An Improved Evans Blue Staining Method for Consistent, Accurate Assessment of Plasmodiophora brassicae Resting Spore Viability

Clubroot caused by Plasmodiophora brassicae is an important disease of brassica crops. The use of vital stains to determine the viability of P. brassicae resting spores can provide useful information regarding spore longevity, inoculum potential, or the efficacy of antimicrobial treatments. Evans blue is one example of a vital stain that has been reported to differentially stain viable and nonviable resting spores. Some previously published protocols using Evans blue to stain P. brassicae resting spores have not provided accurate or consistent results. In this study, we modified the Evans blue method by increasing the staining time to 8 h or more and evaluated P. brassicae resting spores after heat treatment at various combinations of temperature and time. Extending staining times significantly increased the numbers of stained resting spores up to 7 h, after which the numbers of stained spores did not change significantly (R2 = 96.88; P ≤ 0.001). The accuracy of the modified method to discriminate viable and nonviable spores was evaluated in repeated experiments and by comparing the staining data with those derived from inoculation assays and propidium monoazide quantitative PCR (qPCR). The results demonstrated that the modified Evans blue staining method improved the accuracy and consistency of measurement of P. brassicae resting spore viability. Additionally, it was equivalent to the qPCR method for differentiating viable and nonviable spores (R2 = 99.84; P ≤ 0.001) and confirmed in canola infection bioassays.

The case for using the Most Probable Number (MPN) method in ballast water management system type approval testing

Recently, the U.S. Coast Guard (USCG) rejected the Serial Dilution Culture-Most Probable Number (SDC-MPN) method for enumerating viable phytoplankton cells in ballast water discharge as an alternate to their prescribed method — the Environmental Technology Verification (ETV) Protocol. This method distinguishes living from dead organisms using vital stains and motility. Succinctly, the USCG position has been that the ETV Protocol is a reliable and repeatable efficacy test and the SDC-MPN method is not. New evidence and an expanded consideration of published research supports a fundamentally different assessment. A peer-reviewed quantitative evaluation of ETV vital stains for 24 species of phytoplankton has conclusively established that the ETV Protocol, even with observations of motility, is not reliable for all species. In contrast, published results suggest that errors in the method were small for the limited number of locations studied to date. It is possible that the communities tested in these were dominated by species that can be classified accurately using vital stains. Even so, it must be acknowledged that the reliability and accuracy of vital stains is untested for thousands of species of phytoplankton. Introduced in 1951, the SDC-MPN method for phytoplankton is an established approach for use with multi-species communities. As applied to ballast water testing, SDC-MPN is much less vulnerable to methodological uncertainties than has been assumed. Notably, all species of phytoplankton need not be cultured in the conventional sense. Rather, a single viable cell in a dilution tube need grow only enough to be detected — a requirement known to have been met by otherwise uncultured species. Further, delayed restoration of viability after treatment with ultraviolet radiation (UV) is not a problem: organisms repair UV damage quickly or not at all, consistent with the assumptions of the test. Two critical methodological failures could compromise protection of the environment in ballast water testing: living organisms that do not stain or move, and viable organisms that do not grow to detection in the MPN cultures. These can be assessed with complementary measurements, but importantly, the relative protection of each method can be evaluated by comparing counts of living cells from the ETV Protocol with counts of viable cell from SDC-MPN in untreated samples. Available evidence provides no basis for concluding that either method is consistently less protective. However, as applied in ballast water testing, the statistical estimate of MPN is less precise. On this basis, SDC-MPN is worse for a single test. But, counter-intuitively, it is more protective of the environment when five consecutive tests must be passed for type approval, because the likelihood of one false rejection out of five tests is higher and five false passes would be exceedingly rare. Addressing only the science, we conclude that both the ETV Protocol and the SDC-MPN method, though imperfect, are currently appropriate for assessing the efficacy of ballast water management systems in a type-approval testing regime. In closing, we show proof of concept for a rapid assay of viability, benchmarked against SDC-MPN, that could be well suited for routine assessment of treatment system performance.