Screening And Analysis Of Antagonist Peptides From Bacillus Species To Inhibit Bacterial Wilt Disease

Bacterial wilt is a dangerous plant disease caused by Ralstonia solanacearum,responsible for extensive crop losses worldwide.Control of this disease depends largely on the use of chemical-based pesticides that cause serious threats to the environment and human health.Control of bacterial wilt by antagonist peptides is considered as a good alternative to chemicals.Bacillus antagonist peptides are given a particular interest due to their wide spectrum activity and the endospore-forming and growth promoting abilities of Bacillus species.This present study aimed to isolate antagonist peptides from Bacillus species with potential biocontrol activity against bacterial wilt.To fulfill this purpose,15 soil samples from Yuxi,Fujian,China,were screened for Bacillus species that produce antagonist peptides inhibitors of Ralstonia solanacearum.Isolate with the highest antimicrobial activity was characterized and submitted to further experiments,including characterization of the antagonist peptides and maximization of culture conditions and medium components for improvement of strain growth and antagonist peptides production.Furthermore,preliminary mode of action of the compounds was tested and the ability of the isolate to control bacterial wilt in the greenhouse was investigated.1.Screening for antagonist peptides producers.Screening of antagonist Bacillus species was done by drop test and Agarwell diffusion assay.Fusarium oxysporum was used as fungus control pathogen to evaluate the antagonist peptide spectrum of activity.In total,15 isolates exhibited antagonist activity against Ralstonia solanacearum and Fusarium oxysporum.Strain FJAT-42192 with the highest antimicrobial activity(at least 20 mm)was identified as Bacillus methylotrophicus based on 16S rRNA gene sequencing,morphological,biochemical and physiological characteristics.2.Isolation and identification of the antagonist peptidesFJAT-42192 crude peptides methanol extract was tested for antagonist activity against R.solanacearum by Agar-well diffusion assay and F.oxysporum by dual culture.Results showed that FJAT-42192 has antibacterial and antifungal activity.PCR amplification of strain FJAT-42192 DNA revealed the presence of surfcatin,iturin and fengycin biosynthesis gene cluster.LC-MS/MS analysis of FJAT-42192 methanol extract confirmed the coproduction of surfactin,iturin and fengycin peptides homologs.They consist of C12,C13,C14,C15 surfactin homologs represented by[M+Na]+at m/z 1016-1030-1044 and 1058;C14,C15,C16 iturin homologs represented by[M+Na]+at m/z 1065,1079 and 1093 and two fengycin isoforms C17 A and C17.B.Fengycin structure were confirmed by the protonated molecules[M+H]+at m/z 1477.9 and 1506.These two fengycin isoforms had each a C16 homolog[M+H]+at m/z 1492.9 and 1463.9.3.Antimicrobial peptides characterization.Temperature and pH stability test on the lipopeptides methanol extract declare that FJAT-42192 compounds are active at 60℃ for 30min,1h,and 2h with slight decrease compare the untreated compounds(methanol extract at room temperature).It was also active at 80℃,100℃ for 30 min,1h and 2hours with significant decreases after lhour.The antagonist compounds were also active at pH values ranging from pH=2.2 to pH=8.To identity the mode of action of FJAT-42192 lipopeptides,R.solanacearum cells treated with the crude extract were further analyzed by Transmission electron microscopy(TEM)and compared to the untreated cell.Results revealed that the lipopeptides act by damaging Ralstonia cells structure.4.Maximization of culture conditions and medium constituents for the growth and antimicrobial activity of strain FJAT-42192Influence of growth conditions was tested by culturing strain FJAT-42192 in different pH,temperature for different incubation time.Results showed that the optimum growth and antimicrobial activity were displayed when the strain was cultured at 25℃ or 30℃;pH 6.0 for 24h.Culture medium was found to influence both growth and antimicrobial activity.Results showed that optimum growth was obtained when Tryptic soy broth was used whereas,optimum antimicrobial activity was registered when Casamino-acid glucose peptone(CPG)was used.Results on the variation of medium constituents showed that optimum growth was obtained when tryptone(or peptone(20g/l)),starch(20g/l),NaCl 3%(30g/l)were present in the medium.Whereas,the highest antimicrobial activity was generated when tryptone(20g/l),Fructose(20g/l),and NaCl(10g)were used.5.Biocontrol activity of FJAT-42192 against Ralstonia solanacearumBiocontrol efficacy of FJAT-42192 in pot experiment was realized.Obtained results reported that FJAT-42192 reduced bacterial wilt disease incidence by 50%and considerably slowed down the disease progression.Henceforth this study reports the potential biocontrol activity of B.methylotrophicus FJAT-42192 antagonist peptides in vitro and in vivo.This study has a great significance,in modern agricultural sustainability and in the agro-economy.