A Real-Time PCR Assay for the Quantification of Plasmopara viticola Oospores in Grapevine Leaves

Abstract

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 (R 2 = 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% noninfested 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.

The Study of the Germination Dynamics of Plasmopara viticola Oospores Highlights the Presence of Phenotypic Synchrony With the Host

“…Even if alternative forms of survival have been recently postulated, thanks to the discovery of special P. viticola structures inside grapevine tissues ( Fröbel and Zyprian, 2019 ), the sexual spores are considered to be the main survival structure of the pathogen. The oospores are large (25–50 μm diameter) thick-walled sexual spores that form, with different densities, within leaf tissues ( Lehoczky, 1956 ; Si Ammour et al, 2020 ). They overwinter on the soil surface and germinate in the following grapevine-growing season, producing the inoculum for primary infections.…”

Exploring the association between latent Plasmopara viticola infection and downy mildew epidemic in commercial vineyards: Application of qPCR assay

“…This study used a well-established qPCR assay to accurately quantify P. viticola in asymptomatic leaves from commercial vineyards, enabling a comprehensive assessment of GDM progression. The employed qPCR methods were initially designed by Valsesia et al (2005) specifically for P. viticola and have been extensively validated in previous research (Ammour et al, 2020;Yang et al, 2023).…”

“…The multiplex quantitative PCR (qPCR) assay has emerged as highly sensitive and specific, making it a reliable tool for the targeted screening of specific fungal DNA. This assay facilitates the detection of potential pathogens within mixed populations and allows quantification of very low infection levels (Ammour et al., 2020; Chu et al., 2019; Yang et al., 2023). The qPCR assay outperforms traditional PCR assays in detecting and quantifying DNA from various sources, including soilborne fungi (Luo et al., 2009), airborne spores (Heger et al., 2022), oomycetes (Sanzani et al., 2014) and plant tissues (Ammour et al., 2020).…”

“…This assay facilitates the detection of potential pathogens within mixed populations and allows quantification of very low infection levels (Ammour et al., 2020; Chu et al., 2019; Yang et al., 2023). The qPCR assay outperforms traditional PCR assays in detecting and quantifying DNA from various sources, including soilborne fungi (Luo et al., 2009), airborne spores (Heger et al., 2022), oomycetes (Sanzani et al., 2014) and plant tissues (Ammour et al., 2020). Furthermore, the quantitative and rapid detection capabilities of the qPCR make it invaluable for epidemiological studies.…”

Use of a real-time PCR method to quantify the primary infection of Plasmopara viticola in commercial vineyards in northwestern China

“…2007; Rossi et al 2011; Wong et al 2001). During the winter, oospores reach morphological maturity but are prevented from germinating due to dormancy; when dormancy is broken, oospores are considered physiologically mature and capable of germinating under favorable environmental conditions and are formed macro-sporangia (Ammour et al 2020;Brischetto et al 2020; Kennelly et al 2007; Rossi and Ca . 2007).…”