Development of a real-time PCR protocol for the specific detection and quantification of Penicillium digitatum in lemons

Abstract

Penicillium digitatum is the main species responsible for postharvest losses in citrus fruit and, therefore, rapid and accurate methods are needed for its early detection. For this purpose, a new procedure based on real-time quantitative PCR (qPCR) was developed to detect and quantify P. digitatum in lemons. The procedure included a rapid extraction of fungal DNA using the commercial Quick-DNA Fungal/Bacterial Miniprep Kit; the design of a specific primers pair by selecting the conserved region of the calmodulin A (cmdA) gene; and the design and evaluation of a qPCR system based on SYBR-Green dye detection chemistry. The functionality of the developed method was demonstrated by the high linear correlation of the standard curve constructed over the entire range of fungal DNA concentrations used (R2 > 0.99), indicating precision and accuracy of the qPCR. The protocol was developed aiming to evaluate the biotechnological potential of the biocontrol yeast Clavispora lusitaniae AgL21, by quantifying the fungus P. digitatum in lemon wounds. The yeast was shown to restrict the pathogen growth, reaching fungal DNA concentrations of up to 0.038 ng/wound after 4 days of incubation, compared to 173.06 ng/wound when the fungus was alone. No inhibition of qPCR due to food matrices was observed. The developed qPCR protocol could be considered as a suitable tool to detect P. digitatum in food products and can be used to specifically test the ability of new biological control microorganisms against green mold in lemons.