The microbial world is a microcosm whose activities are of central importance to the biosphere. Microbial products contribute to environment, plant, public, and soil health. There is a striking diversity of microorganisms in their ecological and physiological specialisations. They have evolved to cope with and flourish in almost every niche, no matter how inhospitable. Microorganisms also form a range of associations with other microbes and with other plants and animals. They can be pathogens, parasites, symbionts, commensales and saprophytes, and thus, their ecological influence infiltrates into all trophic levels of life and gamut of possible ecosystems. Microbes have proved an exceptionally rich source of new products, and there is every indication that they will continue to be so in the future. Therefore, exploration of biodiversity for novel microbes which are ecologically significant or are of economic value is of importance. This has prompted microbiologists to continue to search for novel useful microbes from sources that remain uncharacterised.

India is one of the few countries in world, which has contributed richly to the International livestock gene pool and improvement of animal population in world. Cattle and buffalo contribute nearly 15% of the gross national income. The country possesses 23% of world bovine population. According to Hindu mythology as well as the Indian traditional medical practices (both the classical systems like Ayurveda and Siddha and the oral practices of the rural villagers) cow's milk has rejuvenatory health protecting and health promotery properties and hence has been said as the best one among vitalisers [Caraka-Samhita, Editor-translator P. Sharma, Chaukhambha Orientalia, Varanasi, India, volume 1, p. 213 (1981)]. Milk may be defined as the normal secretion of the mammary gland of the mammals. Milk as it is secreted by the gland of the mammals is free of microorganisms. However, microorganisms associated with the teat move up the teat canal and into the interior of the udder [J.C. Olsen and G. Mocquot. Milk and milk products. In: International commission on microbiological specifications for foods. Microbial ecology of foods. Food commodities. Vol. 2. New York: Academic Press (1980) pp. 470-486]. This causes even aseptically drawn milk to contain microorganisms, mostly bacteria. Bacteria in aseptically drawn milk are usually limited in number and include mostly micrococci, lactococci, staphylococci, streptococci, and bacillus [F.L. Bryan, Journal of Food Protection, Volume 46, pp. 637-649 (1983); R.A. Ledford. Raw milk and fluid milk products. In: Applied dairy microbiology. Eds. E.H. Marth and J.L. Steele, New York: Marcel Dekker, Inc. (1998) pp. 55-64].

It has been known for the past more than four decades that many of the bacteria that occur commonly in milk find it a relatively unfavourable medium and it would thus appear that milk has pronounced selective properties [T. Gibson and Y.A. Abd-El-Malek, Canadian Journal of Microbiology, Volume 3, pp. 203-213, (1957)]. Thus the bacterial flora that have invaded in the teat and/or udder must have the persistence ability for survival and multiplication, under these suboptimal conditions. Therefore, work on the milk described pertains to bacterial flora persisting in the teat and/or udder, which have gained entrance into the aseptically drawn milk, in our attempt to search for novel microbes, from an ecological niche that remain uncharacterised.

Improving soil fertility is one of the most common tactics to increase agricultural and forest production. We have isolated plant beneficial bacteria from cow milk. Inoculation of seeds or soil with beneficial microorganisms for crop improvement has been practised for a number of years. A variety of mechanisms have been identified as being responsible for such plant growth promoting activity. For example, certain microorganisms indirectly promote plant growth by inhibiting the growth of deleterious microorganisms; or directly enhance plant growth by producing growth hormones; and/or by assisting in the uptake of nutrients by the crops, e.g., phosphorus (P) [C.S. Nautiyal et al., FEMS Microbiology Letters, Volume 182, pp. 291-296  (2000)]. However, a major factor in the unsuccessful commercialisation of bioinoculants has been the inconsistency of field test results as their establishment and performance are severely effected by environmental factors especially under stress conditions encountered in soil e.g., salt, pH, and temperature. Therefore, it would be desirable to provide stress tolerant bacterial strains as bioinoculants [C.S. Nautiyal, Biocontrol of plant diseases for agricultural sustainability. In: Biocontrol potential and its exploitation in sustainable agriculture. Volume 1, Eds. R.K. Upaahyay, K.G. Mukerji, and B.P. Chamola, Kluwer Academic/Plenum Publishers, New York (2000) pp. 9-23]. Plant growth promoting microorganisms include but are not limited to Rhizobium, Pseudomonas, Azospirillum, and Bacillus  etc.

While work on microbiology of the milk so far has been on psychrotrophic bacteria because of their importance in milk and dairy products [M.A. Cousin. Journal of food protection. Volume 45, pp. 172-207 (1982); R.A. Ledford. Raw milk and fluid milk products. In: Applied dairy microbiology. Eds. E.H. Marth and J.L. Steele, New York: Marcel Dekker, Inc. (1998) pp. 55-64], no bacterial strain has been previously found from cow which has the ability to control phytopathogenic fungi, promote plant growth, tolerance for abiotic stresses, solubilise phosphate under abiotic stress conditions. Accordingly, there has been no clear indication heretofore that any bacteria isolated from cow might act as a biocontrol agent, and certainly no showing of direct, bacterial-mediated stimulation of plant growth per se. Nevertheless, a bacterial strain capable of promoting plant growth, tolerance for abiotic stresses, solubilise phosphate under abiotic stress conditions, if one were isolated, could find immediate application, e.g., in soils affected by phytopathogens, poor nutrient availability like phosphorus, and environment stresses etc., did not result in a desired improvement in crop development, additionally, no procedure for the selection of such bacterial strain has been reported. We have found by direct comparison on a variety of plant types that the unique combination of selected bacterial strains is effective in the enhancement of plant growth and health.

Our work relates to method for screening useful bacteria from the milk of human, Sahiwal cow, Holestien cow and buffalo and application thereof for promoting plant growth. Six hundred bacterial strains were screened for their ability to inhibit growth of plant pathogenic fungi Colletotrichum falcatum, Sclerotium rolfsii, Alternaria solani, Penicillium sp., Pythium aphanidermatum, Phytopthora palmivora, Curvularia lunata, Sclerotinia sclerotiorum, and Aspergillus niger under in vitro  conditions as follows: Four single bacterial colonies on NA plates were streaked around the edge of a 90-mm diameter petri plate and the plates were incubated it at 28oC for two days. An agar plug inoculum of the fungi to be tested (5-mm square) was then transferred to the centre of the plate individually from a source plate of the fungi. After incubation for 5 to 7 days inhibition zones were readily observed in the case of bacterial strains having the biocontrol activity as the fungal growth around the streak was inhibited. While in case of bacterial strains not having biocontrol activity, fungal growth around the streak was not inhibited and the fungi grew towards the edge of the plate. We have discovered that % of bacterial strains showing biocontrol activity against phytopathogenic fungi was maximum in Sahiwal cow, followed by human, Holestien cow and buffalo (Table 1). From this parameter milk of Sahiwal cow was superior to human, Holestien cow and buffalo. The 3 strains Bacillus lentimorbus  NBRI0725, Bacillus subtilis  NBRI1205, and Bacillus lentimorbus  NBRI3009 isolated from Sahiwal cow milk have the ability to control phytopathogenic fungi and promote plant growth under field conditions, tolerance for abiotic stresses, and solubilise phosphate under abiotic stress conditions.  

India has the largest area under sugarcane among cane growing countries of the world. Its sugar industry ranks as the second major agro-industry in the country. Press mud is a "waste" product obtained during sugar manufacture. Many of these sugar mills uses yeast to ferment molasses for producing ethyl alcohol. Spent wash, a distillery effluent of the fermentation process along with press mud are considered as pollutants and therefore can not be disposed off into the environment. We have invented a process of manufacturing plant growth enhancer which utilises press mud and/or spent wash as a raw material. Fermentation of press mud and/or spent wash, using bacteria isolated from Sahiwal cow milk results into an value addition product, useful for enhancing growth of plants. Our plant growth promoting microbes propagates well in the fermented press mud. The following procedures were performed to utilise sugar factory sulphitation press mud and distillery spent wash as carrier for preparing at commercial scale, after its fermentation using consortium of of our novel microbes. About 300 tons of fresh sulphinated press mud, obtained while clarifying sugarcane juice with lime and sulphur dioxide, is laid out on cemented floor with width of 2.5 meter, 1.5 meter tall, and length of 150 meter windrows. The press mud was churned and homogenised, either manually or by the help of an aero tiller before adding 150 kg consortium i.e., 500 gm of the consortium/ton press mud, and mixing again. Within 2-3 days, temperature of the windrow goes up to 70-75^oC. Thereafter, the windrows are churned twice a day and spent wash is sprayed on daily basis to maintain 55-65% moisture, for up to 40 days. After about 40 sprays the spraying of spent wash is stopped and windrows regularly turned for 3-5 days to reduce the moisture of the fermented product to about 30%. Usually after 45 days, the temperature of the windrow goes down to 40-45^oC. The product at this stage is totally fermented and ready for its application. The procedures for fermenting sugar factory carbonation press mud as carrier for preparing at commercial scale, using consortium is same as described for sulphinated press mud, except water was used instead of spent wash, to maintain the moisture during fermentation

Press mud like any other organic manure affects the physical, chemical and biological properties of the soils. It also helps to increase water stable aggregates in soils. Our product significantly enhances plant growth of wide range of plants representing economically important horticulture, floriculture, and agronomic crops in the range of 10-60%. Table 1 show increases in the number of tillers, plant height, girth of cane, millable cane, and cane yield inoculated with our consortium, compared with un-inoculated control. We have developed the technology using novel microbial synergistic mixture of microbes isolated from Sahiwal cow's milk and cost effective manufacturing procedures.  The technology has been exclusively developed in view of the usage of raw materials available at sugar factory which makes our procedure sugar factory friendly and highly cost effective. Global patent application (NF404/2001) has been filed by Council of Scientific & Industrial Research for our invention. Agricultural and environmental industry would therefore clearly benefit from our simple, less expensive method of microbial inoculants for plants, seeds and soil as it may help in economising crop production and maintenance of soil structure, fertility and healthy ecosystem. Collectively these activities will determine the ability of the soil to sustain plant production.
 

Table 1. Screening of bacterial strains isolated from milk under in vitro conditions for ability to suppress plant pathogenic fungi
                                                                                

 Table 2. Effect of bioinoculant on growth and yield of sugarcane var. Co 89003

 * Data analysed by sugar mill. Data on juice was not provided, however due to enhanced yield it is obvious that juice must have been appropriately more in inoculated sugarcane, compared with uninoculated control.