Antigungal Substances Probuced By Bacillus Velezensis HN-Q-8 Inhibiting Rhizoctonia Solani In Potato

Potato black scurf is a soil-borne fungal disease caused by Rhizoctonia solani,which is hard to prevent and control,seriously affecting the potato production.At present,thifluzamide and azoxystrobin are main chemical agents to prevent and control Rhizoctonia solani.But,these chemicals are harmful to natural environment and can destroy the structure of microbial community of soil.Bacillus with the characteristics of strong bacteriostasis and environmental friendly,which has been widely used to control plant disease.In this study,Bacillus velezensis HN-Q-8,which showed strong inhibitory effect on potato black scurf,isolated from soil was used to explore its inhibition spectrum of cell-free fermented supernatant(CFFS)and stability.Also,the biocontrol factors and main antifungal active substances of HN-Q-8 were purified and identified.The main results are as follow:1.The CFFS of Bacillus velezensis HN-Q-8 showed the characteristics of broad antifungal spectrum and strong stability.The CFFS of strain HN-Q-8 exhibited strong inhibitory activity against different kinds of plant fungal pathogens,including Rhizoctonia solani,Botrytis cinereu,Alternaria Alternaria f.sp mali,Fusarium solani,Alternari solani,Phytophthora infestanms and Streptomyces scabies.The inhibition rates ranged from 50%to 90%.The inhibition rate of R.solani?B.cinerea and S.scabies was over 85%,However,the inhibition rate to Fusarium oxysporum was only 24.17%,but not to Erwinia carotene.The inhibition rates of CFFS retained 74%after 35 min exposure to UV,and 92%after10-hour exposure to natural light,respectively.The CFS retained 98%antifungal activity after thermal treatment at 100? for 30 min.In addition,the antifungal activity of CFFS was not affected by trypsin or protease K.However,extreme acid and alkali environments could inhibit the activity of CFFS,and the suitable pH of it ranged from 4.0 to 10.0.2.The biocontrol factors of HN-Q-8 were determined.It has been found that strain HN-Q-8 can secrete proteases,amylases,cellulases,pectinases,siderophore and surfactin.Also,it showed the capacity to fix nitrogen,but no chitinase secretion ability.AntisMASH software was used to predict the secondary metabolites produced by strain HN-Q-8.The genome of strain HN-Q-8 contains the gene clusters of fengycin and surfactin.Five genes which encoding main antifungal active substances were tested in strain HN-Q-8,and 4 of them were found in HN-Q-8 including fenD,ituD,srfAB and yndJ,but no sboA was detected.3.Fengycin was the main antifungal substance produced by HN-Q-8.The lipopeptides produced by HN-Q-8 were purified by acid precipitation method,which showed strong inhibitory effect against Rhizoctonia solani,Botrytis cinerea,Fusarium solani,Alternaria Alternaria f.sp mall,Fusarium oxysporum,and Alternaria solani Sorauer.The width of bacteriostatic zone ranged from 6 mm-17 mm.The scanning electron microscope(SEM)results proved that purified lipopeptides could cause the shrink,bend and even cytoplasm leaking of R.solani hypha.By matrix assisted laser desorption ionization time of flight ionization mass spectrometry,the specific type of lipopeptides identified.The results showed that fengycin and surfactin are the two components in the purified lipopeptides.Among them,fengycin was the main antifungal substance.