Assessing The Impact Of Biocontrol Agent Pseudomonas Fluorescens CPF10on The Fungal Community Diversity In Soil Of Watermelon Rhizosphere

Biopesticides have been widely used for controling plant diseases. Most of the current studies focused on the interaction of biocontrol and pathogenic microorganisms, while relatively few reports on the effects of soil fungal communities. This article combined the methods of plate counting, T-RFLP, PCR-DGGE and Pyrosequencing to assess the influence of the pesticides of Pseudomonas fluorescens CPF10on soil fungal community in watermelon rhizosphere as the basic of the safety assessment of the exogenous microorganisms into soil.Through the recycling of fluorescent labeling of biocontrol agents found Pseudomonas fluorescens CPF10in the field decreased from107CFU/g dry soil to105CFU/g dry soil at the fourth week, only maintained at about103CFU/g dry soil at the seventh week, while the total number of soil fungi maintained105CFU/g dry soil, CPF10survived in the soil significantly inferiored to the indigenous fungi. Series of dilution culture counting method found that two weeks before the applied biocontrol strain significantly promoted the growth of culturable fungi. biocontrol colonization CPF10had certain inhibition of soil culturable fungi. The inhibition maintained a month or so, disappeared between the seventh weeks and eighth weeks.The principal component of the T-RFLP analysis using restriction enzymes Hinf I and Taq I digested the results of PCR production showed that from the first week, biocontrol CPF10treated group compared to the control group, fungal community had a significant difference, this difference continued until the seventh week, especially the seventh week. After the seventh week, fungal communities by biocontrol treated began to recover the level of the control group that means normal level. By PCR-DGGE analysis, biocontrol CPF10applied watermelon rhizosphere soil had the impact on soil fungal communities maintained seven weeks or so. From the third week to the sixth week, the abundance of the dominant fungi genus such as Alternaria, Fusarium, Chaetomium genus decreased. Fusarium, Mortierella, Wallemia sebi and uncultured soil Zygomycetes had the most dramatic changes, and the seventh week later, these genus began to recover the blank control was insignificant.Nested PCR-DGGE results showed that the effect of biocontrol agents CPF10on soil mycorrhizal fungi compared indigenous fungi were relatively small. From the first week to the third weeks of biocontrol agents applied, biocontrol strain had little effect on soil mycorrhizal fungi. But until the fourth week, biocontrol agents CPF10began to promote the growth of mycorrhizal fungi which continued for three weeks or so. Compared to NCBI Genbank, the vast majority of arbuscular mycorrhizal fungi were Glomus sp. Until the eighth week, this effect was not obvious, without any differences with the blank control.454pyrosequencing technology found a significant increase in the abundance and diversity of fungi in the seventh week of biocontrol applied. Under high temperature, biocontrol agents became the dominant bacteria in the soil as indigenous fungi decreased, thus, it was easier to biocontrol agent play the role in fungal community. The plant pathogens of Colletotrichum, Glomerella, Leptosphaeria, Nectria, Valsa and Verticillium disappeared after biocontrol applied. The dominant fungi Emericella had a significant reduction, while non-dominant species such as Cladonia, Xylaria and Rhizophydium which were benefit for soil function became the dominant fungi.In summary, compared with climate, plant growth stage, ecological microenvironment, the influence of biocontrol strain P. fluorescens CPF10on soil fungal communities was short-lived, especially on arbuscular mycorrhizal fungi which lasted for only three weeks or so, would not pose a threat to long-term stability of the soil fungal communities. Therefore, anti-bacteria P. fluorescens CPF10had good ecological security.This study combined traditional culture method and molecular biology of the T-RFLP, DGGE, pyrosequencing technology evaluating the impact of biocontrol agent on soil microecosystem comprehensively and detailedly which provide a novel and effective system for the safety biopesticides used.