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RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS OF PCR-AMPLIFIED NIFH SEQUENCES FROM WETLAND PLANT RHIZOSPHERE COMMUNITIES
Citation:
Chelius, M. K., J. E. Lepo, AND D. E. Weber. RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS OF PCR-AMPLIFIED NIFH SEQUENCES FROM WETLAND PLANT RHIZOSPHERE COMMUNITIES. APPLIED AND ENVIRONMENTAL MICROBIOLOGY 20(8):883-889.
Description:
We describe a method to assess the community structure of N2-fixing bacteria in the rhizosphere. Total DNA was extracted from Spartina alterniflora and Sesbania macrocarpa root zones by bead-beating and purified by CsCl-EtBr gradient centrifugation. The average DNA yield was 5.5 ug g-1 of soil, and was of sufficient purity for PCR amplification of nifH. (a-32P)dCTP was incorporated into the PCR reaction and nifH PCR products were restriction digested. Restriction Fragment Length Polymorphism (RFLP) analysis of the amplified sequences revealed differences in the community structure of N2 -fixing rhizobacteria of the salt marsh plant, Spartina alterniflora, and a laboratory grown, Sesbania macrocarpa. Soil inoculation experiments were used to determine the efficiency of the methods, and amplified nifH DNA could be detected when 104 cells each of Vibrio natriegens and Azotobacter vinelandii were added per gram of soil. Restriction patterns produced by each species were detected at 106 cells g-1 soil. These results indicate that RFLP analysis of amplified nifH sequences from rhizosphere communities may provide information on species composition and reveal shifts in diversity.