Symbiotic nitrogen fixation, involving legumes and rhizobia is of considerable economic and ecological significance. Rhizobia are a group of soil bacterial species that infect leguminous plants leading to the formation of special type of organs called nodules, where nitrogen fixation takes place. The nitrogenase enzyme complex of bacterioids i.e. symbiotic form of rhizobia supplies constant source of reduced nitrogen to the legume host and intern, receives the nutrients and energy. This symbiotic process occurs on all continents and accounts for a fourth of the nitrogen fixed annually on earth [1] , has become important component of sustainable organic agriculture.

The nitrogen fixing genes are located in Rhizobia and symbiotic establishment and regulation of nitrogenase activity in the nodules depends on the genes of host plant. Host plant genotype compatibility and differences in nitrogen fixation potential of Rhizobial strains often results in variation in symbiotic effectiveness [2] . Establishment of effective symbiosis depends on cross talk between plant host and rhizobia at different stages, starting from the initial recognition of partners, through functional nodule formation and nitrogen reduction [3] .

Rhizobium is also a plant growth promoting rhizobacteria (PGPR) exhibit a variety of characteristics responsible for influencing plant growth and performance. As a symbiotic partner in addition to nitrogen supply, Rhiz- obium also improves nutritional uptake by promoting the growth of plant root system through production of Indole acetic acid (IAA) [4] and as a rhizospheric microbe solubilises phosphorus and various mineral nutrients [5] .

Legume crops, which provide dietary proteins, fats and fodder, are preferred in semi-arid agriculture system due to their inherent adaptation to the existing characteristic ecological features and symbiotic nitrogen fixation potential. Climatic changes experienced during the recent years further aggravating ecological problems of arid and semiarid legume cropping areas leading to prolonged dry season, rise in temperature, increase in soil salinity and soil pH alteration. These typical environmental stresses are problematic for the establishment of effective symbiosis and survival of rhizobia in the soil [6] . Further, the requirement for fertilizer-N is increasing and predicted a need for more than 190.4 million tons by 2015, to supply the world’s food. The economic and ecological costs of fertilizer usage will eventually become prohibitive in future due to current technologies employed for fertilizer production and application respectively.

Genetic variability for the environmental stress factors in the commercial inoculants/strains and Rhizobial populations in the agriculture fields most often very limited due to monoculture of legume crops and competition between Rhizobial populations in the soil favours colonization of certain genotypes and co-evolution of symbiotic partners.

Enormous diversity exists in rhizobia due to their wide geographical distribution, different plant hosts and niches they occupy all over the globe [7] . The major challenge in agricultural system, to exploit the enormous potential of symbiotic nitrogen fixation requires the selection and development of efficient inoculant strains compatible to legume host and the target edaphic environment in which their fullest genetic potential of nitrogen fixation expressed. Occurrence of rhizobia in varied ecosystems and their significance for sustainable organic agriculture emphasizes the need for the exploration of non-agricultural areas, indigenous soils and nodulating wild plants to know the rhizobial diversity and their economic utilization. Selection and development of efficient compatible Rhizobial partner for a specific crop is a potential strategy due to their simple genetic system compared to complexity of nodulation genotype of legume plants.

Nodulated wild legumes with their biodiversity and adaptation to arid/semi-arid climatic conditions are vital to the soil fertility and nitrogen cycle [8] and are an important source for elite rhizobial strains. The natural symbiotic rhizobia of these wild legumes exhibit higher tolerance to prevailing adverse conditions like salt stress, elevated temperature and drought, which can be important for increasing productivity of legume crops when cross inoculated.

There were few reports on phenotypic characterization of rhizobia from wild legumes of arid/semiarid regions viz, Rajasthan, India [9] , Saudi Arabia [10] and Tunicia [11] .

The knowledge on genetic and functional diversity of root-nodule bacteria associated with wild legume flora of arid and semiarid ecosystems of the continents is largely unexplored. This study aimed at evaluating the phenotypic diversity, stress tolerance and plant growth promoting characteristics of rhizobial isolates from wild legumes of semiarid habitat, Tirupati, India.

Isolation of rhizobia: Rhizobial isolates from fifteen nodulating native wild legumes were selected to study morphological, biochemical and plant growth promoting characters. Wild legume hosts include Albizia lebbeck, Alysicarpus monilifer, Chamaecrista absus, Cassia tora, Clitoria ternatea, Crotalaria hebecarpa, Desmodium trifolium, Indigofera linnaei, Indigofera mysorensis, Indigofera trifoliata, Millettia pinnata, Mimosa pudica, Rhynchosia minima, Tephrosia purpurea and Tephrosia tinctoria and cultivated Arachis hypogea for comparison. From carefully uprooted roots, soil was removed by gentle washing with tap water, separated from the stem and placed in polythene covers, kept in cool box and brought to the laboratory. By leaving small piece of root above and below the nodules, roots were cut. Root pieces containing nodules were first subjected to surface sterilization with 0.01% HgCl₂ for 30 sec. Traces of HgCl₂ were removed by repeated washings with sterile distilled water and then incubated in 70% ethanol for 3 min and final washings were made with sterile distilled water for 3 times. Randomly selected ten nodules were separated from root part of each species, crushed with sterile glass rod and squeezed for white coloured milky secretion into 0.5 ml of sterile YEM broth. From this suspension loopful of inoculum was streaked onto YEMA medium (Composition per litre: Yeast extract 1 gr, Mannitol 10 gr, Dipotassium phosphate 0.5 gr, Magnesium sulphate 0.2 gr, Sodium chloride 0.1 gr, Agar 20 gr, pH 6.8) containing 0.0025% Congo red [12] . For each isolate three replicates were kept. Fractions from the last washed distilled water were also streaked onto YEMA medium to check the effect of surface sterilization and plates were incubated at 28˚C. Cultures were observed after 3 - 6 days and were restreaked onto YEMA medium to obtain single colonies. A single colony reinoculated into YEM broth and maintained on shaker at 200 rpm for the stock culture and use. All cultures were maintained in triplicates and data was recorded after 96 hours of inoculation. The isolates were given codes by taking the first capital letter of genus and first two letters of the species name of the respective original legume host.

Morphological and Biochemical characterization: Isolates were characterized for colony and cell morphology, biochemical characteristics includes (Melanin production test, Ammonia production test, Phosphate solubilisation test, Organic acid production test, Oxidase test, Catalase test, Indole production, Gelatin hydrolysis, Starch hydrolysis) was carriedout following standard methods described by Somasegaran and Hoben [13] . Exopolysaccharides isolation [14] and estimation [15] , Siderophore production test [16] were employed. For carbon and nitrogen utilization tests, 10% sugars or organic acids were prepared; filter sterilized and was added to the basal medium to a final concentration of 1% (w/v). Stress tolerance studies including intrinsic antibiotic resistance (IAR), resistance to salinity, pH and temperature were carriedout by the standard method [17] .

Statistical and Cluster analysis: Cluster analysis was performed for rhizobial isolates. Analysis of variance and correlation coefficient was calculated using SPSS version 12 statistical software which was used to estimate the simple matching similarity coefficient (Ssm) of each strain and to generate a similarity matrix [18] computed with Jaccard coefficient. The similarity matrix was used for cluster analysis to construct a phenogram using the unweighted pair group method with averages (UPGMA) [18] .

Study site: The study site, S. V. University campus is located in Tirupati (13˚62'N and 79˚40'E) at foothills of Seshachalam (Tirumala) hill range covering nearly 200 acres (Figure 1) and represent typical semi-arid temperate climate. The mean average annual rainfall is 950 mm followed by prolonged dry season with mean annual maximum temperature 40˚C ± 2˚C and a minimum of 20˚C ± 2˚C. Soil is red loamy with poor water holding capacity and organic contents. The site of Arachis hypogea’s collection is having similar soil profile but under cultivation for many years.

Morphological characteristics: Bacteria that form nitrogen-fixing nodules in the roots of legumes have different growth rates, morphologies, biosynthetic pathways, catabolic capabilities and habitats depending on host species, edaphic and climatic conditions of habitats. Generally morphological features of the colony i.e, form, elevation and margins vary with rhizobia species/strain [13] . The fifteen isolates from the wild legumes and isolate from cultivated Arachis hypogea displayed variation in colony morphological features including, form, elevation, margin and opacity (Table 1).

Morphology of the colony formed by Cte, Tpu, and Tti isolates was irregular and the colonies of remaining isolates were regular. The colony elevation was flat in Che, Itr, Imy and Tti, raised in Cab, Cto, Cte, Ili, Rmi and Tpu, and convex in the remaining isolates. Margin of the colony was lobate in Cte, Che, Rmi and Tpu and in the remaining 11 isolates it was entire. The circular and irregular colonies are in agreement with characteristic colony forms of rhizobia [13] . The colonies were creamy coloured and mucose. Eight isolates are categorized as slow growers and remaining six isolates as fast growers based on the colour change of YMA-BTB medium to

Note: All isolates exhibit characteristic colony colour creamy on YEMA, white on YEMA with Congo red, blue and yellow on YEMA with BTB. Mucose in appearance, aerobic and non-spore forming in nature.

blue and yellow (Table 1) depending on acid and alkaline production respectively as reported earlier by Saeki et al. [19] .

When the light transmittency of the colony is considered, it is transparent in Cab, Cto, Dtr, Imy, Rmi, Tpu and Tti, and opaque in the remaining isolates. All isolates were aerobic, rod shaped, gram negative and motile. The discriminative power of colony morphology characters in revealing genetic diversity among the isolates was realised when the combination of four characters (form, elevation, margin and opacity) were considered. The isolates formed into 11 groups of which 9 were loners; one group represented by 5 isolates and another with 2 isolates.

Biochemical characters: The isolates are positive to Ammonia, Catalase, Indole, Melanin, Oxidase and H₂S production tests. Further, isolates are also positive to MR-VP test, Starch hydrolysis, and negative to gelatin hydrolysis test (Table 2).

Phenotypic characteristics: Several techniques for characterization, identification and assessment of metabolic diversity of strains have been developed, such as Carbon, Nitrogen sources utilization and Intrinsic Antibiotic Resistance pattern. Of 11 sugars tested all the isolates were able to utilise fructose, galactose, glucose, mannose, sucrose, starch, succinate, rhamnose, malate, citrate and they fail to utilise dulcitol as carbon source (Table 3). In the case of nitrogen sources utilization all the isolates were able to utilise glutamate, ammonium chloride, aspartate, tryptophan, glycine and unable to utilise potassium nitrate and casein hydrolysate (Table 3). Similar response of isolates to carbon and nitrogen utilization may be due to lack of variation in the genes operating in these pathways. The results of biochemical tests, carbon and nitrogen sources utilization pattern of isolates compared with Bergey’s Manual of Determinative Bacteriology [20] and were confirmed as Rhizobia.

The rhizobial isolates showed variation in colony morphological features including form, elevation, margin and opacity, and Intrinsic Antibiotic Resistance. The isolates tolerated moderate level of salinity, temperature and showed optimum growth at neutral pH. Majority of rhizobia are significantly high IAA producers, and all isolates displayed other plant growth promoting EPS, siderophore production and phosphate solubilisation. Molecular characterization for the species/strain identification is in progress. Functional diversity of isolates can be utilised for the improvement of nitrogen fixation in legume crops through cross inoculation.

Y.Bhargava,J. S. R.Murthy,T. V. RajeshKumar,M. NarayanaRao, (2016) Phenotypic, Stress Tolerance and Plant Growth Promoting Characteristics of Rhizobial Isolates from Selected Wild Legumes of Semiarid Region, Tirupati, India. Advances in Microbiology,06,1-12. doi: 10.4236/aim.2016.61001