Screening Endophytic Actinomyces Against Banana Fusarium Wilt And Effect Studies

Fusarium wilt of banana is a typical Fusarium oxysporum.f sp.cubense Fusarium infection caused by the typical soil vascular bundle disease, causing serious harm to the banana industry in China, and there is no effective prevention and treatment methods at present. In order to prevent the disease, the antagonistic strains against Fusarium oxysporum f.sp.tritici were selected as the key factor for the success of bio-control.Plant endophytic actinomycetes can produce a variety of secondary metabolites with biological activity, and have great potential in biological control of plant diseases, new fungicide development,etc. It is a kind of highly valuable microbial resources. In this paper, Fusarium oxysporium f. sp. cubense 4 (FOC 4) was used as the target bacteria to isolate endophytic actinomycetes from Lobelia clavata plants in Yunnan, China. According to the morphological characteristics, cultural characteristics, physiological and biochemical characteristics, 16S rDNA sequence analysis, the strains were identificated, and the fermentation parameters were optimized. All the work have done above can provide technical supports and theoretical basis for the popularization and application of the strain.The results are as follows:(1) Isolation and screening of Endophytic Actinomycetes. According to the colony shape and color of the strains in the separation medium, 126 strains of endophytic actinomycetes were purified by the method of dilution plate. Two lines of actinomycetes with larger diameter of inhibition zone were obtained by plate-to-plate culture, which were identified as DJ15 and XLG-1.(2) Identification of the strain. The 16S rDNA sequences of the DJ15 and XLG-1 strains were compared by EzTaxon and GenBank. The results showed 21 strains were the most homologous to strain DJ15 and XLG-1. Constructing phylogenetic tree showed that DJ15 strain had the highest homology with Streptomyces yatensis NBRC 101000T(AB249962), which was 99.96%. The strain XLG-1 and Streptomyces kasugaensis M338-M1T (AB024441)?Streptomyces celluloflavus NRRL B-2493T (JOEL01000102)had the highest homology of 99.09% and were all in the same branch. According to morphological, cultural and physiological and biochemical characteristics, the strain DJ15 was identified as Streptomyces yatensis, and XLG-1 Streptomyces celluloflavus.(3) Inhibitory activity of Endophytic Actinomycetes. The experiment was carried out by using the obtained strain DJ15 and XLG-1 against 11 kinds of plant pathogens. Results show that: The inhibitory rates of DJ15 and XLG-1 against 11 plant pathogens mycelium was above 51%, and the inhibition rate against banana fusarium wilt was 54.46% and 70.12% respectively, among which XLG-1 shows the strongest inhibition, which was74.99%. The activity experiment was carried out by using strains DJ15 and XLG-1 against Fusarium oxysporum f. sp. cubense race 4 conidia with different concentrations of fermentation liquids. The secondary metabolites of XLG-1, DJ15, were used to carry out a broad spectrum antibiotic experiment. The antimicrobial activity of DJ15 and XLG-1 against Banana Fusarium Wilt No. 4 was 19.89mm and 23.99mm,respectively. The inhibitory effect of DJ15 on pathogen of banana tree was the strongest, and the inhibition bandwidth was 21.11mm. XLG-1 had the strongest inhibitory effect on pathogens of banana cigarette butts, and the inhibition bandwidth was up to 26.24mm.(4) Optimization of culture medium and fermentation conditions. Optimization of culture medium formula for strain fermentation and orthogonal experiments were carried out, studying the influence of different carbon source, nitrogen source and inorganic salt type and concentration on Fermentation by strain DJ15. Optimal medium was 1.5%glucose, soybean powder 1%, peptone 3%, ammonium chloride, 1% sodium chloride and 0.4% calcium carbonate, 0.3% Magnesium Sulfate 0.1%, 0.1% potassium sulfate. The optimization of fermentation conditions screening experiments were carried out, result showed that he optimum culture conditions were pH 6, inoculation 15%, shaking speed 200r / min and culture time 120h.(5) The control effect of the DJ15 broth on banana plant and the effect of promoting growth. By using the antibiotic marker method on the antagonistic DJ15, stable characters of the mutant strain was obtain, and pot inoculation test showed that antagonistic bacteria DJ15 can colonize in banana seedlings. The number of DJ15 could be up to CFU/g soil.The antagonistic bacterium DJ15 fermentation liquid could reduce the incidence of banana wilt disease, the control effect was 81.91%, the control effect was significantly higher than that of other treatments, including Fenaminosulf agents, etc. DJ15 fermentation liquid in the potted plant condition, can not only significantly improve control effect of banana Fusarium wilt, but also improve the plant height, stem diameter and fresh weight of banana,and promote the growth of banana, and had a certain effect on the enzyme activity of banana plant.