Molecular identification and characterization of some novel halo tolerant rhizobacteria containing ACC-deaminase for growth promotion of Phaseolus vulgaris L. under salt stress

Abstract

Soil salinity is one of the factors that most severely affect plant growth and production around the world. The rhizosphere supports the development and activity of a huge and diversified microbial community, including microorganisms capable to promote plant growth. Bacterial strains have variable ability to tolerate the salt stress and some salt tolerant rhizobium strains can grow at NaCl concentration up to 500 mM. Such bacterial strains are halophiles, with distinctive physiological structure and hereditary properties, salt-tolerant bacteria go through some morphological, metabolic and structural modifications to tolerate salt stress. These halo tolerant rhizobacteria colonize roots of monocots and dicots, and enhance plant growth by direct and indirect mechanisms. Modification of root system architecture by plant growth promoting bacteria implicates the production of phytohormones and other signals that lead, mostly, to enhanced lateral root branching and development of root hairs. Plant growth promoting bacteria also modify root functioning, improve plant nutrition and influence the physiology of the whole plant. Such halophilic microorganisms have captivated the attention of scholars at home and abroad. Our country has large salt deposits providing suitable condition for growth of salt loving microbes. Such salt deposits are best source for isolation of halo tolerant bacteria. In this project plants were collected from Karak salt mines (Bahadur Khel, Jatta Ismail Khel) and Khewra salt mines of Pakistan. The salt deposits of Khewra and Karak are enormously saline and the microbial communities in such areas have not been explored yet. This study reports isolation, identification and characterization of halo tolerant bacteria. This report also represents novel bacteria that require NaCl for its growth. Total sixty four bacterial strains were isolated from the rhizosphere of plants collected from Karak salt mines (Bahadur Khel, Jatta Ismail Khel) and Khewra salt mines of Pakistan belonging to fourteen different genera. Such bacterial strains can grow in media with different salt concentrations. Morphological and biochemical characteristics of these strains were studied by optimizing their growth conditions such as pH range 5-10 and NaCl range 0-30%. High microbial growth was observed at low salt concentration. Bacterial strains were identified on the basis of 16S rRNA gene sequence and phylogenetic analysis demonstrated that these are closely related to species belonging to different genera: Halomonas, Kushneria, Alcanivorax, Brevibacterium, Planococcus, Staphylococcus, Zhihengluella, Bacillus, Tenuibacillus, Thalassobacillus, Salinicola, Brachybacterium, Piscibacillus and Halobacillus. NCCP-934 is candidate novel strain in this study. Based upon phylogenetic analysis, physiological and chemotaxonomic data and DNA-DNA hybridization values, strain NCCP-934T belong to the genus Kushneria and showed the highest similarity as 98.9% with Kushneria marisflavi SW32T (AF251143), 98.7% with K. indalinina CG2.1T (AJ427627), 98.4% with K. avicenniae MW2aT (DQ888315) and less than 95% with the other species of the genus Kushneria and other taxa of the related genera. Strain NCCP-934T was characterized as Kushneria pakistanensis. Cells of strain NCCP-934T are Gram-stain negative, motile, non-spore forming rods and strictly aerobic. The optimal growth conditions occur at 30–33ºC and a pH of 7.0–9.0. Strain NCCP-934T tolerated to 30% NaCl with optimum growth at 3–9% (w/v) NaCl and thus, it can be considered as a moderately halophilic microorganism. DNA-DNA relatedness between strain NCCP-934T and other related strain was less than 30%. The data supported the affiliation of NCCP-934T with genus Kushneria. On the basis of results strain NCCP-934T distinguished from closely related strains and represented a novel species in the genus Kushneria, for which the name Kushneria pakistanensis sp. nov. is proposed with the type strain, NCCP-934T (=LMG 28525T = KCTC 42082T = JCM 18802T) is AB970675. Isolated bacterial strains and novel strain were characterized for their plant growth promoting properties. For this purpose PCR amplification of nifH and acdS genes was done. Bacterial strains with nifH gene and acdS genes were inoculated to Phaseolus vulgaris under sodium chloride stress. Inoculated bacterial strains showed positive results indicating that such bacterial strains can be utilized for growth promotion of different plants under salt stress.