Impact Of Biocontrol Strains Bacillus Subtilis B99-2,Pseudomonas Fluorescens CPF10 And Trichoderma Harzianum T4 On Watermelon(Citrullus Lanatus) Rhizosphere Soil Bacterial Communities

Biocontrol agents are a new kind of pesticides to protect the plant diseases by the biocontrol strain itsself or their secondary metabolites. It has the advantages of high specificity, no pollution to the environment and no harm to human body and livestock.In resent years, many kinds of biocontrol agents were used in filed plant diseases and pests protection. However, to use biocontrol agents for the controling the plant diseases in agriculture filed may cause significant affect to the local microbal communities and destroy the microecological balance. The important roles of bacterial community diversity in soil to the microecology system are the formation of the soil, degradation of the contaminating material and the circulation of the materials. So, the microecological risk of the biocontrol agents should be evaluated before it was used abundantly in the field. At present, few researchers concern about the side effect of biocontrol agents on the aboriginal bacterial communities.In present study, the effect of three excellent biocontrol agent strains Bacillus subtilis B99-2, Pseudomonas fluorescens CPF10 and Trichoderma harzianum T4 on the watermelon rhizosphere soil bacterial communities and AOB communities were detected by plate counting, PCR-denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) synthetically. In addition, we used pyrosequencing method to study the effect of CPF10 on the rhizosphere soil bacterial communities. Our research could form a preliminary theoretical and technical system to assess the application microecological risk of the biocontrol agents.To determine the rhizosphere soil properties in the greenhouse, the results showed that all three biocontrol strains have no significant affect on soil pH and moisture content. Meanwhile, the total nitrogen, organic carbon, ammonium-N and nitrate-N were also not been affected by the inoculating the three strains respectively. Plate count method showed that the colonization of CPF10 on the plant soil rhizosphere was not very well. After inoculation of a month, the number of CPF10 in the soil was decreased sharply and it was just 104cfu·g-1 dry soil mass at the end of the watermelon growth period. Two weeks after the inoculation of CPF10 to the soil, the cultureable bacteria was increased slightly, while B99-2 and T4 have no significant influence on the soil bacterial communities.Certain regions of the 16S rDNA were amplified from soil metagenomic for the further analysis. Both T-RFLP and DGGE results showed that all three biocontrol strains had markable influence on the aboriginal soil bacterial communities in first month after the inoculation. Later, this influence phenomenon gradually became tiny. In detail, B99-2 had lasting influence on soil bacterial communities for 4 weeks. The influence of CPF10 on the soil bacterial communities began at second week and lasted about three weeks. This may be caused by the colonization period of CPF10 in the rhizosphere soil. Biocontrol strain T4 had more obvious effect on the soil bacteria and lasted much longer, but in general, the effect in first four weeks was more notable than later ones. In addition, all three biocontrol strains had no significant influence on the soil AOB communities. This may caused by the low abundance of AOB in soil and slow growth rate.The shifted bands in the DGGE finger profile were sequenced and identified. The application of B99-2 inhibited some bacterial communities like Pseudomonas、Hydrogenophaga、Saccharococcus、Brevibacterium、Enterobacter et al. While CPF10 suppressed Bacillus、Escherichia and Klebsiella at the same time. It had enhancement on Mesorhizobium、Ochrobactrum、Sinorhizobium and Brucella melitensis. At last, T4 promoted some communities such as Pseudomonas、Bacillus、Ochrobactrum Mesorhizobium et al in certain degree.The effect of biocontrol strain CPF10 on the rhizosphere soil bacterial communities was measured with pyrosequencing. To amplify the V4 region of 16S rDNA and sequence about 15000 reads per sample, the results were clustered in 97%similarity.PCA analysis of the different OTU showed that the disturbance to soil bacterial communities caused by CPF10 happened in the second week after the inoculation. This result also was demonstrated by the Shannon index of different time. And the application of CPF10 promoted some group of bacterial communities such as Clostridium、Bacillus、Shinella、Sphingomonas et al. While for other group like Thiobacillus、Sporosarcina、Lactococcus、Arenimonas、Steroidobacter、Anaeromyxobacter、Elizabethkingia, CPF10 showed depressant effect in different degrees.Biocontrol strain Bacillus subtilis B99-2, Pseudomonas fluorescens CPF10 and Trichoderma harzianum T4 were safe for the microecology to a certain degree. Their application may not significantly affect the soil intrinsical bacterial for a long period.