This study was designed to characterize seed-colonizing microbial communities that were previously shown to be involved in the suppression of seedling disease caused by Pythium ultimum in a municipal biosolids compost. Selective microbial inhibitors were employed to inactivate portions of the microbial community associated with seed germinated in a compost medium to evaluate their impact on disease suppression. After initial screenings for toxicity to both cucumber and P. ultimum, six selective inhibitors were eventually used to assess the impact of seed treatment on the reduction of bacterial and fungal populations and on disease suppression. Rifampicin was the most effective inhibitor for inactivating disease suppression. Bacterial communities that colonized cucumber seed sown in compost medium for 8 h and seed sown in compost medium for 8 h followed by a 3-h treatment of either rifampicin at 500 ppm or water were dislodged from seed surfaces and subjected to RNA extraction and reverse transcription to cDNA. Differences in the composition of seed-colonizing bacterial communities were assessed using terminal restriction fragment length polymorphisms (T-RFLP) of polymerase chain reaction-amplified 16S rDNA genes. T-RFLP profiles revealed a diversity of distinct bacterial taxa, a number of which dominate seed surfaces within 8 h of sowing. Analysis of similarity (ANOSIM) using terminal restriction fragment (T-RF) presence or absence showed that community profiles of nontreated and water-treated seed were quite similar whereas community profiles from rifampicin-treated seed were distinct. Differences in community profiles based on T-RF abundance (peak height and peak area) indicated that all treatments were unique (ANOSIM, all pairwise comparisons P < 0.05) Peaks heights and areas of relatively few T-RFs were reduced to zero following rifampicin treatment and 34 T-RFs explained 85% of the observed difference between treatments. Tentative taxon assignments for each of the T-RFs that contributed to the treatment differences revealed a preponderance of sequences with affinities to the α-, β-, and γ-Proteobacteria and Firmicutes. Limited sequencing of clones associated with water-treated and rifampicin-treated seed revealed the presence of similar taxa dominated by members of the γ-Proteobacteria. Many species within these taxa (such as Pseudomonas spp., Enterobacter spp., and Bacillus spp.) are known to be suppressive to Pythium diseases. Results of our study have confirmed that Pythium disease suppression in a municipal biosolids compost is mediated by compost-associated bacteria that colonize seed within hours after sowing. By focusing on actively growing microbes in the infection court during important stages of pathogen infection, we believe we can more efficiently determine the mechanisms of disease suppression and the microbes involved. Although specific to this pathosystem and compost, our results have a much broader scope of inference and illustrate the utility of such a targeted approach in identifying a rela...

A versatile microfluidic platform for the evolving molecular diagnostics industry is described. It incorporates low cost Rheonix CARD® (Chemistry and Reagent Device) technology to analyze a variety of clinical specimens. A patented lamination process incorporates all pumps, valves, microchannels and reaction compartments into an inexpensive disposable plastic device. Once an untreated clinical specimen is introduced, all assay steps, including cell lysis, nucleic acid purification, multiplex PCR, and end-point analysis, are automatically performed. Three distinct CARD assays are described which utilize either a low density microarray for multiplex detection of amplicons or an integrated primer extension assay to detect single nucleotide polymorphisms of interest. The STI (Sexually Transmitted Infections) CARD® is able to simultaneously detect four sexually transmitted infectious agents (N. gonorrhoeae, C.trachomatis, T. pallidum and T. vaginalis). Human C33A cervical epithelial cells were spiked with different levels of genomic DNA from the four species of interest, singly or in combination, and applied to the CARD device. Using multiplex PCR amplification of the targets followed by microarray detection, the CARD device was able to correctly detect a minimum of 10 copies of each of the four pathogens. The HPV (Human Papillomavirus) CARD® was able to detect and distinguish 20 different clinically relevant HPV types using cloned HPV DNA. In addition, the HPV CARD could identify HPV types in vaginal specimens previously demonstrated to contain high or low risk HPV using a currently commercially available testing method. Finally, the detection of specific single nucleotide polymorphisms (SNP) associated with warfarin dosing sensitivity was achieved on the Warfarin Genotyping CARD® by analyzing human buccal swabs. Once multiplex PCR was completed, the SNPs were detected using a primer extension assay.

The EncompassMDx workstation (Rheonix, Inc, Ithaca, New York) is a molecular detection system that can automatically determine the genotypes of individuals in an unattended manner. Considerably less technical expertise was required to achieve results identical to those obtained using more complex, time-consuming, and expensive bidirectional DNA sequencing. This optimized system may dramatically simplify and reduce the costs of pharmacogenomics testing, thus leading to more-widespread use.

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