ROLE OF ROOT ASSOCIATED BENEFICIAL BACTERIA FOR NUTRIENT MOBILIZATION IN SUNFLOWER

Abstract

Sunflower is an improtant oil seed crop that have potential to bridge up the huge gap between oil production and consumption in Pakistan. Present study was carried out to evaluate the potential of beneficial root associted bacteria with ultimate objective to improve sunflower growth, nutrient uptake, yield and oil contents. Total 67 bacterial isolates were purified from four locations of Pakistan and characterized for plant beneficial traits like nitrogen fixation, phosphate solubilization and indole-3-acetic acid (IAA) production. The isolates showed the production of IAA (0.06 to 10.65 μg mL-1), solubilization of inorganic phosphate (9.71 to 37.34 μg mL-1) and nitrogenase activity of 34.96 to 146.33 ηMol mg-1 protein h-1. Ten potent PGPR strains exhibiting more than one plant beneficial traits in vitro were found metabolically diverse (in phenotypic microarray analysis) showing their wide-range of environmental adaptibility. The screened isolates were identified through 16S rRNA gene sequencing and they were found to belong to Pseudomonas, Bacillus, Enterobacter and Alcaligenes genera. The nitrogen fixing strains were confirmed for the property through nifH gene sequencing with 96-98 % similarity to different strains. Phosphate solubilization and organic acid production (gluconic, lactic, malic and tartaric) ability of selected strains is positivly correlated with decrease in pH of growth medium (r = 0.904 to 0.997). Only three out of eight P-solubilizing bacteria showed the presence of pqqE gene which were also able to produce gluconic acid. The pqqE gene sequence of Pseudomonas marginalis Fs-3, Enterobacter sp. Fs-11 and Pseudomonas sp. Ms-16 showed 96%, 98% and 98% identity with Gluconobacter oxydans, Enterobacter cloacae and Klebsiella pneumoniae, respectively. A comparative phylogenetic analysis based on 16S rRNA, nifH and pqqE genes revealed considerable phylogenetic differneces at species as well as genus level. Nitrogen fixing Pseudomnas trivialis Fs-9, Pseudomonas sp. Fs-15 and P- solubilizing Enterobacter sp. Fs-11 were checked for root colonization through transmission electron microscopy. The results showed that these strains were intense colonizers of sunflower rhizosphere. Moreover, sunflower seedlings, inoculated with yellow flourescent protein (yfp) labelled strain Fs-11, were obsrved under confocal laser scanning microscope (CLSM) which confirmed its potential of colonizing roots. Plant inoculation experiment with nitrogen fixing strains (Pseudomonas trivialis Fs-9, Pseudomonas sp. Fs-15, Bacillus sp. Ms-7 and Alcaligenes faecalis Ms-8) significantly (P≤ 0.05) increased sunflower growth and nitrogen uptake or nitrogen fixed by Pseudomonas and Alcaligenes spp. as compared to un-inoculated plants. In another pot experiment, phosphate solubilizing strains (Pseudomonas marginalis Fs- 3, P. trivialis Fs-9, Enterobacter sp. Fs-11, Pseudomonas sp. Fs-13, Bacillus sp. Ms- 7, Pseudomonas sp. Ms-16, Alcaligenes faecalis Ss-2 and Bacillus sp. Ps-5) significanlty (P≤ 0.05) enhanced plant growth and phosphate uptake as compared to un-treated plants. In green house experiment conducted in soil to determine the colonization potential and effect on sunflower growth of rifampicin resistant derivatives of Enterobacter sp. Fs-11 and Pseudomonas sp. Fs-15, strain Fs-11 and Fs-15 were sucessfully recovered from sunflower rhizosphere and rhizoplane up to 30 days after transplanting at a cell density of 6-8 cfu g-1of root fresh wt. or rhizosphere soil. Both the strains significantly (P≤ 0.05) augmented sunflower growth parameters in single inoculation, but the effect on plants inoculated with mixed inoculum (Fs-11 + Fs-15) was found non-significant as compared to un-inoculated plants. Three field experiments were conducted at Faisalabad, Peshawar and Rawalakot and inoculation of strains Fs-9, Fs-11 and Fs-15 significantly (P ≤ 0.05) increased sunflower growth, nutrient uptake, yield and oil contents as compared to un- inoculated treatment with half dose and without fertilizer. Sunflower achene yield and oil contents were significantly (P ≤ 0.05) correlated with growth and physiological parameters of sunflower. On the basis of in vitro and in vivo studies, it is concluded that strains Fs-9, Fs-11 and Fs-15 are potential PGPR that can enhance sunflower yield and oil contents by increasing nutrient uptake and IAA production. These strains can be used as bio-inoculats for sunflower crop along with the reduced dose of nitrogen and phosphorus fertilizers.