Study On Microbial Community Structure In The High-yield Apple Orchard Of Weibei Loess Plateau

Microbial molecular ecology techiniques based on cultivation-independent methods were used in this work,and they can overcome drawbacks of the conditional-dependent tachniques.The idea is based on the fact that the diversity of microbial genemic DNA extracted directly from soil which can reflect the microorganisms,and this can give a more comprehensive way of microbial diversity in soil.In order to find the optimal method,ine methods of extracting soil total DNA were developed,which include direct extraction method,indirection method and commercial extraction product.Application of DGGE methods enables the detection of diverse members of soil microbial consortia,even including those microorganism not yet cultivated,but the extraction of DNA from soil is not that simple.Based on nine improved methods,four kinds of soils in Weibei Loess Plateau were used to analysis for the best method.The V3 domain sequences of 16SrDNA from soil microbial consortia were amplified by PCR.The optimum condition of DGGE in this work was confirmed that electrophreses were run for 7h at 150V on 8.0% acrylamide/bis-acrylamide(37.5:1)gel with 30%-70% denaturant gradient,and this was run at a constant tempreture of 60℃.We studied the soil microbial community of different areas(50cm,100cm from the center of the tree,and alleyway),and the relation between nitrogen rate and soil microbial community,apple leaves.The results were as the followings:The band in 23kb was obtained in all those methods, while those indirect methods of extraction was more efficient than direct mehods.Though the yield of direct methods were ten times as the indirect methods,the microbial community obtained by indirect method was more comprehensive than the other.The bands of microbial community in soil applified by three direct methods to some extent similar as by using kits.Two methods were found to be the best,and the pH of the extraction buffer and the lyase tye were discussed in this work,which was suggested that the extraction method with pH=9.0 and proteinase K could reflect a more comprehensive result to some extent.The right way in collecting soil was to pick the site with 10cm up layer around 50cm away to the apple tree's center.The application of nitrogen(N) and organic fertilizer increased significantly the amount of the microbial community in apple orchard.The nitrogen at high concentration inhabited certain microbes'growth.