Identification Of Strain S17-377 And Studying Its Mechanism Against Rice Sheath Blight

This study focused on screening biocontrol bacteria from rhizosphere soil of healthy rice plants against rice sheath blight,as well as its taxonomic status,optimization of fermentation conditions and its antimicrobial mechanism.The main experimental results are as follows:A total of 390 bacterial isolates were isolated from 45 soil samples of rice rhizosphere collected from 9 regions of China.Twenty-eight strains with inhibition activity against Rhizoctonia solani were picked out by dual culture method.Bacterium with the strongest antifungal effect was strain S17-377 with inhibition zone diameter of 10 mm.Based on the morphological and molecular biological characteristics,S17-377 was identified as Burkholderia pyrrocinia.The growth inhibition rates of S17-377 supernatant against rice sheath blight pathogen were 77.14%,86.92%and 98.26%when the ratio of supernatant and PDA medium was 1:20,1:10 and 1:5 respectively.It can also inhibit the formation of sclerotia.The supernatant could cause some hyphae of R.solani to swell or collapse.The antagonistic activity of S17-377 against 14 kinds of other pathogenic fungi such as Botrytis cinerea,Magnaporthe grisea and Verticillium dahliae Kleb was also determined.The results showed that the inhibition rate of S17-377 against these pathogenic fungi was between 22.50%and 90.90%.The effects of S17-377 on citrus canker pathogen?Xanthomonas axonopodis pv.citri?,konjac soft rot pathogen?Ervinia carotovora?,cucumber angular spot pathogen?Pseudomonas syringae pv.lachrymans?and ginger bacterial wilt pathogen?Ralstonia solanacearum?were determined.The results demonstrated that the inhibition zone of S17-377 against the pathogenic bacteria was from 9.75 mm to 13.14 mm.The results of in vitro leaves experiment indicated that the rice leaves had no damage after treatment with strain S17-377 only,and the area of disease in the treatment group was reduced by 84%compared with the control.The results of pot experiment proved that the control effect of the strain S17-377 on rice sheath blight reach 65.93%after treating the rice plants with it for three times.The strain S17-377 showed the strongest antagonistic activity when it was cultivated in the basal salt medium with the pH of 5.0 and the culture time of 120 h or 144 h.The strain S17-377 fermentation supernatant had a strong induction effect on rice disease resistance.After the rice plants were treated with the fermentation supernatant,the rice PAL and POD activities showed an upward trend,while the PPO activities increased at the initial stage and then decreased.In addition,the strain S17-377 could activate the expression of rice defense genes?NH1?PR1a?PR10?PAL/ZB8?LOX?AOS₂?.The strain S17-377 had the ability to produce protease,chitinase and siderophores,but could not produce cellulase.Strain S17-377 could form biofilm,and the formation strength was related to the concentration of bacteria.The biofilm strength at the concentration of OD₆₀₀=0.6 was significantly greater than that at the concentration of OD₆₀₀=0.8 or 1.0.The labled strain S17-377-GFP was obtained by transformation the plasmid carring with the gfp gene into the strain S17-377.The strain S17-377GFP and the original strain S17-377 had the same morphology,no obvious growth difference,and the genetic stability was over 75%.They showed similar antagonistic ability against R.solani.Fluorescence microscope observation revealed that the strain S17-377GFP could colonize in the root tissues,leaf epidermis,mesophyll cells,and vascular bundles of rice.The antimicrobial bioactive substance from strain S17-377 was not sensitive to temperature.The active components were successively extracted by the methods of acid precipitation,ammonium sulfate precipitation and organic solvent extraction.It was found that the inhibition rates of the n-butanol extract,crude protein extract and crude lipopeptide extract with concentration of 50?g/mL were 95.23%,94.43%,and75.11%,respectively.Among them,the EC₅₀ of the n-butanol extract against R.solani was 8.95?g/mL.In addition,the n-butanol extract was analyzed by GC-MS and found to contain 38 compounds,of which there were seven compounds[Pyridine,3-?1-methyl-2-pyrrolidiny??s?-],[Nonadecane,9-methyl-],[Pyrrolo[1,2-a]P yrazine-1,4-doine,hexahydro-],Heptadecane,Dibutyl phthalate,n-hexadecanoic acid,[Bis?2-ethylhexyl?phthalate]with a matching degree of over 90%.