Studies On The Antibacterial Activity And Mechanism Of DIMBOA And Its Derivatives Against Ralstonia Solanacearum

Tobacco bacterial wilt is the most serious soil-borne disease caused by the Ralstonia solanacearum,which could be suppressed by rotating with Poaceae cro ps during the tobacco production.Hydroxamic acids,firstly found in Poaceae cr ops like Secale cereale L.,Triticum aestivum L.and Zea mays L,exhibit phytot oxic activities,play a significant role in plant defense against fungi,bacteria,and insects,and participate in allelopathy mechanisms.2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one,commonly known as DIMBOA,is one of the principal cyclic hydroxamic acids in aqueous extracts of maize.The present study aims to isolate the cyclic hydroxamic acid 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one(DIMBOA)from maize tissues(Zea mays L.),and evaluate the antibact erial activity of DIMBOA and its derivatives against R.solanacearum.We also r esearched about the effect of corn cropping and DIMBOA on the the soil micro bial community.The results as following:1.The preliminary result of corn rotation and intercropping tobacco showed that corn rotation can partly reduce the disease index of tobacco bacterial wilt.The disease incidence of corn rotation and tobacco monocropping on tobacco bacterial wilt were66.67%,88.89% respectively at 15 days.The index of corn rotation and intercropping were 2.67,3.06 respectively,which were significantly lower than the tobacco monocropping treatment.2.DIMBOA was extracted from etiolated maize seedlings(Zea Mays L.).Th en we evaluated the antibacterial activity of the isolated DIMBOA and its deriva tives 2-benzoxazolinone(BOA),6-chloro-2-benzoxazolinone(CDHB),and 2-merca ptobenzothiazole(MBT)against R.solanacearum.MBT showed the strongest anti bacterial activity,followed by CDHB and DIMBOA,with minimum inhibitory co ncentrations(MICs)of 50,100 and 200 mg/L,respectively,better than the BOA with 300 mg/L.The minimum bactericidal concentrations(MBCs)of MBT,CD HB and DIMBOA against R.solanacearum were 400,500,and 500 mg/L respec tively.The MBCs of BOA which exceeded the test concentration of 1000 mg/L.These compounds also significantly affected bacterial growth,reduced biofilm fo rmation,and inhibited swarming motility within 24 h.The antibacterial activity o f DIMBOA and its derivatives against R.solanacearum increased with dosage.T he results also showed that hydrosulphonyl and chlorine substituents enhanced th e bactericidal activity against R.solanacearum.Hence,DIMBOA and its derivati ves are antibiotics eliciting modest toxicity to R.solanacearum.3.Detected by qRT-PCR,the results showed that DIMBOA and its derivatives could significantly inhibit expression of the motility related gene FilA 、 MotB at concentrations 50 mg/L.MBT showed the best inhibited effects.They may induced the expression of the flagellum gene FlgM、FlhC、FlhD,but BOA and MBT can not affect the expression of FlhC.DIMBOA could induce the expression of the chemotaxis gene cheY approximately 3 to 7 times and CDHB showed 7-fold improvement.DIMBOA could significantly induce the expression of cheA,cheY gene several-fold higher than housekeeping gene,approximately 3.3,5.5 times,respectively.4.DIMBOA and its derivatives have no toxicity to tobacco seedlings at 7 days,14 days after root irrigation.Unexpectedly,BOA and CDHB enhanced the fresh weight and root length.Biocontrol bacteria were tolerant to DIMBOA.In vitro growth of Bacillus subtilis is not affected by up to 100 mg/L DIMBOA within 24 h,whereas Paenibacillus polymyxa is increasingly affected at concentrations of 10 mg/L DIMBOA and greater.The result of greenhouse experiments showed that DIMBOA and its derivatives(10 mg/L)could decrease the disease index of tobacco bacterial wilt significantly.The controlled efficiency of DIMBOA and MBT on tobacco bacterial wilt was 27.24% and 9.55% at 15 days,respectively.5.DIMBOA and corn cropping could significantly increased soil microbial metabolic activity,diversity,evenness indices and numbers,especially the bacteria.The treatment of corn continuous cropping could increase the available K partly.In conclusion,the treatment of DIMBOA and corn cropping improved the metabolic activity and functional diversity of the soil microbial community and then changed the rhizosphere.The change of rhizosphere microorganisms against Ralstonia solanacearum and then decrease the disease index of tobacco bacterial wilt.