Pyrenopeziza Brassicae Research Articles

Pathogen lifestyle determines host genetic signature of quantitative disease resistance loci in oilseed rape (Brassica napus)

Key messageUsing associative transcriptomics, our study identifies genes conferring resistance to four diverse fungal pathogens in crops, emphasizing key genetic determinants of multi-pathogen resistance.Crops are affected by several pathogens, but these are rarely studied in parallel to identify common and unique genetic factors controlling diseases. Broad-spectrum quantitative disease resistance (QDR) is desirable for crop breeding as it confers resistance to several pathogen species. Here, we use associative transcriptomics (AT) to identify candidate gene loci associated with Brassica napus constitutive QDR to four contrasting fungal pathogens: Alternaria brassicicola, Botrytis cinerea, Pyrenopeziza brassicae, and Verticillium longisporum. We did not identify any shared loci associated with broad-spectrum QDR to fungal pathogens with contrasting lifestyles. Instead, we observed QDR dependent on the lifestyle of the pathogen—hemibiotrophic and necrotrophic pathogens had distinct QDR responses and associated loci, including some loci associated with early immunity. Furthermore, we identify a genomic deletion associated with resistance to V. longisporum and potentially broad-spectrum QDR. This is the first time AT has been used for several pathosystems simultaneously to identify host genetic loci involved in broad-spectrum QDR. We highlight constitutive expressed candidate loci for broad-spectrum QDR with no antagonistic effects on susceptibility to the other pathogens studies as candidates for crop breeding. In conclusion, this study represents an advancement in our understanding of broad-spectrum QDR in B. napus and is a significant resource for the scientific community.

Novel gene loci associated with susceptibility or cryptic quantitative resistance to Pyrenopeziza brassicae in Brassica napus

Key messageQuantitative disease resistance (QDR) controls the association of the light leaf spot pathogen with Brassica napus; four QDR loci that were in linkage disequilibrium and eight gene expression markers were identified.Quantitative disease resistance (QDR) can provide durable control of pathogens in crops in contrast to resistance (R) gene-mediated resistance which can break down due to pathogen evolution. QDR is therefore a desirable trait in crop improvement, but little is known about the causative genes, and so it is difficult to incorporate into breeding programmes. Light leaf spot, caused by Pyrenopeziza brassicae, is an important disease of oilseed rape (canola, Brassica napus). To identify new QDR gene loci, we used a high-throughput screening pathosystem with P. brassicae on 195 lines of B. napus combined with an association transcriptomics platform. We show that all resistance against P. brassicae was associated with QDR and not R gene-mediated. We used genome-wide association analysis with an improved B. napus population structure to reveal four gene loci significantly (P = 0.0001) associated with QDR in regions showing linkage disequilibrium. On chromosome A09, enhanced resistance was associated with heterozygosity for a cytochrome P450 gene co-localising with a previously described locus for seed glucosinolate content. In addition, eight significant gene expression markers with a false discovery rate of 0.001 were associated with QDR against P. brassicae. For seven of these, expression was positively correlated with resistance, whereas for one, a HXXXD-type acyl-transferase, negative correlation indicated a potential susceptibility gene. The study identifies novel QDR loci for susceptibility and resistance, including novel cryptic QDR genes associated with heterozygosity, that will inform future crop improvement.

Azole fungicide sensitivity and molecular mechanisms of reduced sensitivity in Irish Pyrenopeziza brassicae populations.

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. 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. 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.

Quantitative Trait Locus Mapping for Resistance Against Pyrenopeziza brassicae Derived From a Brassica napus Secondary Gene Pool.

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.

Pyrenopeziza brassicae (light leaf spot)

This datasheet on Pyrenopeziza brassicae covers Identity, Overview, Distribution, Dispersal, Hosts/Species Affected, Diagnosis, Biology & Ecology, Seedborne Aspects, Natural Enemies, Impacts, Prevention/Control, Further Information.

Black Leg and Chlorotic Leaf Spot Occurrence on Brassicaceae Crop and Weed Hosts.

Black leg (caused by Plenodomus lingam and P. biglobosus) and chlorotic leaf spot (caused by Pyrenopeziza brassicae) are economically important fungal diseases of Brassicaceae crops. Surveys of seed fields and weed hosts were conducted to understand the distribution and prevalence of these diseases in Oregon after black leg and chlorotic leaf spot outbreaks occurred in Brassicaceae crops in 2014. Postharvest black leg ratings were conducted in seed fields of canola, forage rape, and turnip in 2015 and 2016. The incidence of black leg was greater for turnip (51%) than for canola (29%) and forage rape (25%). The overall average disease incidence was greater for seed crops harvested in 2015 (46%) than for crops harvested in 2016 (28%). A disease survey of wild Brassicaceae plants was conducted along Interstate 5 in Oregon. Brassicaceae weed population sites were identified and 40 sites were sampled for these diseases. Black leg and chlorotic leaf spot were present in 60 and 45%, respectively, of the sampled sites. Both species of Plenodomus were detected in weed populations, with P. lingam being the predominant species recovered (95%). The northernmost sample site with black leg was <32 km from the Oregon-Washington border, and the southernmost site with black leg was within 32 km of the Oregon-California border. Chlorotic leaf spot was detected <32 km from the Oregon-Washington border, whereas the southernmost site where it was detected was approximately 164 km from the Oregon-California border. Based on this study, infected crop residues and weed hosts may facilitate the persistence and spread of these pathogens.

Contrasting Responses of Rhizosphere Bacterial, Fungal, Protist, and Nematode Communities to Nitrogen Fertilization and Crop Genotype in Field Grown Oilseed Rape (Brassica napus)

The rhizosphere microbiome is considered to play a key role in determining crop health. However, current understanding of the factors which shape assembly and composition of the microbiome is heavily biased toward bacterial communities, and the relevance for other microbial groups is unclear. Furthermore, community assembly is determined by a variety of factors, including host genotype, environment and agricultural management practices, and their relative importance and interactions remain to be elucidated. We investigated the impact of nitrogen fertilization on rhizosphere bacterial, fungal, nematode and protist communities of 10 contrasting oilseed rape genotypes in a field experiment. We found significant differences in the composition of bacteria, fungi, protist and nematode communities between the rhizosphere and bulk soil. Nitrogen application had a significant but weak effect on fungal, bacterial, and protist community composition, and this was associated with increased relative abundance of a complex of fungal pathogens in the rhizosphere and soil, including Mycosphaerella sp. and Leptosphaeria sp. Network analysis showed that nitrogen application had different effects on microbial community connectivity in the soil and rhizosphere. Crop genotype significantly affected fungal community composition, with evidence for a degree of genotype specificity for a number of pathogens, including L. maculans, Alternaria sp., Pyrenopeziza brassicae, Olpidium brassicae, and L. biglobosa, and also potentially beneficial Heliotales root endophytes. Crop genotype had no significant effect on assembly of bacteria, protist or nematode communities. There was no relationship between genetic distance of crop genotypes and the extent of dissimilarity of rhizosphere microbial communities. Field disease assessment confirmed infection of crops by Leptosphaeria sp., P. brassicae, and Alternaria sp., indicating that rhizosphere microbiome sequencing was an effective indicator of plant health. We conclude that under field conditions soil and rhizosphere nutrient stoichiometry and crop genotype are key factors determining crop health by influencing the infection of roots by pathogenic and mutualistic fungal communities, and the connectivity and stability of rhizosphere microbiome interaction networks.

A phylogenetically distinct lineage of Pyrenopeziza brassicae associated with chlorotic leaf spot of Brassicaceae in North America.

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.

A real-time PCR assay for detection of light leaf spot on Brassica crops in the Pacific Northwest of the USA

Outbreaks of light leaf spot (Pyrenopeziza brassicae), a disease new to North America, occurred along with the diseases blackleg (Leptosphaeria spp.) and white leaf spot (Mycosphaerella capsellae) on Brassica crops in western Oregon. Disease symptoms in seed fields planted in the fall of 2013 were observed in the spring of 2014. Field surveys were conducted for disease during 2014 and 2015 to determine the geographic range of light leaf spot, blackleg, and white leaf spot within the Willamette Valley of western Oregon. Infected seed crops included turnip and oilseed rape in Benton, Lane, Linn, Marion, Polk, and Yamhill counties. New protocols that are more sensitive for detection of light leaf spot were developed for the pathogen population in the Pacific Northwest of the USA. Pyrenopeziza brassicae was detected when samples also contained L. maculans or M. capsellae. The test did not react with a range of other fungi evaluated. Sensitivity of the PCR protocol reported is robust at 10 pg of genomic DNA. Standard and real-time PCR screenings developed in this research increase sensitivity for detection of P. brassicae in affected Brassica hosts, which is critical for timely diagnosis and management of this newly-introduced disease in Brassica crops in the Pacific Northwestern region of the USA.

Multispectral imaging for presymptomatic analysis of light leaf spot in oilseed rape

BackgroundThe 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.ResultsThe 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.ConclusionsThe 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.

Differences in the fungal communities nursed by two genetic groups of the alpine cushion plant, Silene acaulis.

Foundation plants shape the composition of local biotic communities and abiotic environments, but the impact of a plant's intraspecific variations on these processes is poorly understood. We examined these links in the alpine cushion moss campion (Silene acaulis) on two neighboring mountain ranges in the French Alps. Genotyping of cushion plants revealed two genetic clusters matching known subspecies. The exscapa subspecies was found on both limestone and granite, while the longiscapa one was only found on limestone. Even on similar limestone bedrock, cushion soils from the two S. acaulis subspecies deeply differed in their impact on soil abiotic conditions. They further strikingly differed from each other and from the surrounding bare soils in fungal community composition. Plant genotype variations accounted for a large part of the fungal composition variability in cushion soils, even when considering geography or soil chemistry, and particularly for the dominant molecular operational taxonomic units (MOTUs). Both saprophytic and biotrophic fungal taxa were related to the MOTUs recurrently associated with a single plant genetic cluster. Moreover, the putative phytopathogens were abundant, and within the same genus (Cladosporium) or species (Pyrenopeziza brassicae), MOTUs showing specificity for each plant subspecies were found. Our study highlights the combined influences of bedrock and plant genotype on fungal recruitment into cushion soils and suggests the coexistence of two mechanisms, an indirect selection resulting from the colonization of an engineered soil by free‐living saprobes and a direct selection resulting from direct plant–fungi interactions.

Abstracts of Presentations at the 2017 APS Annual Meeting.

Light leaf spot (LLS), caused by Pyrenopeziza brassicae, is an important disease of Brassica napus (canola and oilseed rape) and B. oleracea (vegetable brassicas) in Europe (EU) as well as New Zealand and Australia (Oceania, OC). LLS was first reported in North America (NA) on B. juncea, B. napus, and B. rapa in six counties in western Oregon in 2014; and on B. juncea cover crops and wild B. rapa in three counties in northwestern Washington in 2016. Multi-locus sequence analysis (ITS ribosomal DNA, beta-tubulin, and elongation factor 1-α sequences) and comparison of the mating type genes (MAT1-1 and MAT1-2) grouped isolates from the EU (n = 28) and OC (n = 4) with the P. brassicae type specimen, IMI 204290, whereas isolates from NA (n = 16) represented a novel genotype. Sexual compatibility of NA and EU strains of complementary MAT1-1 and MAT1-2 genotypes is being determined to assess if NA isolates represent a distinct evolutionary lineage or a cryptic sibling species. Fungicide resistance has been documented in some EU populations of P. brassicae, but none of the NA isolates possessed amino acid substitutions E198A and L240F in the beta-tubulin sequences that confer resistance to benzimidazole fungicides; comparison of these sequences for the NA isolates revealed 100% identity to wild type EU P. brassicae isolates and the closely related fungus Rhynchosporium commune; and 98 and 99% identities to Sclerotinia sclerotiorum and Venturia inaequalis, respectively.

Loop‐mediated isothermal amplification (LAMP) assays for rapid detection of Pyrenopeziza brassicae (light leaf spot of brassicas)

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.

Host–pathogen interactions in relation to management of light leaf spot disease (caused by Pyrenopeziza brassicae) on Brassica species

Light leaf spot, caused by Pyrenopeziza brassicae, is the most damaging disease problem in oilseed rape (Brassica napus) in the United Kingdom. According to recent survey data, the severity of epidemics has increased progressively across the UK, with yield losses of up to £160M per annum in England and more severe epidemics in Scotland. Light leaf spot is a polycyclic disease, with primary inoculum consisting of airborne ascospores produced on diseased debris from the previous cropping season. Splash-dispersed conidia produced on diseased leaves are the main component of the secondary inoculum. Pyrenopeziza brassicae is also able to infect and cause considerable yield losses on vegetable brassicas, especially Brussels sprouts. There may be spread of light leaf spot among different Brassica species. Since they have a wide host range and frequent occurrence of sexual reproduction, P. brassicae populations are likely to have considerable genetic diversity, and evidence suggests population variations between different geographic regions, which need further study. Available disease-management tools are not sufficient to provide adequate control of the disease. There is a need to identify new sources of resistance, which can be integrated with fungicide applications to achieve sustainable management of light leaf spot. Several major resistance genes and quantitative trait loci have been identified in previous studies, but rapid improvements in the understanding of molecular mechanisms underpinning B. napus–P. brassicae interactions can be expected through exploitation of novel genetic and genomic information for brassicas and extracellular fungal pathogens.

Control of light leaf spot and clubroot in brassica crops using defence elicitors

Plant defence elicitors are compounds that can induce host defence responses against plant pathogens and offer a novel strategy for disease management. Disease control by elicitors can be inconsistent and is often dependent on the crop, the variety and the environment. The use of foliar application of defence elicitors to control light leaf spot (LLS) disease caused by Pyrenopeziza brassicae in the brassica crops winter oilseed rape (WOSR) and Brussel sprouts was evaluated in field trials across multiple years. Elicitor responses in WOSR varied between years. Yield benefits were also inconsistent and did not reflect the level of disease control. Results with Brussel sprouts were more consistent although variation between variety, trial site and year were observed. In particular the salicylic acid analog Acibenzolar-S-Methyl, in the commercial product Bion®, demonstrated good disease control across the field trial sites in the early maturing Brussel sprout variety Cobus. Levels of LLS were consistently reduced when Bion® was alternated within a standard fungicide programme, applied as an individual spray or in combination with other defence elicitors. When applied as a root drench or seed soak Bion® also reduced symptom development of the soil-borne brassica disease clubroot, caused by Plasmodiophora brassicae, in WOSR. These results indicate that defence elicitors such as Bion® can be used as an additional disease management tool alongside host resistance and standard fungicide programmes to protect brassica crops.

Milestones in plant sulfur research on sulfur-induced-resistance (SIR) in Europe.

Until the 1970's of the last century sulfur (S) was mainly regarded as a pollutant being the main contributor of acid rain, causing forest dieback in central Europe. When Clean Air Acts came into force at the start of the 1980's SO2 contaminations in the air were consequently reduced within the next years. S changed from an unwanted pollutant into a lacking plant nutrient in agriculture since agricultural fields were no longer “fertilized” indirectly by industrial pollution. S deficiency was first noticed in Brassica crops that display an especially high S demand because of its content of S-containing secondary metabolites, the glucosinolates. In Scotland, where S depositions decreased even faster than in continental Europe, an increasing disease incidence with Pyrenopeziza brassicae was observed in oilseed rape in the beginning 1990's and the concept of sulfur-induced-resistance (SIR) was developed after a relationship between the S status and the disease incidence was uncovered. Since then a lot of research was carried out to unravel the background of SIR in the metabolism of agricultural crops and to identify metabolites, enzymes and reactions, which are potentially activated by the S metabolism to combat fungal pathogens. The S status of the crop is affecting many different plant features such as color and scent of flowers, pigments in leaves, metabolite concentrations and the release of gaseous S compounds which are directly influencing the desirability of a crop for a variety of different organisms from microorganisms, over insects and slugs to the point of grazing animals. The present paper is an attempt to sum up the knowledge about the effect of the S nutritional status of agricultural crops on parameters that are directly related to their health status and by this to SIR. Milestones in SIR research are compiled, open questions are addressed and future projections were developed.

Arable Crop Disease Control, Environmental Change and Food Security

Crop diseases cause yield losses, yet crop productivity needs to increase by > 70% over the next 35 years to accommodate population growth and changing dietary preferences1. Crop plants and pathogens interact in response to changes in environmental conditions. Recent predictions made by combining climate model data with an oilseed rape growth model and a phoma stem canker model suggest that yield will increase in the UK, especially in Scotland, whereas potential losses from phoma stem canker will be greatest in southern England2. Oilseed rape production in China is threatened by the possible arrival of Leptosphaeria maculans, a more virulent causal agent of phoma stem canker than the existing species Leptosphaeria biglobosa3. A strong correlation found between sulphur dioxide concentration and the ratio of Phaeosphaeria nodorum to Zymoseptoria graminicola in archived wheat samples at Rothamsted Research (1844 to date) suggests that subtle environmental changes can also cause changes in pathogen populations4. Ensuring future food security under environmental change is challenging and will require increased understanding of in planta disease processes. Effector-triggered defence (ETD) operates against extracellular fungal pathogens by R gene-encoded cell surface receptor-like proteins that trigger cell death only after a period of endophytic pathogen growth5. An example of this is resistance in oilseed rape against Pyrenopeziza brassicae that operates by preventing asexual sporulation. Exploiting such understanding and developing strategies to control biotic stressors are important to ensure adequate food supply for the future6.

Alterations in the predicted regulatory and coding regions of the sterol 14α‐demethylase gene (CYP51) confer decreased azole sensitivity in the oilseed rape pathogen Pyrenopeziza brassicae

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.

Detection and molecular characterisation of Pyrenopeziza brassicae isolates resistant to methyl benzimidazole carbamates

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. 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. 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.

Detection and quantification of airborne inoculum of Pyrenopeziza brassicae in Polish and UK winter oilseed rape crops by real-time PCR assays

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.