Harper Adams University College, Newport, Shropshire, UK.
J Appl Microbiol. 2010 Jan;108(1):81-7. doi: 10.1111/j.1365-2672.2009.04410.x.
To evaluate competitive PCR assays for quantifying seed-borne Microdochium and Fusarium seedling blight pathogen DNA and to determine test and year repeatability and sources of variability.
Relationships between DNA and plate counts were significant for Fusarium and Microdochium seedling blight pathogens in 152 seed batches from 3 years. Coefficient of determinations, however, differed greatly (Fusarium; R(2) = 0.25, P = 0.029, Microdochium; R(2) = 0.73, P < 0.001). Significant differences between years were observed in the regression slopes for Microdochium. Pathogen DNA quantified in 16 extractions after sampling was highly correlated to results following storage for 1-2 years (R(2) > 0.90). Residual maximum likelihood analysis showed that the least and greatest variance components of the testing procedure were DNA extraction subsampling and PCR assay respectively.
Amount of pathogen DNA is a useful estimator of seed batch contamination for Microdochium but not Fusarium seedling blight pathogens. Although reproducible over time, improvements to the testing procedure should focus on repeated PCR amplifications to reduce assay variability.
Replacing plate counts with competitive PCR for determining the severity of seed batch contamination is feasible in areas where Microdochium seedling blight pathogens predominate.
评估竞争 PCR 检测方法，用于定量种子传播的 Microdochium 和镰刀菌幼苗枯萎病病原菌 DNA，并确定检测和年度重复性以及变异源。
在来自 3 年的 152 批种子中，病原菌 DNA 与平板计数之间的关系对于镰刀菌和 Microdochium 幼苗枯萎病病原菌具有显著意义。然而，决定系数差异很大（镰刀菌；R²=0.25，P=0.029，Microdochium；R²=0.73，P<0.001）。在 Microdochium 中，还观察到回归斜率在年份之间存在显著差异。在采样后进行的 16 次提取中定量的病原菌 DNA 与存储 1-2 年后的结果高度相关（R²>0.90）。残差最大似然分析表明，检测程序的最小和最大方差分量分别是 DNA 提取的亚采样和 PCR 检测。
病原菌 DNA 的数量是 Microdochium 种子批次污染的有用估计值，但不是镰刀菌幼苗枯萎病病原菌的估计值。尽管随着时间的推移具有可重复性，但测试程序的改进应侧重于重复的 PCR 扩增，以减少检测变异性。
在 Microdochium 幼苗枯萎病病原菌占主导地位的地区，用竞争 PCR 代替平板计数来确定种子批次污染的严重程度是可行的。
