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Sharma and Kumawat 2011/12 1 (3) 383-394
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: email@example.com
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
1999). crop yield and biological control (Day et al.. MATERIAL AND METHODS Isolation and identification of Bradyrhizobia B. In context of increasing international concern for food and environmental quality. 2004. enhanced growth. Zakhia 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. 2004). and as a food plant it forms a part of routine diet of the people (Tiwari. the need of ecofriendly and cost-effective agricultural practices are the prime attention (Dube and Maheshwari. 2004.. For economically viable and environmentally prudent farming practices. Soybean has played a significant contribution to yellow revolution in India (Chauhan and Joshi. 1995).. Li and Alexander. 2008). 2003. 2000a. Herman et al. The objective of our study was to evaluate the possible role of indigenous PGP fungal and B. the use of PGP (Plant Growth Promoting) microbes for reducing chemical inputs in agriculture is a potentially important issue.japonicum isolates on plant growth in terms of nitrogen fixation.. 1968. 2005). Root nodules were washed under a gentle 384 . 2008). The ultimate option is to go for sustainable agriculture practices... successful management of nitrogen inputs through Biological Nitrogen Fixation (BNF) with the help of legumes is essential (Bohlool and Schmidt. 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. japonicum was isolated from the freshly nodules collected from the field and identified by different morphological and biochemical identification following Buchanan and Gibbons (1984). Giongo et al. 1986. 1988). For nodule collection. Vance and Graham. Thakuria et al. Most of the strains of Bradyrhizobium are associated with soybean species (Sameshima et al. Symbiotic bacteria such as Rhizobium. Bradyrhizobium... 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. Soybean plants were excavated carefully avoiding any injury and were brought in the laboratory. 2006). PGP microbes have been used in various crops for seed emergence.JMBFS / Sharma and Kumawat 2011/12 1 (3) 383-394 INTRODCUTION In present scenario where use of chemical fertilizers is excessive.
05 and P<0. seeds were transferred in Petri dishes (10 cm) lined with two circles of cellulose filter papers. (1959) and identified using A Manual of Soil Fungi by Gillman (1962). After proper growth of both the microbes (1×108cells ml-1 for bacteria and 1×104cells ml-1 fungi. moistened with about 4 ml distilled water. Six kilograms of soil was filled in the earthen-pots already treated with 5% CuSO4 solution.JMBFS / Sharma and Kumawat 2011/12 1 (3) 383-394 stream of water. 15 seeds were placed about 2-3 cm deep in soil. isolated from rhizospheric soil of same soybean cultivar (JS-335) following Johnson et al. and SPSS (Statistical Package. Apparently healthy germinated seeds were selected and transferred in the previously prepared pots. Sufficient amount of water and fertilizer were added. 385 . niger Aspn1 strain. shoot and root samples were dried overnight at 72°C. root dry weight and numbers of root nodules per plant. Version 10. Isolation of the bacteria was performed following Subba Rao (1984). healthy and pinkish root nodules were selected for the bacterial isolation.1% HgCl2. shoot dry weight. Statistical analyses The statistical analyses were done using Gomez and Gomez (1968). 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.. Young.01 level. japonicum that were isolated from root nodules of local soybean cultivar JS-335 were grown in the YEM (yeast extract mannitol) broth (HiMedia Lab. Twenty surface sterilized seeds were placed in the sterilized Petri plates and incubated at 30°C. After rinsing with water sufficiently. The variables were subjected to ANOVA and were tested for significance at P<0.0). was grown in potato dextrose broth. Zar (2004). Seedlings were observed appropriately for development and watered whenever needed. In each pot. Mumbai) and A. root length. Various isolated and identified strains of B. Ujjain and were surface-sterilized with 0. For dry weight estimation. measured using Haemocytometer). Pot arrangement Soil for the pots was colleted from the fields in polythene bags and sterilized in autoclave at 15Lb/inch2. a liquid inoculum (culture) was prepared and mixed with soil in the ratio of 1: 60. Seed germination Seeds of JS-335 Soybean cultivar procured from the JawaharLal Nehru Agriculture Centre.
32±2.56±2.02±2. The shoot dry weight of plant harvested in floral initiation is the generally accepted criteria for nitrogen fixing effectiveness.10 10.9 22. 2008) and Rhizobium inoculation of legumes is one of the success stories of world agriculture (Herridge.60 3.2±1.24±0.50±3.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.2 06.8 13.14±2.50±2.0 B1. grown only with distilled water) Control-2 (Seedlings inoculated with only B.60 11..3 24.50 14.64±4. japonicum Isolates + A.23±2.6 26.5 10.31±3. japonicum and A. japonicum co-inoculated with A.6 10. japonicum only No. japonicum inoculum) ± Standard Deviation. Table-1 and Table-2 reveal the status and effect of treatment of various indigenous bacterial (B. Table 1 Effect of co-inoculation of B.56±1.24±1.95±2.25±1.0 11.86±3. japonicum) co-inoculated with A.7 13. shoot and root length.36±1.75±2.1 4. and their dry weight respectively. plantlets were removed at the stage of flowering initiation.02±2.45±2. niger Aspn1 B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 Control 1 (Uninoculated) Control 2 Inoculated with B.52±1.5 24.80 8. After 32 days of normal growth with different treatments of cultivar JS-335.8 14.6±2.50±3.2 Root length (cm) 11.2 13.25±3. B3…. but nodule number and dry weight may also be employed for more refined evaluation (Somasegaran and Hoben. 2008). B2.20 10.04±2.2±0.3±2.50±2.60±1.00±1. different isolates of B.4 25. In the present study.25±3. niger Aspn1 on number of root nodules.3 26.9 11. 386 .30 13.5 14.30 11.15±2. 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. B.30 10. niger Aspn1 Control-1 (Seedlings with no bacterial and fungal inoculum. of root nodules 23.8 22.8 13.80 11.6 26.0 13. length of shoot and root in JS-335 soybean cultivar after 32 days of sowing in Black Cotton soil.60±3. niger Aspn1 on number of root nodules.00±2.4 Shoot length (cm) 14.3 15.28±1.B10. 1985).32±1.7 25.
B10.69±0. niger Aspn1 on root nodules. grown only with distilled water) Control-2 (Seedlings inoculated with only B.15 0. niger Aspn1 Control-1 (Seedlings with no bacterial and fungal inoculum.10 0.54±0. combination of A.69±0.71±0. Tiwari and Agrawal. 1975.91±0.71±0. niger Aspn1 and the bacterial isolates increased number of root nodules when compared with control treatments.12 0. also reported positive interaction between Rhizobium and fungi inoculation on nodulation.52±0.14 0..88±0.89±0.08 0.26 0.18 0.98±0.16 0.02 Root dry weight (g) 0.41±0.87±0.09 0.15 1. japonicum inoculum) ± Standard Deviation.06 0. In our study. there was a significant difference among isolates over controls was observed.19 1.11 1.08 0.48±0.99±0.18 0.073 0.08 Shoot dry weight (g) 0. japonicum and A.2 0.72±0.25 1. However.17 0.88±0.38±0.10 0. There is a strong positive relationship between number and dry weight of nodules and yield and per plant in soybean (Roy and Mishra.57±0.29±0.02±0. B3….06 0.16 0.009 0.13 0. we have grouped the isolates as excellent for more than 25 nodules 387 .68±0. Root nodules dry weight (g) 1.14 1.12 0.21 1.69±0.08 Root nodules and dry weight As evident from the Table-1.92±0.23 1.72±0. different isolates of B.46±0. japonicum co-inoculated with A.68±0.89±0. Tarafdar and Rao (2001). the response of co-inoculation treatment varied with different bacterial strains.63±0.10 0. dry weight of shoot and root in JS-335 soybean cultivar after 32 days of sowing in Black Cotton soil B.30±0.09 1.10 1.4±0. japonicum Isolates + A.41±0.69±0.75±0.05 0.87±0.23 0. B2. japonicum only B1.86±0.JMBFS / Sharma and Kumawat 2011/12 1 (3) 383-394 Table 2 Effect of co-inoculation of B. niger Aspn1 B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 Control 1 (Uninoculated) Control 2 Inoculated with B. Based on the nodulation. 1977).05±0.
Further. B5. Similar results were obtained in the case of shoot dry weight. (1997) and Nandasena et al. japonicum (Sevilla et al.. 2001. While in few other cases where certain rhizobial cultures have been reported. 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. Many researches confirmed that inoculation of effective rhizobial strains increase plant height (shoot) and dry matter production as well as seed yield. B6 and B9 were showing good nodulation (Table-1). nodulation and shoot growth.. When the crop is inoculated with the isolates capable of IAA production. Ca and Mg was reported by Farzana and Radizah (2005). (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. 2004.. (2001) also demonstrated that inoculation of soybean with native and well-adapted rhizobia resulted in a better response than inoculation with exotic. significant increase in uptake of N. isolate B3. Therefore. Similar results were obtained by Abaidoo et al. bacterial isolates with A. Rastogi et al. isolate B2. Several workers have demonstrated that inoculation with indigenous Bradyrhizobium strains favours the formation of nodules on primary root (Milic et al. Soliman et al.. Increase in shoot length was significantly higher in all the bacterial isolates over controls in combination with A. P. Kumutha et al. B4 and B8 shows excellent nodulation. to exhibit better nodulation and to produce 388 .JMBFS / Sharma and Kumawat 2011/12 1 (3) 383-394 per plant. B7 and B10 come under very good nodulation category while other isolates viz. (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. B1. According to Fuhrmann and Wollum (1989). Isolates producing IAA have stimulatory effect on the plant growth. Abril et al. non-adapted strains. 1992. (2000) with native Bradyrhizobium strains where increase in nodulation was observed in host plants. K. japonicum strains significantly increase the soybean crop yield. niger significantly increase the dry weight of root nodules over both the control treatments (Table 2). 2005). niger. some plant growth promoting microorganisms may promote plant growth indirectly by affecting symbiotic N2 fixation. Dudde and Raut. 1996).. very good for 24-25 nodules per plant while less than 24 nodules grouped as good. 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. 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). Bhat et al.
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. (2008) that expression of nodulation and increase in shoot is directly related to the Rhizobium inoculum used. dry matter accumulation. (2010). 1988. Pahalwan and Tripathi 1999. 2003). (1998). 2000. Therefore.. (1997) and Ito et al. all ten isolates showed increase in shoot length and shoot dry weight over controls. 389 . 2004). Provorov et al. 2005). (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. 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. Although. accumulation and redistribution of nitrogen in plant parts specially root part (Konde et al. 2000. 1995. and Adeghipour et al. Kumar et al. 2007).. 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. Similar results have been reported by Provorov et al. (2008). Kumutha et al. japonicum isolates (Table-1 and 2). 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.. Mansoor. Hayat et al. In present study. 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%. 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). Anjum et al. 2006.. 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. 2002... Sharma et al.. Hazarika et al.. Musiyiwa et al. Sharma. 1998.. higher availability of nitrogen. 1991. Root length and root dry weight A.. Thakur and Panwar. significantly increased uptake. Chanway et al. 2001. Results of present study come in agreement with the observations of Harper et al. 1998.
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