Identification Of Active Compounds Suppressing Banana Fusarium Wilt Of Symbiotic Actinomycetes Isolated From Soft Corals Of The South China Sea And Study Of Their Antifungial Mechanism

Banana（Musa spp.）is an important commercial and food crop in the tropics and subtropics.Fusarium wilt of banana（also known as Panama disease）caused by Fusarium oxysporum f.sp.cubense（Foc）is one of the most disastrous diseases,especially,tropical race 4（Foc TR4）.In recent years,the biocontrol is considered as a promising strategy.Coral is importantly composed of marine coral reef ecosystem in the rainforests of oceans,harboring abundantly a various of microbial communities.It has been reported that coral’s microbial communities are partially species-specific.Marine Actinobacteria,particularly coral-associated Actinobacteria,have drawn attention.In this study,five kinds of soft corals from Xisha islands in the South sea of China were selected as research objects to explore the diversity of symbiotic and epiphytic microorganisms.The actinomycetes with antifungal activity were isolated and screened by a special method.The compounds with antifungal activity were isolated and identified from the fermentation broth of actinomycetes.The broad-spectrum antifungal activity and antibacterial mechanism of compounds were analyzed.In addition,the pot culture was used to verify the prevention and control efficiency and evaluate its effect on the soil micro ecological environment in order to obtain a novel unique,highly active and safe biological control agent.1.Symbiotic and epiphytic actinomycetes from five soft corals（Lobophytum sp.,Sinlaria sp.,Iciligorgia sp.,Rumphella aggregata,Menella woodin）were isolated and identified from the Xisha Islands in the Sounth sea of China.A total of 132 morphologically different actinomycetes were isolated from five soft corals,belonging to Streptomyces,Ochrobactrum,Nocardiopsis,Pseudonocardia,Marinobacter,Glycomyces,Blastococcus,Saccharomonospora,Kitasatospora,Microbispora and Stenotrophomonas.The dominant genera were Streptomyces and saccharomonospora,accounting for 71.97%and 10.61%,respectively.Symbiotic and epiphytic actinomycetes from five soft corals were significantly different.This study will help us to understand the community structure of microorganisms in soft coral and provide a number of culturable actinomycete resources for the screening secondary metabolites.2.A total of 132 isolates were screened for their antifungal activity using a conventional spot inoculation method and a TLC bioautography.Out of all of them,49 isolates had antifungal activity during a preliminary experiment,especially secondary metabolites of strain 2-6,2-11 and H-7 exhibiting stronger antifungal activity against the tested phytopathogenic fungi.According to the analysis of 16S r RNA sequence,morphological,culture profiles as well as physiological and biochemical characteristics,strain 2-6,2-11 and H-7 were identified as Streptomyces sp.Besides,strain 2-11 exhibited a low similarity with Streptomyces rapamycinicus NRRL B-5491（T）（EF408733）,in accordance with the principle of polyphasic taxonomy,a systematic identification had been conducted using genome data.Strain 2-11 represented a novel species of genus Streptomyces,named as Streptomyces xishaensis 2-11 sp.nov.To obtain high active secondary metabolites of Streptomyces sp.2-6,2-11 and H-7,M1 medium was selected from five fermentation media.The fermentation conditions were optimized through the response surface methodology.After optimization,the antifungal activity of strain 2-6,2-11 and H-7 were 20.35 mm,22.56mm and 24.73 mm,respectively,and 19.35%,21.56%and 23.73%higher than that before optimization.3.Three compounds with high antifungal activity were isolated from the fermentation broth of three Streptomyces by a positive and reverse silica gel column chromatography,a Sephadex LH-20 gel chromatography and a high-performance liquid chromatography.The structures of compounds were identified by NMR,HR-MS,IR and other modern structural identification techniques.Three compounds were identified as terrein（A7）,niphimycin C（M1）and tetramycin a（H1）.Their antifungal activity against Foc TR4 were 33.67 mm,30.33mm and 26.33 mm,respectively.The compound A7 was isolated and reported firstly from actinomycetes.Similarly,the antifungal activity of the compound M1 was also reported firstly.Although the compound H1 has been reported for many plant pathogenic fungi,but Banana Fusarium Wilt and other soil borne diseases were reported firstly.4.Foc TR4 was used as the target fungi to study the antifungal mechanism of A7,M1and H1.These results showed that three compounds could significantly inhibit the growth of mycelia of Foc TR4 with EC₅₀ of 20.8μg/m L,20.8μg/m L and 20.8μg/m L,respectively.Scanning electron microscopy（SEM）of the mycelia and spores of Foc TR4 treated with three compounds revealed morphological alteration,including deformation,shrinkage,collapse,tortuosity,and broken,leading to the prominent shape loss and integrity of spores.Morphology changes of Foc TR4 were detected by transmission electron microscope（TEM）.In the control group,the cell wall and membrane were intact and well defined.The organelles such as mitochondria and endoplasmic reticulum were structurally well defined.On the contrary,cells treated with three compounds showed that some organelles in the cytoplasm were disintegrated,cell membrane was dissolved and the mitochondria abnormally increased.Mitochondria from cells exhibited a collapsed surface,anomalous shape,unordered structure and rough surfaces.Three compounds induced activation of the target fungal chitinase,interfering with the synthesis of cell wall.These compounds could also inhibit the biosynthesis and induce oxidative stress and apoptosis of Foc TR4.After treatment,the content of total sugar,total protein and fat in Foc TR4 gradually decreased gradually with the increase of the treatment concentration.Three compounds decreased enzyme activities related to the electron transport chain（ETC）and the tricarboxylic acid（TCA）cycle.Accumulation of active oxygen was promoted,the oxidative stress was caused,the structure and function of mitochondria were damaged,the cell metabolism was disordered,and then cell necrosis of Foc TR4 was caused.5.Because the antifungal activities of compounds A7 and M1 were reported firstly,the control effect of strain 2-6 and 2-11 on Foc TR4 were further verified by pot experiments.The results showed that strains 2-6 and 2-11 inhibited the entry of Foc TR4 into banana roots and vascular bundles,in addition reduced the number of Foc TR4 in soil.The growth and reproduction of Foc TR4 pathogens were effectively controlled.The results of high-throughput sequencing showed that the relative abundances of Bacillus,Lactobacillus,Podospora,Plectosperella,Subulicystidium and Ophiocordyceps increased significantly.However,the relative abundance of Fusarium decreased continuously by 49.03%and 46.66%（P<0.05）in the later stage of treatment,respectively.Furthermore,Streptomyces sp.2-6 and2-11 had a growth-promoting effects on banana plants.Compared with treatment of without Streptomyces sp.2-6 and 2-11,the chlorophyll content showed 27.10%and 51.07%improvement,the plant height showed 30.46%and 12.48%improvement,the pseudostem girth showed 14.52%and 15.21%improvement,leaf area showed 117.32%and 111.42%improvement,and blade thickness showed 13.73%and 17.70%improvement,respectively.The fresh weight showed 38.42%and 31.07%improvement,and the dry weight showed41.61%and 39.05%improvement,respectively.Thus,Streptomyces sp.2-6 and 2-11 are important microbial resources as a biological control agent against plant pathogenic fungi and for promoting banana growth.