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

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

Effects of fungicide on growth of Leptosphaeria maculans and L. biglobosa in relation to development of phoma stem canker on oilseed rape (Brassica napus)

Controlled‐environment and field experiments were done to investigate effects of the fungicide Punch C (flusilazole plus carbendazim) on growth of Leptosphaeria maculans and L. biglobosa in oilseed rape. In controlled‐environment experiments, for plants inoculated with L. maculans, fungicide treatment decreased lesion size and amount of L. maculans DNA in leaves; for plants inoculated with L. biglobosa, fungicide did not affect lesion size or amount of pathogen DNA. When release of ascospores was monitored using a Burkard spore sampler, the timing and pattern of ascospore release differed between the four seasons. In 2006/2007, the majority of ascospores released were L. maculans, whilst in 2007/2008 the majority were L. biglobosa; in both seasons L. maculans ascospores were released before L. biglobosa ascospores. In field experiments in 2002/2003 and 2003/2004, fungicide treatment decreased severity of stem canker on cv. Apex, but gave no significant yield response. In 2006/2007 and 2007/2008, fungicide treatment decreased phoma leaf spot incidence in autumn and stem canker severity at harvest, and increased yield. Fungicide treatment decreased stem canker severity more on cv. Courage, with a good yield response, than on cv. Canberra. In 2002/2003 and 2003/2004, fungicide treatment decreased the frequency of spread of L. maculans into stem pith tissues and in 2006/2007 fungicide decreased the amount of L. maculans DNA in stem tissues (measured by quantitative PCR). These results are used to suggest how effects of fungicides on interactions between L. maculans and L. biglobosa might affect severity of phoma stem canker and yield response.     

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.     

Possibilities for integrated control of winter oilseed rape diseases in Latvia

The sowing area of winter oilseed rape in Latvia has been rapidly increasing during the last 10 years, therefore oilseed rape diseases have become an important risk factor. The present paper reports 4‐year data (2008–2011) on different fungicide application systems (including forecasting systems) tested in field trials in Central Latvia. The development of stem canker (Leptosphaeria spp.) and white stem rot (Sclerotinia sclerotiorum) was evaluated depending on fungicide application schemes. Stem canker was found to be a widespread disease, and coexistence of both L. maculans and L. biglobosa was established in Latvia. There was no epidemic of white stem rot observed during the investigation period (incidence of disease was only 0.7–4.5%). Application of fungicides significantly (P < 0.05) decreased severity of stem canker. However, substantial differences between different treatments were not detected. Yield of oilseed rape fluctuated at about 4.5 tonnes ha^?1, and use of fungicides did not increase the yield. It was found that DaCom Plant Plus program had overestimated the risk of white stem rot in years with low disease pressure, whereas the Swedish model of forecasting, based on the risk‐point system, had produced acceptable forecasts.     

Epidemiology of root rot caused by <Emphasis Type="Italic">Leptosphaeria maculans</Emphasis> in <Emphasis Type="Italic">Brassica napus</Emphasis> crops

The infection of above-ground tissues of Brassica napus by Leptosphaeria maculans is well understood. However, root infection (root rot) under field conditions, the development of root rot over time and its relationship to other disease symptoms caused by L. maculans has not been described. A survey of B. napus crops was conducted in Australia to investigate the incidence and severity of root rot. Additionally, the pathway of root infection was examined in field experiments. Root rot was present in 95% of the 127 crops surveyed. The severity and incidence of root rot was significantly correlated with that of crown canker; however, the strength of this relationship was dependent on the season. Root rot symptoms appeared before flowering and increased in severity during flowering and at maturity, a pattern similar to crown canker suggesting that the infection of the root is an extension of the crown canker phase of the L. maculans lifecycle. All isolates of L. maculans tested in glasshouse experiments caused root rot and crown canker in B. napus and Brassica juncea. In the field, the main pathway of root infection is via invasion of cotyledons or leaves by airborne ascospores, rather than from inoculum in the soil. Root rot was present in crops in fields that had never been sown to B. napus previously, in plants grown in fumigated fields, and in glasshouse-grown plants inoculated in the hypocotyl with L. maculans.     

Characterization of accessions and species of Macadamia to stem infection by Phytophthora cinnamomi

Phytophthora cinnamomi is a major pathogen in most macadamia plantations worldwide. Due to stem lesions, stem cankers and leaf defoliation, it results in loss of productivity and tree death. This study examined accessions of the four Macadamia species and their hybrids, produced via rooted stem cuttings or germinated seeds, for susceptibility to stem canker and necrotic lesions caused by P. cinnamomi. Plants were wound‐inoculated with agar containing P. cinnamomi. The symptoms produced in inoculated plants were used to characterize host susceptibility variation within and among the population. Lesion length and severity of stem canker were recorded. The four species and hybrids differed significantly in stem canker severity (P < 0.001) and lesion length (P = 0.04). Macadamia integrifolia and M. tetraphylla hybrids were the most susceptible. Macadamia integrifolia had the greatest stem canker severity and the most extensive lesions above and below the site of inoculation. Restricted lesion sizes were observed in M. ternifolia and M.  jansenii. The effects of basal stem diameter and the method of propagation either from cuttings or from seed were not significant. The genetic variation in the reaction of macadamia accessions to stem infection by P. cinnamomi is discussed.     

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 flooding on the survival of Leptosphaeria spp. in canola stubble

This study used a versatile temperature‐control device to assess the effect of temperature (12–40°C) and duration (2–12 weeks) of flooding on the survival of Leptosphaeria spp. in canola (Brassica napus) stubble. Canola basal stems with blackleg symptoms were submerged in water in small glass jars containing 20 cm³ soil on a thermogradient plate capable of simultaneously maintaining up to 96 independent temperature regimes. Flooded stems were sampled at 2‐week intervals, surface‐sterilized, and incubated on V8‐juice agar for 10 days to recover the pathogen. Flooding for 2 weeks substantially reduced pathogen recovery relative to non‐flooded controls and the pathogen was not recovered after 6 weeks of flooding, irrespective of temperature. The pathogen was eliminated slightly more rapidly at flooding temperatures >20°C than at 12–16°C. There was no difference between Leptosphaeria maculans and L. biglobosa in their ability to survive flooding. Stem tissues degraded most rapidly during the first 2 weeks of flooding, corresponding to a quick decline in pathogen survival during the same period. These results indicate that a paddy rice crop following winter rapeseed may minimize the impact of blackleg by eradicating the inoculum of Leptosphaeria spp. in stubble.     

<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.     

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.     

Relating plant and pathogen development to optimise fungicide control of phoma stem canker (<Emphasis Type="Italic">Leptosphaeria maculans</Emphasis>) on winter oilseed rape (<Emphasis Type="Italic">Brassica</Emphasis><Emphasis Type="Italic">napus</Emphasis>)

In winter oilseed rape experiments at Rothamsted in 2000/01 to 2002/03 growing seasons, the severity of phoma stem canker epidemics in summer depended on the timing of phoma leaf spot epidemics in the previous autumn, and hence on the timing of Leptosphaeria maculans ascospore release. The first major release of L. maculans ascospores was earlier in 2000 (26 September) and 2001 (18 September) than in 2002 (21 October). Consequently, the autumn phoma leaf spot epidemic was also earlier in 2000 and 2001 than in 2002. The resulting stem canker epidemics were severe by harvest (July) in 2001 and 2002 but not in 2003. No correlation was found between the severity or duration of phoma leaf spotting (lesion days or lesion °C-days) and the subsequent severity of phoma stem canker epidemics. Rates of leaf production and loss were similar in the three growing seasons. Out of ca. 25 leaves produced on plants during each season, leaf numbers 10–14 generally remained on plants for the longest. Treatment with flusilazole + carbendazim in autumn decreased the severity of phoma leaf spotting for several weeks after treatment, decreased the severity of stem canker the following summer and increased yield significantly in 2001 and 2002 but not in 2003. The most effective timings for flusilazole + carbendazim application were when leaves 7–11 were present on most plants and at least 10% of plants were affected by phoma leaf spot. Two half-dose applications of fungicide reduced phoma stem canker and increased yield more than a single full dose application when phoma leaf spot epidemics were early (<800 °C-days after sowing).     

Dissemination of Plasmodiophora brassicae in livestock manure detected by qPCR

The present study was performed to investigate whether Plasmodiophora brassicae can be disseminated by livestock manure. A quantitative PCR (qPCR) assay was developed and used to detect and quantify P. brassicae in manure samples from naturally and artificially infested chickens and pigs. In naturally infested manure, quantifiable levels of infestation were observed in 7 out of the 28 samples, ranging from 10³ to 10⁷ resting spores per g of manure. The vast majority of the resting spores (76–91%) were viable, as determined by a dual fluorescence viability assay. Clubroot symptoms developed on plants inoculated with P. brassicae resting spores isolated from all seven qPCR‐positive samples. Artificially infested manure samples were produced by feeding chickens and pigs on P. brassicae‐contaminated feed. The levels of infestation were 10³ resting spores per g of manure for both chicken and pig manure sampled 24 and 48 h after feeding, respectively. Spore viability was >80% for both samples, and the disease severity indices were both >40 as indicated by bioassay. This showed that resting spores can survive the digestive tracts of chickens and pigs, and retain strong pathogenicity. The findings indicate that manure dissemination is possible for P. brassicae. Farmers should avoid feeding livestock on P. brassicae‐contaminated feed or applying infested manure as fertilizer on land intended for crucifer crops.     

Resistance to a highly aggressive isolate of Sclerotinia sclerotiorum in a Brassica napus diversity set

Sclerotinia stem rot (SSR) of oilseed rape (OSR, Brassica napus), caused by Sclerotinia sclerotiorum, is a serious problem in the UK and worldwide. As fungicide‐based control approaches are not always reliable, identifying host resistance is a desirable and sustainable approach to disease management. This research initially examined the aggressiveness of 18 Sclerotinia isolates (17 S. sclerotiorum, one S. subarctica) on cultivated representatives of B. rapa, B. oleracea and B. napus using a young plant test. Significant differences were observed between isolates and susceptibility of the brassica crop types, with B. rapa being the most susceptible. Sclerotinia sclerotiorum isolates from crop hosts were more aggressive than those from wild buttercup (Ranunculus acris). Sclerotinia sclerotiorum isolates P7 (pea) and DG4 (buttercup), identified as ‘aggressive’ and ‘weakly aggressive’, respectively, were used to screen 96 B. napus lines for SSR resistance in a young plant test. A subset of 20 lines was further evaluated using the same test and also in a stem inoculation test on flowering plants. A high level of SSR resistance was observed for five lines and, although there was some variability between tests, one winter OSR (line 3, Czech Republic) and one rape kale (line 83, UK) demonstrated consistent resistance. Additionally, one swede (line 69, Norway) showed an outstanding level of resistance in the stem test. Resistant lines also had fewer sclerotia forming in stems. New pre‐breeding material for the production of SSR resistant OSR cultivars relevant to conditions in the UK and Europe has therefore been identified.     

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.     

Dissemination of Information About Management Strategies and Changes in Farming Practices for the Exploitation of Resistance to Leptosphaeria maculans (Phoma Stem Canker) in Oilseed Rape Cultivars

The management of phoma stem canker (blackleg disease, caused by Leptosphaeria maculans) is an integral component of oilseed rape production. In this paper, we discuss the information about management strategies that is disseminated in Europe and Australia. New cultivars have been introduced with improved resistance to disease, but sometimes this resistance has been overcome as new races of the pathogen have emerged. When cultivars with single major gene resistance have been introduced into areas with high inoculum concentrations, significant economic damage has been caused by new races of L. maculans within 2–3 years. Quantitative or polygenic resistance has also been used successfully against stem canker and offers more durable disease resistance if plant breeders and farmers deploy this resistance more effectively. Strategies to improve the durability of resistance need to be developed and tested in practice. New information on the occurrence of virulence and avirulence genes in populations of Leptosphaeria maculans and modelling of the durability of resistance provide opportunities for plant breeders, specialist technical organisations, cooperatives, advisory services and farmers to collaborate and better exploit cultivar resistance. Changing economic and environmental factors influence cropping practices and, if to be considered successful, management strategies must show clear financial benefits. Technology transfer will need to address all aspects of managing stem canker and other diseases of oilseed rape and using effective written, verbal and electronic methods of communication.     

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.     

Identifying seedling and adult plant resistance of Chinese Brassica napus germplasm to Leptosphaeria maculans

Blackleg disease of canola/rapeseed (Brassica napus), caused by the devastating fungal pathogen Leptosphaeria maculans, can significantly influence B. napus production worldwide, except for China, where only the less aggressive L. biglobosa has been found associated with the disease. The aim of this study was to characterize both seedling resistance (major gene resistance, R gene resistance) and adult plant resistance (APR) from a collection of Chinese B. napus varieties/lines (accessions) to L. maculans. Evaluation of seedling resistance was carried out under a controlled environment, using 11 well‐characterized L. maculans isolates as differentials. The identification of APR was performed under multiple field environments in western Canada. R genes were detected in more than 40% of the accessions tested. Four specific R genes, Rlm1, Rlm2, Rlm3 and Rlm4 were identified, with Rlm3 and Rlm4 being the most common genes, while Rlm1 and Rlm2 were detected only occasionally. Results of field evaluation indicated significant variations among field locations as well as accessions; a large portion of the B. napus accessions, regardless of the resistance level observed at the seedling stage, showed high to moderate levels of APR under all environments tested. This study highlights that both R gene resistance and APR are present in Chinese B. napus germplasm and could be potential sources of resistance against blackleg caused by L. maculans if the pathogen ever becomes established in China.     

Variations in relative humidity modulate <Emphasis Type="Italic">Leptosphaeria</Emphasis> spp. pathogenicity and interfere with canola mechanisms of defence

Blackleg (phoma stem canker), caused by Leptosphaeria spp., is an important disease of canola (oilseed rape, Brassica napus). Control strategies rely on the use of resistant cultivars, chemical and disease-reducing cropping practices. In Canada, the pathogen population is represented by L. maculans and L. biglobosa, which are considered to be highly and weakly aggressive, respectively. It is largely admitted that L. biglobosa isolates are not able to cause a significant amount of stem canker and develop on the plant only when it becomes senescent, late in the season. The prevalence of L. maculans over L. biglobosa has been considered to be linked to the low aggressiveness of the latter. However, in this study, we show that L. biglobosa isolates could become highly aggressive in terms of lesion appearance on cotyledons, if the right conditions of temperature and relative humidity (RH) are provided. Percent germination of inoculated pycnidiospores was not affected by the RH regimes tested. This is the first study to show the importance of RH as a factor conditioning the pathogenicity of L. biglobosa isolates on canola cotyledons. Concurrent changes in the host defence mechanisms against L. biglobosa isolates in response to variations in the RH were also investigated. Under high RH, the increase in disease caused by the weakly aggressive isolates coincided with a reduced accumulation of lignin at the early stages of infection.