Effects Of Two Biocontrol Bacterial Strains On Banana Fusarium Wilt Control And Rhizosphere Soil Microbial Community

Banana is a major food and economic source in many developing countries and is the fifth most important agricultural product in world trade.However,the emergence and widespread occurence of Banana Fusarium wilt have brought significant economic losses to the global banana planting industry.At present,biological control is the most promising measure to control Banana Fusarium wilt,and the previous research mainly has focused on the screening of biocontrol agents,pot experiments,the field application and the prevention mechanism of biocontrol agents.Bacillus amyloliquefaciens(XP1)and Trichoderma viride(XP2)are highly effective strains against Fusarium oxysporum f.sp.cubense Race4 GFP-FOC4,which were screened independently in the early stage of this project.The inhibition circles of plate confrontation experiment were 46.3 mm and 62.5 mm,respectively,better than those reported in the literature Circles(27.0+8.7 mm,n=26),which were increased by 1.7 times and 2.3times,respectively.In order to verify the control effect and mechanism of two biocontrol bacteria XP1 and XP2 on Banana Fusarium wilt,field experiments were conducted to quantitatively determine the number of pathogenic bacteria and biocontrol bacteria in rhizosphere soil of each treatment by means of real-time fluorescence quantitative PCR and Illumina Hiseq 2500 high-throughput sequencing platform.And the effects of two biocontrol bacteria XP1 and XP2 on the diversity of rhizosphere microbial community structure were also analyzed.The main findings of the present research are as follows:(1)Both XP1 and XP2 showed the potential of preventing and controlling the occurrence of banana wilt,and the effect was highly significant(P<0.01).No disease was found in banana plants treated with B1 and F1 in simulated healthy soil.The incidence rates of B2 and F2 banana plants inoculated with GFP-FOC4 were higher by 95.0%and 85.6%,respectively.The incidence of banana plants treated with B3(inoculated with XP1 on the basis of GFP-FOC4inoculation)and F3(inoculated with XP2 on the basis of GFP-FOC4 inoculation)were only13.3%and 12.8%,respectively,and the control effects were 84.2%and 78.7%,respectively.(2)Real-time PCR and laser confocal scanning microscopy showed that biocontrol bacteria XP1 and XP2 can colonize and proliferate in the rhizosphere soil of banana plants,and inhibit the growth of GFP-FOC4.Fluorescent-labeled pathogenic bacteria GFP-FOC4 were not observed in rhizosphere soil treated with B1 and F1.The fluorescent-labeled pathogenic bacteria GFP-FOC4 in B3 and F3 treatments were significantly less than those in B2 and F2treatments.The number of copies of XP1 in B3 treatment was 5 orders higher than that in B2treatment(1.42×10~2 copies/g),and that of FOC4 amplified by FOC4 specific primers were 4orders lower than B2 treatment(4.67×10~7 copies/g).The number of copies of XP2 in F3treatment were 4 orders higher than in F2 treatment(3.76×10~2 copies/g),and that of FOC4 was3 orders lower than that in F2 treatment(4.34×10~4 copies/g).(3)Microbial community diversity in rhizosphere of banana plants was analyzed by Illumina Hiseq 2500 high throughput sequencing platform.A total of 88 6890 valid sequences were obtained by sequencing in B1,B2,and B3 treatments,and 61 660 OTUs(Operational Taxonomic Units)were obtained by clustering at 97%similarity level.All the high-quality sequences of the three treatments were classified into 40 phyla,145 classes,285 orders,444families and 710 genera by cluster annotation.Proteobacteria,Acidobacteria and Firmicutes were the dominant bacteria in B1,B2 and B3 treatments.Proteobacteria accounted for the largest proportion of B3 treatment and was up to 53.78%.Acidobacteria accounted for a large proportion of B1 treatment and was 18.50%.Firmicutes accounted for 14.63%of the B2treatment and was higher than B1 treatment and B3 treatment.At the genus level,the proportion of Bacillus in the B3 treatment inoculated with XP1 was the largest,accounting for 32.04%,followed by B2 treatment,accounting for 1.35%;whereas,B1 treatment was the smallest,accounting for 0.52%.When the LDA value was more than 4.0,Bacillus was the most important species that caused the difference between the three treatments at the genus level.At the same time,in terms of bacterial community diversity index,XP1 increased the number of bacterial species in rhizosphere soil,and the Chao1 index and Observed-species index increased by 7.3%and 0.7%,respectively,compared with the single inoculation of pathogenic bacteria.The bacterial community structure in rhizosphere soil gradually evolves to the direction of bacterial community structure in healthy soil.(4)Based on the diversity analysis of fungal community for F1,F2 and F3 treatments,there were 611653 effective sequences.After clustering at 97%similarity level,4737 OTUs,8fungi phyla,27 classes,82 orders,164 families and 276 genera were obtained.Ascomycota was the dominant bacteria in F1,F2,and F3 treatments,accounting for 74.36%,99.53%and 99.36%of the species with relative abundance more than 1%,respectively.Basidiomycota and Zygomycota with relative abundance(?1%)accounted for 7.40%and 5.73%in F1 treatment,respectively,and was less than 1%in F2 and F3 treatments.At the genus level,Trichoderma accounted for 97.13%of F3 treatment inoculated with XP2,15.89%of F1 treatment and 0.15%of F2 treatment.The dominant position of Trichoderma in F3 treatment Fungi Community was highly significant(P<0.01)after inoculation with XP2,and the proportion of Fusarium in F3treatment Fungi Community was reduced by 15.97%compared with F2 treatment.(5)Redundancy analysis between microbial community characteristics and environmental factors in rhizosphere soil under different treatments showed that at the genus level,the abundance of Bacillus and Trichoderma was positively correlated with soil pH(P<0.05).The results showed that the proper regulation of soil pH can adjust the proportion of bacteria in the soil.In conclusion,the biocontrol bacteria XP1 and XP2 used in the experiment can colonize and proliferate in banana rhizosphere soil,and inhibit the growth of pathogenic bacteria of Banana Fusarium wilt through the interaction of metabolic production of bacteriostatic substances and niche occupancy,biocontrol bacteria-pathogenic bacteria-microbial community diversity.