Fermentation Culture Of Endophytic Bacterium ZJ1 From Buddleja Lindleyana Fort And The Study Of Antifungal Activity

An endophytic bacteria named ZJ1 was isolated from the stem of Buddleja lindleyana Fort in this paper.A variety of tested plant pathogens were used as indicator bacteria to test the antibacterial activity and we found that the endogenous bacterium had a inhibitory effect on all of them in various degree.Systematic research was conducted on the strain’s bacteriostatic properties,taxonomic status,fermentation conditions,metabolite stability,antibacterial substance types,and growth-promoting characteristics,which is beneficial to provide new strain information for the development and utilization of green pesticides.Here are the main contents:1.The four-point agar disk diffusion method and growth rate method were used to determine the antibacterial effect of ZJ1 strain and its fermentation broth on 10 tested pathogenic fungi.The results showed that the endogenous bacterium,ZJ1,isolated from the stem of Buddleja lindleyana Fort had a inhibitory effect on all of them in various degree.Among them,the endogenous bacterium had a very good inhibitory effect on M.fructicola,B.cinerea,L.libanotis and M.Laxa.2.Based on morphological observation,physiological-biochemical Identification,Molecular biological identification and sequence alignment of 16 S r RNA,the ZJ1 strain was100% homologous to Bacillus sp.B-1-30 and Bacillus sp.A-3-7B,which were closest.Therefore,the strain ZJ1 can be initially identified as Bacillus subtilis.3.The turbidimet method was used to determine the growth curve of endophytic bacteria ZJ1.As a result,it was found that the growth amount of the strain ZJ1 showed a trend of rapid increase and then decline.Then,the fermentation conditions were optimized with a single factor.After screening,YSP basic medium was the best basic culture solution,lactose was the best C source for strain ZJ1,beef extract with yeast powder and peptone was the best N source,and 28³0℃ was the best fermentation temperature.Combined with the variance analysis of and cost though orthogonal test,the optimal fermentation conditions of ZJ1 strain were selected as p H-7.0,liquid volume-100/500 mL,inoculum volume-1%,speed-190 r·min^-1and fermentation time-72 h.4.The stability of ZJ1 strain metabolites was determined.As a result,it was found that the antibacterial substance of strain ZJ1 had good thermal stability,acid-base stability,insensitive to ultraviolet light and natural light irradiation,which could also be stored for a long time.5.The oil drainage and droplet collapse characteristics of endophytic bacteria ZJ1 metabolites were measured.The results showed that ZJ1 had the ability to drain oil and droplet collapse.It was speculated that the metabolites of the endogenous bacteria ZJ1 contained lipopeptides.6.Microscopic observation and conductivity method were taken to determine the growth status of B.cinerea hyphae and cell membrane permeability.The results showed that after treatment with strain ZJ1 metabolites,B.cinerea mycelia seldom spawned,grew abnormally and even broke.As time passed,the permeability of B.cinerea cell membrane increased,osmotic substances flowed out,and the conductivity increased.7.The seed germination test and root-irrigation method were used to determine the growth-promoting effect of ZJ1 strain metabolites on tomato seedlings.It was found that when the fermentation broth of ZJ1 strain was diluted 100 X,the germination rate of tomato seeds could be improved.When the fermentation broth was diluted 50 X,it had the best effect on plant growth and protective enzyme activity induction.We also found that after the tomato fruit was isolated,ZJ1 strain fermentation broth also had certain preventive effects on A.solani.