J. Microbiol. Biotechnol. (2011), 21(6), 545–555 doi: 10.4014/jmb.1012.12022 First published online 18 April 2011 Diversity Analysis of Diazotrophic Bacteria Associated with the Roots of Tea (Camellia sinensis (L.) O. Kuntze) Arvind, Gulati1*, Swati Sood1, Praveen Rahi1, Rishu Thakur1, Sunita Chauhan1,2, and Isha Chawla nee Chadha1 1Plant Pathology and Microbiology Laboratory, Hill Area Tea Science Division, Institute of Himalayan Bioresource Technology, Council of Scientific and Industrial Research, P.O. Box 6, Palampur (H.P.)-176 061, India 2Department of Microbiology, College of Basic Sciences, CSK Himachal Pradesh Krishi Vishvavidyalaya, Palampur (H.P.)-176 062, India Received: December 16, 2010 / Revised: February 27, 2011 / Accepted: March 21, 2011 The diversity elucidation by amplified ribosomal DNA of atmospheric N into utilizable forms by plants and restriction analysis and 16S rDNA sequencing of 96 indirectly through the secretion of phytohormones that associative diazotrophs, isolated from the feeder roots of enhance plant growth [53]. Nitrogen-fixing ability is widely tea on enriched nitrogen-free semisolid media, revealed distributed among phylogenetically diverse bacteria such as the predominance of Gram-positive over Gram-negative Acetobacter, Arthrobacter, Azoarcus, Azospirillum, Azotobacter, bacteria within the Kangra valley in Himachal Pradesh, Bacillus, Burkholderia, Enterobacter, Herbaspirillum, India. The Gram-positive bacteria observed belong to two Klebsiella, and Pseudomonas associated with some taxonomic groupings; Firmicutes, including the genera agronomically important crops [11, 13, 15, 23, 47, 61]. Bacillus and Paenibacillus; and Actinobacteria, represented Diazotrophic bacteria are considered to be possible by the genus Microbacterium. The Gram-negative bacteria alternatives to inorganic nitrogen fertilizers for promoting included α-Proteobacteria genera Brevundimonas, Rhizobium, crop growth and yield [25, 28, 51]. and Mesorhizobium; γ-Proteobacteria genera Pseudomonas The sustained productivity of the world’s most popular and Stenotrophomonas; and β-Proteobacteria genera Azospira, non-alcoholic beverage, tea, relies heavily upon the use of Burkholderia, Delftia, Herbaspirillum and Ralstonia. The nitrogen [37, 50]. Leaching, the harvesting of shoots, and low level of similarity of two isolates, with the type strains the skiffing and pruning of bushes as an essential husbandry Paenibacillus xinjiangensis and Mesorhizobium albiziae, practice, are the major limiting factors for the maintenance suggests the possibility of raising species novum. The of nitrogen levels suitable for the sustainability of tea bacterial strains of different phylogenetic groups exhibited productivity [38]. The intensive use of chemical fertilizers distinct carbon-source utilization patterns and fatty acid creates a highly selective environment and adversely affects methyl ester profiles. The strains differed in their nitrogenase microbial diversity. This phenomenon is even more pronounced activities with relatively high activity seen in the Gram- in the monocultural conditions of tea plantations, where the negative strains exhibiting the highest similarity to Azospira perennial nature of bushes also exerts a strong rhizosphere oryzae, Delftia lacustris and Herbaspirillum huttiense. effect on the soil microflora [20, 29, 39]. The introduction Keywords: Camellia sinensis, diazotrophic bacteria, acetylene of non-resident microorganisms into such environments is reduction assay, 16S rDNA sequencing, FAME analysis, problematic and necessitates the selection of indigenous carbon-source utilization microorganisms well-adapted to the environmental conditions [49, 55]. Information about diazotrophs in tea is limited to knowledge of the presence of Azospirillum, Azotobacter, Achromobacter, Bacillus, Mycobacterium, Clostridium, and Nitrogen (N), considered an essential macronutrient for plant Beijerinkia, reported in the Republic of Georgia [34]. The growth, is often a yield-limiting factor in crop productivity aim of the present study is to broaden the current knowledge owing to the loss of a major component of mineral nitrogen base through an assessment of the genetic diversity of from the soil through run-off and leaching [8]. Diazotrophic associative tea diazotrophs in the Kangra valley, with a bacteria influence plant growth directly through the fixation view towards developing eco-friendly bioformulations based on the native microorganisms. The Kangra valley’s century- *Corresponding author Phone: +91-1894-230411; Fax: +91-1894-230433; old tea plantations, with bushes of nearly 60 years average E-mail: [email protected]; [email protected] age, cover about 2,300 hectares and are located between 546 Arvind et al. 32.03o and 32.20oN and 76.37o and 76.80oE at 1,000- water. The restriction mixtures were incubated for 2 h with AluI, o o 1,700 m above mean sea level in the mid-hills of the HpaII, and HinfI at 37 C and at 65 C with TaqI. The reaction Western Himalayas in Himachal Pradesh in India. The products of ARDRA resolved on 2% (w/v) agarose gel, prepared in regional soil has been characterized as acidic with a low 1× TBE buffer, were stained with ethidium bromide (10 µg/ml). The availability of nutrients [54]. The valley endures somewhat banding patterns were visualized and photographed with Digidoc (BioRad, CA, USA) under UV light. The band sizes were estimated hot and humid summers, with a mean temperature maximum through a comparison with a 100 bp DNA ladder (Fermentas, Vilnius, of 36oC and a minimum of 16.5oC, and cool winters, with a o o Lithuania). Fingerprints generated by restriction analysis were recorded mean temperature maximum of 20 C and a minimum of 0 C. in a binary form, where 1=presence of a band and 0=absence of the The annual potential transpiratory demand of 1,100 mm is band. The dissimilarity dendrogram was generated using the hierarchic met fully by an annual mean rainfall of 2,500 mm [57]. Unweighted Pair Group Means Average (UPGMA) method employing TREECON software ver. 1.3b [59]. MATERIALS AND METHODS 16S rDNA Sequence Analysis The representative strains of various rDNA types generated by Sample Collection ARDRA were subjected to sequence elucidation of 16S rDNA. The The feeder roots of tea bushes under cultivation were sampled from DNA from the gel slice was eluted by employing a Quick Gel 17 different locations: Baijnath, Banoi, Ballah, Bir, Choubin, Chauntra, Extraction Kit (Invitrogen, CA, USA). The gel-purified 16S rDNA Drang, Gopalpur, Joginder Nagar, Kathak, Pathiar, Palampur, Sakri, was ligated into the pGEM-T easy vector and transformed into Rajpur, Sungal, Yol and 61 Miles, which cover the various tea chemically competent Escherichia coli strain DH5-α cells. Plasmid growing zones of the Kangra valley. The soil was dug to a 30 cm DNA for sequencing was purified by using a Plasmid GenElute depth and young feeder roots were identified by tracing them through Miniprep Kit (Sigma-Aldrich Corp, MO, USA). The quality of the their main roots to their respective bushes. Fifty-one samples of purified plasmid DNA was checked on 1% (w/v) agarose gel these young feeder roots were then collected, in sum representing prepared in a 1× TAE buffer. The sequences of the inserts were three samples for each location. determined using a Big-Dye Terminator Cycle Sequencer and ABI Prism 310 Genetic Analyzer (Applied Biosystems, CA, USA). The Enrichment and Isolation of Diazotrophs sequencing PCR reaction, of a total volume of 5 µl, included 1 µl of N-free semisolid malate (NFb) enrichment media and Rojo Congo 5× sequencing buffer, 1 µl of Big-Dye Terminator Premix, 1 µl of agar were employed for the isolation of diazotrophs from the root primer (5 pmol), and 2 µl of plasmid. Thermal cycling conditions samples [5, 44]. The feeder roots were washed with sterile water consisted of an initial denaturation at 96oC for 3 min, followed by and treated with 1% chloramine-T for 2 min and again washed with 30 cycles of 94oC for 10 s, 50oC for 40 s, and 60oC for 4 min. The sterile water, cut into 1-2 mm length pieces and placed individually unincorporated dye terminators were removed from the sequencing o into tubes containing a 5 ml NFb medium and incubated at 28±1 C reaction using a Montage SEQ96 Sequencing Reaction clean up kit for 72 h. A loopful of white, dense, undulating and diffuse pellicles, (Millipore Corp, Billerica, MA, USA). The purified sequencing developing 1-4 mm undersurface, were streaked on Rojo Congo products were then transferred into the injection plate for sequence agar and incubated at 28±1oC. The light pink or colorless colonies elucidation. which turned scarlet after 72 h were purified by repeated subculturing Sequences were compared with the GenBank database using the on Rojo Congo agar. Blastn (http://www.ncbi.nlm.nch.gov) facility of the National Center for Biotechnology Information and aligned to the nearest neighbors in 16S rDNA Amplification of Diazotrophic Bacteria ClustalW (http://align.genome.jp/). The evolutionary distances amongst Genomic DNA was extracted using a Qiagen DNeasy Plant Mini the bacterial strains and their related taxa were calculated with a Kit (Qiagen, Valencia, CA, USA). The quality of genomic DNA was TREECON software package, ver. 1.3b [59], using Kimura’s two- checked on 0.75% agarose gel prepared in 1× TAE buffer. Amplification parameter model [24]. The 16S rDNA sequences of the diazotrophs of 16S rDNA was performed using fD1 (5-AGAGTTTGATCCTGG were then submitted to the GenBank (Accession No. in Table 1). CTCAG-3) and rP2 (3-ACGGCTACCTTGTTACGACGT-5) primers [60]. The 50 µl PCR reaction mixture contained 200 µM dNTPs, Carbon-Source Utilization Pattern (BIOLOG) 50 µM of each primer, 1× PCR buffer, 3U Taq DNA polymerase Carbon-source utilization pattern for 95 carbon sources, by the (Invitrogen, CA, USA) and 100 ng of genomic DNA. The thermocycling diazotrophs representing rDNA types, was studied using a BIOLOG procedure involved an initial denaturation at 94oC for 3 min, followed system (Microstation, Microbial Identification System, 1998, Biolog by 35 cycles at 94oC for 1 min, 52oC for 1 min, and 72oC for 2 min, Inc, CA, USA).