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1 Research article CO-INOCULATION EFFECT OF PLANT GROWTH PROMOTING RHIZOBACTERIA AND PLANT GROWTH PROMOTING ENDOPHYTIC BACTERIA ON SESBANIA GRANDFLORA (L.)PERS R.Rajamani 1, Ghanshyam Srivastava 2, Bharat Jondhale 3, Maathangi Murali 4. 1 Co-principal investigator, Sri Sri Institute of Advanced Research (A Division of Ved Vignan Maha Vidya Peeth), 21 st KM, Kanakapura Road, Udayapura, Bangalore Head of the Department, Sri Sri Institute of Advanced Research (A Division of Ved Vignan Maha Vidya Peeth), 21 st KM, Kanakapura Road, Udayapura, Bangalore Research scholar, Sri Sri Institute of Advanced Research (A Division of Ved Vignan Maha Vidya Peeth), 21 st KM, Kanakapura Road, Udayapura, Bangalore ABSTRACT: Plant Growth Promoting Rhizobacteria (PGPR) is found in the rhizosphere of root surfaces and in association with roots. PGPR helps to reduce the plant stress and it enhances the plant growth by increasing the synthesis of plant growth promoting hormones such as Auxin, Cytokinin and Giberrelins. Use of both ectophytic and endophytic bacteria were found to be ecofriendly and increased the plant growth. Plant Growth Promoting Endophytic Bacteria (PGPE) along with Plant Growth Promoting Rhizobacteria (PGPR) acts as a Biocontrol agent for several plant diseases. It has been found that Sesbania grandflora treated with Bacillus subtilis- IN937b + PGPR mixture showed highest vigor index of 3957 when compared to other strains. Similarly Sesbania grandflora treated with Bacillus subtilis- IN937b+ PGPR mixture showed highest total plant fresh weight 97.22% and highest total plant dry weight 88.72%. 4 Research scholar, Sri Sri Institute of Advanced Research (A Division of Ved Vignan Maha Vidya Peeth), 21 st KM, Kanakapura Road, Udayapura, Bangalore corresponding author raj @gmail.com Keywords: Auxin, Cytokinin, Giberrelins, Plant Growth Promoting Rhizobacteria (PGPR), Plant Growth Promoting Endophytic Bacteria (PGPE), Sesbania grandflora, Biocontrol agent.

2 INTRODUCTION Agriculture depends on the use of chemical fertilizers, growth factors and pesticides for increasing yield. Use of bio source to replace chemicals and artificial growth factors is of increasing importance. A large number of microorganisms such as bacteria, fungi, yeast, algae, and protozoa play an important role in soil enrichment. Bacteria are widely available and rapidly multiplying organisms which can act as a tool to provide sufficient nutrients, carbon and nitrogen sources. Bacteria protect and promote plant growth by colonizing and multiplying along the surface of the inoculated plants (Mishra et al., 2008). Plant Growth Promoting Rhizobacteria (PGPR) is a heterogeneous group of bacteria that are found in the rhizosphere at the root surfaces and in association with roots. PGPR reduces plant stress associated with phytoremediation strategies (Ana et al., 2010). PGPR enhances the plant growth by reducing the Ethylene production (Glick et al., 1998), Nitrogen fixation (Khan et al., 2005), producing or changing the concentration of growth regulators (Ahmad et al., 2008), raising the colonization of nutrients (Glick et al., 1995), Production of phytohormones such as Auxin, Cytokinin and Giberrelins (Glick et al., 1995) and synthesis of antibiotic and other pathogen depressing substances (Kamnev and lelie, 2000). Use of both ecto and endophytic bacterial consortium has been ecofriendly and effective on plant growth. The PGPR has the ability to Synthesize fungal cell walllysing enzyme (Protease) or Hydrogen cyanide (HCN) which suppress the growth of fungal pathogens and promotes successful compete with pathogens for nutrients or specific niches on the root surface (Persello-cartileaux et al, 2003). Further Plant Growth Promoting Endophytic Bacteria(PGPE), and Plant Growth Promoting Rhizobacteria (PGPR) strains have been developed commercially as a bio fertilizer and tested on several crops to control disease and as well as growth promotion (Rajendran et al., 2006). Therefore the designed PGPR work is based on the selection of exact composition of PGPR for enhancing the growth of Sesbania grandflora (L.) pers. MATERIALS AND METHODS Preparation of Bacterial Suspension Seven strains of endophytic Bacillus species were selected for plant growth promotion activity. Seven species are reported (Table 1).The seven bacterial strains were grown onto the culture medium Tryptic Soya Agar (TSA) at 28 o C for 48 hours. The single colony from each culture was suspended in Trypsin soya broth and incubated at 28 0 C for 24 hours. The concentration was adjusted to 10 8 CFU/mL by UV-Vis Spectrophotometer at 580nm. These suspensions were used directly for seed bacterialization in pot culture experiment (Shouan et al., 2010). Pot Culture Experiment Seeds were surface sterilized with 5% Sodium Hypochloride for 3 mins and rinsed thoroughly in Sterile distilled water for three times.the excess water was removed by sterile filter paper. In parallel, a pot mixture composed of two parts of Cocopeat and red soil were prepared and autoclaved for 30 mins at 120 o C. The sterile pot mixture was distributed in nursery tray and seeds were placed along with 10 ml bacterial endophytes (10 6 /CFU) suspension. 5 day old seedlings were again treated with bacterial endophytes suspension before transplanting to the pots. The PGPR s such as Azotobacter, Azospirllum, Pseudomonas, Trichoderma and phosphate solublizing bacteria were used as a consortium mixture and treated at the rate of 7gm per pot on 15 days old seedlings. For each endophytic bacterial strain 7 pots containing one seed on each pot were used. Treatments were in a factorial experiment based on complete randomized design. The control pots were treated with only consortium mixture without endophytes. Growth parameters were recorded after 45 days of sowing (Ana et al., 2010).

3 Table 1: Selected PGPR and PGPE Strains. Sr.No PGPE* PGPR (commercial products)** 1 Bacillus pumilus-t4 Phosphate Solubilizing Bacteria 2 Bacillus pumilus-se34 Azotobacter sp 3 Bacillus subtilis-gbo3 Azospirllum sp. 4 Bacillius pumilus-inr7 Cow pea Rhizobium 5 Brevibacillus brevi -IPC11 Pseudomonas fluorescens 6 Bacillus amyloliquefaciens-in937a Trichoderma viridae 7 Bacillus subtilis-in937b *Obtained from Department of Entomology and Plant Pathology Auburn University, Auburn, AL, USA **Obtained from University of Agriculture science, Department of Agriculture Microbiology, GKVK, Bangalore. Measurement of Plant Growth Parameters To evaluate the plant growth parameters, standing plants were uprooted and aerials parts and roots were separated (Abdul Baki and Anderson, 1973). Length of the stem and root, fresh and dry weight of shoot and root of each plant was determined. The vigor index was determined using the formula: Vigor Index = mean root length + (mean shoot length X germination %). RESULTS Treatment of seeds with PGPE and PGPR combination significantly increases seed germination, stem height, lateral roots, fresh and dry weight of aerial parts and roots. However the rate of growth and biomass enhancement varied with endophytic bacterial strains. When comparing the vigor index, treatment of Bacillus subtilis- IN937b+ PGPR mixture (3957) and Bacillus pumilus-t4 + PGPR mixture (3818) were found to stimulate the plant growth much higher when compared to other strains (Table 2, Graph 1). It was found that Bacillus subtilis- IN937b (Total plant fresh weight 97.21% and Total plant dry weight 88.72%) and Bacillus pumilus-inr7+pgpr mixture (Total fresh weight 82.61% and Total dry weight 77.12%) showed a good increase in total plant weight when compared to other species (Table 3, Graph 2). Shoot biomass was compared and it was found that Bacillus pumilus-t4+ PGPR mixture (Shoot fresh weight 1.36 gm and Shoot dry weight 0.19 gm) and Bacillus subtilis- GBO3 + PGPR mixture (Shoot fresh weight gm and Shoot dry weight 0.16 gm) was found to have higher shoot biomass when compared to other species (Table 4, Graph 3 and Figure 1). Root biomass was compared and it was found that Brevibaccillus brevis-ipcii + PGPR mixture (Root fresh weight 1.011gm and Root dry weight 0.17 gm) and Bacillus subtilis GBO3 + PGPR mixture (Root fresh weight -1.18gm and Root dry weight 0.16 gm) showed higher Root biomass when compared to other species (Table 5, Graph 4 and Figure 2).

4 Table 2: Effect of PGPR and PGPE on Plant Growth Promotion using pot culture. Treatment No T 0 T 1 PGPE + PGPR Strains Shoot Length (cm) Root Length (cm) Vigor Index Control Bacillus pumilus-t T 2 T 3 T 4 T 5 T 6 T 7 Bacillus pumilus-se34 Bacillus subtilis-gb03 Bacillus pumilus-inr-7 Brevibaccillus brevis- IPCII Bacillus amyloliquefaciens- IN937a Bacillus subtilis-in937b Graph 1: Effect of PGPR and PGPE on plant growth promotion using pot culture.

5 Table 3: Effect of PGPR and PGPE on total plant weight. Sr.No Culture Code Total plant fresh weight (gm) Difference (gm) % Difference Total plant dry weight (gm) Difference (gm) % Difference 1 CONTROL T SE GBO INR IPC IN937a IN937b Graph 2: Effect of PGPR and PGPE on total plant weight.

6 Table 4: Effect of PGPR and PGPE on Shoot Biomass. Sr.No Culture Code Shoot Fresh weight(gm) Shoot Dry weight(gm) 1 Control Bacillus pumilus-t4 Bacillus pumilus-se34 Bacillus subtilis-gb03 Bacillus pumilus-inr7 Brevibaccillus brevis-ipcii Bacillus amyloliquefaciens IN937a Bacillus subtilis-in937b Graph 3: Effect of PGPR and PGPE on Shoot Biomass.

7 Figure 1: Effect of PGPR and PGPE on Shoot Biomass (A- Control, B- GBO3 treated crops, C- T4 treated crops). " # $ Table 5: Effect of PGPR and PGPE on Root Biomass. Sr.No Culture Code Root Fresh Weight (gm) Root Dry Weight (gm) 1 Control Bacillus pumilus-t4 Bacillus pumilus-se34 Bacillus subtilis-gb03 Bacillus pumilus-inr7 Brevibaccillus brevis-ipcii Bacillus amyloliquefaciens IN937a Bacillus subtilis-in937b

8 Graph 4: Effect of PGPR and PGPE on Root Biomass. Figure 2: Effect of PGPR and PGPE on Root Biomass (A- Control, B- IPC II treated crops, C- GBO 3 treated crops). " # $

9 DISCUSSION Perhaps, there is the reason for initial slow growth of seeds and it was thought to be growth character of the seeds. When treated with PGPR and PGPE, plant grows vigorously and the difference is perceptible from the beginning. Since this is an applied research, not much scientific data could be collected on the mechanism of growth promotion. All tested PGPE were able to enhance sesbania grandflora growth rate, Root biomass and Shoot biomass. Nevertheless, it appears that some PGPE were found to be more effective than others. The vigor index of Bacillus subtilis- IN937b+ PGPR mixture Treated plants (3957) can be compared to that of EPCO16+Pf1 Strains ( ) (S. Subramaniam and ponnusamy, 2012). The Shoot biomass of Bacillus subtilis-gbo3 + PGPR mixture (Shoot fresh weight gm and Shoot dry weight 0.16 gm) which is found to be similar to that of seed germination in Triticum aestivum treated with SMA4 Strains (Shoot fresh weight mg and Shoot dry weight 62.1 mg) and Root Biomass of Bacillus subtilis GBO3 + PGPR mixture (Root fresh weight -1.18gm and Root dry weight 0.16 gm) can be compared to that of seed germination in Triticum aestivum treated with SMA4 Strains (Root fresh weight 67.2mg and Root dry weight- 30.5mg) (Meena and Balijeet, 2013). Similar improvement of seed germination parameters due to PGPR has been reported in maize (Jarak et al., 2012). CONCLUSION Therefore, it can be concluded that the PGPR and PGPE mixture enhanced the growth rate of Sesbania grandflora. The application of PGPR and PGPE solves multifaceted problems of plant disease and growth. It gives immense knowledge to select right PGPR and PGPE mixture to enhance the growth rate of crops. ACKNOWLEDGEMENT We are grateful to Mr.Prasanth S Nair,Director,VVMVP, Art Of Living International Center,Bangalore,India.We also thank A.K.Sivaraj- QA &QC,Manager and Dr.Hari Venkatesh -R&D Sri Sri Ayurveda Trust (SSAT) for providing facilities and keen interest for completing the work. REFERENCE: Aref A, Abdul-Baki and James D. Anderson, (1973). Vigor Determination in soyabean seed by Multiple criteria. Crop Science. 13:6: Ahmad F. I., Ahmad, M.S Khan, (2008). Screening of free living rhizospheric bacteria for the multiple plant growth promoting activities. Microbiological Research. 163: Ana P.G.C. M, P.Carlos, M.Helena, R.O.S.S Antonio and C.M.L. Paula, (2010). Assessment of the plant growth promotion abilities of six bacterial isolates using zea mays as indicator plant. Soil Biology and Biochemistry. 42: Glick B.R., (1995). The enhancement of plant growth by free living bacteria. Canadian journal of microbiology. 41: Glick B.R, D.M, Penrose, J, Li., (1998). A model for the lowering of plant ethylene concentrations by plant growth promoting bacteria. Journal of theoretical biology. 190: Jarak M., N,Kovacki D, Bjelic, J.T, Hajnal, D.Stamenov, (2012). Effect of plant growth promoting rhizobacteria on maize in green house and field trail. African Journal of Microbiology research. 6(27): Kamnev A.A, D Lelie, (2000). Chemical and Biological parameters as tools to evaluate and improve heavy metal phytoremediation. Bioscience reports. 20: Khan A.G, (2005). Role of Soil microbes in the rhizospheres of plants growing on trace metal contaminated soils in phytoremediation. Journal of trace elements in medicine and biology.18:

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