A quantitative real-time PCR assay for early detection and quantification of Ramularia mali , an emerging pathogen of apple causing dry lenticel rot.

i is an emerging pathogen of apple () causing dry lenticel rot. No preventive measures have been adopted due to the lack of knowledge about the life cycle and epidemiology of this pathogen. In a preliminary survey to identify the agents of dry lenticel rot of apple, was constantly associated to the disease. Using isolates from this survey, a SYBR Green qPCR assay was developed, using calmodulin as target gene, for the detection and quantification of in apple fruits. The qPCR assay was validated in terms of specificity, sensitivity, repeatability and reproducibility following the international EPPO standard PM 7/98. The primers amplified a region of 237 bp specific to R. mali. The specificity was validated with 20 fungal species commonly found on apple, 36 strains of and closely related species of the species complex. Positive amplifications were obtained only with DNA of and no cross-reaction was detected with the other fungal species. Sensitivity was assessed with serial dilutions of target DNA and the limit of detection was 100 fg. No influence of host DNA was observed when target DNA was diluted on the DNA of 'Ambrosia' and 'Golden Delicious' apple. The assay permitted to detect and quantify R. mali in symptomatic and asymptomatic fruits. The presence of on asymptomatic 'Ambrosia' and 'Golden Delicious' apples was demonstrated, and the presence of the pathogen was reported for the first time on 'Jeromine', 'Gala', 'Opal' and 'Story Inored' fruits. This assay could be useful to clarify the life cycle of this pathogen in order to build up an effective disease management strategy. Furthermore, the early detection of the pathogen on asymptomatic apples could be used to forecast the development of dry lenticel rot, supporting the packinghouse operators in deciding the storage length of apple lots.