Ceratocystis platani is the causal agent of canker stain of plane trees, a lethal disease, able to kill mature trees in one or two successive growing seasons. The pathogen is a quarantine organism and has a negative impact on anthropogenic and natural populations of plane trees. Contaminated sawdust produced during pruning and sanitation fellings can contribute to disease spread. The goal of this study was to design a rapid, real-time quantitative PCR assay to detect C. platani airborne inoculum. Airborne inoculum traps (AITs), were placed in an urban in the city of Florence (Italy), where the disease was present. Primers and TaqMan® MGB probes were designed to target cerato-platanin (CP) and internal transcribed spacer (ITS2) genes. The detection limit of the assay was 0.05 pg/?l and 2 fg/?l of fungal DNA for CP and ITS, respectively. Pathogen detection directly from AITs demonstrated specificity and high sensitivity for C. platani detecting DNA concentrations as low as 1.2-1.4 x10-2 pg/?l, corresponding to approximately 10 conidia per ml. Airborne inoculum traps were able to detect C. platani inoculum within 200 meters of the closest symptomatic infected plane tree. The combination of airborne trapping and real-time quantitative PCR assay provides a rapid and sensitive method for the specific detection of C. platani inoculum. This technique may be used to identify the period of highest risk of pathogen spread in a site, thus helping the disease management.
Rapid detection of Ceratocystis platani inoculum by quantitative real-time PCR assay
Luchi N;Ghelardini L;Santini A
2013
Abstract
Ceratocystis platani is the causal agent of canker stain of plane trees, a lethal disease, able to kill mature trees in one or two successive growing seasons. The pathogen is a quarantine organism and has a negative impact on anthropogenic and natural populations of plane trees. Contaminated sawdust produced during pruning and sanitation fellings can contribute to disease spread. The goal of this study was to design a rapid, real-time quantitative PCR assay to detect C. platani airborne inoculum. Airborne inoculum traps (AITs), were placed in an urban in the city of Florence (Italy), where the disease was present. Primers and TaqMan® MGB probes were designed to target cerato-platanin (CP) and internal transcribed spacer (ITS2) genes. The detection limit of the assay was 0.05 pg/?l and 2 fg/?l of fungal DNA for CP and ITS, respectively. Pathogen detection directly from AITs demonstrated specificity and high sensitivity for C. platani detecting DNA concentrations as low as 1.2-1.4 x10-2 pg/?l, corresponding to approximately 10 conidia per ml. Airborne inoculum traps were able to detect C. platani inoculum within 200 meters of the closest symptomatic infected plane tree. The combination of airborne trapping and real-time quantitative PCR assay provides a rapid and sensitive method for the specific detection of C. platani inoculum. This technique may be used to identify the period of highest risk of pathogen spread in a site, thus helping the disease management.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
