Indian Journal of Biotechnology Vol 13, October 2014, pp 540-543 Diversity of diazotrophs in tropical rice field under the influence of organic and nitrogen fertilization Alaknanda Sarkar, K Manjunath and Pranjali Vishwakarma* Department of Microbiology and Biotechnology, Bangalore University Jnana Bharthi Campus, Bangalore 560 056, India Received 10 February 2013; revised 3 July 2013; accepted 28 August 2013 The diversity of diazotrophic community of two different rice field of typical tropical rice agroecosystem under long-term application of nitrogen and organic fertilizer was studied. Most probable number (MPN) result revealed that organic fertilizer amended rice field (O) have higher diazotroph population than nitrogen fertilizer amended rice field (N). NifH gene based restriction fragment length polymorphism (RFLP) analysis revealed presence of uncultured diazotroph, Anabaena, Rhizobium, Nostoc, Azospirllium, Methylobacter, Leptolyngbya and Sinorhizobium. Diversity index of diazotroph was found higher in O soil. The study suggested that application of nitrogen fertilizer adversely affected community composition of diazotroph in comparison to organic fertilizer and the differences were found significant. Keywords: Diazotroph, diversity, fertilizer, nifH gene, RFLP, rice Biological nitrogen fixations by diazotrophs play a significant role in nitrogen cycling and have drawn the attention of researchers because they represent one of the largest sources of nitrogen input to soil 1 . Diazotrophs are present in the aerobic soil layers, rhizosphere, roots and stem bases of rice agro-ecosystems. They have the capacity to fix molecular nitrogen under micro-aerophilic conditions as a growth promoting substrate in nitrogen deficient environment. All the known diazotrophs possess nifH gene, which encodes the iron protein subunit of nitrogenase enzyme, and has been frequently used as a molecular marker to detect diazotrophs in environmental samples 2,3 . The sequence-based nifH phylogeny correlates well with the 16S rRNA gene based phylogeny and is considered the ideal marker to characterize soil diazotrophic communities and to assign specific generic and species affiliations to diazotrophs 3 . In general, diazotrophs are well studied in rice fields as bio-fertilizers. Biological nitrogen fixation by diazotrophic bacteria is an important process affecting fertility of agro-ecosystem. It has been well documented that physico-chemical factors and management practices can influence microbial populations. Therefore, it is inevitable that amendment of synthetic nitrogen fertilizer could affect the activity of diazotrophs. Nitrogen fixed by free living nitrogen fixers is also typically important for soil C:N ratio and these processes are fundamental to the formation of soil aggregates 4 , which in turn affect soil physical properties. In such circumstances, we hypothesise that load of nitrogen fertilizer can lead to alteration in diazotrophic community. The present study is an attempt to assess the composition and structure of N 2 -fixing bacteria associated with soil under the effect of organic and nitrogen fertilizers. To accomplish this, the molecular diversity of nifH gene was investigated using RFLP. Study sites were two rice field of the Cauvery plain (Table 1). Chemical fertilizer in the form of urea was applied at the rate of 30 Kg N ha -1 in two split doses in nitrogen fertilizer amended rice field (N). Rice straw fermented for 5 months was used as organic compost in organic fertilizer amended rice field (O). Field moist samples in triplicates, collected at reproductive stages of rice plant, were mixed, sieved and stored in polyethylene bags at 4°C and -20°C for subsequent analysis. Enrichment of diazotrophs from soil samples was done in semisolid N-free NFb medium 5 . The number of diazotrophs was obtained by MPN technique using McCrady’s table. In brief, 10 g fresh soil was suspended in saline solution (NaCl 0.8%) and serially diluted in the range from 10 -1 to 10 -9 were inoculated in triplicates in N-free NFb medium. After 7 d of incubation at 37°C, population size was estimated by MPN technique. Extraction of DNA from soil samples were carried out using Himedia soil DNA extraction Kit as per manufacturer’s protocol and purified using the QIA quick® gel extraction kit (QIAGEN). PCR amplification of nifH gene was carried out using a degenerate primer pair PolF (50-TGCGAYCCSAARGCBGACTC-30) and PolR —————— *Author for correspondence: E-mail: pvsci36@gmail.com