulation for biological control of soil borne pathogens has water soluble carrier. Also, the formulation for biological control of soil borne pathogens is stable and effective. Furthermore, the formulation for biological control of soil borne pathogens promotes plant growth.

In one aspect, the present invention provides a formulation for biological control of soil borne plant pathogens and a method of preparation thereof. The formulation includes any one or combination of Trichoderma viride and Bacillus subtilis strains mixed with a water soluble carrier. (Dried spore of T.viride and lyophilized cells of B.subtilis 0.50g and 2g (Minimum) per Kg of dextrose).
In preferred embodiment, the water soluble carrier is dextrose. However, it may be evident to those skilled in the art to use other water soluble carrier known in the art.

Dextrose used herein as a carrier makes the formulation most suitable for soil drenching and foliar application. Dextrose acts as a source of carbohydrate for microbes when goes into the field.

Specifically, the formulation comprises dried spores of Trichoderma viride strain. Further, the formulation comprises lyophilized spores of the Bacillus subtilis strain.

The Trichoderma viride and Bacillus subtilis secretes many lytic enzymes which control soil pathogens and produce plant growth hormones, solubilize minerals, increases systemic resistance in plant and improve overall quality and yield.

Specifically, the formulations uses is dried viable spore of Trichoderma viride strain and lyophilized spores of Bacillus subtilis strain which makes formulation stable and effective and both the formulations promotes plant growth in a realistic manner. Because of drying of spore of Trichoderma viride at ambient temperature, spores become resistance to hostile environmental condition such as heat, dryness, pH and nutrient condition. Both formulations are suitable for seed treatment, drenching and foliar spray as well as for soil application for the control of Fusarium, Rhizoctonia, Macrophomina spp, Pythium spp and pest like Heliothis that affecting the plants.

In another aspect, the present invention provides a method for preparing the formulation of Trichoderma viride for biological control of soil borne pathogens, and pest as described above. The method includes preparing mother culture of Trichoderma viride strain for conidial mass production in liquid medium containing dextrose 10g, yeast extract 5g, K2HPO4 2g and MgSO4 1g per liter of water. Further, the method includes washing and sterilizing of substrate sorghum grains in 200 gauge polypropylene bags. Furthermore, the method includes inoculating 72 hrs old mycelium broth of Trichoderma viride in polypropylene bags filled with sterilized sorghum grains followed by incubation for at least 5 days at room temperature. The 72 hrs old mycelium broth of Trichoderma viride used as inoculums which ultimately decrease the incubation period (from 10 days to 5 days) for maximum spore yield.

Thereafter, the method includes separating spores from sorghum grain base substrate using spore harvesting liquid and purifying spores of Trichoderma viride by centrifugation. Finally, the method includes drying the spores of Trichoderma Viride at 38±10C for 48 hrs in hot air oven, followed by preparation of fine powder of spores for formulation with water soluble carrier.

In an embodiment, the spore harvesting liquid includes sterilized 0.1% between 80 solution. The water soluble carrier used herein is dextrose.

In yet another aspect, the present invention provides a method for preparing formulation of Bacillus subtilis for biological control of soil borne pathogens, as described above. The method includes preparing mother culture of Bacillus subtilis strain in liquid medium containing nutrient broth. Further, the method includes, inducing nutrient stress on Bacillus subtilis in a fermenter for maximum spore formation followed by harvesting of cells/spore by centrifugation. Furthermore, the method includes freeze drying of Bacillus subtilis cells followed by viability testing for formulation with water soluble carrier.

The dextrose sugar used as a carrier in present invention is best substitute for conventional carriers of bio agents. Antagonistic microbes survive very well in dextrose formulation upto 1 year without losing its bio efficacy and dextrose is an easily available renewable source. Such types of bio pesticides are better alternative for chemical pesticides as well as conventional bio pesticides under eco friendly and sustainable agriculture.

Following examples provides detailed process of preparation of formulations containing Trichoderma viride and Bacillus subtilis in suitable water soluble carrier such as dextrose.

Surface sterilization of substrate:

Surface sterilization of substrate, for example, sorghum grains was performed for the production of contamination free conidia. For surface sterilization, about 5 Kg of sorghum grain were filled in green color shade net bags. The sorghum grains were washed by manual squeezing under running tap water. About ten liter tap water was taken and soaked all such bags for 30 minutes. The bags were removed out of the tap water and hanged so that all water dripped out. The left over water was discarded.

Thereafter, about 10 liter of 10% chlorine (with 6% active ingredient) in distilled water was prepared using a wide-mouth air-tight drum. The washed sorghum grains contained in shed net bags were dipped in the chlorine solution for 30 minute followed by three successive washing with sterilized water. All the bags were hanged so that all water was dripped out. Thereafter, 10 liters of sterilized water was taken in another drum and all bags were dipped for another 30 minute. The bags were hanged till all the water was dripped. These drip-free and treated grains (about 400gm) were filled in 200 gauge polypropylene bags (21x14cm) and autoclaved at 1210C for 20 minute.

Strain revival and Inoculums preparation:
Standard strain of Trichoderma viride was used for experimental studies which was received from NBAII (National Bureau of Agriculturally Important Insect) formally Project Directorate of Biological Control (PDBC) Bangalore. The cultures were maintained on Potato Dextrose Agar slants (PDA) at 40 C. Fungal spores of Trichoderma viride from reference culture slant was inoculated in potato dextrose broth and incubated at 28±20Cfor 72 hrs. After 72 hrs of incubation, this mycelia broth was used for inoculation in sorghum grains contained in poly bags, 10 ml for each bag. Similarly, standard strain of Bacillus subtilis was used for experimental studies was received from ICRISAT (International Crops Research Institute for the Semi Arid Tropics) Hyderabad. The culture was maintained on Luria Bertani (LB) slant at 40 C. Bacterial cells of B.subtilis were inoculated in LB broth and incubated under shaking condition at 120 rpm for 48 hrs at room temperature.

Inoculation, Incubation and Harvesting of conidia:

After cooling, 10 ml of 72 hrs old mycelia broth of Trichoderma viride was used as inoculums for each polypropylene bag separately, under laminar air flow chamber. All bags were sealed manually and incubated at 28±20C for 5 days for maximum conidiospore formation. Followed by maximum sporulation, each bag was cut at corner and around 400 ml previously sterilized spore harvesting liquid (0.1% between 80 solution) was added in each bag. The bags were squeezed manually, so the maximum spores were separated from grains. The spore liquid was then filtered through sterilized muslin cloth and centrifuged using tubular tower centrifuge at 15000 RPM. The spores were separated from the bowl of centrifuge, collected on plastic sheet that was spread on stainless still tray and all such trays were kept for drying in hot air oven at 38±10C for 48 hrs. After drying, fine powder of spores was made manually under laminar air flow unit and stored in air tight sterilized plastic bottle. CFU (Colony forming unit) of dried conidia was determined by serial dilution technique.

Submerge fermentation of Bacillus Subtilis

Mass multiplication of Bacillus subtilis was made in shake flasks followed by fermentation using a commercial nutrient broth medium. The fermentation was carried out in 100 lit capacity pilot scale fermenter containing nutrient broth medium which was inoculated by 48 hours old vegetative inoculums. The vegetative inoculums were developed from standard culture slants. During fermentation nutrient stress was given on Bacillus cells for maximum spore formation. Followed by fermentation, broth was centrifuged at 15000 RPM. The spores were harvested and collected in sterilized glass bottles. Followed by harvesting, spores were freeze dried and stored in air tight glass bottles. Lyophilized Bacillus spores were used for dextrose formulation development, its CFU (colony forming unit) was taken at each months of interval upto 12 months for determination of shelf life.

Dextrose formulation development of Trichoderma viride and Bacillus subtilis

Before preparing the formulation, dextrose was tested for contamination, pH and moisture then used for formulation development. Zero contamination was recorded in dextrose having neutral pH range, and moisture is not more that 1%. The dextrose base formulation of Trichoderma viride was prepared using dried spore of Trichoderma viride in homemade mixer. Its CFU (colony forming unit) was taken and multiple samples were kept for shelf life upto 1 year. Similarly for dextrose base formulation of Bacillus subtilis was prepared using freeze dried cells which was previously stored in air tight glass bottle. Same homemade mixer model was used for formulation of Bacillus subtilis, its CFU was taken and multiple samples were kept for shelf life upto 12 months. For dextrose base formulation of Trichoderma viride and Bacillus subtilis0.50g dried spore and 2g lyophilized cells per kg of dextrose were used, respectively.

Pot Trial:

Bio efficacy of Dextrose base Trichoderma viride, against soil borne pathogen of Brinjal (Fusarium oxysporum ):

Experimental details:
In the present invention dextrose based formulation of Trichoderma viride was used for bio efficacy testing against Fusarium oxysporum in comparison with same strength of talc base formulation of Trichoderma viride in pot trial. The pot trial was conducted in growing season of 2012. Performance of dextrose based Trichoderma viride was compared with chemical fungicide (Thiram) and talc based bio fungicide of Trichoderma viride against above said plant pathogens. The pot soil was infested with 1x107/ml fungal spore suspension of Fusarium spp. prior to start of the seed sowing; all seeds were moistened with water, so that all formulations were adhered to seeds. All the seeds were treated with following 7 types of treatments.
T1- Control (Untreated)
T2- Seeds treated with talc base formulation of T.viride (10g/kg of seeds.)
T3-Seeds treated with dextrose base formulation of T.viride (2.5 g/kg of seeds.)
T4-Seeds treated with dextrose base formulation of T.viride (5g/kg of seeds.)
T5-Seeds treated with dextrose base formulation of T.viride (7g/kg of seeds.)
T6 -Seeds treated with dextrose base formulation of T.viride (10g/kg of seeds.)
T7- Seeds treated with chemical fungicide (Thiram 2g/Kg)

Germination percentage, Root and shoot length, percent disease control and Phytotoxic effect were recorded.
Observation &Results:
Effect on Growth Parameters:
The perusal of data (Table 1) reveled that all treatments exhibited significantly higher seed germination, root length and shoot length over untreated control. However, seed treatment with dextrose base T.viride (10g/kg) of seed (T6) recorded significantly the highest seed germination (92.66%), root length (7.97cm) and shoot length (14.90 cm). This was followed by the seed treatment (T7) with fungicide Thiram at 2g/Kg of seed (89.33%, 6.97 cm and 13.95 cm. Seed treatment (T2) with talc base T.viride at 10g/Kg seed (82.33%, 5.66cm, and 11.80cm) with seed germination, root length and shoot length respectively. Rest of the treatments also recorded maximum seed germination (range 70.33 to 76.33), root length (range: 4.22 to 5.44), shoot length range: 8.43 to 9.88cm) as against significantly least seed germination. (56.33%, root length (3.59cm), shoot length (6.11cm) in untreated control. Following Table 1 shows the effect of dextrose base T.viride seed treatments on seed germination, root length and shoot length in Brinjal.
Table 1
Tr. No. Treatments Seeds germination (%) Root length (cm) Shoot length (cm)
T1 Control (Untreated) 56.33 3.59 6.11
T2 Seed treatment with talc base T.viride (10g/kg of seed) 82.33 5.66 11.80
T3 Seed treatment with Dextrose base T.viride (2.5 g/kg of seed) 70.33 4.22 8.43
T4 Seed treatment with dextrose base T.viride (5 g/kg of seed) 71.66 4.83 8.74
T5 Seed treatment with dextrose base T.viride (7g/kg of seed) 76.33 5.44 9.88
T6 Seed treatment with dextrose base T.viride (10g/kg of seed) 92.66 7.97 14.90
T7 Seed treatment with Thiram (2g/kg of seed) 89.33 6.97 13.95

Wilt disease incidence and disease control:

The data (Table 2) reveled that all the treatments recorded significantly minimum wilt incidence and maximum disease control as compare to untreated control. However, the seed treatment with dextrose base T.viride at 10g/Kg of seed (T6) recorded significantly least wilt incidence (16.87%), and highest reduction in wilt incidence (72.71%). This was followed by seed treatment (T7) with Thiram at 2g/Kg of seed (19.29%) and reduction in wilt incidence (68.80%). Seed treatment with Talc base T.viride (T2) 10g/Kg of seed (21.30%) and 65.55% wilt incidence. Rest of the treatment also recorded comparatively minimum wilt incidence. Further following Table 2 shows the effect of dextrose base T.viride seed treatments on wilt incidence and disease control in Brinjal.
Table 2

Tr. No. Treatments Disease incidence (%) Disease control (%)
T1 Control (Untreated) 61.83 -
T2 Seed treatment with talc base T.viride (10g/kg of seed) 21.30 65.55
T3 Seed treatment with Dextrose base T.viride (2.5 g/kg of seed) 33.74 45.42
T4 Seed treatment with dextrose base T.viride (5 g/kg of seed) 33.40 46.35
T5 Seed treatment with dextrose base T.viride (7g/kg of seed) 31.40 49.33
T6 Seed treatment with dextrose base T.viride (10g/kg of seed) 16.87 72.71
T7 Seed treatment with Thiram (2g/kg of seed) 19.29 68.80

Phytotoxicity:

The results (Table 3) reveled that T.viride (Talc and Dextrose base) seed treatment at various concentration did not express any phytotoxic effect or abnormalities on Brinjal crop which indicate that T.viride seed treatment even at higher dose at 10g/Kg is safe to use as Bio fungicide for the management of wilt disease in Brinjal. Following Table 3 shows the phytotoxic effect of dextrose base T.viride seed treatment in Brinjal
Table 3

Tr. No. Treatments Phytotoxicity (%)
Epinasty /Hyponasty /Necrosis /Chlorosis

T1
Control (Untreated) 1 3 5 7 10
Nil Nil Nil Nil Nil
T2 Seed treatment with talc base T.viride (10g/kg of seed) Nil Nil Nil Nil Nil
T3 Seed treatment with Dextrose base T.viride
(2.5 g/kg of seed) Nil Nil Nil Nil Nil
T4 Seed treatment with dextrose base T.viride (5 g/kg of seed) Nil Nil Nil Nil Nil
T5 Seed treatment with dextrose base T.viride (7g/kg of seed) Nil Nil Nil Nil Nil
T6 Seed treatment with dextrose base T.viride (10g/kg of seed) Nil Nil Nil Nil Nil
T7 Seed treatment with Thiram (2g/kg of seed) Nil Nil Nil Nil Nil

Conclusion:
Base on results it could be concluded that, seed treatment with dextrose base T.viride at 10g/Kg of seed (T6) can effectively managed the wilt disease caused by Fusarium oxysporum in Brinjal. The second and third best treatment found were seed treatment (T7) with Thiram at 2g/Kg and seed treatment (T2) with talc based T.viride at 10g/Kg of seed.

Bio efficacy of dextrose base B.subtilis against soil borne pathogens of Brinjal (Fusarium oxysporum):

Experimental details:

In the present attempt dextrose based formulation of Bacillus subtilis was used for bio efficacy testing against Fusarium oxysporum in comparison with same strength of talc base formulation of Bacillus subtilis in pot trial. The pot trial was conducted in growing season of 2012. Performance of dextrose based Bacillus subtilis was compared with chemical fungicide (Thiram) and talc based bio fungicide of Bacillus subtilis against above said plant pathogens. The pot soil was infested with 1x107/ml fungal spore suspension of Fusarium spp. prior to start of the seed sowing; all seeds were moistened with water, so that all formulations were adhered to seeds. All the seeds were treated with following 7 types of treatments.
T1- Control (Untreated)
T2- Seeds treated with talc base formulation of B.subtilis (10g/kg of seeds.)
T3-Seeds treated with dextrose base formulation of B.subtilis (2.5 g/kg of seeds.)
T4-Seeds treated with dextrose base formulation of B.subtilis (5g/kg of seeds.)
T5-Seeds treated with dextrose base formulation of B.subtilis (7g/kg of seeds.)
T6 -Seeds treated with dextrose base formulation of B.subtilis (10g/kg of seeds.)
T7- Seeds treated with chemical fungicide (Thiram 2g/Kg)

Germination percentage, Root and shoot length, percent disease control and Phytotoxic effect were calculated.

Observation &Results:

Effect on Growth Parameters:
The perusal of data (Table 4) reveled that all treatments exhibited significantly higher seed germination, root length and shoot length over untreated control. However, seed treatment with dextrose base B.subtilis (10g/kg) of seed (T6) recorded significantly highest seed germination (94.10%), root length (8.47cm) and shoot length (15.23 cm). This was followed by the seed treatment (T7) with fungicide Thiram at 2g/Kg of seed (91.09%,7.30 cm and 13.07 cm. Seed treatment (T2) with talc base B.subtilis at 10g/Kg seed (83.92%, 6.89cm, and 12.27cm with seed germination, root length and shoot length respectively. Rest of the treatments also recorded maximum seed germination (range 69.44 to 73.35), root length (range: 4.89 to 5.78), shoot length range: 8.10 to 9.31cm) as against significantly least seed germination. (50.81%, root length (4.20cm), shoot length (5.83cm) in untreated control. Following Table 4 shows the effect of dextrose base B.subtilis on seed germination, root length and shoot length in Brinjal
Tr. No. Treatments Seeds germination (%) Root length (cm) Shoot length (cm)
T1 Control (Untreated) 50.81 4.20 5.83
T2 Seed treatment with talc base B.subtilis (10g/kg of seed) 83.92 6.89 12.27
T3 Seed treatment with Dextrose base B.subtilis (2.5 g/kg of seed) 69.44 4.89 8.10
T4 Seed treatment with dextrose base B.subtilis (5 g/kg of seed) 72.29 5.13 8.90
T5 Seed treatment with dextrose base B.subtilis (7g/kg of seed) 73.35 5.78 9.31
T6 Seed treatment with dextrose base B.subtilis (10g/kg of seed) 94.10 8.47 15.23
T7 Seed treatment with Thiram (2g/kg of seed) 91.09 7.30 13.07
Table 4

Wilt disease incidence and disease control:

The data (Table 5) reveled that all the treatments recorded significantly minimum wilt incidence and maximum disease control as compare to untreated control. However the seed treatment with dextrose base B.subtilis at 10g/Kg of seed (T6) recorded significantly least wilt incidence (16.72%), and highest reduction in wilt incidence (73.41%). This was followed by seed treatment (T7) with Thiram at 2g/Kg of seed (18.54%) and reduction in wilt incidence (70.88%). Seed treatment with Talc base B.subtilis (T2) 10g/Kg of seed (18.81%) and 70.14% wilt incidence. Rest of the treatment also recorded comparatively minimum wilt incidence. Following Table 5 shows the effect of dextrose base B.subtilis seed treatment on wilt incidence and disease control in Brinjal.

Table 5
Tr. No. Treatments Disease incidence (%) Disease control (%)
T1 Control (Untreated) 62.96 -
T2 Seed treatment with talc base B.subtilis (10g/kg of seed) 18.81 70.14
T3 Seed treatment with Dextrose base B.subtilis (2.5 g/kg of seed) 36.01 42.80
T4 Seed treatment with dextrose base B.subtilis (5 g/kg of seed) 33.77 46.35
T5 Seed treatment with dextrose base B.subtilis (7g/kg of seed) 33.12 47.40
T6 Seed treatment with dextrose base B.subtilis (10g/kg of seed) 16.72 73.41
T7 Seed treatment with Thiram (2g/kg of seed) 18.54 70.80

Phytotoxicity:

The results (Table 6) reveled that B.subtilis (Talc and Dextrose base) seed treatment at various concentration did not express any phytotoxic effect or abnormalities on Brinjal crop which indicate that B.subtilis seed treatment even at higher dose at 10g/Kg is safe to use as Bio fungicide for the management of wilt disease in Brinjal. Following Table 6 shows the phytotoxic effect of dextrose base B.subtilis seed treatment in Brinjal.

Table 6

Tr. No. Treatments Phytotoxicity (%)
Epinasty /Hyponasty /Necrosis /Chlorosis

T1
Control (Untreated) 1 3 5 7 10
Nil Nil Nil Nil Nil
T2 Seed treatment with talc base B.subtilis (10g/kg of seed) Nil Nil Nil Nil Nil
T3 Seed treatment with Dextrose base B.subtilis (2.5 g/kg of seed) Nil Nil Nil Nil Nil
T4 Seed treatment with dextrose base B.subtilis (5 g/kg of seed) Nil Nil Nil Nil Nil
T5 Seed treatment with dextrose base B.subtilis (7g/kg of seed) Nil Nil Nil Nil Nil
T6 Seed treatment with dextrose base B.subtilis (10g/kg of seed) Nil Nil Nil Nil Nil
T7 Seed treatment with Thiram (2g/kg of seed) Nil Nil Nil Nil Nil

Conclusion: Base on results it could be concluded that seed treatment with dextrose base B.subtilis at 10g/Kg of seed (T6) can effectively managed the wilt disease of Fusarium oxysporum in Brinjal. The second and third best treatments found were seed treatment (T7) with Thiram at 2g/Kg and seed treatment (T2) with talc based B.subtilis at 10g/Kg of seed.

Shelf Life studies of dextrose base formulation of T.viride:

The experiment was conducted at three different locations of Maharashtra, using Dextrose base formulations of T.viride at three different temperatures 50C, 150C and 300C. The samples were analyzed at interval of three months for active ingredient content, physical, chemical and biological properties. Moisture content of formulation and pH was calculated using recommended method of BIS. Colony forming units of T.viride was estimated on potato dextrose agar medium by serial dilution technique.1gm of T.viride dextrose formulation was transfer in 9 ml sterilize distilled water and mixed thoroughly. Pipette out 1ml suspension from 1st dilution and serially diluted up to 10-9. Two replication of each dilution was maintained. Last three dilutions were selected and 0.1ml suspension from each dilution was transfer on Potato dextrose agar (PDA) containing plates, spread uniformly and all plates were incubated at 300C for 72 hrs. The number of colonies per gram of formulation was calculated. Similarly other microbial concomitant also recorded. Further following Table 7 shows the shelf life results of dextrose base T.viride.
Table 7
Parameters 0 Day analysis Analysis after 3 month Analysis after 6 month Analysis after 9 month Analysis after 12 month
Colour Off white Powder Off white Powder Off white Powder Off white Powder Off white Powder
Odour Odourless Odourless Odourless Odourless Odourless
Moisture Content % 1.510 1.525 1.552 1.577 1.594
pH 6.53 6.53 6.57 6.57 6.61
CFU/g
(2x 106 ) 2.72 2.68 2.63 2.56 2.10
Other contamination Nil Nil Nil Nil Nil

An analysis result shows that there is no significant change in the active ingredient content and other physic-chemical properties of dextrose base formulation of T.viride after 12 months of storage.

Shelf Life studies of dextrose base formulation of B.subtilis:

The experiment was conducted at 3 different locations of Maharashtra, using Dextrose base formulations of B.subtilis at 3 different temperatures 50C, 150C and 300C. The samples were analyzed at interval of three months for active ingredient content, physical, chemical and biological properties. Moisture content of formulation and pH was calculated using the method of BIS. Colony forming units of B.subtilis was estimated on nutrient agar medium by serial dilution technique. 1gm of B.subtilis dextrose formulation was transfer in 9ml sterilize distilled water and mixed thoroughly. Pipette out 1ml suspension from 1st dilution and serially diluted up to 10-9 . Two replication of each dilution was maintained. Last three dilutions were selected and 0.1ml suspension from each dilution was transfer on nutrient agar (NA) containing plates, spread uniformly and all plates were incubated at 300Cfor 48 hrs. The number of viable spore or cells per gram of formulation was calculated. Similarly other microbial concomitant also recorded. Further following Table 8 shows the shelf life results of dextrose base B.subtilis.
Table 8
Parameters 0 Day analysis Analysis after 3 month Analysis after 6 month Analysis after 9 month Analysis after 12 month
Colour Off white Powder Off white Powder Off white Powder Off white Powder Off white Powder
Odour Odourless Odourless Odourless Odourless Odourless
Moisture Content % 1.0047 1.0069 1.0086 1.0104 1.0138
pH 6.52 6.52 6.55 6.56 6.59
CFU/g
(2x 108 ) 2.853 2.850 2.845 2.841 2.836
Other contamination Nil Nil Nil Nil Nil

An analysis result shows that there is no significant change in the active ingredient content and other physic-chemical properties of dextrose base formulation of B.subtilis after 12 months of storage.

Advantages of the invention

The formulation of Trichoderma viride and Bacillus subtilis with water soluble carrier such as dextrose enhances the efficacy of as well as promotes the plant growth by producing various enzymes, siderophores, plant growth promoting compound. Further, the formulation includes dextrose as a carrier which improves the shelf life of products and act as source of carbohydrate for microbes when goes into the field which ultimately enhances seed germination. Also, the formulations are most suitable for drip as well as foliar application. Furthermore, the formulations are stable and effective and promote plant growth as well. Moreover dextrose is an easily available and renewable source.

The foregoing object of the invention is accomplished and the problems and shortcomings associated with prior art techniques and approaches are overcome by the present invention described in the present embodiment. Detailed descriptions of the preferred embodiment are provided herein; however, it is to be understood that the present invention may be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the present invention in virtually any appropriately detailed system, structure, or matter. The embodiments of the invention as described above and the methods disclosed herein will suggest further modification and alterations to those skilled in the art. Such further modifications and alterations may be made without departing from the spirit and scope of the invention.