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A Real-Time PCR Assay for the Quantification of Plasmopara viticola Oospores in Grapevine Leaves.
Frontiers in Plant Science ( IF 4.1 ) Pub Date : 2020-07-24 , DOI: 10.3389/fpls.2020.01202
Melissa Si Ammour 1 , Federica Bove 1 , Silvia Laura Toffolatti 2 , Vittorio Rossi 1
Affiliation  

Grapevine downy mildew caused by Plasmopara viticola is one of the most important diseases in vineyards. Oospores that overwinter in the leaf litter above the soil are the sole source of inoculum for primary infections of P. viticola; in addition to triggering the first infections in the season, the oospores in leaf litter also contribute to disease development during the season. In the current study, a quantitative polymerase chain reaction (qPCR) method that was previously developed to detect P. viticola DNA in fresh grapevine leaves was assessed for its ability to quantify P. viticola oospores in diseased, senescent grapevine leaves. The qPCR assay was specific to P. viticola and sensitive to decreasing amounts of both genomic DNA and numbers of P. viticola oospores used to generate qPCR standard curves. When the qPCR assay was compared to microscope counts of oospores in leaves with different levels of P. viticola infestation, a strong linear relationship (R2 = 0.70) was obtained between the numbers of P. viticola oospores per gram of leaves as determined by qPCR vs. microscopic observation. Unlike microscopic observation, the qPCR assay was able to detect significant differences between leaf samples with a low level of oospore infestation (25% infested leaves and 75% non-infested leaves) vs. samples without infestation, and this ability was not influenced by the weight of the leaf sample. The results indicate that the qPCR method is sensitive and provides reliable estimates of the number of P. viticola oospores in grapevine leaves. Additional research is needed to determine whether the qPCR method is useful for quantifying P. viticola oospores in grapevine leaf litter.



中文翻译:

实时PCR检测葡萄叶中葡萄毛囊虫的孢子定量。

葡萄霜霉病引起的 葡萄纤维单胞菌是葡萄园中最重要的疾病之一。在土壤上方的枯枝落叶中越冬的卵孢子是最初感染烟草的唯一接种源。葡萄; 除了引发本季节的首次感染外,枯枝落叶中的卵孢子还有助于季节内疾病的发展。在当前的研究中,定量聚合酶链反应(qPCR)方法以前被开发用于检测葡萄 评估新鲜葡萄叶中的DNA定量能力 葡萄患病的,衰老的葡萄叶片中的卵孢子。qPCR检测特定于葡萄 对减少基因组DNA的数量和 葡萄卵孢子用于产生qPCR标准曲线。当将qPCR分析与显微镜下不同水平的叶片中卵孢子的计数进行比较时葡萄的侵扰,之间的数字之间获得了很强的线性关系(R 2 = 0.70)葡萄通过qPCR vs.显微镜观察确定的每克叶子的卵孢子。与显微镜观察不同,qPCR分析能够检测出卵孢子侵染水平较低(25%受侵害叶片和75%非侵害叶片)与未侵染的叶片样品之间的显着差异,并且该能力不受细菌侵染的影响。叶样品的重量。结果表明,qPCR方法灵敏,可提供可靠的估计数量。葡萄葡萄叶中的卵孢子。需要进行其他研究以确定qPCR方法是否可用于定量葡萄 葡萄叶凋落物中的卵孢子。

更新日期:2020-08-08
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