The Antimicrobial Activity Substances And Disease Prevention Mechanism Of The Endophytic Antagonistic Bacterium N6-34 From Poplar

Poplar is an important man-made forest species in industrial plantation in China.The productivity of woodland is decreasing generation by generation and the poplar disease is frequent because of long-term continuous cropping,which seriously affects the stability and sustainable development of poplar production.Poplar is the main greening tree species in China and the economic value produced by poplar is higher every year.It has caused great economic losses bacause of the harm caused by poplar disease.Poplar fungal canker is an important disease that seriously affects poplar production.This disease significantly reduces the economic and ecological benefits of poplar.Chemical control method,which uses all kinds of chemical fungicides to kill or inhibit the pathogen of poplar fungal canker,has been used for many years to control the poplar fungal diseases.But this method can easily cause the resistance of pathogenic fungi,thus reducing the effect on the prevention and control of poplar fungal canker,and also causing environmental pollution.With the rapid development of economic society and the enhancement of people’s awareness of environmental protection,more and more attention has been paid to the biological control of poplar diseases.In this study,an endophytic antagonistic bacterium N6-34 was isolated from poplar bark by using Botryosphaeria dothidea as a target pathogen.The previous study showed that the strain had excellent fermentation characteristics,good antagonism and broad spectrum of antifungal activity,and had strong antagonism to various pathogenic fungi such as Botryosphaeria dothidea,Fusarium oxysporum and Rhizoctonia solani.In this study,the strain N6-34 was identified through the analysis of the 16 S rDNA and gyrB gene sequences,and the antifungal active substance of N6-34 strain fermentation liquid was isolated and purified.The structure of the active substances was identified by amino acid analysis combined with ESI-MS and ESI-MS/MS.The physico-chemical properties of C17 fengycin B were studied.The inhibition spectrum of N6-34 strain toward the common pathogenic fungi was tested.Transmission electron microscopy and scanning electron microscopy were used to study the pathogen prevention mechanism.Finally,the effect of N6-34 strain on the microbial diversity of rhizosphere soil and rhizoplane soil was studied by high throughput sequencing.The main results are as follows:(1)Identification of N6-34 strainThe N6-34 strain was identified as Bacillus subtilis by colony and cell property,physiological and biochemical characteristics,16 S rDNA sequence analysis and gyrB gene sequence analysis.(2)Isolation and purification of active substances from N6-34 strainFourteen components were separated after 70% ammonium sulfate precipitation,methanol extraction,DEAE Sepharose Fast Flow ion exchange column chromatography,G25 molecular sieve chromatography and analytical high performance liquid chromatography.All the components were collected by semi-preparative high performance liquid chromatography and tested the activity and purity.Thirteen antifungal active components with the purity >95% were obtained.(3)Structure identification of active substances from N6-34 strainThe molecular weight of the component 2-14 was obtained as 1463,1491,1491,1505,1447,1475,1475,1477,1505,1491,1477,1489 and 1489 Da respectively by ESI-MS detection.The two proton ion peaks were selected as the precursor ion for ESI-MS/MS detection.The components 2,6,7,8,9 and 12 obtained the marker ion fragments 966 and 1080 of fengycin A,and the components 3,4,5,10,11,13 and 14 obtained the marker ion fragments 994 and 1108 of fengycinB.Component 5 and 6 were used as the representative components(belong to fengycin A and fengycinB respectively)to identify their structure.The analysis of amino acid composition showed that the amino acid composition of component 5 was Glx:Orn:Tyr:Thr:Val:Pro:Ile=3:1:2:1:1:1:1,and the amino acid composition of component 6 was Glx:Orn:Tyr:Thr:Ala:Pro:Ile=3:1:2:1:1:1:1.The series of ions dissociated from component 5 and component 6 by ESI-MS/MS were further analyzed.The complete sequence of the two components was as follows:Component 5: FA-Glu-Orn-Tyr-Thr-Glu-Val-Pro-Gln-Tyr-IleComponent 6: FA-Glu-Orn-Tyr-Thr-Glu-Ala-Pro-Gln-Tyr-IleThe complete sequence of the other components can be deduced in the same way.(4)Physico-chemical properties of active substance C17 fengycinBThe physico-chemical properties of the active component 5(C17 fengycin B)were studied.The results showed that C17 fengycin B was stable at 120℃ and less stable in the environment with pH greater than 6.It was not sensitive to UV at different intensities and protease.Fusarium oxysporum was more sensitive to C17 fengycin B,and the MIC and MFC were 6.25μg/m L and 12.5μg/m L respectively.Botryosphaeria ribis(Tode)was least sensitive to C17 fengycin B,and the MIC and MFC were 25μg/m L and greater than 50μg/m L respectively.(5)Medium optimization of antifungal substances fermentation broth by response surface methodologySingle factor test showed that the best available carbon source,slow-release carbon source,organic nitrogen source,inorganic nitrogen source and inorganic salt in N6-34 strain fermentation medium were glucose,corn flour,soybean powder,ammonium sulfate and potassium dihydrogen phosphate respectively.Through the PB design,the steepest ascent design and the BBD design,the final optimized medium formula was as follows: glucose 13.0g/L,corn flour 25g/L,soybean powder 5.0g/L,potassium dihydrogen phosphate 0.7g/L,and ammonium sulfate 10.0g/L.The titer of the optimized medium increased by 250% compared with that of the basal medium.(6)The study on the antifungal mechanism of N6-34 strainN6-34 strain has different inhibitory effects on the common plant pathogenic fungi preserved in our laboratory.They have stronger inhibitory effect on the pathogen of Botryosphaeria dothidea,Alternaria alternata,Fusarium oxysporum and Exserohilum rostratum,while the antifungal effect on Botryosphaeria ribis(Tode),Streptosporangium sp.is weak.The inhibitory effect of N6-34 strain on the growth of Fusarium oxysporum is observed under ordinary optical microscope.The results showed that the hyphae of the control fungus were stronger and the hyphae interval was obvious.There were many mature spores among the hyphae.The inhibited hyphae had a deep color in the growth process and the hyphae had irregular bifurcation.The hyphae was fine,the hyphae interval was irregular,and the immature spores were observed occasionally.The inhibition effect of the N6-34 strain antifungal substances on the spore germination of Fusarium oxysporum was tested.The results showed that the germination rate of spores declined sharply with the increase of treatment concentration.The germination rate and inhibition rate of spore were 14%,11% and 83.69% and 88.69% respectively when treated with the antifungal substances of 10mg/L and 20mg/L,and the difference of spore germination rates was not significant,but the rate of spore inhibition was significantly different.The activities of amylase,cellulase,protease,lipase,chitinase and β-1,3-glucanase in N6-34 fermentation broth were detected.(7)Colonization of N6-34 strainThe colonization of N6-34 strain with GFP marker in poplar rhizosphere soil,root,stem and leaf was studied.The results showed that the colonization concentration of N6-34-GFP strain in poplar rhizosphere soil,root,stem and leaf increased first and then declined after a period of stability.Colonization position of N6-34 strain in roots,stems and leaves was observed by laser confocal microscopy.The labelled strains were colonized mainly in the meristem region and elongated region in the roots.The colonization sites in the stems were mainly in the vascular bundles.The labelled strains were colonized in the transverse and longitudinal sections in the leaves.The inhibitory effect of N6-34 strain on Fusarium oxysporum was confirmed by fluorescence quantitative PCR.(8)The influence of N6-34 strain on fungal community composition of poplar rhizosphere and rhizoplane soilThrough high throughput sequencing analysis of the poplar rhizosphere and rhizoplane soil,the results showed that the diversity and richness of the poplar rhizosphere and rhizoplane soil after N6-34 strain treatment had been significantly changed,and the control and treatment of rhizosphere soil showed significant difference in the fungal community structure,while the control and treatment of the rhizoplane soil were not significant in the fungal community structure.In addition,there was a significant difference in microbial diversity between the control and treatment of rhizosphere soil and rhizoplane soil.