Analysis of bacterial and fungal community structure in replant strawberry rhizosphere soil with denaturing gradient gel electrophoresis

Abstract

High quality DNA is the basis of analyzing bacterial and fungal community structure in replant strawberry rhizosphere soil with the method of denaturing gradient gel electrophoresis (DGGE). DNA of soil  microorganisms was extracted from the rhizosphere soil of strawberries planted in different replanted  years (0, two, six and seven), respectively, and crude DNA was purified after extraction. Three methods  were established to evaluate the effects of cetyl trimethylammonium bromide (CTAB),  polyvinylpolypyrolidone (PVPP), proteinase K and bacteriolytic enzymes on DNA extraction. DNA  fragments above 23 kb in size were isolated well by method 1 (1% CTAB, proteinase K, no PVPP, no  bacteriolytic enzyme) and method 3 (no CTAB, no proteinase K, 3% PVPP, bacteriolytic enzyme). Method 3 got the best yields 43.06 ìg/g, and A260/A280 and A260/A230 were 1.1623 and 0.8135, respectively,  which could ensure the veracity of subsequent DGGE analysis. Method 2 (3% CTAB, no proteinase K, no PVPP, no bacteriolytic enzyme) could not extract enough DNA to do the next PCR-DGGE analysis.  F341/R534 and FR1/FF390 primers were used to amplify the 16S rDNA V3 region of bacteria and 18S rDNA of fungi, and the expected fragments of 230 bp 16S rDNA V3 region and 390 bp 18S rDNA were amplified. The results of DGGE analysis showed that there were common and specific bacterial and fungal  communities in different replant soils of strawberry. There were 84 and 54% similarity of bacterial and  fungal communities between different replant soils. The numbers of both bacterial and fungal communities increased in the replant strawberry soil, they were positively correlated with the replant years. As the  number of replant years increased from two to seven years, while the ratio of bacteria/fungi was  decreased from 2.29 to 1.46 in the rhizosphere soils planted with strawberries.

Key words: Rhizosphere soil, bacterial community, fungal community, replant strawberry, fruiting fields.