Screening And Functional Characterization Of Nematode-pathogenic Factors From Pseudomonas Syringae MB03

Nematodes are widely distributed and the most abundant invertebrates on earth.The diverse habitats of nematodes include soil,fresh water and sea.Some species are parasitic to animals,plants and humans.The root knot nematode,Bursaphelenchus xylophilus,soybean cyst nematode and stem nematode are the main plant parasitic nematodes,causing serious harm to the agriculture,forestry and economy.The physical methods for the control of nematodes,are difficult to eradicate disease because the plant parasitic nematodes are widely distributed in the soil,root and other hidden places.Therefore,biological control has become an important method of the control for the plant parasitic nematodes.In this regard,the development and utilization of microbial resources have become an emerging research topic.Previously,it has been reported that the laboratory strain Pseudomonas syringae MB03 effectively killed Caenorhabditis elegans and Meloidogyne incongnita.In this study,candidate nematicidal proteins were heterologously expressed in Escherichia coli and different bioassays were conducted to elucidate the effects of purified proteins on the growth and reproduction of C.elegans.In total,120 candidate toxic genes were identified by applying bio informatics tools.These include protease,adhesion factors,Rhs family protein and pilus proteins.Based on those predicted proteins,recombinant strains were constructed successfully for the expression of three proteins.Moreover,six previously constructed bacterial strains were also used for purification of proteins.In total,nine proteins were expressed and bioassays were performed to evaluate pathogenicity of recombinant bacterial strains.Out the basis of bioassays,three proteins were purified which include 2-methyl-aconitate isomerase,neutral zinc metalloprotease and a Rhs family proteins.LC50 of 2-methyl-aconitate isomerase was found to be 155.3(123.4?176.6)?g/mL,as linear regression equation(linearly dependent coefficient)was Y=-35.55+16.27X(0.942).Similarly,LT50 was also determined and it was found as 3.72(1.64-4.85)days.L1 grow assay showed that the purified protein at a concentration of 450 ?g/mL showed a reduction of 28%in the size of worm compared to control.A reduction of 32%in brood size was observed when purified protein was used at a concentration of 180 ?g/mL.Compared to control group,when the concentration was 450 ?g/mL,the amplitude of the body wave was reduced approximately by 1.52-fold and the wavelength was reduced by 1.05-fold,respectively.FITC and pathological observation showed that the protein might damage gut of Celegans.In addition,the recombinant bacterial strains that expressed neutral zinc metalloproteinases and Rhs family protein showed high toxicity on the PG agar after 14 days as the mortality rates were 56.71%and 66.25%,respectively.Mortality of C elegans was found as 29.22%and 30.01%when the concentrations of the neutral zinc metalloproteinases and Rhs family protein were 67 ?g/mL and 83 ?g/mL,respectively.Moreover,effect of purified neutral zinc metalloproteinases and Rhs family protein on the size of C.elegans was also investigated and compared to control,a reduction of 19%and 11%was observed after 72 h when assay was conducted at the concentrations of 94.8 and 198.5 ?g/mL,respectively.Brood size assay showed that neutral zinc metalloproteinases could caused a reduction of 22%,the Rhs family protein have a reduction of 25%when the used at concentrations of 180 ?g/mL.Neutral zinc metalloprotease also effected the movement of C.elegans.Compared to control group,when concentration was 180 p,g/mL,the amplitude of the body was reduced approximately by 3.04-fold and the wavelength was reduced by 1.12-fold,respectively.When the concentration of Rhs family protein was 180 ?g/mL,the amplitude of the body was reduced approximately by 1.27-fold and the wavelength was reduced by 1.01-fold,respectively.The neutral zinc metalprotease has high toxicity against M.incongnita.Mortality was 80%when the protein was used at concentrations of 80 ?g/mL.Mortality was 38.94%when the concentration of the Rhs family protein is 200 ?g/mL.Mortality is 34.90%when the concentration of 2-methyl-aconitate isomerase is 250 ?g/mL,respectively.