A spectrophotometric method for determining the viability of sporangia and zoospores of the oomycete Plasmopara viticola (causal agent of grapevine downy mildew) is described and evaluated to overcome the limitations of currently available methods for assessing propagule viability. Sporangia produced on leaf discs in the laboratory were harvested at different days after the initiation of sporulation (DAS) to yield differences in sporangium viability. Sporangia were suspended in sterile water, the suspensions were placed in a cuvette, and sporangium germination was monitored in a spectrophotometer (λ = 600 nm) at 2‐ to 3‐min intervals for 5 hr. Absorbance started to increase after sporangia were suspended in water for ~30–60 min followed by major peak(s) for younger sporangia (1–3 DAS), whereas low to no increase in absorbance was observed for senescent sporangia (>7 DAS). Microscopic observation confirmed that the increase in absorbance corresponded to the release and active swimming of zoospores, whereas absorbance decreased when zoospores encysted and settled. A positive correlation (r = .839, p = .0365) was observed when the time to the initial increase in absorbance was plotted against the age of sporangia. The time to the absorbance peak (marking the time of maximum zoospore movement) was shortest for immature sporangia (0 DAS), longest for young sporangia (2 DAS) and decreased for mature and senescent sporangia. A similar pattern was observed for the standardized area under the absorbance curve (indicating the overall quantity of zoospores released), for which values were lowest for immature and senescent sporangia, highest for young sporangia and intermediate for mature sporangia. Consistent patterns obtained across two independent experiments suggest that the method is reproducible and may be further developed for other zoospore‐releasing pathogens.

中文翻译：

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一种测定葡萄孢霉孢子囊和游动孢子活力的分光光度法

描述并评估了一种用于确定卵菌 Plasmopara viticola（葡萄霜霉病的病原体）孢子囊和游动孢子活力的分光光度法，以克服目前可用的评估繁殖体活力方法的局限性。在孢子形成 (DAS) 开始后的不同天数收获在实验室叶盘上产生的孢子囊，以产生孢子囊活力的差异。孢子囊悬浮在无菌水中，将悬浮液置于比色皿中，并在分光光度计（λ = 600 nm）中以 2 至 3 分钟的间隔监测孢子囊萌发 5 小时。孢子囊在水中悬浮约 30-60 分钟后，吸光度开始增加，随后出现年轻孢子囊（1-3 DAS）的主要峰值，而对于衰老的孢子囊（> 7 DAS）观察到吸光度的增加很少或没有增加。显微镜观察证实，吸光度的增加与游动孢子的释放和活跃游动相对应，而当游动孢子成囊和沉降时吸光度下降。当将吸光度初始增加的时间与孢子囊的年龄作图时，观察到正相关（r = .839，p = .0365）。吸光度峰值的时间（标志着最大游动孢子运动的时间）对于未成熟孢子囊（0 DAS）最短，对于年轻孢子囊（2 DAS）最长，成熟和衰老孢子囊减少。吸光度曲线下的标准化面积（表示释放的游动孢子的总量）观察到类似的模式，未成熟和衰老孢子囊的值最低，幼孢子囊最高，成熟孢子囊中等。在两个独立实验中获得的一致模式表明该方法是可重复的，并且可以进一步开发用于其他释放游动孢子的病原体。