Resistance to Leptosphaeria maculans (phoma stem canker) in Brassica napus (oilseed rape) induced by L. biglobosa and chemical defence activators in field and controlled environments

Effects of pretreatment of Brassica napus leaves with ascospores of Leptosphaeria biglobosa or chemical defence activators [acibenzolar- S -methyl (ASM) or menadione sodium bisulphite (MSB)] on infection by ascospores of Leptosphaeria maculans (phoma stem canker) and development of disease were studied in controlled-environment (phoma leaf spot) and field (phoma leaf spot and stem canker) experiments. In controlled-environment experiments, pretreatment of oilseed rape leaves (cv. Madrigal) with L. biglobosa , ASM or MSB delayed the appearance of L. maculans phoma leaf spot lesions. These pretreatments also decreased the phoma leaf spot lesion area in both pretreated leaves (local effect) and untreated leaves (systemic effect). In winter oilseed rape field experiments in the 2002/03 and 2003/04 growing seasons, pretreatment with L. biglobosa or ASM in October/November decreased not only the number of phoma leaf spot lesions per leaf caused by L. maculans in autumn/winter, but also the severity of phoma stem canker in the subsequent spring/summer. Effects were greater in 2002/03 (when natural L. maculans ascospore release began in September 2002) than in 2003/04 (when ascospore release began in December following a period of dry weather in August/September 2003). These results suggest that pretreatment with biological or chemical defence activators can induce local and systemic resistance to L. maculans , with both short-term effects on the development of phoma leaf spotting and long-term effects on the development of stem canker 8 months later.     

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.     

Relationship between severity of blackleg (Leptosphaeria maculans/L. biglobosa species complex) and subsequent primary inoculum production on oilseed rape stubble

The relationship between severity of blackleg, or phoma stem canker ( Leptosphaeria maculans/L. biglobosa ), and subsequent primary inoculum production on oilseed rape ( Brassica napus ) stubble was investigated at two sites in France over 3 years. The quantity of primary inoculum produced in the following year increased with canker severity, from 1·9 to 10·8 pseudothecia cm⁻² on stubble with the least and most severe cankers, respectively. Stubble incubated at Le Rheu (cooler, more rain) had 1·7 times more pseudothecia than stubble incubated at Grignon. Stubble collected at Grignon had 2·7 times more pseudothecia than that collected at Le Rheu. The use of Darmor, a cultivar with a good level of quantitative resistance, reduced the severity of canker in the field, but not the subsequent inoculum production for stubble of the same canker severity class. At both sites, maturation of pseudothecia occurred after 63–75 days of incubation and increased with canker severity with a mean of 0·5 and 3% mature pseudothecia appearing per favourable day, on stubble with the least and most severe cankers, respectively. A simplified procedure for pseudothecial quantification proved satisfactory: for all three observers, most (91–96%) of the fructifications counted as pseudothecia were real pseudothecia. Only a few (4–14%) of the fructifications considered as non-pseudothecia were in fact pseudothecia of L. maculans . The total area occupied by pseudothecia, which was simpler and faster to evaluate, was correlated (coefficient of determination, R ² = 71%) with the number of counted pseudothecia. The results presented here make it possible to forecast the quantity of available primary inoculum for a given disease severity.     

Influence of relative humidity and temperature on development of Didymella rabiei on chickpea debris

Didymella rabiei grew saprophytically on pieces of artificially and naturally infected chickpea stem debris under artificial incubation conditions, and formed pseudothecia and pycnidia. The extent of growth was not significantly affected by temperature of incubation within the range 5–25°C, but was significantly reduced as relative humidity (RH) decreased from 100% to 86%, when no growth occurred. Pseudothecia matured at 10°C and constant 100% RH, or at 5 and 10°C and alternating 100%/34% RH. Under these conditions, pseudothecial maturation, assessed by a pseudothecia maturity index, increased over time according to the logistic model. For temperatures higher than 10°C or RH lower than 100%, pseudothecia either did not form ascospores, or ascopores did not mature and their content degenerated. When pseudothecia that initially developed to a given developmental stage were further incubated at a constant 100% RH, temperature became less limiting for complete pseudothecial development as the developmental stage was more advanced. Pycnidia of the fungus developed and formed viable conidia in all environmental conditions studied, except at 86% RH. However, the density of pycnidia formed and the number of viable conidia per pycnidium were significantly influenced by temperature, RH and the type of debris (artificially or naturally infected) used.     

Maturation of pseudothecia and discharge of ascospores of <Emphasis Type="Italic">Leptosphaeria maculans</Emphasis> on oilseed rape stubble

The effects of temperature, wetness and darkness on formation of pseudothecia and the effect of temperature on the release of ascospores of L. maculans on oilseed rape stubble were studied in a controlled environment in South Australia. Pseudothecia of L. maculans developed at 5–20°C and the time taken to reach maturity and discharge ascospores decreased from 58 days at 5°C to 22.2 days at 15°C. The optimum temperature of those tested for pseudothecium maturation was between 15°C and 20°C but fewer pseudothecia were observed at 20°C than at 15°C. Exposure to a 12 h photoperiod enhanced pseudothecium formation on the stubble compared with continuous darkness. No pseudothecia formed on stubble moistened once a day at 15°C, whereas three sprays of water per day decreased maturation time in comparison with two sprays per day. More ascospores were released for a longer duration at 20°C than at 5–15°C, although peak sporulation occurred earlier at 5–10°C than at 20°C. These findings highlight the importance of moisture, temperature and light for production and release of inoculum from stubble. This information, combined with field data, may help to predict the onset of inoculum release.     

Phenology of Didymella rabiei Development on Chickpea Debris Under Field Conditions in Spain

ABSTRACT The development of Didymella rabiei on debris of naturally infected chickpea was investigated in four chickpea-growing areas with different climatic conditions in Spain during 1987 to 1992. D. rabiei extensively colonized chickpea debris and formed pseudothecia and pycnidia. Differentiation of pseudothecial initials occurred regularly across experimental locations by November, 1 month after placement of debris on the soil. Ascospore maturation occurred mainly from late January to late March, depending on location and year. Maximum ascospore discharge from sampled debris pieces placed under suitable environmental conditions occurred 2 to 4 weeks after ascospore maturation, after which ascospore release decreased sharply. Pseudothecia were exhausted, due to ascospore discharge, by the beginning of summer. New asci did not develop in empty pseudothecia and no pseudothecia formed in tissues after the first season. Ascospore maturation and liberation in cooler locations were more uniform and occurred later compared to maturation in warmer locations. Also, production of asci and ascospores per pseudothecium was much higher in cooler than in warmer locations. A similar relationship was found for density of pseudothecia and pycnidia and conidia production per pycnidium. The percentage of mature pseudothecia increased according to the logistic model, with the cumulative number of Celsius degree days calculated by computing the mean of the maximum and minimum daily air temperatures on rainy days from the date of debris placement on the soil. There were significant differences among model parameter estimates between cooler and warmer locations, but minor differences were found among parameters for locations with similar environmental conditions. There was an inverse linear relationship between the average temperature during the period of pseudothecia maturation and the number of asci produced per pseudothecium.     

Survival of Leptosphaeria maculans and associated mycobiota on oilseed rape stubble buried in soil

Leptosphaeria maculans , the causal agent of phoma stem canker on oilseed rape, is an important pathogen in oilseed rape growing regions of the world, including Australia. Survival of L. maculans and associated mycobiota on oilseed rape stubble buried for 13 months in field soil and in sandy soil was studied under South Australian environmental conditions. Stubble weight decreased significantly by the end of the burial period, more so in field (53·7%) than in sandy soil (22%). Pseudothecia did not develop on stubble buried in field soil and few formed when buried in sandy soil. Moist incubation of stubble following retrieval from both media generated pseudothecia; however, pseudothecial development ceased on stubble that had been buried for 10 and 12 months in field and sandy soil, respectively. In total, 20 and 36 genera of fungi were isolated from stubble before and after burial, respectively. Alternaria spp., L. maculans and Stemphylium botryosum were isolated from 81·7, 70 and 60% of stubble pieces before burial, respectively. Isolation frequency of these species decreased significantly throughout the burial period in both media. Conversely, isolation frequency of Stachybotrys chartarum , Fusarium spp. and Trichoderma spp., having pre-burial frequencies of 26·7, 16·7 and 2·5%, respectively, increased over the burial period regardless of soil type. These findings suggest that inoculum production of L. maculans decreases with the increasing burial duration in field soil over 10 months, before ceasing, and this may be due to associated mycobiota.     

Environmental Factors Affecting Pseudothecial Development and Ascospore Production of Mycosphaerella citri, the Cause of Citrus Greasy Spot

ABSTRACT Mycosphaerella citri, the cause of citrus greasy spot, produces pseudothecia and ascospores in decomposing leaf litter on the grove floor. In laboratory studies, the effect of wetting and drying and temperature on the formation, maturation, and production of pseudothecia and ascospores was evaluated on mature, detached grapefruit leaves. Production of pseudothecia was most rapid when leaves were soaked five times per week for 2 h per day, but pseudothecial density and total ascospore production were greatest when leaves were soaked three times per week for 2 h per day. In duration of wetting studies, 3 h per day, 3 days per week brought about the most rapid production, but 10 to 30 min per day resulted in production of the most pseudothecia and ascospores. Pseudothecia and ascospore production were greatest at 28 degrees C and declined rapidly at lower and higher temperatures. Maturation of pseudothecia was slow at 20 and 24 degrees C, but production was high at 24 degrees C; at 32 degrees C, pseudothecia matured rapidly, but degenerated quickly. No mature pseudothecia were produced on leaves maintained continuously under wet conditions. In field studies, leaves were placed on the grove floor monthly from April 2000 to September 2001. Pseudothecia production was rapid during the summer rainy season from June to September. Pseudothecia produced on leaves placed in the grove from October to May developed and matured more slowly but were produced in much larger numbers than in summer. The number of days to first pseudothecial initials, 50% maturation, first discharge of ascospores, leaf decomposition, as well as pseudothecial density and incidence, were negatively related to average temperature. Total ascospore production was unrelated to temperature.     

Strategies to prevent spread of Leptosphaeria maculans (phoma stem canker) onto oilseed rape crops in China; costs and benefits

Field experiments in Europe have shown that Chinese cultivars of winter oilseed rape ( Brassica napus ) are very susceptible to the pathogen Leptosphaeria maculans (cause of phoma stem canker). Climatic and agronomic conditions in China are suitable for L. maculans since the closely related but less damaging pathogen L. biglobosa occurs on the winter and spring oilseed rape crops there. Major gene resistance to L. maculans is not durable; when introduced into commercial oilseed rape cultivars it is rapidly rendered ineffective by changes in the pathogen population. The threat to Chinese oilseed rape production from L. maculans is illustrated by the way in which L. maculans has spread into other areas of the world where previously only L. biglobosa was present, such as Canada and Poland. Models were developed to describe the spread (in space and time) of L. maculans across Alberta province, Canada, based on survey data collected over a 15-year period. These models were used to estimate the potential spread of L. maculans across the Yangtze river oilseed rape growing areas of China and its associated costs. Short-term strategies to prevent occurrence of severe phoma stem canker epidemics in China include training of extension workers to recognise symptoms of the disease and use of PCR-based diagnostics to detect the pathogen on imported seed. Long-term strategies include the introduction of durable resistance to L. maculans into Chinese oilseed rape cultivars as a component of an integrated disease management programme. The costs of such strategies in relation to costs of a phoma stem canker epidemic are discussed.     

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.     

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.     

Occurrence of a new subclade of Leptosphaeria biglobosa in Western Australia

Stem canker of crucifers is caused by an ascomycete species complex comprising of two main species, Leptosphaeria maculans and L. biglobosa. These are composed of at least seven distinct subclades based on biochemical data or on sequences of internal transcribed spacer (ITS), the mating type MAT1-2 or fragments of actin or beta-tubulin genes. In the course of a wide-scale characterization of the race structure of L. maculans from Western Australia, a few isolates from two locations failed to amplify specific sequences of L. maculans, i.e., the mating-type or minisatellite alleles. Based on both pathogenicity tests and ITS size, these isolates were classified as belonging to the L. biglobosa species. Parsimony and distance analyses performed on ITS, actin and beta-tubulin sequences revealed that these isolates formed a new L. biglobosa subclade, more related to the Canadian L. biglobosa 'canadensis' subclade than to the L. biglobosa 'australensis' isolates previously described in Australia (Victoria). They are termed here as L. biglobosa 'occiaustralensis'. These isolates were mainly recovered from resistant oilseed rape cultivars that included the Brassica rapa sp. sylvestris-derived resistance source, but not from the susceptible cv. Westar. The pathogenicity of L. biglobosa 'occiaustralensis' to cotyledons of most oilseed rape genotypes was higher than that of L. biglobosa 'canadensis' or L. biglobosa 'australensis' isolates.     

Effects of environmental factors on discharge and germination of ascospores of Venturia nashicola

Experiments were conducted under controlled environmental conditions to study the effects of temperature, duration of wetness, relative humidity (RH) and light on the discharge and germination of ascospores of Venturia nashicola , the causal agent of pear scab in China. Discharge of ascospores from pseudothecia required free water or 100% RH. A period of soaking in water as short as 10 s was sufficient to initiate the discharge of ascospores. Temperatures from 10 to 30°C did not significantly affect the temporal trend of ascospore discharge. A greater proportion of ascospores was discharged under light than in the dark. However, a period of light as short as 10 min, either during the initial wetting of pseudothecia or interrupting the darkness, was sufficient to reduce the inhibitory effect of darkness on ascospore discharge. Ascospores were discharged within 10 min after pseudothecia were wetted and most ascospores ( c. 80%) were discharged within the first hour. The temporal pattern of ascospore discharge could be well described by a logistic model, which estimated that 50% of ascospores were discharged within half an hour of wetting. Ascospores germinated over a wide range of temperatures from 5 to 30°C, with an optimum at c . 20°C. Temporal dynamics of ascospore germination at six temperatures (5, 10, 15, 20, 25 and 30°C) were satisfactorily described by logistic models.     

Latent infection of winter oilseed rape by Leptosphaeria maculans

Plants of oilseed rape, cultivars Primer and Jet Neuf, were grown in a glasshouse and inoculated at G.S. 2.4–2.7 with pycnidiospores or ascospores of Leptosphaeria maculans. The plants were kept for a further 2–4 weeks at 14°C and then transferred, together with uninoculated plants, to a polythene tunnel in winter. The majority of stems of inoculated plants did not have macroscopic symptoms of L. maculans infection 6 weeks after inoculation. Examination of whole mounts of peripheral tissue and transverse sections of fixed and embedded portions of these stems revealed intercellular septate fungal hyphae, often deep in non-necrotic cortical tissue, in symptomless inoculated plants but not in uninoculated plants. L. maculans was recovered following surface sterilization of adjacent portions of the same stems. When symptomless inoculated plants were transferred to a glasshouse at 18–20°C, cankers soon developed. The significance of these latent mycelial infections to canker development in the field is discussed.     

Geostatistical analysis of the distribution of Leptosphaeria species causing phoma stem canker on winter oilseed rape (Brassica napus) in England

In June/July 2001, 2002, 2003 and 2006, regional variation in distribution of the pathogens Leptosphaeria maculans and L. biglobosa that are causally associated with phoma stem canker was surveyed on winter oilseed rape crops in England. In 2001–2003, when isolates from basal cankers were visually identified as L. maculans or L. biglobosa based on cultural morphological characteristics, 70% were L. maculans and 30% L. biglobosa . In 2001, 2002, 2003 and 2006, when amounts of DNA of each species in basal cankers were determined by quantitative PCR, the abundance of L. maculans DNA was greater than that of L. biglobosa DNA in 77% of samples. When regional differences in amounts of L. maculans and L. biglobosa DNA were mapped geostatistically, quantities of L. maculans DNA were greater in cankers from southern England and those of L. biglobosa DNA were greater in northern England. A comparison with geostatistically mapped predictions made using a weather-based model describing stages in development of phoma stem canker epidemics suggested that these differences in Leptosphaeria populations may have been a consequence of differences in temperature after onset of leaf spotting between northern and southern England. Both PCR and morphological evidence suggested that the abundance of L. maculans in England has increased since the last surveys in the 1980s. Implications of these surveys for control of phoma stem canker are discussed.     

The effect of winter weather conditions on the ability of pseudothecia of Leptosphaeria maculans and L. biglobosa to release ascospores

Leptosphaeria maculans and L. biglobosa are damaging pathogens of oilseed rape. The infection of plants occurs predominantly in early autumn or spring by spores produced in pseudothecia. The aim of this study was to investigate whether pseudothecia formed in the autumn are still viable in the spring and to what extend they are destroyed by winter frosts. The studies presented here demonstrated that winter frosts can render pseudothecia unable to release spores. Nevertheless, ascospores present in pseudothecia unable to discharge ascospores, were fully capable of germination, regardless of the incubation temperature. No significant differences were found between the studied Leptosphaeria species in their response to frost. A multiple regression equation has been elaborated to forecast the ability of pseudothecia to release ascospores, based on winter temperatures. Considerable correlation was found between the ascospore release in the autumn and the ability of pseudothecia to release ascospores over the winter period and the subsequent symptoms of stem canker before harvest. We have demonstrated that the potential and the survival of inoculum can have a large impact on the success of the pathogen. This may be particularly important in the light of forecasted climate change. Higher winter temperatures may increase the ability of pseudothecia to release ascospores and the discharge of ascospores of L. maculans and L. biglobosa into the air, and cause early plant infections. This in turn will increase the number of infected plants, the disease incidence at harvest, and reduce the yield of oilseed rape.     

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.     

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 Leptosphaeria maculans – Leptosphaeria biglobosa species complex in the American continent

Stem canker of oilseed rape (canola, Brassica napus ) is associated with a species complex of two closely related fungal species, Leptosphaeria maculans and L. biglobosa . Of these, L. maculans is the most damaging and develops gene-for-gene relationships with the host . Here, a wide scale analysis of the L. maculans - L. biglobosa species complex was performed throughout the American continent (23 locations from Chile to Canada) plus several locations in Western Australia for comparison purposes, based on a collection of 1132 isolates from infected tissues of a susceptible cultivar. Fungal species were discriminated on the basis of morphological, phytopathological and molecular criteria and showed that L. biglobosa was closely associated with L. maculans in most of the locations. Multiple gene phylogeny using sequences of ITS, actin and β-tubulin confirmed the prevalence of the L. biglobosa 'canadensis' sub-clade in Canada, whereas up to three different sub-clades of L. biglobosa were found in Georgia (USA). Race structure of L. maculans was investigated using a combination of pathogenicity tests and PCR amplification of avirulence alleles AvrLm1 , AvrLm4 and AvrLm6 . Three contrasting situations were observed: (i) race structure in Ontario, Chile and Georgia was related to that of European and Western Australian populations, with a low race diversity; (ii) only one race was found in Mexico, and not found outside of this country; (iii) a large diversity of races was observed in central Canada (Manitoba, Alberta and Saskatchewan) with very specific features including maintenance of avirulence alleles absent from Europe, absence of the AvrLm7 allele common in Europe (or eastern Canada) and wide location-to-location variability.