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: 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 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 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. © 2023 Society of Chemical Industry.

Effect of climate change on sporulation of the teleomorphs of Leptosphaeria species causing stem canker of brassicas.

Leptosphaeria maculans and L. biglobosa are closely related sibling fungal pathogens that cause phoma leaf spotting, stem canker (blackleg) and stem necrosis of oilseed rape (Brassica napus). The disease is distributed worldwide, and it is one of the main causes of considerable decrease in seed yield and quality. Information about the time of ascospore release at a particular location provides important data for decision making in plant protection, thereby enabling fungicides to be used only when necessary and at optimal times and doses. Although the pathogens have been studied very extensively, the effect of climate change on the frequencies and distributions of their aerially dispersed primary inoculum has not been reported to date. We have collected a large dataset of spore counts from Poznan, located in central-west part of Poland, and studied the relationships between climate and the daily concentrations of airborne propagules over a period of 17 years (1998-2014). The average air temperature and precipitation for the time of development of pseudothecia and ascospore release (July-November), increased during the years under study at the rates of 0.1 °C and 6.3 mm per year. The day of the year (DOY) for the first detection of spores, as well as the date with maximum of spores, shifted from 270 to 248 DOY, and from 315 to 265 DOY, respectively. The acceleration of the former parameter by 22 days and the latter by 50 days has great influence on the severity of stem canker of oilseed rape.

Molecular screening for avirulence alleles AvrLm1 and AvrLm6 in airborne inoculum of Leptosphaeria maculans and winter oilseed rape (Brassica napus) plants from Poland and the UK.

A combination of staining, light microscopy and SYBR green- and dual-labelled fluorescent probe-based qPCR chemistries with species- and gene-specific primers was employed to evaluate fluctuations in the aerial biomass of Leptosphaeria maculans spores captured by volumetric spore trappings in Poznan, Poland (2006, 2008) and Harpenden, UK (2002, 2006). Arising from these surveys, DNA samples extracted from Burkard spore-trap tapes were screened for fluctuation patterns in the frequencies of AvrLm1 and AvrLm6, the most prominent of the 15 genes that code for avirulence effectors in this Dothideomycete cause of the destructive phoma stem canker disease of oilseed rape worldwide. In Poznan, very low frequencies of AvrLm1 allele were found in the autumn of both 2006 and 2008, reflecting significantly increased cultivation of rape seed with Rlm1-based resistance. In contrast, at least six folds-higher frequencies of AvrLm6, which were also confirmed by end-point PCR bioassays on phoma-infected leaves from the same region of Poland, were obtained during both years. In the UK, however, relatively higher AvrLm1 allele titres were found in L. maculans spores captured in air samples from the autumn of 2002 on the experimental fields of Rothamsted Research, Harpenden, that were historically sown to genetically heterogeneous B. napus cultivars. In the 2006 screen these levels had plummeted, to a 1:4 ratio, in favour of frequencies of the AvrLm6 allele. Patterns of fluctuations in erg11 (CYP51) fragments coding for sterol 14α-demethylase suggest October as the month with the most viable wind-dispersed L. maculans propagules of each season of the screens.