WO2021130735A1 - Organic fungicide derived through microbial fermentation and efficient formulation - Google Patents

Abstract

The present invention provides an efficient organic fungicide composition, and a method to prepare the same. The fungicide composition is derived through microbial composition and active against pathogens. The composition comprises microbial extract, Tween 80 and Menthol. The organic fungicide of the present invention is effective at a minimum dose of 0.01 ml/ lt.

Description

Title of the Invention

Organic Fungicide derived through microbial fermentation and efficient formulation

Field of the Invention

[001] The present invention generally relates fungicide, more specifically it is related to organic fungicide which is derived through microbial fermentation.

Background of the invention

[002] Plant diseases cause major economic losses for farmers worldwide. The Food and

Agriculture Organization estimates that pests and diseases are responsible for about 25% of crop loss. Control of plant diseases is crucial in crop management. Downy mildew is a major pathogenic disease that is caused mostly by organisms that belong to either the Peronospora or Plasmopara genus parasitic organisms that are more closely related to algae.

Phytophthora and fusarium are other pathogenic fungus that causes severe damage on various crops .These pathogens cause severe damage on vegetable crops, grape vine, pepper etc. They can appear in different ways, depending on what kinds of plants it is infecting.

Most often, an infection of these group of fungus will also include a fuzzy, soft looking growth that can be white, grey, brown or purple. This growth is most commonly seen on the lower leaves of the plant. Infected plants may appear less vigorous than usual with yellowish or pale green foliage and mild, inconspicuous mottling. As the disease progresses, more obvious symptoms may include stunted plant growth, wilting, downward curling or distorted leaves, severe leaf drop and plant collapse.

[003] In India, downy mildew epidemics caused substantial yield losses during 1970s and

1980s. Grain yield losses of 10 to 60% have been reported. The yield reducing potential of downy mildew is very high, and this was dramatically recorded in HB 3, a popular hybrid, when pearl millet grain production in India was reduced from 8 million tons in 1970-71 to

5.3 million tons in 1971-72. This reduction was due to a downy mildew epidemic, in which yields in some fields were reduced by 60 to 70%. Many fungicides are available in market which contains chemicals as a part of the product or may be main ingredient like

Mefenoxam, Azoxystrobin, Potassium Phosphite etc. These chemicals have side effects on humans like nausea, vomiting, stomach pain, diarrhoea, bone or joint pain, headache, dizziness, tired feeling, muscle pain or weakness etc. on prolonged use. Organic control of downy mildew can be done by eliminating moisture and humidity around the impacted plants. Watering from below, such as with a drip system, and improved air circulation through selective pruning. In enclosed environments, like in the house or in a greenhouse, reducing the humidity will help as well. These types of methods are very time consuming and occurrence of pathogen will prevail over a period of time.

[004] Improper use of synthetic chemical pesticides in crop production causes several problems such as nonselective toxicity, accumulation of toxic compounds and outbreak of pathogens resistant to the pesticides. One way to handle these problems is to develop bio- pesticides comprising antagonistic microorganisms. Bio-pesticides can be safer, more biodegradable, and less expensive to develop than synthetic chemical pesticides.

Antagonistic microorganism effectively controls various kinds of plant diseases through several mechanisms such as competition, parasitism, antibiosis and induced resistance, and produces antibiotic compounds.

Summary of the Invention [005] One embodiment of the present invention provides a novel fungicide composition comprising; a) a supernatant or an extract of strain selected from Bacillus aryabhata strain or

Streptomyces strain, b) Tween 80 and c) Menthol.

[006] Another embodiment of the present invention provides a novel fungicide composition comprising; a) a supernatant or an extract of strain selected from Bacillus aryabhata strain or

Streptomyces strain, b) 10% Tween 80 and c) Menthol.

[007] Another embodiment of the present invention provides a novel fungicide composition comprising; a) a supernatant or an extract of strain selected from Bacillus aryabhata strain or

Streptomyces strain, b) 10% Tween 80 and c) 0.01 to 5% Menthol.

[008] Another embodiment of the present invention provide a method of controlling or inhibiting pathogens in a plant comprising applying an effective amount of the novel composition comprising Bacillus aryabhata strain or Streptomyces strain containing a supernatant or an extract thereof, Tween 80 and menthol.

[009] Another embodiment of the present invention provide a method of controlling or inhibiting pathogens in a plant comprising applying an effective amount of the novel composition comprising Bacillus Aryabhata strain or Streptomyces strain containing a supernatant or an extract thereof, Tween 80 and menthol, wherein the effective amount is

O.OlmLZ lt.

Brief Description of Drawings [010] Fig. 1 illustrates zones of inhibition

[011] Fig. 2 illustrates zones of inhibition for shortlisted cultures

[012] Fig. 3 illustrates the growth of pathogen

[013] Fig. 4 illustrates biochemical and microbiological analysis of isolated strain

[014] Fig. 5 illustrates biochemical and microbiological analysis of isolated strain [015] Fig. 6 illustrates the efficacy of the formulation on a wide range of pathogens.

Detailed Description of the Invention

[016] In a preferred embodiment, a fungicide which is organic in nature and derived from microbial fermentation and stabilized using Tween 80 (stabilizer) at 10% concentration. The active ingredient of the invention is of microbial origin and is capable of preventing growth inhibition and withering of the plant caused by plant pathogens when applied with an effective amount thereof either to soil where the plants grow or to the plants themselves.

[017] In an implementation of the present invention, the following process is used to identify the microorganisms that would form part of the active ingredient of the invention. Samples collected from different sources were screened to determine their anti-fungal property.

Temperature and pH while collecting the samples were estimated to be 30°C and 6 respectively The soil samples were taken and diluted serially up to 10-6 unit. About 0.1ml of serially diluted sample is taken and the spread plate technique was used having yeast peptone dextrose agar plate with ampicillin of pH-7 (2% dextrose, 1% peptone, 0.5% yeast extract, ampicillin 0.01%). The inoculated plates were incubated for 48 hours at 30°C. a. Subculture technique and media optimization^

[018] The 10 isolates of microbes obtained were sub-cultured on a suitable nutrient such as agar and yeast peptone dextrose broth media, and incubated at 30°C for 48hrs.

[019] The microbe isolated was grown in different media with the following composition:

[020] The following media compositions were used:

Media 1:

Maltose -3%

Yeast Extract - 1 % Peptone - 2%

Soya - 1% pH-6

All the 10 isolates were grown aerobically under room temperature at 120 rpm for a period of 5 days. The colony count was optimized spectrophotometrically at OD greater than 9 at 600nm wavelength.

Media 2: Sucrose-2% Yeast extract - 1%

NaCl -1%

Dipolssium hydrogen phosphate - 0.5% All the 10 isolates were grown aerobically at 120 rpm or 5 days and measured spectrophotometrically.

Media 3:

SD broth /Glucose Yeast extract - 5 g/1

Molasses - 20 g/1

Vitamins - 4 g/1

Boric acid - 500 μg/l

Copper sulphate - 40 μg/l Potassium iodide - 100 μg/l

Ferric chloride - 10 μg/l

Manganese sulfate- 10 μg/l

All the 10 isolates were grown in the above said media for period of 5 days at 120 RPM aerobically. Purified cultures were routinely maintained on nutrient agar kept at 4°C. b. Screening for antifungal property

[021] The isolated microbes were then separated based on morphological characteristics on a standard specific media and then incubation period was extended for 10 days. Among the

10 isolates 7 were found to be bacteria and 3 were Actinomycetes by morphological characteristics. These microbes were then grown on different specific media like actinomycetes on YMDA and Bacteria on Luria bertani (LA) agar.

Media Composition (YMDA) Yeast extract-0.4%

Malt extract- 1%

Dextrose-0.4%

Agar- 1.9% Media Composition (LA)

Peptone- 1%

Yeast-0.5%

Nacl-0.5%

Agar- 1.9%

[022] These media were weighed in 250ml flask and were autoclaved at 121° centigrade for

ISminutes at 15PSI. Then the media were poured into autoclaved petriplates to solidify.

When the media is solidified these cultures were streaked to get isolated colonies and incubated for 2 to 3 days. Once isolated colonies were obtained these culture were subjected to grow on liquid broth media for antifungal metabolite production. For metabolite production bacterial cultures were inoculated in specific media and grown for 5 days @ 30° centigrade whereas actinomycetes culture were grown on YMDB for 7-8 days @ room temperature.

[023] Once after 5 to 8 days of incubation the cultures were extracted with different solvents like Ethyl acetate, methanol at 3:1 ratio. Extraction was done overnight. Once extraction is done these were tested against Fusarium, Pythium, Phytophthora. 3 ml of pathogen spore suspension was added to 10ml of the solid agar media and then plated. 10μ1 of the each extract was spotted on the plate containing pathogen. These plates were incubated at room temperature for 24 to 72hours for the zone of inhibition.

[024] Cultures were shortlisted based on best/more zone of inhibition as illustrated in Fig. 1

[025] Zone of inhibition was rated as †, †† & †††

[026] Single + indicates: Good (+)

[027] Double† indicates: Better (††)

[028] Triple+ indicates: Best (+++)

[029] Among 10 isolates two bacterial isolates (AFB 04, AFB 07) and one Actinomycetes

(AFA10) were shortlisted which showed good zone of inhibition i.e., Triple †††.

[030] These 3 cultures were further subjected to media trials in order to increase the yield of the metabolite. The media composition was varied in order to find the best one which gives high yield/good zone of inhibition. Ethyl acetate was chosen as the solvent. Extraction was done by ethyl acetate. The media composition was as follows for bacteria Media composition (A)

Peptone- 1%

Yeast-0.5%

Nacl-0.5% Media composition (B) Peptone-2%

Yeast-1%

Nacl-0.5% Media composition(C)

Peptone-0.5%

Yeast-0.5%

Nacl-0.25% Media composition (D)

Peptone- 1%

Yeast-0.5%

Nacl-0.5% And media Composition for actinomycetes were varied

Media Composition (A)

Yeast extract-0.4%

Malt extract- 1% Dextrose-0.4%

Media Composition (B)

Yeast extract- 1%

Malt extract- 1% Dextrose-0.4% Media Composition(C)

Yeast extract- 1%

Malt extract-0.5% Dextrose- 1%

Media Composition (D)

Yeast extract-0.5%

Malt extract-0.5% Dextrose-0.5%

[031] These media were weighed in 250ml flask and autoclaved at 121°C for ISminutes at 15psi. After autoclaving the media these flasks were cooled and then inoculated with respective cultures and incubated at 30°C for 5days for bacteria and at room temperature for

7 days for actinomycetes. After incubation period these cultures were subjected to extraction with Ethyl acetate in order to extract the metabolite @ 1:3 ratio. Extraction was done for overnight. Once extraction was done the immiscible supernatant part was then collected and vacuum evaporated up to 30X times. The concentrate obtained was spotted against all the 3 pathogens. Among the shortlisted cultures and media optimisation bacterial with media A

(AFB7 culture) showed highest zone of inhibition as illustrated in Fig. 2

[032] The culture AFB7 was further grown in large quantities (2Liter) flasks and then the same protocol was followed and extraction was done with shortlisted solvent at 3:1 ratio and extracted overnight. The solvent was then concentrated by vacuum evaporation up to

30X times by vacuum evaporation. The concentrate obtained was then tested against the pathogen. This was done by adding the concentrate to the media and then plated. To this plate a 2days grown pathogen was placed by taking the pathogen in disc borer. This plate was incubated for 5days along with control plate in order know the zone of inhibition by the concentrate along with the control plate where there has no product in it. The results are in centimetres of diameter of pathogen on the plate for 4 days as illustrated in Fig. 3.

[033] Based on the above results found AFB7 culture had given the best score based on the culture as well as media trials. This culture along with shortlisted media was taken into consideration. b. Phenotypic characterizations of shortlisted isolate:

[034] The morphological and biochemical characterizations of the selected strain AF-B7 were carried out as described in Bergey’s Manual of Systematic Bacteriology. The isolates were plated in sterile petri plates by spread plate method and plates incubated at 28 °C. For cell and colony morphology (shape, size, color, motility) determination of the isolates, they were studied under microscopes.

[035] Identification of bacteria bv sequencing of thel6S rRNA gene:

[036] DNA extraction and PCR Amplification Genomic DNA was isolated according to the following procedure. 50 ml LB broth was inoculated with a single bacterial colony and grown to an absorbance 600 nm of 0.5-1.0 and cells were collected by centrifugation at

5000 rpm, at 4°C, for 10 min. The genomic DNA was isolated from the given organism.

Amplification of the 16s rRNA gene was performed using the universal primers.

[037] Genomic DNA extraction and sequencing of the partial 16S rRNA gene: [038] Isolation of genomic DNA was performed according to the modified isoamyl alcohol - chloroform method. The partial 16S rRNA gene of the selected strain was amplified by

Polymerase chain reaction. The sequences of two oligonucleotides used as PCR primers were 5 " GAG TAA TGT CTG GGA AACTGC CT-3" (forward primer) and 5 " CCA GTT

TCGAAT GCA GTT CCC AG-3" (reverse primer). Amplification was performed in a 25 μΐ reaction mixture containing the template DNA, 0.02 μg; each primer 0.2 μΜ; dNTPs, 200 μΜ; Taq DNA polymerase, 1.5 U and 10 x Tag buffer, 2.5 μl. The mixture was subjected to the following amplification conditions: 5 min at 95°C, 30 cycles of 40 s at 95°C, 60 s at53°C, 1 min at 72°C and one final step of 10 min at72°C. The PCR products were electrophoresed on l%w/v agarose gels and the target band was excised and purified with a

DNA purification kit. These DNA of the isolates was sent for sequencing.

[039] Identification and Phylogenetic analysis of the partial 16S rRNA sequences:

[040] Biochemical and microbiological analysis was performed to characterize the screened strain. According to the sequence comparisons based on the partial 16S rRNA gene of the strain in this study with sequences of the nearest type species retrieved from the ribosomal database project (RDP), this strain showed high homology with Bacilli Aryabhata.

Following the physiological and biochemical characteristics and comparison of its 16S rRNA gene sequence, the selected strain was identified as Bacillus Aryabhata as illustrated in Fig. 4 and Fig. 5.

[041] Formulation:

[042] The shortlisted extracted culture was formulated using Tween 80 @ 10% which is a stabilizer to enhance the stability of the formulated product. Further addition of Menthol 0-

5%, preferably 0.01-5% to the above increased the efficacy of the product in terms of controlling wider range of pathogens that includes colletotrichum, altemaria, botrytis and sclerotia at a lower dose of 0.01ml compared to earlier dosage of lml/lt. Fig. 6 shows the efficacy of the formulations on a wide range of pathogens.

[043] Sample Collection

[044] 10 samples were collected from foot of Nandi Hill region of Bangalore, Karnataka.

[045] Soil samples were collected from 5-25 cm depth by sterile method from various locations visited throughout this scientific expedition to Nandi hills area and transported aseptically to the Microbiology lab of Agferm Innovations Private Limited, Bangalore.

[046] Fusarium was procured from KAU, Kerala. The fungus was cultured and sub-cultured using potato dextrose agar medium and kept in refrigerator at 4oC for further testing.

[047] Isolation of Actinomycetes:

[048] Calcium carbonate enrichment methods were used to isolate Actinomycetes, the soil samples were mixed with CaC03 at the ratio of 10:1, and were incubated under moisture rich conditions for seven days at room temperature (Hayakawa et al., 2004). The soil was further held in a water bath at 50°C to further destroy other vegetative microorganisms [5].

Isolation and Enumeration of Actinomycetes were performed by the soil dilution plate technique using starch casein agar medium (gfi: Starch-10 grams, Casein-0.3 grms,KN03-

2 grams,Nacl-2garms, K2HP04-2grams,MgS04.7H20-0.05grams,CaCo3-

0.02grams,FeS04.7H20-0.01 grams, and Agar 18grams)[7]. To minimize the fungal and bacterial growth, actdione 20 mg/L, and nalidixic acid 100 mg/L were added. The plates were incubated at 30°C for 10 days. After an incubation period, the plates were examined for the presence of actinomycetes colony. The suspected colonies were picked up and purified on media and incubated at room temperature for about 7 days. [049] Characterization of the Isolates

[050] Morphological, physiological and biochemical characterization of actinomycetes

[051] Identification of Actinomycetes to genus level was conducted by first using morphological and chemical criteria according to Bergey’s Manual of Determinative

Bacteriology. (10) Cultural characteristics of the isolates was studied based on the intensity of the growth, growth pattern, colony colour along with colour of aerial mycelia on Tryptone yeast agar (ESP medium 1), yeast extract malt extract agar (ISP Medium 2), Oat meal agar

(ESP Medium 3), Inorganic salt starch agar (ISP Medium 4), Glycerol Asparginine agar (ISP

Medium 5), Peptone yeast extract iron agar (ESP Medium 6), Tyrosine agar (ISP Medium

7) as described by Shirling and Gottlieb, 1966 and also on Starch casein agar, Potato

Dextrose agar, Kuster’s agar and CzapekDox agar. Gram staining and spore surface morphology was examined by scanning electron microscopy (SEM). The arrangement of spore and sporulating structures were examined microscopically by using cover slip culture method (9). The mycelium structure, color and arrangement of conidiophores and arthro spore on the mycelium were observed through the oil immersion (lOOOx) microscope.

[052] Biochemical characterization of actinomycetes

[053] The isolates were inoculated into ESP-2 medium Ml medium. Incubation of the slide was done at 37°C for 7 d. Staining with methylene blue was done followed by observation of the slides under the microscope after staining with methylene blue stain. Based on physical parameters 2 isolates were selected and were studied further.

[054] Diagnostic tests of actinomycetes strains were performed based on macro and microscopic features according to Bergey’s Manual of Systematic Bacteriology

[Goodfellow et al. 2012] and Crawford et al. [1993]. The genus of 2 selected actinomycetes was ascertained using above methods and were found to be of genus Streptomyces. These suspected pure actinomycetes cultures were inoculated on ISP-2 slants, after the incubation period the slants were taken for antifungal screening. The stock culture was preserved in

50% glycerol.

[055] Fermentation and extraction of Secondary metabolite

[ 056] Spores (10⁷ /ml) of the 2 isolates were used to inoculate 1000 ml Erlenmeyer flasks containing 200 ml of actinomycetes media each. After six days of incubation at 30°C in an orbital incubator shaker at 200rpm, the flask was opened and Ethyl Acetate was added to the broth in ratio 1:1 and was again kept for shaking for 24hrs. This whole solution was evaporated at 50°C in a rotary vacuum evaporator to eliminate the solvent fully and concentrate the extracted metabolite broth up to 10 folds. A final volume of 20ml of secondary metabolite concentrate was retrieved and was tested against plant pathogen

Fusarium solani.

[057] Fungal growth inhibition test/poisoned food technique.

[058] To determine the effect of two microbial extracts on growth of fungus at different concentrations diluted with acetone in 1:1 ratio, was added into Potato dextrose agar media at 0.1, 0.25, 0.5, 0.75, 1, 2.5, 5 % concentration. Treated media (20 ml) was then poured into the petri plate and allowed to solidify. Mycelial plugs (3 mm in diameter) of pure culture of Fusarium solani were incubated in the center of each PDA plate (9 cm diameter). All the experimental transfers were performed aseptic ally in laminar air flow. These plates inoculated with fungus were incubated at 28°C and 70% RH for 5-7days. Mycelial growth was measured every day until control plates were completely colonized with mycelium.

Plates with only media and with extracts were used as control. A solvent control was also set up with media and solvent. The experiments were done in triplicates.

[059] Statistical analysis [060] All statistical analyses were carried out using ANOVA. Analysis of variance

(ANOVA) was performed on all experimental data and means were compared using

Duncan’s multi range test. The significance level was p<0.05.

[061] Results and Discussion:

[062] The antifungal activity of the two extracts was studied using growth inhibition assay or poisoned food technique. The extracts were added in different concentration 0.1, 0.25, 0.5,

0.75, and 1% in the media. The results of the antifungal tests showed that the one of the extracts treated media inhibited the fungal mycelial growth at varying levels and exhibits more potent antifungal activity than the other one. Effect of extract 1 and extract 2 is shown in Table 1 and 2 respectively.

Table -1 showing results of Extract 1

Mean± Standard Deviation (SD) on three determinations, Values (means of 3 replicates) in each column not sharing a common letter differ significantly (p<0.05) from each other

(Duncan’s multirange test). Table-2 showing results of Extract 2

Mean± Standard Deviation (SD) on three determinations, Values (means of 3 replicates) in each column not sharing a common letter differ significantly (p<0.05) from each other

(Duncan’s multirange test).

[063] Different concentrations of Extract 1 and 2 were tested against the fungal growth. It was found that extract 2 was not much effective in inhibiting fungal growth at lower concentration. All the concentrations allowed the growth of fungi except the higher concentrations (0.75 to 5%). Controls allowed good growth of fungal mycelium (table 1).

[064] Extract lwas found to be most effective in controlling fungal growth. All the concentrations were able to control the growth of pathogen. In comparison control showed growth (table 2).

[065] The growth of pathogen was not restricted completely with extract 2 at lower concentrations: 0.1 - 0.5% not able to inhibit the growth of fungal mycelium. Fungi grew in control and solvent control plates.

[066] Example 1:

[067] The following is an example of the effectiveness of the current invention on

Phytophthora infestans. In the detached leaf assays the commercial preparations showed a significant effect on the level of infestation by P. infestans. In order to take curative and protective modes of action into account, the test substances were applied 24 h before, or 90 min after inoculation with P. infestans. Generally, better effects were obtained when the applications were made 24 h before inoculation. For defining the optimum time of application, potted plants were treated 72 or 24 h before, and 1 and 24 h after inoculation with P. infestans. The invention showed best results when applied 1 day before inoculation.

[068] Example 2:

[069] Fusarium wilt is an important disease of tobacco. The invention was evaluated under in vivo conditions. The product was found to be more promising and affected 60.9% control over check. Among chemicals Carbendazim and Propiconazole treatments resulted control measure to the extent of 24.53 and 31.77% respectively. The management strategy gave an Incremental Cost Benefit Ratio (ICBR) of 1: 7.35 and 1: 7.38 in chemical and bioagent respectively.

[070] Example 3:

[071] Altemaria -late blight is an important disease of tomato. The product was evaluated under in vivo conditions. The product was found to be more promising and affected 60.9% control over check. Among chemicals Carbendazim and Propiconazole treatments resulted control measure to the extent of 24.53 and 31.77% respectively. The management strategy gave an Incremental Cost Benefit Ratio (ICBR) of 1: 7.35 and 1: 7.38 in chemical and bioagent respectively.

[072] In vitro studies

[073] The antagonists tested in vitro conditions revealed the suppression of the pathogen. The product were found to be very effective in controlling the all pathogen and hence selected for in vivo screening.

[074] Example 4:

[075] Four seed samples each of sorghum [Sorghum bicolarL. (Moench.)], maize ( Zea mays

L.) and rice Oryzae sativa (L.) were collected directly from field. Associated fungi were identified based on growth characteristic, mycelial morphology, spore morphology and other important characters using standard manuals. The fungi were frequently associated in higher percentage in sorghum, maize and rice which served as test fungi. The selected ten species of Aspergillus and three species of Penicillium were sub-cultured using Malt extract-

Salt- Agar (MESA) medium and eight species of Fusarium, two species of Drechslera and a species of Altemaria were sub-cultured using Czapek-Dox-Agar (CDA) medium. The selection of media was based on the standard recommendation for culturing these fungi. [076] Example 5:

[077] Anti-fungal activity assay:

A. Determination of percent mycelial inhibition bv dry mycelial weight technique:

[078] The invention was taken at 1% concentration in the liquid medium. 50 ml of this was taken in a 100 ml Erlenmeyer conical flask and sterilized at 121°C, 15 lb/inch2 pressure for

15 minutes and allowed to cool. The liquid medium without any product served as control.

The flasks were inoculated with 5 mm diameter mycelia disc of Fusariumsolani and

Aspergillusflavus taken from 7 days old culture and incubated for 7 days at 22 ± 1 degree C temperature under alternate cycles 12 h. light and 12 h. darkness. After incubation the content of each flask were poured into a pre- weighed Whatman No. 1 filter paper. The filter paper with the mycelial mat was dried in an oven at 60 degree C until a constant weight was reached. The dry weight of the mycelia was determined by subtracting the weight of the filter paper from the total weight of the filter paper with mycelia. Three replicates were maintained for each treatment. The percent inhibition of mycelial growth was calculated using the formula:- Percent inhibition = C - T / C X 100 where C = Mycelial weight in control and T = Mycelial weight in treatment. Data were subjected to statistical analysis and compared with Turkey HSD at 0.5 subset.

[079] Example 6:

[080] B. Determination of percent inhibition of spore germination bv cavity slide method

[081] Conidial suspension of F. solani and A. flavus were prepared in sterile distilled water and spore concentrations which adjusted to 1.0 X 104 spores/ml. 90 μΐ of placed in large

Petri dishes laden with moist blotter paper to maintain moisture level. 10 μΐ of product were placed in separate conidial suspended cavity slide and mixed well. 10μ1 of distilled water placed in 90 ml of conidia suspended media served as control. The setup was incubated for

12 hours at 22 ± 1 degree C. Germination of spore was counted under compound microscope

(Singh and Tripathi, 1999). The percent of inhibition was calculated using the formula: -

Percent inhibition = C - T / C X 100 where C = Number of spores germinated in control

(Average of 10 microscopic field) T= Number of spores germinated in treated (Average of

10 microscopic field). Data were subjected to statistical analysis and compared with Turkey

HSD at 0.5 subset.

[082] Screening/evaluation of the product showed significant inhibition of test pathogens.

Thus the invention lead to the development of a potent fungicide against major plant pathogens at low dosage of O.Olml/lt

[083] In this invention, a consortium of microbial extracts is identified which plays a significant role in controlling the pathogen as well as avoiding its future persistence.

Microorganism and its secretions, are used as curative as well as preventive for future occurrence. Through this invention the product which formulated was made of organic and the active ingredient is from microbial origin.

[084] Many microbial isolates were tested for antagonist activity against the plant pathogens.

[085] The microbial agent of the present invention can be useful in preventing growth inhibition and withering of the plant caused by plant pathogens when applied with an effective amount thereof either to soil where the plants grow or to the plants themselves. [086] Standardisation methods and invitro on media trails and triggering the specific active metabolite ingredient to be produced and right period of extraction and its down streaming makes this product a novelty. Formulation also plays an important role.

Claims

I Claim:

1. A novel fungicide composition comprising; a supernatant or an extract of strain selected from Bacillus Aryabhata strain or

Streptomyces strain,

Tween 80 and

Menthol.

2. The novel fungicide composition according to the claim 1 , wherein Tween-80 is present in an amount of 10%.

3. The novel fungicide composition according to the claim 1, wherein menthol is present in an amount of 0.01 to 5%.

4. The novel fungicide composition according to the claim 1, wherein the composition is effective against pathogens at 0.01 mlV It.

5. A novel fungicide composition comprising; a supernatant or an extract of strain selected from Bacillus Aryabhata strain or

Streptomyces strain,

10% Tween 80 and

Menthol.

6. A novel fungicide composition comprising; a supernatant or an extract of strain selected from Bacillus Aryabhata strain or

Streptomyces strain,

10% Tween 80 and

0.01 to 5% Menthol.

7. A method of controlling pathogens in a plant comprising applying an effective amount of the novel composition according to claims 1-6, wherein the effective amount is

O.OlmLZ lt.

8. The method according to claim 7, wherein the composition is applied on the plant or in the soil directly.

9. The method according to claim 7 wherein the pathogens are selected from Fusarium,

Pythium, Phytophthora, Altemaria, Botrytis and Sclerotia.