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.
Highlights
Meloidogyne spp. [root-knot nematodes (RKNs)] are one of the major constraints of vegetable cultivation worldwide, mainly in light soil and warm regions; their losses reached about US$125 billion yearly (Chitwood 2003)
Several results suggest that chitinase interfered with the hatching of Meloidogyne sp. eggs resulting in the early emergence of juveniles that were less able to survive in soil (Mercer et al 1992 and Woo-Jin et al 2002)
The aim of this study was to improve the nematicidal potential of Bacillus amyloliquefaciens and Lysinibacillus sphaericus using protoplast fusion technique and assess their potential as biological control agents against M. incognita under laboratory and greenhouse conditions
Summary
Meloidogyne spp. [root-knot nematodes (RKNs)] are one of the major constraints of vegetable cultivation worldwide, mainly in light soil and warm regions; their losses reached about US$125 billion yearly (Chitwood 2003). Biological control, using rhizosphere bacteria, has been reported to be effective in improving plant growth and affect nematodes’ reproduction through different mechanisms involving production of plant growth hormones, enhancing nitrogen fixing ability and mineral availability in soil (Saharan and Nehra 2011), and producing metabolites and enzymes that act directly against nematodes (Becker et al 1988). The enzymatic digestion or deformation of the chitin component of nematode organisms by chitinase could be an effective method for their control. Several results suggest that chitinase interfered with the hatching of Meloidogyne sp. Studies on optimization of chitinases have been reported earlier with the effects of different medium ingredients on its production. Different types of substrates, which contain chitin, have been tried for the production of chitinase (Sudhakar and Nagarajan 2010)
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