Genetic diversity and beneficial role of plant growth promoting rhizobacteria in oil seed producing Sunflower (Helianthus annuus L.) crop of Azad Jammu and Kashmir

Abstract

The use of plant growth promoting rhizobacteria is a promising strategy for sustainable agriculture production. The aims of the present study were to isolate, characterize and identify sunflower associated beneficial bacteria and to evaluate their inoculation and colonization potential towards sunflower. Therefore, sixteen sites with varying altitudes of Himalayan Mountain region of Dhirkot (subdivision), Azad Jammu and Kashmir have been selected. A total of 163 isolates were obtained from rhizosphere (97) and root interior (66 putative endophytes) of sunflower to evaluate the potential of these beneficial root associated bacteria and their root colonization potential to improve sunflower growth, nutrient uptake, yield and oil contents. Out of 163 screened isolates, 44 % were found positive for phosphate solubilization (9.51 to 48.80 µg mL-1), 24 % for IAA production (1.13-24.6 µg mL-1), 20 % for nitrogen fixation (28.68-137.84 nmoles mg-1 protein h-1) and 12% for biocontrol properties against Fusarium oxysporum detected by using standard microbiological and biochemical procedures. Most of the phosphate solubilizing isolates were able to produce a variety of organic acids dominated by gluconic acid (G.A) ranging between 2.17 µg mL-1 to 15.44 µg mL 1. The isolates exhibiting multiple plant growth promoting traits in vitro were identified as species of the genus Azospirillum, Bacillus, Enterobacter, Citrobacter, Pseudomonas, Serratia, Stenotrophomonas and Lysinibacillus, Cellulosimicrobium, Staphylococcus, Chryseobacterium showing 99% homology of 16S rRNA gene sequence. Major population was dominated by Bacillus species followed by Pseudomonas and Enterobacter. Phylogenetic analysis did not show any correlation or distribution of specific species/genera at specific sites indicating that the distribution of PGPR is independent of the surrounding topography. Eleven potential PGPR strains exhibiting at least 3 of the above mentioned plant beneficial traits were further tested for intrinsic antibiotic resistance through disc diffusion method and found to be resistant against most of the tested antibiotics. The bacterial strains were then tested as inoculant on sunflower (cv. FH331) in soil-free medium (growth pouches) and in sterilized soil (pots) under controlled conditions for their N2-fixing and P-solubilizing abilities separately, as well as in field under natural conditions at two locations i.e., Rawalakot, AJK, and Faisalabad, Pakistan. All the eleven bacterial strains (belonging to 8 genera) promoted the sunflower growth under controlled environmental conditions and improved N and P uptake over non-inoculated control treatment. Out of these 11 strains, Azospirillum brasilense AF-22, Enterobacter cloacae AF-31, Pseudomonas sp. strain AF-54 and Citrobacter freundii AF-56 were found more effective and potent strains in augmenting sunflower growth, yield and oil contents and NP uptake compared with 50 % (of their recommended dose) N and P fertilizers treatments. These four strains exhibiting multiple plant growth promoting traits i.e., N2-fixation, P-solubilization, IAA production, organic acid production and metabolic versatility, performed well in both experimental locations at Rawalakot and Faisalabad. Principal component analysis indicated that inoculation with these selected PGPR had better response at Rawalakot. To confirm the efficiency of these bacterial strains for sunflower, their host specificity and colonization potential was extensively studies in vitro and in vivo. Bacterial population dynamics were observed at different time intervals to check the strain persistency in sunflower rhizosphere. All the strains showed strong association with sunflower roots up to 45 days. Their colonization potential was confirmed through a series of high throughput microscopy techniques including yfp-labelling technique, fluorescent antibody (FA) labelling, fluorescent in situ hybridization (FISH) techniques coupled with confocal laser scanning microscopy and by ultrastructural and immunogold labelling technique through transmission electron microscopy (TEM). These biomarkers confirmed the host specificity of the applied strains in both sterilized and natural conditions. Transmission electron microscopic studies also showed the localization of Azospirillum brasilense AF-22 and Citrobacter freundii AF-56 both in the rhizosphere and root interior, confirming their endophytic association with sunflower. Based on the results of this study, it is concluded that the potential PGPR strains namely A. brasilense AF-22, E. cloacae AF-31, Pseudomonas sp. strain AF-54 and C. freundii AF-56 can be used as biofertilizer for sunflower crop for enhancing yield and to minimize the use of chemical (NP) fertilizers. It is further recommended that the inoculum should be checked for the cross inoculation potential on other oil seed crops in field.