Search

BACKGROUND: Light leaf spot, caused by Pyrenopeziza brassicae, is amongst the most damaging diseases of winter oilseed rape (Brassica napus), and currently the sterol 14α‐demethylase (CYP51) inhibitors (azoles) represent the main class of fungicides used to control light leaf spot development. However, a shift in sensitivity to azole fungicides in P. brassicae populations has been observed in different European countries, including Ireland. RESULTS: To assess the sensitivity status of Irish P. brassicae populations to azole fungicides, three collections of P. brassicae from 2018–2020 were tested in vitro against tebuconazole and prothioconazole‐desthio, and the PbCYP51 gene targeted by this class of fungicides was genotyped in different isolates. A change in sensitivity to azole fungicides was observed and differences in sensitivity to tebuconazole between Irish populations were present. There were two substitutions within PbCYP51 (G460S and S508T) and inserts of different sizes in its promoter region. The presence of the G460S/S508T double mutant was reported for the first time, and the diversity in insert size was greater than previously known. Compared to wild type isolates, those carrying G460S or S508T were less sensitive to both fungicides and, where inserts were also identified, they further reduced sensitivity to azole fungicides. CONCLUSIONS: The results of this study suggest that azole fungicides are still very effective in controlling light leaf spot in Ireland. However, using azole fungicides in mixtures of fungicides with different modes of action is recommended. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Use of host resistance is the most economical and environmentally safe way to control light leaf spot disease of oilseed rape (Brassica napus). The causal organism of light leaf spot, Pyrenopeziza brassicae, is one of the most economically damaging pathogens of oilseed rape in the United Kingdom and it is considered to have a high potential to evolve due to its mixed reproduction system and airborne ascospores. This necessitates diverse sources of host resistance, which are inadequate at present to minimize yield losses caused by this disease. To address this, we screened a doubled haploid (DH) population of oilseed rape, derived from a secondary gene pool (ancestral genomes) of B. napus for the introgression of resistance against P. brassicae. DH lines were phenotyped using controlled-environment and glasshouse experiments with P. brassicae populations obtained from three different geographic locations in the United Kingdom. Selected DH lines with different levels of resistance were further studied in a controlled-environment experiment using both visual (scanning electron microscope – SEM) and molecular (quantitative PCR) assessment methods to understand the mode/s of host resistance. There was a clear phenotypic variation for resistance against P. brassicae in this DH population. Quantitative trait locus (QTL) analysis identified four QTLs with moderate to large effects, which were located on linkage groups C1, C6, and C9. Of these, the QTL on the linkage group C1 appeared to have a major effect on limiting P. brassicae asexual sporulation. Study of the sub-cuticular growth phase of P. brassicae using qPCR and SEM showed that the pathogen was able to infect and colonise both resistant and susceptible Q DH lines and control B. napus cultivars. However, the rate of increase of pathogen biomass was significantly smaller in resistant lines, suggesting that the resistance segregating in this DH population limits colonisation/sporulation by the pathogen rather than eliminating the pathogen. Resistance QTLs identified in this study provide a useful resource for breeding cultivar resistance for effective control of light leaf spot and form a starting point for functional identification of the genes controlling resistance against P. brassicae that can contribute to our knowledge on mechanisms of partial resistance of crops against pathogens.

Use of host resistance is the most economical and environmentally safe way to control light leaf spot disease of oilseed rape (Brassica napus). The causal organism of light leaf spot, Pyrenopeziza brassicae , is one of the most economically damaging pathogens of oilseed rape in the United Kingdom and it is considered to have a high potential to evolve due to its mixed reproduction system and airborne ascospores. This necessitates diverse sources of host resistance, which are inadequate at present to minimize yield losses caused by this disease. To address this, we screened a doubled haploid (DH) population of oilseed rape, derived from a secondary gene pool (ancestral genomes) of B. napus for the introgression of resistance against P. brassicae. DH lines were phenotyped using controlled-environment and glasshouse experiments with P. brassicae populations obtained from three different geographic locations in the United Kingdom. Selected DH lines with different levels of resistance were further studied in a controlled-environment experiment using both visual (scanning electron microscope – SEM) and molecular (quantitative PCR) assessment methods to understand the mode/s of host resistance. There was a clear phenotypic variation for resistance against P. brassicae in this DH population. Quantitative trait locus (QTL) analysis identified four QTLs with moderate to large effects, which were located on linkage groups C1, C6, and C9. Of these, the QTL on the linkage group C1 appeared to have a major effect on limiting P. brassicae asexual sporulation. Study of the sub-cuticular growth phase of P. brassicae using qPCR and SEM showed that the pathogen was able to infect and colonise both resistant and susceptible Q DH lines and control B. napus cultivars. However, the rate of increase of pathogen biomass was significantly smaller in resistant lines, suggesting that the resistance segregating in this DH population limits colonisation/sporulation by the pathogen rather than eliminating the pathogen. Resistance QTLs identified in this study provide a useful resource for breeding cultivar resistance for effective control of light leaf spot and form a starting point for functional identification of the genes controlling resistance against P. brassicae that can contribute to our knowledge on mechanisms of partial resistance of crops against pathogens. [ABSTRACT FROM AUTHOR]

Abstract Background The use of spectral imaging within the plant phenotyping and breeding community has been increasing due its utility as a non-invasive diagnostic tool. However, there is a lack of imaging systems targeted specifically at plant science duties, resulting in low precision for canopy-scale measurements. This study trials a prototype multispectral system designed specifically for plant studies and looks at its use as an early detection system for visually asymptomatic disease phases, in this case Pyrenopeziza brassicae in Brassica napus. The analysis takes advantage of machine learning in the form of feature selection and novelty detection to facilitate the classification. An initial study into recording the morphology of the samples is also included to allow for further improvement to the system performance. Results The proposed method was able to detect light leaf spot infection with 92% accuracy when imaging entire oilseed rape plants from above, 12 days after inoculation and 13 days before the appearance of visible symptoms. False colour mapping of spectral vegetation indices was used to quantify disease severity and its distribution within the plant canopy. In addition, the structure of the plant was recorded using photometric stereo, with the output influencing regions used for diagnosis. The shape of the plants was also recorded using photometric stereo, which allowed for reconstruction of the leaf angle and surface texture, although further work is needed to improve the fidelity due to uneven lighting distributions, to allow for reflectance compensation. Conclusions The ability of active multispectral imaging has been demonstrated along with the improvement in time taken to detect light leaf spot at a high accuracy. The importance of capturing structural information is outlined, with its effect on reflectance and thus classification illustrated. The system could be used in plant breeding to enhance the selection of resistant cultivars, with its early and quantitative capability.

Light leaf spot, caused by the ascomycete Pyrenopeziza brassicae, is an established disease of Brassicaceae in the United Kingdom (UK), continental Europe, and Oceania (OC, including New Zealand and Australia). The disease was reported in North America (NA) for the first time in 2014 on Brassica spp. in the Willamette Valley of western Oregon, followed by detection in Brassica juncea cover crops and on Brassica rapa weeds in northwestern Washington in 2016. Preliminary DNA sequence data and field observations suggest that isolates of the pathogen present in NA might be distinct from those in the UK, continental Europe, and OC. Comparisons of isolates from these regions using genetic (multilocus sequence analysis, MAT gene sequences, and rep‐PCR DNA fingerprinting), pathogenic (B. rapa inoculation studies), biological (sexual compatibility), and morphological (colony and conidial morphology) analyses demonstrated two genetically distinct evolutionary lineages. Lineage 1 comprised isolates from the UK, continental Europe, and OC, and included the P. brassicae type specimen. Lineage 2 contained the NA isolates associated with recent disease outbreaks in the Pacific Northwest region of the USA. Symptoms caused by isolates of the two lineages on B. rapa and B. juncea differed, and therefore "chlorotic leaf spot" is proposed for the disease caused by Lineage 2 isolates of P. brassicae. Isolates of the two lineages differed in genetic diversity as well as sensitivity to the fungicides carbendazim and prothioconazole. [ABSTRACT FROM AUTHOR]

Pyrenopeziza brassicae (anamorph Cylindrosporium concentricum) is an ascomycete fungus that causes light leaf spot ( LLS) disease of brassicas. It has recently become the most important pathogen of winter oilseed rape ( Brassica napus) crops in the UK. The pathogen is spread by both asexual splash-dispersed conidia and sexual wind-dispersed ascospores. Such inoculum can be detected with existing qualitative and quantitative PCR diagnostics, but these require time-consuming laboratory-based processing. This study describes two loop-mediated isothermal amplification ( LAMP) assays, targeting internal transcribed spacer ( ITS) or β-tubulin DNA sequences, for fast and specific detection of P. brassicae isolates from a broad geographical range (throughout Europe and Oceania) and multiple brassica host species ( B. napus, B. oleracea and B. rapa). Neither assay detected closely related Oculimacula or Rhynchosporium isolates, or other commonly occurring oilseed rape fungal pathogens. Both LAMP assays could consistently detect DNA amounts equivalent to 100 P. brassicae conidia per sample within 30 minutes, although the β-tubulin assay was more rapid. Reproducible standard curves were obtained using a P. brassicae DNA dilution series (100 ng-10 pg), enabling quantitative estimation of amounts of pathogen DNA in environmental samples. In planta application of the β-tubulin sequence-based LAMP assay to individual oilseed rape leaves collected from the field found no statistically significant difference in the amount of pathogen DNA present in parts of leaves either with or without visible LLS symptoms. The P. brassicae LAMP assays described here could have multiple applications, including detection of symptomless host infection and automated real-time monitoring of pathogen inoculum. [ABSTRACT FROM AUTHOR]

The incidence and severity of light leaf spot epidemics caused by the ascomycete fungus Pyrenopeziza brassicae on UK oilseed rape crops are increasing. The disease is currently controlled by a combination of host resistance, cultural practices and fungicide applications. We report decreases in sensitivity of modern UK P. brassicae isolates to the azole (imidazole and triazole) class of fungicides. By cloning and sequencing the P. brassicae CYP51 ( PbCYP51) gene, encoding the azole target sterol 14α-demethylase, we identified two non-synonymous mutations encoding substitutions G460S and S508T associated with reduced azole sensitivity. We confirmed the impact of the encoded PbCYP51 changes on azole sensitivity and protein activity by heterologous expression in a Saccharomyces cerevisiae mutant YUG37: erg11 carrying a controllable promoter of native CYP51 expression. In addition, we identified insertions in the predicted regulatory regions of PbCYP51 in isolates with reduced azole sensitivity. The presence of these insertions was associated with enhanced transcription of PbCYP51 in response to subinhibitory concentrations of the azole fungicide tebuconazole. Genetic analysis of in vitro crosses of sensitive and resistant isolates confirmed the impact of PbCYP51 alterations in coding and regulatory sequences on a reduced sensitivity phenotype, as well as identifying a second major gene at another locus contributing to resistance in some isolates. The least sensitive field isolates carry combinations of upstream insertions and non-synonymous mutations, suggesting that PbCYP51 evolution is ongoing and the progressive decline in azole sensitivity of UK P. brassicae populations will continue. The implications for the future control of light leaf spot are discussed. [ABSTRACT FROM AUTHOR]

BACKGROUND Methyl benzimidazole carbamate ( MBC) fungicides are used to control the oilseed rape pathogen Pyrenopeziza brassicae. Resistance to MBCs has been reported in P. brassicae, but the molecular mechanism(s) associated with reductions in sensitivity have not been verified in this species. Elucidation of the genetic changes responsible for resistance, hypothesised to be target-site mutations in β-tubulin, will enable resistance diagnostics and thereby inform resistance management strategies. RESULTS P. brassicae isolates were classified as sensitive, moderately resistant or resistant to MBCs. Crossing P. brassicae isolates of different MBC sensitivities indicated that resistance was conferred by a single gene. The MBC-target encoding gene β-tubulin was cloned and sequenced. Reduced MBC sensitivity of field isolates correlated with β-tubulin amino acid substitutions L240F and E198A. The highest level of MBC resistance was measured for isolates carrying E198A. Negative cross-resistance between MBCs and the fungicides diethofencarb and zoxamide was only measured in E198A isolates. PCR-RFLP was used to screen isolates for the presence of L240F and E198A. The substitutions E198G and F200Y were also detected in DNA samples from P. brassicae populations after cloning and sequencing of PCR products. The frequencies of L240F and E198A in different P. brassicae populations were quantified by pyrosequencing. There were no differences in the frequencies of these alleles between P. brassicae populations sampled from different locations or after fungicide treatment regimes. CONCLUSIONS The molecular mechanisms affecting sensitivity to MBCs in P. brassicae have been identified. Pyrosequencing assays are a powerful tool for quantifying fungicide-resistant alleles in pathogen populations. © 2013 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Following a sudden outbreak of light leaf spot disease in Polish winter oilseed rape crops at the beginning of the last decade, volumetric spore traps installed around the country were used for the detection of ascospores, which constitute the primary inoculum of Pyrenopeziza brassicae, the causal organism. In 2004–2008, weather conditions that were adverse to the development of the pathogen appeared to have hindered the macroscopic signs of light leaf spot disease symptoms on oilseed rape, even in Pomerania, a region in north Poland, which cultivates the crop intensively and is favourable for the disease. Recently, however, the development of β-tubulin-based primers and dual labelled fluorescent probes has enabled the detection of minute quantities of P. brassicae DNA that correlated highly and positively with low abundance of inoculum of the pathogen in air samples. Depending on the season, the Spearman's correlation rank between spore abundance obtained by light microscopy and P. brassicae DNA yield determined by quantitative polymerase chain reaction (qPCR) on extracts from spore sampler tapes varied from 0.256 to 0.562. These primers and probe also detected P. brassicae in DNA extracted from tapes of spore traps operated in the autumn of 2006 and 2007 within field sown winter oilseed rape crops in Harpenden, southern UK. [ABSTRACT FROM AUTHOR]

Weather data generated for different parts of the UK under five climate change scenarios (baseline, 2020s low CO2 emissions, 2020s high emissions, 2050s low emissions, 2050s high emissions) were inputted into weather-based models for predicting oilseed rape yields and yield losses from the two most important diseases, phoma stem canker and light leaf spot. An economic analysis of the predictions made by the models was done to provide a basis to guide government and industry planning for adaptation to effects of climate change on crops to ensure future food security. Modelling predicted that yields of fungicide-treated oilseed rape would increase by the 2020s and continue to increase by the 2050s, particularly in Scotland and northern England. If stem canker and light leaf spot were effectively controlled, the value of the crop was predicted to increase above the baseline 1980s value by £13 M in England and £28 M in Scotland by the 2050s under a high CO2 emissions scenario. However, in contrast to predictions that phoma stem canker will increase in severity and range with climate change, modelling indicated that losses due to light leaf spot will decrease in both Scotland and England. Combined losses from both phoma stem canker and light leaf spot are predicted to increase, with yield losses of up to 40% in southern England and some regions of Scotland by the 2050s under the high emission scenarios. For this scenario, UK disease losses are predicted to increase by £50 M (by comparison with the baseline losses). However, the predicted increases in fungicide-treated (potential) yield and phoma stem canker/light leaf spot yield losses compensate for each other so that the net UK losses from climate change for untreated oilseed rape are small. [ABSTRACT FROM AUTHOR]

Data from surveys of winter oilseed rape crops in England and Wales in growing seasons with harvests in 1987–99 were used to construct statistical models to predict, in autumn (October), the incidence of light leaf spot caused byPyrenopeziza brassicaeon winter oilseed rape crops the following spring (March/April), at both regional and individual crop scales. Regions (groups of counties) with similar seasonal patterns of incidence (percentage of plants affected) of light leaf spot were defined by using principal coordinates analysis on the survey data. At the regional scale, explanatory variables for the statistical models were regional weather (mean summer temperature and mean monthly winter rainfall) and survey data for regional light leaf spot incidence (percentage of plants with affected pods) in July of the previous season. At the crop scale, further explanatory variables were crop cultivar (light leaf spot resistance rating), sowing date (number of weeks before/after 1 September), autumn fungicide use and light leaf spot incidence in autumn. Risk of severe light leaf spot (> 25% plants affected) in a crop in spring was also predicted, and uncertainty in predictions was assessed. The models were validated using data from spring surveys of winter oilseed rape crops in England and Wales from 2000 to 2003, and reasons for uncertainty in predictions for individual crops are discussed. [ABSTRACT FROM AUTHOR]

Background: The use of spectral imaging within the plant phenotyping and breeding community has been increasing due its utility as a non-invasive diagnostic tool. However, there is a lack of imaging systems targeted specifically at plant science duties, resulting in low precision for canopy-scale measurements. This study trials a prototype multispectral system designed specifically for plant studies and looks at its use as an early detection system for visually asymptomatic disease phases, in this case Pyrenopeziza brassicae in Brassica napus. The analysis takes advantage of machine learning in the form of feature selection and novelty detection to facilitate the classification. An initial study into recording the morphology of the samples is also included to allow for further improvement to the system performance. Results: The proposed method was able to detect light leaf spot infection with 92% accuracy when imaging entire oilseed rape plants from above, 12 days after inoculation and 13 days before the appearance of visible symptoms. False colour mapping of spectral vegetation indices was used to quantify disease severity and its distribution within the plant canopy. In addition, the structure of the plant was recorded using photometric stereo, with the output influencing regions used for diagnosis. The shape of the plants was also recorded using photometric stereo, which allowed for reconstruction of the leaf angle and surface texture, although further work is needed to improve the fidelity due to uneven lighting distributions, to allow for reflectance compensation. Conclusions: The ability of active multispectral imaging has been demonstrated along with the improvement in time taken to detect light leaf spot at a high accuracy. The importance of capturing structural information is outlined, with its effect on reflectance and thus classification illustrated. The system could be used in plant breeding to enhance the selection of resistant cultivars, with its early and quantitative capability. [ABSTRACT FROM AUTHOR]