Multiplex qPCR for detection and quantification of Sphaerulina musiva in Populus stems

Abstract

Septoria canker is one of the major limiting factors in the use of hybrid poplar in agroforestry. The use of qPCR for quantification of fungal biomass in host tissue following infection can improve the speed and accuracy of both detection and phenotyping, potentially improving the management of this disease. This study reports the use of a multiplexed hydrolysis probe qPCR assay to detect and quantify the pathogen Sphaerulina musiva in artificially inoculated and naturally infected Populus trees. Species‐specific probes and primers were designed based on conserved gene sequences of S. musiva β‐tubulin and Populus spp. eukaryotic initiation factor IV (eIF4AII). Experiments were performed on greenhouse‐inoculated poplar stems at 1, 3 and 7 weeks post‐inoculation. The qPCR assay detected significant differences among clones and isolates in symptomless samples after 1 week. These differences were not apparent by visual phenotyping. The assay also detected differences among isolates, collected from a range of geographic locations, 3 weeks post‐inoculation. For naturally infected samples, the S. musiva detection rate ranged from 92% to 100%. The primers also proved robust for direct endpoint PCR‐based detection of S. musiva spores, with a minimum detection threshold of 10 spores. The primers proved to be sensitive and specific for S. musiva detection, making them a useful tool for diagnostics and comparisons among isolates and clones in the early stages of infection.