Isolation And Antimicrobial Activity Of Endophytic Actinomycetes Against Tomato Bacterial Wilt And Study On Their Metabolites

Tomato bacterial wilt is caused by the pathogenic bacteria Ralstonia solanacearum.The pathogen is severely endangered by infecting more than 200 plants all around the world.Because of the long-term survival of tomato wilt pathogens in soil,water,and diseased plants,it can infect other plants at any time once the opportunity arises.So the incidence of tomato bacterial wilt is high,which is difficult to prevent and cure.As main agricultural economic crop,a serious decline production of tomato was caused by suffering from the disaster of bacterial wilt every year.At present,the control of bacterial wilt is mainly based on chemical pesticides.However,the resistance of host plants has been improved with a mass of useful chemical pesticides,while environment was also damaged seriously.Therefore,it is an urgent measure that researching of new-shaped,low-toxic and harmless antibiotics are used to control bacterial wilt in agricultural production.The aim of the study is mainly to screen antagonistic actinomycetes against bacterial wilt and isolate their secondary metabolites.It will provide more antagonistic actinomycetes and new-shaped antibiotic for biological control of tomato bacterial wilt.In this study,endophytic actinomycetes were isolated from roots of three groups healthy and infected wilt bacterial tomato plants by using five different screening media.We took some measures to deal with endophytic actinomycetes obtained such as combination of repeated strains,genus classification of all isolates,statistics of the number of strains and the richness analysis of different strains.Tablets antibacterial activity against pathogenic bacterial R.solanacearum in vitro were launched respectively,which test the inhibition activity of the endophytic actinomycetes isolated from the tomato roots.The fermentation broth of antagonistic actinomycetes with great antibacterial activity were further tested the inhibition activity against bacterial wilt and separated the secondary metabolites.The molecular features and morphological culture characteristics of antagonistic actinomycetes were described.It provided new resources for the biological control tomato bacterial wilt in the future.In addition,this experiment also carried out polyphasic taxonomic identification of two novel actinomycetes isolated from tomato roots,its providing the new resource for rare actinomycetes taxonomy.The results are as follows:(1)We isolated 1465 actinomycetes from the roots of healthy and diseased tomato plants.We obtained 256 actinomycetes by rearranging and combining repetitive strains.85 strains only existed in infected roots;49 isolates only existed in the roots of healthy tomato plants and 122strains were found both diseased plants and healthy in tomato roots.On the whole,the richness of endophytic actinomycetes of healthy samples was higher than diseased.There are more kinds of actinomycetes in diseased tomato plants than those isolated from healthy roots.(2)All isolated actinomycetes were tested for the inhibition activity against bacterial wilt.We obtained 23 strains which can inhibit bacterial wilt pathogens.7 strains isolated from normal roots and the inhibitory diameters ranged from 17.64 to 22.04 mm;13 strains of actinomycetes were isolated from diseased tomato roots and the inhibitory diameters ranged from 18.54 to 28.06 mm;3isolates was found both diseased and healthy roots and the inhibitory diameter ranged from 17.80to 20.12 mm.Two strains NEAU-JGH122(healthy tomato)and NEAU-BGG209(diseased tomato)with large inhibitory effects on the bacterial wilt pathogen,and also effects on the other two pathogenic bacteria.Furthermore,it is detected that strain NEAU-BGG209 also had inhibitory effect on other 8 pathogenic fungi.(3)The strains NEAU-BGG209 and NEAU-JGH122 belong to genus Streptomyces determined by molecular biology and morphological characteristics.(4)The secondary metabolites of strains NEAU-BGG209 and NEAU-JGH122 were isolated.NEAU-BGG209 got three compounds:1)4,5-dimethoxybenzene-1,3-diol 2)9-methylstreptimide 3)methylgrisorixin.NEAU-JGH122 isolated two compounds:5-prenyltryptophol and nocardamines.Methylgrisorixin,5-prenyltryptophol and nocardamines can inhibited tomato bacterial wilt when concentrations were 0.08 mg/mL,10 mg/mL and 2 mg/mL respectively.(5)NEAU-DE8(1)~T and NEAU-HE1(2)characterized by using a polyphasic approach.Similarity of NEAU-DE8(1)~T and NEAU-HE1(2)was up to 99.6%based on 16S rRNA gene sequences analysis.Morphological and chemotaxonomic characteristics showed the two strains are extremely similar.We regarded the two strains as a indentical strain.Two strains were distinguished from the closest relatives by physiological and biochemical characteristics.In addition,DNA–DNA relatedness values between NEAU-DE8(1)~T/NEAU-HE1(2)and their closest strains was less than 70%.NEAU-DE8(1)~T and NEAU-HE1(2)represent a new species of the genus Nonomuraea and designated Nonomuraea lycopersici.