Clavibacter michiganensis subsp. sepedonicus (Cms) multiplies very rapidly, passing through the vascular strands and into the stems and petioles of a diseased potato. Therefore, the rapid and specific detection of this pathogen is highly important for the effective control of the pathogen. Although several PCR assays have been developed for detection, they cannot afford specific detection of Cms. Therefore, in this study, a computational genome analysis was performed to compare the sequenced genomes of the C. michiganensis subspecies and to identify an appropriate gene for the development of a subspecies-specific PCR primer set (Cms89F/R). The specificity of the primer set based on the putative phage-related protein was evaluated using genomic DNA from seven isolates of Cms and 27 other reference strains. The Cms89F/R primer set was more specific and sensitive than the existing assays in detecting Cms in in vitro using Cms cells and its genomic DNA. This assay was also able to detect at least 1.47×102 copies/μl of cloned-amplified target DNA, 5 fg of DNA using genomic DNA or 10−6 dilution point of 0.12 at OD600 units of cells per reaction using a calibrated cell suspension.
Among pre-harvest environmental factors, increasing attention has been paid to the effects of chemical and microbiological factors on fresh produce. The occurrence and prevalence of these factors have been usually studied with regard to the final products at the post-harvesting stage and/or when they are sold in the market. However, the origin and routes of transmission of both factors remain to be clarified. In the present study, we examined the contamination levels of food-borne pathogens and chemical factors such as pesticide residues and heavy metals in 83 and 43 samples, respectively, including various soil, water, and fertilizer samples, as well as post-harvested and processed samples. Among the organic farming samples, only one pesticide, dimethomorph, was detected in the soil sample, however no pesticides were observed from any other samples in organic farming system. Thus, it was thought that might be contaminated from conventional farm land in the vicinity. Whereas many pesticide residues were detected in conventional farming systems such as soil, fertilizer, water, and fresh produce as expected. Furthermore, heavy metals detected from all tested samples did not shown contamination levels higher than the standard limit. We comparatively assessed the levels of contamination by food-borne pathogens on the samples from organic and conventional farming systems, and found aerobic bacteria at approximately 7 log CFU/g, with no significant differences observed between the two systems. Coliforms were present at lower levels than aerobic bacteria. No human pathogens were present among the coliforms detected, indicating that these bacteria are saprophytes without the ability to cause food-borne illnesses. In contrast, among the high-risk food-borne pathogens, only sporadic cells of Bacillus cereus were found on samples of organic farming system. These data extend previous findings that the most prevalent food-borne pathogen is B. cereus and demonstrate that it spreads to whole living plants via soil.
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