DESC:Field of Invention
The present invention is related to biopesticide such as fungicide; more specifically it is related to organic fungicide which is derived through process of microbial fermentation.
Background of Invention
Plant diseases such as Downy mildew cause major economic loss to farmers worldwide. According to the Food and Agriculture Organization; pests and diseases are responsible for 25% of crop loss every year. Thus, Control of plant diseases is crucial in crop management.
Downy mildew is a pathogenic disease that is caused mostly by organisms that belong to either genusPeronospora or genus Plasmoparawhich are parasitic organisms that are more closely related to algae.Phytophthoraand fusarium are other pathogenic fungus that also causes severe damage on various vegetable crops, grape vine, pepper etc. The infection appears on plant in white, grey, brown or purple colour growth mostly on lower leaves of the plant. It further causes stunted plant growth, wilting, downward curling or distorted leaves, severe leaf drops and finally plant collapse.
In India, the reduction in yield due to a downy mildew epidemic were reduced by 60 to 70%.
Many fungicides are available in market which contains chemicals as a part of the product such asMefenoxam, Azoxystrobin, PotassiumPhosphite etc. On prolonged use, 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. 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.
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. However, these methods are very time consuming and occurrence of pathogen will prevail over a period of time.
One way to handle these problems is to develop bio-pesticides comprising antagonistic microorganisms.Bio-pesticides are safer, more biodegradable, and less expensive to develop than synthetic chemical pesticides. Antagonistic microorganismeffectively controls various kinds of plant diseases through several mechanisms such as competition, parasitism, antibiosis and induced resistance, and produces antibiotic compounds.
U.S. Pat. No. 6,060,051 discloses a method of treating or protecting plants more specifically protecting plant roots from fungal and bacterial infections such as corn rootworm by applying an effective amount of the antibiotic/metabolite producing Bacillus subtilis. The novel metaboliteproducingBacillus subtilis can be provided as a suspension in a whole broth culture or as a metabolitecontaining supernatant or a purified metabolite obtained from a whole broth culture of the microorganism. It also provides compounds such asagrastatins produced by the strain AQ713 and a combination of compounds comprising inturin A, a plipastatin, a surfactin and an agrastatin.
U.S. Pat. No. 6,103,228 is related to the findings that a strain of Bacillus subtilis, AQ713, can inhibit a broad range of fungal and bacterial plant diseases and also have activity against insects. The patent describes fungicidal and bactericidal compositions comprising this Bacillus strain and the antibiotics and metabolites produced by this strain. The use of this metabolites can be either alone, or in combination with other chemical and biological pesticides.
U.S. Pat. No. 8,025,875 describes methods of inducing pathogen resistance in plants, such as inducing systemic acquired resistance to infection in plants by applying a Bacillus control agent comprising Bacillus mojavensis isolate 203-7 and/or Bacillus mycoides isolate BmJ to one or more plants.
Patent application WO1998021964, describes B. subtilis strain ATCC No. 55614, which is effective in inhibiting growth of plant pathogenic bacteria and fungi. The application describes a method for protecting a plant against fungal or bacterial infection by applying to the plant a B. subtilis strain ATCC 55614, a supernatant obtained from a culture or a bioactive extract.
The patent application WO2000058442 describes a biologically pure culture of a strain having all the identifying characteristics of a Bacillus pumilus strain designated NRRL No. B-30087, or mutants thereof, that have fungicidal activity.
The infecting agents such as bacteria or fungi develop resistance to fungicides. Thus, there was a need to develop new fungicide with strong action and more versatile range.

Object of Invention
The object of the invention is to identify a new microbial strainhaving antifungal or antibacterial activity.

Summary of the invention
The present invention provides a method of protecting or treating plants from fungal or bacterial infection, comprising applying an effective amount of composition of Bacillus aryabhatastrain containing a supernatant or an extract thereof, to said plants.
The invention further provides aprocess of identifying Bacillus aryabhata strain composition which comprises:
i. Isolation of organism from the sample obtained from source
ii. Culturing the sample in media with ampicillin for 48 hours
iii. Sub-culturing the sample obtained from step ii for 5 days
iv. Isolated colonies obtained by growing step iii into specific media such as YMDA or LB agar for 2-3 days.
v. Isolated colonies from step v are grown in liquid broth media for 5 to 8 days.
vi. Extraction of cultures to test against Fusarium, Pythium, Phytophthora.
vii. screening the fractions for antimicrobial activity
viii. Shortlisting of strain based on good zone of inhibition.
ix. Biochemical and microbiological analysis to characterize the screened strain

Brief Description of Drawings
Figure 1 illustrates zones of inhibition
Figure 2 illustrates zones of inhibition for shortlisted cultures
Figure 3 illustrates the growth of pathogen
Figure 4 illustrates biochemical and microbiological analysis of isolated strain. The figure is distribution of 129 blast hits on the query sequence
Figure 5 illustrates biochemical and microbiological analysis of isolated strain

Detailed description
Definitions
The term "bacteria" includes any prokaryotic organism that does not have a distinct nucleus.
The term “fungi” or "fungus" includes a wide variety of nucleated spore-bearing organisms that are devoid of chlorophyll. Examples of fungi include yeast, moulds, mildews, rusts, and mushrooms.
The term “fungicide” refers to a composition comprising one or more chemical substances or biological organisms capable of killing or inhibiting both true fungi and their spores as well as oomycete pathogens, usually in a selective way. Fungicides are used both in agriculture and to fight fungal infections in animals. Fungicide can be either contact or systemic. In agriculture, a contact fungicide kills fungi by direct contact; a systemic fungicide spreads internally through the plant, thereby killing the fungi.
The term “isolates” refer to a culture of microorganisms isolated for study.
The term “microorganisms” or “microbe” or “microbes” refer to a microscopic organism(s). The microbes can be multi-cellular or single-celled organisms and include bacteria, protozoa, and some fungi and algae. Microbes can be found in every type of environment and can be either beneficial or harmful to plants.
The term “media” refers to a growth medium or culture medium is a solid, liquid or semi-solid designed to support the growth of microorganisms or cells, or small plants.
The term “plant” refers to any living organism belonging to the kingdom Plantae (i.e., any genus/species in the Plant Kingdom). This includes familiar organisms such as but not limited to trees, herbs, bushes, grasses, vines, ferns, mosses and green algae. The term refers to both monocotyledonous plants also called monocots, and dicotyledonous plants also called dicots.
The present invention provides a fungicide which is organic in nature and is derived from process of microbial fermentation. The fungicide is further formulated using Tween 80 (stabilizer) at 10% concentration.
The active ingredient of fungicide of the present invention is of microbial origin and is capable of preventing ‘growth inhibition’and ‘withering’ of the plant(s) caused by plant pathogens when applied with an effective amount in soil as a soil drench or directly on the plant body.
The present invention describes a process to identify the microorganisms that would form the active anti-microbial ingredient of the fungicide composition.

Isolation of microorganism:
The microorganism of the present invention was obtained from samples collected from different sources such as soil. The samples were later screened to determine their anti-fungal property. While collecting the sample(s) from the source the temperature was 30°C and pH of the source was approximately 6.
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 30ºC temperature.

Subculture technique and media optimization:
As mentioned in section 4.1, initially the 10 isolates of the micro-organisms obtained from various sources were cultured on a suitable nutrient such as agar and yeast peptone dextrose broth media, and incubated at 30ºC for 48hrs.
The microbes isolatedfurtherwere grown/ sub-cultured in different media with the following composition(s):
Media 1:
All the 10 isolates were grown aerobically under room temperature in medium 1 at 120 rpm for a period of 5 days. The colony count was optimized spectrophotometrically at OD greater than 9 at 600nm wavelength.
Maltose -3%
Yeast Extract – 1%
Peptone – 2%
Soya – 1%
pH-6
Media 2:
All the 10 isolates were grown aerobically in medium 2 at 120 rpm for 5 days and measured spectrophotometrically at OD of 0.8 at 600nm wavelength.
Sucrose-2%
Yeast extract – 1%
Nacl -1%
Dipotssium hydrogen phosphate – 0.5%
pH-6.5-7
Media 3:
All the 10 isolates were grown in the media 3 for time period of 5 days at 120 RPM aerobically. Purified cultures were routinely maintained on nutrient agar kept at 4ºC.measured spectrophotometrically at OD of 0.9 at 600nm wavelength.
SD broth /Glucose- 10g/lt
Yeast extract – 5 g/
Molasses – 20 g/l
Vitamins – 4 g/l
Boric acid – 500 µg/l
Copper sulfate – 40 µg/l
Potassium iodide – 100 µg/l
Ferric chloride – 10 µg/l
Manganese sulfate-10 µg/l

Screening for antifungal property:
Upon sub-culturing of the micro-organisms in different media, the microbes furtherseparated based on morphological characteristics on a standard specific media and then incubation period were extended for 10 days.
Among the 10 isolates, by morphological characteristics, 7 isolates were found to be bacteria and 3isolates were Actinomycetes. These identified microbes were then grown on different specific media like actinomycetes on Yeast Malt Dextrose Agar (YMDA) and Bacteria on Luria Bertani(LB)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%
The media were weighed in 250ml flask and were autoclaved at 1210 centigrade for 15 minutes at 15PSI. Further the media were poured into autoclaved Petri-plates to solidify. Upon media solidification these cultures were streaked to get isolated colonies and incubated for 2 to 3 days.

Metabolite Production:
The isolated colonies grown on liquid broth media for antifungal metabolite production.
(A) Bacterial cultures were inoculated in specific media and incubated/grown for 5 days at 300C.
(B) Actinomycetes culture were incubated/grown onYeast Malt Dextrose Broth (YMDB) for 7-8 days at room temperature (370C).
Upon incubation the cultures were extracted with different solvents such as Ethyl acetate, methanol at 3:1 ratio. Extraction was done overnight. The extractedcultureswere tested against Fusarium, Pythium, Phytophthora. 3ml of pathogen spore suspension was added to 10ml of the solid agar media and then plated. 10µl of each extract was spotted on the plate containing pathogen. These plates were incubated at room temperature (370C) for 24 to 72 hours for the zone of inhibition.
Cultures were shortlisted based on best/more zone of inhibition as illustrated in Fig. 1 Zone ofinhibition was rated as +, ++ & +++.
Single + indicates: Good(+)
Double+ indicates: Better(++)
Triple+ indicates: Best(+++)
Among 10 isolates two bacterial isolates (MIKB 04, MIKB 07) and one Actinomycetes (MIKA10) were shortlisted which showed good zone of inhibition i.e. Triple +++ .
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. Extraction was done by ethyl acetate.
The media composition for bacteria were as follows:
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%
Media composition for actinomycetes were varied, as follows:
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%
These media were weighed in 250ml flask and autoclaved at 1210C for 15 minutes at 15psi. After autoclaving the flask were cooled and then inoculated with respective cultures and incubated at 300C 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 at 1:3 ratio. Extraction was done 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 (MIKB7 culture) showed highest zone of inhibition as illustrated in Fig. 2.
The culture MIKB7 was further grown in large quantities (2 Litre) 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. Thiswas 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.

Phenotypic characterizations of shortlisted isolate:
The morphological and biochemical characterizations of the selected strain MIK-B7 were carried out as described in Bergey’s Manual of Systematic Bacteriology. The isolates wereplated in sterile petri plates by spread plate method and plates incubated at 280C. For cell and colony morphology (shape, size, colour, motility) determination of the isolates, they were studied under microscopes.
Identification of bacteria by sequencing of the16S rRNA gene:
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.
Genomic DNA extraction and sequencing of the partial 16S rRNA gene:
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 25µl reaction mixture containing the template DNA, 0.02 µg; each primer 0.2µM; dNTPs, 200µM; TaqDNA polymerase, 1.5 U and 10 × Taqbuffer, 2.5µl. The mixture was subjected to the followingamplification 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 1%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.
Identification and Phylogenetic analysis of the partial 16S rRNA sequences:
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 Bacillisaryabhata. 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.
Following examples show the embodiments of the present invention of the fungicide with anti-microbial active ingredient.
Example 1
The following is an example of the effectiveness of the current invention on Phytophthorainfestanswhich an amoeba protozoa microorganism, specifically an oomycete, which causes the serious potato and tomato disease known as late blight or potato blight. In order to take curative and protective modes of action into account, the testsubstances were applied 24 hours before, or 90 min after inoculation with P. infestans. Fordefining the optimum time of application, potted plants were treated 72 or 24 hours before, and 1 and 24 hours after inoculation with P. infestans.
The invention showed best results when applied 1 day before inoculation.

Example 2
Fusarium wilt is an important disease of tobacco and caused by Fusarium oxysporum. 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.

Example 3
Isolation of important phytopathogenic fungi from sorghum, maize and rice seeds
Four seed samples each of sorghum [Sorghum bicolar L. (Moench.)], maize (Zea mays L.) and rice Oryzaesativa (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 Alternaria were sub-cultured using Czapek-Dox-Agar (CDA) medium. The selection of media was based on the standard recommendation for culturing these fungi.

Example 4
Anti-fungal activity assay:
Determination of percent mycelial inhibition by dry mycelial weight technique:
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 Fusarium solani and Aspergillus flavus taken from 7 days old culture and incubated for 7 days at 22 ± 10C 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 600C 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. Thepercentinhibition of mycelial growth was calculated using the formula: - Percent inhibition = C – T / C ? 100 where C = Mycelial weight in control and T = Mycelial weight in treatment. Datawere subjected to statistical analysis and compared with Turkey HSD at 0.5 subset.

Example 5
Anti-fungal activity assay
Determination of percent inhibition of spore germination by cavity slide method
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 µl of placed in large Petri dishes laden with moist blotter paper to maintain moisture level. 10 µl of product were placed in separate conidial suspended cavity slide and mixed well. 10µl of distilled water placed in 90 ml of conidia suspended media served as control. The setup was incubated for 12 hours at 22± 10C. 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 ? 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.
Screening/evaluation of the product showed significant inhibition of test pathogens.
,CLAIMS:1) A method of protecting or treating plants from fungal or bacterial infection, comprising applying an effective amount of composition of Bacillus aryabhatastrain containing a supernatant or an extract thereof, to said plants.

2) The method of claim 1, wherein said composition of Bacillus aryabhatastrain is applied as a wettable powder, granules, aqueous flowable, dry flowable or is microencapsulated in a suitable substance.

3) The method of claim 1, wherein the roots of the plants or soil around the roots are treated.

4) The method of claim 1, wherein, microbe is selected from the group consisting of Aspargillus, Fusarium, Pythium, Phytophthora, Alternaria, Penicillium and Drechslera.

5) A method of inhibiting Fusarium wilt infection in a plant, comprising: applying a composition of claim 1 to said plant in an amount effective to inhibit growth of Fusarium.

6) A process ofidentifyingBacillusaryabhatastraincompositionwhich comprises:
i. Isolation of organism from the sample obtained from source
ii. Culturing the sample in media with ampicillin for 48 hours
iii. Sub-culturing the sample obtained from step ii for 5 days
iv. Isolated colonies obtained by growing step iii into specific media such as YMDA or LB agar for 2-3 days.
v. Isolated colonies from step v are grown in liquid broth media for 5 to 8 days.
vi. Extraction of cultures to test against Fusarium, Pythium, Phytophthora.
vii. screening the fractions for antimicrobial activity
viii. Shortlisting of strain based on good zone of inhibition.
ix. Biochemical and microbiological analysis to characterize the screened strain

7) The process of claim 10, wherein the sample is collected from the soil.

8) The process of claim 10, wherein, the Actinomycetes are grown on YMDB.

9) The process of claim 10, wherein the extraction is performed with ethyl acetate in 3:1 ratio.

10) The process of claim 10, wherein the extractedculture exhibits antifungal activity.