Development of diagnostic tools for quantitative detection of Apiospora marii using qPCR and ddPCR technologies

Apiospora marii (sin. Arthrinium marii) is an ascomycete recently associated with olive tree dieback in Italy and Spain. With this study, quantitative (q)PCR and digital droplet (dd)PCR protocols were developed and validated to detect and quantify A. marii. Two primers/probe sets (AM135 and AM158) were designed on the ITS sequences of the fungus. The optimization of the PCR conditions allowed to identify 60 °C as the best annealing temperature and 500/250 nM as the best primers/probe concentration in both qPCR and ddPCR. Under these conditions, up to 1 fg μL-1 of DNA of the A. marii DiSSPA_A1 was detected in qPCR (Cq=34 for both AM135 and AM158). The same DNA concentration was the lowest one detected in ddPCR after 40 cycles of amplification and corresponded to 0.20 and 0.12 DNA copies μL-1, respectively for AM135 and AM158. The specificity of both primer/probe sets was tested against a panel of microorganisms commonly associated with olive wood of several olive varieties. Untargeted amplifications were observed after 35 cycles, using the DNA of Armillaria mellea, Pseudophaeomoniella oleae, and Verticillium dahliae, and in accordance with the qPCR – LoD was Cq 35. For the assay validation, healthy and A. marii artificially-inoculated olive plants were analysed simultaneously in qPCR assay and on potato dextrose agar medium, confirming a sound performance of the qPCR assay. Results achieved in this work let to improve monitoring and surveillance of this new olive pathogen.