Effect of neutral protease overproduction in Bacillus subtilis 168 via site-directed mutation against Meloidogyne incognita infecting eggplant under greenhouse conditions

Abstract

Abstract Root-knot nematodes (RKN), Meloidogyne incognita, caused significant problems for many important crops. Measuring control with low environmental impact has been required since EU legislation revised pesticide laws for crops. Bacteria-based control methods reduce pollutants and stabilise ecological changes, which makes them promising for controlling plant pathogens. In this study, the derivative of Bacillus subtilis168, termed Bs118, was generated by replacing the native promoter of the extracellular neutral metalloprotease-encoding gene (nprE) with a constitutive promoter of the repU gene responsible for replication of the Staphylococcus aureus plasmid pUB110. As a result, protease production increased to twice that of the wild type. Results revealed that the overproduction of neutral metalloprotease conferred Bs118 high nematocidal activity by inducing 98% mortality in the M. incognita J2 in vitro study. Bs118 stated its priority in affecting root-knot nematode reproduction under greenhouse conditions. The soil drench treatment was more promising than root dipping in controlling M. incognita compared with the untreated control treatment. The same trend happened in the eggplant growth parameters, where Bs118 improved plant health more than Bs168. In conclusion, site-directed mutation via homologous recombination to replace the native promoter with another constitutive one is a promising approach to constructing modified strains with higher protease production that can be employed as an efficient biocontrol agent against root-knot nematodes in addition to the positive impacts on plant growth.