Novel primers and PCR protocols for the specific detection and quantification of Sphingobium suberifaciens in situ.

The pathogen causing corky root on lettuce, Sphingobium suberifaciens, is recalcitrant to standard epidemiological methods. Primers were developed from 16S rDNA sequences to be useful for the specific detection and quantification of S. suberifaciens. Quantitative PCR (qPCR) protocols specifically amplified DNA from the type strain of S. suberifaciens (LMG 17323) and other members of this species but not from other members of the Sphingomonadaceae. The detection limit was as little as 100 fg DNA (equivalent to 2 × 10(2) cells) in the qPCR. Detection was successful from soils inoculated with as little as 1 × 10(3) CFU/g soil. DNA isolated from naturally infested soils and diseased lettuce roots was amplified and sequenced fragments were identical or nearly identical to 16S rDNA sequences from S. suberifaciens. In growth chamber experiments, there was a positive correlation between disease severity and S. suberifaciens population levels in roots and soil, as detected by qPCR. Detection levels were below population levels of the pathogen necessary for disease development.

Comparison of soil bacterial communities under diverse agricultural land management and crop production practices.

The composition and structure of bacterial communities were examined in soil subjected to a range of diverse agricultural land management and crop production practices. Length heterogeneity polymerase chain reaction (LH-PCR) of bacterial DNA extracted from soil was used to generate amplicon profiles that were analyzed with univariate and multivariate statistical methods. Five land management programs were initiated in July 2000: conventional, organic, continuous removal of vegetation (disk fallow), undisturbed (weed fallow), and bahiagrass pasture (Paspalum notatum var Argentine). Similar levels in the diversity of bacterial 16S rDNA amplicons were detected in soil samples collected from organically and conventionally managed plots 3 and 4 years after initiation of land management programs, whereas significantly lower levels of diversity were observed in samples collected from bahiagrass pasture. Differences in diversity were attributed to effects on how the relative abundance of individual amplicons were distributed (evenness) and not on the total numbers of bacterial 16S rDNA amplicons detected (richness). Similar levels of diversity were detected among all land management programs in soil samples collected after successive years of tomato (Lycopersicon esculentum) cultivation. A different trend was observed after a multivariate examination of the similarities in genetic composition among soil bacterial communities. After 3 years of land management, similarities in genetic composition of soil bacterial communities were observed in plots where disturbance was minimized (bahiagrass and weed fallow). The genetic compositions in plots managed organically were similar to each other and distinct from bacterial communities in other land management programs. After successive years of tomato cultivation and damage from two major hurricanes, only the composition of soil bacterial communities within organically managed plots continued to maintain a high degree of similarity to each other and remain distinct from other bacterial communities. This study reveals the effects of agricultural land management practices on soil bacterial community composition and diversity in a large-scale, long-term replicated study where the effect of soil type on community attributes was removed.

Fungal-specific PCR primers developed for analysis of the ITS region of environmental DNA extracts.

BACKGROUND: The Internal Transcribed Spacer (ITS) regions of fungal ribosomal DNA (rDNA) are highly variable sequences of great importance in distinguishing fungal species by PCR analysis. Previously published PCR primers available for amplifying these sequences from environmental samples provide varying degrees of success at discriminating against plant DNA while maintaining a broad range of compatibility. Typically, it has been necessary to use multiple primer sets to accommodate the range of fungi under study, potentially creating artificial distinctions for fungal sequences that amplify with more than one primer set. RESULTS: Numerous sequences for PCR primers were tested to develop PCR assays with a wide range of fungal compatibility and high discrimination from plant DNA. A nested set of 4 primers was developed that reflected these criteria and performed well amplifying ITS regions of fungal rDNA. Primers in the 5.8S sequence were also developed that would permit separate amplifications of ITS1 and ITS2. A range of basidiomycete fruiting bodies and ascomycete cultures were analyzed with the nested set of primers and Restriction Fragment Length Polymorphism (RFLP) fingerprinting to demonstrate the specificity of the assay. Single ectomycorrhizal root tips were similarly analyzed. These primers have also been successfully applied to Quantitative PCR (QPCR), Length Heterogeneity PCR (LH-PCR) and Terminal Restriction Fragment Length Polymorphism (T-RFLP) analyses of fungi. A set of wide-range plant-specific primers were developed at positions corresponding to one pair of the fungal primers. These were used to verify that the host plant DNA was not being amplified with the fungal primers. CONCLUSION: These plant primers have been successfully applied to PCR-RFLP analyses of forest plant tissues from above- and below-ground samples and work well at distinguishing a selection of plants to the species level. The complete set of primers was developed with an emphasis on discrimination between plant and fungal sequences and should be particularly useful for studies of fungi where samples also contain high levels of background plant DNA, such as verifying ectomycorrhizal morphotypes or characterizing phylosphere communities.