In this study, we report on the development of two quantitative PCR assays for the detection and quantification of the soilborne plant pathogen Phytophthora cactorum causing root and crown rot of strawberry. The assays rely on the use of SYBR Green I chemistry and use the internal transcribed spacer region 2 (ITS2) and the Ras-related protein gene Ypt1 as detection markers. An extensive list of Phytophthora isolates was included in the study to evaluate assay specificity. High specificity was obtained when Ypt1 was used as detection marker, with cross-reactions only occurring with the closest relatives of P. cactorum, including P. hedraiandra, P. idaei and P. pseudotsugae. In contrast, the ITS-based assay was less specific, resulting in a larger number of cross-reactions (eight species when tested at 10 pg DNA). In sensitivity tests, P. cactorum DNA was detected down to 10 fg using the ITS-based assay, while the limit of detection (LOD) for the Ypt1-based assay was 1 pg DNA, irrespective of the presence of background DNA from strawberry or soil. When strawberry, growth substrate and soil samples were spiked with a 10-fold dilution series of P. cactorum zoospores, the LOD for the ITS-based assay was 10 zoospores g⁻¹ plant material, growth substrate or soil. The limit of quantification (LOQ) of the assay was 109 zoospores g⁻¹ plant material, 15.8 zoospores g⁻¹ growth substrate, and 106 zoospores g⁻¹ soil. For the Ypt1-based assay, the LOD and LOQ values were 1000 and 5910 zoospores g⁻¹ plant material, 1000 and 1818 zoospores g⁻¹ growth substrate, and 1000 and 3823 zoospores g⁻¹ soil, respectively. In terms of detection, analysis of field samples suggested that the Ypt1-based assay almost performed equally well compared to the ITS-based assay, especially for plant and growth substrate samples. Further, our results showed that both qPCR assays outperformed classical plating, illustrating their power to be used for diagnostic purposes. Interestingly, P. cactorum was also detected in soil and growth substrate samples from areas with no symptoms, suggesting that the assays can also aid in early pathogen detection before the disease manifests.

在这项研究中，我们报告了两种定量 PCR 测定法的发展，用于检测和量化引起草莓根腐病和冠腐病的土壤传播植物病原体Phytophthora cactorum 。该检测依赖于 SYBR Green I 化学的使用，并使用内部转录间隔区 2 (ITS2) 和 Ras 相关蛋白基因Ypt1作为检测标记。研究中包括了大量疫霉分离株，以评估检测特异性。当获得高特异性Ypt1用作检测标记物，具有交叉反应只与近亲发生P.恶疫霉，包括P. hedraiandra，P. idaei和P. pseudotsugae。相比之下，基于 ITS 的检测特异性较低，导致更多的交叉反应（在 10 pg DNA 下测试时有 8 个物种）。在敏感性测试中，使用基于 ITS 的测定法检测到的P. cactorum DNA 低至 10 fg，而基于Ypt1的测定法的检测限 (LOD) 为1 pg DNA，无论是否存在来自草莓或土壤。当草莓、生长基质和土壤样品加入 10 倍稀释系列的P. cactorum游动孢子时，基于 ITS 的测定的 LOD 是 10 个游动孢子 g ^-1植物材料、生长基质或土壤。该测定的定量限 (LOQ) 为 109 个游动孢子 g^-1植物材料、15.8 个游动孢子 g ^-1生长基质和 106 个游动孢子 g ^-1土壤。对于基于Ypt1的测定，LOD 和 LOQ 值分别为 1000 和 5910 个游动孢子 g ^-1植物材料、1000 和 1818 个游动孢子 g ^-1生长基质以及 1000 和 3823 个游动孢子 g ^-1土壤。在检测方面，对田间样品的分析表明，与基于 ITS 的检测相比，基于Ypt1的检测几乎表现得同样好，尤其是对于植物和生长基质样品。此外，我们的结果表明，这两种 qPCR 检测都优于经典电镀，说明它们可用于诊断目的。有趣的是，在来自无症状地区的土壤和生长基质样本中也检测到了P. cactorum，这表明该检测还有助于在疾病出现之前进行早期病原体检测。