Journal of Microbiology and Biotechnology Research

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J. Microbiol. Biotech. Res., 2012, 2 (6):888-893

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ISSN : 2231 3168 CODEN (USA) : JMBRB4

Studies on the effect of different native strains of Azospirillum on paddy (Oryza sativa L.)
Senthil Kumar. R and Panneerselvam.A Dept of Botany and Microbiology, A.V.V.M. Sri Pushpam College (Autonomous), Poondi, Thanjavur Dt, Tamilnadu, India
_____________________________________________________________________________________________ ABSTRACT An attempt was made to study the effect of different native strains of Azospirillum inoculation on the paddy variety of ADT-36. To find out the best strain in terms of seedling characters such as seed germination, biomass and phytomass yield under controlled conditions. In the case of seed germination, the percentage of seed germination was higher in Azospirillum treated seed than in control. Similarly, shoot and root lengths and fresh and dry weights of paddy variety ADT-36 treated with Azospirillum inoculation showed better response than the untreated plants due to the secretion of plant growth hormones by Azospirillum. The biochemical parameters of total chlorophyll and carotenoid were also increased to varying level in Azospirillum treated plants. The overall studies indicated that the growth of Azospirillum treated paddy seedling excelled over the untreated ones due to biofertilizer effect upon nitrogen fixation. Among the 10 native Azospirillum strains (TNM01, TVM02, TKV03, TNM04, TVR05, TKC06, TMG07, TKR08, TTP09 and TMP10) treated the TMP10 and TNM5 strains showed good response. Key words: Azospirillum, Paddy, Biofertilizer, Biomass and Chlorophyll _____________________________________________________________________________________________ INTRODUCTION Biofertilizers form an important part of Integral Plant Nutrient Supply System (IPNS) and organic farming which constitutes the present as well as future mandate of Indian agriculture. Biofertilizers manufactured in India presently are carrier based, in general and suffer from short shelf life, poor quality, high contamination and low and unpredictable field performances. Biofertilizers are preparations containing agriculturally useful microorganisms, which help in mobilizing plant nutrients through their biological activity. Biofertilizers generally are defined as preparation containing live or latent cells of efficient strains of N- fixing, P- solublizing or cellulolytic microorganisms used for the application to seed or soil (Motsara et al., 1995). Rice (Oriza sativa L.) is the stable food for half of the worlds population especially in oriental countries. In India, about 2500 varieties of rice are being cultivated, from which more than 1500 varieties are in southern India which are preferred over others, sowing to their yield, good quality and quantity of grain, short duration of growth and resistance against pest and diseases. A large number of experiments have been conducted in several countries to investigate the effect of inoculation of various grasses (Smith et al., 1976; Watanable et al., 1981). The aim of the application of Azospirillum is to get fast growth, better health of the plant and higher yield. It is known to be very active nitrogen fixing bacteria viz., Azotobacter, Nitrosomonas and Azospirillum to increase yield under controlled conditions.

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Senthil Kumar. R et al J. Microbiol. Biotech. Res., 2012, 2 (6):888-893 ______________________________________________________________________________

Balasubramaniam and Kumar, (1987); Wani, (1990); Bashan and Holguin, (1995) investigated that Azospirillum treatment showed remarkable increase in the grain and the straw yield in sorghum, Wheat, Maize, paddy and other food and fodder crops. Local strain Azospirillum brasilense B-4485 possesses a high N2-fixing activity and a significant hormonal effect. Strain A. brasilense B-4485 proved to be an effective inoculant for barley, wheat and perennial grasses (Nesterenko et al., 1995). Seed treatment with PGPR strains improved seed germination, seedling vigor, seedling emergence and seedling stand over the control. Similar improvement of seed germination parameters by rhizobacteria has been reported in other cereals such as sorghum and pearl millet (Niranjan et al., 2004). The inoculation of plants with Azospirillum can result in a significant change in various growth parameters in different cereals such as an increase in plant biomass, nutrient uptake, tissue N-content, plant height, leaf size, tiller numbers, root length and volume (Salantur et al., 2006). Improved plant growth by A. brasilense has been attributed both to production of plant hormones, especially growth promoters and by supplying combined nitrogen (Cohen et al., 2007). The yield responses caused by Azospirillum inoculation may be due to biological nitrogen fixation (Hartmann et al., 1983). Split application of biofertilizer inoculation through seed, seeding and soil gave the highest grain, straw yield, plant height and number of productive tillers in rice (Gopalswamy and Vidhyasekaran, 1988). The objective of present study was to investigate the effect of different native strains of Azospirillum inoculation on the ADT-36 paddy variety to find out the best strain. The observation were made to record the seedling characters such as seed germination, biomass and phytomass yield of different strains under controlled conditions. MATERIALS AND METHODS Isolation of Azospirillum (Dobereiner et al., 1976) The soil samples were collected from 10 agronomical divisions of Thiruvarur district, Tamilnadu, South India for isolation of Azospirillum. From the collected soil samples, 1 g was taken and serially diluted using sterile distilled water up to 10-8 dilutions. One ml of diluted sample from 10-6 to 10-8 dilutions was taken and 0.1ml of aliquot was inoculated in test tube containing Nfb (Nitrogen free bromothymol) semisolid media. All the tubes were incubated at 32C for 48 h and observed the growth by the formation of pellicles. The pellicles were streaked on Nfb solid media and incubated at 32C for 24 h. Morphologically divergent Azospirillum colonies (white, yellow and pink) were picked from the plates and streaked on basal minimal salt agar medium and incubated at 32C for 24 h. after attained sufficient growth, all the isolates were preserved and used for further studies. Pot culture experiment Healthy, viable paddy seeds of ADT-36 variety were procured from Indian Rice Research Insititute, Aaduthurai. The 50 seeds were selected for each treatment and dressed well with paste (Azospirillum culture mixed with gum Arabic substance) of 250 mg of native strains of Azospirillum individually in water. These seeds were dried under the shade condition and transferred after sowing to an earthen pot containing 2Kg of sterilized garden soil. The pots were watered regularly and being maintained under controlled condition. A control setup was also made by following the same conditions except the addition of biofertilizer. Five seedlings were selected at random from each pot and the following observations were made on the 10th, 20th and 30th day of sowing. The seedlings were uprooted gently without causing any damage to the root and shoot systems and washed well with water. The shoot and root lengths were measured with a metric scale. The shoot and root fresh weights were determined using an electronic balance. The total chlorophyll and carotenoid were estimated (Horborne, 1973). RESULTS AND DISCUSSION In the present study, totally ten different native strains of Azospirillum viz, TNM01- Nannilam, TVM02Valangaiman, TKV03- Kudavasal, TNM04- Needamangalam, TVR05- Thiruvarur, TKC06- Koradacherry, TMG07Mannargudi, TKR08- Kottur, TTP09-Thiruthuraipoondi and TMP10- Muthupet were isolated and identified from paddy fields of 10 agronomical divisions of Thiruvarur district, Tamilnadu, South India. Cristyakova and Kalininskaya, (1984) reported that bacteria of the genus Azospirillum are widespread in the soils of various regions and usually occur in the rhizosphere of vascular plants. In pot culture experiment, the results indicated that the growth of Azospirillum treated paddy seedlings excelled over the untreated ones. The seed germination studies revealed that the percentage germination of seeds were higher in Azospirillum treated seeds than in control (Table-

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Senthil Kumar. R et al J. Microbiol. Biotech. Res., 2012, 2 (6):888-893 ______________________________________________________________________________

1). Among the 10 different native strains of Azospirillum TMP10 strain showed a considerable increase in the seed germination than the other strains under same experimental conditions. The reason for this may be due to the tremendous pressure developed inside the seeds, which was responsible for breaking of the seed coat quickly (Sifton, 1959). This pressure may be induced by phytohormones especially auxin, indole acetic acid, cytokinin and giberellic acid like substances secreted by Azospirillum (Okon, 1984; Okon and Kapulnik 1986). The observations made on 10th, 20th and 30th days of sowing revealed that Azospirillum treated seeds had higher productivity than control. The seedlings from this particular biofertilizer treated seeds had no longer shoot and root lengths than the untreated ones. Among the 10 different native strains of Azospirillum TMP10 and TNM04 strains showed a considerable increase in the biomass than the other strains under same experimental conditions. The seed dressing by the biofertilizer induces the production of plant growth promoting substances and leads to the increase of shoot and root length (Table-1). Secretion of plant growth hormones by Azospirillum was reported in several cereals and grasses (Balasubramanian and Kumar, 1987: Bashan and Holguin, 1995). Bottini et al., (1989) also reported that the specific capability of the host plant to attract the bacteria and modify the rhizosphere and or to respond to some bacterial activity and benefit from it.
Table-1 Effect of different strains of Azospirillum on shoots and root lengths of paddy variety ADT-36. Shoot length (cm) Root length (cm) Number of days Number of days 10th 20th 30th 10th 20th 30th Control 64.6 13.9 16.7 18.9 5.9 11.3 13.7 TNM01 76.4 14.2 18.2 20.4 6.4 12.7 14.9 TVM02 79.5 14.6 17.9 20.9 6.6 12.9 15.2 TKV03 72.1 14.4 17.5 21.1 6.3 12.6 15.1 TNM04 87.4 15.2 19.1 22.6 7.3 13.9 16.2 TVR05 86.2 14.9 18.6 22.2 7.1 13.5 16.0 TKC06 80.2 14.6 18.4 21.8 6.9 13.3 15.9 TMG07 79.3 14.3 17.8 21.4 6.5 13.2 15.7 TKR08 76.7 14.5 17.6 21.7 6.7 12.9 15.3 TTP09 78.6 14.7 18.2 21.9 6.9 13.0 15.5 TMP10 92.4 15.9 20.3 24.2 7.9 14.8 16.9 Values are the mean standard deviation of five replicates % of germination

Treatment

Table-2 Shoot and root fresh weight between control and different strains of Azospirillum treated paddy variety ADT-36. Shoot weight (mg) Root weight (mg) Number of days Number of days 10th 20th 30th 10th 20th 30th Control 180 437 690 20.3 40.1 69.5 TNM01 191 461 718 29.5 47.2 90.4 TVM02 192 459 722 29.8 48.6 91.8 TKV03 195 455 730 28.9 47.7 92.1 TNM04 210 470 741 30.5 49.6 96.9 TVR05 199 465 738 30.1 49.3 95.4 TKC06 194 464 735 30.0 49.1 94.9 TMG07 190 460 733 29.6 48.8 92.6 TKR08 193 458 736 29.4 48.2 92.2 TTP09 196 462 727 29.9 48.5 92.4 TMP10 219 479 749 31.3 51.2 99.7 Values are the mean standard deviation of five replicates Treatment

The fresh and dry weights of root and shoot system of paddy treated by various strains of Azospirillum were also found to be increased to a considerable extent in Azospirillum treated seedlings particularly TMP10 Azospirillum strains treated (Table-2 and 3). This may be due to the formation and development of numerous root branching, root hairs and primary and secondary lateral roots which increases the nutrient uptake capacity of roots (Gopalswamy and Vidhyasekaran, 1988: Hartmann et al., 1983). This effect on the root system as well as more root colonization and root proliferation are probably due to the growth hormones secreted by the bacteria and also nitrogen fixation by it. The increased nitrogen uptake from the soil might have correspondingly increased the biomass to some extent. The changes in root functions due to Azospirillum treatment in different wheat cultivars were also reported (Kapulnik et al., 1981). These growth enhancing effects are of interest because of their potential

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Senthil Kumar. R et al J. Microbiol. Biotech. Res., Res. 2012, 2 (6):888-893 ______________________________________________________________________________

significance for yield increases in agronomic systems in which the use of fertilizers was the limiting factors for their development (Sarig et al., 1984).
Table-3 3 Shoot and root dry weight between control and different strains of Azospirillum treated paddy variety ADT-36 ADT Shoot weight (mg) Root weight (mg) Number of days Number of days 10th 20th 30th 10th 20th 30th Control 19.2 39.5 81.3 4.6 10.2 16.5 TNM01 20.7 48.7 87.6 5.9 11.0 18.5 TVM02 20.8 49.1 88.2 6.1 11.1 18.7 TKV03 20.9 49.6 88.9 6.2 11.3 18.8 TNM04 22.2 51.2 90.5 7.3 12.1 19.2 TVR05 21.5 50.6 89.4 7.2 11.9 19.1 TKC06 21.4 50.4 89.3 7.0 11.6 19.1 TMG07 21.2 50.2 89.1 6.9 11.4 18.6 TKR08 21.3 49.9 88.6 6.6 11.2 18.7 TTP09 21.7 50.6 90.1 7.3 11.8 18.9 TMP10 23.1 52.3 91.7 7.6 12.1 19.6 Values are the mean standard deviation of five replicates Treatment

Kannan and Ponmurugan, (2010) studied s the he biochemical parameters such as total chlorophyll, carotenoid were increased to varying level in Azospirillum treated plants when compared to control ones (Fig-1, (Fig 2). Very high contents of biochemical constituents in TMP10 strain treated and very less in TNM01 strain treated. treated
Fig- 1 Chlorophyll content of control and Azospirillum treated paddy seedlings.

1.4 1.2 1 0.8 10th day 0.6 0.4 0.2 0 20th day 30th day

The results clearly showed that TMP10 Azospirillum strain accounted well followed by TNM04 and TTP09. Other strains showed poor response. From these observations, it can be concluded that among the 10 Azospirillum strains TMP10 strain showed good response tested with ADT ADT-36. 36. It had high phytomass accumulation accumula and biochemical parameters. It may be due to nitrogen uptake from the soil and proper utilization. The beneficial effect of Azospirillum strains varies itself depending up on microbial strains, method of inoculation and environmental factors particularly rly soil temperature, pH, EC, moisture content and water holding capacity.

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Senthil Kumar. R et al J. Microbiol. Biotech. Res., Res. 2012, 2 (6):888-893 ______________________________________________________________________________

Fig- 2 Carotenoid content of control and Azospirillum treated paddy seedlings.

1.5

1 10th day 0.5 20th day 30th day 0

Acknowledgement The authors express sincere thanks to Secretary and correspondent and the Principal of A.V.V.M. Sri Pushpam College (Autonomous) Poondi for providing facilities and moral support to conduct this work. REFERENCES [1] Balasubramanian, A. and Kumar, K. 1987. IRRN 12: 43. [2] Bashan, Y. and Holguin, G. 1995. Microbial. Ecology 29: 269-281. [3] Bottini, R., Fulcheri, M., Pearce, D. and Pharis, R. P. 1989. Plant physiol. 90: 45-47. [4] Cohen, A.T., Mariela, P., Rubn, B. and Patricia, P. 2007. Azospirillum brasilense and ABA Improve Growth in Arabidopsis. International Plant Growth substances substances Association 19th annual meeting Puerto Vallarta, Mexico-July Mexico 21-25. [5] Cristyakova. I.K and Kalininskaya, T.A., 1984. Ser.Biol., 1: 149-153. [6] Dobereiner, J., Marriel, J.E. and Nevy, M., 1976. Can. J. Microbiol., 22: 1464 1473. [7] Gopalswamy, G. and Vidhyasekaran, P. 1988. IRRN 12: 56-57. [8] Hartmann, A., Mahavir, S. and Kligmaller, W. 1983. Can. J. Microbiol. 29: 916-922. [9] Horborne, J. B. 1973. Phytochemical methods. A guide to modern techniques of plant analysis. PP 277. Chapman and Hall, London. [10] Kannan, T. and Ponmurugan, P. 2010. J. Phytol, 2(6): 08-13. [11] Kapulnik, Y., Sarig, S., Nur, I., Okon, Y., Kiel, J. and Henis, Y. 1981. Expl. Agric. 17: 179-187. [12] Motsara, M.R., Bhattacharyya, P. and Srivastava, B. 1995. Biofertilizer technology, Marketing and usageusage A source book-cum- glossary. Fertilizer development and consultation organisation, New Delhi, India. 37-39. 37 [13] Niranjan, S. R., Shetty, N. P. and Shetty, H. S. 2004. J.pest.manage.50(1): 41-48. [14] Nesterenko V., Mikhailouskaya N. and Barashenko, T.1995. Soil Sci. Fert. Appl., Minsk, 23: 261167. [15] Okon, Y. 1984. Trends in Biotech. 3: 223-228. [16] Okon, Y. and Kapulnik, Y. 1986. Plant and soil. 90: 3-16. [17] Salantur, A., Ozturk, R. and Akten, S. 2006. Growth and yield response of spring wheat (Triticum aestivum L.) to inoculation with rhizobacteria. Plant Soil Environ. 52: 111118. [18] Sarig, S., Kapul nik, Y., Nur, I. and Okon, Y. 1984. Expl. Agric.20: 59-66. [19] Sifton, H. B. 1959. Can. J. Bot. 37: 719-741.

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[20] Smith, R. L., Bouton, R. H., Schank, S. C., Quessenberry, K. S., Tyer, M. E., Milam, J. R., Garkins, M. H. and Little, R. 1976. Curr. Sci. 48: 133. [21] Wani, S. P. 1990. Indian. J. Microbiol. 30: 363-393. [22] Watanable, I., Cabrera, D. and Barraquio, W. L. 1981. Plant and soil 59: 391-398.

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