India is an agricultural country. Population explosion is posing serious pressure on food supply. Agriculture has become an occupation and that too a much needed one. Various modern techniques are adopted in agriculture to fulfill our food requirements. 

Soil resources and natural inputs available in the world in the earlier stages, primitive agricultural practices were more than sufficient to satisfy the requirements of primitive farmers. Soils provide the physical support the root system and also serve as the reservoir of air, water and nutrients for the plant growth.

Bio-fertilizers are carrier based preparations con- taining beneficial microorganisms in a viable state intended for seed or soil application and designed to improve soil fertility and help plant growth by increasing the number and biological activity of desired microorganisms in the root environment (Subbarao, 1999). Azotobacter chroococcum has present Peritrichous flagella moderate slime and produce black brown insoluble pigment (Bagyaraj and Menge, 1978; FAO, 1982). Azospirillium sp isolated from different soils and rhizophere of different cultivars of rice exhibited wide variation in the amount of nitrogen fixed (Rao et al., 1981).

Rhizobium inoculation enhanced the growth (6%~133%) bioms yield (30%~109%) total nitrogen and total soluble sugars. In a nodulating legume nitrogen fixation process requires sufficient photo- synthetate supply. Nitrogen fixation and co2 fixation are interdependent process (Phillips, 1978). Positive effect of Rhizobium so inoculation on grain yield in chickpea was observed to increase different growth attributes (Ghag et al., 1982; Shendue, 1986). The ability to fix atmospheric nitrogen, Azotobacter is also known to synthesize biologically active substances such as B. Vitamins indole avetic acids and Gibbe- rrelines in pure culture (Mishustin, 1969; Dart et al., 1972; Rao, 1973). 

Application of rice straw enhance nitrogen fixation where addition ammonium sulphate (combined nitrogen) retard nitrogen fixation (Kalininskaya, 1978). Azospirillum sp. is an associative microaerophilic diazotroph isolated from the root and aerial parts of a variety of crop plants (Cohen et al., 1980; Hara et al., 1981; Tilak and Murty, 1981; Patriquim et al., 1983; Pacovsky et al., Agarwal and Tilak, 1988).

Plant growth promotion by Azotobacter and Azospirillum may also be attributed to other mechanisms such as ammonia excretion (Shende et al., 1977; Narula et al.,1981; Martinez et al.,1988; Suneja et al.,1994).

There is numerous soil microorganisms actively involved in the synthesis of auxins in pure culture and in soil (Arshad and Frankenberges, 1991). Azotobacter chroococcum (Brown and Walker, 1970) produce plant growth regulators in nitrogen free media. Interactions between Azospirillum and other inocu- lants has been reviewed (Rao et al., 1989; Sexena and Tilak, 1994). 

The advantages of bio-fertilizers over the use of chemical fertilizers are low cost, enhancement of soil fertility, no change in the soil texture, no health hazards and retention in the soil for longer period.

1 Results and Discussion
In the present study, the leguminous crop plants such as Clitoria ternashia, Arachis hypogea and Vigna mungo are analysed for rhizobial population in root nodules. The isolation of Azotobacter sp, and Azospirillium sp, from the soil sample was done pour plate method morphological and biochemical characteristics of isolated bio-inoculants.

Table 1 represents the biochemical characteristics of Azotobacter sp, Azospirillium sp and Rhizobium sp, respectively. Ashby’s medium used for the isolation of Azotobacter sp, developed white colour colonies. Blue colour colonies developed on nitrogen free semisolid Malate medium used for the isolation of Azospirillium sp. Bio-fertilizers are eco friendly and cannot at any rate replace chemical fertilizers that are indispensable for getting maximum yield of crops. There products can at best minimize the use of chemical fertilizers not exceeding 40 kg n/ha under ideal agronomic and pest free conditions (Subba Rao, 1999).

Table 1 Biochemical characteristic for the identification of Azotobacter sp., Azospirillium sp., and Rhizobium sp.

The impact of bio-inoculants on plant was assessed on the basis of physical and biochemical parameters. This includes the analytical parameter of plant such as root length, plant height, leaf area and inter-nodal distance and chemical profile includes the pigment, protein, nitrogen, carotenoids pesticides.

The treatment pot inoculated with combined inoculants resulted in an effective increase in plant height cluster beans (32.1 cm) and chilly (22.0) over control and further observations at 40^th day, the maximum height of plants was recorded (Table 2; Table 3). This observation clearly indicated the limitation of combining bio-inoculants that may result in faster exhaustion of soil nutrient, there by making the soil less nutritive for plants.

Table 2 Effect of bio-inoculants on the morphological characteristics of cluster beans after 30 days

Table 3 Effect of bio-inoculants on the morphological characteristics of chilly plant after 40 days

This well known combination of bio-inoculants such as Rhizobium sp., Azotobacter sp., and Azospirillium sp., secretes a variety of plant hormones that would be the reason for sharp increase in the height of the plant.

Photosynthesis is an important physical activity that determined the ultimate organic constituent of plant cell. This is evident from the protein analysis especially in the case of plant that was supplemented with mixed inoculants in which protein content was very high cluster beans (0.92 mg) over the control (0.61) and chilly (0.74) control (0.44) (Table 4; Table 5).

Table 4 Effect of bio-inoculants on carotenoid, nitrogen and protein content in cluster beans after 40 days

Table 5 Effect of bio-inoculants on carotenoid, nitrogen and protein content in chilly after 40 days

The nitrogen analysis especially in the case of plant that was supplemented with mixed inoculants in which nitrogen content was very high in cluster beans (0.16) over the control (0.06) and chilly (0.19) over the control (1.1) (Table 4; Table 5).

 

The carotenoids analysis especially in the case of plant that was supplemented with mixed inoculants in which carotenoids content was very high in cluster beans (0.021) over the control (0.012) and chilly (0.78) and over the control (0.43) (Table 6; Table 7).

Table 6 Effects of bio-inoculants on Chlorophyll A, B and Total chlorophyll content of cluster beans, after 40 days

Table 7 Effects of bio-inoculants on Chlorophyll A, B and Total chlorophyll content of chilly after 40 days

The chlorophyll analysis especially in the case of plant that was supplemented with mixed inoculants in which chlorophyll content was very high in cluster beans (0.95) over the control (0.66) and chilly (0.71) and over the control (0.35) (Table 6; Table 5).

It can also be seen that the antimicrobials activity of the chemical extract on Nureelle and Hinoson is highest pesticide effect. It is also observed from these result that the chemical extract (20 µg) have antibacterial activity against both gram positive and negative stains under with highest activity (Table 8).

Table 8 Effect of pesticide activity in bio inoculants Azospiriilum sp., Azotobacter spand Rhizobium sp.

In India, The use of inoculam was started gaining momentum only in late 1980s. bacterization with Azotobacter sp increased the yield of potato by 3.45% to 11.5% (Shende et al.,1986); that of onion by 18.6% to 22.0% (Mehrota and Lehri, 1971).

Tomoto, cabbage and cauliflower by 19.0%, 40% and 28%~33.8%, respectively (Verma, 1998); and that of brinjal by 1.42 percent (Mehrota and Lehri, 1971). Maximum grain yield of was 6.45 t/ha and 5.68 t/ha in kuruvai and with more pronounced residual effect in thaladi seasons with 4.07 t/ha and 3.79 t/ha which were 20 and, 5 percent increase over 150kg N/ha level (Gopalaswamy and Anthoniraj et al., 1997).

Inoculation of Azospirillum either applied individually or in combination with Sesbania rostrata increase the plant height total as well as productive tillers at 75kg Nha. Application of Azospirillum in general increased the yield (Gopalaswamy et al., 1989).

This dual inoculation improved plant growth more that the single inoculation and un-inoculated control (Summana and Bagyaraj, 2002) rhizobium inoculation resulted in a substantial increase in leaf area of both Vigna mungo and Vigna radiata when compared to un-inoculated control (Krizek et al., 1997). The beneficial role of Azospirillium in enhancing N₂ fixation in combination with rhizobium is further strengthened by the observation that when mutants defective in nodulation and N₂ fixation, perform equal to the parent when used in combination with Azospirillium (Kundu, 1988).

Interaction between Azospirillam and other inoculants has been reviewed (Saxene and Tilak, 1994) dual inoculation with Azospirillam lopoferum and Gigaspora calospora positively influence the growth and Chlorophyll content (Rao et al., 1989).

In India, the use of inoculums was started gaining momentum only in late 1980s. Bacterization with Azotobacter sp increased the yield of potato by 3.45 to 11.5 percent (Shende et al., 1986); that of onion by 18.6% to 22.0% (Mehrota and Lehri, 1971). Tomoto, cabbage and cauliflower by 19.0%, 40% and 28%~33.8% respectively (Verma, 1998); and that of brinjal by 1.42% (Mehrota and Lehri, 1971).

Hence keeping all these points in mind, a preliminary attempt was made to study the effect of bio-inoculants on the growth of Capsicum annum and Cymposis tetragonoloba was analyzed based on various parameters. The growth was analyzed based on physical parameters such as plant height, shoot length, root length, number of leaves, leaf area inter-nodal distance etc. and also on the basis of biochemical parameters such as chlorophyll, protein, carotenoid, nitrogen and pesticide effect.

2 Material and Methods

2.1 Sampling area

The sampling area selected for the isolation of Rhizobium sp., Azotobacter sp., Azospirillum sp., for the present investigations were the fertile cultivable lands of Alwarkurichi and Kalyanipuram of Tirunelveli district. Major crops cultivated in these areas are paddy, chilly, cluster beans, brinjal, tomoto, groundnut, banana, soybean etc. The beneficial microorganisms such as nitrogen fixers were added with seeds, during seed preparation.

2.2. Sample collection

In the present study Rhizobium sp., Azotobacter sp., Azospirillum sp., was isolated from various plants and also from the Rhizosphere soil. Rhizobium sp., was isolated from following leguminants plants, (1) Clitoria ternashia-Sangupuspam; (2) Arachis hypogea- Ground nut; (3) Vigna mungo-Black gram. Azoto- bacter sp. and Azospirillum sp were isolated from the rhizophere soil.

2.3 Sample preparation

The root nodules of the above mentioned plants were collected using sterile knife and transferred into gamma irradiated polythene bags. Rhizophere soil samples were collected using sterile spatula and transferred in to sterile polythene bags. After collection of the sample, they were placed in an ice box and brought to the laboratory immediately for microbiological analysis. The time taken for the collection of nodules, soil samples and plating were complete under four hours.

2.4 Microbiological studies

2.4.1 Isolation of Rhizobium sp. from root nodule

Pour plate technique was employed for the isolation of Rhizobium sp. from the leguminous root nodule. One gram of nodules was surface sterilized using 0.1% mercuric chloride solution and 70% ethanol and washed with distilled water. The surface sterilized nodules were homogenized in a surface sterilized mortar and pestle.

One ml of the homogenized samples was transferred to 99 mL of sterile distilled water blank and mixed thoroughly in a rotator shaker for uniform distribution of the bacterial cells. Further, serial dilutions were made upto 10~8 using 9 mL of sterile distilled water blanks. From these dilutions, 1 mL of sample was pipetted out and pour plated along with sterile yeast extract Mannitol agar (yema) medium. The inoculated plates were then incubated at room temperature, at (28±20)℃ for 3~10 days depending on appearance of bacterial out growth. The colonies of Rhizobium sp., appeared as white, translucent, glistening and elevated with entire margin. The well isolated Rhizobium sp., colonies were purified by streaking on to nutrient agar plates and isolated colonies were streaked on nutrient agar slants and stored at 4℃ for further biochemical studies.

2.4.2 Isolation of Azotobacter sp., and Azospirillium sp., from rhizophere soil

One gram of rhizophere soil samples were transferred separately in 99 mL of sterile distilled water blank and mixed thoroughly in a rotatory shaker for the uniform distribution of bacterial cells. Serial dilutions were using 9 mL sterile distilled water blanks. The samples were serially diluted upto 10~6 dilution.

Pour plate technique was employed for the isolation of Azotobacter sp. from the serially diluted tubes, 1 mL of sample was pipetted out and poured in sterile petriplates followed by 20 mL of sterile Ashbhy’s medium and nitrogen free semi solid malate agar medium plates. After solidification, the plates were incubated at 37℃ for 24~96 hours. After the incubation the isolated colonies were transferred to nutrient agar slants and stored at 40℃for further identification tests. The bacterial isolates were subjected to various biochemical, identification tests and observed. 

2.4.3 Effect of bio-inoculants on cluster beans and chilly

In the present investigation, the isolated cultures of Rhizobium sp., Azotobacter sp., and Azospirillium sp., were used as mono and co-inoculants in pot trails. The following combination of bio-fertilizer and chemical fertilizer tried to study the effect of bio-inoculants on the growth of Cyamopsis tetragonoloba (cluster beans) and Capsicum annum (chilly).

2.4.4 Seed treatment

Approximately 4 grams of healthy seeds of cluster beans and chilly were surface sterilized using 0.1% mercuric chloride, 70% ethanol, sterile distilled water and germinated and inoculated with 5 mL culture of different combination of bio-inoculants in 100 mL conical flasks and kept for 12 hour (Table 9). Then the seeds were grown to 24 hour and allowed to germinate in respective pots.

Table 9 Treatment schedule

2.4.5 Effect of bio-inoculants on the growth of Cyamopsis tetragonoloba (Cluster beans) and Cap- sicum annum (Chilly)

After 15 days and 30 days sowing the seeds in the pots, the effect of bio-inoculants such as Rhizobium sp., Azotobacter sp., Azospirillum sp., on the growth of Cyamopsis tetragonoloba (cluster beans) and Cap- sicum annum (chilly) were inoculated. The following physical, and biochemical parameters were observed in the plants.

2.4.6 Chlorophyll estimation (a, b)

The chlorophyll content of the leaves of the expe- rimental and control plants were estimated by following the method of Sadhasivam and Manickam (1991). 

2.4.7 Estimation nitrogen

The total nitrogen in the leaves of the control and experimental plant samples were estimated by Kjeldhal method (Jayaraman, 1999).

2.4.8 Preparation of pesticide concentration

The pesticide such as Reeva, Hinoson, Nurelle, Fujione were used to study the effect of these recalcitrant xenobiotics on the growth of the bio- inoculants such as Rhizobium sp., Azotobacter sp., Azospirillium sp., in sterile YEMA, Ashby’s, n.f.s.s. malate medium agar plates the pesticides were inoculated at different concentrations (5 µ, 10 µ, 15 µ, 20 µ) prepared (incorporated on sterile discs and the discs were impregnated). Plates were incubated at 37℃ for 24 hours. Inhibitory patterns were observed and zones of inhibition were measured and tabulated.

Acknowledgement

The authors wish to acknowledge and thanks to Dr.A.J.A.Ranjit Singh, Principal, Sri Paramakalyani College, Alwarkurichil. Tirunelveli, Tamilnadu-627 412 and Department of Science and Technology for their support and encouragement.

References

Agarwal R., and K.V.B.R. Tilak, 1998, Colonization of Azospirillum in leaves of Eleusine coracana and Setaria italic, Zantralbl. Microbial, 143: 537

Arshad M., and W.T. Frankenberger Jr., 1991, Microbial production of plant hormones, Plant soil, 133: 1-8

Bagyaraj D.J., and J.A. Menge, 1978, Interactions between a VA Mycorrhizal fungus and Azotobacter and their effects on rhizophere micorflora and plant growth, New phytol., 80: 567-573

Brown M.E., and N. Walker, 1970, Indole 3-acetic acid formation by Azotobacter chroococcum, Plant soil, 32: 250-253

Cohen E., Y. Okon., J. Nur., and Y. Hennis, 1980, Increase in dry weight and total nitrogen content in Zea mays and Setaria italica associated with nitrogen fixing Azospirillum sp., Plant Physiology,66: 746-749

Dart P.J., D. Harris, and J.M. Day, 1972, Assay of nitrogenous activity by acetylene reduction. Reprints, Rothamsted Experiment station, Part, 2: 87-100

FAO, 1982, FAO Soil Bulletin 49, Application of nitrogen fixing systems in Soil Management, FAO Rome

Ghag M.S., B.K. Korde, and R.B. Deshmukh, 1982, Effect of Rhizobium sp., Seed inoculation of growth attributes yield of chick pea. Pulse crop Newsletter 2: 61:63

Gopalaswamy G., and S. Anthoniraj., 1997, Integrated use of Azospirillum and Sebanea rostrata on rice productivity, Madras agric J., 84(2): 87-89

Jayaraman J., 1999, Laboratory manual in bio-chemistry, sixth edition, pp: 75-77

Kalinishaya T.A., V.R. Rao, T.N. Volkova, and Lippolitov, 1973, Nitrogen fixing activity of soil under rice crop studied by acetylene reduction assay, Microbiologiya,42: 481-485

Krizek K.J., G.F. Kramen, and R.M. Mirecke, 1997, Influence on UV-Radiation and putresine on shoot and root growth of cucumber seedling grown in nutrients solution, Nut., 20: 613-623

Kundu B.S., 1988, Effect of dual inoculation with Azospirillium and rhizobium on nodulation and nitrogen fixation in Pigeon pea, Indian J Microbial, 28(3): 233-237

Mehrotra O.L., and C.K. Lehri, 1971, Effect of Azotobacter inoculation on crop yield, J and Soil Sci., 19: 243-48

Mishustin E.N., and V.K. Shilnikova, 1969, The biological fixation of atmospheric nitrogen by free-living bacteria. In Soil Biology, Review of Research, pp. 72-109, UNESCO, Paris

Okerake G.U., and D-Unaegnu, 1992, Nodulation and biological nitrogen fixation of 80 soybean cultivates in symbiosis with indigenous Rhizobia, World J. Microbial Biotechnol, 8: 171-174

Pacovsky R.S., E.A.Pawar., and G.J. Benthlenfalvay, 1985, Nutrition of sorghum plants fertilized with nitrogen or inoculated with A. brasilense, Plant and Soil, 85: 145-148

Phillips D.A., G.J. Bethlenfalvay., and S.S. Abu-Shatra, 1978, Interdependence of nitrogen nutrition and photosynthesis in Pisum sativum, Plant Physiol, 62: 131-133

Rao V.R., 1973, Non-symbiotic nitrogen fixation on paddy fields, Doctoral Theses, Academy of Cines, U.S.S.R. Moscow

Shende S.T., O.P. Verma., and N.S. Bisht, 1986, Azotobacter sp a bio-fertilizer for potato seed and farms. 12: 41-43

Shende S.T., R.G. Apte., and T. Singh, 1977, Influence of Azotobacter sp., on germination of rice and cotton seed, Curr. Sci., 46: 675-676.

Subba Rao N.S., 1999, Soil Microbiology, pp. 381-382, pp. 135

Suneja S., K. Lakshinaraya., and P.P. Gupta, 1994, Role of Azotobacter chroochoccum siderosphores in control bacterial rot and Sclerotinia rot of mustard, Indian J. Mycol Plant Pathol., 15: 121-124

Synaba D.A., and D.J. Bagyaraj, 2002, Selection of efficient VA Mycorrhizal fungi for neem (Azadiracha indica, A. Juso)

Tilak K.V.B.R., 1993, Rhizobium inoculant. Bacterial fertilizer. Indian council of Agricultural Research Krishi Anusandhar Bahvan, New Delhi, pp 4-34

Tilak K.V.B.R., and B.N. Murty, 1981, Occurrence of Azospirillium sp., in association with roots and stems of different cultivates of barley (Hordeum vulgare L.), Curr. Sci.,50: 496-498