Screening And Identification Of Nematicidal Bacteria And Preliminary Separation Of Lipopeptide

Root-knot nematode disease is one of the most important soil-borne diseases that seriously threaten the world's vegetable industry,causing plant diseases and huge yield losses.This study screened 2 strains of biocontrol bacteria with good biocontrol potential against root knot nematode disease.In this study,we conducted a systematic study on the identification of the two biocontrol bacteria,optimization of fermentation conditions,extraction of active substances,and separation and purification.The result is as follows:1.According to the indoor contact test,I screen out two highly effective bacterial strains which is Sneb709 and Sneb811.The fermentation broth has significant activity on the growth of second-instar larvae(J2)and eggs of Meloidogyne incognita.The corrected mortality rate of poisoning of southern root-knot nematode J2 by Sneb709 and Sneb811 fermentation broth is 91.10%(24 h)and 98.09%(48 h);93.15%(24 h)and98.17%(48 h),respectively.The relative inhibition rates of eggs hatching were 75.00%(24 h)and 62.5%(48 h);100%(24 h)and 75.00%(48 h).After that,the fermentation broth is applied to indoor pots and greenhouse field trials to test its disease-preventing effect.The experimental result shows that both fermentation broths not only has good control effect against M.incognita disease,but also promote the growth and development of tomato plants,even fruit quantity and weight.2.The high-efficiency bacteria Sneb709 and Sneb811 are identified by single colony culture,gram staining,transmission electron microscopy,physiological and biochemical reactions,and 16 S rDNA biological identification.The results showed that Sneb709 is Bacillus amyloliquefaciens;Sneb811 is Pseudomonas fragi.3.The fermentation conditions of B.amyloliquefaciens Sneb709 are determined by preliminary studies: the initial pH value is neutral,the liquid volume is loaded with 150 mL fermentation broth per 250 mL erlenmeyer flask,and the rotation speed is 180 r/min,and the fermentation time is 24 h.the fermentation medium consists of 1% glucose,0.5% yeast extract,0.3% NaCl,and 0.2% MgSO4.The fermentation conditions of P.fragi Sneb811 are: the initial pH value is neutral,the liquid volume is loaded with 150 mL fermentation broth per 250 mL erlenmeyer flask,and the rotation speed is 160 r/min,and the fermentation time is 48 h.The fermentation medium consists of 1% sucrose,0.5% peptone,0.3% NaCl and 0.2% MgSO4.4.Through the fermentation broth by centrifugation,suction filtration,methanol extraction,rotaryevaporation and high performance liquid chromatography(HPLC)and other methods to systematically explore the active substance of B.amyloliquefaciens Sneb709,preliminary determination of the isolated active substance is a lipopeptide.metabolites.However,lipopeptide antibiotics don't have an ideal lethal effect,and its corrected mortality rate to the M.incognita J2 is only about 34.83%.Therefore,it is inferred that the lipopeptide metabolites of the active substance in Sneb709 may not be the main active substances for its killing of M.incognita.