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

Melissa Si Ammour,Vittorio Rossi,Silvia Laura Toffolatti,Federica Bove

doi:10.3389/fpls.2020.01202

Melissa Si Ammour, Vittorio Rossi + Show 2 more

Open Access

https://doi.org/10.3389/fpls.2020.01202

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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 (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.

Highlights

Diseases represent a constant threat to grapevine production and may cause considerable yield and economic losses
The germination of oospores requires a minimum temperature of 12–13°C (Laviola et al, 1986; Gessler et al, 2011), and requires the moistening of the leaf litter by rainfall or water flux from the atmosphere (Arens, 1929; Rossi et al, 2008a; Rossi et al, 2013)
In late May of 2018, sporangia of P. viticola were collected from leaves showing typical downy mildew (DM) lesions with fresh sporulation on the abaxial surface of the leaf blade

Summary

Introduction

Diseases represent a constant threat to grapevine production and may cause considerable yield and economic losses. Oospores represent the sexual stage of P. viticola and are formed after the fusion of an antheridium and an oogonium in the affected leaf tissue from mid-summer to autumn (Burruano, 2000; Rossi et al, 2013). The formation of oospores does not require particular temperatures but seems to be favoured by dry conditions (which impede asexual sporulation) or by leaf senescence (Gessler et al, 2011). Germination of morphologically mature oospores is prevented by dormancy (Galet, 1977; Rossi and Caffi, 2007), a process regulated by the environment, nutrient permeability, and endogenous inhibitors. When the dormancy is broken, oospores are considered physiologically mature and are able to germinate under favorable environmental conditions (Rossi and Caffi, 2007). Some oospores remain dormant but viable for an entire season or even for several years (Kennelly et al, 2007; Caffi et al, 2011)

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