Analyses of air samples for ascospores of Leptosphaeria maculans and L.biglobosa by light microscopy and molecular
J Kaczmarek1, M Jedryczka, B D L Fitt
1Institute of Plant Genetics, Polish Academy of Sciences, Poznan, Poland. jkac@igr.poznan.pl
Abstract:
Spores of many fungal pathogens are dispersed by wind. Detection of these airborne inocula is important in forecasting both the onset and the risk of epiphytotics. Species-specific primers targeted at the internal transcribed spacer (ITS) region of Leptosphaeria maculans and L. biglobosa - the causal organisms of phoma stem canker and stem lesions of Brassica spp., including oilseed rape - were used to detect DNA extracted from particles deposited on tapes obtained from a spore trap operated in Rarwino (northwest Poland) from September to November in 2004 and 2006. The quantities of DNA assessed by traditional end-point PCR and quantitative real-time PCR were compared to microscopic counts of airborne ascospores. Results of this study showed that fluctuations in timing of ascospore release corresponded to the dynamics of combined concentrations of DNA from L. maculans and L. biglobosa, with significant positive correlations between ascospore number and DNA yield. Thus the utilization of PCR-based molecular diagnostic techniques enabled the detection, identification, and accurate quantification of airborne inoculum at the species level. Moreover, real-time PCR was more sensitive than traditional PCR, especially in years with low ascospore numbers.
Insights
Detecting airborne fungal spores using DNA analysis is key for disease forecasting in crops like oilseed rape. Molecular techniques, especially real-time PCR, accurately quantify fungal pathogens, improving epiphytotic risk assessment.
Area of Science:
- Plant Pathology
- Molecular Biology
- Aerobiology
Background:
- Airborne fungal spores are critical for plant disease epiphytotics.
- Accurate detection of airborne inoculum aids in disease forecasting.
- Leptosphaeria maculans and L. biglobosa cause significant Brassica spp. diseases.
Purpose of the Study:
- To compare traditional PCR and real-time PCR for detecting airborne fungal DNA.
- To correlate DNA quantification with microscopic spore counts.
- To assess the utility of molecular methods for species-level identification of airborne pathogens.
Main Methods:
- DNA extraction from spore trap samples.
- Species-specific PCR targeting the ITS region of L. maculans and L. biglobosa.
- End-point PCR and quantitative real-time PCR for DNA quantification.
- Comparison with microscopic ascospore counts.
Main Results:
- Ascospore release timing correlated with L. maculans and L. biglobosa DNA concentrations.
- Significant positive correlation between ascospore number and DNA yield.
- Real-time PCR demonstrated higher sensitivity than end-point PCR, particularly in low-spore conditions.
Conclusions:
- PCR-based methods enable accurate, species-level detection and quantification of airborne fungal inoculum.
- Real-time PCR is a sensitive tool for monitoring airborne pathogens.
- This approach enhances forecasting of plant disease risk.
