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BACKGROUND: Phoma stem canker is caused by two coexisting pathogens, Leptosphaeria maculans and L. biglobosa. They coexist because of their temporal and spatial separations, which are associated with the differences in timing of their ascospore release. L. maculans produces sirodesmin PL, while L. biglobosa does not. However, their interaction/coexistence in terms of secondary metabolite production is not understood. RESULTS: Secondary metabolites were extracted from liquid cultures, L. maculans only (Lm only), L. biglobosa only (Lb only), L. maculans and L. biglobosa simultaneously (Lm&Lb) or sequentially 7 days later (Lm+Lb). Sirodesmin PL or its precursors were identified in extracts from 'Lm only' and 'Lm+Lb', but not from 'Lm&Lb'. Metabolites from 'Lb only', 'Lm&Lb' or 'Lm+Lb' caused significant reductions in L. maculans colony area. However, only the metabolites containing sirodesmin PL caused a significant reduction to L. biglobosa colony area. When oilseed rape cotyledons were inoculated with conidia of 'Lm only', 'Lb only' or 'Lm&Lb', 'Lm only' produced large gray lesions, while 'Lm&Lb' produced small dark lesions similar to lesions caused by 'Lb only'. Sirodesmin PL was found only in the plant extracts from 'Lm only'. These results suggest that L. biglobosa prevents the production of sirodesmin PL and its precursors by L. maculans when they grow simultaneously in vitro or in planta. CONCLUSION: For the first time, L. biglobosa has been shown to inhibit the production of sirodesmin PL by L. maculans when interacting simultaneously with L. maculans either in vitro or in planta. This antagonistic effect of interspecific interaction may affect their coexistence and subsequent disease progression and management. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

BACKGROUND: Phoma stem canker is a damaging disease of oilseed rape caused by two related fungal species, Leptosphaeria maculans and L. biglobosa. However, previous work has mainly focused on L. maculans and there has been little work on L. biglobosa. This work provides evidence of the importance of L. biglobosa to stem canker epidemics in the UK. RESULTS: Quantification of L. maculans and L. biglobosa DNA using species‐specific quantitative PCR showed that L. biglobosa caused both upper stem lesions and stem base cankers on nine oilseed rape cultivars in the UK. Upper stem lesions were mainly caused by L. biglobosa. For stem base cankers, there was more L. maculans DNA than L. biglobosa DNA in the susceptible cultivar Drakkar, while there was more L. biglobosa DNA than L. maculans DNA in cultivars with the resistance gene Rlm7 against L. maculans. The frequency of L. biglobosa detected in stem base cankers increased from 14% in 2000 to 95% in 2013. Ascospores of L. biglobosa and L. maculans were mostly released on the same days and the number of L. biglobosa ascospores in air samples increased from the 2010/2011 to 2012/2013 growing seasons. CONCLUSION: Effective control of L. maculans increased infection by L. biglobosa, causing severe upper stem lesions and stem base cankers, leading to yield losses. The importance of L. biglobosa to phoma stem canker epidemics can no longer be ignored. Effective control of phoma stem canker epidemics needs to target both L. maculans and L. biglobosa. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

BACKGROUND: Phoma stem canker is an economically important disease of oilseed rape, caused by two co‐existing fungal pathogen species, Leptosphaeria maculans (Plenodomus lingam) and Leptosphaeria biglobosa (Plenodomus biglobosus). Leptosphaeria maculans produces a phytotoxin called sirodesmin PL. Our previous work showed that L. biglobosa has an antagonistic effect on the production of sirodesmin PL if it is simultaneously co‐inoculated with L. maculans. However, the effects of sequential co‐inoculation on interspecific interactions between the two pathogens are not understood. RESULTS: The interactions between L. maculans and L. biglobosa were investigated in liquid culture by inoculation with L. maculans first, followed by L. biglobosa sequentially at 1, 3, 5 or 7 days later and vice versa; the controls were inoculated with L. maculans only, L. biglobosa only, or L. maculans and L. biglobosa simultaneously. The results showed that L. biglobosa inhibited the growth of L. maculans, the production of both sirodesmin PL and its precursors if L. biglobosa was inoculated before, or simultaneously with, L. maculans. However, the antagonistic effects of L. biglobosa were lost if it was co‐inoculated 5 or 7 days after L. maculans. CONCLUSION: For the first time, the results of this study provided evidence that the timing when L. maculans and L. biglobosa meet significantly influences the outcome of the interspecific competition between them. Leptosphaeria biglobosa can inhibit the production of sirodesmin PL and the growth of L. maculans if it is inoculated before L. maculans or less than 3 days after L. maculans in liquid culture. There is a need to further investigate the timing of co‐inoculation on interactions between L. maculans and L. biglobosa in their host plants for improving the control of phoma stem canker. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

BACKGROUND: Leptosphaeria maculans, the cause of stem canker of oilseed rape, develops gene‐for‐gene interactions with its host and shows a high evolutionary potential to 'break down' novel resistance genes (R, Rlm) deployed in cultivars over large areas. For optimal management of R genes, updated knowledge of the population structure of the pathogen is needed. In France, large‐scale surveys have been done at 10‐year intervals since 2000. Here we report the characterization of a large L. maculans population collected in France in 2019–2020. RESULTS: A total of 844 isolates were collected from 11 sites in ten French departments and were phenotyped for their virulence against nine Brassica napus R genes. All isolates were virulent toward Rlm2 and Rlm9. Very few isolates were avirulent on Rlm1 (1.8%) and Rlm4 (0.6%). Avirulent isolates toward Rlm7 ('AvrLm7') varied from 67% to 11.3%, depending on the site sampled, illustrating the ongoing breakdown of Rlm7. The decrease of AvrLm7 isolates (29.2% at the national level) compared to the 2010 survey (96.5%) was accompanied by an increase of avirulent isolates on Rlm3 (0% in 2010; 54% in 2019–2020). However, virulent isolates on both Rlm3 and Rlm7, previously rarely detected, were found in all sites with a frequency of 17.3%. Finally, most or all isolates were avirulent on Rlm11 (96.1%), LepR2 (RlmS, 99.8%), and Rlm6 (100%), suggesting these three genes still effectively control the disease. CONCLUSION: These data will help guide strategies for breeding and deploying resistant oilseed rape varieties against L. maculans in France. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

The fungal phytopathogen Leptosphaeria maculans, which causes stem canker (blackleg) of rapeseed (Brassica napus), is mainly contiolled worldwide by genetic resistance, which includes major resistance genes (Rim). This model is one of those for which the highest number of avirulence genes (AvrLm) has been cloned. In many systems, including the L. maculans-B. napus interaction, intense use of resistance genes exerts strong selection pressure on the corresponding avirulent isolates, and the fungi may rapidly escape resistance through various molecular events which modify the avirulence genes. In the literature, the study of polymorphism at avirulence loci is often focused on single genes under selection pressure. In this study, we investigate allelic polymorphism at 11 avirulence loci in a French population of 89 L. maculans isolates collected on a trap cultivar in four geographic locations in the 2017--2018 cropping season. The corresponding Rim genes have been (i) used for a long time, (ii) recently used, or (iii) unused in agricultural practice. The sequence data generated indicate an extreme diversity of situations. For example, genes submitted to an ancient selection may have either been deleted in populations (AvrLml) or replaced by a single-nucleotide mutated virulent version (AvrLm2, AvrLm5-9). Genes that have never been under selection may either be nearly invariant (AvrLm6. AvrLmlOA, AvrLmlOB), exhibit rare deletions (AvrLml 1, AvrLml4), or display a high diversity of alleles and isoforms (AvrLmS-Lep2). These data suggest that the evolutionary trajectory of avirulence/virulence alleles is gene-dependent and independent of selection pressure in L. maculans. [ABSTRACT FROM AUTHOR]

Leptosphaeria maculans is one of the major pathogens of oilseed rape (B. napus). It causes blackleg disease, which accounts for significant yield losses worldwide. Using cultivars that harbor major resistance (R) genes is one of the most effective control methods. However, the efficacy of major R genes is related to the frequency of the corresponding avirulence (Avr) genes in a L. maculans population. In this paper, we report the Avr profiles of L. maculans populations and the ratio of its mating types in Northern and Central regions of Germany. Eleven Avr genes in five-hundred and seventy-four isolates were characterized either by applying cotyledon tests on a B. napus differential set or by amplifying avirulence gene-specific PCR markers. Fifty-two races were determined, among which the most dominant race was Avrlm6, -7, -11, AvrlepR1, -R2. Results showed that the resistance gene Rlm2 is 100% ineffective, some other major R genes such as Rlm1, Rlm3, Rlm4 and LepR3 are partially effective (with corresponding Avr frequencies ≤ 42%), while LepR1, LepR2, Rlm6, Rlm11 and Rlm7 can still provide relatively effective resistance in the German fields investigated (with corresponding Avr frequencies of 63–100%). Sexual reproduction is a factor that enhances the potential of L. maculans to evolve under selection pressure. Mating types of the L. maculans populations did not deviate from the ratio of 1:1 in the examined regions, indicating that sexual reproduction and ascospores play central roles in the L. maculans lifecycle. Overall, this study provides an important dataset for the establishment of a strategic plan to preserve the efficacies of major R genes in Germany by applying cultivar rotations of oilseed rape. [ABSTRACT FROM AUTHOR]

Cultivar resistance is an important tool in controlling pathogen-related diseases in agricultural crops. As temperatures increase due to global warming, temperature-resilient disease resistance will play an important role in crop protection. However, the mechanisms behind the temperature-sensitivity of the disease resistance response are poorly understood in crop species and little is known about the effect of elevated temperatures on quantitative disease resistance. Here, we investigated the effect of temperature increase on the quantitative resistance of Brassica napus against Leptosphaeria maculans. Field experiments and controlled environment inoculation assays were done to determine the influence of temperature on R gene-mediated and quantitative resistance against L. maculans ; of specific interest was the impact of high summer temperatures on the severity of phoma stem canker. Field experiments were run for three consecutive growing seasons at various sites in England and France using twelve winter oilseed rape breeding lines or cultivars with or without R genes and/or quantitative resistance. Stem inoculation assays were done under controlled environment conditions with four cultivars/breeding lines, using avirulent and virulent L. maculans isolates, to determine if an increase in ambient temperature reduces the efficacy of the resistance. High maximum June temperature was found to be related to phoma stem canker severity. No temperature effect on stem canker severity was found for the cultivar ES Astrid (with only quantitative resistance with no known R genes). However, in the controlled environmental conditions, the cultivar ES Astrid had significantly smaller amounts of necrotic tissue at 20°C than at 25°C. This suggests that, under a sustained temperature of 25°C, the efficacy of quantitative resistance is reduced. Findings from this study show that temperature-resilient quantitative resistance is currently available in some oilseed cultivars and that efficacy of quantitative resistance is maintained at increased temperature but not when these elevated temperatures are sustained for a long period. [ABSTRACT FROM AUTHOR]

Cultivar resistance is an important tool in controlling pathogen-related diseases in agricultural crops. As temperatures increase due to global warming, temperature-resilient disease resistance will play an important role in crop protection. However, the mechanisms behind the temperature-sensitivity of the disease resistance response are poorly understood in crop species and little is known about the effect of elevated temperatures on quantitative disease resistance. Here, we investigated the effect of temperature increase on the quantitative resistance of Brassica napus against Leptosphaeria maculans. Field experiments and controlled environment inoculation assays were done to determine the influence of temperature on R gene-mediated and quantitative resistance against L. maculans; of specific interest was the impact of high summer temperatures on the severity of phoma stem canker. Field experiments were run for three consecutive growing seasons at various sites in England and France using twelve winter oilseed rape breeding lines or cultivars with or without R genes and/or quantitative resistance. Stem inoculation assays were done under controlled environment conditions with four cultivars/breeding lines, using avirulent and virulent L. maculans isolates, to determine if an increase in ambient temperature reduces the efficacy of the resistance. High maximum June temperature was found to be related to phoma stem canker severity. No temperature effect on stem canker severity was found for the cultivar ES Astrid (with only quantitative resistance with no known R genes). However, in the controlled environmental conditions, the cultivar ES Astrid had significantly smaller amounts of necrotic tissue at 20°C than at 25°C. This suggests that, under a sustained temperature of 25°C, the efficacy of quantitative resistance is reduced. Findings from this study show that temperature-resilient quantitative resistance is currently available in some oilseed cultivars and that efficacy of quantitative resistance is maintained at increased temperature but not when these elevated temperatures are sustained for a long period.

Leptosphaeria maculans is one of the major pathogens of oilseed rape (B. napus). It causes blackleg disease, which accounts for significant yield losses worldwide. Using cultivars that harbor major resistance (R) genes is one of the most effective control methods. However, the efficacy of major R genes is related to the frequency of the corresponding avirulence (Avr) genes in a L. maculans population. In this paper, we report the Avr profiles of L. maculans populations and the ratio of its mating types in Northern and Central regions of Germany. Eleven Avr genes in five-hundred and seventy-four isolates were characterized either by applying cotyledon tests on a B. napus differential set or by amplifying avirulence gene-specific PCR markers. Fifty-two races were determined, among which the most dominant race was Avrlm6, -7, -11, AvrlepR1, -R2. Results showed that the resistance gene Rlm2 is 100% ineffective, some other major R genes such as Rlm1, Rlm3, Rlm4 and LepR3 are partially effective (with corresponding Avr frequencies ≤ 42%), while LepR1, LepR2, Rlm6, Rlm11 and Rlm7 can still provide relatively effective resistance in the German fields investigated (with corresponding Avr frequencies of 63–100%). Sexual reproduction is a factor that enhances the potential of L. maculans to evolve under selection pressure. Mating types of the L. maculans populations did not deviate from the ratio of 1:1 in the examined regions, indicating that sexual reproduction and ascospores play central roles in the L. maculans lifecycle. Overall, this study provides an important dataset for the establishment of a strategic plan to preserve the efficacies of major R genes in Germany by applying cultivar rotations of oilseed rape.

In the Czech Republic, demethylation inhibitors (DMIs) are used both as fungicides in controlling phoma stem canker and as growth regulators. This heavy use can result in the development of resistant isolates. A total of 45 and 286 Leptosphaeria maculans isolates were tested in vitro, using the mycelial growth and microtiter plate assays, respectively. The objective was to determine the sensitivity of L. maculans isolates collected in the Czech Republic to the fungicides tetraconazole, metconazole, and prochloraz. The mean EC50 values with the mycelial growth plate method were 1.33, 0.78, and 0.40 µg mL−1 for tetraconazole, metconazole, and prochloraz, respectively. The mean EC50 values for the microtiter plate assay were 3.01, 0.44, and 0.19 µg mL−1 for tetraconazole, metconazole, and prochloraz, respectively. All three fungicides also had high variation factors that may be due to inserts in the ERG11 promoter region. In addition, cross sensitivity among the three fungicides was observed. Overall, the high variation factors and the PCR (polymerase chain reaction) results observed in this study could signify the presence of resistant isolates in L. maculans Czech populations, especially in isolates tested for sensitivity to tetraconazole.

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Background: The fungal pathogen Leptosphaeria maculans (Lm). causes blackleg disease on canola/rapeseed in many parts of the world. It is important to use resistant cultivars to manage the disease and minimize yield losses. In this study, twenty-two Lm isolates were used to identify resistance genes in a collection of 243 canola/rapeseed (Brassica napus L.) accessions from Canada and China. These Lm isolates carry different compliments of avirulence genes, and the investigation was based on a genome-wide association study (GWAS) and genotype-by-sequencing (GBS). Results: Using the CROP-SNP pipeline, a total of 81,471 variants, including 78,632 SNPs and 2839 InDels, were identified. The GWAS was performed using TASSEL 5.0 with GLM + Q model. Thirty-two and 13 SNPs were identified from the Canadian and Chinese accessions, respectively, tightly associated with blackleg resistance with P values < 1 × 10− 4. These SNP loci were distributed on chromosomes A03, A05, A08, A09, C01, C04, C05, and C07, with the majority of them on A08 followed by A09 and A03. The significant SNPs identified on A08 were all located in a 2010-kb region and associated with resistance to 12 of the 22 Lm isolates. Furthermore, 25 resistance gene analogues (RGAs) were identified in these regions, including two nucleotide binding site (NBS) domain proteins, fourteen RLKs, three RLPs and six TM-CCs. These RGAs can be the potential candidate genes for blackleg resistance. Conclusion: This study provides insights into potentially new genomic regions for discovery of additional blackleg resistance genes. The identified regions associated with blackleg resistance in the germplasm collection may also contribute directly to the development of canola varieties with novel resistance genes against blackleg of canola. [ABSTRACT FROM AUTHOR]

• Fungicide resistant Leptosphaeria maculans strains were isolated. • All resistance is due to insertion of DNA into the erg11 promoter. • A new pipeline for estimating frequencies of insertions was developed. • Frequency of azole resistance in populations is as high as 33%. Continued use of fungicides provides a strong selection pressure towards strains with mutations to render these chemicals less effective. Previous research has shown that resistance to the demethylation inhibitor (DMI) fungicides, which target ergosterol synthesis, in the canola pathogen Leptosphaeria maculans has emerged in Australia and Europe. The change in fungicide sensitivity of individual isolates was found to be due to DNA insertions into the promoter of the erg11 / CYP51 DMI target gene. Whether or not these were the only types of mutations and how prevalent they were in Australian populations was explored in the current study. New isolates with reduced DMI sensitivity were obtained from screens on DMI-treated plants, revealing eight independent insertions in the erg11 promoter. A novel deep amplicon sequencing approach applied to populations of ascospores fired from stubble identified an additional undetected insertion allele and quantified the frequencies of all known insertions, suggesting that, at least in the samples processed, the combined frequency of resistant alleles is between 0.0376% and 32.6%. Combined insertion allele frequencies positively correlated with population-level measures of in planta resistance to four different DMI treatments. Additionally, there was no evidence for erg11 coding mutations playing a role in conferring resistance in Australian populations. This research provides a key method for assessing fungicide resistance frequency in stubble-borne populations of plant pathogens and a baseline from which additional surveillance can be conducted in L. maculans. Whether or not the observed resistance allele frequencies are associated with loss of effective disease control in the field remains to be established. [ABSTRACT FROM AUTHOR]

Using resistant cultivars is a common approach to managing blackleg of canola/rapeseed caused by Leptosphaeria maculans (Lm). Quantitative resistance (QR), as opposed to major-gene resistance, is of interest because it is generally more durable, due to its multi-genetic basis. However, the mechanisms and genes underlying QR are mostly unknown. In this study, potential QR modes of action in “74-44 BL” was explored. This Canadian canola cultivar showed moderate but consistent race-nonspecific resistance at the cotyledon and adult-plant stages. A susceptible cultivar, “Westar”, was used as a control. After inoculation, the lesions developed more slowly on the cotyledons of 74-44 BL than those of Westar. We used RNA sequencing (-RNA-seq) to identify genes and their functions, putatively related to this resistance, and found that genes involved in programmed cell death (PCD), reactive oxygen species (ROS), signal transduction or intracellular endomembrane transport were most differentially expressed. ROS production was assessed in relation to Lm hyphal growth and lesion size; it occurred beyond the tissue colonized by Lm in 74-44 BL and appeared to trigger rapid cell death, limiting cotyledon colonization by Lm. In contrast, Lm grew more rapidly in Westar, often catching up with the ring of ROS and surpassing lesion boundaries. It appears that QR in 74-44 BL cotyledons is associated with limited colonization by Lm possibly mediated via ROS. The RNA-seq data also showed a link between ROS, signal transduction, and endomembrane vesicle trafficking, as well as PCD in the resistance. These results provide a starting point for a better understanding of the mechanisms behind QR against Lm in canola.

Abstract: Genetic, physiological and physical homogenization of agricultural landscapes creates ideal environments for plant pathogens to proliferate and rapidly evolve. Thus, a critical challenge in plant pathology and epidemiology is to design durable and effective strategies to protect cropping systems from damage caused by pathogens. Theoretical studies suggest that spatio‐temporal variation in the diversity and distribution of resistant hosts across agricultural landscapes may have strong effects on the epidemiology and evolutionary potential of crop pathogens. However, we lack empirical tests of spatio‐temporal deployment of host resistance to pathogens can be best used to manage disease epidemics and disrupt pathogen evolutionary dynamics in real‐world systems. In a field experiment, we simulated how differences in Brassica napus resistance deployment strategies and landscape connectivity influence epidemic severity and Leptosphaeria maculans pathogen population composition. Host plant resistance, spatio‐temporal connectivity [stubble loads], and genetic connectivity of the inoculum source [composition of canola stubble mixtures] jointly impacted epidemiology (disease severity) and pathogen evolution (population composition). Changes in population composition were consistent with directional selection for the ability to infect the host (infectivity), leading to changes in pathotype (multilocus phenotypes) and infectivity frequencies. We repeatedly observed decreases in the frequency of unnecessary infectivity, suggesting that carrying multiple infectivity genes is costly for the pathogen. From an applied perspective, our results indicate that varying resistance genes in space and time can be used to help control disease, even when resistance has already been overcome. Furthermore, our approach extends our ability to test not only for the efficacy of host varieties in a given year, but also for durability over multiple cropping seasons, given variation in the combination of resistance genes deployed. [ABSTRACT FROM AUTHOR]

Because epidemics of successive cropping seasons are not independent, epidemiological studies need to encompass the processes occurring during the transmission of epidemics from one season to the next. With Leptosphaeria maculans, infected stubble allows carry‐over of the fungus. Generation experiments using recurrent selection on field plots are a useful means of comparing the effects of selection pressures. However, the full life cycle of the fungus, from plant infection to the next generation of ascospores, has not yet been achieved under controlled conditions. Studies were undertaken to achieve an experimental set‐up with sexual reproduction under controlled conditions. Cankered oilseed rape stems were produced under controlled conditions, after inoculation with a mixture of 12 isolates across both mating types. Stems were cut longitudinally and attached to styropore plates. Stem halves were incubated outside or in climate chambers regularly soaked in tap water to ensure maturation. Incubation was stopped when mature pseudothecia were observed. In all three independent experiments, more stem halves had pseudothecia when incubated under controlled conditions (30–100%) than incubated outside (0–80%). To the authors’ knowledge, this is the first study achieving the full life cycle of the fungus under controlled conditions, from infection of the plant to mature pseudothecia. This opens up the prospect of running experiments year‐round to better understand inoculum production, to compare fungal fitness, or to run generation experiments with exotic pathogen populations. [ABSTRACT FROM AUTHOR]

Summary: Blackleg disease of Brassica napus caused by Leptosphaeria maculans (Lm) is largely controlled by the deployment of race‐specific resistance (R) genes. However, selection pressure exerted by R genes causes Lm to adapt and give rise to new virulent strains through mutation and deletion of effector genes. Therefore, a knowledge of effector gene function is necessary for the effective management of the disease. Here, we report the cloning of Lm effector AvrLm9 which is recognized by the resistance gene Rlm9 in B. napus cultivar Goéland. AvrLm9 was mapped to scaffold 7 of the Lm genome, co‐segregating with the previously reported AvrLm5 (previously known as AvrLmJ1). Comparison of AvrLm5 alleles amongst the 37 re‐sequenced Lm isolates and transgenic complementation identified a single point mutation correlating with the AvrLm9 phenotype. Therefore, we renamed this gene as AvrLm5‐9 to reflect the dual specificity of this locus. Avrlm5‐9 transgenic isolates were avirulent when inoculated on the B. napus cultivar Goéland. The expression of AvrLm5‐9 during infection was monitored by RNA sequencing. The recognition of AvrLm5‐9 by Rlm9 is masked in the presence of AvrLm4‐7, another Lm effector. AvrLm5‐9 and AvrLm4‐7 do not interact, and AvrLm5‐9 is expressed in the presence of AvrLm4‐7. AvrLm5‐9 is the second Lm effector for which host recognition is masked by AvrLm4‐7. An understanding of this complex interaction will provide new opportunities for the engineering of broad‐spectrum recognition. [ABSTRACT FROM AUTHOR]

In the period 2009-2011 monitoring of the incidence of phoma stem canker was carried out at selected sites of the Czech Republic (Šumperk and Opava regions in North Moravia). The risk of infection was evaluated by assessing the autumn release of Leptosphaeria spp. ascospores and with the proPlant prediction model. In recent years, the incidence of phoma stem canker has been relatively low and this corresponded with the total count of ascospores released in the autumn period but not with an increased level of infection risk announced by the proPlant model. During the monitored period the ascospore concentration reached maximally 2 ascospores/1 m3 per day. Maximum ascospore release was recorded in 2010 as a result of favourable weather conditions. The first incidence of phoma leaf spot has been observed in mid-October. The higher values of ascospores concentration were recorded in the Opava region, but the total number of the trapped ascospores was more often higher in the Šumperk region.

New genotypes of winter oilseed rape bred under the Czech breeding program were subjected to performance tests within the framework of inter-institutional pre-tests. At four monitored localities in the Czech Republic, field trials were carried out on 50 genotypes in the years 2015 to 2017. In these trials, evaluations were made on infestation by the fungal pathogens Leptosphaeria maculans, Leptosphaeria biglobosa, and Sclerotinia sclerotiorum, as well as the level of infestation caused by phytoplasmas. The evaluation of new oilseed rape genotypes confirmed a medium to high level of resistance to phoma stem canker and Sclerotinia stem rot compared to the resistant reference varieties. The most severe infestations caused by Leptosphaeria spp. were observed in Chlumec nad Cidlinou in 2015 (mean severity score 22.38%) and in Opava in 2016 (mean severity score 20.72%). The same trend was observed for infestations caused by S. sclerotiorum, where the most damage to trial plots were observed at the same localities, in 2015 in Chlumec nad Cidlinou (mean severity score 36.85%) and in 2016 in Opava (mean severity score 18.84%). PCR determination of Leptosphaeria spp. showed that 98% of sampled leaves in autumn season showing symptoms were infected by L. maculans, while the remaining 2% were identified as bearing L. biglobosa. Molecular detection of infected parts of oilseed rape plants with symptoms of phyllody confirmed in all cases the presence of phytoplasma isolates from the 16SrI Aster Yellows group.

In the UK, fungicides are often used to control phoma stem canker on winter oilseed rape. Field trials were established near Boxworth, Cambridgeshire for four cropping seasons (2011/2012, 2012/2013, 2013/2014 and 2014/15) to test the efficacy of a new fungicide mixture Refinzar® (penthiopyrad + picoxystrobin) by comparison to an existing fungicide Proline 275® (prothioconazole) against phoma stem canker ( Leptosphaeria spp.) and the effect on winter oilseed rape (cv. Catana) yield. In each season, weather data were collected from a weather station at Boxworth and the release of ascospores was monitored using a nearby Burkard spore sampler. The patterns of ascospore release differed between seasons and related to weather conditions. Fungicides penthiopyrad + picoxystrobin and prothioconazole were applied in October/November when 10 % of plants had phoma leaf spotting (T1, early), 4/8 weeks after T1 (T2, late) or at both T1 and T2 (combined). When phoma leaf spot symptoms were assessed in autumn/winter, penthiopyrad + picoxystrobin and prothioconazole both decreased numbers of phoma leaf spots caused by L. maculans; there were few leaf spots caused by L. biglobosa. Penthiopyrad + picoxystrobin and prothioconazole both reduced phoma stem canker severity before harvest compared to the untreated control but did not increase yield in these seasons when epidemics were not severe. In 2013/2014, the presence of L. maculans and L. biglobosa in upper stem lesions or stem base cankers was determined by species-specific PCR. The proportions of stems with L. maculans DNA were much greater than those with L. biglobosa DNA for both upper stem lesions and basal stem cankers. These results suggest that both penthiopyrad + picoxystrobin and prothioconazole can decrease phoma stem canker severity on winter oilseed rape in severe disease seasons. [ABSTRACT FROM AUTHOR]

Blackleg (Phoma stem canker), caused by the Leptosphaeria species complex L. maculans and L. biglobosa, is a severe and economically important disease in most oilseed production areas worldwide. In this study, the virulence structure of a population of 115 L. maculans isolates from six counties in Alberta, Canada, was determined on three differential canola cultivars, Westar, Quinta and Glacier. Based on the differential interactions as assessed on a 0-9 severity scale, the isolates were classified into four pathogenicity groups: PG-2, PG-T, PG-3, and PG-4. When the differential interactions were assessed by analysis of variance of lesion size, the isolates were classified as into PG-2, PG-T and PG-4, among which PG-4 was predominant comprising 59% of the isolates, followed by PG-T at 37% and PG-2 at 4%. The presence or absence of five avirulence genes, AvrLm1, AvrLm4-7, AvrLm6, AvrLmJ1 and AvrLm11, in each of the isolates was evaluated by PCR analysis. AvrLm1 was absent, while AvrLm4-7 and AvrLm6 were present, in all of the 115 isolates. AvrLmJ1 and AvrLm11 were present in most isolates but absent in three isolates collected from Ponoka County. AvrLm11 also was absent in another two isolates collected from Strathcona County. This study describes the current status of virulence in L. maculans population in Alberta, which may provide guidance for blackleg resistance breeding efforts and cultivar deployment. [ABSTRACT FROM AUTHOR]

Current modelling of inoculum transmission from a cropping season to the following one relies on the extrapolation of kernels estimated on data at short distances from punctual sources, because data collected at larger distances are scarce. We estimated the dispersal kernel of Leptosphaeria maculans ascospores from stubble left after harvest in the summer previous to newly sown oilseed rape fields, using phoma stem canker autumn disease severity. We built a dispersal model to analyse the data. Source strengths are described in the spatial domain covered by source fields by a log-Gaussian spatial process. Infection potentials in the following season are described in the space consisting of the target fields, by a convolution of sources and a power-exponential dispersal kernel. Data were collected on farmers' fields considered as sources in 2009 and 2011 (72 and 39 observation points) and as targets in 2010 and 2012 (172 and 200 points). We applied the Bayesian approach for model selection and parameter estimation. We obtained fat tail kernels for both data sets. This estimation is the first from data acquired over distances of 0 to 1000 m, using several non-punctual inoculum sources. It opens the prospect of refining the existing simulators, or developing disease risk maps. [ABSTRACT FROM AUTHOR]

Blackleg, caused by the Leptosphaeria maculans/L. biglobosa, is one of the most damaging diseases of Brassica spp. Isolates of L. maculans and L. biglobosa were recovered from oilseed rape, cabbage and broccoli in Lithuania. All isolates from cabbage were identified as L. biglobosa, while both fungal species were detected on broccoli and oilseed rape. Amplified fragment length polymorphism (AFLP) analysis was used to assess the genetic diversity among 68 isolates of L. maculans and L. biglobosa. Cluster analysis divided isolates into two groups of L. maculans and L. biglobosa species. Analysis of molecular variance attributed 57.9% of molecular variance to differences among isolates within the population of L. maculans and 26.6% within the population of L. biglobosa. This is the first study that demonstrates the coexistence of both Leptosphaeria species on B. oleracea in Lithuania. Analysis of molecular variance showed that the populations of Leptosphaeria spp from different host plants were genetically differentiated. [ABSTRACT FROM AUTHOR]

Phoma stem canker (blackleg), caused by the fungi Leptosphaeria maculans and L. biglobosa, is the most devastating disease in oilseed rape (canola) production worldwide. In this study, 84 Leptosphaeria isolates were collected in China from rapeseed ( Brassica napus) plants with blackleg symptoms and identified using multiplex PCR and rDNA-ITS sequence analysis. Results showed that all these isolates are L. biglobosa and no L. maculans was detected. A total of 99 isolates was analyzed, including 15 other L. biglobosa isolates from the UK, Canada and Poland using rDNA-ITS sequences, and all isolates were found to belong to the L. biglobosa 'brassicae' subclade. The genetic variation of these 99 isolates was tested using ISSR (inter simple sequence repeats) markers using 24 polymorphic primers pairs that generated 241 bands by ISSR-PCR amplification. A dendrogram based on weighted pair group mean analysis of these bands revealed that L. biglobosa isolates from China clustered into different groups from those obtained from Canada and Europe. Furthermore, Chinese isolates from seven provinces, except those from Jiangsu, could be placed within the same cluster, based on geographical location. Nei's gene diversity ranged from 0.25 in Jiangsu to 0.11 in Sichuan, and Shannon's information index from 0.16 to 0.37. Genetic identity ranged from 0.784 to 0.834 when isolates from China were compared with those from Canada and the UK, respectively. Analysis of gene flow among populations (Nm) indicated that almost no gene exchange has occurred between L. biglobosa in China and the populations of the fungus from the UK or Canada. [ABSTRACT FROM AUTHOR]

Airborne sexual spores of Leptosphaeria maculans and L. biglobosa are primary inoculum of fungi causing phoma stem canker of oilseed rape. In this study ascospore release of these two species was compared between Lithuania (of autumn 2010 and 2011) and Poland (autumns of 2009 till 2012), using identical equipment and methods. Dynamics of L. maculans and L. biglobosa ascospore dispersal was investigated using volumetric samplers followed by microscopic and molecular methods of detection. In Lithuania, the earliest detection of Leptosphaeria ascospores, using microscopy was on 1st September (2010) and on 2nd September (2011), whereas in Poland the earliest detection was on 5th September (2009 and 2010) and the latest on 11th October (2011), which demonstrates that differences between the seasons may exceed a month (36 days). In the case of molecular detection performed for the Polish samples, the dates of the earliest and the latest release of the first ascospores ranged from 5th September to 15th October (40 days). The number of days with ascospores detected in the air samples ranged from 6 (2012) to 25 (2009). At earliest, the detection of the highest concentration of ascospores was on 6th September (2010) and the latest - on 28th October (2009). These results demonstrate big differences between the results of spore monitoring between the years and the importance of aerobiological studies to follow the development of the pathogen in natural infection. The fluctuations in quantities of spores and fungal deoxyribonucleic acid (DNA) strongly depended on the weather conditions, mainly rainfall, air temperature and relative humidity. Ascospore release was observed immediately after the rainfall events, but not later than two days after the rainfall. On days without rain, spores were present in the air samples when average relative air humidity exceeded 90% and day temperature was below 15°C. In 2009, molecular detection revealed the presence of both fungal species in the air samples, whereas in 2010-2012 only L. biglobosa DNA was found. Molecular detection with a quantitative real-time polymerase chain reaction (qPCR) was identified as a fast and accurate method of pathogen detection and identification from air samples. The highest coefficient of correlation between microscopic and molecular detection of Leptosphaeria species was 0.828 (2009) and the lowest was 0.483 (2011); in all years both techniques of detection were statistically correlated. We have demonstrated that the correlation coefficient highly depended on the number of ascospores in air samples. Based on the studies performed in Lithuania and Poland, we have shown that climatic conditions in the northern part of Central Europe are favourable for the spread of Leptosphaeria spp. by ascospores; however, the patterns of ascospore release greatly differ between the both countries. [ABSTRACT FROM AUTHOR]

A combination of staining, light microscopy and SYBR green- and dual-labelled fluorescent probe-based qPCR chemistries with species- and gene-specific primers was employed to evaluate fluctuations in the aerial biomass of Leptosphaeria maculans spores captured by volumetric spore trappings in Poznan, Poland (2006, 2008) and Harpenden, UK (2002, 2006). Arising from these surveys, DNA samples extracted from Burkard spore-trap tapes were screened for fluctuation patterns in the frequencies of AvrLm1 and AvrLm6, the most prominent of the 15 genes that code for avirulence effectors in this Dothideomycete cause of the destructive phoma stem canker disease of oilseed rape worldwide. In Poznan, very low frequencies of AvrLm1 allele were found in the autumn of both 2006 and 2008, reflecting significantly increased cultivation of rape seed with Rlm1-based resistance. In contrast, at least six folds-higher frequencies of AvrLm6, which were also confirmed by end-point PCR bioassays on phoma-infected leaves from the same region of Poland, were obtained during both years. In the UK, however, relatively higher AvrLm1 allele titres were found in L. maculans spores captured in air samples from the autumn of 2002 on the experimental fields of Rothamsted Research, Harpenden, that were historically sown to genetically heterogeneous B. napus cultivars. In the 2006 screen these levels had plummeted, to a 1:4 ratio, in favour of frequencies of the AvrLm6 allele. Patterns of fluctuations in erg11 ( CYP51) fragments coding for sterol 14α-demethylase suggest October as the month with the most viable wind-dispersed L. maculans propagules of each season of the screens. [ABSTRACT FROM AUTHOR]

In the period 2009-2011 monitoring of the incidence of phoma stem canker was carried out at selected sites of the Czech Republic (Šumperk and Opava regions in North Moravia). The risk of infection was evaluated by assessing the autumn release of Leptosphaeria spp. ascospores and with the proPlant prediction model. In recent years, the incidence of phoma stem canker has been relatively low and this corresponded with the total count of ascospores released in the autumn period but not with an increased level of infection risk announced by the proPlant model. During the monitored period the ascospore concentration reached maximally 2 ascospores/1 m3 per day. Maximum ascospore release was recorded in 2010 as a result of favourable weather conditions. The first incidence of phoma leaf spot has been observed in mid October. The higher values of ascospores concentration were recorded in the Opava region, but the total number of the trapped ascospores was more often higher in the Šumperk region. [ABSTRACT FROM AUTHOR]

The genetic diversity of Leptosphaeria biglobosa, causing phoma stem canker on oilseed rape, was analyzed using ISSR (inter simple sequence repeats) marker. 84 blackleg isolates were collected from China and 15 from the United Kingdom, Canda and Poland were used as control. A total of 245 bands generated by the ISSR - PCR with 24 selected primers were polymorphic. The dendrogram constructed with 99 individuals using UPGMA ( unweighted pair group mean analysis ) showed that Chinese isolates were different from Canada and Europe. Meanwhile, Chinese isolates from 7 provinces were almost divided into 5 groups according to their geographical lo-cations only isolates from the Jiangsu Province distributed into other groups. Nei ' s gene diversity ranged from 0.252 7 (in Jiangsu) to 0. 108 1 (in Sichuan), with the order of Jiangsu > Hubei > Anhui > Inner Mongolia > Shanghai > Sichuan. Shannon' s information index were from 0. 157 5 to 0. 372 6. The Nei ' s gene identity were 0.783 9 between the China and Canada isolates, and 0.834 3 between China and the United Kingdom isolates. [ABSTRACT FROM AUTHOR]

Abstract: Phoma stem canker is a worldwide disease of oilseed rape, responsible for major economic losses. The main control methods are the use of resistant cultivars, cropping practices and spatial territory organization, involving large-scale spatial processes. A participatory approach can be useful for dealing with this problem, which requires stakeholders' coordination as regards to the timing and spatial layout of cropping practices. The scenario concept is used to plan possible future cropping systems and to reveal their main drivers. In this paper, we test a method to build quantitative land use scenarios and to test them with an existing spatially-explicit model to assess the effects of various scenarios on phoma stem canker management. It was built from previous participatory methods. The method we tested is composed of six steps: stakeholder identification, building with them a common vision of the disease behaviour, collective scenario design, and discussion with stakeholders of model-based scenario evaluations. We tested this method on a regional case study application in France. This application revealed benefits of the method with respect to the diversity of the designed scenarios (predictive and exploratory scenarios; driven by local or global context change) and the diversity of results on phoma stem canker management. Based on this application, recommendations for participation improvement and model acceptance are made: (1) build a partnership with a key local stakeholder; (2) describe and discuss precisely model functioning with stakeholders (avoid the “black box”); and (3) facilitate interpretation of scenario assessment by adapting model outputs. This method, combining a participatory approach (qualitative and quantitative construction of scenarios and their evaluation with an existing model) highlights the potential benefits of involving stakeholders in attempting to solve a local problem, in this case, phoma stem canker management. [Copyright &y& Elsevier]

In western Canada, canola is traditionally grown in rotation once every 4 years to restrict losses due to pests. Recently, growers have begun to produce canola more intensively due to market opportunities and cultivar improvements. This study was initiated to investigate the consequences of more intensive production of canola in rotations, integrated with currently available disease management practices, including blackleg resistant cultivars and fungicides. A 4 replicate split-plot experiment was established at Scott and Melfort, Saskatchewan with canola rotation treatments ranging from yearly to every second, third or fourth season in combination with pea, wheat and flax using a blackleg resistant and a blackleg susceptible cultivar. Sub-plots were fungicide treatments for blackleg and sclerotinia stem rot diseases. Blackleg incidence and severity was increased in rotations comprising more than one canola crop every four years, regardless of cultivar, although the blackleg resistant cultivar was much less affected than the blackleg susceptible cultivar. The yield of the blackleg resistant canola cultivar was similar among rotations that included canola every 2, 3 and 4 years. Fungicide application was of limited value to maintain canola yield. The results suggest that canola cultivars with strong blackleg resistance can be grown more intensively than once every 4 years with limited yield reduction. However, the increased severity of infection and amount of infested residue produced as canola rotations are intensified, which occurs even with resistant cultivars, increases the risk of inoculum carry-over, resistance breakdown and yield loss. Therefore, it would be prudent for western Canadian canola growers to adhere to less intensive inclusion of canola in rotations, such as one canola crop in 4 years, as an effective blackleg management strategy. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Leptosphaeria maculans and L. biglobosa are fungal pathogens able to cause allergic reactions in humans and infect plants of Brassica species. The rate of their development and subsequent spore release depend on weather conditions. The aim of this paper was to pinpoint the exact meteorological conditions triggering the release of L. maculans and L. biglobosa ascospores in central and eastern Poland. Multiple regressions indicated that the frequency and amount of rainfall over short periods were important in mediating spore release. The first ascospore event depended mainly on the number of rainy days during the first 10 days of July and the cumulative precipitation during July and September. The most important variables for maximum spore release were cumulative rainfall in the beginning of July and the end of September, as well as the number of days with precipitation events in the first 10 days of August. The results highlighted for the first time the importance of the days preceding the collection of oilseed rape plants from the field. Higher moisture content of senescing but still living stems play a crucial role in the early start of the ascospore season and the maximum release of ascospores. This was not yet considered to date. [ABSTRACT FROM PUBLISHER]

Development of phoma leaf spot (caused by Leptosphaeria maculans) on winter oilseed rape (canola, Brassica napus) was assessed in two experiments at Rothamsted in successive years (2003–04 and 2004–05 growing seasons). Both experiments compared oilseed rape cultivars Eurol, Darmor, Canberra and Lipton, which differ in their resistance to L. maculans. Data were analysed to describe disease development in terms of increasing numbers of leaves affected over thermal time from sowing. The cultivars showed similar patterns of leaf spot development in the 2003–04 experiment when inoculum concentration was relatively low (up to 133 ascospores m−3 air), Darmor developing 5.3 diseased leaves per plant by 5 May 2004, Canberra 6.6, Eurol 6.8 and Lipton 7.5. Inoculum concentration was up to sevenfold greater in 2004–05, with Eurol and Darmor developing 2.4 diseased leaves per plant by 16 February 2005, whereas Lipton and Canberra developed 2.8 and 3.0 diseased leaves, respectively. Based on three defined periods of crop development, a piece-wise linear statistical model was applied to the progress of the leaf spot disease (cumulative diseased leaves) in relation to appearance (‘birth’) and death of leaves for individual plants of each cultivar. Estimates of the thermal time from sowing until appearance of the first leaf or death of the first leaf, the rate of increase in number of diseased leaves and the area under the disease progress line (AUDPL) for the first time period were made. In 2004–05, Canberra (1025 leaves ×°C days) and Lipton (879) had greater AUDPL values than Eurol (427) and Darmor (598). For Darmor and Lipton, the severity of leaf spotting could be related to the severity of stem canker at harvest. Eurol had less leaf spotting but severe stem canker, whereas Canberra had more leaf spotting but less severe canker. [ABSTRACT FROM AUTHOR]