Effects of temperature on the development of light leaf spot (Pyrenopeziza brassicae) on oilseed rape (Brassica napus)

The effects of temperature on the development of light leaf spot (Pyrenopeziza brassicae) on winter oilseed rape were investigated in controlled-environment experiments. The proportion of conidia which germinated on leaves, the growth rate of germ tubes, the severity of light leaf spot and the production of conidia increased with increasing temperature from 5 to 15 C. The time to 50% germination of conidia and the incubation and latent periods of light leaf spot lesions decreased when temperature increased from 5 to 15°C. At 20°C, however, light leaf spot severity and production of conidia were less and the incubation and latent periods were longer than at 15 C. There were differences between P brassicae isolates and oilseed rape cultivars in the severity of light leaf spot, the production of conidia and the length of the incubation period but not in the length of the latent period. The responses to temperature for lesion severity and incubation and latent periods appeared to be approximately linear over the temperature range 5-15°C and could be quantified using linear regression analysis.     

Visual and PCR assessment of light leaf spot (Pyrenopeziza brassicae) on winter oilseed rape (Brassica napus) cultivars

Methods to assess light leaf spot ( Pyrenopeziza brassicae ) on winter oilseed rape cultivars were compared in laboratory, controlled-environment and field experiments. In controlled-environment experiments with seedling leaves inoculated at GS 1,4, the greatest differences in percentage area affected by P. brassicae sporulation were observed with inoculum concentrations of 4 × 10³ or 4 × 10⁴ spores mL⁻¹, rather than 4 × 10² or 4 × 10⁵ spores mL⁻¹, but older leaves had begun to senesce before assessment, particularly where they were severely affected by P. brassicae . In winter oilseed rape field experiments, a severe light leaf spot epidemic developed in 2002/03 (inoculated, September/October rainfall 127·2 mm) but not in 2003/04 (uninoculated, September/October rainfall 40·7 mm). In-plot assessments discriminated between cultivars best in February/March in 2003 and June in 2004, but sometimes failed to detect plots with many infected plants (e.g. March/April 2004). Ranking of cultivar resistance differed between seedling experiments done under controlled-environment conditions and field experiments. The sensitivity of detection of P. brassicae DNA extracted from culture was greater using the PCR primer pair PbITSF/PbITSR than using primers Pb1/Pb2. P. brassicae was detected by PCR (PbITS primers) in leaves from controlled-environment experiments immediately and up to 14 days after inoculation, and in leaves sampled from field experiments 2 months before detection by visual assessment.     

Modelling the progress of light leaf spot (Pyrenopeziza brassicae) on winter oilseed rape (Brassica napus) in relation to leaf wetness and temperature

A compartmental model was developed to describe the progress with time of light leaf spot ( Pyrenopeziza brassicae ) on leaves of winter oilseed rape ( Brassica napus ) during the autumn in the UK. Differential equations described the transition between the four compartments: healthy susceptible leaves, infected symptomless leaves, sporulating symptomless leaves and leaves with necrotic light leaf spot lesions, respectively. The model was fitted to data on the progress of light leaf spot on winter oilseed rape at a single site during the autumn of the 1990–1991 season. Model parameters were used to describe rates of leaf appearance, leaf death, infection by airborne ascospores (primary inoculum) and infection by splash-dispersed conidiospores (secondary inoculum). Infection was dependent on sufficient leaf wetness duration. The model also included delay terms for the latent period between infection and sporulation and the incubation period between infection and the appearance of necrotic light leaf spot lesions. This modified SEIR model formulation gave a reasonable fit to the experimental data. Sensitivity analysis showed that varying the parameter accounting for the rate of infection by ascospores affected the magnitude of the curves after the start of the epidemic, whilst including a parameter for conidiospore infection improved the fit to the data. Use of ascospore counts from different sites and different years showed variation in spore release patterns sufficient to affect model predictions.     

Early development of light leaf spot (Pyrenopeziza brassicae) on winter oilseed rape (Brassica napus) in relation to temperature and leaf wetness.

In controlled environment experiments to study early development of light leaf spot, lesions developed with leaf wetness durations of 16 to 48 h after inoculation of oilseed rape with conidial suspensions of Pyrenopeziza brassicae at 12 or 18°C, but not with leaf wetness durations of 0 to 13h. The incubation period was 21 to 22 days at 12°C and 14 to 18 days at 18°C for leaf wetness durations of 16 to 48 h. The latent period was 21 to 23 days at 12°C and 18 to 19 days at 18°C, and the total number of lesions increased with increasing leaf wetness duration at both temperatures. In field experiments, light leaf spot always developed on oilseed rape with a leaf wetness duration of 48 h after inoculation in both 1990/1991 and 1991/1992, but the percentage leaf area affected was less on plants placed in an oilseed rape crop than on those placed in a glasshouse. Plants moved to an oilseed rape crop immediately after inoculation nearly always developed light leaf spot symptoms when they were inoculated between 19 October 1990 and 1 March 1991 or between 27 September 1991 and 14 February 1992, but plants inoculated between 31 August and 16 October 1990 or on 20 September 1991, when estimated leaf wetness duration was less than 16 h for several days after they were placed in crops, did not develop symptoms. The latent period of light leaf spot on plants transferred to the oilseed rape crop was 15 to 40 days, and there was an approximately linear relationship between 1 (latent period) and mean temperature during this period. The accumulated temperature during the latent period ranged from c. 150 to 250 day-degrees. The severity of lesions on these plants increased with increasing temperature from 5 to 15°C.     

Predicting light leaf spot (Pyrenopeziza brassicae) risk on winter oilseed rape (Brassica napus) in England and Wales, using survey, weather and crop information

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 by Pyrenopeziza brassicae on 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.     

Effects of R gene‐mediated resistance in Brassica napus (oilseed rape) on asexual and sexual sporulation of Pyrenopeziza brassicae (light leaf spot)

The phenotype of the R gene‐mediated resistance derived from oilseed rape (Brassica napus) cv. Imola against the light leaf spot plant pathogen, Pyrenopeziza brassicae, was characterized. Using a doubled haploid B. napus mapping population that segregated for resistance against P. brassicae, development of visual symptoms was characterized and symptomless growth was followed using quantitative PCR and scanning electron microscopy on leaves of resistant/susceptible lines inoculated with suspensions of P. brassicae conidia. Initially, in controlled‐environment experiments, growth of P. brassicae was unaffected; then from 8 days post‐inoculation (dpi) some epidermal cells collapsed (‘black flecking’) in green living tissue of cv. Imola and from 13 to 36 dpi there was no increase in the amount of P. brassicae DNA and no asexual sporulation (acervuli/pustules). By contrast, during this period there was a 300‐fold increase in P. brassicae DNA and extensive asexual sporulation in leaves of the susceptible cv. Apex. However, when leaf tissue senesced, the amount of P. brassicae DNA increased rapidly in the resistant but not in the susceptible cultivar and sexual sporulation (apothecia) was abundant on senescent tissues of both. These results were consistent with observations from both controlled condition and field experiments with lines from the mapping population that segregated for this resistance. Analysis of results of both controlled‐environment and field experiments suggested that the resistance was mediated by a single R gene located on chromosome A1.     

Comparative epidemiology of Pyrenopeziza brassicae (light leaf spot) ascospores and conidia from Polish and UK populations

Despite differences in climate and in timing of light leaf spot epidemics between Poland and the UK, experiments provided no evidence that there are epidemiological differences between populations of Pyrenopeziza brassicae in the two countries. Ascospores of Polish or UK P. brassicae isolates germinated on water agar at temperatures from 8 to 24°C. After 12 h of incubation, percentages of ascospores that germinated were greatest at 16°C: 85% (Polish isolates) and 86% (UK isolates). The percentage germination reached 100% after 80 h of incubation at all temperatures tested. The rate of increase in germ tube length increased with increasing temperature from 8 to 20°C but decreased from 20 to 24°C, for both Polish and UK isolates. Percentage germination and germ tube lengths of UK P. brassicae ascospores were less affected by temperature than those of conidia. P. brassicae produced conidia on oilseed rape leaves inoculated with ascospores or conidia of Polish or UK isolates at 16°C with leaf wetness durations from 6 to 72 h, with most sporulation after 48 or 72 h wetness. Detection of both mating types of P. brassicae and production of mature apothecia on leaves inoculated with mixed Polish populations suggest that sexual reproduction does occur in Poland, as in the UK.     

Sporulation of Pyrenopeziza brassicae (light leaf spot) on oilseed rape (Brassica napus) leaves inoculated with ascospores or conidia at different temperatures and wetness durations

In controlled environment experiments, sporulation of Pyrenopeziza brassicae was observed on leaves of oilseed rape inoculated with ascospores or conidia at temperatures from 8 to 20°C at all leaf wetness durations from 6 to 72 h, except after 6 h leaf wetness duration at 8°C. The shortest times from inoculation to first observed sporulation ( l ₀), for both ascospore and conidial inoculum, were 11–12 days at 16°C after 48 h wetness duration. For both ascospore and conidial inoculum (48 h wetness duration), the number of conidia produced per cm² leaf area with sporulation was seven to eight times less at 20°C than at 8, 12 or 16°C. Values of Gompertz parameters c (maximum percentage leaf area with sporulation), r (maximum rate of increase in percentage leaf area with sporulation) and l ₃₇ (days from inoculation to 37% of maximum sporulation), estimated by fitting the equation to the observed data, were linearly related to values predicted by inserting temperature and wetness duration treatment values into existing equations. The observed data were fitted better by logistic equations than by Gompertz equations (which overestimated at low temperatures). For both ascospore and conidial inoculum, the latent period derived from the logistic equation (days from inoculation to 50% of maximum sporulation, l ₅₀) of P. brassicae was generally shortest at 16°C, and increased as temperature increased to 20°C or decreased to 8°C. Minimum numbers of spores needed to produce sporulation on leaves were ≈25 ascospores per leaf and ≈700 conidia per leaf, at 16°C after 48 h leaf wetness duration.     

The production and release of ascospores of Pyrenopeziza brassicae on oilseed rape

The results of observation for three seasons in oilseed rape crops of the production and release of ascopores of Pyrenopeziza brassicae , the cause of light leaf spot disease of brassicas, are presented. Large apothecia (1–2 mm in diameter) take at least 3 weeks to develop on leaf petioles after infected leaves die, but small apothecia (50–200 um) may form on leaf lamellae after about 15 days. Apothecia were found on all 12 oilseed rape varieties examined. Spore trapping experiments in infected crops show that ascospore release is associated with rain, but most ascospores are released after rainfall when the crop debris bearing apothecia are wet. Ascospores can be released for up to 5 days after rain. Laboratory measurements show that apothecia can continue to release ascospores for up to 3 weeks even when they are subjected to wet and dry cycles. The consequences of the widespread occurrence of the sexual stage of P. brassicae are discussed and possible cycles of disease and spore production in oilseed crops are suggested.     

Factors affecting the incubation period of dark leaf and pod spot (Alternaria brassicae) on oilseed rape (Brassica napus)

Experiments to investigate the factors affecting the incubation period of dark leaf and pod spot (Alternaria brassicae) on leaves and pods of oilseed rape (Brassica napus) were done in controlled environment (constant temperatures) and glasshouse conditions (fluctuating temperatures). The length of the incubation period of dark leaf and pod spot decreased as infection and incubation temperatures increased from 6 to 20 °C. The incubation period decreased as wetness period increased from 2 to 12 h, as inoculum concentration increased from 80 to 2 × 10³ spores ml^–1 and as leaf age increased from 4 to 10 days. Asymptotes of leaf age and inoculum concentration, above which the length of the incubation period did not decrease, were 10 days and 2 × 10³ spores ml^–1, respectively. The shortest and longest incubation periods were 1 and 11 days. The mechanism by which the infection conditions influenced the incubation period of dark leaf and pod spot on oilseed rape seemed to be linked to lesion density. Usually, the length of the incubation period decreased greatly with increasing lesion density.     

Winter oilseed rape with high levels of resistance to Pyrenopeziza brassicae derived from wild Brassica species

Oilseed rape cultivars possess inadequate levels of resistance to light leaf spot disease, caused by the ascomycete Pyrenopeziza brassicae Sutton & Rawlinson. High levels of resistance to this disease were found within wild accessions of Brassica oleracea and B. rapa . This resistance was introgressed into agronomically acceptable winter oilseed rape breeding lines. Seedling resistance was determined by two genes. One of these, derived from B. rapa and positioned on linkage group N1, resulted in no apparent symptoms following infection, while the other, derived from B. oleracea and positioned on N16, resulted in black necrotic flecks and a reduced amount of sporulation compared with standard cultivars. Several agronomically acceptable double haploid lines were developed which expressed very high levels of adult plant resistance.     

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.     

Assessment of benomyl resistance and mating type in field isolates of Pyrenopeziza brassicae, cause of light leaf spot of brassicas

In 1987, 254 isolates of Pyrenopeziza brassicae from 27 winter oilseed rape crops in East Anglia were tested for their resistance to benomyl and for their mating type. All of the isolates were found to be susceptible to benomyl. Both the known mating types were represented amongst the isolates from 74% of the fields although in 33% of the fields 70% or more of the isolates were of one mating type. In two fields, only one mating type (MA T1-l) was present. Some isolates were of neither of these mating types.     

Detection of airborne inoculum of Leptosphaeria maculans and Pyrenopeziza brassicae in oilseed rape crops by polymerase chain reaction (PCR) assays

The potential use of DNA-based methods for detecting airborne inoculum of Leptosphaeria maculans and Pyrenopeziza brassicae , both damaging pathogens of oilseed rape, was investigated. A method for purifying DNA from spores collected using Hirst-type spore samplers and detecting it using polymerase chain reaction (PCR) assays is described. For both pathogens, the sensitivities of the DNA assays were similar for spore-trap samples and pure spore suspensions. As few as 10 spores of L. maculans or P. brassicae could be detected by PCR and spores of both species could be detected against a background of spores of six other species. The method successfully detected spores of P. brassicae collected using spore traps in oilseed rape crops that were infected with P. brassicae. Leptosphaeria maculans spores were detected using spore traps on open ground close to L. maculans -infected oilseed rape stems. The potential use of PCR detection of airborne inoculum in forecasting the diseases caused by these pathogens is discussed.     

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.     

Effects of temperature and wetness duration on conidial infection, latent period and asexual sporulation of Pyrenopeziza brassicae on leaves of oilseed rape

Experiments in controlled environments were carried out to determine the effects of temperature and leaf wetness duration on infection of oilseed rape leaves by conidia of the light leaf spot pathogen, Pyrenopeziza brassicae . Visible spore pustules developed on leaves of cv. Bristol inoculated with P. brassicae conidia at temperatures from 4 to 20°C, but not at 24°C; spore pustules developed when the leaf wetness duration after inoculation was longer than or equal to approximately 6 h at 12–20°C, 10 h at 8°C, 16 h at 6°C or 24 h at 4°C. On leaves of cvs. Capricorn or Cobra, light leaf spot symptoms developed at 8 and 16°C when the leaf wetness duration after inoculation was greater than 3 or 24 h, respectively. The latent period (the time period from inoculation to first spore pustules) of P. brassicae on cv. Bristol was, on average, approximately 10 days at 16°C when leaf wetness duration was 24 h, and increased to approximately 12 days as temperature increased to 20°C and to 26 days as temperature decreased to 4°C. At 8°C, an increase in leaf wetness duration from 10 to 72 h decreased the latent period from approximately 25 to 16 days; at 6°C, an increase in leaf wetness duration from 16 to 72 h decreased the latent period from approximately 23 to 17 days. The numbers of conidia produced were greatest at 12–16°C, and decreased as temperature decreased to 8°C or increased to 20°C. At temperatures from 8 to 20°C, an increase in leaf wetness duration from 6 to 24 h increased the production of conidia. There were linear relationships between the number of conidia produced on a leaf and the proportion of the leaf area covered by 'lesions' (both log₁₀-transformed) at different temperatures.     

Effects of wetness period and temperature on development of dark pod spot (Alternaria brassicae) on oilseed rape (Brassica napus)

In controlled environment experiments, when oilseed rape pods or leaves were inoculated with spore suspensions of Alternaria brassicae, the maximum disease incidence (proportion of pods or leaves diseased) increased as wetness period after inoculation increased from 4 to 24 h and as temperature increased to 20°C. There was a clear relationship between disease incidence on pods and incidence on leaves with the same wetness/temperature conditions. Logistic equations described the effects of wetness period after inoculation on disease incidence (number of pods or leaves infected) or disease severity (number of lesions on pods or leaves) using temperature-dependent and tissue-dependent parameters. The time from inoculation to the appearance of the first lesions was shorter on pods than on leaves at temperatures ≤15°C and wetness periods ≤12 h. Two-dimensional response surface equations or simple interpolations from one-dimensional equations were used to develop contour maps of expected disease incidence and severity, respectively, on leaves or pods to estimate the effects of different combinations of wetness period during infection and temperature on disease development.