Synergistic bioinoculant composition comprising bacterial strains of accession nos. NRRL B-30486, NRRL B-30487, and NRRL-B 30488 and a method of producing said composition thereof

Abstract

A synergistic composition useful as bioinoculant includes bacterial strains of accession Nos. NRRL B-30486, NRRL B-30487, and NRRL-B 30488, individually or in all possible combinations, and optionally a carrier, with each of the strains showing plant promoting activity, phytopathogenic fungi controlling activity, abiotic stress conditions tolerating capability, phosphate solubilization capability under abiotic stress conditions; and further, a method of producing the composition thereof, and in addition, a method of isolating the bacterial strains from cow ‘Sahiwal’.

1 . An in vitro method of isolating bacterial strains of accession nos. NRRL B-30486, NRRL B-30487, and NRRL-B 30488, which comprises: 
 (a) collecting milk from cow ‘Sahiwal’,    (b) plating the milk on a culture medium,    (c) incubating the culture medium at a temperature of 25-35° C. for about 1-3 days,    (d) selecting all morphologically distinct bacteria from the culture medium,    (e) screening said strains selected in step (d) which will suppress phytopathogenic fungi by showing a zone of inhibition of at least 2 mm, by incubating at 30-35° C. for 20-35 days,    (f) screening said strains selected in step (e) for plant growth promoting bacteria showing at least a 5% increase in dry weight of a plant, by growing plants in a presence of selected bacteria at a concentration of bacteria ranging between about Log 6 to 10 CFU/seed or about Log 6 to 8 CFU/gram of soil,    (g) screening said strains selected in step (f) at 4-8% salt stress tolerance for further selection,    (h) screening said strains selected in step (g) at pH 4-10 stress tolerance for further selection,    (i) screening said strains selected in step (h) at 50-60° C. temperature stress tolerance for further selection,    (j) screening said strains selected in step (i) for ability to solubilize phosphate under abiotic stress conditions of high salt, pH, and temperature for further selection, and    (k) isolating the bacterial strains.    
   
   
       2 . The method as claimed in  claim 1 , wherein a plant for growth promoting activity is selected from the group consisting of  Zea mays, Abelmoschus esculentus, Luffa cylindrica, Lycopersicon esculentum, Abelmoschus esculentus , and  Cucumis sativus.    
   
   
       3 . The method as claimed in  claim 1 , wherein culture medium is Nutrient Agar, said medium comprising 2-10 gms beef extract, 5-15 gms peptone, 2-10 gms sodium chloride, 10-20 gms agar, and about 1.0 L distilled water, with pH ranging between 7.0-7.4.  
   
   
       4 . The method as claimed in  claim 1 , wherein pH tolerance is tested at 30° C.  
   
   
       5 . The method as claimed in  claim 1 , wherein soil moisture is ranging between 1-30%.  
   
   
       6 . The method as claimed in  claim 1 , wherein a salt for salt stress tolerance is NaCl.  
   
   
       7 . The method as claimed in  claim 1 , wherein the strains are grown on Nutrient Broth (NB) medium consisting of 0.5% beef extract, 1% peptone, 0.5% NaCl and distilled water, with pH of the medium being 7.2.  
   
   
       8 . The method as claimed in  claim 1 , wherein pathogenic fungus for control testas are at least one selected from the group consisting of  F. moniliforme, C. falcatum, F. oxysporum  f sp.  ciceri, R. solani, Pythium  sp.,  Phoma sorghii, Sclerotium rolfsii, alternaria solani, curvularia lunata, sclerotinia sclerotiorum , and  aspergillus niger.    
   
   
       9 . The method as claimed in  claim 1 , wherein a concentration of the strains is ranges between 4-10 CFU/ml.  
   
   
       10 . The method as claimed in  claim 1 , wherein phosphate solubilization increases by about 428% upon a combined increase of temperature, salt, and pH.  
   
   
       11 . The method as claimed in  claim 1 , wherein phosphate solubilization increases by about 160% with increase in salt concentration.  
   
   
       12 . The method as claimed in  claim 1 , wherein phosphate solubilization increases by about 130% with increase in pH.  
   
   
       13 . The method as claimed in  claim 1 , wherein strains are selected for high pH stress tolerance at pH 9.  
   
   
       14 . The method as claimed in  claim 1 , wherein strains are selected for 55° C. temperature stress tolerance.  
   
   
       15 . The method as claimed in  claim 1 , wherein selecting bacteria demonstrating best results in terms of less seedling mortality, and better seedling germination, plant height, number of pods and seed dry weight.  
   
   
       16 . The method as claimed in  claim 1 , wherein plant promoting activity goes up by 3-400%.  
   
   
       17 . The method as claimed in  claim 1 , wherein a concentration of fungi ranges between 4-7 spores/ml of culture medium.  
   
   
       18 . The method as claimed in  claim 1 , wherein abiotic stress conditions for solubilization of phosphate comprise pH ranging between 7-9, temp ranging between 30-45, and salt concentration ranging between 0.1-4%.  
   
   
       19 . A method of preparing plant growth promoting formulation comprising at least one of bacterial strains of accession Nos. NRRL B-30486, NRRL B-30487, and NRRL-B 30488, and a carrier, comprising: 
 (a) growing bacteria in a culture, individually to a concentration of about Log 8 to 11 cfu/ml, preferably log 9 to 10 cfu/ml, followed by mixing the cultures in equal ratio if preparing a consortium,    (b) diluting the culture with water in the ratio of 1:50 to 1:150, preferably 1:100, containing approximately Log 8-9 cfu/ml of bacteria,    (c) spraying the culture on freshly homogenized carrier at about 1-3 liter of the culture/ton of freshly homogenized carrier and mixing,    (d) churning windrows daily at least twice a day for about 2 days, to increase the temperature of the windrows up to 70-75° C.,    (e) spraying spent wash or water into the churning windrow for about 40 days to maintain a moisture level of about 55-65%,    (f) churning the windrows further for another 3-5 days, now to reduce the moisture and temperature of fermented product to about 30% and 40-45° C., and    (g) packaging plant growth promoting bioinoculant ready for application.    
   
   
       20 . The method as claimed in  claim 19 , wherein the carrier is selected from the group consisting of fresh sulphinated press mud and carbonation press mud.  
   
   
       21 . The method as claimed in  claim 19 , wherein a culture medium is NB medium.  
   
   
       22 . The method as claimed in  claim 19 , wherein the mixture is homgenized manually and/or by using an aero tiller.  
   
   
       23 . The method as claimed in  claim 19 , wherein the formulation demonstrates maximum viability under varied storage, greenhouse or field conditions.  
   
   
       24 . The method as claimed in  claim 19 , wherein a ratio of the said strains is about 1:1:1.  
   
   
       25 . The method as claimed in  claim 19 , wherein the formulation is applied on plants, seeds or soil.  
   
   
       26 . The method of promoting plant growth, comprising: 
 applying a bioinoculant formulation comprising at least one of bacterial strains of accession Nos. NRRL B-30486, NRRL B-30487 or NRRL-B 30488, and a carrier, in a liquid or dry form to seeds, plants, or soil.    
   
   
       27 . The method as claimed in  claim 26 , wherein the bioinoculant also contains gums or sugars to improve adhesion.  
   
   
       28 . The method as claimed in  claim 26 , wherein the strains are in a ratio of 1:1:1.  
   
   
       29 . The method as claimed in  claim 26 , wherein the formulation demonstrates maximum viability, under varied storage or greenhouse or field condition.  
   
   
       30 . The method as claimed in  claim 26 , wherein the carrier is selected from the group consisting of fresh sulphinated press mud and carbonation press mud.  
   
   
       31 . The method as claimed in  claim 26 , wherein plants are fieed tested for plant growth promotion in a presence of the bacteria in a concentration of about Log 7-9 cfu/seed or about Log 6-8 cfu/gram of soil.  
   
   
       32 . The method as claimed in  claim 26 , wherein applying the formulation is alone or in combination with other chemicals which are harmless to growth and survival of bacteria.  
   
   
       33 . The method as claimed in  claim 32 , wherein the chemicals are selected from the group consisting of pesticides, fertilizers, nematicides, and herbicides, with or without for example lime pelleting to limit the severity of the effect of these materials.  
   
   
       34 . A method of making a composition useful as bioinoculant, said composition comprising one or more of bacterial strains of accession Nos. NRRL B-30486, NRRL B-30487, or NRRL-B 30488 and a carrier, the method comprising: 
 (a) culturing said bacterial strains in a growth medium to log phase,    (b) diluting the said culture with water in a ratio ranging between 1:10 to 1:100000,    (c) mixing the diluted culture with an inert powdered carrier, with a moisture level of the mixture ranging between 20-40 on a wet basis,    (d) incubating the mixture for at least about two days, while maintaining constant moisture level in the mixture,    (e) increasing a bacteria count in the said mixture to a range of about log 4-10 CFU/g of carrier, and    (f) monitoring a survival rate of the bacteria over a period of at least one year in the said composition, wherein the bacterial strains are present in a range from about Log 7 to 9 CFU/g of carrier, showing long survival rate of microbes as inoculate.    
   
   
       35 . The method as claimed in  claim 34 , wherein the carrier is at least one selected from the group consisting of vermiculite, charcoal, a mixture of fermented sugar factory sulphitation press mud and distillery spent wash, sugar factory carbonation press mud, rice husks, carboxymethyl cellulose, peat, perlite, talc, and polyvinyl pyrrolidone.  
   
   
       36 . The method as claimed in  claim 34 , wherein carriers are selected from the group consisting of vermiculite, charcoal and fermented press mud.  
   
   
       37 . The method as claimed in  claim 34 , wherein the bacterial count is about Log 8 CFU/g of carrier.  
   
   
       38 . The method as claimed in  claim 34 , wherein plants for growth promotion are selected from the group consisting of chickpea,  A. esculentus, C. sativus, Z. mays, Triticum aestivum, Glycine max, Pisum sativum  and  Impatiens balsamina.    
   
   
       39 . The method as claimed in  claim 34 , wherein a ratio of strains is about 1:1:1, in case of a consortium.  
   
   
       40 . The method as claimed in  claim 34 , wherein a growth medium is NB medium.  
   
   
       41 . The method as claimed in  claim 34 , wherein bacteria are individually grown to a concentration of about Log 9 to 10 CFU/ml followed by mixing the cultures at a ratio of about 1:1:1.  
   
   
       42 . The method as claimed in  claim 34 , wherein the culture is diluted with water at a ratio of 1:10, containing approximately Log 8-9 cfu/ml.  
   
   
       43 . The method as claimed in  claim 34 , wherein moisture of the product is regulated with windrows.