Thermo-neutrophilic cellulases and chitinases characterized from a novel putative antifungal biocontrol agent: Bacillus subtilis TD11.

Cellulose and chitin are the most abundant naturally occurring biopolymers synthesized in plants and animals and are used for synthesis of different organic compounds and acids in the industry. Therefore, cellulases and chitinases are important for their multiple uses in industry and biotechnology. Moreover, chitinases have a role in the biological control of phytopathogens. A bacterial strain Bacillus subtilis TD11 was previously isolated and characterized as a putative biocontrol agent owing to its significant antifungal potential. In this study, cellulase and chitinase produced by the strain B. subtilis TD11 were purified and characterized. The activity of the cellulases and chitinases were optimized at different pH (2 to 10) and temperatures (20 to 90°C). The substrate specificity of cellulases was evaluated using different substances including carboxymethyl cellulose (CMC), hydroxyethyl cellulose (HEC), and crystalline substrates. The cellulase produced by B. subtilis TD11 had a molecular mass of 45 kDa while that of chitinase was 55 kDa. The optimal activities of the enzymes were found at neutral pH (6.0 to 7.0). The optimum temperature for the purified cellulases was in the range of 50 to 70°C while, purified chitinases were optimally active at 50°C. The highest substrate specificity of the purified cellulase was found for CMC (100%) followed by HEC (>50% activity) while no hydrolysis was observed against the crystalline substrates. Moreover, it was observed that the purified chitinase was inhibitory against the fungi containing chitin in their hyphal walls i.e., Rhizoctonia, Colletotrichum, Aspergillus and Fusarium having a dose-effect relationship.

Biological control of fungal pathogens of tomato (Lycopersicon esculentum) by chitinolytic bacterial strains.

The need to increase food production and to reduce the pollution caused by synthetic chemicals has led to a search for biocontrol agents against plant pathogens. In the present study, a total of 37 chitinolytic bacteria were isolated from the rhizospheric soil of tomatoes using a chitin agar medium. In vitro bacterial isolates, that is, TD9, TD11, TD15, and TD24 showed strong antagonistic and enzymatic activities against Rhizoctonia (8%-55%), Fusarium (31%-48%), Colletotrichum (24%-49%), and Aspergillus on a dual culture plate and enzyme assay. Furthermore, these putative antagonistic bacterial isolates were identified as Pantoea agglomerans (TD9), Bacillus subtilis (TD11), Bacillus cereus (TD15 and TD24) using 16S rRNA sequence analysis. Additionally, in culture filtrate in vivo assay, the isolates TD11 and TD15 inhibited the growth of Rhizoctonia solani about 40% and Fusarium oxysporum about 80%. However, in the pot trials, these two bacterial isolates (TD11 and TD15) considerably suppressed the disease rate in tomatoes caused by Fusarium and Rhizoctonia fungal species. Moreover, it was concluded that B. subtilis (TD11) was found to be the most promising putative biocontrol agent, inhibiting the fungal diseases of tomatoes by 50% and showing versatile antagonistic potential.

Role of halotolerant and chitinolytic bacteria in phytoremediation of saline soil using spinach plant.

Novel technologies are required for rapid reclamation of saline soils. The halotrophic and chitinolytic bacterial strains were used for phytoremediation of saline soils using spinach plants (Spinacia oleracea L.). The previously isolated chitinolytic bacteria showed high antifungal potential against Fusarium oxysporum, and Alternaria spp. The halotolerant bacterial strains were previously isolated showing a salt tolerance of up to 20% in culture media. Specially designed microcosms were used here to investigate the reclamation of saline soil by bacteria. The soil salinity was reduced by both types of bacteria (from 6.5 to 2 dS/m). A decline in Na contents from 22-24 to 9-12 meq/L and in sodium adsorption ratio from 10-11 to 7-8 was also observed in saline soils. The Ca/Mg contents increased from 24 to 30-33 meq/L. The bioassays were performed to evaluate the effect of the bacteria on the phytoremediation. The shoot, root weights (both fresh (1.927 g, 0.244 g) and dry (0.387 g, 0.104 g)) increased by bacterial inoculation as compared to control in saline soils. The Na/K ratio decreased in plant tissues. Here we report the increased efficacy of phytoremediation by combined inoculation of chitinolytic and halotolerant bacterial strains in soil which has never been reported before.