Molecular Detection Of Bacillus Subtilis B908 In Soil

Bacillus subtilis B908 ,which was isolated from soil of rice rhizosphere by Department of Plant Pathology,China Agriculture University.It is antagonistic to other fungi,include important pathology in soil. Department of plant pathology (Yunnan agriculture univerisity), Biological control team (Department of plant pathology,China agriculture university) and Yunnan Xingyao biological product factory had researched to manufacture a microorganism agrochemical product whose main and valid composition was "Bacillus subtilis B908".The release of bicontrol agents into enviroment has created the demand for monitoring them .A simply PCR procedure was developed for direct detection of B908 released into field soil.1.The randomly amplified polymorphic DNA(RAPD),the cloning of lap-14 gene and arbitrarily primed PCR(AP-PCR) were used to screen B908 , other Bacillus subtilis strain ,other bacteria and fungi.Strain-specific marker was identified for strain B908.The marker was sequenced and converted into a sequence-characterized amplified region (SCAR), and a specific PCR primer pair was designed.Eighteen strains were tested and this specificity was confirmed.2.The effectiveness of two extract DNA protocols(lysozyme/sodium dodecyl sulfate[SDS]/freeze-thaw lysis,NaI) and two purify DNA methods(chloroform /isoamyl alcohol, glass milk)were compared and evaluated based on the yield of DNA ,and removal of PCR inhibitors.lysozyme/SDS/freeze-thaw lysis and glass milk purify was found to be best for successful PCR amplifications.Three soil(sandy soil,greenhouse soil and agricultural soil) were test by this procedure and were successful for PCR amplifications.3. The SCAR primers could detect B908 inoculated in soil.The lower limit of detectiond is 106 cfu per gram of soil.And the primers detected B908 for 20 days. The fate of B908 population in the soil was known.A molecular detection procedure to B908 released in soil was developed.