Differentiating A and B groups of <i>Leptosphaeria maculans</i>, causal agent of stem canker (blackleg) of oilseed rape

Williams, Roger H.; Fitt, Bruce D.L.

doi:10.1046/j.1365-3059.1999.00333.x

Cited by 109 publications

(95 citation statements)

References 73 publications

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“…Additionally, microscopy is unreliable for detection of the small, nondescript spores produced by many fungi, while cultural techniques are unsuitable for detection of spores that are slow growing, or nonculturable in vitro and the choice of medium may influence which species can grow. These difficulties have restricted the use of routine air sampling in the study of plant, animal, and human diseases.Recently, however, molecular methods have been used in the development of diagnostic tests for a variety of fungi involved in plant diseases (8,25,26,28,30). While the potential of these techniques for detection of airborne spores has been recognized for some time (12,14), there have been few reports on progress in this area.…”

mentioning

confidence: 99%

Methods for Integrated Air Sampling and DNA Analysis for Detection of Airborne Fungal Spores

Williams

Ward

McCartney

2001

Appl Environ Microbiol

155

102

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Integrated air sampling and PCR-based methods for detecting airborne fungal spores, using Penicillium roqueforti as a model fungus, are described. P. roqueforti spores were collected directly into Eppendorf tubes using a miniature cyclone-type air sampler. They were then suspended in 0.1% Nonidet P-40, and counted using microscopy. Serial dilutions of the spores were made. Three methods were used to produce DNA for PCR tests: adding untreated spores to PCRs, disrupting spores (fracturing of spore walls to release the contents) using Ballotini beads, and disrupting spores followed by DNA purification. Three P. roqueforti-specific assays were tested: single-step PCR, nested PCR, and PCR followed by Southern blotting and probing. Disrupting the spores was found to be essential for achieving maximum sensitivity of the assay. Adding untreated spores to the PCR did allow the detection of P. roqueforti, but this was never achieved when fewer than 1,000 spores were added to the PCR. By disrupting the spores, with or without subsequent DNA purification, it was possible to detect DNA from a single spore. When known quantities of P. roqueforti spores were added to air samples consisting of high concentrations of unidentified fungal spores, pollen, and dust, detection sensitivity was reduced. P. roqueforti DNA could not be detected using untreated or disrupted spore suspensions added to the PCRs. However, using purified DNA, it was possible to detect 10 P. roqueforti spores in a background of 4,500 other spores. For all DNA extraction methods, nested PCR was more sensitive than single-step PCR or PCR followed by Southern blotting.Conventional methods for identifying and enumerating airborne fungi and other microorganisms rely on microscopic or cultural techniques and, as a consequence, are time-consuming and laborious. Additionally, microscopy is unreliable for detection of the small, nondescript spores produced by many fungi, while cultural techniques are unsuitable for detection of spores that are slow growing, or nonculturable in vitro and the choice of medium may influence which species can grow. These difficulties have restricted the use of routine air sampling in the study of plant, animal, and human diseases.Recently, however, molecular methods have been used in the development of diagnostic tests for a variety of fungi involved in plant diseases (8,25,26,28,30). While the potential of these techniques for detection of airborne spores has been recognized for some time (12,14), there have been few reports on progress in this area. The use of immunoassay in the detection of airborne plant pathogenic fungi has been investigated (10,20,21). However, the application of these methods is restricted by the difficulties of developing antibodies showing the required specificity. DNA-based detection methods offer greater potential for sensitive and specific detection, and some progress has been made in the detection of airborne bacteria using these techniques (1, 2, 9, 15, 17). Progress with fungi has been slower, although dete...

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mentioning

confidence: 99%

Methods for Integrated Air Sampling and DNA Analysis for Detection of Airborne Fungal Spores

Williams

Ward

McCartney

2001

Appl Environ Microbiol

155

102

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“…Leptosphaeria maculans and L. biglobosa differ in their rates of spread within agricultural fields sown with winter oilseed rape and also in sensitivity to fungicides. The former species is more aggressive and it incites severe cankers at the stem base resulting in economic yield loss arising from occluded and dysfunctional vascular tissues, premature pod ripening and lodging (Williams and Fitt 1999). Lesions caused by L. biglobosa are mainly superficial necrosis on upper parts of stems.…”

Section: Discussionmentioning

confidence: 99%

Monitoring blackleg (Leptosphaeria spp.) ascospore release timing and quantity enables optimal fungicide application to improved oilseed rape yield and seed quality

Brachaczek¹,

Kaczmarek

Jędryczka

2016

Eur J Plant Pathol

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Stem canker is a damaging disease of winter oilseed rape, caused by ascomycetous fungi Leptosphaeria maculans and L. biglobosa. The initial source of plant infection is ascospores, formed in pseudothecia on stubble from the previous growing season. The aim of this work was to evaluate the effectiveness of fungicide treatments at different times of application in the autumn on quality and quantity of seed yield, in relation to the concentration of ascospores of L. maculans and L. biglobosa in the air. Field experiments were done in Lower Silesia, the region of intensive oilseed rape cultivation, located in south-west Poland. We have demonstrated that, in case of high disease pressure, fungicide treatment against stem canker was most efficient when done 4-11 days after the highest concentration of pathogen ascospores in air. The seed yield, oil and protein content increased by 17.7-18.8 %, 1.60-1.72 % and 0.8-0.9 % respectively. In low disease pressure the optimal spray time was more flexible, but the highest yield was always following the maximum ascospore release. In some cases it also coincided with the increase of oil, without adverse effect on protein content. Furthermore, it coincided with the increase of indole glucosinolates, such as glucobrassicine and 4-hydroxyglucobrassicine, and -besides one studied year -it was also connected with decreased amounts of alkenyl glucosinolates. Both Leptosphaeria species were present in air and in infected plants during their vegetation. The ratio between both species depended on seasons; L. maculans prevailed on leaves in the autumn, whereas L. biglobosa dominated on stems before harvest.

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“…The disease can cause yield losses at harvest of about 30-50%. Leptosphaeria maculans is regarded as more damaging than L. biglobosa (Williams, Fitt, 1999). In general, L. maculans is related with stem base canker, while L. biglobosa induces upper stem lesions (Salam et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

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

Piliponytė-Dzikienė

Kaczmarek

Petraitienė

et al. 2014

Zemdirbyste-Agriculture

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Airborne sexual spores of Leptosphaeria maculans and L. biglobosa are primary inoculum of fungi causing phoma stem canker of oilseed rape. In this study ascospore release of these two species was compared between Lithuania (of autumn 2010 and 2011) and Poland (autumns of 2009 till 2012), using identical equipment and methods. Dynamics of L. maculans and L. biglobosa ascospore dispersal was investigated using volumetric samplers followed by microscopic and molecular methods of detection. In Lithuania, the earliest detection of Leptosphaeria ascospores, using microscopy was on 1 st September (2010) and on 2 nd September (2011), whereas in Poland the earliest detection was on 5th September (2009 and and the latest on 11 th October (2011), which demonstrates that differences between the seasons may exceed a month (36 days). In the case of molecular detection performed for the Polish samples, the dates of the earliest and the latest release of the first ascospores ranged from 5 th September to 15 th October (40 days). The number of days with ascospores detected in the air samples ranged from 6 (2012) to 25 (2009). At earliest, the detection of the highest concentration of ascospores was on 6 th September (2010) and the latest -on 28 th October (2009). These results demonstrate big differences between the results of spore monitoring between the years and the importance of aerobiological studies to follow the development of the pathogen in natural infection. The fluctuations in quantities of spores and fungal deoxyribonucleic acid (DNA) strongly depended on the weather conditions, mainly rainfall, air temperature and relative humidity. Ascospore release was observed immediately after the rainfall events, but not later than two days after the rainfall. On days without rain, spores were present in the air samples when average relative air humidity exceeded 90% and day temperature was below 15°C. In 2009, molecular detection revealed the presence of both fungal species in the air samples, whereas in 2010-2012 only L. biglobosa DNA was found. Molecular detection with a quantitative real-time polymerase chain reaction (qPCR) was identified as a fast and accurate method of pathogen detection and identification from air samples. The highest coefficient of correlation between microscopic and molecular detection of Leptosphaeria species was 0.828 (2009) and the lowest was 0.483 (2011); in all years both techniques of detection were statistically correlated. We have demonstrated that the correlation coefficient highly depended on the number of ascospores in air samples. Based on the studies performed in Lithuania and Poland, we have shown that climatic conditions in the northern part of Central Europe are favourable for the spread of Leptosphaeria spp. by ascospores; however, the patterns of ascospore release greatly differ between the both countries.

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Differentiating A and B groups of Leptosphaeria maculans, causal agent of stem canker (blackleg) of oilseed rape

Cited by 109 publications

References 73 publications

Methods for Integrated Air Sampling and DNA Analysis for Detection of Airborne Fungal Spores

Methods for Integrated Air Sampling and DNA Analysis for Detection of Airborne Fungal Spores

Monitoring blackleg (Leptosphaeria spp.) ascospore release timing and quantity enables optimal fungicide application to improved oilseed rape yield and seed quality

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

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