Field efficiency of Brassica napus specific resistance correlates with Leptosphaeria maculans population structure

Field experiments were conducted in Versailles, France, to assess blackleg resistance of Brassica napus cultivars Quinta and Glacier under natural infection conditions. Blackleg disease severity was assessed twice during growth of B. napus. Quinta resistance was highly expressed as only 13% to 18% of the plants exhibited leaf symptoms in December, whereas Glacier and other cultivars displayed more than 80% of infected plants. In June (harvest), 70% (first year) to 41.5% (second year) of Quinta plants were canker-free. In contrast, Glacier was as infected as the susceptible control cultivars, with more than 88% of plants displaying canker. The Leptosphaeria maculans population structure was examined in parallel. Based on soluble protein patterns, 9% of the 299 fungal isolates collected were characterized as Tox⁰ species, and belonged to the NA1 sub-group. All but two of Tox⁰ isolates were isolated from atypical dark necrotic leaf lesions, mainly occurring on Quinta. In contrast, the Tox⁺ isolates were recovered from typical leaf lesions. Following a cotyledon inoculation test on the differential set Westar, Quinta and Glacier, 92 to 95% of Tox⁺ isolates collected on susceptible cultivars were characterized as PG3 isolates, i.e. avirulent on Quinta. The remaining Tox⁺ isolates belong to PG4, i.e. virulent on the three cultivars. No PG2 isolate, i.e. avirulent on both Quinta and Glacier, was identified in the sampling. The present study suggests that specific resistance expressed at the cotyledon level can be efficient under field conditions where the corresponding avirulent races of the pathogen are prevalent.     

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.     

Splash dispersal of Leptosphaeria maculans pycnidiospores and the spread of blackleg on oilseed rape

The fungus Leptosphaeria maculans causes blackleg (phoma stem canker), one of the most serious diseases of oilseed rape. The role of pycnidiospores produced during asexual reproduction is poorly documented and limits the understanding of the pathogen's population dynamics. The objectives of this study were to assess rain-splash dispersal of pycnidiospores of L. maculans from phoma leaf spots, and transmission of the disease from oilseed rape stubble carrying pycnidia. The work was conducted in still air with either a drop generator or a rain simulator. The impact of simulated incident drops on phoma leaf spots resulted in the dispersal of L. maculans pycnidiospores within splash droplets. Ninety per cent of the spores were collected within 14 cm of the source and a few were regularly observed up to 40 cm. Pycnidiospores produced on oilseed rape stubble and dispersed by simulated rain infected oilseed rape trap plants in a spatial pattern that matched the spatial dispersal of the pycnidiospores. In the field, rain-splash dispersal of pycnidiospores could increase the pathogen population and may enhance sexual reproduction by facilitating the mating of initially spatially separated isolates of opposite mating type.     

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.     

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.     

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.     

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.     

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.     

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.     

Reaction of Brassica juncea (Indian Mustard) Lines to Australian Isolates of Leptosphaeria maculans under Glasshouse and Field Conditions

Brassica juncea (Indian mustard) lines from diverse geographical locations around the world and from Australian breeding programs were screened for resistance to the blackleg fungus, Leptosphaeria maculans, in both glasshouse and field trials. The five Australian L. maculans isolates used in glasshouse trials could be classified into two groups; those that attacked all B. juncea lines, and those that attacked none. All these isolates caused lesions on cotyledons of B. napus cultivars including Westar, Glacier and Quinta, suggesting that they are in Pathogenicity Group 4 as described by Koch et al. (1991). The two isolates that attacked B. juncea also attacked B. napus lines to a similar extent, but did not attack the two B. carinata lines tested. Brassica lines were sown in a blackleg disease nursery at Lake Bolac, Victoria, Australia, and five indicators of blackleg disease were measured (survival rate, disease rating, disease incidence, external and internal lesion length). All 92 B. juncea lines developed blackleg symptoms. Although they displayed a high disease incidence in the field, almost all of the B. juncea lines were more blackleg-resistant than a B. napus cultivar, Dunkeld, which is amongst the most resistant cultivars in commercial production in Australia. Four B. carinata lines were more resistant than any of the B. juncea lines, suggesting that this species may be a useful source of blackleg resistance in B. napus breeding programs.     

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.     

Race structure and frequency of avirulence genes in the western Canadian Leptosphaeria maculans pathogen population,the causal agent of blackleg in brassica species

Leptosphaeria maculans is the causal agent of blackleg, a serious disease on canola/rapeseed in western Canada, Australia and Europe. Genetic resistance and extended crop rotation provided effective disease control in western Canada for years but the emergence of new pathogen races has reduced the effectiveness of current management strategies. The objective of this study was to analyse L. maculans isolates derived from canola stubble in commercial fields collected in 2010 and 2011 across western Canada for the presence and frequency of avirulence (Avr) genes. A total of 674 isolates were examined for the presence of Avr alleles AvrLm1, AvrLm2, AvrLm3, AvrLm4, AvrLm6, AvrLm7, AvrLm9, AvrLepR1, AvrLepR2 and AvrLmS using a set of differential host genotypes carrying known resistance genes or PCR amplification of AvrLm1, AvrLm6 and AvrLm4–Lm7. Certain alleles were more prevalent in the pathogen population, with AvrLm6 and AvrLm7 present in >85% of isolates, while AvrLm3, AvrLm9 and AvrLepR2 were present in <10% of isolates. A total of 55 races (different combinations of Avr alleles) were detected, with the two most common ones being AvrLm2–Lm4–Lm6–Lm7 and AvrLm2–Lm4–Lm6–Lm7–LmS. Races carrying as many as seven and as few as one known Avr allele were detected. Selection pressure from the race‐specific resistance genes carried in canola cultivars has probably played a significant role in the current Avr profile, which may have also contributed to the recent increase in blackleg observed in western Canada.     

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     

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.     

Cytological responses in the hypersensitive reaction in cotyledon and stem tissues of Brassica napus after infection by Leptosphaeria maculans

A hypersensitive response (HR) occurs after infection of cotyledon, leaf and stem of Brassica napus cv. Surpass 400 by an avirulent strain of Leptosphaeria maculans. The cotyledon or the sixth true-leaf stages of plants were inoculated with pycnidiospores of L. maculans (strain UWA P11). For the first time in this specific pathosystem and its HR, we report condensation of cytoplasm, shrinkage in cell size and nuclear DNA fragmentation in cotyledon tissues, and in stem tissues, shrinkage and condensation of the cytoplasm, chromatin fragmentation and lobing of the nucleus.     

Methods for Sampling and Assessment in Relation to the Spatial Pattern of Phoma Stem Canker (Leptosphaeria maculans) in Oilseed Rape

A sound assessment of phoma stem canker symptoms is needed to develop epidemiological, agronomical and physiological studies on the pathosystem. A specific analysis was therefore carried out to: (i) compare four methods of crown canker assessment; (ii) test the among and within assessor repeatability of one of the methods compared; (iii) characterise the spatial pattern of the disease; and (iv) define the sample size required to achieve a given level of disease assessment precision. The methods compared examined the symptoms with different procedures and graded the plants observed into six severity classes. A disease index (DI) summarised the severity distribution observed. Examination of crown cross-sections was the most precise method for assessing crown cankers. The method was repeatable, though an 'assessor effect' was apparent. The disease generally had a random pattern although significant spatial correlations were detected for four out of the fifteen plots studied at the scales examined. A relationship between the coefficient of variation of the DI and the sample size was established, evaluated with experimental field data and exemplified for typical severity distributions.     

In vitro evaluation of fungal isolates for their ability to influence leaf rheology,production of pseudothecia,and ascospores of Venturia inaequalis

As part of an ongoing research project on biological control of apple scab, this study presents a novel approach for the in vitro selection of potential antagonists of the saprophytic phase of Venturia inaequalis. A collection of forty-two fungal isolates were tested for their in vitro ability to degrade apple leaf tissue, inhibit pseudothecia, and ascospore production. The inhibition of ascospore production cannot always be linked reliably with leaf degradation or the evaluation of pseudothecia production. Consequently, ascospore production was retained as the most useful screening parameter. Six isolates proved to significantly reduce the ascospore production of Venturia inaequalis. Two were as effective as Athelia bombacina, a previously reported antagonist of pseudothecia formation and inhibited over 98% of the ascospore production. These new organisms are now available for future field tests. Future selections from a large collection of fungal and bacterial saprophytes can now be based on a reliable and simple in vitro screening method.     

Mechanisms of resistance in Brassica carinata,B. napus and B. juncea to Pseudocercosporella capsellae

Studies were undertaken to compare susceptible and resistant host responses to Pseudocercosporella capsellae in cotyledons of Brassica carinata, B. juncea and B. napus in order to define the mechanisms of resistance in these three species. On both resistant and susceptible hosts, hyphal penetration was always through stomatal openings and without infection pegs or appressoria. On resistant B. carinata ATC94129P, up to 72% of spores disintegrated and, generally, germination (<22%) and germ tube lengths (<25 μm) were comparatively low. Resistant B. napus Hyola 42 had the lowest germination (8%) and susceptible B. carinata UWA#012 had the highest (51%). On resistant B. carinata ATC94129P, germ tube extension was impeded across 24–60 h post‐inoculation (hpi) and percentage stomatal penetration lower (4%) at 60 hpi compared with susceptible B. carinata UWA#012 (26%). Stomatal densities (stomata/14 757 μm²) on resistant B. juncea Dune (2·12) and B. napus Hyola 42 (1·62) were lower than for susceptible B. juncea Vardan (2·40) and B. napus Trilogy (2·03). Resistant B. carinata ATC94129P had greater stomatal density (1·89) than susceptible B. carinata UWA#012 (1·58). Overall, B. juncea had greater stomatal density (2·26) compared with B. napus (1·83) and B. carinata (1·74). In resistant B. carinata ATC94129P, P. capsellae induced 28% stomata to close, while in susceptible B. carinata UWA#012 no such closure was induced. Epicuticular wax crystalloids were present only on resistant B. carinata ATC94129P and probably also contribute towards resistance.