The Isolation,Screening Of Endophytic From Ligusticum Jeholense And Condition Optimization Of Available Endophytes

The plant of Ligusticum jeholense Nakai et Kitag is a herb of the genus Umbelliferae,medicated with dried roots and rhizomes,and it is a common Chinese medicine in the provinces of Northeast China.It has the functions of expelling wind,dispersing cold,dehumidifying,and analgesic.It is a commonly used Chinese medicinal material for the treatment of colds,colds,and pain.It mainly contains ferulic acid,ligustilide and myristyl ether.Endophytes of medicinal plants are a kind of microorganisms that live in healthy plant tissues for a long time and establish a harmonious coexistence relationship with the host plant,but will not cause obvious damage to the plants.They can produce antibacterial,antitumor,antiviral and promote Rich secondary metabolites,such as plant growth,have potential application value in the fields of bioengineering pharmaceuticals,industrial product fermentation,and agricultural crop pest control.Pesticide residues in traditional Chinese medicinal materials are currently facing a serious safety problem.Chemical pesticides are mainly used in production to prevent and cure medicinal plant diseases,which leads to excessive pesticide residues in medicinal materials,which affects their quality.High-efficiency,low-toxicity,low-residue biocontrol agents have gradually attracted attention.People have gradually transformed plant disease control from pesticide control to biological control,and antagonism against plant diseases has also been applied to industrial production.Therefore,the research on the use of endophytic bacteria in the development of biological agents has important significance and potential application value.It has been reported that the endophytes isolated from medicinal plants can produce the same or similar chemical components as the host plants.The endophytes present in L.jeholense had important positive effects on plant growth and metabolism.However,the research on endophytic bacteria of L.jeholense was still in its infancy.In this paper,the endophytic bacteria of the genus Apiaceae belonging to L.jeholense were isolated,purified and identified,and the fermentation broth conditions of the endogenous strains ZHAB63 and ZHAF1 with good antagonism and broad spectrum were optimized.It is of great significance to screen out the most suitable growth medium components and fermentation conditions,provided a methodological basis for the in-depth development and utilization of native endophytic resources,and further develop biological agents that can prevent and cure common medicinal plant diseases in Northeast China.The research content of this article was as follows:(1)The different concentrations of sodium hypochlorite and mercuric chloride were used in surface sterilizing with different treatments time,disinfection on the roots and rhizomes of L.jeholense,and to determine the most appropriate disinfection scheme:L.jeholense roots surface is 0.10% mercuric chloride treated 15 min;the stem surface disinfection was 0.10% mercuric chloride treated 12 min.(2)Screening Botrytis cinerea,Fusarium solani,Alternaria panax,Phytophthora cactorum and Cylindrocarpon destructans of ginseng,Botrytis cinerea and Fusarium oxysporum of Saposhnikovia divaricata,Alternaria tenuissima,Sclerotinia asari and Mycocentrospora accerina of Asarum,and Atractylodes arborea have endophytic bacteria that have inhibitory effects.After screening,23 strains of endophytic bacteria and 11 strains of endophytic fungi can significantly inhibit at least one phytopathogenic bacterium.Among them,ZHAB63 and ZHAF1 in the stem have a broad spectrum of 11 phytopathogenic bacteria.The bacteriostasis effect,especially when inhibiting Sclerotinia asari of Asarum had the best antagonism effect,and the bacteriostasis rates were 91.84% and 72.22%,respectively,reaching a significant difference.(3)Isolation of endophytic bacteria in L.jeholense,block method was used,with morphology and molecular biology to identify 102 isolates of endophytes,the results showed that 32 species of 14 genera of endophytes were isolated.Endophytic bacteria include Bacillus,Pseudomonas,Enterobacter,Serratia,Achromobacter,Agrobacterium,Mycobacterium,Luteibacter,Paenibacillus,Sphingobacterium,Pantoea,and total 11 genera.Endophytic fungi were identified in three genera,including Fusarium,Alternaria,and Aspergillus.Among them,Bacillus and Fusarium were the most endophytic,accounting for 15.625% and 18.75% of the total endophytes,respectively.The endophytic fungus ZHAF1 with better antagonism is Fusarium solani,and the endophytic ZHAB63 strain is Bacillus amyloliquefaciens.(4)The optimal medium for liquid fermentation of endogenous fungi ZHAF1 in L.jeholense Province is: glucose 15 g/L,yeast extract 2.5 g/L,magnesium sulfate 5.0g/L;the optimal fermentation conditions are: temperature 27℃,p H 7.0,rotation speed200 r/min,culture 5d.After optimizing the culture conditions,the bacteriostatic rate was significantly higher than before optimization,and the bacteriostatic rate of Sclerotinia asari of Asarum was increased from 91.84% before optimization to95.36%.(5)The optimal medium for liquid fermentation of endophytic bacteria ZHAB63 strain is: sucrose 15.0 g/L,yeast powder 20.0 g/L,peptone 20.0 g/L,magnesium sulfate 5.0 g/L;the optimal fermentation conditions are: 30℃for 48 h,p H 6.5,rotating speed 180r/min.After the optimization of the culture conditions,the inhibition rate of Sclerotinia asari of Asarum was increased from 72.22% before optimization to 84.99%.In this study,102 endophytic bacteria were isolated from the medicinal plant L.jeholense for the first time,from which strains ZHAB63 and ZHAF1,which had broad-spectrum antibacterial activity against 11 common plant pathogens in Northeast China,were screened.ZHAB63 strains were isolated in vitro The ZHAF1 strain and its fermentation broth have a strong inhibitory effect on Sclerotinia asari of Asarum.The two strains were identified as Bacillus amyloliquefaciens and Fusarium solani,respectively.This study provided a theoretical basis for the biological control of fungal diseases of medicinal plants,and provided a scientific basis for the development of potentially reliable new medicinal compounds.