Use of low cost natural resources for enhanced chitinase production and optimization using CCD and RSM: a new initiative for bio-control of plant pathogen

Abstract

A total number of 160 native fluorescent Pseudomonads were isolated from rhizospheric soil of tomato cultivated in different agro-ecological regions of West Bengal. Out of them 25 (15.63%) were found to be chitinase positive by semi-quantitative spot culture assay on chitinase detection agar media. By quantifying the chitinase production based on spectrophotometric assay, best performing isolate Pseudomonas aeruginosa FPK22 (Gene bank accession number: KY575332) was selected for further study of media optimization. Ten different media compositions were designed with cheap and readily available nutrient and substrate sources viz. aqueous extract of cow dung manure, aqueous extract of vermi compost, aqueous extract of de-oiled neem cake, sugar, sugarcane molasses, Baker’s yeast powder and crab shell powder in their different combinations. The media I, comprising with aqueous extract of vermi compost (V), sugarcane molasses (M), Baker’s yeast (Y) and crab shell powder (C) exhibited maximum biomass and highest chitinase production of the strain FPK22. The media was named, based on its composition, as VMYC media. In VMYC media chitinase production of the strain was enhanced by 1.15 fold than from the standard chitinase detection media. The composition of the VMYC media was then optimized by a four factor (A, B, C, D) central composite design (CCD) and response surface methodology for the response (Y) chitinase activity. By using this design a total of 30 runs were tested and the data were fitted. The regression analysis showed good fit of the experimental data to the second order polynomial model with coefficient of determination (R2) value of 0.9093 and model F value 10.74. The optimum concentrations of vermi wash (250 g/l), sugarcane molasses (20.7 g/l), Baker’s yeast (15 g/l), crab shell powder (5.2 g/l) were recorded from desirability function with a predicted value of chitinase production of 1.125 EU/ml. Ten repetitive runs were tested under the optimized condition of the four variables and observed that they yielded 92.9–98.7% of the predicted chitinase production. Two soil borne fungal pathogens R. solani and S. rolfsii were co-inoculated with the strains P. aeruginosa FPK22 and P. monteilii FPK4 in the optimized VMYC media in dual culture study. The enhancement of inhibition of mycelial growth of the fungal pathogens was also observed in optimized VMYC media compared to PDA media. The mycelial percent inhibition of R. solani challenged with FPK22 and FPK4 were increased by 1.17 and 1.13 fold respectively, besides, the mycelial percent inhibition of S. rolfsii co-inoculated with FPK22 and FPK4 were observed to be enhanced by 1.04 and 1.12 fold, respectively. The field evaluation study with the strains FPK22 and FPK4 grown in optimized VMYC media exhibited that the consortia of the strains could efficiently check the damping off disease, 69.2% and 62%, over control in case of tomato and chilli respectively.