Crop Diversification Centre South, Alberta Agriculture and Forestry, Brooks, AB T1R 1E6, Canada.
Crop Diversification Centre North, Alberta Agriculture and Forestry, Edmonton, AB T5Y 6H3, Canada.
Plant Dis. 2019 Sep;103(9):2330-2336. doi: 10.1094/PDIS-05-18-0855-RE. Epub 2019 Jul 12.
Clubroot caused by is an important disease of brassica crops. The use of vital stains to determine the viability of 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 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 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 ( = 96.88; ≤ 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 resting spore viability. Additionally, it was equivalent to the qPCR method for differentiating viable and nonviable spores ( = 99.84; ≤ 0.001) and confirmed in canola infection bioassays.
由 引起的根肿病是十字花科作物的一种重要病害。使用活体染料来确定休止孢子的活力可以提供有关孢子寿命、接种体潜力或抗菌处理效果的有用信息。吖啶橙是一种已被报道可区分活的和非活的休止孢子的活体染料的例子。一些以前发表的使用吖啶橙染色 休止孢子的方案没有提供准确或一致的结果。在这项研究中，我们通过将染色时间延长至 8 小时或更长时间来改进吖啶橙方法，并评估了在不同温度和时间组合下热处理后的 休止孢子。将染色时间延长至 7 小时以上，显著增加了染色休止孢子的数量，之后染色孢子的数量没有显著变化（ = 96.88； ≤ 0.001）。通过重复实验和将染色数据与接种试验和吖啶橙单染定量 PCR（qPCR）得出的数据进行比较，评估了改进方法区分活孢子和非活孢子的准确性。结果表明，改进的吖啶橙染色方法提高了休止孢子活力测量的准确性和一致性。此外，它与 qPCR 方法在区分活孢子和非活孢子方面等效（ = 99.84； ≤ 0.001），并在油菜感染生物测定中得到了验证。
