Journal of Microbiology, Biotechnology and Food Sciences

Sharma and Kumawat 2011/12 1 (3) 383-394

REGULAR ARTICLE

CO-INOCULATION STUDY OF BRADYRHIZOBIUM JAPONICUM AND ASPERGILLUS NIGER IN SOYBEAN FOR NITROGEN FIXATION

Manish K. Sharma* and D.M. Kumawat

Address*: Manish K. Sharma, Vikram University, Faculty of Life Sciences, School of Studies in Environment Management, 456010 Ujjain Madhya Pradesh, India. Email: mks_ujjain@yahoo.com

ABSTRACT

Present study was aimed to evaluate the liquid inoculation of ten Bradyrhizobium japonicum with fungi Aspergillus niger (plant growth promoting) in pot experiments for determination of nitrogen fixation potential. Commonly grown JS-335 cultivar of Soybean around Ujjain region was used as host plant. Nodule number, shoot length, root length and their dry weight were taken as criteria for nitrogen fixation. Inoculation in pot along with fertilizer significantly increase the nodulation, shoot and root length at 5% of probability over inoculation of only B. japonicum and uninoculated. Total 12 treatments (10 tests + 2 controls) with three replicates were set and total 36 pots were arranged. Results revealed that B. japonicum increases the nitrogen fixation in combination with A. niger and induces the plant growth directly like phosphate solubilization, through IAA production, ammonia production and indirectly by inhibiting the pathogenic fungi. Co-inoculation was found suitable and effective over single inoculums. The present study supports that liquid inoculum of more than one organism results in increase in nodulation, and can effectively increase the crop productivity.

Keywords: Yeast Extract Mannitol Broth, Potato Dextrose Broth, Nodule Occupancy Per Plant

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2003. Li and Alexander. enhanced growth. Soybean plants were excavated carefully avoiding any injury and were brought in the laboratory.. Vance and Graham. 2008). the need of ecofriendly and cost-effective agricultural practices are the prime attention (Dube and Maheshwari. 2004). crop yield and biological control (Day et al. 2004.. 2004. 2005). The objective of our study was to evaluate the possible role of indigenous PGP fungal and B. 2006). MATERIAL AND METHODS Isolation and identification of Bradyrhizobia B. In context of increasing international concern for food and environmental quality. successful management of nitrogen inputs through Biological Nitrogen Fixation (BNF) with the help of legumes is essential (Bohlool and Schmidt. Symbiotic bacteria such as Rhizobium. Root nodules were washed under a gentle 384 . Thakuria et al.. Co-inoculation studies with plant growth promoting rhizobacteria (PGPR) and Bradyrhizobium have shown increased plant nodulation and nitrogen fixation under normal growth conditions (Verma et al.. Herman et al. The economic and environmental costs of the heavy use of chemical fertilizers in agriculture are of a global concern and there is an urgent need to seek some alternatives to fertilizers.. 1995). 1988). Most of the strains of Bradyrhizobium are associated with soybean species (Sameshima et al. 2008). 1968. Soybean has played a significant contribution to yellow revolution in India (Chauhan and Joshi.. japonicum was isolated from the freshly nodules collected from the field and identified by different morphological and biochemical identification following Buchanan and Gibbons (1984). and Sinorhizobium belonging to the group rhizobia (from the Latin ‘root living’) play an important role in the nutrition of leguminous plants by fixing atmospheric nitrogen in root nodules (Howieson et al.. For economically viable and environmentally prudent farming practices. The ultimate option is to go for sustainable agriculture practices. Zakhia et al. and as a food plant it forms a part of routine diet of the people (Tiwari.JMBFS / Sharma and Kumawat 2011/12 1 (3) 383-394 INTRODCUTION In present scenario where use of chemical fertilizers is excessive.. PGP microbes have been used in various crops for seed emergence. 2000a. Bradyrhizobium.japonicum isolates on plant growth in terms of nitrogen fixation. 1999). For nodule collection. the use of PGP (Plant Growth Promoting) microbes for reducing chemical inputs in agriculture is a potentially important issue. 1986. Giongo et al.

After 32 days of normal growth (period for onset of flowering in JS-335 cultivar) ten plants from each pot were used for the estimation of shoot length.01 level. Zar (2004). Statistical analyses The statistical analyses were done using Gomez and Gomez (1968). Seed germination Seeds of JS-335 Soybean cultivar procured from the JawaharLal Nehru Agriculture Centre. a liquid inoculum (culture) was prepared and mixed with soil in the ratio of 1: 60. Young. healthy and pinkish root nodules were selected for the bacterial isolation. The variables were subjected to ANOVA and were tested for significance at P<0. measured using Haemocytometer). Ujjain and were surface-sterilized with 0. isolated from rhizospheric soil of same soybean cultivar (JS-335) following Johnson et al. Seedlings were observed appropriately for development and watered whenever needed. and SPSS (Statistical Package. niger Aspn1 strain.JMBFS / Sharma and Kumawat 2011/12 1 (3) 383-394 stream of water. root length. (1959) and identified using A Manual of Soil Fungi by Gillman (1962). Six kilograms of soil was filled in the earthen-pots already treated with 5% CuSO4 solution. Sufficient amount of water and fertilizer were added. Twenty surface sterilized seeds were placed in the sterilized Petri plates and incubated at 30°C.1% HgCl2. japonicum that were isolated from root nodules of local soybean cultivar JS-335 were grown in the YEM (yeast extract mannitol) broth (HiMedia Lab. shoot and root samples were dried overnight at 72°C. shoot dry weight. After rinsing with water sufficiently. 385 . In each pot. Mumbai) and A. was grown in potato dextrose broth. moistened with about 4 ml distilled water.. Apparently healthy germinated seeds were selected and transferred in the previously prepared pots.0). Version 10. 15 seeds were placed about 2-3 cm deep in soil. Isolation of the bacteria was performed following Subba Rao (1984).05 and P<0. Pot arrangement Soil for the pots was colleted from the fields in polythene bags and sterilized in autoclave at 15Lb/inch2. Various isolated and identified strains of B. seeds were transferred in Petri dishes (10 cm) lined with two circles of cellulose filter papers. root dry weight and numbers of root nodules per plant. After proper growth of both the microbes (1×108cells ml-1 for bacteria and 1×104cells ml-1 fungi. For dry weight estimation.

The response of inoculum with respect to nodule formation (NOPP)/ root and shoot length and their dry weight was reflected in dry matter production as well.52±1.7 13.0 11.60 11.1 4.02±2.86±3. shoot and root length.9 11. but nodule number and dry weight may also be employed for more refined evaluation (Somasegaran and Hoben.10 10.24±0.50 14. japonicum) co-inoculated with A. niger Aspn1 on number of root nodules. length of shoot and root in JS-335 soybean cultivar after 32 days of sowing in Black Cotton soil.14±2. different isolates of B.0 13. Table 1 Effect of co-inoculation of B.6 10.36±1.20 10.95±2.00±1.5 24. The shoot dry weight of plant harvested in floral initiation is the generally accepted criteria for nitrogen fixing effectiveness.32±2.0 B1.2±1.15±2.50±2.2 13.5 14.B10.50±2.31±3.30 13. niger Aspn1 on number of root nodules.45±2.8 22. niger Aspn1 B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 Control 1 (Uninoculated) Control 2 Inoculated with B.3 15.5 10. niger Aspn1 Control-1 (Seedlings with no bacterial and fungal inoculum.6 26.9 22.3 26.4 25.25±1.80 8.30 11. Table-1 and Table-2 reveal the status and effect of treatment of various indigenous bacterial (B.56±1.6 26.56±2. In the present study.2 Root length (cm) 11.28±1.8 13.32±1.25±3. After 32 days of normal growth with different treatments of cultivar JS-335. japonicum co-inoculated with A. 1985). and their dry weight respectively.8 13.60±3.4 Shoot length (cm) 14.50±3.3 24.60 3. japonicum Isolates + A.80 11.00±2. B3….3±2. 2008) and Rhizobium inoculation of legumes is one of the success stories of world agriculture (Herridge. japonicum inoculum) ± Standard Deviation. of root nodules 23. plantlets were removed at the stage of flowering initiation.30 10.JMBFS / Sharma and Kumawat 2011/12 1 (3) 383-394 RESULTS AND DISCUSSION Nitrogen fixation is one of most important biological processes on earth (Graham. B.25±3.6±2. B2.75±2.2±0.50±3.7 25.02±2.23±2. grown only with distilled water) Control-2 (Seedlings inoculated with only B.24±1.8 14.04±2. 2008).64±4. japonicum and A.. 386 .2 06.60±1. japonicum only No.

98±0.JMBFS / Sharma and Kumawat 2011/12 1 (3) 383-394 Table 2 Effect of co-inoculation of B. japonicum Isolates + A.06 0.08 0..12 0. japonicum co-inoculated with A.68±0.21 1. there was a significant difference among isolates over controls was observed.88±0.69±0.08 0.02 Root dry weight (g) 0.89±0. japonicum inoculum) ± Standard Deviation.02±0.4±0.15 1.073 0.11 1.18 0.25 1.19 1. 1977).63±0.26 0.38±0. Based on the nodulation.05 0.16 0.23 1.86±0.12 0.41±0. niger Aspn1 and the bacterial isolates increased number of root nodules when compared with control treatments. japonicum only B1. the response of co-inoculation treatment varied with different bacterial strains.16 0.10 1.009 0.30±0. dry weight of shoot and root in JS-335 soybean cultivar after 32 days of sowing in Black Cotton soil B.88±0.69±0.75±0. Tiwari and Agrawal.72±0.91±0.09 1. niger Aspn1 on root nodules.87±0.09 0.71±0. combination of A.10 0.54±0.71±0.17 0.18 0.06 0.10 0.41±0.08 Root nodules and dry weight As evident from the Table-1.2 0. In our study. 1975.14 1.99±0. also reported positive interaction between Rhizobium and fungi inoculation on nodulation.68±0.05±0.B10.23 0. grown only with distilled water) Control-2 (Seedlings inoculated with only B.10 0.15 0.72±0.92±0.29±0. niger Aspn1 B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 Control 1 (Uninoculated) Control 2 Inoculated with B.13 0. However.08 Shoot dry weight (g) 0. There is a strong positive relationship between number and dry weight of nodules and yield and per plant in soybean (Roy and Mishra. B2.52±0.14 0. different isolates of B. we have grouped the isolates as excellent for more than 25 nodules 387 .69±0.46±0.57±0. Tarafdar and Rao (2001).87±0.69±0. B3….48±0. japonicum and A.89±0. Root nodules dry weight (g) 1. niger Aspn1 Control-1 (Seedlings with no bacterial and fungal inoculum.

isolate B3. B6 and B9 were showing good nodulation (Table-1).. japonicum (Sevilla et al. Increase in shoot length was significantly higher in all the bacterial isolates over controls in combination with A. (2000) with native Bradyrhizobium strains where increase in nodulation was observed in host plants. Similar results were obtained in the case of shoot dry weight. B4 and B8 shows excellent nodulation.. While in few other cases where certain rhizobial cultures have been reported.JMBFS / Sharma and Kumawat 2011/12 1 (3) 383-394 per plant. When the crop is inoculated with the isolates capable of IAA production. 2004. The reason behind such type of results might be the production of some kind of plant growth promoting phytohormones by the fungal isolate and synergistic effect with B. 2005). B1. Dudde and Raut. (1997) and Nandasena et al. Soliman et al. (1981) concluded that there was a significant increase in number of root nodule and dry matter of plants when Rhizobium culture was applied to the seedlings. Abril et al. (2011) also reproduce the data on nodulation and revealed that the inoculation with Rhizobium along with fungi significantly increased the nodule dry weight per plant. 1996).. Several workers have demonstrated that inoculation with indigenous Bradyrhizobium strains favours the formation of nodules on primary root (Milic et al.. 2001. Kumutha et al. (2001) also demonstrated that inoculation of soybean with native and well-adapted rhizobia resulted in a better response than inoculation with exotic. non-adapted strains. Shoot length and shoot dry weight Effect of the bacterial and fungal combination was also studied on the shoot length and shoot dry weight (Table-1 and 2). niger significantly increase the dry weight of root nodules over both the control treatments (Table 2). some plant growth promoting microorganisms may promote plant growth indirectly by affecting symbiotic N2 fixation. japonicum strains significantly increase the soybean crop yield. According to Fuhrmann and Wollum (1989). Many researches confirmed that inoculation of effective rhizobial strains increase plant height (shoot) and dry matter production as well as seed yield. significant increase in uptake of N. isolate B2.. There was a strong positive correlation between number of root nodules and crop yield which suggest that optimization of root nodulation by inoculating compatible and effective B. Therefore. bacterial isolates with A. Similar results were obtained by Abaidoo et al. niger. nodulation and shoot growth. Bhat et al. Further. B7 and B10 come under very good nodulation category while other isolates viz. Ca and Mg was reported by Farzana and Radizah (2005). 1992. to exhibit better nodulation and to produce 388 . Rastogi et al. K. Isolates producing IAA have stimulatory effect on the plant growth. P. very good for 24-25 nodules per plant while less than 24 nodules grouped as good. B5.

and Adeghipour et al.. 2005). 1998. (2008) that expression of nodulation and increase in shoot is directly related to the Rhizobium inoculum used.. (1998). 1991. (1997) and Ito et al. dry matter accumulation. 2006. 2001.. Sharma et al. In present study. 2003). significantly increased uptake. 1988. japonicum isolates (Table-1 and 2). Anjum et al. niger showed significant increase in root length and dry weight when co-inoculated with B.JMBFS / Sharma and Kumawat 2011/12 1 (3) 383-394 considerable increase in shoot and root length as well as shoot and root weight of seedlings in comparison to control (Brar and Lal. Sharma. 2000.... Musiyiwa et al. Similar results have been reported by Provorov et al. there was no significant difference observed in shoot length between the ten bacterial isolate treatments but a significant difference was observed when compared to respective controls (Table-1). accumulation and redistribution of nitrogen in plant parts specially root part (Konde et al. 2004). Therefore. Kumar et al. Kumutha et al. all ten isolates showed increase in shoot length and shoot dry weight over controls.. Chanway et al. Hayat et al.. (1989) and Yanni (1992) worked with native rhizobial strains and obtained similar kind of results where increase in nodulation and shoot length was observed in field trial as well as in Leonard Jar assembly. higher availability of nitrogen. 2002. 1995.. Dhami and Prasad (2009) concluded that inoculation of Rhizobium increase the accumulation of N in the root and results in increase in root length and also reported that nitrogen content in soil of inoculated experimental pots increased up to 13-33%. Root length and root dry weight A. Provorov et al. Results of present study come in agreement with the observations of Harper et al. 389 . (2008). It is known that indigenous rhizobial populations fix N2 with different efficiency depending of their density and activity and affect the root part and nodulation (Mpepereki et al. Thakur and Panwar. Although.. Though nitrogen status and its partitioning in crop plants was not studied in present work but possibly it was at par with the reported earlier findings that is reflected in over all growth of soybean crop. Mansoor. Pahalwan and Tripathi 1999. 2007). 2000. Hazarika et al. 1998. translocation of nutrients during reproductive stage which in turn improved yield attributes that affected the grain and straw yield at the end (Patel et al. Same type of results were obtained when a field experiment was conducted with soybean to find out the effect of inoculation and scheduling of irrigation on nodulation. (2010).

ANJUM.A.I. . phosphorus and molybdenum on yield and its components in mungbean. – SIDDIQUE. M.SINGH. Ujjain. J. p. Sci. Vol. 2010. It is clearly revealed that seedling growth in soybean can be enhanced by co-inoculating the seeds with the effective rhizobial strains along with plant growth promoting fungi.BANGROO. 2006. Vikram University.B. it is clear that the combination of B. In J. p. 2. In Int. – LAL. 17-20. Acknowledgements: Authors acknowledge the Head of Department. A. – MONEM. Sci. Madhya Pradesh for the necessary laboratory facilities provided. 10. A. 1012-1014. Recent developments in Indian agriculture.A. Effect of Rhizobium inoculation and nitrogen fertilizer on yield and yield components of mungbean.. . Research Journal of Agricultural Sciences.S. Vol. Biol. Vol.) as affected by nitrogen and phosphorus fertilizer application. 1. The microbial strains should preferably be indigenous ones. – RAFIQUE. 390 . – CHAUDHRY.A.A.YADAV. In growth and yield response of mungbean (Vigna radiata L. B. BRAR. – AHMED. BOHLOOL. Wilczek) under Temperate Conditions. R. . – AZIZ. Nonspecific staining: its control in immunefluorescence examination of soil. G. P. japonicum and A. 879-880. School of Studies in Environment Management. 29. J. niger is showing an increased nitrogen fixation.JMBFS / Sharma and Kumawat 2011/12 1 (3) 383-394 CONCLUSION From all six parameters tested in the present study. M. G. 1999. Biol.S. 2. In Combined Effects of Rhizobium and Vesicular Arbuscular Fungi on Green Gram (Vigna radiata L.B. – MALIK.I. S. p. 2. 1968.TAHIR. 1997. 1999.. 1991. BHAT. Vol. J.A. ALI. O. C. BHALLA. 2011. 1991.SCHMIDT E. In Science. Production of Mungbean (Vigna radiata L. S. In Indian Agriculturist . 67–69. p. . REFERENCES ADEGHIPOUR. M. a state level analysis. 162. – TAJALI. Pak.. p. A. 238-240.S. Effect of Rhizobium inoculation. 1997. 2011.R. In Econom.35. 2006. Vol. Vol. Agric. Z. – RAUF. M. Appl. M. 843-847.) to different seed rates and levels of phosphorous. M. 2010. A 2-18.A. Vol.S. p. 1968.L. p. & Political Weekly.A. M.

. – BARBERA.) Nitrogen-fixing Leguminous Symbioses.WOLLUM. In Dilworth.G. In Plant Cell Physiology. 2005. In Pak.A. – RAUT. FARZANA. 15. Vol. E.7. 1989. A. 1956. 2008. John Wiley and Sons.T. Sprent JI. FUHRMANN. 2. K. S. p. no. 315-318. HERRIDGE. T. no.E. .C. – HYNES. 23-43. 1956. Leguminous Nitrogen-fixing Symbioses. (eds. 2.). C. 49. Oxford and IBH publishing Co. S. M. 506-512. Newton WE (Eds. Estimation of N2-fixation of mung bean and mash bean through xylem ureide technique under rainfed conditions. HARPER. – KATO. mung bean.C. Published by Sprinker. DUBE. Vol. 2006. p. p. W. Application. Inoculation technology for legumes. L. p. 1984. 1984. N.M.A. K. 301-309. In: Dilworth MJ. 723-734. D. Y. T.K. GRAHAM. In A manual of Soil fungi (II eds). GOMEZ.C. – OHTAKE. T. Sprent.B. The autoregulation of nodulation mechanism is related to leaf development. J. p. M. Vol. Vol. NY 178-182. R. J. 2008. – CORRIGAN. S. 1242–1246. – GOMEZ.S.H. Kluwer. I and Newton. . Nitrogen Fixation: Orgins.K. R. M. A.A. ITO.S. – SUEYOSHI. James. p. 368-372. and hyacinth bean is controlled by a common shoot signal. p. GILMAN. 1997. p.ABD-ALLA. Combined effects of Rhizobium and VA-mycorrhiza inoculation on groundnut. James EK.ALI. Bot. O. 391 . 1 2005. 2008.F.IJAZ. Vol. J. 176-179. In Crop Science. Netherlands. 2008. In Journal of Soils and Crops Vol.H. 2005. 77–81. 1989. In Statistical procedure for agricultural research. p. 1989.121–125. J. p. 40. 37. R. 511-517. Volume 7. R. 3. 1989. 2005. – RADIZAH. – MAHESHWARI. 2008. – SIDDIQUE. D. K. In On Line Journal of Biological Science. Ecological of the root-nodule bacteria of legumes. 2008. J. 2008. Nodulation competition among Bradyrhizobium japonicum strains as influenced by rhizosphere bacteria and iron availability. Hypernodulation of soybean. p. K. 220-230. 21. 2008. J.P. 108–112.JMBFS / Sharma and Kumawat 2011/12 1 (3) 383-394 CHANWAY. Vol. K. – CHATHA. A. Biochem.J. In Biology and Fertility of Soils. 1997. p.H. In Soil Biol. Influence of rhizobacterial inoculation on growth of the sweet potato cultivar. HAYAT. Plant growth promoting rhizobacteria: effects on growth and nitrogen fixation of lentil and pea. .E. .OHYAMA. – NELSON. P. DUDDE. In A text book of Microbiology (Ed II) 2006.

2001. S. Effect of inoculation. 392 . M.) grown at Uzbekistan. Effect of insoluble phosphate and dual inoculation on soybean. P. 108. – SONANI. Wilczek). Vol. 569-575. – SARIC.R.R. Yield performance of Mungbean (Vigna radiata L. Azotobacter and Rhizobium on growth and nutrition of cowpea. p. Effect of seed inoculation and different fertilizer levels on the on the growth and yield of mungbean (Vigna radiata L. Effect of rhizobia inoculation on the seed (herbage) production of mungbean (Phaseolus aureus Roxb. J. Arid Environ. 37. K. M. Symbiotic effectiveness and host ranges of indigenous rhizobia nodulating promiscuous soybean varieties in Zimbabwean soils. – ALEXANDER. 1998.B. B. no. – KOZHEMYAKOV. Vol. 1998. – TALUKDER. G. V.KURBANOV. – MPEPEREKI. Braydrhizobium japonicum capacity to synthesize growth regulators affecting nodulation and nitrogen uptake by soybean. PROVOROV. In Soil Biol. Kumar and K. K. p. – AHMAD. no. D. J.U. 2004. 17-22. 3. Sci.P. . Z. In Plant and Soil.A. 1988. K. 1988. 2002. MPEPEREKI. – DAVIS. 2004.JAVAHERI. Agron. p. p. In J. 5.137-149. 1p. F. N and P fertilization on yield and yield attributes of green gram var. – SAIMNAZAROV. – JALALI. 2000. D. R. Vol. NADEEM.L.A. N. RAHMAN. A. 40-42. – SANTHONAKRISHNAN. K. p. – SASANI. p. N. In Mikrobiologija Beograd. PATEL. 28. A. In Pak.A. – SEMPAVALAN. A. p. G. MILLIC. – HASSAN. – PULATOVA. V. – MRKOVACKI.P. 211219. – AHMAD. 14. – ISLAM. 1988. In Field Crops Res. S. Vol.V. 1988. Kannaiyan. M. Biochem. 2. – GILLER.Z. – PATEL. In Journal of Mycology and Plant Pathology.K. N. Vol. Govindaraj and scientific Publishers (India) Jodhpur. Vol. 145-152. J..VOROSBARANYI L. 354-358. P. Gujarat-1 (Vigna radiate L. – GUPTA. KUMUTHA. 1992.JMBFS / Sharma and Kumawat 2011/12 1 (3) 383-394 KUMAR. Wilczck) cv. M. 2005. R. 146-148. 65. .V. 2001. Barimung-4 as Influenced by Rhizobium inoculation and NPK Fertilizer. 1169-1176. P.S. K.M. 2004.M.R. 2004 p. S. Vol. In Gujarat Agricultural University Research Journal. I. LI. M.).A. U. Biol. R. 39. 38-41. In J. 2005. Co-inoculation with antibiotic-producing bacteria to increase colonization and nodulation by rhizobia. Vol. – ISLAM. Impact of VAM. – BAHROMOV. In Biofertilizer technology Eds. p. MUSIYIWA.E. Soybeans and sustainable agriculture: promiscuous soybeans in Southern Africa. 3. 31. Vol.E. 2002. 2000. 1992. – GILLER.

1995. ZAR. TARAFDAR. 2001. Inter.) Kluwer Academic Publishers. 1. 29-31. 1995. – PUROHIT.UPADHYAYA. 112-115.K. – PANWAR. 2. IA Tikhonovich.GHANDOUR I. physiological and yield aspects of mungbean (Vigna radiata) as affected by inoculation treatment by different strains of Bradyrhizobium culture. Response of cluster bean to Glonus mossea and Rhizobium in an arid soil fertilized with nitrogen. D. Vol. p. 1995.JMBFS / Sharma and Kumawat 2011/12 1 (3) 383-394 SHARMA.P.S. In Journal of the Indian Society of Soil Science Vol. 1986. In Mol. J.H. p.) under field conditions. SOLIMAN. Plant Mol. 1995. 2003. 1999. Plant Pathol. C. SEVILLA. 1986. yield and economics of mungbean (Vigna radiata L. .197. 77-86 p. p. – STANLEY. Performance of chickpea. In World J. (eds. S. p. Vol. et al. P. 51-61. p. In Indian Journal of Agricultural Research Vol. 2003. 62-65. 2.H. – ABBADY. M. C. In Indian J. A. – FORTIN. Res.C. H.. – GRAHAM. 751-755. cobalt and molybdenum fertilization schedules. In I. Comparison of benefit to sugarcane plant growth and 15 N2 incorporation following inoculation of sterile plants with Acetobacter diazotrophicus wildtype and Nif-mutant strains.8. Vol. 37. Biostatistical analysis (Ed IV) Pearson Education Inc. Plant-Micr. 607. 212-218.K. Growth.A. V.).D. – MORRISON. Crop Vol. – RAMESHWAR. SHARMA. – KENNEDY. K. C.S. 52-55. p. N. Dordrecht. 2001. Response on growth. . Biol. R. S. boron.K. Microbiol.P.G. THAKUR. 41. Hill Farming. S. M. Wilczek under rain fed and mid hill conditions of Himachal Pradesh. SHARMA. 2003. N. 1996. J. 49. 2001. p. In Nitrogen Fixation: Fundamentals and Applications. J. 1992. Effect of nitrogen and phosphorus supply and of Rhizobium and VAM fungus inoculants on dinitrogen fixation in soybean. VERMA. – SARMA. S. – GUNAPLA. . 393 . 1992. R. Y. .SHARMA.G. p.RAO. p. In Folia Microbiological. 2003. lentil and lupine nodulated with indigenous or inoculated rhizobia micro-partners under nitrogen. Vol. Biotechnol. VANCE. In Nodulins and nodulin genes of Glycine max: A perspective. 1999. New Delhi. A.613.V. 12.200. – MAURO. 14. J. – BURRIS. S.A.G. 38.P. Effect of different production factors on growth. phosphorus and farm yard manure. 7. 2001. YANNI. 358-366. In Indian J. 2001. 1996. physiological parameters and yield of Vigna radiate L. p. R. Vol.H. 2001. Vol. Effect of Rhizobium -VAM interactions on growth and yield in mungbean (Vigna radiata L. C.