Egg-hatching Suppression Of Meloidogyne Incognita By Lecanicillium Attenuatum And Cloning Of Chitinase Gene LACHI1from The Fungus

Root-knot nematodes (Meloidogyne spp.) have been reported as one of the most important plant pathogens, which threaten the yields in China. Chemical nematicides had caused serious environmental pollution and also have negative impact on human healty. Using of extensive chemicals made the resistance of plant parasitic nematodes strengthen, reducing its control effect The releases of antagonists of nematodes give a new way to control root-knot nematodes.Nematophagous fungi are an important group of soil microorganisms that can suppress the populations of plant-parasitic nematodes. The nematode egg-parasitic fungus CGMCC5328was isolated from cysts of Heterodera glycines infecting soybean in Heilongjiang Province. Based on morphological characters and molecular analysis of ITS-rDNA, the strain was identified as L. attenuatum. The fungus was a chitinolytic-nematophagous microorganism. The chitinolytic systems and exochitinase produced by L. attenuatum CGMCC5328were measured by means of NAG(N-acetyglucosamine) and pNP(p-nitrophenol), respectively. The activity of chitinolytic systems reached the highest level of16.90U/mL on the fourth day; The exochitinase activity reached the highest level of0.59U/mL on the sixth day and then plateared until14days. The molecular weights of chitinase were estimated as79.6kDa,65.6kDa,54.1kDa,42.1kDa,32.9kDa on SDS-PAGE analysis with0.01%glycol chitin. Different concentrations of crude chitinase from L. attenuatum CGMCC5328caused lysis of the eggshell of Meloidogyne incognita, and the maximum inhibitory rate of eggs hatching achieved83.17%on the seventh day by100%concentration following treatment. The fungus parasitized the eggs of M. incognita and the rate of parasitism was85.10%on the sixth day after egg inoculation.To clone chitinolytic genes from L. attenuatum and provide theoretical basis for the inhibition of the chitinases on egg-hatching of root knot and cyst nematode. The chitinase gene LACHI1from L. attenuatum was cloned using the degenerate PCR primers and RACE techniques. The analysis of the gene LACHI1and its amino acid sequence was by biology software. We have cloned a chitinase gene LACHI1from I. attenuation for the first time. The gene is1743bp in length and contains three putative introns. The ORF of LACHI1is1272bp in size with encoding protein of423aa, molecular mass of45.9kD and pI of5.90. The chitinase deduced by LACHI1belongs to glycosyl hydrolase family18chitinase. Comparison of the chitinase amino acid sequence with other chitinases from entomopathogenic fungi and nematophagous fungi revealed that the enzymes were highly similar.These results demonstrated that L. attenuation CGMCC5328with the strong chitinolytic activity and egg-parasitic capability has a great biocontrol potential against M. incognita.