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Biomolecular Characterization of Diazotrophs Isolated from the Tropical Soil in Malaysia

Umme Aminun Naher
Principal Scientific Officer
Soil Science Division, Bangladesh Rice Research Institute

Radziah Othman
Associate Professor
Land Management Depertment Universiti Putra Malaysia

Abstract/ Executive Summary:

This study was conducted to evaluate selected biomolecular characteristics of rice root-associated diazotrophs isolated from the Tanjong Karang rice area of Malaysia. A total of 28 diazotrophs were isolated using a nitrogen-free medium. The biochemical properties of the isolated bacteria, such as nitrogenase activity, indoleacetic acid production and sugar utilization, were measured. According to a cluster analysis of Jaccard’s similarity coefficients, the genetic similarities among the isolated diazotrophs ranged from 10 to 100%. A dendrogram constructed using the unweighted pair-group method with arithmetic mean showed that the isolated diazotrophs clustered into 11 groups. The genomic DNA rep-PCR data were subjected to a principal component analysis, and the first four principal components (PC) accounted for 52.46% of the total variation among the 28 diazotrophs. Ten diazotrophs that tested highly positive in the acetylene reduction assay (ARA) were identified as Bacillus spp. (9 diazotrophs) and Burkholderia sp. (Sb16) using partial 16S rRNA gene sequence analysis. In the analysis of the biochemical characteristics, 3 principal components accounted for 85% of the total variation among the identified diazotrophs. The examination of root colonization using scanning and transmission electron microscopy proved that two of the isolated diazotrophs (Sb16 and Sb26) were able to colonize the surface and interior of rice roots and fixed 22-24% of the total tissue nitrogen from the atmosphere. In general, the tropical soils (Inceptisols) of the Tanjong Karang rice area in Malaysia harbor a diverse group of diazotrophs that exhibit a large variation of biomolecular characteristics.

Keywords: Rice soil, Diazotrophs, 16S rRNA gene sequence analyses, Principal component analyses.

Location: Universiti Putra Malaysia, Malaysia

Start Date: 30-01-2005

End Date: 30-09-2009

Program Area: Crop-Soil-Water Management

Commodity: Bio fertilizer

Objectives:

To identify potential efficient indigenous diazotrophs using molecular approaches
To evaluate their diversity, colonization efficiency and nitrogen fixation.

Methodology:

Plant and soil samples were collected from different rice field. All of the soils were considered Inceptisols. Diazotrophs were isolated from the rhizosphere and non-rhizosphere soil using a nitrogen-free (Nfb) semi-solid malate medium. The nitrogenase activity was assayed using the acetylene reduction assay (ARA). The concentration of IAA was determined using a spectrophotometer. Cellulase activity was evaluated using Jensen’s agar plates with 0.1% carboxymethyl cellulose. For sugar production, carbon and nitrogen-free nutrient culture broth used was modified from Egener et al. (1999) After 36 hours of incubation, the cultures were filtered through 0.2 µM pore syringe filters, and sugars were determined using high-performance liquid chromatography (HPLC, Jasco Borwin software). The amount of sugar consumption was determined by subtraction from the initial substrates. The bacterial genomic DNA was extracted from the pure bacterial culture and Rep-PCR was performed using the primers: REP IR, 5′-IIIICgICgICATCIggC-3′, and REP 2I, 5′-ICgITTATCIggCCTAC-3′. The strain diversity was evaluated by rep-PCR genomic fingerprinting. Ten isolates with high ARA values were selected for partial 16S rRNA gene sequencing analysis. The DNA was extracted from pure isolates using the QIAamp Genomic DNA Mini Kit UK AS. The rep-PCR was performed using the forward primer 8F, 5'-AGA GTT TGA TCC TGG CTC AG-3' and the reverse primer 1492R, 5'-GGT TAC CTT ACG ACT T-3'. The primers selected for this study amplify sequences from both symbiotic and free-living nitrogen-fixing bacteria. The PCR products were purified and sequenced by First BASE laboratories, Malaysia. The partial gene sequences were aligned using the ClustalW package. An unrooted phylogeny tree was constructed using the Neighbor-Joining method. Partial sequences for the strains (Bacillus sp., GenBank JN695718.1, Burkholderia sp. GenBank AF219125.1 and Stenotrophomonas maltophilia, GenBank HQ219979.1) were obtained as references. The topology of the distance tree was tested by re-sampling the data using 1000 bootstrap replicates. The phylogenetic analyses were conducted in MEGA4. The sequences obtained were deposited in the European Molecular Biology Laboratory data bank (accession number JQ820251 to JQ820260). Two strains, Sb16 and Sb26, were selected for biological nitrogen fixation (BNF) assessment in a greenhouse study. The popular modern rice varieties were inoculated with the selected diazotrophs, and the N₂fixation rate was estimated using the ¹⁵N isotope dilution method. Root colonization was studied using scanning electron microscopy (Philips XL 30 ESEM) and transmission electron microscopy (Leo 912AB EFTEM). The biochemical analyses and glass house experiments were arranged in completely randomized designs with 5 replicates. The quantitative results were subjected to an ANOVA, and significance at the 5% level was tested by Tukey’s studentized range using SAS statistical program version 9.1. The genomic DNA was analyzed using NTSYS-pc software to assess the genetic relatedness of the 28 isolated diazotrophs. The coefficients of genetic similarity for all of the pair-wise comparisons were computed using the Jaccard’s coefficient, and the similarity matrix was subjected to a cluster analysis using the unweighted pair-group method with arithmetic mean (UPGMA) to produce a dendrogram. A principal component analysis (PCA) analysis was also performed for the isolates and their biochemical properties.

Source of Information: Inter. J. Mol. Sci. 14: 17812-17829. (2013)

Article Link (Online Journal):

Funding Source:

1.

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The tropical rice soils of Malaysia are dominated by β-subclass Proteobacteria: 9 diazotrophs were identified as Bacillus spp. and one was identified as a Burkholderia sp. (Sb16). Isolates Sb16 and Sb26 (Bacillus spp.) were able to colonize the rice rhizosphere and the interior of the root tissue. Scanning and transmission electron micrographs clearly showed that both of the diazotrophs were able to colonize the root surface and interior. These strains had the potential to fix atmospheric nitrogen and contributed approximately 22-24% atmospheric nitrogen to the total plant tissue nitrogen. In addition, the ¹⁵N isotope analysis also proved that both diazotrophs can fix approximately 10-12 kg N ha^-1 within a 60 day period. Four principal components were identified among the 28 isolated diazotrophs and accounted for 52.46% of the total genetic variation, confirming that the evaluated soil harbors a diverse group of diazotrophs.

Knowledge Management: Journal