Exploring the Role of β-glucanase in Biopesticide Activity of Indigenous Rhizobacteria

Abstract

Exploring the Role of β-Glucanase in Biopesticide Activity of Indigenous Rhizobacteria Biopesticides consist of active microbes and/or their metabolites capable to suppress pests and control diseases. Plant growth promoting rhizobacteria are being ubiquitously used as biopesticide which exhibit broad spectrum activity through their dual mechanism of action i.e. suppressing the pathogens and enhancing soil fertiltiy. The consistent efficacy of PGPR based biopesticide depends upon their metabolic diversity. Glucanases are important enzymes produced by a number of rhizobacteria that enables them to survive in competitive environment of rhizosphere and hydrolyze the fungal cell wall. In current study, glucanase producing bacteria were isolated from the rhizosphere/endosphere of different crops viz. sugarcane (Saccharum officinarum L.) grown in mono-cropping system, Wheat (Triticum aestivum L.) and Maize (Zea mays L.) grown as multi-cropping system, in Pakistan. In first phase, a total of 45 bacteria were isolated from the rhizosphere and endosphere of different sugarcane varieties growing at the different farmer’s fields of Punjab, Pakistan. Out of 45 isolates, 3 were able to produce hydrolytic enzyme glucanase. The glucanase producing ability of bacterial strains was variable with solubilization zone 5.5–13.8 mm and quantity (0.1–0.3 U/mL of culture filtrate) on different substrates. All the glucanase producing bacteria significantly inhibited the economically important pathogens of sugarcane, i.e., Fusarium moniliforme (45–56%) and Colletotrichum falcatum (52–63%), and pathogens of other crops, i.e., Fusarium oxysporum (58–63%), Rhizoctonia solani (42–53%) and Macrophomina phaseolina (53–61%). The glucanase producing bacteria significantly induced the activities of enzymes involved in ROS scavenging, viz. SOD, POD, CAT and PPO by 1.4–2.0-fold. The glucanase producing bacteria MAZ 3SR was identified as Bacillus amyloliquefaciens, while MAZ 10SR and MAZ 29SR were identified as Bacillus subtilis by 16S rRNA sequence comparison. The low prevalence of glucanolytic bacteria effective against sugarcane diseases lead us to isolate further strains from the other crops/cropping systems. xii In second phase, a total of 178 isolates were obtained from the rhizosphere and endosphere of wheat-maize growing under arid/semi-arid and irrigated areas of Pakistan. Out of 178 isolates from multi-cropping systems, 10 exhibited glucanolytic activity. The ability of bacterial strains to produce β-1, 3 and β-1, 4 glucanase was found highly variable (solubilization zone 7.33 – 14.44 mm). A significant positive correlation was observed between qualitative and quantitative assay of glucanolytic actvity. The glucanolytic bacteria significantly inhibited the economically important pathogens viz. Fusarium moniliforme (45–56%), Fusarium oxysporum (58–63%), Rhizoctonia solani (42–53%) and Macrophomina phaseolina (53–61%). The glucanase producing bacteria also colonized the tomato rhizosphere (8.8 X 107) and reduced the Fusarium wilt disease (59%) under net house conditions. The glucanase producing bacteria prevailing in multi cropping system were found to be diverse i.e. MAZ 51 (B. subtilis), MAZ 73 (Bacillus magaterium), MAZ 13 (Achromobacter mucicolins), MAZ 48 (Serratia marcescens), MAZ 18 (Enteriobacter cloacae), MAZ 165 (Bacillus subtilis), MAZ 117 (B. subtilis), MAZ 32 (B. subtilis) and MAZ 151 (Bacillus mycoides) based on 16S rDNA identification. In silico analysis depicted, that endoglucanase gene is highly diverse even at species/strain level. A 850 bp endo glucanase gene was amplified in Bacillus subtilis strains MAZ 51, MAZ 117 and MAZ 165. Finally, we evaluated the comparative efficacy of glucanolytic bacteria and their crude extract on wheat and chickpea. In net house study, the glucanolytic bacteria exhibited greater biocontrol activity against the chickpea black rot/stem rot (M. phaseolina) and wheat root rot (F. oxysporum) as compared to that of crude enzyme extract. However a consistency was observed in crude extract. Conclusively, the glucanolytic bacteria and/ or their crude enxyme extract could be ideal bio inoculants for the disease management of different crops.