The Effect Of Biocontrol Agents On Soil Fungal Community In Cucumber Rhizosphere Using Traditional And Molecular Methods

Fungi play an important role in soil ecosystem, such as degradation of plant residue, inhibition of pathogen, etc. The diversity and richness of soil fungal community could even be a significant index in evaluation of soil microbial health. Biopesticides have a broad application prospect, but there was little research about the impact of biopesticides on soil microbial community, especially on fungi. In this thesis, traditional cultural method combined with molecular methods was used to investigate the effect of biocontrol agent Pseudomonas fluorescens 2P24 and Bacillus subtilis BS908 on soil fungal community in cucumber rhizosphere. It would provide important information and critical technologies for assessing the environmental safety of using microbial pestcides.P. fluorescens 2P24 strain was gfp labeled with plasmid pSMC21 (gfp, Ap+, Kn+). Through further study, it showed that there was little discrimination of growth rate between the normal strain and gfp-labeled strain. Therefore, it could be used in field experiment instead of normal strain.The fermentation broth of 2P24 and BS908 was diluted and inoculated into cucumber rhizosphere. The survival of 2P24 was monitored weekly, and KB medium with ampicillin, kanamycin was used to detect survival of 2P24 recovery. During a month, the amount decreased from 108 to 10 CFU/g dry soils, and remained steady to 105 CFU/g dry soils.The amount of soil fungi was sampled and detected regularly through traditional cultural method. It indicated that the amount of soil fungi had a significant increase, initially applied with 2P24. It may be related to the growth-promoted effect of 2P24. After three weeks, this impact was gradually decreased. The amount of soil fungi was inhibited by BS908, and this influence lasted about two weeks.DNA of soil samples was extracted and amplified with universal fungal primers ITS IF and ITS4. The PCR products were purified and digested with enzyme Hinf I and Taq I. After detected by T-RFLP method, it implied that the soil fungal community was greatly influenced after the inoculation of 2P24. However, the influence of 2P24 became not obvious after a month. Effect of Bacillus subtilis BS908 on soil fungal community was similar to 2P24.The soil DNA was also amplified with fungal universal primers NS1 and GCfung. The PCR product was purified and electrophoresised through DGGE method. The result showed that the DGGE bands of 2P24 treatments were significantly decreased. But four weeks later, this situation was totally changed and there was little difference between the treatments and controls. The effect of BS908 lasted about 35 days through cluster analysis.Overall, the survival of 2P24 gradually decreased after inoculated into the soil of cucumber rhizosphere. It significantly influenced the soil fungal community at the beginning. However, the influence of 2P24 reduced gradually, especially after its concentration decreasing to 106 CFU/g dry soils. It implicated that the effecting time was about one month, which also sugguested that the biocontrol time of 2P24 may just last one moth.