The Effect And Mechanism For Biocontrol Bacteria Sneb159 And Sneb517 Against Heterodera Glycines

Soybean cyst nematode(SCN)cause one of the most destructive diseases in soybean.Currently,more attention was focused on biological control because of its eco-friendly,low toxicity and residue,and safe to humans and mammals.In this study,two bacterial strains were selected in the screening experiments,which control SCN disease.The stability and efficiency of the two strains were assessed in pot and field experiments.The induced resistance was responsible for the mode-of-action of Sneb159.A nematicidal active compound was isolated from Sneb159.The components of volatile organic compounds(VOCs)were identified using headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry(HS-SPMEGC-MS),and the nematicidal compounds were determined.The main results of this study are shown as follows:1.Screening and identification of bacterial strains.Sneb159 and Sneb517 had the highest antagonistic activity against Heterodera glycines cysts on the root among 804 bacterial strains in two screening experiments.Sneb159 was identified as Microbacterium maritypicum,and Sneb517 was identified as Bacillus aryabhattai based on the results of morphological,physiological and biochemical,BIOLOG and molecular methods.2.Evaluation of biological effect of bacterial strains on H.glycines.There is no effect of Sneb159 and Sneb517 on soybean seeds germination.In the pot experiments,Sneb159 and Sneb517 effectively decreased the population densities and penetration of juveniles in root,and also reduced cysts in pot.In separate field experiments,Sneb159 and Sneb517 effectively decreased the number of juveniles inside the roots,and cysts in rhizosphere soil.However,the number of cysts on the root was not different significantly.There was no significant effect of Sneb159 and Sneb517 on soybean growth and yields(pods,seeds,and 100-seed weight).3.Sneb159 induced resistance to SCN disease.In the split-root assay,the autoclaved culture of Sneb159 induced local and systemic resistance to H.glycines.The root was inoculated with Sneb159 and juveniles on the same side was named B+N,and the H.glycines infected root with Sneb159 inoculated on the different side was named B/N.The number of the second-stage juveniles(J2s)in the infection stage,and third-and fourth-stage juveniles in development stage was significantly decreased in B+N and B/N.The effect of Sneb159 on the movement of J2 s was determined via a linked twin-pot chamber system.The results showed that the number of J2 s in Sneb159 treated roots was significantly less than that in sterilized water treated roots.The transcript level of soybean genes related to defense enzyme,lignin,SA and JA was evaluated at the stage of H.glycines infection,syncytium formation,and development.The results suggested that the expression of POD in defense enzyme,C4 H in lignin synthesis,PR2 in SA and PR3 b in JA was induced significantly in B+N or B/N in comparison with control,and these pathways were triggered by Sneb159.4.Separation,purification and,structural determination of nematicidal compounds of Sneb159.Culture of Sneb159 and Sneb517 inhibited the juveniles and eggs hatch.In the organic solvent extraction test of Sneb159,the highest nematicidal activity compounds were found dissolved in ethyl acetate.The components were separated further using silica column chromatography,and the component 20-23 performed best.The compound A6 was isolated using semi-preparative high-performance liquid chromatography(HPLC),and the purity was 90.27% determined by HPLC.The compound was identified as phenylacetamide(C8H9NO)according to the results of nuclear magnetic resonance.5.Determination of nematicidal VOCs.The VOCs of Sneb159 and Sneb517 suppressed the H.glycines J2 s and eggs hatch.The components of their VOCs were determined using HS-SPME-GC-MS,and the results showed that nitrogen-containing compounds,ketone compounds,sulfur compounds,siloxane compounds,hydrocarbons and esters were included.Seven commercial compounds were selected to determine their effect on J2 s and eggs hatch by soaking and fumigating.Significant suppression was detected in dimethyl disulfide,dimethyl trisulfide,6-methyl-2-heptanone,and 1-phenylbutan-2-one.