A quantitative real-time polymerase chain reaction assay for Botrytis aclada in onion bulb tissue

Abstract

A real-time polymerase chain reaction (PCR) assay has been developed for the detection and quantification of Botrytis aclada (Fresenius), a causal agent of neck rot in onion (Allium cepa L.) bulbs. The assay uses TaqMan probe-based chemistry to detect an amplicon from the L45-550 region of B. aclada while using a DNA sequence from the onion serine acetyl transferase gene (SAT1) as a control. The assay detection limits for B. aclada and onion were 10 pg·μL-1 of genomic DNA. The detection limit for lyophilized B. aclada mycelium was 1 μg. The presence of onion tissue in the samples did not affect the performance of the real-time PCR assay. The assay distinguished among different amounts of B. aclada mycelium growing on onion disks that were inoculated with 0, 102, or 104 B. aclada conidia. Visual observations during the incubation period corresponded with changes in real-time PCR results. This assay could be used to determine the amount of B. aclada mycelium in bulbs during growth, harvest, and storage, thus giving researchers an objective and efficient tool by which to quantify the growth rate and virulence of B. aclada strains in vivo.