Screening Of An Acid-resistant Rhizosphere Antagonistic Bacterium And Its Biocontrol Effects On Tobacco Bacterial Wilt

Tobacco bacterial wilt caused by R.solanacearum is a devastating soil-borne disease tobacco bacterial wilt in the continuous cropping and acid soil.Biological control can reduce the chemical agent application and has broad application prospects.At present,there is little research on the biological control of tobacco bacterial wilt in acid soil,so it is very necessary to study on acid-resistant biocontrol bacteria application to control tobacco bacterial wilt in acid soil.In this study,an acid-resistant biological bacterium CLP6 against R.solanacearum was screened under acidic condition and identified.The antibacterial mechanism of strain CLP6 was preliminarily studied through analysis of antagonistic substance,rhizosphere colonization and soil remediation ability;Combined the indoor and field experiments to verify the effects of disease prevention and growth promotion.The main research results were as follows:1.A biocontrol bacterium CLP6 with good antagonistic activity against R.solanacearum was screened under pH 5.5 condition was obtained from rhizosphere soil of healthy tobacco planting acid field where tobacco bacterial wilt is severe in Cili County,Zhangjiajie City,Hunan Province.Strain CLP6 had acid resistance,and its growth and antagonistic activity were the strongest under pH 5.5 acid condition.Result of transmission electron microscopy was shown that the extracellular substances secreted by strain CLP6 could cause R.solanacearum cell deformity and inhibit the growth of R.solanacearum;Strain CLP6 also could significantly reduce the R.solanacearum gene copy numbers in rihzosphere soil and stem of tobacco.Combining BIOLOG GEN?and 16S rDNA gene sequence analysis,the acid resistant antagonistic bacterium CLP6 was identified as Pseudomonas protegens.2.P.protegens CLP6 could secrete proteases,2,4-diacetylresorcinol?2,4-DAPG?and volatile organic compound?VOCs?,respectively.The crude extracts of intracellular and extracellular proteins obtained by cell lysis and?NH₄?₂SO₄ sediment had antagonistic activity against R.solanacearum.Strain CLP6 was defected that could secrete 2,4-DAPG only at pH 5.5 acid condition based on the analysis of ethyl acetate extraction,Thin Tayer Chromatography?TLC?and High Performance Liquid Chromatography-mass spectrometry?HPLC-MS?,and the yield was 30?g·L^-1.The 2,4-DAPG antagonistic R.solanacearum and the diameter of the inhibition zone was 9mm.Strain CLP6 could secrete VOCs with a broad antagonistic spectrum and strong antagonistic activity against many plant pathogenic fungi and bacteria.The antagonistic ability to R.solanacearum was positively correlated with the inoculation concentration,inoculation amount and fumigation time.Based on the detection technology of Headspace Solid Phase Microextraction-Gas Chromatography-Mass Spectrometry?HS-SPME-GC-MS?,VOCs of strain CLP6 was contained nine components:S-methyl thioacetate,methyl thiocyanate,dimethyl disulfide,1-decene,2-ethylhexanol,1,4-undecadiene,1-undecene,benzothiazole,2,5-dimethylpyrazine.Among them,the contents of S-methyl thioacetate,1-decene,and 2-ethylhexanol of VOCs at pH 5.5 cultured condition was higher than those at pH 7.0,and S-methyl thioacetate was produced only at pH 5.5.The inhibition rates?IRs?of benzothiazole and 2-ethylhexanol to R.solanacearum were 100%,and the IRs of methyl thiocyanate and dimethyl disulfide were 88.91%and 67.64%;The IRs of S-methyl thioacetate and 2,5-dimethylpyrazine were 53.29%and 43.98%,respectively,and the antagonistic activities of other three components were weak.3.P.protegens CLP6 could colonize in the rhizosphere of tobacco and had the ability to improve soil fertility of continuous cropping.The strain CLP6-GFP was constructed by the green fluorescent protein gene labeling technique.The growth rate was basically the same as that of strain CLP6 and the plasmid was stable.The strain CLP6-GFP had no significant difference in the antagonistic ability of the R.solanacearum,indicating that the next step of the experiment can be carried out;Observed by laser confocal microscopy,strain CLP6 colonized in the epidermal cell gaps of tobacco root tip.P.protegens CLP6 could increase the activities of soil phosphatase,urease and catalase,and their enzyme activities were the strongest at 60 d,60 d and 20 d after application of strain CLP6,respectively.The index of soil nutrient content was increased after application of strain CLP6.Among them,the content of soil available phosphorus,nitrate nitrogen and organic matter reached the highest level at 20 d after application of strain CLP6,the highest content of ammonium nitrogen at 40 d after application of strain CLP6,and available potassium at 60 d after application of strain CLP6.4.The pot and field experiments results showed that strain CLP6 had a good control effect on tobacco bacterial wilt and promoted the tobacco growth.The pot test results indicated that after 216h of inoculation with R.solanacearum,the control effect of strain CLP6 treatment and the commercial Bacillus agent of"Bijian"in acid soil were 78.66%and 74.00%,respectively,the neutral soil control effect was 72.61%and 76.99%.The field disease control test results indicated that the control effect of strain CLP6 on tobacco bacterial wilt was 69.49%at 60 d after application of strain CLP6.Tobacco biomass accumulation both indoors and in field conditions,including tobacco plant height,whole plant fresh weight,root dry weight,effective leaf number and maximum unit leaf area were increased by strain CLP6.The whole plant dry weight and plant height were increased significantly,and the growth rates were 70.15%and 74.24%,respectively.