Screening And Characterization Of Nematode-pathogenic Proteins From Pseudomonas Syringae MB03 And Pseudomonas Aeruginosa

More than 4000 species of plant parasitic nematodes have been found in the world,accounting for about 10% of all nematodes.Plant-parasitic nematodescan be parasitic on plant roots,stems,leaves and other parts,absorb plant nutrients through the mouth needle stab into the cell tissue.The economic losses of agriculture have reached over one hundred billion caused by plant parasitic nematodes in the world every year.The traditional methods of nematode control mainly include the physical and chemical control and the most effective method is chemical pesticide.But these chemicals are difficult to degrade,can cause irreversible damage to the environment,and pesticide residues in crops can also cause harm to humans.Therefore,the methods of biological prevention and control which are harmless to environment and havegained much interest of scientific community.At present,the main biological control microorganisms including edible nematode fungi,Bacillus thuringiensis,Pseudomonas aeruginosa and so on.Pseudomonas syringae MB03 is a strain isolated from frozen plants and has a high toxicity to Caenorhabditis elegans and Meloidogyne incongnita.P.syringae MB03 is a plant pathogenic bacterium that produces ice nucleation proteins that cause freeze injury in plants.However,there are little report about its pathogenicity to animals so far.The research between P.aeruginosa and C.elegans pathogenic has been very deeply and many important virulence genes have been found.But there are few report about toxic substances in P.aeruginosa effect on M.incongnita.To identify the MB03 of nematode virulence genes and study their pathogenic mechanisms in our laboratory using pUT mini-Tn5Km2 transposon constructs library including 1256 mutant and using liquid insecticidal screened 12 strains that nematode killing decreased.7 mutant strains corresponding to transposable gene were identified by thermal asymmetric PCR.A total of 5 proteins were purified.Using the above 5 purified proteins to check the nematicidal activity to C.elegans,we got the results as follows:the LC₅₀ values of VT47₀6935,VT47₁9690 and VT47₀6900 were 259.7 μg/ml,268.5μg/ml,147.3μg/ml,respectively.And the oviposition rate of VT47₁9645 decreased by18%,32.4% and 14% respectively at a concentration of 2₅₀μg/ml.The results showed that the amplitudes of VT47₀6935,zapE and VT47₁9645 proteins decreased by 29%,22%,14%,respectively at a concentration of 200μg/ml.In addition The fluorescenceobservation showed that VT47₀6935 protein distribution and nematode body,suggesting that nematode death may be caused by intestinal damage while zapE and VT47₁9645 are mainly distributed with intestinal nematode,indicating that it is not caused by damaging the intestinal nematode death mechanism specific results need further validation.Finally,we check the nematicidal activity to M.incongnita,it was regretted that only zapE had a 38.5% mortality at 2₅₀μg/ml after 5days.And VT47₁9645 and VT47₀6935 have no effect.For P.aeruginosa,the P.aeruginosa ATCC 15442 and CMCC（B）10104 as the research object,selected which may play a direct nematicidal toxicity gene and construct therecombinant plasmid.A total of 11 genes have been constructed and 6 strains induced the target proteins,namely lasB,exoS,clpA,clpP,plcH,pepP.And all the corresponding target proteins were purified except the lasB.The results showed that exoS and clpA weak toxicity on M.incongnita at the concentration of 200μg/ml after 5 days mortality rate were 30.2% and 34.2%,while pepP protein toxicity is relatively high,with about60.1%mortality under the same conditions.