Improving the nematicidal potential of Bacillus amyloliquefaciens and Lysinibacillus sphaericus against the root-knot nematode Meloidogyne incognita using protoplast fusion technique

Abstract Root-knot nematodes (RKNs) are one of the major constraints of vegetable cultivation worldwide. Chemical nematicides, the primary management tool for over 50 years, have a negative impact on the environment and the ineffectiveness after prolonged use. Biological control using eco-friendly rhizosphere bacteria antagonistic to nematodes is one of the alternative approaches. The objective of this study was to improve the nematicidal activity of Bacillus amyloliquefaciens subsp. plantarum SA5 and Lysinibacillus sphaericus Amira strain against RKN Meloidogyne incognita, using the protoplast technique. Their fusants were tested for their chitinase and nematicidal activity using bioassay and greenhouse experiments. The selected fusants from the two bacterial strains were more effective in killing M. incognita J2 under laboratory conditions. Percentage mortality after 24 h of exposure were 70.85, 84.69, 95.56, 94.99, 100, and 89.46% due to the parental strains B. amyloliquefaciens and L. sphaericus and the fusants Bas3, Bas6-2, Bas8, and Bas11, respectively. There was a positive correlation between the chitinase production and the nematicidal effect of the bacterial strains. Under greenhouse conditions, Bas8 which produced the highest amount of chitinase induced the greatest reduction in nematode counts and gave the best results in shoot length and fresh and dry weights as compared to control. Chitinase production of fusant was much higher under solid-state fermentation (SSF) than submerged fermentation conditions. The recorded chitinase produced by B. amyloliquefaciens, L. sphaericus, and Bas8 were 0, 1393, and 3399 units (μg NAG/ml enzyme/h), respectively, under solid-state fermentation and 90, 85, and 143 units (μg NAG/ml enzyme/h), respectively, under submerged fermentation conditions. Protoplast fusion was a powerful technique in improving nematicidal activity. Chitinase production is an important factor in improving the nematicidal activity of such microorganisms. The obtained improved fusant could be used as a biological control agent for M. incognita