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.     

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.     

Systemic growth of Leptosphaeria maculans from cotyledons to hypocotyls in oilseed rape: influence of number of infection sites, competitive growth and host polygenic resistance

The influence of competitive effects between two isolates, of the number of infection sites on cotyledons and of host polygenic resistance on the systemic growth of Leptosphaeria maculans , the cause of phoma stem canker in oilseed rape ( Brassica napus ), were investigated. Controlled-condition experiments were conducted with two oilseed rape doubled haploid lines, one susceptible and the other with a high level of polygenic resistance, inoculated via wounded cotyledons with conidial suspensions obtained from two isolates. Expression of cankers in plants was enhanced by exposing inoculated plants to low temperature (6°C) followed by warm temperature (20°C). The fungus was detected by PCR amplifications of three minisatellite markers in all stems with visible canker symptoms and also in the stems of 14 of the 59 plants without visible cankers on the hypocotyls. Disease severity increased with the number of infection sites on cotyledons: in one of the three replicate experiments, the mean external necrosis length on the hypocotyl ranged from 6·47 to 35·3 mm for one and eight infections sites on cotyledons, respectively. The probability of an isolate reaching the hypocotyl from inoculated cotyledons decreased with increasing competing inoculum load on cotyledons: for instance, for isolate A290v it decreased from 1 when inoculated alone to 0·28 when coinoculated with six drops of competing isolate P27d. Polygenic resistance significantly reduced disease incidence and severity. For instance, in one of the three replicate experiments, disease incidence ranged from more than 74% in susceptible plants to 16% in resistant ones, while mean external necrosis length was up to 35·3 and 6·5 mm on susceptible and on resistant plants, respectively. This study offers new possibilities for assessing levels of polygenic resistance to stem canker in B. napus and studying the aggressiveness of L. maculans isolates.     

Factors affecting the severity of bacterial canker of pear caused by Pseudomonas syringae pv. syringae

Several factors affecting the severity of bacterial canker of pear were studied. In the orchard, infection of shoots by Pseudomonas syringae pv. syringae occurred only when the inoculum dose exceeded 10⁶ colony-forming units/shoot. However, under favourable conditions in a growth chamber, cankers formed on detached shoots inoculated with 5 cfu/shoot. A second-order polynomial relationship was established between log₁₀ transformed canker length and log₁₀ transformed inoculum dose. In orchard and growth chamber experiments, shoots were susceptible from the time of bud swell until after fruit harvest. The severity of Pseudomonas canker of detached shoots increased if they were frozen at – 10°C for 24 h before inoculation. Shoots were most susceptible when inoculated immediately after wounding, and no cankers developed in the orchard when 3-day-old wounds were inoculated. Additionally, no cankers resulted from inoculation of leaf scars at leaf drop. Actively growing, current-season shoots were more susceptible than shoots that had set a terminal bud. The practical implications of these results are discussed as a basis for control of bacterial canker of pear.     

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.     

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.     

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.     

Potential for using host resistance to reduce production of pseudothecia and ascospores of Leptosphaeria maculans, the blackleg pathogen of Brassica napus

Pseudothecial density of the blackleg fungus Leptosphaeria maculans and discharge of ascospores was measured from stubble of a range of Brassica species, including Brassica napus (canola) cultivars, with a range of blackleg resistance. Since ascospores are the primary inoculum, these parameters reflect inoculum potential for blackleg. Stubble from a representative line of each of B. carinata , B. nigra , Sinapis alba and B. napus cv. Surpass 400 (incorporates blackleg resistance from B. rapa ssp. sylvestris ) had lower pseudothecial density and discharged fewer ascospores than stubble of other B. napus cultivars (Karoo, Oscar, Emblem, Dunkeld and Columbus). These latter B. napus cultivars and a representative B. juncea line had higher pseudothecial densities and discharged higher numbers of ascospores. If this trait of low blackleg inoculum from stubble could be introgressed into commercial canola cultivars, blackleg disease severity could be substantially reduced, resulting in higher and more stable canola yields. However, the trait of reduced ascospore discharge may not be stable, as demonstrated by the B. rapa ssp. sylvestris -derived resistance already being overcome by the blackleg fungus in Australia.     

Dynamics of soilborne Rhizoctonia solani in the presence of Trichoderma harzianum: effects on stem canker, black scurf and progeny tubers of potato

Stem canker and black scurf are diseases of potato caused by the fungus Rhizoctonia solani . Spatiotemporal experimentation and empirical modelling were applied for the first time to investigate the effect of antagonistic Trichoderma harzianum on the dynamics of soilborne R. solani on individual potato plants. Trichoderma harzianum reduced the severity of symptoms, expressed as 'rhizoctonia stem lesion index' (RSI), during the first 7 days post-inoculation when the inoculum of R. solani was placed at certain distances (30–60 mm) from the host. For example, with inoculum at 40 mm from the host, RSI was 6 and 40 with and without T. harzianum , respectively. At later observation times, the antagonistic effect was overcome. Trichoderma harzianum reduced the severity of black scurf on progeny tubers. Furthermore, the mean number of progeny tubers per potato plant was reduced by the biocontrol treatment (means of 6·5 ± 1·1 and 9·9 ± 2·7 tubers per plant with and without T. harzianum , respectively), as was the proportion of small (0·1–20·0 g) tubers (48% and 66% with and without T. harzianum , respectively). Additionally, there were fewer malformed and green-coloured tubers in pots treated with T. harzianum than in those without T. harzianum .     

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.     

Biological diversity of Rhizoctonia solani (AG-3) in a northern potato-cultivation environment in Finland

A total of 119 isolates of Rhizoctonia were collected from stem canker lesions, stolon and root lesions, hymenia on stems, or from black scurf on tubers of potato plants ( Solanum tuberosum ) in Finland (latitudes 60–67°N). All isolates except three belonged to anastomosis group 3 (AG-3) of R. solani , as determined by phylogenetic analysis of the internal transcribed spacer sequences (ITS1 and ITS2) of ribosomal RNA (rRNA) genes. Sensitivity of the 119 isolates to the fungicide flutolanil was tested in vitro (EC₅₀ values 0·14–0·75  µ g active ingredient mL⁻¹). The isolates also varied considerably in growth rate (5·1–14·8 mm day⁻¹). The severity of disease caused by 99 isolates was determined based on the proportion of potato sprouts affected by lesions, discoloration or death, which was c . 1–60%. Only two isolates that were able to cause severe symptoms showed particularly low sensitivity to the fungicide and rapid growth rate. One isolate each of anastomosis groups AG-2-1 and AG-5 and an unknown, binucleate Rhizoctonia sp. were detected. The AG-5 isolate and the binucleate isolate caused mild symptoms on potato sprouts, whereas the AG-2-1 isolate was not pathogenic. Taken together, AG-3 of R. solani was the predominant causal agent of the stem canker and black scurf diseases of potato in Finland and showed considerable variability in disease severity, fungicide sensitivity and growth rate in vitro .     

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 temperature on maturation of pseudothecia of Leptosphaeria maculans and L. biglobosa on oilseed rape stem debris

Effects of temperature on maturation of pseudothecia of Leptosphaeria maculans and L. biglobosa , closely related species which coexist on UK oilseed rape, were investigated. Stages in pseudothecial maturation on naturally infected oilseed rape debris were examined, both in controlled environments (5, 10, 15 or 20°C) under continuous wetness and in natural conditions (debris exposed in September and December 2000, and July, September and November 2002). Pseudothecia sampled weekly were assigned to maturation classes A (asci undifferentiated), B (asci differentiated), C (ascospores differentiated) or D (ascospores mature). Progress in pseudothecial maturation (assessed by time until 50% of pseudothecia reached each class) was similar for L. maculans and L. biglobosa at 15–20°C, but L. biglobosa matured more slowly at < 10°C. Maturation time decreased almost linearly with temperature from 5 to 20°C under continuous wetness but was longer in natural conditions, especially when periods of dry weather occurred. Differences in pseudothecial maturation are likely to contribute to epidemiological differences between L. maculans and L. biglobosa , which may explain their coexistence. It is appropriate to use the degree-day approximation to assess pseudothecial maturation at temperatures between 5 and 20°C, providing debris is wet.     

Factors affecting the incidence and severity of Spongospora subterranea infection and galling in potato roots

The incidence and severity of root infection and root galling caused by Spongospora subterranea were assessed in potato plants (cv. Estima) grown under controlled environmental conditions. The effects of temperature, soil type, soil moisture regime and soil inoculum level on infection and root gall development were determined by molecular and visual methods at two plant growth stages. Root gall severity was scored at harvest, after which DNA was extracted from the roots and quantified in a real-time polymerase chain reaction (PCR) assay specific for S. subterranea . Root galling was severe at 17°C, with a disease score of 3·1 on a 0–4 scale, low (0·6) at 12°C, and did not occur at 9°C. The level of inoculum in soil, in the form of artificially added sporosori, had no effect on the incidence and severity of visual symptoms, with 21%, 41% and 33% incidence observed at 5, 15 and 50 sporosori g⁻¹ soil, respectively. Incidence of infection, as detected by the real-time PCR assay, was greater with increasing soil inoculum concentrations, ranging from 48% at 5 sporosori g⁻¹ to 59% (15 sporosori g⁻¹) and 73% (50 sporosori g⁻¹) of plants infected at maturity, but this effect was not statistically significant. No correlation was found between the occurrence of galls on roots and powdery scab on tubers of the same plants.     

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.     

Factors influencing infection of mechanical wounds by Fusarium circinatum on Monterey pines (Pinus radiata)

Pitch canker, caused by the fungus Fusarium circinatum , is a disease affecting many pine tree species. In California, Pinus radiata (Monterey pine) is the principal pine host affected by pitch canker. This investigation into factors affecting infection frequency by F. circinatum of P. radiata examined the influence of: (i) wound size; (ii) relative humidity; (iii) time of inoculation; (iv) inoculum density; and (v) wound age. Wounded branches sustained significantly more infections when large-diameter (1·6 mm) rather than small-diameter (0·5 mm) wounds were made. Infection frequencies tended to be higher at 100% RH than at ambient humidity, although these differences were not statistically significant. Infection frequencies were significantly higher on branches inoculated after 17·00 h than on branches inoculated before noon. Infection frequencies were significantly higher for wounded branches spray-inoculated with 5 × 10⁷ rather than 1 × 10⁷ spores mL⁻¹. Infection frequencies of pruning wounds declined as wounds aged.     

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.     

Relative significance of nursery infections and orchard inoculum in the development and spread of apple canker (Nectria galligena) in young orchards

Three nurseries produced apple rootstocks (M9) and budwood (cv. Royal Gala), which they exchanged at the end of the first year. Each nursery then budded its own budwood onto the rootstocks it had produced and that from the other two nurseries. Budded trees were grown on for a further year before being planted at HRI, East Malling in southern England; NIHPBS, Loughgall in Northern Ireland; and ADAS, Rosemaund in the West Midlands of England. Canker development was monitored twice a year. The position of the infected trees within the orchard was recorded, as was the position of the canker on each tree (main-stem or peripheral). Nectria galligena was isolated from representative cankers and analysed using molecular techniques. At the sites in Northern Ireland and HRI there was a strong positional effect, especially of peripheral cankers, indicating that most of the inoculum was external and had been spread from neighbouring orchards. There was little or no positional effect on main-stem cankers at any of the three sites. The proportions of different isolates taken from peripheral cankers was different in Northern Ireland from that in England, suggesting different populations associated with the geographic areas. In contrast, the populations of N. galligena obtained from main-stem cankers were very similar in England and Northern Ireland. It was concluded that a small proportion of trees developing canker were infected during propagation, with no symptom development until after planting. In a second trial it was demonstrated that trees infected during the propagation phase, and particularly at budding and heading back, could develop canker up to 3 years later. While it is clear that some canker developing in the orchard can be associated with the nursery of production, in climatic conditions conducive to the formation and dissemination of conidia, inoculum from surrounding infected orchards is the primary source of the pathogen. Aerial spread is therefore an essential element of the epidemiology of N. galligena, and its control is a crucial part of any canker-control programme.     

Blackleg sporacle: a model for predicting onset of pseudothecia maturity and seasonal ascospore showers in relation to blackleg of canola

ABSTRACT A simple model has been developed to predict the onset of pseudothecia maturity and seasonal ascospore showers in relation to blackleg disease in canola, caused by the fungus Leptosphaeria maculans. The model considers a combination of two weather factors, daily mean temperature and daily total rainfall, to drive progress of maturity of pseudothecia on the infested canola stubble left from past crops. Each day is categorized as suitable or not suitable for progress of the maturation process. The onset of pseudothecia maturity occurs when approximately 43 suitable days have occurred. Following the onset of maturity, ascospore showers are triggered when daily rainfall exceeds a threshold. The model satisfactorily predicted the timing of the onset of pseudothecia maturity when tested with 3 years of field observations at four locations in Western Australia, which characteristically has a Mediterranean climate. The model also agreed reasonably well with the daily pattern of ascospore release observed in two locations. Sensitivity analysis was performed to show the relative importance of the parameters that describe the onset of pseudothecia maturity.     

Bipolar heterothallism in B-group isolates of Leptosphaeria maculans

Pairwise combinations were carried out between the eight isolates originating from each of three asci of the B-group of Leptosphaeria maculans . Some of the pairings produced mature pseudothecia when incubated on wheat straws at 18°C and under a 12-h photoperiod of blue light. Isolates within a given tetrad could be attributed to one of two sexual compatibility groups, on the basis of combinations of isolates leading to fertile crosses. Within each tetrad, mating type segregated in a 1:1 ratio, suggesting the existence of one mating type gene ( MAT1 ) with two alleles MAT 1–1 and MAT 1–2. Under these same conditions, each individual isolate from an A-group ascus originating from Brassica juncea produced pseudothecia with one or other of two compatible A-group testers originating from B. napus . However, attempts to mate these eight A-group single-ascospore isolates with two B-group single-ascospore isolates of opposite mating types remained unsuccessful. This work is the first successful report of in vitro sexual reproduction between B-group isolates, and provides evidence for bipolar heterothallism in B-group isolates of L. maculans . The implications of this mating protocol for genetic studies of B-group isolates and the consequences of this work for the taxonomy of L. maculans are discussed.