Isolation and Characterization of Plant Growth Promoting Rhizobacteria and their Role in Biocontrol of Fusarium Stalk Rot of Maize (Zea Mays L.)

Abstract

Biocontrol using plant growth promoting rhizobacteria is an eco-friendly, sustainable alternative to chemical pesticides. The present investigation was aimed (i) to isolate and characterize the indigenous antagonistic rhizobacteria which inhibit the infection and proliferation of Fusarium moniliforme, the casual organism for stalk rot, (ii) to evaluate their potential as bio-inoculant in pot experiment under axenic condition as well as under natural conditions of field (iii) to determine the mechanism of action of the PGPR with particular emphasis on antibiotic production. In the first experiment, characterization of 117 rhizobacteria, isolated from the rhizosphere of non-infected and stalk rot infected maize plants grown from Jhang, Yousafwalla and Islamabad territory, were made. The antifungal potential of the PGPR were determined against Fusarium moniliforme, Helminthosporium sativum and Aspergillus flavus. Out of 117 rhizobacteria, 50 rhizobacteria have shown the potential to inhibit the growth of F. moniliforme, Helminthosporium sativum and Aspergillus flavus. These rhizobacteria were further tested for the production of siderophores, antimicrobial secondary metabolites (antibiotics and HCN), production of hydrolytic enzymes (chitinases, proteases, cellulases) and phytohormone production (IAA). On the basis of their efficacy, 18 rhizobacteria were selected as potent biocontrol agent. These selected rhizobacteria were also used as bio-inoculant on maize in an experiment conducted under axenic conditions. Out of these, six rhizobacteria codes as 4nm, NDY, JYR, PTWz, Y5 and Yio have shown higher survival efficiency in soil and significantly improved the growth of maize seedlings. These rhizobacteria were identified by 16S rRNA gene sequencing and two antagonistic rhizobacteria JYR, 4nm, NDY PTWz, Y5 and Yio were identified as Pseudomonas aeruginosa, Pseudomonas sp., Bacillus firmus, Bacillus endophyticus, and Bacillus pumilus, respectively. In the second experiment, the efficacy of antagonistic rhizobacteria was evaluated alone and in combination with fungicide against stalk rot in maize. The experiment was conducted under axenic conditions in pots. All the antagonistic rhizobacteria significantly reduced (up to 61%) stalk rot disease in maize plants. The antioxidant enzymes like superoxidase dismutase, peroxidase, polyphenol oxidase, ascorbate peroxidase, proteases and chitinases were enhanced significantly in the rhizobacteria inoculated maize plants. The combined applications of B.endophyticus, P.aeruginosa JYR and P.aeruginosa 4nm were at par with the full dose (0.2%) of chemical fungicide for controlling the growth of F.moniliforme in maize plants. In the third experiment, the selected rhizobacteria were evaluated as bio- inoculant on maize under natural conditions of field. Four antagonistic rhizobacteria including P. aeruginosa JYR, B .endophyticus Y5, P. aeruginosa 4nm and Pseudomonas sp. NDY exhibited significant decrease (up to 56%) against stalk rot in field. The percentage decrease in disease severity was higher under axenic conditions in pots as compared to that of the field experiment. There were significant increase in enzymes activities, PR proteins and endogenous IAA level in maize leaves. Low concentration (half dose, 0.1%) of fungicide applied in combination with antagonistic rhizobacteria augmented the effect of antagonistic rhizobacteria by 1.36 folds. In fourth experiment, the antagonistic rhizobacteria were characterized for the production of antibiotics 2, 4, diacetylphloroglucinol (DAPG), pyrrolnitrin (PRN), Phenazine (Phz), and Zwittermicin A and the genes involved in the biosynthesis of antibiotics were detected by PCR. The phenazine and pyrrolnitrin biosynthestic genes were found in three Pseudomonas strains P. aeruginosa JYR, P. aeruginosa 4nm and Pseudomonas sp. NDY while, zwittermicin A biosynthetic gene was found in Bacillus endophyticus. The production of phenazine and the expression of its biosynthesis genes by Pseudomonas strains wee quantified by high performance liquid chromatography (HPLC) and RT-PCR, respectively. It is inferred from the results that P. aeruginosa JYR, B. endophyticus and P. aeruginosa 4nm are the most efficient and consistent antagonist PGPR. Three Pseudomonas strains produce antibiotic and their expression of genes possibly correlate with their activity as biocontrol agent.