Screening,Identification And Antifungal Mechanism Of Efficient Biocontrol Strains Against Banana Fusarium Wilt

Banana wilt is a fungal soil-borne disease caused by Fusarium oxysporum f.sp.cubense（FOC）,which is widespread,epidemic and difficult to control.It also brings a serious hazard to banana cultivation.Biological control has the advantages of being safe for humans and animals and not polluting the environment,so screening biocontrol strains and study their inhibition mechanism have important theoretical value and practical significance for efficient control of banana Fusarium wilt.In this study,Chromeazurol S（CAS）screening plate was used to screen out strains with the activity of siderophore-producing activity,and two biocontrol strains with high antifungal activity against FOC were obtained using plate confrontation.Then the two biocontrol strains were subjected to identification,antifungal spectrum determination,growth-promoting activity detection and pot test of banana seedlings,to determine their classification status and biocontrol effect against banana wilt.In addition,the yield of siderophore of strain Gxun-2 was optimized,and the mechanism of disease prevention was further explored from the separation of antifungal substances,the transcriptome interacting with pathogenic fungi,and the diversity of microbial community in rhizosphere soil.The main research results are as follows:1.Screening and identification of biocontrol strains in banana rhizosphere soilAfter primary screening of CAS medium,60 siderophore strains were isolated from the soil,and then five strains with strong antagonistic effects were obtained by using the plate confrontation method with FOC as the indicator fungi.Among them,the average inhibition rate of strains Gxun-1 and Gxun-2 were 74.45%and 65.4%,respectively,and both of them had broad-spectrum antifungal activity against other common plant pathogens.They also had the ability to produce IAA,protease,phosphorus solubilization,potassium and other plant proliferation.Then combined with the results of colony morphology,physiological and biochemical and16S r DNA evolutionary tree analysis,strain Gxun-1 was identified as Bacillus Siamese and strain Gxun-2 as Pseudomonas aeruginosa.The results of the pot experiment showed that both strains Gxun-1 and Gxun-2had good disease control and growth promotion effects on banana seedlings,with control effects up to 72.7%and 84.21%,respectively;fresh weight of banana seedlings increased by 37.02%and 41.91%,respectively,compared with the control;plant height increased by 25%and 32.31%,respectively.Based on the result of plate confrontation,pot experiment disease prevention and growth promotion,strain Gxun-2 had the best disease prevention effect,and the fermentation conditions should be further optimized.2.Optimization of strain Gxun-2 siderophore-producingThe medium and culture conditions affecting the siderophore production were optimized by single factor experiment at the flask level.Four significant factors affecting the siderophore production were further screened out by PB test.The optimal level of the important factors was determined by orthogonal test.Finally,the optimal condition was obtained as follows:sodium succinate 2 g/L,glycerol 10 g/L,ammonium sulfate 1g/L,initial p H 6.8,temperature 35℃,inoculum size 1%（v/v）,and liquid loading 120/250 m L.After optimization,the siderophore unit of strain Gxun-2 was 81.2%,82.84%higher than that before optimization.The antifungal rate against Fusarium oxysporum reached was 52.68%higher than that before the optimization condition.3.Study on antifungal substances of strain Gxun-2The antifungal substances of Gxun-2 were preliminarily studied.By using the separation methods of extraction,silica gel column chromatography,gel column chromatography,thin layer chromatography（TLC）and high performance liquid chromatography（HPLC）,it was preliminarily determined that the main antifungal substance of Gxun-2 was the phenazine.The stability results of antifungal active substances of strain Gxun-2 showed that temperature,natural light and ultraviolet had little effect on the stability of antifungal substances.The oxidizing agent and reducing agent had significant effects on the activity of antifungal substances.With the increase of concentration of Na₂SO₃,the activity of antifungal substances was gradually decreased.Different metal ions had different effects on the activity of antibacterial substances,and EDTA-Na₂and Na⁺had little effect on the stability of antibacterial substances.Mg²⁺,Mn²⁺,Ni²⁺,Ca²⁺and K⁺had significant effects on the stability of antifungal substances,making them almost lose their antifungal activity.However,Cu²⁺and Fe³⁺had very obvious effects on the activity of antifungal substances,making them almost completely lose their antifungal activity.4.Study on biological control mechanism of strain Gxun-2In order to explore the molecular mechanism of biocontrol effect of Gxun-2,the transcriptome analysis of Fusarium oxysporum and the changes of microbial community in banana rhizosphere soil applying biocontrol strain were conducted by using transcriptome sequencing and high-throughput sequencing technology.The FOC transcriptome data treated with Gxun-2 mainly reflect the significant changes in four aspects:synthesis of pathogenic fungus cell wall and cell membrane,antioxidant damage,detoxification,and autophagy process to cope with a series of damages caused by Gxun-2.The results of microbial diversity analysis in banana rhizosphere soil showed that the application of pathogenic fungus and biocontrol agents changed the soil fungal community structure and improved the richness and diversity of the fungal community structure.In addition,the proportion of pathogenic fungus Fusarium in the soil community significantly decreased with the introduction of biocontrol agent Gxun-2.The application of pathogenic fungus and biocontrol strain had no significant effect on the abundance and diversity of soil bacterial communities.