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.     

World-Wide Importance of Phoma Stem Canker (Leptosphaeria maculans and L. biglobosa) on Oilseed Rape (Brassica napus)

Phoma stem canker is an internationally important disease of oilseed rape (Brassica napus, canola, rapeseed), causing serious losses in Europe, Australia and North America. UK losses of €56M per season are estimated using national disease survey data and a yield loss formula. Phoma stem canker pathogen populations comprise two main species, Leptosphaeria maculans, associated with damaging stem base cankers, and Leptosphaeria biglobosa, often associated with less damaging upper stem lesions. Both major gene and quantitative trait loci mediated resistance to L. maculans have been identified in B. napus, but little is known about resistance to L. biglobosa. Leptosphaeria maculans, which has spread into areas in North America and eastern Europe where only L. biglobosa was previously identified, now poses a threat to large areas of oilseed rape production in Asia. Epidemics are initiated by air-borne ascospores; major gene resistance to initial infection by L. maculans operates in the leaf lamina of B. napus. It is not clear whether the quantitative trait loci involved in the resistance to the pathogen that can be assessed only at the end of the season operate in the leaf petioles or stems. In countries where serious phoma stem canker epidemics occur, a minimum standard for resistance to L. maculans is included in national systems for registration of cultivars. This review provides a background to a series of papers on improving strategies for managing B. napus resistance to L. maculans, which is a model system for studying genetic interactions between hemi-biotrophic pathogens and their hosts.     

Improved Resistance Management for Durable Disease Control: A Case Study of Phoma Stem Canker of Oilseed Rape (Brassica napus)

Specific resistance loci in plants are generally very efficient in controlling development of pathogen populations. However, because of the strong selection pressure exerted, these resistances are often not durable. The probability of a resistance breakdown in a pathosystem depends on the evolutionary potential of the pathogen which is affected by: (i) the type of resistance (monogenic and/or polygenic), (ii) the type of reproduction of the pathogen (sexual and/or asexual), (iii) the capacity of the pathogen for dispersal, (iv) the resistance deployment strategy (pyramiding of specific resistances, mixture of cultivars, spatio-temporal alternation), (v) the size of the pathogen population, which is affected by control methods and environmental conditions. We propose the concept of Integrated Avirulence Management (IAM) to enhance the durability of specific resistances. IAM involves a strategy to limit the selection pressure exerted on pathogen populations and, at the same time, reduce the size of pathogen populations by combining cultural, physical, biological or chemical methods of control. Several breakdowns of resistance specific to Leptosphaeria maculans, the causal agent of phoma stem canker have occurred in Europe and in Australia. This review paper examines control methods to limit the size of L. maculans populations and discusses how this limitation of population size can enhance the durability of specific resistances. It proposes pathways for the development of a spatially explicit model to define IAM strategies. Simulation results are presented to demonstrate the potential uses of such a model for the oilseed rape/L. maculans pathosystem.     

Effect of defoliation by livestock on stem canker caused by Leptosphaeria maculans in Brassica napus

Brassica napus (canola, oilseed rape), an important break crop for cereals across the Australian wheat belt, is being rapidly adopted as a dual‐purpose (forage and grain) crop in mixed farming systems. Stem canker caused by the fungus Leptosphaeria maculans is the most important disease of B. napus in Australia. The primary source of inoculum is airborne ascospores released during autumn/winter which coincides with the grazing of dual‐purpose crops. Field experiments were defoliated by sheep to determine the effect of grazing on blackleg stem canker severity at plant maturity in B. napus cultivars differing in their resistance level and grazed at different times. One cultivar was sown on different dates to investigate the impact of grazing at the same time, but at different growth stages. Defoliation by mowing was compared to defoliation by livestock. Similar amounts of dry matter remained after defoliation by machinery (0·66 t ha^?1) or livestock (0·52 t ha^?1). However, stem canker severity was higher in the grazed (40% of crown cross‐section diseased) compared with the mown (25%) treatment, which was higher than the ungrazed control (9%). Stem canker severity generally increased with grazing, but the increase was eliminated or reduced in cultivars with good resistance. Grazing during vegetative plant growth minimized the increase in stem canker severity compared with grazing during reproductive growth. Currently, cultivars with good L. maculans resistance are recommended in high disease situations. To avoid excessive yield loss in dual‐purpose B. napus crops due to L. maculans it is recommended that such cultivars are grown even in low‐moderate disease situations.     

Geographic variation in severity of phoma stem canker and <Emphasis Type="Italic">Leptosphaeria maculans/ L. biglobosa</Emphasis> populations on UK winter oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis>)

Phoma stem canker, caused by Leptosphaeria maculans and L. biglobosa, is the most important disease of oilseed rape in Europe. Differences between L. maculans and L. biglobosa in their life-cycles enable the two species to co-exist on oilseed rape crops over a cropping season. This review considers the factors affecting geographic variation in the severity of phoma stem canker epidemics and in the structure of the population of the pathogens in the UK, where the most severe epidemics occur in the south of England and cankers do not develop in Scotland. It is concluded that this variation is directly related to differences in climate, since weather-based models show that stem canker severity increases with increasing winter/spring temperature and temperatures are greater in the south of the UK. It may also be related to differences in pathogen populations, since surveys showed that the proportion of the more damaging L. maculans in stem cankers was greatest in southern England, with most L. biglobosa in northern England. Regional variation in agronomic practices such as cultivar choice and fungicide use may also indirectly influence phoma stem canker severity. Differences in cultivar choice result in differences in L. maculans race structure, which may influence the severity of epidemics. Differences in fungicide use may also influence pathogen populations, since L. maculans and L. biglobosa differ in their sensitivities to different azole fungicides. These factors are discussed in relation to strategies for sustainable production of oilseed rape by adaptation to threats posed by climate change.     

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.     

A 10-year Survey of Populations of Leptosphaeria maculans in France Indicates a Rapid Adaptation Towards the Rlm1 Resistance Gene of Oilseed Rape

Leptosphaeria maculans, the cause of stem canker of oilseed rape (OSR), exhibits gene-for-gene interactions with its host plant. The race structure of L. maculans was assessed on the basis of the analysis of 1011 isolates collected in France between 1990 and 2000, with regards to three AVR genes, AvrLm1, AvrLm2 and AvrLm4. The effect of selection pressure, due to large-scale cropping of Rlm1 cultivars, on the evolution of races of the fungus was also evaluated. The results revealed a scarcity or complete absence of isolates harbouring AvrLm2, whereas isolates harbouring AvrLm4 were present at a variable level, that was as high as 17.2–31.2% depending on the sample year and location. When obtained from rlm1 cultivars, isolates harbouring AvrLm1 always represented more than 83% of the populations until the 1997–1998 growing season. As a consequence, the Rlm1 cultivars had been highly efficient at controlling the disease and were grown on an estimated 43.7% of the total French acreage in OSR in 1998–1999. However, the increased commercial success of Rlm1 cultivars was paralleled by a decrease in the proportion of isolates harbouring AvrLm1 in 1997–1998 and 1998–1999. This resulted in less than 13% of isolates harbouring AvrLm1 in populations being collected from rlm1 cultivars in 1999 and 2000, and contributed to the loss of efficiency of the Rlm1 resistance in the field. The present study is an illustration of one round of a `boom and bust' cycle that occurred for a pathosystem where it has never been reported before. These data and the high evolutionary potential of L. maculans are fully supportive of one pathogen species with a high risk of breaking down resistance genes in OSR and suggest that the development of integrated strategies aiming at maximising the durability of novel resistance is now a priority for this pathosystem.     

Determining frequencies of avirulent alleles in airborne Leptosphaeria maculans inoculum using quantitative PCR

Phoma stem canker (blackleg disease) of Brassica napus (oilseed rape, canola) is caused by the fungus Leptosphaeria maculans. Frequencies of avirulent alleles for loci where virulence can be associated with gene deletion (AvrLm1 and AvrLm6) were determined in samples of L. maculans airborne ascospore inoculum using quantitative PCR. The accuracy, reproducibility and limitations of detection were determined. Changes in the frequency of avirulent alleles were determined for the 2006/2007, 2007/2008 and 2008/2009 growing seasons for winter oilseed rape in the UK. The frequency of AvrLm1 remained small (between 9% and 16%), whilst the frequency of AvrLm6 fluctuated between 35% and 66%. Estimation of frequencies of avirulent alleles in airborne pathogen inoculum gives an efficient and unbiased method to assess the potential of crop cultivars with corresponding resistance genes being at risk of disease.     

Effects of fungicides on in vitro spore germination and mycelial growth of the phytopathogens Leptosphaeria maculans and L. biglobosa (phoma stem canker of oilseed rape)

BACKGROUND: Phoma stem canker, caused by the coexisting related fungal pathogens Leptosphaeria maculans (Des.) Ces. & de Not and L. biglobosa Shoemaker & H Brun, is a major disease of winter oilseed rape in the UK. Annually, over 90% of UK crops receive at least one foliar application of fungicide, but little is known about the sensitivity of the more damaging L. maculans and the less damaging L. biglobosa to these fungicides. The effects of flusilazole, tebuconazole and Methyl Benzimidazole Carbamate (MBC) fungicides (benomyl and carbendazim) on the germination of ascospores, conidia and germ tube growth of both species were examined. Isolates collected from different oilseed rape crops in England and Wales were assessed for their mycelial growth on fungicide‐amended medium, and ED₅₀ values were calculated. RESULTS: Leptosphaeria maculans and L. biglobosa differed in their sensitivity to fungicides. Conidial germination of L. maculans was more sensitive to these fungicides than that of L. biglobosa. Isolates of L. maculans had smaller ED₅₀ values for mycelial growth for all fungicides tested than isolates of L. biglobosa. CONCLUSION: These results suggest that fungicide applications might affect the structure of L. maculans/L. biglobosa populations in UK winter oilseed rape crops. Copyright © 2009 Society of Chemical Industry     

Major Gene and Polygenic Resistance to Leptosphaeria maculans in Oilseed Rape (Brassica napus)

The most common and effective way to control phoma stem canker (blackleg) caused by Leptosphaeria maculans in oilseed rape (Brassica napus) is through the breeding of resistant cultivars. Race specific major genes that mediate resistance from the seedling stage have been identified in B. napus or have been introgressed from related species. Many race specific major genes have been described and some of them are probably identical in B. napus (allotetraploid AACC) and the parental species B. rapa (diploid AA). More work is needed using a set of well-characterised isolates to determine the number of different major resistance genes available. In some B. napus cultivars, there is resistance which is polygenic (mediated by Quantitative Trait Loci) and postulated to be race non-specific. Many of these major genes and Quantitative Trait Loci for resistance to L. maculans have been located on B. napus genetic maps. Genes involved in race specific and polygenic resistance are generally distinct.     

Effect of rotation of canola (Brassica napus) cultivars with different complements of blackleg resistance genes on disease severity

Blackleg disease (phoma stem canker) caused by the fungus Leptosphaeria maculans is a major disease of canola (oilseed rape, Brassica napus) worldwide. Canola plants in pots were exposed to blackleg‐infested stubble of canola with different complements of resistance genes and then assessed for disease. Plant mortality was reduced when plants were exposed to stubble from a cultivar with a different complement of resistance genes compared to stubble of a cultivar with the same resistance gene. These findings were consistent with 7 years of field surveys, which showed that changes in selection pressure as a result of extensive sowing of cultivars with major‐gene resistance, termed ‘sylvestris resistance’, dramatically influenced the frequency of virulent isolates in the population towards particular resistance genes, and therefore disease severity. All these data were supported by PCR‐genotyping surveys of fungal populations whereby the frequency of virulence alleles of avirulence genes AvrLm1 and AvrLm4 changed significantly depending on the resistance gene present in the cultivar from which the isolates were cultured. This is the first example of a study showing that sowing of canola cultivars with different complements of resistance genes in subsequent years, i.e. rotation of resistance genes, minimizes disease pressure by manipulating fungal populations. This approach provides a valuable disease management strategy for canola growers and is likely to be applicable to other plant diseases.     

DNA polymorphism in Leptosphaeria maculans

DNA polymorphism in Leptosphaeria maculans is described for the first time. Two distinct genetic groups, differing in repetitive DNA sequences, were detected by gel electrophoresis and by probing Southern blots with mitochondrial, ribosomal or random genomic DNA sequences. These two genetic groups correspond with groupings based on ability to accumulate extra-cellular pigments in culture but do not give a perfect correlation with stem canker potential on oilseed rape.It is concluded that there are major genetical differences between the two sub-groups within the population of L. maculans.     

Evaluation of oilseed rape seed yield losses caused by <Emphasis Type="Italic">Leptosphaeria biglobosa</Emphasis> in central China

Phoma stem canker of oilseed rape (Brassica napus), caused by Leptosphaeria maculans/L. biglobosa is a globally important disease. Severe phoma stem canker symptoms have been observed on winter oilseed rape in China but the seed yield loss caused by this disease remains unknown. In May 2012 and May 2013, 17 and 13 crops were surveyed, respectively, in seven counties of Hubei Province, central China. Stems with phoma stem canker disease symptoms were sampled for pathogen isolation and identification. Only L. biglobosa was identified by culture morphology and species-specific PCR; no L. maculans was found. To evaluate the yield losses, yield components (number of branches per plant, number of pods per plant, 1000-seed weight, number of seeds per pod) were assessed on healthy and diseased plants sampled from crops in four counties and on plants from inoculated pot experiments (plants of three cultivars were inoculated at the green bud stage by injecting L. biglobosa conidia into the stem between the first and second leaf scars). Results of the field surveys showed that diseased plants had 14–61% less branches and 32–83% less pods than healthy plants, respectively. The estimated seed yield loss varied from 10% to 21% and from 13% to 37% in 2012 and 2013, respectively. In the pot experiments, there were no differences in numbers of branches or pods but there were differences in number of seeds per pod between inoculated and control plants. For the three cultivars tested, the inoculated plants had yield losses of 29–56% compared with the control. This study indicates that L. biglobosa could cause substantial seed yield loss in China.     

<Emphasis Type="Italic">Brassica napus</Emphasis> plants infected by <Emphasis Type="Italic">Leptosphaeria maculans</Emphasis> after the third to fifth leaf growth stage in south-eastern Australia do not develop blackleg stem canker

Blackleg (Phoma stem canker) caused by Leptosphaeria maculans is the most damaging disease of Brassica napus (canola, rapeseed, colza) worldwide and is controlled by sowing blackleg resistant cultivars and crop management strategies that reduce exposure to inoculum and fungicide application. In experiments in south-eastern Australia, canola cultivars inoculated after the three to five leaf growth stage did not develop stem canker. Although mature canola plants are known to be less susceptible to blackleg than seedlings, this highlights for the first time the specific importance of protecting seedlings up to the three to five leaf growth stage in Australia. This would typically correspond to a period of four to six weeks after emergence. Canola plants are likely to be significantly less vulnerable to infection after this growth stage. However, this timing may vary due to the influence of environmental conditions.     

Automated image processing framework for analysis of the density of fruiting bodies of Leptosphaeria maculans on oilseed rape stems

Understanding the transmission of plant pathogen inoculum during the periods when the host plants are not present is crucial for predicting the initiation of epidemics and optimizing mitigation strategies. However, inoculum production at the end of the cropping season, survival during the intercrop period, and the emergence or release of inoculum can be highly variable, difficult to assess, and generally inferred indirectly from symptom data. As a result, a lack of large datasets hampers the study of these epidemiological processes. Here, inoculum production was studied in Leptosphaeria maculans, the cause of phoma stem canker of oilseed rape. The fungus survives on stubble left in the field, from which ascospores are released at the beginning of the next cropping season. An image processing framework was developed to estimate the density of fruiting bodies produced on stem pieces following incubation in field conditions, and a quality assessment of the processing chain was performed. A total of 2540 standardized RGB digital images of stems were then analysed, collected from 27 oilseed rape fields in Brittany over four cropping seasons. Manual post-processing removed 16% of the pictures, e.g. when moisture-induced darkening of the oilseed rape stems caused overestimation of the area covered with fruiting bodies. The potential level of inoculum increased with increasing phoma stem canker severity at harvest, and depended on the source field and the cropping season. This work shows how image-based phenotyping generates high-throughput disease data, opening up the prospect of substantially increased precision in epidemiological studies.     

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.     

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.     

Frequency of Avirulence Alleles in Field Populations of Leptosphaeria maculans in Europe

This paper describes the first large-scale Europe-wide survey of avirulence alleles and races of Leptosphaeria maculans. Isolates were collected from the spring rape cultivar Drakkar, with no known genes for resistance against L. maculans, at six experimental sites across the main oilseed rape growing regions of Europe, including the UK, Germany, Sweden and Poland. Additionally in Poland isolates were collected from cv. Darmor, which has resistance gene, Rlm9. In total, 603 isolates were collected during autumn in 2002 (287 isolates from Germany and the UK) and 2003 (316 isolates from Poland and Sweden). The identity of alleles at eight avirulence loci was determined for these isolates. No isolates had the virulence allele avrLm6 and three virulence alleles (avrLm2, avrLm3 and avrLm9) were present in all isolates. The isolates were polymorphic for AvrLm1, AvrLm4, AvrLm5 and AvrLm7 alleles, with virulence alleles at AvrLm1 and AvrLm4 loci and avirulence alleles at AvrLm7 and AvrLm5 loci predominant in populations. Virulent avrLm7 isolates were found at only one site in Sweden. Approximately 90% of all isolates belonged to one of two races (combinations of avirulence alleles), Av5-6-7 (77% of isolates) or Av6-7 (12%). Eight races were identified, with four races at frequencies less than 1%. The study suggested that Rlm6 and Rlm7 are still effective sources of resistance against L. maculans in oilseed rape in Europe. The results are comparable to those of a similar survey done in France in autumn 2000 and 2001.     

Major gene resistance in Brassica napus (oilseed rape) is overcome by changes in virulence of populations of Leptosphaeria maculans in France and Australia

Resistance of Brassica napus (oilseed rape, canola) conferred by three different major resistance genes has been overcome by changes in virulence of Leptosphaeria maculans populations in France and Australia. In South Australia where B. napus cultivars with major gene resistance derived from Brassica rapa ssp. sylvestris were grown extensively, resistance was rendered ineffective within 3 years of commercial release of the cultivar. Disease severity was higher on cultivars with sylvestris-derived resistance than cultivars with polygenic resistance. This Australian situation is compared to that in France, where resistance conferred by the Rlm1 gene was overcome nation-wide in 5 years under commercial cropping practices, and also where a source of resistance introgressed into B. napus from B. juncea was rendered inefficient in 3 years in experimental field plots near Rennes.