SummaryCultivation-independent studies suggest that roots of rice ( Oryza sativa ) are colonized by a diverse community of nitrogen-fixing bacteria. Here we report for the first time mRNA-based profiling of nitrogenase ( nifH ) genes, to study the impact of lowland-rice genotypes at the cultivar level on the functional diversity of rootassociated diazotrophs. Root RNA extracts from all plants contained nifH mRNA at levels detectable by reverse transcription polymerase chain reaction (RT-PCR). Terminal restriction fragment length polymorphism (T-RFLP) analysis of RT-PCR products showed only small interplant variations. However, RNA-and DNA-based profiles obtained from the same root extractions differed from each other, suggesting that presence of diazotrophs did not necessarily coincide with active transcription of nif genes. Application of N-fertilizer at planting had a long-term effect on the profile of expressed nitrogenase genes. Phylogenetic analysis of a clone library constructed for nifH fragments expressed in wild species of rice roots indicated that active diazotrophs were not related to cultured strains. The composition of active diazotrophic communities was compared for six related cultivars of O. sativa , wild species Oryza brachyantha , and a genetic cross between it and cv. IR56, grown under identical conditions in rice field soil in the Philippines without N-fertilizer application. Remarkable varietal differences in root associated nifH -gene expressing communities were detected. This underlines the importance of mRNA-based approaches to study functional diversity and eventually identify key diazotrophs in a particular environment.

Significance and Impact of the Study: The choice of commercial kits (FD, INS, NSP) has been of great importance regarding the quantity and purity of DNA extracted from paddy soils in this study. The composition of the cell lysis buffer represented a key component for successful extractions of DNA from different soils. The possibility of adjusting the lysis buffer to the soil of interest as well as the reproducibility of DGGE banding patterns makes the recently introduced NSP kit a strong competitor to the well-established FD kit for the extraction of DNA from paddy soils. AbstractThe objective of this study was to compare the extraction efficiency of commercial DNA kits by evaluating the quantity and purity of DNA extracts obtained from paddy soils. DNA was extracted from three paddy soils using the FastDNA ® SPIN kit for soil (FD), the innuSPEED soil DNA kit (INS) and the NucleoSpin ® soil kit (NSP). DNA extracts were analysed by agarose gel electrophoresis and UV spectroscopy. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analyses were conducted to evaluate the potential bias of the DNA extractions on fingerprinting techniques. Regarding the quantity and the purity of the extracted DNA, the NSP kit was detected superior to the FD kit, while the INS kit failed to extract detectable amounts of DNA. DGGE fingerprints generated from PCR products (FD, NSP) showed high levels of similarity for the amplified 16S rRNA genes of methanogenic archaea (>95%) and bacteria (up to 100%) in each soil. This study suggested that the recently introduced NSP kit allowed for the adjustment of the lysis buffer composition to the soil of interest and is at least equivalent to the well-established FD kit for the extraction of DNA from paddy soils.

Plants shape distinct, species-specific microbiomes in their rhizospheres. A main premise for evaluating microbial communities associated with root-soil compartments is their successful separation into the rhizosphere (soil-root interface), the rhizoplane (root surface), and the endosphere (inside roots). We evaluated different approaches (washing, sonication, and bleaching) regarding their efficiency to separate microbial cells associated with different root compartments of soil-grown rice using fluorescence microscopy and community fingerprinting of 16S rRNA genes. Vigorous washing detached 45% of the rhizoplane population compared to untreated roots. Additional sonication reduced rhizoplane-attached microorganisms by up to 78% but caused various degrees of root tissue destruction at all sonication intensities tested. Treatment with sodium hypochlorite almost completely (98%) removed rhizoplane-associated microbial cells. Community fingerprinting revealed that microbial communities obtained from untreated, washed, and sonicated roots were not statistically distinguishable. Hypochlorite-treated roots harbored communities significantly different from all other samples, likely representing true endospheric populations. Applying these procedures to other root samples (bean and clover) revealed that treatment efficiencies were strongly affected by root morphological parameters such as root hair density and rigidity of epidermis. Our findings suggest that a careful evaluation of separation strategies prior to molecular community analysis is indispensable, especially when endophytes are the subject of interest.

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