| Under the mode of monoculture year after year,the Fusarium oxysporum f.sp.momordicae,the cause of Fusarium wilt of bitter gourd,gradually accumulates in the soil and survives through the winter in various ways,has caused occurrence of this devastating polyetic disease.The use of biological control means to control the disease have less destructive to the soil and more in line with the requirements of sustainable agricultural development,compared with chemical control means.The rhizosphere of plants contains a wide variety of microbial resources,among which the number of actinomycetes are only less than the soil bacteria,and the rhizosphere of healthy plants is a treasure bank for the accumulation of beneficial microorganisms.In this study,actinomycetes were isolated from the rhizosphere soil of healthy bitter gourd plants in the area with high incidence of Fusarium wilt.Firstly,the antimicrobial activity of the isolates was screened.Secondly,the inhibition potential of antagonistic actinomycetes,the fungicidal effect of its fermentation filtrate on fungal pathogen and the molecular mechanism of antifungi were investigated.Then the candidate antagonistic strains were identified by multiphase classification to determine their scentific name.Finally,to verify its growth-promoting and disease-preventing effects,the candidate antagonistic strains was applied to experiments of bitter gourd in the potting and in the field.Through measuring the ability of actinomycetes were inoculated to induce the disease-related defense enzymes of bitter gourd plants and the changes of physiological indexes related to plant growth and disease prevention in the potting experiment to analysis the mechanism of these strain in preventing and curing wilt.The main results of the study are as follows:110 actinomycete strains were isolated from the tested soil,with the pathogen of bitter gourd wilt was targeted strain,through the inhibition of these strains on a growth and conidial germination of Fusarium wilt pathogen were tested in vitro.The results showed that inhibition percentage of mycelia growth of Fusarium wilt of bitter gourd by strains 0213 and0250 were 62.41% and 69.10%,respectively,and the inhibitory effects of aseptic fermentation filtrate on the growth of mycelia of pathogen were 76.40% and 78.27%,respectively,while the inhibitory percentage on the spores germination were above 95%.Two strains were identified as Streptomyces rhizosphaericus by observing their morphological characteristics cultured on different media,analyzing their physiological and biochemical activities,with conducting average nucleotide identity(ANI)and building an evolutionary tree based on single copy genes.Both strains had the best growth on oat agar medium.When they were cultured on Gause’s synthetic agar,they did not produce pigment,the aerial mycelia were white and dense,spores were spherosome,and the basal mycelia were vertical branches.Both strains had good melanin production and enzyme production capacity,with only a small difference in strength between each other.At present,according to the literatures,the Streptomyces rhizosphaericus has not been used to control plant diseases at home and abroad.Inoculation of two strains of actinomycetes can significantly increase the plant height,fresh weight and dry weight of bitter gourd plants in potting,so as to promote development of roots and increase the biomass of the plants,the control effects on Fusarium wilt were52.99% and 66.97% respectively.It was found that 51.50% and 62.11% of Fusarium oxysporum were inhibited in the potting soil.Two strains of actinomycetes were applied to the soil without F.oxysporum in the field,and the yield of bitter gourd could be increased by10.86% and 21.84%,respectively.Similarly,two strains of actinomycetes reduced the incidence of Fusarium wilt of bitter gourd by 58.26% and 27.56%,respectively in the field.Under the challenge of pathogen,in the leaves of bitter gourd seedlings inoculated with two strains of actinomycetes,the activities of PAL,POD and CAD which play key roles in lignin biosynthesis,as well as the activities of pathogenesis-related proteins(β-1,3-GA)were improved.After inoculation with strain 0250,the enzyme activity of the above mentioned enzymes reached the maximum difference with the control on the 9th day,the 6th day,the 3th day and the 9th day,respectively,with the increase of 42.05%,82.22%,73.47% and 106.25%,respectively.All kinds of enzyme activity of plant treated by inoculation of strain 0213 was slightly lower than that treated by inoculated strain 0250,but it still showed a significant increase compared with the control.The results showed that the actinomycetes suspension could induce resistance to Fusarium wilt of bitter gourd seedlings,and the plant resistance might be related to the increase of related defense enzyme activity.Chlorophyll content and root activity are physiological indexes of healthy plant growth.Compared with the treatment of pathogen control,the change of chlorophyll content in plant treated with two strains of actinomycetes was the largest on the third day.With the extension of sampling time,the change of chlorophyll content of plant in each treatment was stable and the difference became smaller and smaller.The root activity of bitter gourd plants treated by actinomycetes was 1.70 and 1.87 times of that treated by pathogens.Strains 0213 and 0250 produced 18.90 and 26.70 μg/m L auxin,respectively,and their auxin production increased significantly after exogenous tryptophan was added.In addition,the antibiotic biosynthesis genes of six compounds of the two strains of actinomycetes were PCR amplified.The results founded that the actinomycetes species had amplified multiple antibiotic biosynthesis genes at the same time,which to some extent indicated that the actinomycete species had the potential to synthesize certain or even a variety of antibiotics.Transcriptome sequencing analysis was performed on samples of Fusarium oxysporum cultured alone and co-cultured with antagonistic actinomycetes.The results showed that under the antagonistic stress of actinomycetes strain 0213 and 0250,there were 1760 and 2157 genes differentially expressed respectively.GO and KEGG databases were used to annotate the functional and metabolic pathways of all the differentially expressed genes.It was found that the actinomycetes had the greatest effect on the cell membrane of Fusarium oxysporum in co-culture,and the changes of carbohydrate metabolism and amino acid metabolism were significant,this indicated that antagonistic actinomycetes destroyed the cell membrane and cell structure of the fungus.Further analysis revealed that antagonistic strains hindered the DNA replication and repair of Fusarium oxysporum,increased intracellular reactive oxygen species,accelerated the material and energy metabolism of cells,and the signal pathway response of repairing the mechanical damage of cell walls caused by extreme environment. |
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