Effects Of The Introduction Of Biocontrol Agents Pseudomonas Fluorescens 2P24,Pseudomonas Fluorescens CPF10,Trichoderma Harzianum T4 On The Bacterial Community In Cucumber Rhizosphere

The negative effect of synthetic fungicides on ecological environment, beneficial biology and human had increased, biocontrol agents (BCAs) had been used widely as alternatives of chemical pesticide to controll plant disease. As exogenous microbes, a large amount of BCAs released into field might pose risks for natural microflora. At present, numerous studies had been focus on the impact of BCAs on target soil micro-organisms, there were a few papers dealing with the effect of BCAs on the native soil microbial community. The methodological approach that comprised classical microbiological numbering technique, molecular tools based on DGGE and T-RFLP was used in this study.The main objective was to assess the modifications of the bacterial community in cucumber rhizosphere after inoculation with the biocontrol agents P.fluorescens 2P24,P.fluorescens CPF10,T.harzianum T4, and to provide objective biosafety evaluations for the field application of biocontrol agents.The result of recovery of gfp-tagged Pseudomonas fluorescens released in field revealed that the survival ability of 2P24 in cucumber rhizosphere was stronger than CPF10, but the difference was not significant. Inoculation resulted in ca.2.8x107 and 5.0 x 107 CFU per gram dry soil of P.fluorescens 2P24 and P.fluorescens CPF10 separately. The drop of bacterial numbers sped up persistently during the remainder of the monitoring period. At 56th day, the numbers of 2P24 and CPF10 just stayed at levels of ca.1.2×103 and 6×102 CFU per gram dry soil. No remnants and diflusion was detected in soil.The results of plating of soil serial dilutions implicated that Pseudomonas fluorescens 2P24 and CPF10 inhibited bacterium in 14th day rhizosphere soils after introduction. At 56th day samples, inhibition could still be detected, but weakened gradually. The impact of Pseudomonas fluorescens 2P24 and CPF10 on bacterium was not significantly different from each other.The influence of T4 assumed promoting soil bacterium in rhizosphere from 7 to 21 days after inoculation. However, inhibition substituting for promotion was observed after 28 days. On 70th day after T. harzianum T4 introduced, the impact of T4 on soil bacterium decreased obviously.Inoculation of different biological control agents could influence on culturable bacterial populations, but not significantly. Throughout the test period, culturable bacterial populations remained in the same level between different treatment groups.A PCA was performed to reveal and interpret the information contained in these T-RFLP dates. Introduction of P. fluorescens 2P24 or P. fluorescens CPF10 in the soil either resulted in a remarkable modification of the bacterial community structure from 7 days post-inoculation. The permutation test (Monte Carlo) showed that this difference between inoculated and control soils remained significant for 28 days after inoculation. Subsequently, the bacterial community structures of inoculated soils reverted to the structures of non-inoculated soils at 42th day. In the inoculated soils, the bacterial community structures assessed were grouped with each other, the effects of two biocontrol agents were similar. Introduction of T. harzianum T4 in the soil resulted in a significant variation of the bacterial community structures from 14 days post-inoculation.The impact of T4 persisted for along time.On 70th day after T. harzianum T4 introduced, the bacterial community structures of different treatments were similar. Therefore, the impacts of three biocontrol agents on the structures of bacterial communities in cucumber rhizosphere were different.The sequencing of DGGE retrieved bands suggested that Cyanobacterium, Beta-proteobacterium, Staphylococcus, Microbacterium groups were suppressed by Pseudomonas sp.2P24 and CPF10, nevertheless the biocontrol agents could promote the slight growth of Bacillus.The impact of two biocontrol agents on these bacteria was transitory, there was no prominent alteration of bacterial community detected due to inoculation with biocontrol agents later on. However, the impacts of both applied bacterial antagonists on the cucumber rhizosphere-associated bacteria were basically similar, whether on community structure or species, the impacts on certain strains by 2P24 emerged before CPF10. Introduction of T. harzianum T4 in the soil resulted in significant variations of multiple bacterial genera for some time.T.harzianum T4 could restrain effectively Cyanobacterium, Beta-proteobacterium, Serratia, Staphylococcus, Microbacterium, Burkholderia, Acidobacterium and some uncultured bacterium, while it improved the growth of Agrobacterium, Gemmatimonadetes, Bacillus observably, whereas after harvest the bacterial community revert to normal status.In conclusion, the application of three biocontrol agents all exerted only a transient and confined impact on the bacterial community of cucumber roots in systematic field investigation, the perturbation was unable to imperil the bacterial community stability in the long-term. The variations of the bacterial communities due to the introduction of BCAs were less important than the"non-intentional" variations due to other factors, including plant climate changes, growth stage, and ecological environment condition. The three strains were determined briefly to be safe to the environment.