Screening,Identification And Primary Application Of Biocontrol Bacteria And Their Volatile Organic Compounds Against Meloidogyne Incognita

Root-knot nematodes?Meloidogyne spp.?is an important pathogen that causes severe damages on crops.It reduces the yield and quality of crops and causes serious economic losses to global agriculture.Currently,chemical pesticides application is the major measure for controlling root-knot nematodes.However,with the heavy use of chemical,the human and environmental safety are under threat,some chemical nematicides have been restricted or banned.Therefore,it is an urgent need to select the environmentally friendly,safe and pollution-free control methods.Biological measures,especially bacteria,as an environmentally friendly alternative with advantages of satisfactory nematicidal activities and easy to culture has attracted wide attention.In China,B.firmus,B.cereus and B.thuringiensis have been registered for the control of root knot nematodes.However,there are few commercially available strains,which cannot fully meet the demand.It is urgent to screen novel strains with high efficiency against root-knot nematodes.106 bacterial strains were isolated from soil where root-knot nematodes were detected,and 8 of the obtained isolates exhibited satisfactory nematicidal activity against Meloidogyne incognita.Then the 8 strains were identified.The effect of bacterial fermentation broth for controlling root-knot nematode disease was verified.The volatile organic compounds?VOCs?of four strains were identified by SPME-GC-MS/MS.And the nematicidal activity of pure products was verified.The results are summarized as follows:1.The nematode immersion method was used to test the nematicidal nematicity?NA?of bacterial fermentation broth against M.incognita.Seven bacterial strains?DDWA,DDWB,DDWC,DDWD,DDWNEI,DDWWAI and JNC?with strong nematicidal activity?NA?80%?and one strain?JNB?with moderate nematicidal activity?50%?NA<80%?were isolated.Fumigation method was used to determine the laboratory toxicity of volatile organic compounds?VOCs?produced by the strain against M.incognita.Four strains?DDWB,DDWNEI,DDWWAI and JNB?with strong nematicidal activity?NA?80%?were isolated.2.Based on their morphological,physiological,biochemical and molecular?16S rDNA and gyrB gene sequences?characterizations,the 8 strains were identified as B.halotolerans DDWA,B.kochii DDWB,B.oceanisediminis DDWC,B.pumilus DDWD,B.toyonensis DDWNEI,B.cereus DDWWAI,P.aeruginosa JNB and B.pseudomycoides JNC.3.In the pot experiments,B.oceanisediminis DDWC showed the greatest control effect on root-knot nematode disease?69.96%?at 3×10¹² CFU mL^-1,followed by B.toyonensis DDWNEI?66.79%?.In field experiments,the control efficacy of strains was 49.98%-67.47%at 6×10¹² CFU mL^-11 in 2018,and B.kochii DDWB showed the best control effect.In 2019,the control efficacy was 53.40%-66.79%,and B.oceanisediminis DDWC showed the best control effect.After treatment,the yield of tomatoes can be increased by 1.38%to 26.19%.4.The fumigation method was used to determine the presence of volatile active components in four bacterial strains?B.kochii DDWB,B.toyonensis DDWNEI,B.cereus DDWWAI and P.aeruginosa JNB?,and the species and structure of each component were further identified by combining solid-phase microextraction?SPME?and GC-MS/MS.The volatile active components were identified as alkyl,benzene,ester,phenol,aldehyde,alkene,alcohol,ketone,acid and heterocyclic.5.Ten substances were screened from the identified substances and purchased the pure products to verify their nematicidal activity.Among them,4 substances?1-undecanol,methyl octanoate,2-octanone and 4-chlorobutyrophenone?were obtained with concentrations less than12 mmol,which exhibited 100%nematicidal activity against M.incognita.The LC₅₀ of 4 pure compounds against M.incognita were 8.70,51.76,66.85 and 220.26 mg L^-1,respectively.The pot experiments of methyl octanoate,2-octanone and 1-undecanol were performed,and the control effect of 1-undecanol could reach 82.18%at the concentration of 600 L ha^-11 against M.incognita.The control effect of methyl octanoate and 2-octanone were 58.17%and 73.98%at3000 L ha^-1,respectively.