1. Plant growth promoting bioformulation of Rhizobium for use in drought susceptible leguminous plants, said bioformulation comprises Rhizobium aegyptiacum in yeast extract-mannitol broth (YEMB) and one or more self protectant, wherein said bioformulation enhances crop yield naturally, improve productivity per area, mitigate contamination of soil, make crop withstand in drought condition, increase soil fertility, and promote antagonism and biological control of phytopathogenic organisms.

2. The plant growth promoting bioformulation of Rhizobium as claimed in claim 1, wherein said bioformulation comprises Rhizobium aegyptiacum in YEMB and one or more self protectant in the range of 6.3% to 12.7%.

3. The plant growth promoting bioformulation of Rhizobium as claimed in claim 1, wherein said one or more self protectant is selected from PEG, glycerol, lactose or a combination thereof.

4. The plant growth promoting bioformulation of Rhizobium as claimed in claim 1, wherein said bioformulation comprises Rhizobium aegyptiacum in YEMB, PEG in the range of 0.3% to 0.7%, glycerol in the range of 1% to 3%) and lactose in the range of 5% to 9%.

5. The plant growth promoting bioformulation of Rhizobium as claimed in claim 1, wherein said bioformulation comprises Rhizobium aegyptiacum in YEMB, PEG 0.5%, glycerol 2% and lactose 7%.

6. The plant growth promoting bioformulation of Rhizobium as claimed in claim 1, wherein said bioformulation has viable cell count of 1.84xl013 cfu/ml.

7. The plant growth promoting bioformulation of Rhizobium as claimed in claim 1, wherein said bioformulation has viable cell count of 6.2 x 109 cfu/ml after 6 months.

8. The plant growth promoting bioformulation of Rhizobium as claimed in claim 1, wherein said drought susceptible leguminous crop can be selected from drought susceptible variety of Vigna mungo, Cicer arietinum, lens culinaris, Glycine max, Phaseolus vulgaris, Cajanus cajan, Vigna radiata, Vigna unguiculata, Pisum sativum.

9. The plant growth promoting bioformulation of Rhizobium as claimed in claim 1, wherein said bioformulation has shelf life of more than 6 months.

10. A process to obtain the bioformulation as claimed in claim 1, wherein said process comprises (a) isolating Rhizobacteria from the root nodules of a desired drought tolerant leguminous crop cultivar grown in semi-arid region, (b) characterizing the isolated Rhizobacteria for desired characteristics by morphological, biochemical and molecular testing, (c) screening of isolated Rhizobacteria for drought tolerance, (d) cross inoculating a drought susceptible crop cultivar of a desired drought susceptible leguminous crop to examine desiccation tolerance of the screened Rhizobacteria and tested for drought tolerance, (e) culturing the screened isolated Rhizobacteria in YEMA broth containing one or more self protectant, (f) preparing a bioformulation with viable cell count of 1.84xl013 cfu/ml.

11. The process to obtain the bioformulation as claimed in claim 10, wherein said desired drought tolerant leguminous crop cultivar grown in semi-arid region is Vigna mungo var PU-1.

12. The process to obtain the bioformulation as claimed in claim 10, wherein said Rhizobacteria is R. aegyptiacum.

13. The process to obtain the bioformulation as claimed in claim 10, wherein said desired drought susceptible leguminous crop can be selected from drought susceptible variety of Vigna mungo, Cicer arietinum, lens culinaris, Glycine max, Phaseolus vulgaris, Cajanus cajan, Vigna radiata, Vigna unguiculata, Pisum sativum.

14. The process to obtain the bioformulation as claimed in claim 10, wherein said one or more self protectant is selected from PEG, glycerol, lactose or a combination thereof.

FIELD OF THE INVENTION
The present invention relates to the field of agronomy. The present invention relates to plant growth promoting bioformulation of Rhizobium. More specifically, the present invention relates to bioformulation of Rhizobium for leguminous plants, in particular. The bioformulation of the present invention enhances crop yield naturally, improve productivity per area, mitigate contamination of soil, make crop withstand in drought condition, increase soil fertility, and promote antagonism and biological control of phytopathogenic organisms. The present invention also provides a method to obtain plant growth promoting bioformulation of Rhizobium for leguminous plants in particular for arid and semi-arid regions.
BACKGROUND OF THE INVENTION
Global population will strike about 9.6 billion by 2050 and the world have to deal with enormous challenges such as growing demands for energy, inconsistency in climatic conditions, food scarcity to name a few. Due to change in climatic condition and its associated abiotic and biotic stresses, globally crop systems faces serious implication regarding productivity (Yadav et ah, 2015). To face these challenges, sustainable production and consumption is necessary.
Soil is the main source of nutrients for plants. Plants extract nutrients from soil to maintain healthy growth. These nutrients include nitrogen and other micro-nutrients. Nitrogen is an essential element for most plants as it plays a role in the synthesis of amino acids, proteins, nucleotides, nucleic acids, chlorophyll, co-enzymes and in the overall growth and health of the plant. Soil has to be fortified with the deficient elements in order to improve growth rates and yields obtained from plants. Cultivated crops require relatively large amounts of nitrogen with a notable exception to the plants from the legume family.

Legumes play an important role in sustainability development as they have major roles in food systems, nutrition and possess a potential to fix nitrogen with the help of symbiotic microorganism. Nitrogen fixation in the nodules of leguminous plants require a symbiotic relationship between the plant and native bacterial within the soil. Symbiotic microorganisms in legumes are very specific to their host crop. A considerable number of bacterial species, most associated with vegetable rhizosphere, are capable of having some beneficial impact on vegetable growth. Therefore, their use as biofertilizers, as agents controlling other organisms and in the improvement of agriculture, has been the subject of research for several years.
The family Leguminosae constitute about 650-750 genera, 18,000-19,000 species of trees, shrubs, climbers and herbs. Chickpea, mung bean, black gram, lentils, peanuts, pigeon pea, soybean, cowpea are some commonly cultivated legumes. Legumes are the richest source of vegetarian protein as their seeds contain 200-250g protein/kg. Legumes and their products such as food, feed, fertilizers, paper, biodiesel, drink, and medicines contribute significantly to the world's economy. Legumes are best known for their nitrogen fixation activity along with nitrogen fixing bacteria. Fixation of atmospheric nitrogen can be done both symbiotically and non-symbiotically by microbes. Legumes through symbiotic association with nitrogen assimilating bacteria have an utmost activity in assimilating nitrogen, enhancing soil fertility as well as reducing the use chemical fertilizers. Additionally, plant growth promoting rhizobacteria (PGPR) is a heterogeneous group that helps in plant growth and development. PGPR helps in producing numerous plant growth regulators, mineral nutrients solubilization, organic matter decomposition, formation of soil structure, root development and bio control of soil and seed borne plant pathogen.
The second most vital pulse in world is Chickpea (Cicer arientinum) which is cultivated in more than 41 countries and cover about 11 million hectares of land in cultivation. It is regarded as dietary nutrition as its mean protein content is about

24%. It belongs to family Fabaceae, and cool season legume crop. Besides protein, chickpea also contain considerable amount of nutrients such as magnesium, zinc, iron, phosphorus and calcium. It also has a good amount of fibre, P carotene and some unsaturated fatty acids. Chickpea cultivation helps sustaining soil fertility by assimilation of nitrogen. Almost 140kg/ha/year nitrogen is fixed by chickpea.
India is the largest producer as well as consumer of V. mungo (black gram). It produces urad about 1.5 million tonnes annually from about 2.5 million hectares of area with 400 kg/ hectare average productivity. Besides India, it is cultivated in Afghanistan, Myanmar, Pakistan and Bangladesh.
The average yield of black gram in 2013-2014 is only 360 kg/ha as against the recommended yield of the crop i.e., 15-20 quintals/ ha in Rajasthan as reported by the Commissionerate of Agriculture, Govt, of Rajasthan, Jaipur.
However, productivity of both crops chickpea and black gram is reduced due to drought stress. Drought stress is a major abiotic stress affecting in the agricultural economy of India as here more than 68% of people are majorly dependent upon agronomy. In India, Rajasthan is considered as one of the major dryland areas. In chickpea, diseases followed by drought stress are the most significant growth-resisting factor. Black gram (Vigna mungo) being an important rainfed pulse crop in India, suffers a yield loss of 20-30%, because of drought at flowering stage. Drought stress declines growth and significantly reduces the yield.
Legume crops inoculated with beneficial microorganism may enhance the drought tolerance potentiality of plants that grown on arid-semiarid areas. Legumes growing in arid or semiarid regions harbour symbiotic microorganism that shows higher tolerance capability against adverse condition such as salinity, drought, high

temperature, pH. So, selection of effective stress tolerant microorganism strain could help in restoring soil fertility and plant development.
It is well known that within a population of Rhizobium bacteria there is a certain variation of the ability to resist dry and hot conditions, and that this variation depends on the fact that the bacteria are not uniform but appear as different strains with different resistances. Cross nodulation of legumes with more efficient strains is important as they are effective in dealing with the growth of field crop. Therefore, in the area of leguminous plant inoculation, cross nodulation grouping of legumes is very important as they are effective in dealing with the growth of field crop. Cross inoculation restricts the dominance of a particular native strain and permits the inoculation of potential strains.
Rhizobium symbiosis with legumes might play a major function and need to be investigated in plants for enhancing the stress tolerance. Plant interaction with different phosphate solubilizing microorganism, diazotrophs is progressively being understood, that they promote plant development in few different ways including fixation of nitrogen, enhancing phosphate availability, by discharging plant development controllers and additionally by competing with plant pathogens. The symbiotic interaction between leguminous plants and bacteria shows a series of interaction such as signaling through Nod factor, cell-surface interaction, EPS (Extra polysaccharide), phenolic compounds, IAA (indole acetic acid) etc. As a result of stress condition, symbiosis is also influenced thus, reducing symbiotic nitrogen fixation rate under drought conditions. Furthermore, excessive stress conditions like abiotic stresses (drought stress, salt stress, soil alkalinity/acidity and heavy metals) may influence the symbiont characteristics and rhizobia growth. But a few rhizobia strains are more effective to tolerate stress and during stress conditions maintain symbiosis by forming effective nodules. The rhizobial partner is able to create exogenous or endogenous proteins/osmolytes that can improve stress conditions.

Rhizobium sp. produced EPS (extra polysaccharide) might be catabolized under nutrient deficient conditions and protects it from desiccation. EPS additionally plays an important role in signal for formation of nodule along with Nod factors. In addition, it can mitigate drought stress by activating the system of antioxidant enzyme in the case of host plant and form biofilm that support the symbiont from condition of drought stress. Enzymes like peroxidase and catalase having antioxidant properties that alleviate during drought stress and protect plant cell from damage. The rhizobacteria have both enzymatic and non-enzymatic antioxidant system which ameliorating water deficit stresses. In plants, rhizobia inoculation enhances the antioxidant enzyme production under stress condition. The SOD, CAT, POD and APX production elevates though the inoculation of PGPR in plants therefore H2O2 and MDA content level declines under the stress condition. During the activation of non-enzymatic and enzymatic antioxidant system enhances the tolerance capacity of host plant to drought.
In the semi-arid and arid regions, several rhizobacterial strains are stress tolerant which shows good strategies to tolerate higher level of stress and these strains may be utilized for better crop yield. Bacterial strains are extremely adaptive to their specific environment conditions and also are region specific. Identification and characterization of tolerant bacteria is more important for higher crop yield and understanding the diversity and distribution of bacterial population for specific environment. For phylogenetic and taxonomic studies of microbial population, sequence analysis of 16sRNA gene is widely used by molecular technique. So that, tolerant bacteria enhance the plant growth because of enhancement in plant microbe interaction and it can simply regulate to natural environment. Several studies indicate that the interaction between legumes and Rhizobium sp. is species specific other than for maximum nitrogen fixation; same plant species prefer different strains of rhizobia. Rhizobium sp. isolated from drought tolerant leguminous crop grown in the semi-arid

climatic zone may yield important strain, effective in fulfilling the tolerance to drought stress and be adaptive to local climate condition.
Shweta Chaudhary et ah, 2019; Screening of Vigna mungo (L.) Hepper varities under drought stress; Plant Cell Biotechnology and Molecular Biology, 20 (1&2) studied three varieties of V. mungo and screened for their growth under drought stress. Drought stress reduced the plant growth and harvest index. The yield of plants in normal soil is better even under water deficit condition as compared to plants grown in autoclaved soil indicating a definite role of soil microorganisms in drought mitigation.
Shweta Chaudhary et ah, 2019; Cross inoculation with beneficial Rhizobium strain promotes plant growth in Vigna mungo; Vegetos, 32(2) studied effect of cross inoculation of Rhizobium aegyptiacum in Vigna mungo cv. T9 and found that R aegyptiacumcan be successfully cross inoculated in drought susceptible agronomically desirable black gram varieties for better growth.
Renuka Saraf et ah, 2018; Isolation and characterization of osmotolerant Rhizobium sp. from Vigna mungo (L.) Hepper grown in semiarid region; Vegetos 31 (special) isolated bacteria from the root nodule of the Vigna mungo, grown in a semi-arid region. The isolates were cultured at different concentration of NaCl (0-5%) YEMA supplemented medium. Out of several isolates that were able to tolerate high salt concentration, the colony characteristics of only one isolate was identical to Rhizobium. Several biochemical assays were done for characterization. Molecular characterization by 16 SrRNA confirmed its identification. The aim of the isolation of rhizobial bacteria associated with V. mungo included the assessment of osmotolerant Rhizobium sp. so as to improve the productivity in susceptible plants on cross inoculation.

US43 06027 Arelates to Rhizobium strains having good infecting and nitrogen-fixing characteristics and which are resistant to fungicides. These strains are produced by cultivating a Rhizobium strain sensitive to a particular fungicide in the presence of an amount of the fungicide, and for a time less than that sufficient to kill the entire Rhizobium population but sufficient to kill a majority of the population. The remaining Rhizobium population is isolated and recultured in the presence of an increased amount of the fungicide, again an amount and a time less than sufficient to kill the entire Rhizobium population, but sufficient to kill a majority of the population. This procedure is repeated for sufficient passages, with increasing amounts of said fungicide, to provide a Rhizobium strain sufficiently resistant to said fungicide so that the Rhizobium strain multiplies and enters into a nitrogen-fixing symbiosis in the presence of agriculturally effective amounts of the fungicide.
US3616236A discloses the production of Rhizobium strains having good infecting and nitrogen-fixing characteristics by cultivating the strain, then subjecting it to drying whereby those strains which are sensitive to drying are destroyed and those strains which are resistant to drying survive. The surviving strains are then re-cultivated and subjected to at least one more drying treatment.
CN10716426IB discloses the Rhizobium leguminosarum for promoting villose vetch to increase the invention discloses one plant and its application. The bacterial strain number of the Rhizobium leguminosarum is ml-10-3, is CGMCC No. 11877 in the deposit number of China Committee for Culture Collection of Microorganisms's common micro-organisms center. It is demonstrated experimentally that plant height, plant fresh weight are compared and are dramatically increased with the plant height of Rhizobium leguminosarum ACCC16505, plant fresh weight than not meeting bacterium control, inoculation Rhizobium leguminosarum H10 after villose vetch inoculation Rhizobium leguminosarum ml-10-3.

There are very few studies on Rhizobium strains and its application for the manufacture of biofertilizer, in particular for arid and semi-arid regions. The Legume-Rhizobium interaction is very specific. Native strains evolves over time and naturally form symbiotic relationships with particular legume crops growing in that area. However, native strains do not always have optimum plant growth promoting characteristics. Thus, there is a requirement of selection of niche-based locally adapted strains. Cross inoculation of legumes with more efficient strains is restricts the dominance of a particular strain and permits the inoculation of potential strains thus promoting growth and yield. Therefore, under agriculture field condition, capability of different rhizobium strains should be evaluated case by case.
The present invention addresses this issue by providing specifically adapted niche-based novel strains of Rhizobium.
OBJECTS OF THE INVENTION
Accordingly, the main object of the present invention to provide a plant growth promoting bioformulation of Rhizobium.
Another object of the present invention is to provide a plant growth promoting bioformulation of Rhizobium for leguminous plants, in particular, in arid and semi-arid regions.
Yet another object of the present invention is to provide a plant growth promoting bioformulation of Rhizobium for leguminous plants, in particular, in arid and semi-arid regions that enhances crop yield naturally, improve productivity per area, consume smaller amounts of energy, mitigate contamination of soil, make crop withstand in drought condition, increase soil fertility, and promote antagonism and biological control of phytopathogenic organisms.

Yet another object of the present invention is to provide a method to obtain plant growth promoting bioformulation of Rhizobium for leguminous plants, in particular, in arid and semi-arid regions.
SUMMARY OF THE INVENTION
It will nevertheless be understood that no limitation of the scope of the invention is thereby intended by way of embodiments and examples. Such alterations and further modifications in the present invention, and such further applications of the principles of the invention as would normally occur to those skilled in the art are to be construed as being within the scope of the present invention.
It will be understood by those skilled in the art that the summary of the invention provided herein is exemplary and explanatory of the invention and are not intended to be restrictive thereof. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. The composition, methods, and examples provided herein are only illustrative and not intended to be limiting.
The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such a process or method. Similarly, one or more steps of method or components preceded by "comprises... a" does not, without more constraints, preclude the existence of other, steps or components. Appearances of the phrase "in a preferred embodiment", "in an embodiment", "in another embodiment" and similar language throughout this specification may, but not necessarily do, all refer to the same embodiment.

The present invention provides a plant growth promoting bioformulation of Rhizobium. The bioformulation of the present invention enhances crop yield naturally, improve productivity per area, consume smaller amounts of energy, mitigate contamination of soil, make crop withstand in drought condition, increase soil fertility, and promote antagonism and biological control of phytopathogenic organisms.
The invention provides a bioformulation of Rhizobium for leguminous crops in particular in arid and semi-arid regions.
In a preferred embodiment, the invention provides a bioformulation of Rhizobium for black gram and chickpea in arid and semi-arid regions.
In another preferred embodiment, the present invention provides bioformulation of Rhizobium aegyptiacum for black gram and chickpea crop that enhances crop yield naturally, improve productivity per area, consume smaller amounts of energy, mitigate contamination of soil, make crop withstand in drought condition, increase soil fertility, and promote antagonism and biological control of phytopathogenic organisms.
The present invention also provides a method to obtain plant growth promoting bioformulation of Rhizobium for leguminous crops in particular in arid and semi-arid regions. The method comprises of isolating and selecting desired bacterial strains, putting the selected bacterial strains to nodulation test, selecting isolates capable of forming nodules with desired characteristics, characterization of bacterial strains forming nodules with desired characteristics for plant growth promoting characteristics, selecting and culturing drought tolerant Rhizobium isolates, cross inoculating germinated seeds of desired legume with said culture of Rhizobium

isolate, physiological and biochemical testing of cross inoculated seeds and field trials.
The characterization of bacteria is done by various biochemical tests such as catalase test, urease test, gelatine test, HCN production, MRVP, indole test, citrate test, carbohydrate test. The selected bacteria also possess PGPR properties like phosphate solubilization, siderophore production, IAA production, EPS production and have antifungal activity against certain disease occurred in pulses. The selected strain have also been screened in PEG for its drought tolerance capability to select desiccation tolerant strain.
In a non-limiting embodiment, root nodules of black gram varieties are processed for Rhizobium strain isolation and selection. Selection for drought tolerance is done on YEMA plates containing PEG. The best growing isolates are selected and put to nodulation tests. After re-inoculation, some isolates gave ineffective white nodules in plants, few isolates fail to form nodules and some isolates provide viable plant.
After the isolation process, bacterial isolates are confirmed by biochemical and molecular characterization. PGPR characterization have been carried out and observed that the selected bacteria have capability to produce siderophore, EPS, HCN and also help in phosphate solubilization. The selected bacteria also possess an antifungal property and help in reduction of disease infestation.
After characterization, the selected drought tolerant Rhizobium isolate are used for cross inoculation by pot experiment. For cross inoculation, earthen pots are filled with the mixture of autoclaved soil and sterile, germinated seeds of black gram are treated with bacterial culture and placed in the pots. Physiological and biochemical experiments are conducted after cross inoculation. Quantitative RTPCR study of

candidate genes reported in drought tolerance in response to drought stress in black gram are conducted to check the gene expression level.
Rhizobium aegyptiacum sequence has been submitted to NCBI MK333259. After submission of bacterial sequence, the bacterial culture has been submitted at NCCS National Centre for Cell Science NCCS, Pune and strain designation number BVVM-78 Processing Reference Number -PRN: D_FEB_19_051. After the 16s rRNA, taxonomic position confirmation of Rhizobium aegyptiacum has been done with atpD, glnA and recA gene and confirmed the Rhizobium sp. and these gene sequences submitted at Banklt NCBI submission. GenBank accession number for atpD gene sequenced from Rhizobium aegyptiacum is MK774656, glnA gene sequenced from Rhizobium aegyptiacum is MK848284 and recA gene sequenced from Rhizobium aegyptiacum is MK830999.
The field trials have been conducted at Krishi Vigyan Kendra KVK, Banasthali Vidyapith. Bacterial inoculants have been applied to black gram seeds and cross inoculated black gram seeds were grown in the field. The cross inoculated seeds showed 23% yield increment of black gram as compare to control.
In another non-limiting embodiment, isolated desiccation tolerant Rhizobium sp. from Vigna mungo PU-1 was cross inoculated in cultivar of chickpea. To check the effect of cross inoculation in chickpea, re-isolation and characterization of same bacteria was done. It was observed that Rhizobium aegyptiacum was successfully cross inoculated in Cicer arientinum variety GNG 1958. This isolated bacteria from chickpea is further sequenced and submitted as Rhizobium aegyptiacum strain BVVM-78 to NCBI with accession no. MT980913.

BRIEF DESCRIPTION OF DRAWINGS
Fig. 1A shows effect of different PEG concentration of bacterial isolate at a
concentration rate of 0, 10, 24% respectively. Fig. IB, 1C, ID shows biochemical,
molecular and PGPR means on 0, 10, 24% PEG concentration, respectively using
FESEM (Field emission scanning electron microscope) of the bacterial isolate.
Fig. 2 depicts the morphological characterization of selected bacterial spp.
Rhizobium. Fig. 2A shows bacterial colony on YEMA supplemented with congo red
dye. Fig. 2B shows bacterial colony on YEMA supplemented with BMB dye.
Fig 3A, 3B, 3C shows PGPR characterization of R aegyptiacum based on IAA
production, phosphate solubilization and EPS production.
Fig. 4 shows comparative growth in uninoculated, uninoculated, Bradyrhizobium
inoculated and Rhizobium aegyptiacum inoculated plants in well watered condition,
drought condition and recovery stage after drought condition in Vigna mungo cv. T9.
Fig. 5 shows effect of drought stress and cross inoculation on biochemical parameters
of Vigna mungo cv. T9 leaves on (A) Chlorophyll a, (B) Chlorophyll b, (C) Total
chlorophyll, (D) H2O2 content (u mol/g), (E) Total phenolics (ug/mg fresh weight
(fw)), (F) Lipid peroxidation (uM MDA reduced/gm fw), (G) Proline content (ug/g
fw). (H) PAL activity (Picokat/ mg protein), (I) SOD activity (U/ mg protein), (J)
APX activity, (K) GPX activity, (L) CAT activity.
Fig. 6 shows quantitative RTPCR study after drought stress based on MD (malate
dehydrogenase), MT (metallothionein), TSN (tryptophan synthase), EREBP (ethylene
responsive element binding protein) and MAPK (mitogen-activated protein kinase
cascades) gene expression.
Fig. 7 shows cross inoculation of selected R aegyptiacum from V. mungo to drought
susceptible C. arietinum in treated non-autoclave, treated autoclave, and untreated
non-autoclave, untreated autoclave plants in pot experiment.
Fig. 8 shows confirmatory test of exogenous and ingenious chickpea's root
nodulating strain.

Fig. 9 shows PGPR characterization of exogenous and ingenious chickpea's root
nodulating strain.
Fig. 10 shows effect of drought stress and cross inoculation on biochemical
parameters of C. arientinum GNG 1958 on (A) H2O2 content (u mol/g), (B) Total
phenolics (ug/mg fw), (C) Proline content, (D) Chlorophyll a, (E) Chlorophyll b, (F)
Total chlorophyll.
Fig. 11 (A-G) shows quantitative RTPCR study of chickpea plants on normal,
drought and recovery stage.
Fig. 12 shows field trial results using bioformulation of the present invention on A)
Black gram, and B) Chickpea.
DETAILED DESCRIPTION OF THE INVENTION WITH NON-LIMITING EMBODIMENTS AND EXAMPLES
Accordingly, the present invention relates to plant growth promoting bioformulation of Rhizobium aegyptiacum for leguminous plants that enhances crop yield naturally, improve productivity per area, mitigate contamination of soil, make crop withstand in drought condition, increase soil fertility, and promote antagonism and biological control of phytopathogenic organisms.
The bioformulation comprises Rhizobium aegyptiacum in yeast extract-mannitol broth (YEMB) and one or more self protectant.
In an embodiment of the invention, the plant growth promoting bioformulation of rhizobium comprises Rhizobium aegyptiacum in YEMB and one or more self protectant in the range of 6.3% to 12.7%.
The one or more self protectant is selected from polyethylene glycol (PEG), glycerol, PVP, sodium alginate, lactose or a combination thereof.

In a preferred embodiment of the present invention, the bioformulation comprises Rhizobium aegyptiacum in YEMB, PEG, glycerol and lactose.
In a specific embodiment of the present invention, the bioformulation comprises Rhizobium aegyptiacum in YEMB, PEG in the range of 0.3% to 0.7%, glycerol in the range of 1% to 3%) and lactose in the range of 5% to 9%.
In a more specific embodiment of the present invention, the bioformulation comprises Rhizobium aegyptiacum in YEMB, PEG 0.5%, glycerol 2%) and lactose 7%.
The bioformulation of the present invention shows viable cell count of 1.84xl013 after 30 days, and 6.2 x 109 cfu/ml after 6 months. Thus, the bioformulation is stable for a period of more than 6 months.
The desired Rhizobacteria is isolated from the root nodules of a desired drought tolerant leguminous crop cultivar grown in semi-arid region. The isolate of Rhizobium sp. is found to be desiccation tolerant. The isolated Rhizobacteria is characterized for the desired characteristics by morphological, biochemical and molecular testing and screened for drought tolerance. The isolated bacteria with drought tolerance characteristics are cross inoculated on a drought susceptible leguminous crop to examine desiccation tolerance of the desired Rhizobium spp.
The desired leguminous crop cultivar to isolate drought tolerant Rhizobacteria is Vigna mungo var. PU-1. The desired leguminous crop to cross inoculate with the Rhizobacteria isolated from Vigna mungo var. PU-1 is the drought susceptible leguminous crop. The drought susceptible leguminous crop can be selected from drought susceptible variety of Vigna mungo, Cicer arietinum, lens culinaris, Glycine max, Phaseolus vulgaris, Cajanus cajan, Vigna radiata, Vigna unguiculata, Pisum sativum.

The isolated bacterial strain of Rhizobium spp. is R. aegyptiacum from the V. mungo var. PU-1. The isolated R. aegyptiacum is grown in a suitable culture media, containing one or more suitable self protectant. The R aegyptiacum in culture media attain a viable cell count of 1.84xl013 cfu/ml in 24-36 hours.
In a preferred embodiment, the suitable culture media is yeast extract-mannitol broth (YEMB).
The one or more self protectant is selected from polyethylene glycol (PEG), glycerol, lactose or a combination thereof.
In a preferred embodiment of the present invention, the bioformulation comprises Rhizobium aegyptiacum in YEMB, PEG, glycerol and lactose.
In a specific embodiment of the present invention, the bioformulation comprises Rhizobium aegyptiacum in YEMB, PEG in the range of 0.3% to 0.7%, glycerol in the range of 1% to 3%) and lactose in the range of 5% to 9%.
In a more specific embodiment of the present invention, the bioformulation comprises Rhizobium aegyptiacum in YEMB, PEG 0.5%, glycerol 2%) and lactose 7%.
Isolation of desired bacterial strain
The crop of Vigna mungo is selected for isolating the drought tolerant Rhizobacterium. The varieties of crop of Vigna mungo used in the invention are PU-1, PU-31, T-9. The plant variety of Cicer arietinum used in the invention is GNG 1958.
Vigna mungo varieties (var.) i.e., PU-1, PU-31 and T9 have been subjected to drought stress by withholding water for 7 days after three weeks of growth for short term

drought stress and watered again and the recovery noted after 24h of watering. V. mungo var. T9 exhibited most prominent wilting symptoms and slower recovery in comparison to V. mungo var. PU-1 and PU-31. The V. mungo var. PU-1 plants have been least affected under drought stress and recovered well as compared to the varieties T9 and PU-31. Also, under stress condition, the V. mungo var. PU-1 has shown better yield index followed by V. mungo var. PU-31 and V. mungo var. T-9. The Harvest Index of V. mungo var. PU-1, V. mungo var. PU-31 and V. mungo var. T-9 based on seed size and seed weight in normal soil and autoclaved soil is shown in Table 1. The Harvest Index in the Table 1 shows that the V. mungo var. PU-1 is drought tolerant and T-9 is drought susceptible variety of V. mungo grown in semi-arid regions.
Table 1: Harvest Index of V. mungo var. PU-1, PU-31 and T9 in normal soil and autoclaved soil

Seed size (cm) 10 seed weight (gm) Harvest index
V. mungo var. PU-1
Normal soil Well Watered 0.45 ±0.001 0.285 ±0.013 31.55 ±0.757

Drought 0.39 ±0.009 0.269 ± 0.004 29.73 ±5.13
Autoclaved soil Well Watered 0.32 ±0.036 0.242 ± 0.020 20.99 ±6.22

Drought 0.29 ±0.002 0.236 ±0.014 17.55 ±0.294
V. mungo var. PU-31
Normal soil Well Watered 0.39 ±0.006 0.277 ± 0.006 28.09 ±5.73

Drought 0.31 ±0.008 0.252 ±0.010 26.83 ±2.21
Autoclaved soil Well Watered 0.27 ±0.007 0.220 ±0.001 10.51 ± 1.04

Drought 0.26 ±0.005 0.213 ±0.005 9.06 ± 1.75
V. mungo var. T9

Normal soil Well Watered 0.21 ±0.003 0.25 ± 0.005 19.35 ± 1.216

Drought 0.13 ±0.010 0.15 ±0.006 16.43 ± 1.153
Autoclaved soil Well Watered 0.093 ±0.011 0.112 ±0.008 10.22 ±0.164

Drought 0.082 ±0.015 0.105 ±0.005 8.76 ±0.86
PU-1 variety of V. mungo has also shown better result in seed index, seed yield, plant height, branches/plants, pod/plant and seeds/pods as compared to T9.
Therefore, the isolation of desired bacteria has been done from root nodules of the drought tolerant V. mungo var. PU-1. The isolated strains have been identified as Rhizobium spp. on the basis of morphological, biochemical, molecular characterization and PGPR means.
Characterization of isolated bacteria
10 bacterial isolates have been obtained from root nodules of the drought tolerant V. mungo var. PU-1 and screened for their drought tolerant capability through Polyethylene glycol (PEG). Screening of the isolates for drought tolerance has been done with different PEG concentrations at a concentration rate of 0, 10, 24% respectively corresponding to water potential of-0.30 MPa to - 0.90 MPa that creates osmotic stress condition in the growth medium.
There has been a decrease in bacterial growth with increase in PEG concentration in the growth medium (Fig. 1A). 21% of PEG tolerant capability has been found in one isolate. Said bacterial isolate showing 21% of PEG tolerant capability is further characterized by biochemical, molecular and PGPR means on 0, 10, 24% PEG concentration, respectively using FESEM (Field emission scanning electron microscope). A concentration dependent reduction in the growth of the bacteria has

been observed with increase in PEG concentration due to decrease in osmotic potential (Fig. IB, 1C, ID).
The isolated bacterial strain with 21% PEG tolerant capability has been identified as Rhizobium sp. on the basis of morphological characterization as circular, mucoid, white in colour, convex, transparent and slime appearance, 2-4mm in diameter (Bergey's Manual of Systematic Bacteriology, 2005). The isolated bacterial strain has not absorbed the red colour on YEMA supplemented with Congo red dye as it absorbs Congo red dye weakly (Fig. 2A). On the basis of acidic reaction on YEMA supplemented with BTB dye containing medium, the selected Rhizobium sp. classified as fast-growing bacteria, Rhizobium (Fig. 2B).
The bacterial strain showing 21% of PEG tolerant capability has been confirmed as Rhizobium aegyptiacum upon molecular characterization using 16S rRNA gene sequencing submitted to NCBI (Ace. No. MK333259). The taxonomic position of Rhizobium aegyptiacum has been confirmed with protein coding genes and the sequences submitted to NCBI, viz., atpD (Ace. No. MK774656), glnA (Ace. No. MK848284) and recA (Ace. No. MK830999).
The selected R aegyptiacum from root nodule of V. mungo var. PU-1 solubilizes phosphate, produced HCN (hydrogen cyanide), EPS (exopolysaccharides), ammonia, siderophore and IAA (indole acetic acid).
The bacterial growth medium has been supplemented with optimal concentrations of mannitol 1.5%, KNO3 0.1%, L-tryptophan 2mg/ml at pH 7. It has been observed that the pH of the medium decreased from 7.0 to 5 at 36 h and 2.1 at 72 h with increase in growth. It shows IAA production by the selected bacterial strain of R aegyptiacum (Fig. 3A). The selected bacterial strain has been inoculated in Pikovskaya's broth in presence of insoluble phosphate. The absorbance has been recorded at 880nm for 12

days. During the growth of selected R. aegyptiacum in the Pikovskaya's broth, it has been observed that the amount of free phosphate increased and reached to maximum on 10th day of the growth for TCP and 10th day of the growth for zinc phosphate (Fig. 3B). FTIR study has been carried out to quantify exopolysaccharides (EPS) extracted from different carbon sources in YEMA media containing the selected bacterial strain. The FTIR spectrum of EPS showed 3600-3400cm-l broad peak, suggesting that these are associated to hydroxyl groups. Primary and secondary amine group have been obtained at 1641 and 1564cm-l. C=0 stretching vibration of carboxylic acid at 1740-1700cm-l revealed the presence of protein in EPS extracted from YEMA media containing different carbon sources such as mannitol, fructose, dextrose, myo-inositol and sucrose. The aldehyde (C=0) and alkene (C=C) groups have also been obtained at 1740 and 1648cm-l. The peak for carbohydrate C-O-C ring (1300- 1000cm-l) has been detected at 1071, 1119, 1159, 1044cm-l regions in EPS extracted from media supplemented separately with fructose, dextrose, sucrose and myo-inositol respectively. When the growth medium has been supplemented with mannitol, three peaks were noted viz., at 1050, at 1146 and at 1217cm-l. Alkanes bend (C-H) (1465-1450cm-l) has been observed at 1459cm-l and glycoside bond of polysaccharide showed peak between 900-1000cm-l (Fig. 3C).
Cross nodulation of V. mungo var. T-9 using isolated desiccation tolerant R. aegyptiacum
The desired bacterial strain of desiccation tolerant R. aegyptiacum showing 21% PEG tolerant capability has been used for cross nodulation to enhance drought tolerance of V. mungo drought susceptible species and black gram spp.
Cross inoculation has been carried out to enhance drought tolerance of V. mungo var. T-9 using desiccation tolerant R aegyptiacum and comparative study with native strain of V. mungo (Bradyrhizobium) has been done by conducting pot trials. V. mungo var. T-9 has been subjected to drought stress by withholding water after three

weeks of growth for one week as a short-term drought stress and then watered again and later, recovery noted after 24h.
To observe successful cross inoculation, physiological, biochemical and RT-PCR tests with drought candidate genes have been performed which showed enhanced growth and increase in total plant biomass in plant treated with R aegyptiacum.
Vigna mungo cv. T9 has been subjected to drought stress by withholding water after three weeks of growth for one week as a short term drought stress and then watered again and later, recovery started after 24h. The plants inoculated with selected R aegyptiacum show less wilting as compared to uninoculated and Bradyrhizobium sp. inoculated plants and recovery was slower in uninoculated plants as compared to Bradyrhizobium sp. and selected R aegyptiacum inoculated plants. The selected R aegyptiacum inoculated plants show early flowering as compared to Bradyrhizobium sp. and uninoculated plants. Under drought condition, wilting has been observed in all experimental plants while it was prominent in uninoculated plants at early growth stage. The physiological properties of Vigna mungo cv. T9 have been determined sixty days after sowing. R. aegyptiacum inoculated plants display better shoot and root length and weight under drought stress conditions as compared to Bradyrhizobium sp. and uninoculated plants (Table 2, Fig. 4).
Table 2: physiological parameters of V. mungo (control (no drought treatment), uninoculated and inoculated plants.

Treatment/
Physiological
response Well watered (control) Uninoculated plants Inoculated plants

Bradyrhizobium sp. Rhizobium aegyptiacum
Plant fresh weight fem) 10.19 ±0.443 2.01 ±0.429 7.86 ±0.430 21.35 ± 0.519
Plant dry weight (gm) 2.27 ±0.174 0.66 ±0.068 1.27 ±0.174 5.89± 0.011
Stem length (cm) 19.43 ±0.066 11.49 ±0.056 16.70 ±0.009 24.26 ± 0.006

Stem diameter (cm) 0.29 ±0.007 0.20 ±0.005 0.28 ±0.008 0.34 ±0.004
Stem fresh weight (gm) 0.72 ±0.013 0.31 ±0.004 0.59 ±0.005 1.58 ±0.007
Stem dry weight fem) 0.069 ±0.002 0.025 ± 0.004 0.038 ±0.003 0.296 ± 0.007
Leaf area (cm2) 48.24 ±0.424 14.20 ±0.737 37.56 ±0.879 69.59 ± 0.948
Number of leaves 18.33 ±0.577 9.00 ±1.000 15.67 ±2.082 34.33 ± 0.577
LeafRWC(%) 39.62 ±2.008 17.97 ± 1.803 35.66 ±0.751 52.89 ± 2.141
Root length (cm) 12.97 ±0.025 6.22 ±0.012 9.95 ±0.016 19.99 ± 0.012
Root FW (gm) 0.66 ±0.016 0.10 ±0.010 0.36 ±0.021 1.78 ±0.042
Root DW (gm) 0.12 ±0.003 0.03 ±0.003 0.08 ±0.006 0.18 ±0.004
Root nodule color Beige - Beige Beige
Number of root nodule 18.00 ± 1.000 - 15.33 ±0.577 33.33 ± 2.081
Root nodule size (cm) 0.220 ±0.008 - 0.190 ±0.012 0.400 ± 0.015
Root nodule FW (gm) 0.060 ±0.002 - 0.040 ±0.009 0.210 ± 0.006
Root nodule DW (gm) 0.008 ±0.001 - 0.005 ± 0.002 0.020 ± 0.010
Number of pod cluster/plant 5.00 ± 1.000 2.00 ±1.000 3.00 ± 1.000 8.00 ±1.000
Number of pods/plant 6.00 ± 1.000 4.33 ±1.155 5.00 ± 1.000 24.33 ± 1.528
Number of seeds/pod 6.67 ±0.577 3.67 ±0.577 5.00 ±0.000 7.00 ±0.000
Number of seeds/plant 36.00 ±6.000 15.66 ±4.042 25 ±5.000 80.00 ± 10.00
Pod length (cm) 3.46 ±0.053 1.60 ±0.346 2.03 ±0.089 4.06 ±0.052
Pod weight (gm) 0.205 ± 0.002 0.115 ±0.009 0.176 ±0.008 0.233 ± 0.002
Seed size (cm) 0.20 ±0.003 0.09 ±0.005 0.14 ±0.006 0.45 ±0.006
10 seed weight (gm) 0.24 ±0.003 0.11 ±0.005 0.14 ±0.005 0.30 ±0.009
Harvest index 19.29 ± 1.307 11.63 ±0.246 16.40± 1.138 36.92 ±
2.327

The biochemical properties of the plants inoculated with R. aegyptiacum has also been determined on various biochemical parameters (Fig. 5).
Chlorophyll a (chl a), chlorophyll b (chl b) and total chlorophyll content is higher in R aegyptiacum inoculated plants while reduction in chlorophyll content was observed in Bradyrhizobium inoculated and the uninoculated plants (Fig. 5 (A,B,C)). Under drought stress condition, there is a high increase in the H2O2 content of uninoculated plants as compared to R aegyptiacum inoculated and Bradyrhizobium sp. inoculated plants. The H2O2 content remains high in uninoculated plants signifying stress which may lead to oxidative damage, whereas the levels go down in R aegyptiacum inoculated plants on re-watering (Fig. 5(D)). The total phenolic content of the R. aegyptiacum inoculated plants is higher than the Bradyrhizobium inoculated and uninoculated plants. On re-watering, the R aegyptiacum inoculated plants recover better as compared to the Bradyrhizobium inoculated and uninoculated plants. (Fig. 5(E)). Lipid peroxidation profile of leaves in the form of MDA (malondialdehyde) content is better in R. aegyptiacum and Bradyrhizobium inoculated plants under well watered condition. After re-watering, the lipid peroxidation levels of the uninoculated plants is higher even while the MDA content of the R. aegyptiacum inoculated plants decreased indicating a better recovery from drought (Fig. 5(F)). Proline content is higher in R aegyptiacum inoculated plants as compared to Bradyrhizobium inoculated and uninoculated plants signifying a better adaptive response of the inoculated plants (Fig. 5(G)). PAL activity in R aegyptiacum inoculated plants is on increasing trend (1.59picokat/mg protein) while on decreasing trend in uninoculated plants (Fig. 5(H)). SOD activity is higher in R aegyptiacum inoculated plants and percentage increase has been 43.66% and 28.29% respectively under drought stress and on recovery (Fig. 5(1)). APX activity is higher in the R aegyptiacum inoculated plants in comparison to the Bradyrhizobium and uninoculated plants under well watered condition. Under drought stress, APX activity increases in both inoculated as well as uninoculated plants (Fig. 5(J)). GPX activity is

higher in R. aegyptiacum inoculated plants as compared to Bradyrhizobium and uninoculated plants under in both the well watered and drought condition (Fig. 5(K)). CAT activity is high in the R. aegyptiacum inoculated plants as compared to Bradyrhizobium as well as uninoculated plants under well watered condition. CAT activity significantly increases in both inoculated as well as uninoculated plants under drought stress (Fig. 5(L)).
MD (malate dehydrogenase), MT (metallothionein), TSN (tryptophan synthase), EREBP (ethylene responsive element binding protein) and MAPK (mitogen-activated protein kinase cascades) gene expression has been upregulated after R. aegyptiacum (cross inoculation) treatment under drought stress, whereas downregulated for ZF (zinc finger) and CAM (calmodulin) in plants under drought stress (Fig. 6 (A-G)). Fig. 6 (A-G) shows gene expression of MAPK, CAM, MT, TSN, ZF, MD and EREBP, respectively. Quantitative RT PCR is done for mitogen-activated protein kinase (MAPK) cascades, calmodulin (CAM), malate dehydrogenase (MD), metallothionein (MT), tryptophan synthase (TSN) and for zinc-finger (ZF) protein, ethylene responsive element binding protein (EREBP) genes after cross inoculation treatment under drought stress. In the present expression of MD, MT, TSN, EREBP and MAPK cascade were induced on cross inoculation treatment as well as against drought stress. Downregulation was observed for CAM (acidic protein) and ZF protein in plants under drought stress (Table.4.15). Higher down regulation in terms of fold changes for calmodulin (CAM) was observed in uninoculated plants (fold change: 0.045) which was followed by Bradyrhizobium inoculated plants (fold change: 0.109) and lower down regulation in terms of fold change was observed in R. aegyptiacum (cross inoculated) inoculated plants after drought stress (fold change: 0.550).
Higher expression of malate dehydrogenase (MD) is observed in cross inoculated plants as well as in Bradyrhizobium inoculated plants under drought stress. Among

uninoculated, Bradyrhizobium spp. and R. aegyptiacum inoculated plants higher upregulation in terms of fold change for MD is observed at R aegyptiacum inoculated plants (cross inoculated plants) after drought stress (fold change: 46.85) and lower upregulation in terms of fold change is observed in uninoculated plants (fold change: 2.24). Higher expression of metallothionein (cysteine rich protein) in cross inoculated plants after drought stress is 23.26 fold. MT is upregulated after cross inoculation under drought. In all the plants, 5.54 fold higher expression of MAPK gene is observed in R aegyptiacum inoculated plants (cross inoculated plants) after drought stress and 0.069 fold lower expression is observed in uninoculated plants. Higher expression of TSN gene is observed in cross inoculated plants (fold change: 1.27) and lower expression is noticed in uninoculated plants on drought stress condition (fold change: 0.31). Higher expression of ZF is observed on cross inoculated plants after drought stress (fold change: 0.243) and lower expression is observed in uninoculated plants (fold change: 0.019). Upregulation of EREBP is observed after cross inoculation under drought stress condition (Fig. 6).
Thus, the selected strain of R. aegyptiacum is able to effectively nodulate susceptible variety of Vigna mungo.
cross nodulation of the selected desiccation tolerant R. aegyptiacum from host V. mungo PU-1 on drought susceptible C. arientinum
The invention further provides cross nodulation of the selected desiccation tolerant R aegyptiacum from host V. mungo on drought susceptible C. arientinum. In a non-limiting example, intra cross inoculation has been performed on drought susceptible C. arientinum GNG 1958.
Four sets of plants have been prepared for Pot experiment. Two sets are treated with selected inoculant R. aegyptiacum, out of which one set is autoclaved and other is

non-autoclaved. Two other sets have not been inoculated with selected inoculant R aegyptiacum, out of which one set is autoclaved and other is non-autoclaved. Pot experiment showed higher growth in plant treated with R. aegyptiacum as compared to uninoculated (Fig. 7).
Confirmation of successful cross inoculation in chickpea is done by reisolating the selected bacterial strain. Total 6 isolates from cross inoculated plant and 4 isolates from native plants have been obtained. One bacterial isolate from the inoculated plant and one from native plants have been used for further biochemical, molecular and PGPR characterization.
Characterization of isolated bacteria from C. arietinum after cross nodulation with Rhizobium spp. from V. mungo
Biochemical studies revealed that the bacterial strains from both the inoculated (a) and uninoculated (b) plants have ability to change BTB color from green to yellow (Fig. 8A (a,b)) and absorb the Congo dyes weakly and appears as white colonies (Fig. 8B (a,b).
In PGPR characterization, both the inoculated (a) and uninoculated (b) plants shows positive result for HCN and IAA Production ((Fig. 9A and 9C (a,b)). However, inoculated plants showed siderophore production while native strain gives negative result (Fig. 9B).
By 16s rRNA sequencing, inoculated bacterial isolate from GNG 1958 Cicer arientinum GNG 1958 has shown 99.45% of similarity with previously isolated R. aegyptiacum MK333259 from Vigna mungo. Isolate from uninoculated or native GNG 1958 C. arientinum have 99.81% similarity with Rhizobium sp.

Further R aegyptiacum isolate of 1109 bp has been submitted at NCBI as strain BVVM-78 with accession no. MT980913. In chickpea grown in semi-arid regions, R aegyptiacum also helps to solubilize phosphate (Table 3), show antagonism effect against fungal causing disease (M phaseolina, A.niger andF. oxysporum) (Table 4), produce siderophore, ammonia, IAA and HCN.
Table 3: Phosphate solubilization by Rhizobium aegyptiacum strain BVVM-78

Isolates Colony Diameter (mm) Halo zone Diameter (mm) PSI PSE (%) LSD, Sig.
R aegyptiacum 11.5±0.5 17.0±1.0 12.9 67.64 4.08, 0. 000
Native 0 0 0 0

There is formation of halo zone by R aegyptiacum shows the ability to solubilize phosphate with phosphate solubilization index (PSI) 12.9 with solubilization efficiency 67.64% while there was no halo zone formed by native isolate.
Table 4: Antifungal Activity

Bacterial Isolates A. niger F. oxysporum M. phaseoli

Zone (cm) Growth inhibition
(%) Zone (cm) Growth inhibition
(%) Zone (cm) Growth inhibition
(%)
R aegyptiacum 1.31±0.08 81.4 0.59±0.35 90.0 1.30±0.012 86.9
Native 1.38 ±0.01 80.2 0.85±0.00 85.7 1.58±0.006 84.2
LSD, Sig. .3738,0.00
Both the isolates, i.e. R. aegyptiacum and native isolate has shown almost similar growth inhibition against Aspergillus niger. Isolate A shows higher 86.9% inhibition rate against M. phaseoli as compared to isolate B which has 84.2% growth inhibition rate.

Pot trails
Pot trials of C. arientinum GNG 1958 under hydration, dehydration and rehydration condition has been performed. Drought susceptible GNG 1958 seeds have been treated with native and exogenous strain of R. aegyptiacum and one set of pot remained untreated. Drought stress increment in proline, chlorophyll, TPC and reduction in H2O2 content has also been observed (Fig. 10).
By conducting RT-PCR, with chickpea drought candidate genes (WRKY, MAN, TUB, DREB2A, CaNAC02, CaNAC16, HSP80) it has been confirmed that selected strain conferred tolerance to drought stress in C. arientinum as response of inoculated exogeneous strain has shown less expression of drought inducible genes in chickpea as compared to indigenous strain and untreated plants (Fig. 11).
Therefore, the experiments demonstrate that select symbiotic microorganisms on successful cross inoculation, provides adequate stimulation and enhance the tolerance of plants in adverse environmental condition. Symbiotic microorganisms in pulses are very specific to their host crop. Rhizobium aegyptiacum BVVM-78 has been isolated from black gram (PU-1) and characterized by several biochemical test such as catalase test, urease test, gelatine test, HCN production, MRVP, indole test, citrate test, carbohydrate test. The selected bacteria also possess PGPR properties like phosphate solubilization, siderophore production, IAA production, EPS production and have antifungal activity against certain disease occurred in pulses. The selected strain was then screened in PEG for its drought tolerance capability. This desiccation tolerant strain is then successfully cross inoculated in black gram variety T9 and chickpea variety GNG 1958 and shows the better performance as compared to their native strains.
11 bioformulation of selected strain of Rhizobium aegyptiacum BVVM-78 are prepared and tested for survivability of viable cell count (Table 5).

Table 5: Bacterial Survivability Assay

Population density CFU/ml of different formulation
Duration
of
Storage
(Days) C
(Control) Tl
(.5% PVP) T2
(1.5% Glycerol) T3
(2% Glycerol) T4
(2.5% Glycerol) T5
(.5% PEG) T6
(1%
PEG) T7
(1.5%
PEG) T8 T9
(.1% Na(7% alginate) lactose) Til
T10 (.5% PEG + (.5% 2% PEG + Glycerol) 2% + 7% Glycerol) lactose)
30 4.25x10" 5.1x10" 6.65xl012 1.08xl013 6.55xl012 8.55xl012 1.685xl013 1.16xl013 3.35xl010 1.045xl013 6.5xl012 1.84xl013
60 3.4xl09 3.5xl010 4.3xl012 6.9xl012 3.15x10" 3.85xl012 1.145xl013 9.85xl012 3.2xl09 3.85xl012 6.5xl012 1.445xl013
90 4.1xl07 4.35xl08 3.45x10" 5.45xl012 3.75x10" 3.7x10" 1.02xl013 7.2x10 12 3.35xl08 3.1x10 12 35.5xl012 1.24xl013
120 3.85xl06 3.25xl08 3.75xl08 3.05x10" 3.95xl09 3.0xl09 3.85x10" 3.45x10" 4.05xl07 3.75xl010 4.15xl010 6.95xl012
150 0 4.05xl05 5.5xl05 4.15x10 9 3.65x10 7 4.25x10 6 4.85x10 9 4.5x10 9 3.35xl03 3.1x10 7 3.8xl09 3.95xl012
180 0 3.7xl03 6.5xl03 4.3x10 6 3.05x10 6 3.1xl05 3.85x10 7 5.25xl06 0 3.4x10 6 3.9x10 6 6.2xl09
The results show the viable cell count of 1.84x10 cfu/ml, which has reduced to 6.2xl09cfu/ml in 6 months. 5
The bioformulation of the present invention is used as biofertilizer for black gram and chickpea crop resulting in enhanced crop yield naturally, improve productivity per area, consume smaller amounts of energy, mitigate contamination of soil, make crop withstand in drought condition, increase soil fertility, and promote 10 antagonism and biological control of phytopathogenic organisms.

We claim:

1. Plant growth promoting bioformulation of Rhizobium for use in drought susceptible leguminous plants, said bioformulation comprises Rhizobium aegyptiacum in yeast extract-mannitol broth (YEMB) and one or more self protectant, wherein said bioformulation enhances crop yield naturally, improve productivity per area, mitigate contamination of soil, make crop withstand in drought condition, increase soil fertility, and promote antagonism and biological control of phytopathogenic organisms.
2. The plant growth promoting bioformulation of Rhizobium as claimed in claim 1, wherein said bioformulation comprises Rhizobium aegyptiacum in YEMB and one or more self protectant in the range of 6.3% to 12.7%.
3. The plant growth promoting bioformulation of Rhizobium as claimed in claim 1, wherein said one or more self protectant is selected from PEG, glycerol, lactose or a combination thereof.
4. The plant growth promoting bioformulation of Rhizobium as claimed in claim 1, wherein said bioformulation comprises Rhizobium aegyptiacum in YEMB, PEG in the range of 0.3% to 0.7%, glycerol in the range of 1% to 3%) and lactose in the range of 5% to 9%.
5. The plant growth promoting bioformulation of Rhizobium as claimed in claim 1, wherein said bioformulation comprises Rhizobium aegyptiacum in YEMB, PEG 0.5%, glycerol 2% and lactose 7%.
6. The plant growth promoting bioformulation of Rhizobium as claimed in claim 1, wherein said bioformulation has viable cell count of 1.84xl013 cfu/ml.
7. The plant growth promoting bioformulation of Rhizobium as claimed in claim 1, wherein said bioformulation has viable cell count of 6.2 x 109 cfu/ml after 6 months.
8. The plant growth promoting bioformulation of Rhizobium as claimed in claim 1, wherein said drought susceptible leguminous crop can be selected from drought susceptible variety of Vigna mungo, Cicer arietinum, lens culinaris, Glycine max,

Phaseolus vulgaris, Cajanus cajan, Vigna radiata, Vigna unguiculata, Pisum sativum.
9. The plant growth promoting bioformulation of Rhizobium as claimed in claim 1, wherein said bioformulation has shelf life of more than 6 months.
10. A process to obtain the bioformulation as claimed in claim 1, wherein said process comprises

(a) isolating Rhizobacteria from the root nodules of a desired drought tolerant leguminous crop cultivar grown in semi-arid region,
(b) characterizing the isolated Rhizobacteria for desired characteristics by morphological, biochemical and molecular testing,
(c) screening of isolated Rhizobacteria for drought tolerance,
(d) cross inoculating a drought susceptible crop cultivar of a desired drought susceptible leguminous crop to examine desiccation tolerance of the screened Rhizobacteria and tested for drought tolerance,
(e) culturing the screened isolated Rhizobacteria in YEMA broth containing one or more self protectant,
(f) preparing a bioformulation with viable cell count of 1.84xl013 cfu/ml.

11. The process to obtain the bioformulation as claimed in claim 10, wherein said desired drought tolerant leguminous crop cultivar grown in semi-arid region is Vigna mungo var PU-1.
12. The process to obtain the bioformulation as claimed in claim 10, wherein said Rhizobacteria is R. aegyptiacum.
13. The process to obtain the bioformulation as claimed in claim 10, wherein said desired drought susceptible leguminous crop can be selected from drought susceptible variety of Vigna mungo, Cicer arietinum, lens culinaris, Glycine max, Phaseolus vulgaris, Cajanus cajan, Vigna radiata, Vigna unguiculata, Pisum sativum.
14. The process to obtain the bioformulation as claimed in claim 10, wherein said one or more self protectant is selected from PEG, glycerol, lactose or a combination thereof.