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 : qPCR detection of Leptosphaeria airborne inoculum

Abstract

BACKGROUND: Detection of the inoculum of phytopathogens greatly assists in the management of diseases, but is difficult for pathogens with airborne fungal propagules. Here, we present experiments to determine 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 in Poznan, Poland and Harpenden, UK. Fungal detection and species discrimination were achieved using a SYBR-Green quantitative polymerase chain reaction (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 fewer than five spores per m 3 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 the specificity and sensitivity of the published SYBR-Green assays were also found, indicating that the Liu primers did not detect L. biglobosa subclade ‘canadensis’, whereas the Mahuku primers detected L. biglobosa subclade ‘canadensis’ and also the closely related Plenodomus dezfulensis. CONCLUSIONS: Comparisons confirmed 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.