Efficient qPCR estimation and discrimination of airborne inoculum of Leptosphaeria maculans and L. biglobosa, the causal organisms of phoma leaf spotting and stem canker of oilseed rape

BACKGROUND: The detection of inoculum of phytopathogens greatly assists in the management of diseases, but it is difficult for those pathogens with airborne fungal propagules. In this paper, we present experiments on the determination of the abundance and distribution frequencies of the ascospores of Leptosphaeria (Plenodomus) species that were collected on the tapes of volumetric Hirst-type traps near oilseed rape fields of Poznan, Poland and Harpenden, UK. Fungal detection and species discrimination were achieved using SYBR-Green qPCR with two different pairs of primers previously reported to differentiate Leptosphaeria maculans (Plenodomus lingam) or L. biglobosa (P. biglobosus). RESULTS: Detection was successful even at less than 5 spores per m3 of air. The primer pairs differed in the correlation coefficients obtained between DNA yields and the daily abundance of ascospores that were quantified by microscopy on duplicate halves of the spore trap tapes. Important differences in specificity and sensitivity of the published SYBR Green assays were also found, indicating that the Liu primers did not detect L. biglobosa subclade ‘canadensis’ while the Mahuku primers detected L. biglobosa subclade ‘canadensis’ and also the closely-related Plenodomus dezfulensis. CONCLUSIONS: Comparisons affirmed that application of qPCR assays to spore trap samples can be used for the early detection, discrimination and quantification of aerially dispersed L. maculans and L. biglobosa propagules before leaf spot symptoms are visible in winter oilseed rape fields. The specificity of the primers must be taken into consideration because the final result will greatly depend on the local population of the pathogen.