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.     

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.     

Effects of Severity and Timing of Stem Canker (Leptosphaeria maculans) Symptoms on Yield of Winter Oilseed Rape (Brassica napus) in the UK

The relationships between yield loss and incidence (% plants with stems affected) or severity (mean stem score, 0–4 scale) of stem canker in winter oilseed rape were analysed using data from experiments at Rothamsted in 1991/92, Withington in 1992/93, Boxworth in 1993/94 and Rothamsted in 1997/98. Critical point models and area under disease progress curve (AUDPC) models were better than multiple point models for describing relationships between yield (tha^–1) and incidence or severity of stem canker for the four experiments. Since yield is influenced by many factors other than disease, % yield loss was calculated and critical point models and AUDPC models relating % yield loss to stem canker were constructed. The critical point models for % yield loss on stem canker incidence for three of the four experiments were similar, but differed from that for Rothamsted in 1991/92. There were also no differences between models of % yield loss on AUDPC of both incidence and severity for these three experiments. Therefore, general models of % yield loss (L) against AUDPC of incidence (X) or severity (S) of stem canker from growth stages 4.8 to 6.4 were derived from the combined data sets for the three experiments: L=–0.76+0.0075X (R²=35%, p<0.001), L=0.26+0.53S (R²=37%, p<0.001). The relationships between % yield loss and % plants with different stem canker severity scores at different growth stages were also analysed; the greatest yield losses were generally associated with the largest severity scores, for plants assessed at the same crop growth stage, and were also associated with the early development of stem lesions. Further analyses showed that % yield loss was related to incidence or severity of both basal stem cankers and upper stem lesions in experiments at Boxworth in 1993/94 and at Rothamsted in 1997/98.     

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

Stem canker or blackleg of brassicas, caused by Leptosphaeria maculans , is one of the most damaging diseases of winter oilseed rape in the UK. Airborne ascospores, released in autumn and winter, initiate leaf infections which may lead to colonization of the petiole and, later in the season, formation of stem lesions and cankers. Although isolates of the pathogen differ in ability to cause damaging stem cankers, this is not readily apparent from leaf spotting or stem lesion symptoms. However, several cultural, biochemical and genetic characteristics appear to be associated with the ability to form damaging stem cankers and isolates can be assigned to one of two groups, termed A and B, on the basis of differences in these characteristics. To investigate the relationship between leaf spotting symptoms and subsequent stem canker formation, and to improve understanding of the epidemiology of this pathogen, it is desirable to differentiate between the stem canker forming A group and the less damaging B group of L. maculans . Characterization of isolate type is also important in seed testing and crop breeding programs, particularly in countries such as Canada and Poland where the A type is not ubiquitous. This article reviews methods, including plant assays, assessments of growth characteristics in vitro , isozyme analyses, secondary metabolite profiling, serology, and nucleic acid analyses, that can be used to differentiate the A and B groups.     

Effects of cultivar resistance and fungicide application on stem canker of oilseed rape (Brassica napus) and potential interseasonal transmission of Leptosphaeria spp. inoculum

Phoma stem canker is a damaging disease of oilseed rape (Brassica napus) that causes annual yield losses to UK oilseed rape growers worth approximately £100 million, despite the use of fungicides. In the UK, oilseed rape is sown in August/September and harvested in the following July. The disease epidemics are initiated by ascospores released from Leptosphaeria spp. pseudothecia (ascocarps) on stem stubble in the autumn/winter. Control of this disease is reliant on the use of cultivars with “field resistance” and azole fungicides. This study investigated the effects of cultivar resistance and application of the fungicide prothioconazole on the severity of stem canker before harvest and the subsequent production of pseudothecia on the infected stubble under natural conditions in the 2017/2018, 2018/2019, and 2019/2020 cropping seasons. The application of prothioconazole and cultivar resistance decreased the severity of phoma stem canker before harvest, and the subsequent production of Leptosphaeria spp. pseudothecia on stubble in terms of pseudothecial density. Results showed that stems with less severe stem cankers produced fewer mature pseudothecia of Leptosphaeria spp. on the infected stubble. This investigation suggests that the most sustainable and effective integrated control strategy for phoma stem canker in seasons with low quantities of inoculum is to use cultivars with medium or good field resistance and apply only one spray of prothioconazole when required.     

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 temperature on ascospore germination and penetration of oilseed rape (Brassica napus) leaves by A- or B-group Leptosphaeria maculans (phoma stem canker)

Ascospores of both A-group and B-group Leptosphaeria maculans germinated at temperatures from 5 to 20°C on leaves of oilseed rape. Germination of ascospores of both groups started 2 h after inoculation and percentage germination reached its maximum about 14 h after inoculation at all temperatures. Both the percentage of A-/B-group ascospores that had germinated after 24 h incubation and germ tube length increased with increasing temperature from 5 to 20°C. Germ tubes from B-group ascospores were longer than those from A-group ascospores at all temperatures, with the greatest difference at 20°C. Hyphae from ascospores of both groups penetrated the leaves predominantly through stomata, at temperatures from 5 to 20°C. A-group ascospores produced highly branched hyphae that grew tortuously, whereas B-group ascospores produced long, straight hyphae. The percentage of germinated ascospores that penetrated stomata increased with increasing temperature from 5 to 20°C and was greater for A-group than for B-group L. maculans after 40 h incubation.     

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.     

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.     

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 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.     

Epidemiology and management of Leptosphaeria maculans (phoma stem canker) on oilseed rape in Australia, Canada and Europe

Phoma stem canker (blackleg), caused by Leptosphaeria maculans , is an important disease on oilseed rape (canola, rapeseed, Brassica napus , Brassica juncea , Brassica rapa ) causing seedling death, lodging or early senescence in Australia, Canada and Europe, but not in China. The two forms of L. maculans (A group and B group) that occur on oilseed rape are now considered to be separate species. The epidemiology and severity of phoma stem canker differs between continents due to differences in the pathogen population structure, oilseed rape species and cultivars grown, climate and agricultural practices. Epidemics are most severe in Australia, where only the A group occurs, and can be damaging in Canada and western Europe, where both A and B groups occur, although their proportions vary within regions and throughout the year. Epidemics are slight in China, where the A group has not been found. Dry climates (Australia, western Canada) lengthen the persistence of infected debris and may synchronize the release of airborne ascospores (after rain) with seedling emergence. L. maculans spreads from cotyledon and leaf infections down petioles to reach the stem, with infections on cotyledons and leaves early in the season producing the most damaging stem cankers at the stem base (crown). Development of both crown cankers and phoma stem lesions higher up stems is most rapid in regions with high temperatures from flowering to harvest, such as Australia and Canada. Breeding for resistance (genetic, disease escape or tolerance), stubble management, crop rotation and fungicide seed treatments are important strategies for control of phoma stem canker in all areas. Fungicide spray treatments are justified only in regions such as western Europe where high yields are obtained, and accurate forecasts of epidemic severity are needed to optimize their use.     

Survival of Leptosphaeria maculans in soil on residues of Brassica napus in South Australia

The survival of Leptosphaeria maculans , which causes phoma stem canker (blackleg), on oilseed rape residues ( Brassica napus ) in South Australia was investigated. Using a quantitative polymerase chain reaction (PCR) assay for L. maculans DNA, the pathogen was mainly detected in the upper 5 cm of the soil profile, including residues on the soil surface. As the size of organic matter particles in the soil decreased, so did the quantity of L. maculans detected in them. To obtain representative data for a field, at least 30 subsamples needed to be collected over the 0·81 ha area studied. In a survey of 49 commercial fields in South Australia, most L. maculans was detected in fields 1 year after oilseed rape had been grown, with less detected after 2 years and negligible amounts 3 years or more after cropping. The diagnostic DNA-based assay for L. maculans reduced the time and cost of studying L. maculans survival in soil and increased the sensitivity and accuracy of results compared with estimates of propagule number of colony-forming units on a semiselective medium.     

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.     

Effectiveness of Rlm7 resistance against Leptosphaeria maculans (phoma stem canker) in UK winter oilseed rape cultivars

The Rlm7 gene in Brassica napus is an important source of resistance for control of phoma stem canker on oilseed rape caused by the fungus Leptosphaeria maculans. This study shows the first report of L. maculans isolates virulent against Rlm7 in the UK. Leptosphaeria maculans isolates virulent against Rlm7 represented 3% of the pathogen population when cultivars with the Rlm7 gene represented 5% of the UK oilseed rape area in 2012/13. However, the Rlm7 gene has been widely used since then, representing >15% of the UK oilseed rape area in 2015/16. Winter oilseed rape field experiments included cultivars with the Rlm7 gene, with the Rlm4 gene or without Rlm genes and took place at five sites in the UK over four cropping seasons. An increase in phoma leaf spotting severity on Rlm7 cultivars in successive seasons was observed. Major resistance genes played a role in preventing severe phoma leaf spotting at the beginning of the cropping season and, in addition, quantitative resistance (QR) in the cultivars examined made an important contribution to control of phoma stem canker development at the end of the cropping season. Deployment of the Rlm7 resistance gene against L. maculans in cultivars with QR in combination with sustainable disease management practices will prolong the use of this gene for effective control of phoma stem canker epidemics.     

Effects of Temperature and Wetness Duration on Infection of Oilseed Rape Leaves by Ascospores of Leptosphaeria maculans (Stem Canker)

In controlled environment experiments, ascospores of Leptosphaeria maculans (stem canker) infected oilseed rape (cv. Nickel) leaves and caused phoma leaf spots at temperatures from 8°C to 24°C and leaf wetness durations from 8 h to 72 h. The conditions that produced the greatest numbers of leaf spot lesions were a leaf wetness duration of 48 h at 20°C; numbers of lesions decreased with decreasing leaf wetness duration and increasing or decreasing temperature. At 20°C with 48 h of leaf wetness, it was estimated that one out of four spores infected leaves to cause a lesion whereas with 8 h of leaf wetness only one out of 300 spores caused a lesion. As temperature increased from 8°C to 20°C, the time from inoculation to the appearance of the first lesions (a measure of the incubation period) decreased from 15 to 5 days but leaf wetness duration affected the length of the incubation period only at sub-optimal temperatures. Analyses suggested that, within the optimal ranges, there was little effect of temperature or wetness duration on incubation period expressed as degree-days; the time until appearance of 50% of the lesions was ca. 145 degree-days. A linear regression of % leaves with lesions (P_l) (square-root transformed) on % plants with lesions (P_p) accounted for 93% of the variance: P_l=1.31+0.061P_p. This relationship was also investigated in winter oilseed rape field experiments in unsprayed plots from October to April in 1995/96 (cv. Envol), 1996/97 (cv. Envol), 1997/98 (cvs Bristol and Capitol) and 1998/99 (cvs Apex, Bristol and Capitol) seasons. The linear regression of % leaves with lesions (square-root transformed) on % plants with lesions accounted for 90% of the variance and had a similar slope to the controlled environment relationship: P_l=0.81+0.051P_p. These results were used to examine relationships between the development of phoma leaf spot on plants in winter oilseed rape crops, the incubation period of L. maculans and the occurrence of infection criteria (temperature, rainfall) in the autumns of 1996, 1997 and 1998.     

Quantitative resistance to symptomless growth of Leptosphaeria maculans (phoma stem canker) in Brassica napus (oilseed rape)

Quantitative resistance to Leptosphaeria maculans in Brassica napus was investigated in field and controlled environments using cultivars Darmor (with quantitative resistance) and Eurol (without quantitative resistance). In field experiments, numbers of phoma leaf spot lesions in autumn/winter and severity of stem canker the following summer were assessed in three growing seasons. There were no differences between Darmor and Eurol in number of leaf lesions in autumn/winter. However, stem cankers were less severe on Darmor than Eurol at harvest the following summer. In controlled-environment experiments, development of leaf lesions at different temperatures (5–25°C) and wetness durations (12–72 h) was investigated using ascospore inoculum; symptomless growth of L. maculans along leaf petioles towards the stem was quantified using quantitative PCR and visualized using GFP-expressing L. maculans ; growth of L. maculans within stem tissues was investigated using GFP-expressing L. maculans . There were more leaf lesions on Darmor than Eurol, although there was no difference between Darmor and Eurol in L. maculans incubation period. There were no differences between Darmor and Eurol in either distance grown by L. maculans along leaf petioles towards the stem or quantity of L. maculans DNA in leaf petioles, but L. maculans colonized stem tissues less extensively on Darmor than Eurol. It was concluded that quantitative resistance to L. maculans operates during colonization of B. napus stems by the pathogen.