Restriction Fragment Length Polymorphism Analysis of PCR-Amplified nifH Sequences from Wetland Plant Rhizosphere Communities

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Abstract
We describe a method to assess the community structure of N2-fixing bacteria in the rhizosphere. Total DNA was extracted from the macrophytic plants Spartina alterniflora and Sesbania macrocarpa root zones by bead beating and was purified by CsCl-EtBr gradient centrifugation. The average DNA yield was 5.5 µg g−1 of soil and was of sufficient purity for PCR amplification of nifH. [α-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 field collected salt marsh plant, Spartina alterniflora, and of a laboratory cultured 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. By examining population shifts within functional microbial groups, such as the nitrogen-fixing bacteria, the method should have particular utility in assessing stress effects on polluted ecosystems or on those undergoing bioremedial treatments.

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