Quantitative PCR analysis of abundance of airborne propagules of Leptosphaeria species in air samples from different regions of Poland

Abstract

When airborne propagules of Leptosphaeria maculans and L. biglobosa were collected in Poland at three ecologically different sites from 1 September to 30 November in 2004 to 2008, using a Hirst-type seven-day volumetric spore trap, there were fluctuations in timing of ascospore release and diverse ratios between airborne propagules of both species depending on season, field location and weather conditions. The detection was done using the microscope as well as quantitative PCR with species-specific primers targeted against fragments of β-tubulin genes and quantified with a duallabelled fluorescent probe approach. This detection chemistry is described for the first time for L. maculans and L. biglobosa. Its advantage over the previous ITS-based SYBR-Green chemistry resides in improved sensitivity and the virtual absence of false positives in the detection of these fungi.There were significant, positive correlations between data obtained using visual assessment of ascospore numbers and DNA concentrations that were measured by qPCR. Climatic differences between the oilseed rape growing regions could have significantly affected the biological processes of pseudothecial maturation and ascospore development of the pathogens. The data suggest that regular rain events of intermediate intensity recorded in the Maritime region favoured the maturation of the pathogen more than the drier weather recorded in the Silesia or Pomerania regions. It was observed that the number of rainy days was of greater importance than the cumulative rainfall to obtain the generative sporulation of the pathogen. Accurate detection of airborne inoculum of pathogenic Leptosphaeria spp. facilitates improved targeting of disease management decisions for oilseed rape protection against phoma stem canker and stem necrosis diseases.

Rapid Detection of Ceratocystis platani Inoculum by Quantitative Real-Time PCR Assay

“…However, qPCR has been also used to quantify the airborne inoculum of fungal pathogens, such as Sclerotinia sclerotiorum, Leptosphaeria spp., Puccinia striiformis, and Botrytis squamosa (38,39,40,41), which cause serious disease in arable crops, and also for the forest tree pathogen Fusarium circinatum (20,42). Many fungal diseases are initiated by airborne inoculum that lands on susceptible hosts under favorable environmental conditions.…”

Phoma stem canker disease on oilseed rape (Brassica napus) in China is caused by Leptosphaeria biglobosa ‘brassicae’

“…Nevertheless, the two species occupy slightly different ecological niches, which enables them to coexist on oilseed rape crops in Europe (Fitt et al 2006c), North America (Dilmaghani et al 2009) and Australia Vincenot et al 2008). Both pathogens are spread by air-borne ascospores (Dawidziuk et al 2012;Kaczmarek et al 2012 ), from 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 3 which they infect the leaves (Biddulph et al 1999;Toscano-Underwood et al 2001) and then spread along the leaf petioles to the stems (West et al 2002). The global importance of phoma stem canker has increased over the last 20 years, with longdistance (e.g.…”

Microscopic and molecular detection of Leptosphaeria maculans and L. biglobosa ascospore content in air samples

“…Another reason of differences between tape halves may arise from picnidiospores; these spores were not counted on tapes due to their small size and eliptic shape typical of numerous spores present in the air. Not just sexual or asexual spores, but other fungal material like hyphal fragments or pycnidia are likely to be present on the cellophane tape as well (Kaczmarek et al, 2012). All this material is difficult to identify under microscope, but the DNA originating from such samples should be amplified with quantitative real-time PCR.…”