A real-time PCR assay for detection and quantification of Botrytis cinerea in Pelargonium x hortorum plants, and its use for evaluation of plant resistance

Abstract

A reliable EvaGreen-based qPCR assay was developed for the relative quantification of fungal and plant DNAs in the Botrytis cinerea-Pelargonium× hortorum pathosystem. Primers, designed on RPB2 (DNA-dependent RNA polymerase subunit II) gene sequences directed the amplification of a specific 149 bp product for B. cinerea. No cross-reactivity was observed in DNA extracted from several nontarget fungi and bacterial, and in pelargonium plants, with the exception of the closely related species Botryotinia pelargonii and Botrytis fabae. This assay allowed for quantification of as little as 1.55 pg fungal DNA and 9.95 pg plant DNA, and detection of the pathogen in both symptomatic and asymptomatic pelargonium plants. The qPCR protocol is suitable for studying cultivar resistance and induced resistance in pelargonium plants, which requires accurate quantification of the pathogen in diseased host tissue. This assay allowed us to establish that the Pelargonium×hortorum cvs. 'XL Boomerang' and 'Eroica 2000' showed high foliar resistance to B. cinerea with respect to the susceptible cv. 'Fernando', and that spray applications of 0.3 mM acibenzolar-S methyl markedly protect pelargonium plants against gray mold. The protection for plants treated with chitosan (0.2 %) did not reach significance.

Metabolic and transcriptional changes associated with the use ofAscophyllum nodosumextracts as tools to improve the quality of wine grapes (Vitis viniferacv. Sangiovese) and their tolerance to biotic stress

“…Prior to their use, the surface of the detached berries was sterilized with 70% ethanol (v/v), rinsed in tap water, and wounded by sterile lancets, following the method advocated by Martinez‐Romero et al . Inoculation was performed with a suspension containing 1 × 10 5 conidia mL −1 of the PVUPG 240 isolate of the pathogen, which was prepared as described by Moretti et al . and sprayed with a hand atomizer.…”

“…27 berries, which were uniform in size, were sampled by cutting at the pedicel on October 8, 2015 (DOY 261) at 1000 h (2 hpt), when they were more susceptible to B. cinerea infection, 34 from those three vines per treatment not harvested. Prior to their use, the surface of the detached berries was sterilized with 70% ethanol (v/v), rinsed in tap water, and wounded by sterile lancets, following the method advocated by Martinez-Romero et al 35 Inoculation was performed with a suspension containing 1 × 10 5 conidia mL −1 of the PVUPG 240 isolate of the pathogen, which was prepared as described by Moretti et al 36 and sprayed with a hand atomizer. Inoculated ANE and NTV berries were incubated in growth chambers (one chamber per vine, each containing 32 berries) at 21 ± 2 ∘ C, 100% relative humidity (RH) and photoperiod of 12 h at 240 μmol m −2 s −1 .…”

A Mechanically Transmitted DNA Mycovirus Is Targeted by the Defence Machinery of Its Host, Botrytis cinerea

“…Unmethylated cytosines were identified as thymines instead of the encoded cytosine. Methylation rates were also determined for an internal control, the ITS region (accession number: KP151604) of B. cinerea [ 24 ].…”

Chip Digital PCR (cdPCR) to Identify and Quantify Botrytis cinerea Infection in Tomatoes

“…Si Ammour et al [14] used the qPCR methodology based on TaqMan chemistry as a reliable tool to quantify B. cinerea in grape bunch trash. An EVAGreen-based qPCR protocol was applied to the early detection of the fungus in pelargoniums (House geraniums) to determine the level of cultivar resistance [15].…”