SUPERIOR SYMBIOTIC PERFORMANCE OF NATIVE BRADYRHIZOBIA ISOLATES OVER FOREIGN INOCULANTS WITH COWPEA CULTIVARS IN INDIA

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1 Legume Res., 37 (2) : , 2014 DOI: /j AGRICULTURAL RESEARCH COMMUNICATION CENTRE SUPERIOR SYMBIOTIC PERFORMANCE OF NATIVE BRADYRHIZOBIA ISOLATES OVER FOREIGN INOCULANTS WITH COWPEA CULTIVARS IN INDIA C. Appunu*, Ravinder Kumar, C. Mahadevaiah, V. Sreenivasa, Devyani Sen and Banshi Dhar Institute of Agricultural Sciences, Banaras Hindu University, Varanasi , India Received: Accepted: ABSTRACT Efficiency of symbiotic interactions of native and foreign inoculants bradyrhizobia isolates with six cowpea varieties (CO1, CO3, CO4, CO6, Paiyur 1 and Vamban 1) were evaluated under field conditions. Based on sequence analysis of 16S-23S rrna intergenic spacer (IGS) region, three native isolates were classified as B. yuanmingense. Cowpea varieties inoculated with bradyrhizobia showed significantly superior nodulation, nitrogenase activity, dry matter accumulation, and seed yield compared to uninoculated treatments. The native B. yuanmingense strains CR03 and CR09 exhibited host-cultivar specificity with CO3, and Paiyur 1, respectively. Plants inoculated with native B. yuanmingense isolates produced higher plant dry matter accumulation and seed yield compared to foreign B. japonicum inoculated plants. Correlation coefficients analysis revealed that plant dry matter accumulation emerged as the best criterion for selection of effective cowpea-bradyrhizobia associations in given physical and biological conditions. Key words: Cowpea, B. yuanmingense, Nitrogen fixation, Symbiotic efficiency. INTRODUCTION Beneficial symbiotic association between legume-endophytic symbionts in for biological nitrogen fixation has been a prime subject of study in the recent past in India and worldwide. It is an important aspect of sustainable and environmental friendly food production and long-term crop productivity (Meghvansh et. al. 2005). One such legume the cowpea [Vigna unguiculata (L.) Walp.] has been an important legume used for human consumption and as livestock feed for over a century in India (Singh, 1999). Cowpea establishes nitrogen fixing root nodules with slow-growers, Bradyrhi zobium spp and fast-growers, Sinorhizobium spp. Legume production in India involves low fertilizer input and legumes like cowpea depends on its symbionts to contribute to 80% its nitrogen nutrient requirements for growth and dry matter production. Therefore, the success of this crop in the country lies on its efficient symbiosis with N 2 - fixing bacteria (Appunu and Dhar 2006). Inoculation with highly effective rhizobia, is a common practice in agricultural production (Meghvansh et. al. 2005), and requires survival and establishment of inoculated rhizobia in the soil environment (Da and Deng, 2003). In India, cowpea is grown throughout the country but majorly in tropical and subtropical regions. Extent of nitrogen fixation by cowpea cultivars also drastically varies from site to site probably due to differences in poor symbiotic effectiveness of rhizobial strains and their compatibility with varietal genotypes. Selections of host cultivar-compatible inoculant have been recognized as an important method for increasing nitrogen fixation in cowpea wherein inoculation of foreign rhizobia isolates are thought to boost crop production (Law et al., 2007). When introduced, the foreign inoculant rhizobia must adapt to the prevailing soil conditions, multiply in the soil and host rhizosphere to form effective symbiosis. However in most cases the inoculants often fail to establish and form nodules (Fening and Danso, 2001). Therefore, an alternative method of improving * Corresponding author s cappunu@gmail.com 1 Division of Crop Improvement Sugarcane Breeding Institute Coimbatore Tamil Nadu,India.

2 176 LEGUME RESEARCH- An International Journal the success of inoculants could be to explore the utility of native strains that are symbiotically effective for nodulation as inoculants. It was observed that the symbiotic nitrogen fixation (SNF) in legume depends upon co-selection of plant and bacterium genotypes (Fall et al., 2003; Appunu et al., 2008; Pule-Meulenberg et al., 2010). Therefore, the present study was undertaken to compare the symbiotic effectiveness of foreign and native isolates and identification of host specific bradyrhizobia, which are superior in nodulation and plant development under field conditions. MATERIALS AND METHODS Isolation and authentication of bradyrhizobia isolates from field grown cowpea: Three native bradyrhizobia isolates (CR03, CR05 and CR09) were obtained by following the procedure detailed by Somasegaran and Hoben (1994) from root nodules of cowpea plants grown under field conditions in Agriculture Farm, Banaras Hindu University (BHU), Varanasi, Uttar Pradesh, India. In addition, two foreign cowpea nodulating B. japonicum isolates (USDA135 and USDA110) received from ARS culture collection, USA were also used in this study. All the isolates were authenticated by plant nodulation test on cowpea cv. Paiyur 1. Single colonies were picked up and maintained on YMA slant at 28 ± 2 o C. Genotypic characterization: Genomic DNA isolation, 16S-23S rrna intergenic spacer (IGS) region amplification and sequencing, were performed as described by Appunu and Dhar (2008). As an initial approach to identify the bacterial isolates, a BLAST search on the GenBank database was performed with the sequences obtained corresponding to the 16S-23S rrna IGS. Symbiotic effectiveness test: Six cowpea varieties (CO1, CO3, CO4, CO6, Paiyur 1 and Vamban 1) were inoculated with three native (CR03, CR05 and CR09) and two foreign (USDA135 and USDA110) isolates. Surface sterilized bold healthy seeds of these varieties were coated by mixing the seeds with gum arabic and bradyrhizobia strain inoculated peat carrier. The coated cowpea seeds were planted in a field experiment at Agriculture Farm, BH U, Varanasi. The experimental layout was a randomized complete block design with treatments distributed in a split block arrangement. Each of the six main plot treatments of cowpea cultivars were subdivided into 6 sub plots treated consisting of randomly distributed treatments of the five bradyrhizobia strains under study while the sixth subplot was the control. Three sets of four replicates were used. Each plot was made up of three rows of 5 m long and 0.6 m wide with seeds planted 20 cm apart. At the full bloom stage, ten plants from each plot were uprooted and nodule dry weight, nitrogenase activity and total dry matter accumulation data were recorded as described previously (Appunu et al., 2008). At maturity, all remaining plants from the middle row were harvested and the seed yield determined. Statistical analysis: Average data recorded over two years was subjected to ANOVA using the statistical analysis systems (SAS, 1988). Means of all treatments were calculated and the differences tested for significance using the least significant differences (LSD) test at 0.05 probability (P) level. Correlation coefficient was calculated to study the associative relations among the measurement traits. RESULTS AND DISCUSSION Based on sequence analysis of 16S-23S rrna intergenic spacer (IGS) region, three native isolates were classified as B. yuanmingense with 100% sequence similarity. Presence of significant proportion of cowpea nodulating B. yuanmingense in Indian soils has already been reported (Appunu et al., 2009). Six varieties were evaluated for their symbiotic performances with five bradyrhizobia strains under field conditions. All isolates induced root nodules and recorded significant (P< 0.05) interaction on nodulation, plant growth and seed yield between host cultivars and inoculation treatment (Table 1). Cowpea cultivars varied in the average nodule dry weight per plant induced. The highest nodule dry weight was noted with variety CO3 in association with CR05, whereas the highest in-vivo nitrogenase expression was observed in variety CO6 treated with strain CR03 followed by variety Paiyur 1 with CR09. On the basis of nitrogenase activity, the efficacy of strains in decreasing order was CR03, CR09, CR05, USDA110 and USDA135. Analysis of data revealed that among the six cultivars, Paiyur 1 recorded

3 Vol. 37, No.2, TABLE 1: Symbiotic performance of bradyrhizobia strains with cowpea cultivars under field conditions. Inoculation treatm ent C ultiv ars CO1 C O3 C O 4 CO6 Paiy ur 1 Vamban 1 N odule dry weight (m g pl ant -1 ) CR CR CR T AL T AL Control LSD Cultiv ars Inoculati ons Interactions N odule nitrogenase acti vity (µmol C 2 H 2 reduced h -1 g -1 fresh nodule) CR CR CR T AL T AL Control LSD Cultiv ars 3.98 Inoculati ons 4.67 Interactions 8.61 T otal dry m atter accumulati on (g plant -1 ) CR CR CR T AL T AL Control LSD Cultiv ars 3.21 Inoculati ons 1.48 Interactions 4.79 Seed yi eld (g plant -1 ) CR CR CR T AL T AL Control LSD Cultiv ars 3.43 Inoculati ons 3.13 Interactions 6.87 highest dry matter accumulation after inoculation with bradyrhizobia strain CR09. The lowest average dry matter production was observed with foreign strain inoculated cultivars. The mean plant dry matter of uninoculated control plants was significantly lower than bradyrhizobia inoculated plants. Significant differences between rhizobial strains for symbiotic parameters such as nodule dry weight, nitrogenase activity and total plant dry weight has been reported (Thies et. al., 1991;Appunu et al., 2008; Sarr et al., 2009; Pule-Meulenberg et al., 2010). Maximum seed yield at harvest was recorded in cv. Paiyur 1 in association with strain CR09. Overall seed yield was highest for cultivars

4 178 LEGUME RESEARCH- An International Journal TABLE 2: Correlation coefficients between symbiotic parameters in cowpea-bradyrhizobia interaction under field conditions. Symbiotic parameters Nitrogenase activity Total plant dry Seed yield (g pl -1 ) (µmol h -1 fr. wt g -1 )` weight (g pl -1 ) Nodule dry weight (mg pl -1 ) 0.599* 0.687* 0.632* Nitrogenase activity 0.696** 0.688* Total plant dry weight 0.902** * and ** are significant at 5% and 1% levels, respectively inoculated with CR09 followed by CR03 inoculated plants whereas, the lowest was with strain USDA135. In agreement with this result, B. yuanmingense isolates had significantly greater nitrogen-fixing potential with cowpea than B. japonicum and B. elkanii strains (Sarr et al., 2009). Genetic variation for symbiotic association for biomass production among cowpea cultivars has already been reported (Fall et. al., 2003; Ampomah et al., 2008; Sarr et al., 2009; Pule-Meulenberg et al., 2010). Comparison of symbiotic performance of cultivars with bradyrhizobia, the most efficient performer was the B. yuanmingense CR09 followed by CR03 and CR05 in that order (Table 1). The plants inoculated with these strains maintained highest average symbiotic performance with regards to plant dry matter accumulation and seed yield compared with plants inoculated with foreign strains. Superior symbiotic performance between the compatible host genotype and rhizobia strain already been reported in cowpea (Thies et. al., 1991; Fall et. al., 2003; Ampomah et al., 2008; Sarr et al., 2009). Under field conditions, the supporting effectiveness of nitrogen fixing rhizobia strains on percent improvement in total plant dry matter accumulation was slightly lower than that observed under laboratory conditions (Botha et. al., 2002; Appunu et al., 2008). This clearly indicates that various climatic and edaphic factors might adversely affect the efficiency of Rhizobium-legume symbiosis when grown under field conditions and this could be one of the reasons why the foreign isolates did not perform to their potential. Further, native rhizobial strains were symbiotically more effective on cowpea cultivars than strains of foreign ori gin. Better symbiotic compatibility between native rhizobia and cowpea cultivars both under laboratory conditions (Fall et. al., 2003; Ampomah et al., 2008; Sarr et al., 2009) and field conditions (Thies et. al., 1991; Musiyiwa et. al., 2005; Pule-Meulenberg et al., 2010) has been observed. Correlation coefficient analysis among symbiotic traits for cowpea-rhizobium system is presented in Table 2. Seed yield correlated positively and significantly with total dry matter accumulation, nodule nitrogenase activity and dry weight in decreasing order. Based on our results and similar such reports on symbiotic performances of nitrogen fixing plants (Appunu et al., 2008; Hefny et al., 2001), it can be concluded that plant dry weight is the best and generally accepted criterion for selection of the most effective legume-rhizobium symbiotic associations. REFERENCES Ampomah, O.Y., Ofori-Ayeh, E., Solheim, B., Svenning, M.M. (2008). Host range, symbiotic effectiveness and nodulation competitiveness of some indigenous cowpea bradyrhizobia isolates from the transitional savanna zone of Ghana. Afr. J. Biotechnol. 7(8): Appunu, C., Sen, D., Singh, M.K., Dhar, B. (2008). Variation in symbiotic performance of Bradyrhizobium japonicum strains and soybean cultivars under field conditions. J. Central European Agri. 9(1): Appunu, C., Dhar B. (2008). Isolation and symbiotic characteristics of two Tn5-derived phage-resistant Bradyrhizobium japonicum strains that nodulate soybean. Curr. Microbiol. 57: Appunu, C., N Zoue, A., Moulin, L., Depret, G., Laguerre, G. (2009). Vigna mungo, V. radiata and V. unguiculata plants sampled in different agronomical-ecological climatic regions of India are nodulated by Bradyrhizobium yuanmingense. Syst. Appl. Microbiol. 32: Botha, W.J., Bloem, J.F., Law, I.J. (2002). Bradyrhizobium sp. (Lupinus) in the winter rainfall region of South Africa. Biol. Fertil. Soils. 36: Da, H.N., Deng, S.P. (2003). Survival and persistence of genetically modified Sinorhizobium meliloti in soil. Appl. Soil Ecol. 22: 1-14.

5 Vol. 37, No.2, 2014 Fall, L., Diouf, D., Fall-Ndiaye, M.A., Badiane, F.A., Gueye, M. (2003). Genetic diversity in cowpea [Vigna unguiculata (L.) Walp.] varieties determined by ARA and RAPD techniques. Afr. J. Biotechnol. 2(2): Fening, J.O., Danso, S.K.A. (2001). Response of cowpea to inoculation with indigenous Bradyrhizobium strains. Trop. Sci. 41: Hefny, M., Dolinski, R., Malek, W. (2001). Variation in symbiotic characters of alfalfa cultivars inoculated with Sinorhizobium meliloti strains. Biol. Fertil. Soils. 33: Law, I.J., Botha, W.F., Majaule, U.C., Phalane, F.L. (2007). Symbiotic and genomic diversity of cowpea bradyrhizobia from soils in Botswana and South Africa. Biol. Fertil. Soils. 43: Musiyiwa, K., Mpepereki, S., Giller, K.E. (2005). Symbiotic effectiveness and host ranges of indigenous rhizobia nodulating promiscuous soybean varieties in Zimbabwean soils. Soil Biol. Biochem. 37: Pule-Meulenberg, F., Belane, A.K., Krasova-Wade, T., Dakora, F.D. (2010). Symbiotic functioning and bradyrhizobial biodiversity of cowpea (Vigna unguiculata L. Walp.) in Africa. BMC Microbiol. 10: Sarr, P.S., Yamakawa, T., Fujimoto, S., Saeki, Y., Thao H.T.B., Myint, A.K. (2009). Phylogenetic diversity and symbiotic effectiveness of root-nodulating bacteria associated with cowpea in the South-West area of Japan. Microbes Environ. 24: SAS Institute, SAS/STAT user s guide (1988). SAS, Cary, N.C. Singh, B.B. (1999). Improved drought tolerant cowpea varieties for the Sahel Project 11. Cowpea-cereal system improvement for the savannas, IITA, Ibadan, Nigeria, p. 36. Somasegaran, P., Hoben, H.J. (1994). Handbook for Rhizobia, Methods in Legume-Rhizobium Technology, p Springer-Verlag, Heidelberg, Germany. Thies, J.R., Bohlool, B.B., Singleton, P.W. (1991). Influence of the size of indigenous rhizobial populations on establishment and symbiotic performance of introduced rhizobia on field-grown legumes. Appl. Environ. Microbiol. 57: Vance, C.P. (2001). Symbiotic nitrogen fixation and phosphorus acquisition. Plant nutrition in a world of declining renewable resources. Pl. Physiol. 127: