Isolation And Identification Of Induced Systemic Resistance Determinants From Bacillus Simplex Sneb545 Against Heterodera Glycines

Soybean cyst nematode disease(Herodera glycines,SCN)is one of the most devastating diseases in soybean production in the world.Biological control has broad prospects in soybean cyst nematode control due to its long-lasting effect,environmental friendliness,low toxicity and low residue.Microorganisms have a variety of action mechanisms in the control process,and their metabolites have a wide range of biological activities,which play an important role in promoting plant growth and inducing plant resistance.Previous studies had proved that the fermentation liquid of Bacillus simplex Sneb545 could stably induce soybean resistance to SCN.In this paper,the metabolites of Bacillus simplex Sneb545 were isolated and purified,and the active substances that induced soybean resistance to Cystic nematode were identified.The pot test was used to investigate the effect of active substances on the control of soybean cyst nematode and analyzed of the expression of disease resistance genes related to salicylic acid and jasmonic acid pathway.The results are as follows:1.In this paper,six components were obtained from B.simplex Sneb545 fermentation broth by systematic separation.A large amount of fermentation broth was cultured in the fermenter,the bacteria were removed by centrifugation,supernatant was concentrated by lyophilisation and was precipitated by ethanol.And using organic solvent gradient extracted to obtain the active crude extract of n-butanol layer.Using silica gel column chromatography,Sephadex LH-20 column chromatography,and semi-preparative high-performance liquid chromatography(semi-HPLC),six active compounds were obtained,respectively,S5-1,S5-2,S5-3,S11-6-1,S11-6-2 and S11-6-3.The purity was identified by HPLC.2.NMR technology was used to identify the structure of active substances,and the structures were determined as cyclic(Pro-Tyr),cyclic(Val-Pro),cyclic(Leu-Pro),uracil,phenylalanine and tryptophan via 1H NMR and 13 C NMR.Six substances were prepared at a concentration of 1 m M to coat soybean seeds for activity of ISR.The results showed that the treatment of soybean seeds with cyclic(Leu-Pro),uracil,and phenylalanine delayed the development the nematodes in the soybean root.And cyclic(Pro-Tyr),cyclic(Val-Pro),and tryptophan can reduce in the number of nematodes and delayed the development of nematodes in the soybean root.3.Quantitative Real-time PCR was used to detect of SA and JA pathway-related disease resistance genes in soybean roots infected by soybean cyst nematode at different stages after soybean seeds coated by cyclic(Val-Pro),tryptophan,and uracil.When inoculated with nematodes at 1 dpi,the expression levels of PR1,PR3 b and PR10 in the roots of the cyclic(ValPro)and tryptophan treatment groups were up-regulated.When inoculated with nematodes at 5 dpi,the expression levels of PR1 and PR3 b in the treatment group and PR3 b in the uracil treatment group were up-regulated significantly.When the nematode was inoculated at 10 dpi,The expression levels of PR2 in the cyclic(Val-Pro)treatment group and PR3a?PR10 in the tryptophan treatment group and PR1?PR10 in the uracil treatment group were up-regulated significantly.The results showed that the treatment of soybean seeds with cyclic(Val-Pro),tryptophan,and uracil can induce the expression of resistance genes in SA and JA pathways,and activate salicylic acid and Jasmonic acid signaling pathway participates in root resistance to nematode stress.