Study On The Biocontrol Role Of Bacillusamyloliquefaciens Strain EDR4 Against Grapevine Downy Mildew

Grapevine(Vitisvinifera),one of the world's major fruit crops,is affected by many diseases.Downy mildew,caused by the oomycete Plasmoparaviticola,especially leads to massive damage and yield losses in this crop if no protective measures are taken.At present,production mainly depends on chemical pesticides to control downy mildew that include Quinone outside Inhibitor fungicides(QoIs),Phenylamides,Carboxylic acid amides(CAAs),among other compounds,although some of them are risky because of the resistance or potential resistance of P.viticola.Therefore,many researchers are currently engaged in identifying efficient biocontrol agents to replace chemical fungicides for limiting downy mildew infection in susceptible cultivars.Plant endophytic Bacillus,as a biocontrol agent for plant disease,is widely used in agricultural production,which have excellent colonization ability on plant rhizosphere,body surface or in vivo tissue to compete for nutrition and space with pathogens,secrete antimicrobial substances to inhibit pathogen growth and induce plant immune system against pathogens invasion.In this study,the plant endophytic Bacillus strain EDR4,which was selected as the biocontrol agent for plant fungi disease,was studied for the inhibitory effect to P.viticola,the biocontrol effect to grape downy mildew and disease biocontrol mechanism.The detailed results listed below.1.B.amyloliquefaciens EDR4 had an excellent inhibitory effect againstP.viticola: Inoculation test of grape in vitro leaves and leaf disks showed that bacterium suspension of B.amyloliquefaciens EDR4 strain(1×108 cells/mL)treated leaf disks,respectively,before and after inoculation of P.viticola,the sporulation index of P.viticola was 1.33 and 1.41,but the treatment with EDR4 germfree filtrate had no sporulation.The control effect of B.amyloliquefaciens EDR4 was about 70% in the field environment for grape downy mildew,and the EDR4 application for three times had a best effect.For 2015 and 2016,the control effect of EDR4 was 76.61% and 70.40%,which was comparable to 80% dimethomorph 1500 fold dilution.2.Aniline blue staining and microscopic observation indicated that infection densities which B.amyloliquefaciens EDR4 bacterium suspension and germfree filtrate treated grape leaves and inoculated P.viticola were 1.3820×104/cm2 and 1.009×104/cm2,respectively,which were significantly smaller than that of the control.After 5 days,the hyphal expansion degree of P.viticola was significantly smaller than that of the control.After 7 days,P.viticola did not form any mature sporulation structure,and the water control had a large number of mature sporangia.3.B.amyloliquefaciens EDR4 enhanced grape resistance against P.viticolathroughISR.After treatment with B.amyloliquefaciens EDR4 strain(1×108 cells/mL),the activity of enzymes related to resistance in grape leaves taken changed.POD,SOD and PPO activities reached the peak at 72 h after inoculation of P.viticola,which were 4.71,1.98 and 2.74 times for control;CAT and PAL activities reached the peak at 96 and 48 h,which were 4.33 and 6.76 times of control,respectively.For treatment of EDR4 germfree filtrate,POD,CAT and PAL activities reached the peak at 72 h after inoculation of P.viticola,which were 6.79,10.95 and 2.32 times of control,respectively;SOD and PPO activities reached the peak at 96 h and 48 h,which were 1.58 and 19.68 times of control,respectively.SA and JA/ET signal pathway-related gene quantitative analysis found that EDR4 bacterium suspension and germfree filtrate could induce SA and JA/ET pathway-related genes up-regulated.After EDR4 treated and inoculated with P.viticola,PR1,PR2,NPR1,NRX-1,LOX gene were up-regulated to reach maximum at 6h or 12 h post inoculation.The bacterium of EDR4 could not induce the accumulation of H2O2 in grape leaves,while the accumulation of H2O2 was observed in advance to 6h after inoculation of P.viticolawhen EDR4 bacterium suspension(1×108 cells/mL)treated.For treatment with EDR4 germfree filtrate,the accumulation of H2O2 was observed at 12 h and could also be observed in advance to 6h after inoculation of P.viticola.4.A crude extract of B.amyloliquefaciens EDR4 lipopeptide was obtained with a concentration of 75% methanol crude extract 2.736 g/L and 95% methanol 0.7494 g/L.The antibacterial activity test found that 75% methanol crude extract had excellent inhibitory effect against plant pathogenic fungi such as V.mali,C.gloeosporioides,B.cinerea et al.,and also had excellent inhibitory effect against plant pathogenic oomycetes P.infestans and P.viticola.From the above,plant endophytic bacteria B.amyloliquefaciens EDR4 can effectively inhibit the invasion and expansion of P.viticola in grape leaves,and control the occurrence of grape downy mildew.The biocontrol effect of EDR4 against downy mildew mainly through ISR to achieve.