Biochemical and Molecular Characterization of Salinity Tolerant PGPR Strains

Abstract

Salinity is the major environmental factor that drastically affects the crop productivity worldwide. Plant growth promoting rhizobacteria (PGPR) are widely used for enhanced soil fertility and crop productivity. Salinity tolerant bacteria have the ability to withstand salinity and promote the growth of plant. The aim of present study was to screen the salinity tolerant bacteria and evaluate their plant growth promotion potential for wheat at high salt conditions. Bacterial strains were obtained from Biofertilizer Resource center (BIRCEN) culture collection NIBGE and screened for salinity tolerance. Moreover, bacteria were isolated from Biosaline Research Station (BSRS-II), Pakka Anna and screened for salinity tolerance. Five out of the total 28 bacteria from BIRCEN culture collection and 22 isolates from BSRS-II were found salt-tolerant. The isolates from BSRS-II were also found halophilic in nature as they could grow upto 65gL-1 salt concentration. Salt tolerance ability of bacterial strains was tested by measuring the optical density as well as number of viable cells in salt-supplemented medium after 24 h growth. Maximum salt tolerance was observed in Ms-3y while minimum was observed in CHW9. Bacterial strain Ms-3y was highly salt tolerant PGPR strain showing maximum viable cell counts (i.e., 4.5x109 at 200 mM and 1.9x102 at 900 mM). The viable counts of this strain remained un-affected upto 200 mM, decreased significantly at 400 mM and drastically up to 900 mM. When bacterial strains from BIRCEN as well as from BSRS-II were evaluated for plant beneficial traits in vitro, it was found that out of total 27 salt-tolerant bacteria, 11 were able to produce indole acetic acid in tryptone-supplemented medium (564.2-18.5 µg/mL), seven were able to solubilize inorganic phosphate (1.5-17.1 µg/mL), one was Zn-solubilizer and 14 bacterial isolates were able to use ACC as sole carbon source showing their ACC-deaminase production ability. Four bacterial strains CHW1, CHW2, CHW4 and 8N-4 from BIRCEN culture collection were positive for nitrogenase activity of which maximum acetylene reduction assay (ARA) was observed in CHW1 (506 nmol of ethylene produced/h/vial) while minimum was observed in 8N-4 (20.3 nmol of ethylene produced/h/vial). Acetylene reduction ability was not detected in any of BSRS-II isolates. These plant beneficial traits (i.e., IAA production, P-solubilization, ACC-deaminase production) were also studied under salt stress conditions. The results showed that bacterial IAA production was un-affected upto 400 mM salt concentration. PGPR strain CHW4 obtained from BIRCEN-culture collection, showed IAA production upto 1000 mM salt concentration while SAL-12 and SAL-15 were able to produce IAA at 300 and 400 mM salt concentration. Isolates SAL-8, SAL-10, SAL-11, SAL-13, SAL-15, SAL-16, SAL-17 and SAL-21 showed P-solubilization ability in the presence of 300 mM NaCl salt conentration. Plant inoculation trials were conducted in lab and green house to evaluate the potential of salinity tolerant bacteria for improvement of wheat growth in vivo. The results showed that all the PGPR isolated from BSRS-II have potential to promote the growth of wheat in saline as well as non-saline conditions. P-solubuilizing PGPR SAL-15 and SAL-16 inoculated to wheat in the presence of inorganic phosphate showed increased growth as compared to un-inoculated control. The bacterial inoculation to wheat with the addition of nitrogen fertilizer showed a non-significant effect of fertilizer x inoculation. The ACC-deaminase producing bacteria inoculated to wheat in saline soil, showed increased root colonization on wheat under salt stress. Selected bacterial isolates were evaluated under field conditions during three consecutive winter seasons (2007-10) to study their growth promotion activity in saline environment using two wheat varieties TJ-83 and SKD-1 in first year and only TJ-83 for further two years. Experiments were conducted using single and/or mix inoculation of CHW2, CHW4, CHW7, Ms-3y in first year and SAL-12, SAL-15, SAL-16, SAL-17 in 2nd and 3rd year with 4 replicates each and compared to un-inoculated and chemical fertilizer controls. Commercially available wheat BioPower was also used as a positive control in all these experiments. Bacterial association of field inoculated wheat root was also studied under electron microscope. Results of field experiment conducted in 2007 and ultrastructure study showed that among all the bacterial strains tested, CHW4 and CHW7 have the ability to colonize wheat roots in saline environment and improve plant growth and yield. The experiments conducted in 2008, 2009 showed that inoculation of salinity tolerant bacteria SAL-12, SAL-15, SAL-16, SAL-17 resulted in increased biomass (up to 62% in 2008-09 and 50% in 2009-10) as well as grain yield (up to 113% in 2008-09 and 77% in 2009-10) compared to the un-inoculated control. Selected salinity tolerant PGPR were identified at molecular as well as morphological levels. On the basis of colony and cell morphology, CHW4 was identified as Alkaligens sp. and CHW7 was identified as Azotobacter sp. While on the basis of 16S rRNA gene sequence similarity, SAL-15 was identified as Planococcus rifitiensis while SAL-12, SAL-16, SAL-17 and SAL-21 as Aeromonas spp. Based on the results, we concluded that the all the PGPR strains screened during this study (having IAA, ACC-deaminase as well as P-solubilizing ability) can be used for plant growth promotion of wheat in non-saline soil while PGPR strains CHW4, CHW7, SAL-15 and SAL-16 can be used as potent bacterial inoculum for improvement of wheat growth in saline soil.