Madurai Kamaraj University, Madurai, India 2 PG Studies & Research Deparment in Botany, Thiagarajar College,

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1 ISSN J. Bio sc i. Re s., 2012.Vol.3(1):76-82 Influence the growth of tree legume Se sb an ia g ran d iflo ra (Pers) by using stress tolerant Rh izo b ium K.KARUPPASAMY 1, J.SUBATHRA DEVI 1, K.SURESH 2, M.DHANIYA 3, AND D. KATHIRESAN 3 * 1 Department of Genetic Engineering, School of Biotechnology, Madurai Kamaraj University, Madurai, India 2 PG Studies & Research Deparment in Botany, Thiagarajar College, Madurai, Tamilnadu, India. 3 PG studies & Research Centre in Botany, Saraswathi Narayanan College, Madurai, Tamilnadu, India.. Abstract Symbiotic nitrogen fixation by legume-rhizobium association play an important role in sustaining crop productivity and maintaining soil fertility, especially on marginal lands. One of the major approaches for the optimization of symbiotic nitrogen fixation is to increase the land area under legumes and enhance their adaptation and resistance to environmental stresses. In present study stress tolerant Rrhizobium was isolated from different stressed soils such as dry land soil (DLS), dye industry effluent soil (DES), saline soil (SS) and calcareous soil (CS) and attempted to use for the growth improvement of tree legume Sesbania grandiflora. Application of rhizobial inoculum was done as seed coating and soil inoculation. The growth and biochemical characteristics were analyzed in Rhizobium treated plants. At the end of 90 day of plant growth, highest increase of 64.50% in shoot length and 45.04% increase root length was recorded using CS-strain and SS-strain respectively. As for the biomass accumulation, the highest increase % in shoot dry weight and % in root dry weight was recorded in SS-T strain treaded plants. Fresh and dry weight of root nodules were analysed, the maximum of 231.4% increase root nodule fresh weight and 220.1% increase nodule dry weight was recorded in CS-strain treated plants. Key words: Rhizobium, Sesbania grandiflora, Stressed soil, Nitrogen fixation For correspondence: kathiresansnc@gmail.com Introduction ecosystem consisting mainly of trees that In developing countries like India buffer the Earth and supports a myriad of forests play an important role in providing life forms. The growing population s high economic wealth maintaining ecological demand for forest resources continued the balance and improving the productivity. destruction and degradation of forests Therefore, improvement of soil fertility and through the 1980s, taking a heavy toll on the afforestation in waste lands are necessary to soil. An estimated 6 million tones of top soil protect the vegetation cover, forest cover is being lost annually. Though the studies on and future life. The forest is a complex rhizobial symbiosis have been emphasized JOURNAL OF BIOSCIENCES RESEARCH 3(1):

2 on crop legumes, in recent years, emphasis is given to tree legumes too. Legumes are well suited for the reclamation of unproductive and barren lands. Legumes are capable of fixing nitrogen in symbiotic association with Rhizobium, Bradyrhizobium and Azorhizobium. Soils that lack adequate number of rhizobia need rhizobial inoculation to establish a successful symbiosis (Sumanmor et al.,1994). Reclamation and improvement of the infertile lands by the rhizobium-legume symbiosis is suggested to be the ideal solution for the improvement of soil fertility and rehabilitation of arid lands and is an important direction for future research. Therefore, it was believed that isolation of rhizobial strains and using them as inoculants for other leguminous plants could improve the growth of these plants. Observation of the development of nodules in the stressed soils leads one to believe that these strains could be highly efficient as they produce root nodules in the legumes under difficult conditions. Since rhizobial symbiosis in leguminous trees plays an important role in nitrogen fixation and improvement of the soil fertility, rhizobia isolated from such environment are useful. With this background, the present paper describes the isolation of rhizobial strains from different stressed soils and using them as inoculants to improve the growth performance of tree legumes in stressed conditions. Materials and Methods Co lle c tio n and analy sis o f so il Saline soil (SS), Calcareous soil (CS), Dye industry effluent soil(des), Dry land soil (DLS) and Garden (red) soil (CON) were collected from different places. The soils were tested for parameters such as ph, Electrical conductivity (according to standard procedures), organic carbon and organic matter (Walkley & Black., 1934). The physico-chemical characters of different soils used are presented in Table-1. Iso latio n and ino c ulatio n Rh izo b ium Stress tollerent strains of rhizobia were selected based on the growth performance of host plants, nodule number and fresh and dry weight of nodules. Rhizobium isolated from Sesbania grandiflora grown on saline soil, calcareous soil and dye industry effluent soil and rhizobia isolated from Erythrina varigata grown on dry land soil was taken for testing their potential on Sesbania grandiflora. For the sake of comparison, a strain of Rhizobium isolated from plants grown in garden soil was also taken. The Rhizobium strains were isolated using healthy and pink nodules only and cultured at 28 0 C on YEM agar with Congo red (Vincent JM.1970). Distinct rhizobial colonies were picked out, purified and maintained. The healthy Sesbania seeds were surface sterilized and seeds were treated with rhizobial inoculums and sown in the polythene bags filled with mixture of red soil and sand (1:1). Different treatments viz., seed treatments and soil treatments were given. Estim atio n o f g ro w th p aram e te rs, p ig m e nts and m e tab o lite s Growth characters were analyzed on the 90 th day of plant growth. Shoot and root length, shoot, root fresh and dry weights and nodule numbers and fresh and dry weight of nodules using data obtained from 10 replicates for each treatment were recorded (Table-2). The following Biochemical characters were analyzed such as chlorophyll (Arnon, 1949), soluble protein (Lowry et al., 1951) and nitrate reductase activity (Muthuchelian,1989) using data obtained from triplicates. All the measurements were made using leaves collected randomly selected plants from each treatment. Results and discussion Plant g ro w th p e rfo rm an c e The morphological characteristics of Sesbania grandiflora, on length, fresh and dry JOURNAL OF BIOSCIENCES RESEARCH 3(1):

3 weight of shoot, root and nodules are given in Table-2. Among the different strains used and different treatments given, the seed and soil treatment increased the length of shoot and root. On a comparative basis the rhizobial isolates of CS increased the shoot length to the maximum of 64.50% and while maximum increase of root length 45.04% recorded in SS-T strain than the untreated control. Though the increase in root length was comparatively lesser than the shoot length, the data on shoot and root length indicate that the difference between performance of the different strains implicates a variation in infection and affective nodules. It is very much evident that the rhizobial strains due to their nitrogen fixing ability have increased the growth of the plant which has resulted in an increase in the shoot length. Sivakumar and Kumutha (2003) in their results found that an increase in shoot length of 17.5, 24.5 and 8.3 percent occurred with the use of TNAU 14, PGR 1 and BGR 1 rhizobial strains respectively. A similar trend was observed in root length also with maximum increase recorded using PGR 1 (8.4%) followed by TNMAU 14 (8.0%). There was no increase in root length using BGR-1. These results are supporting the present. The significant increase in the shoot and root fresh weight occurred in all the treatments. However, the saline soil strain had the maximum influence of shoot (580.52%) and root ( %), when compared to their respective control (Table- 2). The increase in root fresh weight was found to be better than the shoot fresh weight in all the treatments, the difference being 2-3 times. As in the case of shoot fresh weight, the increase was highly significant even when compared to the treated control. The trend observed in the root and shoot fresh weights reflected on the total fresh weight as well. The increase in the fresh weights reflected in an increase in the dry weight also. The increase in shoot, root and total dry weight were all highly significant, not only, when compared to the untreated control but also when compared to the treated control (Table-2). Kantar et al. (2003) reported that inoculation with Rhizobium strains isolated from perennial wild chick peas at high altitudes significantly increased fresh and dry weights of plant and also increased yield components of chick pea (CV.Aziziye-94) in the field compared to the uninoculated control. Singh et al. (2005) reported that more dry matter accumulation with the use of SML 357 strain was due to more leaf area at maturity, while in the case of SML 134 strains, it was due to more growth in terms of plant height. These results are supporting to the present study. The nodulation characters are very much influenced by all the rhizobial isolates from different stressed soils. The maximum increase in nodule number was observed in dry land soil isolates (Table-2). A maximum increase of fresh weight (160.11%) and dry weight (194.12%) of nodule was observed under the influence of saline soil isolates when compared to the respective control. The increase in the fresh and dry weight of the nodule would also result in better nodulation and an increased efficiency in fixing nitrogen. This will certainly increase the availability of nitrogen for an increased synthesis of important biochemical, like protein and nucleic acids. An increased amount of proteins both structural as well as functional will definitely influence better growth. Abdullahi Bala and Giller (2001) found that rhizobial isolates of Calliandra calthyrsus, Gliricida-sepium and Leucaena leucocephala were able to effectively cross nodule each other s hosts as well as Sesbania sesban, Acacia auriculiformis, A.nilotica, Prosopis cineraria and Phaseolus vulgairs. Rewari and Allen (1936) found that cross inoculation of Rhizobium isolated from red sanders formed JOURNAL OF BIOSCIENCES RESEARCH 3(1):

4 effective nodules on Vigna radiata and were similar to Rhizobium phaseolii group. Similarly all the rhizobial isolates of red sanders nodulated Vigna radiata, Dalbergia sissoo and produced ineffective nodules on Leucaena leucocephala (Rajasekhar et al., 2002), these result are agreement with the present results. Pig m e nt fo rm atio n and Nitrate re d uc tase ac tivity The influence of rhizobial isolates of stressed soils on the chlorophyll pigments of Sesbania grandiflora is presented in Fig.1(A). The content of chl.a increased marginally under the influence of calcareous soil and saline soil isolates followed by others. However, chl.b increased significantly in all the treatments. In comparison to the treated control, the performance of the saline and calcareous soil isolates treated plants was better. An increase in the chlorophyll content is a direct indication of the increased rate of photosynthesis which could have occurred in these plants. An increase in the rate of photosynthesis could make more carbon skeletons available for synthesis of amino acid. Increased nitrogen availability would have facilitated this. When there is an increase in the synthesis of amino acids there would be an increase in the protein synthesis also. This could influence all the other related synthetic metabolic pathways which would cause an increase in overall growth. Rachel et al. (2001) observed an increase in the total chlorophyll from 3.75 mg g -1 fr.wt. in control to 4.22 mg g -1 fr.wt. in Rhizobium inoculated Vigna mungo while in Vigna radiata, the total chlorophyll in control was 4.01 mg g -1 fr.wt. and in Rhizobium inoculated plants, it was 4.46 mg g -1 fr.wt. Kantar et al.(2003) in their studies and field trial found that inoculation with HF 274 and HF 177 increased the chlorophyll content. Karuppasamy et al.(2011) abserved two fold increase increase in totel chlorophyll in rhizobium inoculated tree legume Samanea saman. These results are in agreement with the present study. Fig.1(A). mg.g.fr.w t CON- CON-T DLS-T DES-T SS-T CS-T UT (A)Total chlorophyll content Chl.a Chl.b Total-Chl. The increase in the soluble protein content in all the treatments is presented in Fig.1(B). mg.g.fr.w t CON-UT CON-T DLS-T DES-T SS-T CS-T (B) Total protein content Maximum protein content was found in the calcareous soil and saline soilisolate treatment, when compared to treated control. (2011) observed significantly increase in protein level by rhizobium inoculated tree legume Samanea saman. Khanna and Gupta (2005) showed an increase in protein up to 526 per cent in Pisum sativum and increased starch up to 120 per cent by applying Rhizobium. These results are supporting to the present study. The Nitrate reductase activity also increased in all the treatments. The significant increase was observed by treatment with saline soil and calcareous soil strain followed by other strains, when compared to untreated control (Fig.1(C)). (2011) recorded highest increase of root nodules and maximum of Nitrate reductase activity by rhizobium treated tree legume Samanea JOURNAL OF BIOSCIENCES RESEARCH 3(1):

5 Fig.1(C) CON-UT CON-T DLS-T DES-T SS-T CS-T (C) Nitrate recductase activity saman. Polcyn and Lucinski (2001) reported that 97% of nodular NR activity is localized in bacterioid. Symbiotic associations in which NR function is complementary to that of nitrogenase are possible since bacterioids of many rhizobial strains are capable of performing dissimilation of nitrate reduction (Serrano and Chamber, 1990: Chamber- Perez et al. 1997). Sri Sandrasekharan and Muthukkaruppan (2004) revealed that Rhizobium inoculation in soybean increased nitrogenase activity up to 8.5 per cent over the control. Present study suggested that the stress resistant rhizobia isolated from saline soil and calcareous soil may be used for the improvement of growth and physiology of tree legume Sesbania grandiflora and to improve the soil fertility. Acknowledgement The authors express their gratitude to the Principal, Saraswathi Narayanan College and special thanks to Dr. K.K.Natarajan and Dr.C. Sankarana rayanan, Head, PG study and Research Centre in Botany, Saraswathi Narayanan College, Madurai, India, for providing laboratory facilities is acknowledged. References Abdullahi Bala and Ken E Giller (2001). Symbioic specificity of tropical tree rhizobia for host legumes. New Phytol. 149: Arnon DE, (1949) Copper enzymes in isolated chloroplast. Poly phenol oxidase in Beta vulgaris, Plant Physiol, Chamber-Perez MA, Camacho-Martinez M and Soriano-Niebla JJ (1997). Nitrate reductase activities of Bradyrhizobium sp. in tropical legumes: Effects of nitrate on O 2 diffusion in nodules and carbon costs of N 2 fixation. J. Plant Physiol. 150: Karuppasamy, K, Nagaraj,S and Kathiresan, D (2011). Stress Tolerant Rhizobium Enhances the Growth of Samanea saman (JECQ) Merr. African Journal of Basic & Applied Sciences. 3 (6): Kantar F, Elkoca E, Oguteu H and Algur OF (2003). Chick pea yields in relation to Rhizobium inoculation from wild chick pea at high attitudes. J. Agronomy and Crop Science. 189: Lowry OH, Rosenbrough NJ, Farr AL & Randall RJ, Protein measurement with folin phenol reagent,j Biol Chem,13 (1951) Muthuchelian K, Paliwal K and Ganam A (1989). Influence of shading on net photosynthetic and transpiration rates, stomatal diffusive resistance, and nitrate reductase and biomass productivity of wood legume tree species (Erythrina variegata Lam.) Proc. Ind. Aca. Sci. Plant Sci. 99: Polycyn W and Lucinski R (2001). Functional similarities of nitrate reductase from yellow lupine bacteriods to bacterial denitrification system. J. Plant. Physiol. 158: Rachel RD, Santhagru K and Gunasekaran P(2001). Effect of supplementary UV-B Radiation on growth, Nodulation and Nitrogen fixation in Vigna mungo L. and Vigna radiate(l.) Wilzek. Indian.j. Microbiol. 4: Rajasekhar A, Prasad Babu G and Reddy TKK (2002). Characterization of Rhizobium of red sanders (Pterocarpus santalinus L.). An endemic tropical tree legume. Legume Res. 25: JOURNAL OF BIOSCIENCES RESEARCH 3(1):

6 Rewari RP and Bhatnagar RS (1972). Legume inoculants and seed productivity symposium held at Varanasi and Calcutta. Russian Agric. Sci. 37: Serrano A and Chamber M (1990). Nitrate reduction in Bradyrhizobium sp. (Lupinus) strains and its effects on their symbiosis with Lupinus luteus. J. Plant Physiol. 136: Sivakumar V and Kumutha K (2003). Effects of Rhizobium and molybdenum on nodulation, yield and yield contributing characters of groundnut. J. of Ecobiol. 15: Sri Ramachandrasekharan MV and Muthukkaruppan SM (2004). Effect of sulphur and Rhizobium on biological nitrogen fixation, nutrient uptake and seed yield in soybean. J. Ecobiol. 16: Vincent JM (1970). Annual for the practical study of the root nodule bacteria IBP Vol. 15. Oxford. Blackwell Scientific Publications. Walkey &Block, 34(1934). An examination of Degtjareff method for determining soil organic matter and a proposed modification of the chromic acid titration method, Soil sci S.N o Table-1. Physico-chemical characters of different soils used. Soil type ph Electrical Organic Organic matter conductivity carbon (%) (%) (dsm 1 ) 1 Saline soil Calcareous soil Dye Industry effluent soil Dry land soil Red soil Values are means of 10 replicates from each sample JOURNAL OF BIOSCIENCES RESEARCH 3(1):

7 Table -2. Influence of Rhizobial isolates from stressed soils on the growth characteristics and fresh and dry weight of Se sb ania g rand iflo ra. Growth CON- CON- DLS-T DES-T SS-T CS-T parameters UT T Shoot length(cm) (42.34) (47.53) (50.84) (49.93) (64.50) Root length(cm) 40.7 (8.150) (18.18) (36.36) (45.04) (32.41) Shoot Fresh (79.65) (20.70) (428.3) (580.5) (582.94) Root Fresh (172.47) (85.30) ( ) ( ) ( ) Shoot Dry (124.2) (19.76) (451.34) (728.98) (707.32) Root Dry (124.2) (139.4) (117.8) ) ( ) Nodule Fresh (130.43) (4.62) (131.08) (160.11) (118.60) Nodule Dry (143.56) (7.61) (139.45) (194.12) (211.76) Values are means of 10 replicates and figures in parenthesis are percentage increase over the control. (Con-UT: Control Untreated, Con-T: Control with garden soil strain, DLS-T: Dry Land Soil Strain, DES-T: Dye Effluent Soil Strain, SS-T: Saline Soil Strain and CS-T: Calcareous soil strain) JOURNAL OF BIOSCIENCES RESEARCH 3(1):