Search

In order to understand the epidemiological features of rapeseed blackleg in northern China, a 2‐year survey of blackleg on wild mustard (Sinapis arvensis) and rapeseed (Brassica napus) was done in Zhaosu County of Xinjiang Uyghur Autonomous Region of China. Blackleg frequently occurs on both plants in this region, with mean values of disease incidence reaching up to 28% on wild mustard and 14% on rapeseed. A total of 556 pycnidia‐forming fungal isolates were obtained from wild mustard (69) and rapeseed (487). Morphological and molecular identification indicated that all of the 69 isolates from wild mustard and the majority of the isolates from rapeseed (477/487, 97.9%) belonged to Leptosphaeria biglobosa 'canadensis' (Lb‐c); the remaining 10 isolates from rapeseed (10/487, 2.1%) belonged to L. biglobosa 'brassicae' (Lb‐b). Among 239 isolates of Lb‐c (62 from wild mustard and 177 from rapeseed), two mating‐type idiomorphs (MAT1‐1 and MAT1‐2) were detected. Chi‐square test showed that the ratio of MAT1‐1 to MAT1‐2 of isolates was 1:1, implying that the individuals of Lb‐c can frequently conduct sexual reproduction to produce ascospores as the primary airborne infection source. The virulence of 10 Lb‐c isolates from wild mustard and rapeseed and five Lb‐b isolates from rapeseed was determined on both plants. The results showed that the Lb‐c isolates from wild mustard were as virulent as isolates of Lb‐c and Lb‐b from rapeseed. Thus, Lb‐c can act as inoculum of rapeseed blackleg, and eliminating plants of wild mustard may improve efficacy of crop rotation in suppression of rapeseed blackleg. [ABSTRACT FROM AUTHOR]

Blackleg disease, caused by Leptosphaeria spp. fungi, is one of the most important diseases of Brassica napus, responsible for severe yield losses worldwide. Blackleg resistance is controlled by major R genes and minor quantitative trait loci (QTL). Due to the high adaptation ability of the pathogen, R-mediated resistance can be easily broken, while the resistance mediated via QTL is believed to be more durable. Thus, the identification of novel molecular markers linked to blackleg resistance for B. napus breeding programs is essential. In this study, 183 doubled haploid (DH) rapeseed lines were assessed in field conditions for resistance to Leptosphaeria spp. Subsequently, DArTseq-based Genome-Wide Association Study (GWAS) was performed to identify molecular markers linked to blackleg resistance. A total of 133,764 markers (96,121 SilicoDArT and 37,643 SNP) were obtained. Finally, nine SilicoDArT and six SNP molecular markers were associated with plant resistance to Leptosphaeria spp. at the highest significance level, p < 0.001. Importantly, eleven of these fifteen markers were found within ten genes located on chromosomes A06, A07, A08, C02, C03, C06 and C08. Given the immune-related functions of the orthologues of these genes in Arabidopsis thaliana, the identified markers hold great promise for application in rapeseed breeding programs. [ABSTRACT FROM AUTHOR]

Blackleg disease, a major threat to Brassica crops worldwide, is primarily caused by the pathogen Leptosphaeria biglobosa. Investigating the genetic variation of L. biglobosa is crucial for managing and preventing the disease in Brassica napus. To date, there is scarce genomic variation information available for populations of L. biglobosa in China. In this study, 73 L. biglobosa strains of canola stalks were collected from 12 provinces in China and subjected to re-sequencing. The 73 assemblies averaged 1340 contigs, 72,123 bp N50, and 30.17 Mb in size. In total, 9409 core orthogroups and 867 accessory orthogroups were identified. A total of 727,724 mutation loci were identified, including 695,230 SNPs and 32,494 indels. Principal component analysis (PCA) and population structure analysis showed that these strains could be divided into seven subgroups. The strains in most provinces were clustered into a single subgroup, suggesting a strong influence of the geographic environment on strain variation. The average nucleotide diversity (θπ) of all strains was 1.03 × 10−3, indicating important genetic diversity among strains from different regions of China. This study provides valuable resources for future comparative genomics, gives new insights into the population evolution of L. biglobosa, and supports the development of strategies for managing blackleg disease in canola. [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]

Eight fungal strains (P1 to P8) were isolated from rapeseed stems (Brassica napus) infected with the blackleg pathogen Leptosphaeria biglobosa (Lb). They formed pycnidia with similar morphology to those of Lb, and thus were considered as Lb relatives (LbRs). The species-level identification of these strains was performed. Their virulence on rapeseed and efficacy in the suppression of Lb infection were determined, and the biocontrol potential and biocontrol mechanisms of strain P2 were investigated. The results showed that the LbRs belong to two teleomorphic genera in the family Didymellaceae, Didymella for P1 to P7 and Boeremia for P8. Pathogenicity tests on rapeseed cotyledons and stems indicated the LbRs were weakly virulent compared to L. biglobosa. Co-inoculation assays on rapeseed cotyledons demonstrated that P1 to P7 (especially P1 to P4) had a suppressive effect on Lb infection, whereas P8 had a marginal effect on infection by L. biglobosa. Moreover, D. macrostoma P2 displayed a more aggressive behavior than L. biglobosa in the endophytic colonization of healthy rapeseed cotyledons. Cultures of P2 in potato dextrose broth (PDB) and pycnidiospore mucilages exuded from P2 pycnidia showed antifungal activity to L. biglobosa. Further leaf assays revealed that antifungal metabolites (AM) of strain P2 from PDB cultures effectively suppressed infection by L. biglobosa, Botrytis cinerea (gray mold), and Sclerotinia sclerotiorum (white mold). An antifungal metabolite, namely penicillither, was purified and identified from PDB cultures and detected in pycnidiospore mucilages of strain P2. This study suggests that the LbRs are a repertoire for screening biocontrol agents (BCAs) against rapeseed diseases, and D. macrostoma P2 is a multi-functional BCA, a penicillither producer, and an endophyte. [ABSTRACT FROM AUTHOR]

Abstract Background Leptosphaeria maculans “brassicae” (Lmb) and Leptosphaeria biglobosa “brassicae” (Lbb) make up a species complex involved in the stem canker (blackleg) disease of rapeseed (Brassica napus). They coinfect rapeseed together, from the early stage of infection on leaves to the final necrotic stage at the stem base, and both perform sexual crossings on plant residues. L. biglobosa is suggested to be a potential biocontrol agent against Lmb, but there has been no mechanistic investigation of the different types of interactions that may occur between the plant and the two fungal species. Results We investigated the bi- or tripartite interaction mechanisms by (i) confronting Lmb and Lbb in culture conditions or during cotyledon infection, with different timing and/or spore concentration regimes, (ii) performing RNA-Seq experiments in vitro or on the kinetics of infection of cotyledons infected by Lmb and/or Lbb to evaluate the transcriptomic activity and the plant response when both fungal species are inoculated together. Lbb infection of B. napus cotyledons was typical of a necrotrophic behavior, with a very early setup of one pathogenicity program and very limited colonization of tissues. This contrasted with the complex succession of pathogenicity programs of the hemibiotroph Lmb. During simultaneous co-infection by both species, Lmb was strongly impacted in its growth and transcriptomic dynamics both in vitro and in planta, while Lbb was unaffected by the presence of Lmb. However, the drastic inhibition of Lmb growth by Lbb was ineffective in the case of delayed inoculation with Lbb or a lower amount of spores of Lbb compared to Lmb. Conclusions Our data suggest that Lmb growth inhibition by Lbb is the result of a combination of factors that may include competition for trophic resources, the generation by Lbb of an environment unsuitable for the lifecycle of Lmb or/and the effect on Lmb of plant defense responses induced by Lbb. It indicates that growth inhibition occurs in very specific conditions (i.e., co-inoculation at the same place of an equal amount of inoculum) that are unlikely to occur in the field where their coexistence does not prevent any species from completing their life cycle.

Blackleg disease, a major threat to Brassica crops worldwide, is primarily caused by the pathogen Leptosphaeria biglobosa. Investigating the genetic variation of L. biglobosa is crucial for managing and preventing the disease in Brassica napus. To date, there is scarce genomic variation information available for populations of L. biglobosa in China. In this study, 73 L. biglobosa strains of canola stalks were collected from 12 provinces in China and subjected to re-sequencing. The 73 assemblies averaged 1340 contigs, 72,123 bp N50, and 30.17 Mb in size. In total, 9409 core orthogroups and 867 accessory orthogroups were identified. A total of 727,724 mutation loci were identified, including 695,230 SNPs and 32,494 indels. Principal component analysis (PCA) and population structure analysis showed that these strains could be divided into seven subgroups. The strains in most provinces were clustered into a single subgroup, suggesting a strong influence of the geographic environment on strain variation. The average nucleotide diversity (θπ) of all strains was 1.03 × 10−3, indicating important genetic diversity among strains from different regions of China. This study provides valuable resources for future comparative genomics, gives new insights into the population evolution of L. biglobosa, and supports the development of strategies for managing blackleg disease in canola.

Background: Leptosphaeria maculans "brassicae" (Lmb) and Leptosphaeria biglobosa "brassicae" (Lbb) make up a species complex involved in the stem canker (blackleg) disease of rapeseed (Brassica napus). They coinfect rapeseed together, from the early stage of infection on leaves to the final necrotic stage at the stem base, and both perform sexual crossings on plant residues. L. biglobosa is suggested to be a potential biocontrol agent against Lmb, but there has been no mechanistic investigation of the different types of interactions that may occur between the plant and the two fungal species. Results: We investigated the bi- or tripartite interaction mechanisms by (i) confronting Lmb and Lbb in culture conditions or during cotyledon infection, with different timing and/or spore concentration regimes, (ii) performing RNA-Seq experiments in vitro or on the kinetics of infection of cotyledons infected by Lmb and/or Lbb to evaluate the transcriptomic activity and the plant response when both fungal species are inoculated together. Lbb infection of B. napus cotyledons was typical of a necrotrophic behavior, with a very early setup of one pathogenicity program and very limited colonization of tissues. This contrasted with the complex succession of pathogenicity programs of the hemibiotroph Lmb. During simultaneous co-infection by both species, Lmb was strongly impacted in its growth and transcriptomic dynamics both in vitro and in planta, while Lbb was unaffected by the presence of Lmb. However, the drastic inhibition of Lmb growth by Lbb was ineffective in the case of delayed inoculation with Lbb or a lower amount of spores of Lbb compared to Lmb. Conclusions: Our data suggest that Lmb growth inhibition by Lbb is the result of a combination of factors that may include competition for trophic resources, the generation by Lbb of an environment unsuitable for the lifecycle of Lmb or/and the effect on Lmb of plant defense responses induced by Lbb. It indicates that growth inhibition occurs in very specific conditions (i.e., co-inoculation at the same place of an equal amount of inoculum) that are unlikely to occur in the field where their coexistence does not prevent any species from completing their life cycle. [ABSTRACT FROM AUTHOR]

Phoma stem canker (blackleg), caused by the fungi Leptosphaeria maculans (anamorph Phoma lingam) and L. biglobosa, is one of the most devastating diseases in oilseed rape (Brassica napus L.) production worldwide. However, the population structure and genetic variation of L. biglobosa populations in China have rarely been investigated. Here, a collection of 214 fungal strains of blackleg from China (including Shaanxi, Jiangxi, Hubei, Jiangsu, Chongqing, Sichuan, Guangxi, Guizhou, Hunan, and Henan) and Europe (France and Ukraine) was identified as L. biglobosa. Three simple sequence repeat (SSR) markers were developed to characterize their population structure. The results showed that the Nei's average gene diversity ranged from 0.6771 for the population from Jiangsu to 0.3009 for the population from Hunan. In addition, most of the genetic variability (96%) occurred within groups and there were only relatively small amounts among groups (4.0%) (FST = 0.043, p = 0.042 < 0.05). Pairwise population differentiation (FST) suggested that significant genetic differentiation was observed between different L. biglobosa populations. Bayesian and unweighted average method analysis revealed that these L. biglobosa strains were clustered into three branches, and three European strains were similar to those from eastern China. The pathogenicity assay showed that those in Group III were significantly more virulent than those in Group I (t = 2.69, p = 0.016). The study also showed that Group III was dominant in Chinese L. biglobosa populations, which provides new insights for the further study of population evolution and the management of this pathogen. [ABSTRACT FROM AUTHOR]

Eight fungal strains (P1 to P8) were isolated from rapeseed stems (Brassica napus) infected with the blackleg pathogen Leptosphaeria biglobosa (Lb). They formed pycnidia with similar morphology to those of Lb, and thus were considered as Lb relatives (LbRs). The species-level identification of these strains was performed. Their virulence on rapeseed and efficacy in the suppression of Lb infection were determined, and the biocontrol potential and biocontrol mechanisms of strain P2 were investigated. The results showed that the LbRs belong to two teleomorphic genera in the family Didymellaceae, Didymella for P1 to P7 and Boeremia for P8. Pathogenicity tests on rapeseed cotyledons and stems indicated the LbRs were weakly virulent compared to L. biglobosa. Co-inoculation assays on rapeseed cotyledons demonstrated that P1 to P7 (especially P1 to P4) had a suppressive effect on Lb infection, whereas P8 had a marginal effect on infection by L. biglobosa. Moreover, D. macrostoma P2 displayed a more aggressive behavior than L. biglobosa in the endophytic colonization of healthy rapeseed cotyledons. Cultures of P2 in potato dextrose broth (PDB) and pycnidiospore mucilages exuded from P2 pycnidia showed antifungal activity to L. biglobosa. Further leaf assays revealed that antifungal metabolites (AM) of strain P2 from PDB cultures effectively suppressed infection by L. biglobosa, Botrytis cinerea (gray mold), and Sclerotinia sclerotiorum (white mold). An antifungal metabolite, namely penicillither, was purified and identified from PDB cultures and detected in pycnidiospore mucilages of strain P2. This study suggests that the LbRs are a repertoire for screening biocontrol agents (BCAs) against rapeseed diseases, and D. macrostoma P2 is a multi-functional BCA, a penicillither producer, and an endophyte.

Blackleg (Phoma) disease, caused by the ascomycete fungi Plenodomus biglobosus and P. lingam, threatens oilseed rape (OSR; Brassica napus) crops internationally. In many parts of the world, both species co-occur, but in China only P. biglobosus has so far been reported. Plenodomus biglobosus reproduces asexually (pycnidiospores), but also sexually (pseudothecia-yielding ascospores), via a heterothallic mating system requiring MAT1-1 and MAT1-2 genotypes. However, the roles of airborne ascospore inoculum in driving blackleg disease outbreaks in China are less well understood compared to elsewhere in the world. This is despite the very different agronomic cropping practices in parts of China, in which paddy rice and OSR are often grown in rotation; OSR stubble is often submerged under water for long periods potentially affecting pseudothecial development. Here, we indirectly investigate the potential role of sexual reproduction by developing new polymerase chain reaction (PCR) -based mating-type diagnostics for P. biglobosus and subsequently screening an international collection of 59 European and 157 Chinese isolates. Overall, in both Europe and China, P. biglobosus mating types did not deviate from a 1:1 ratio, such as is generally thought to occur under frequency-dependent selection in sexually reproducing pathogen populations. Both mating types were balanced in all the individual European countries tested (Austria, France, Poland, UK). Conversely, in China, mating types were only balanced in the eastern region; in the northern and southwestern regions there were skewed ratios, more typical of predominantly asexual reproduction, towards MAT1-1 and MAT1-2, respectively. The implications of these findings and future research directions for improved understanding of P. biglobosus epidemiology on OSR, particularly in China, are considered. [ABSTRACT FROM AUTHOR]

Phoma stem canker (blackleg), caused by the fungi Leptosphaeria maculans (anamorph Phoma lingam) and L. biglobosa, is one of the most devastating diseases in oilseed rape (Brassica napus L.) production worldwide. However, the population structure and genetic variation of L. biglobosa populations in China have rarely been investigated. Here, a collection of 214 fungal strains of blackleg from China (including Shaanxi, Jiangxi, Hubei, Jiangsu, Chongqing, Sichuan, Guangxi, Guizhou, Hunan, and Henan) and Europe (France and Ukraine) was identified as L. biglobosa. Three simple sequence repeat (SSR) markers were developed to characterize their population structure. The results showed that the Nei’s average gene diversity ranged from 0.6771 for the population from Jiangsu to 0.3009 for the population from Hunan. In addition, most of the genetic variability (96%) occurred within groups and there were only relatively small amounts among groups (4.0%) (FST = 0.043, p = 0.042 < 0.05). Pairwise population differentiation (FST) suggested that significant genetic differentiation was observed between different L. biglobosa populations. Bayesian and unweighted average method analysis revealed that these L. biglobosa strains were clustered into three branches, and three European strains were similar to those from eastern China. The pathogenicity assay showed that those in Group III were significantly more virulent than those in Group I (t = 2.69, p = 0.016). The study also showed that Group III was dominant in Chinese L. biglobosa populations, which provides new insights for the further study of population evolution and the management of this pathogen.

Blackleg, caused by Leptosphaeria maculans, and L. biglobosa, is a major disease that hinders canola (Brassica napus) production worldwide. In this study, 51 Leptosphaeria isolates, and previously deposited sequences were examined for genetic diversity using ITS, β-tubulin, tef and act gene regions. Phylogenetic analyses showed that the isolates fell within two sub-species of brassicae and canadensis. Out of 51 Iranian isolates, 55%, 37%, and 8% were found as L. biglobosa canadensis, L. biglobosa brassicae, and L. maculans, respectively. Accordingly, the canadensis sub-species was found to be the predominant sub-species in Iran. There was no difference between Iranian and foreign brassicae sub-species in terms of ITS nucleotide sequences. This paper confirms the presence of brassicae and canadensis sub-species in Iran and describes the distribution of L. biglobosa in the country. Moreover, Leptosphaeria species were distinguished using a phylogenetic tree based on the tef region. [ABSTRACT FROM AUTHOR]

Leptosphaeria biglobosa is a less virulent Leptosphaeria spp. that causes blackleg disease in canola. Previous studies from our lab have shown that inoculation with the less virulent L. biglobosa can boost the resistance of canola plants against the highly virulent L. maculans. The objective of this study was to confirm the effectiveness of L. biglobosa as a biocontrol agent against L. maculans utilizing morphology, fluorescence microscopy, gene quantification, and transcriptomic analysis. The in planta development of two Leptosphaeria species inoculated at different time points was assessed using fluorescent protein-tagged isolates which are GFP-tagged L. maculans and DsRed-tagged L. biglobosa. The growth inhibition of L. maculans by pre-and co-inoculated L. biglobosa was supported by no lesion development on cotyledons and no or weak fluorescence protein-tagged mycelia under the confocal microscope. The host defense-related genes, WRKY33, PR1, APX6, and CHI, were upregulated in L. biglobosa inoculated Westar cotyledons compared to L. maculans inoculated cotyledons. The quantification of each pathogen through qPCR assay and gene expressions analysis on host defense-related genes by RT-qPCR confirmed the potential of L. biglobosa "brassicae' in the management of the blackleg disease pathogen, L. maculans 'brassicae', in canola. [ABSTRACT FROM AUTHOR]

Blackleg (Phoma) disease, caused by the ascomycete fungi Plenodomus biglobosus and P. lingam, threatens oilseed rape (OSR; Brassica napus) crops internationally. In many parts of the world, both species co-occur, but in China only P. biglobosus has so far been reported. Plenodomus biglobosus reproduces asexually (pycnidiospores), but also sexually (pseudothecia-yielding ascospores), via a heterothallic mating system requiring MAT1-1 and MAT1-2 genotypes. However, the roles of airborne ascospore inoculum in driving blackleg disease outbreaks in China are less well understood compared to elsewhere in the world. This is despite the very different agronomic cropping practices in parts of China, in which paddy rice and OSR are often grown in rotation; OSR stubble is often submerged under water for long periods potentially affecting pseudothecial development. Here, we indirectly investigate the potential role of sexual reproduction by developing new polymerase chain reaction (PCR) -based mating-type diagnostics for P. biglobosus and subsequently screening an international collection of 59 European and 157 Chinese isolates. Overall, in both Europe and China, P. biglobosus mating types did not deviate from a 1:1 ratio, such as is generally thought to occur under frequency-dependent selection in sexually reproducing pathogen populations. Both mating types were balanced in all the individual European countries tested (Austria, France, Poland, UK). Conversely, in China, mating types were only balanced in the eastern region; in the northern and southwestern regions there were skewed ratios, more typical of predominantly asexual reproduction, towards MAT1-1 and MAT1-2, respectively. The implications of these findings and future research directions for improved understanding of P. biglobosus epidemiology on OSR, particularly in China, are considered.

Copyright of Chinese Journal of Oil Crop Sciences is the property of Oil Crops Research Institute of Chinese Academy of Agricultural Sciences and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Chinese Journal of Oil Crop Sciences is the property of Oil Crops Research Institute of Chinese Academy of Agricultural Sciences and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Accurate diagnosis of the differentially aggressive fungi Leptosphaeria maculans 'brassicae' and L. biglobosa 'brassicae' causing blackleg disease in Brassica crops is crucial. Prior to the availability of fungal whole‐genome sequences, gene‐specific markers were designed on specific genes. But, the availability of the whole‐genome sequences of several isolates now offers a greater opportunity to develop more precise and novel markers. The draft genomes of L. maculans and L. biglobosa that are available in the repository were aligned, and species‐specific primers were designed from the variable sequences that encode for effector proteins. Three primer sets namely, Lm1, Lm2 and Lm5 and two primer sets namely Lb1' and Lb3 specifically detected the isolates of target species in PCR assay. The primers Lm5 and Lb1' were multiplexed for detection of L. maculans 'brassicae' and L. biglobosa 'brassicae', respectively, from a single PCR reaction. The markers were highly sensitive in amplifying target species from crude 'pseudothecia and ascospore suspension' without requiring DNA extraction. These markers, solitarily or in combination, could be used for precise, sensitive and rapid detection of L. maculans 'brassicae' and L. biglobosa 'brassicae' species and will be helpful for surveillance, management and transboundary quarantine of the devastating blackleg disease. [ABSTRACT FROM AUTHOR]

Copyright of Chinese Journal of Oil Crop Sciences is the property of Oil Crops Research Institute of Chinese Academy of Agricultural Sciences and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Leptosphaeria biglobosa is a less virulent Leptosphaeria spp. that causes blackleg disease in canola. Previous studies from our lab have shown that inoculation with the less virulent L. biglobosa can boost the resistance of canola plants against the highly virulent L. maculans. The objective of this study was to confirm the effectiveness of L. biglobosa as a biocontrol agent against L. maculans utilizing morphology, fluorescence microscopy, gene quantification, and transcriptomic analysis. The in planta development of two Leptosphaeria species inoculated at different time points was assessed using fluorescent protein-tagged isolates which are GFP-tagged L. maculans and DsRed-tagged L. biglobosa. The growth inhibition of L. maculans by pre-and co-inoculated L. biglobosa was supported by no lesion development on cotyledons and no or weak fluorescence protein-tagged mycelia under the confocal microscope. The host defense-related genes, WRKY33, PR1, APX6, and CHI, were upregulated in L. biglobosa inoculated Westar cotyledons compared to L. maculans inoculated cotyledons. The quantification of each pathogen through qPCR assay and gene expressions analysis on host defense-related genes by RT-qPCR confirmed the potential of L. biglobosa “brassicae’ in the management of the blackleg disease pathogen, L. maculans ‘brassicae’, in canola.

Blackleg is a devastating disease of winter oil seed rape (WOSR) caused by the two species Leptosphaeria maculans and L. biglobosa "brassicae". In this study, Loop-Mediated Isothermal Amplification (LAMP) assays were developed using two primer sets, SirP and PKS5, to identify and monitor L. maculans "brassicae", and one primer set PKS21 to monitor L. biglobosa "brassicae". The three primer sets were tested in real-time LAMP with DNA templates from 38 Leptosphaeria spp. reference isolates. The SirP and PKS5 primer sets exclusively amplified DNA from L. maculans isolates whereas, the PKS21 primers amplified DNA solely from L. biglobosa. Further specificity tests with DNA templates from other ascomycetes showed that both PKS primers were highly specific to Leptosphaeria. The SirP primer set produced in a few cases non-specific amplifications, but they were easily differentiated from the positive reactions by the differences in melting curve temperatures. The detection limits of LAMP assays with PKS21, PKS5 and SirP primers were at 100 fg, 30 pg and 180 fg DNA, respectively. DNA from leaf segments with typical L. maculans lesions was successfully amplified using primer set SirP, as was visually indicated by the colour change of phenol red from pink to yellow. This feature makes the method suitable for simple screening of L. maculans in WOSR leaves and in spores collected by spore traps. [ABSTRACT FROM AUTHOR]

During 2008-2010 the infected Brassica napus plant material was collected from nine localities: Karavukovo, Crvenka, Prigrevica, Subotica, Rimski Šančevi, Srbobran, Beška, Banatsko Karađorđevo, and Srpski Miletić. Infected tissue samples were taken from the root of both upper and basal stems, as well as from the leaf, flower and pods. Two reference isolates from Great Britain were used in these studies for the analysis of 119 isolates from Serbia, using polymerase chain reaction (PCR) and PCR-RFLP. Digestion of PCR products was performed with 5 selected endonucleases: BamHI, HaeIII, Rsal, EcoRII, and AluI. On the basis of PCR analysis, all isolates originating from Serbia belong to L. complex: 111 belong to species Leptosphaeria maculans, and 8 to species Leptosphaeria biglobosa NA1 ('Leptosphaeria biglobosa brassicae), respectively.

Two probe-based qPCR systems, namely P-Lb and P-Lm, specific to the canola blackleg pathogens Leptosphaeria biglobosa and L. maculans , respectively, were developed, and their efficiencies were tested. Each of the two systems targets a single-copy gene exclusively present in the corresponding species. The specificities of the two systems on the species level and their ubiquities on the subspecies level were confirmed by in silico sequence analyses and testing on L. biglobosa (17 strains), L. maculans (10 strains), and other plant pathogens (31 species). For sensitivities, the two systems were tested on synthesized DNA fragments (gBlock) of the targeted regions, from which a standard curve was generated for each system. In addition, standard curves were also generated on gBlocks for duplex qPCR in which the two systems were used in the same reaction. The two systems were further tested in both singleplex and duplex qPCR on DNA samples extracted from fungal spores, inoculated canola cotyledons, and naturally infected canola stubble samples collected from commercial fields. Our data indicated that the two systems are specific to L. biglobosa and L. maculans , respectively, and one reaction could detect as few as 200 spores of either species. When used in duplex qPCR on DNA samples with various origins, the two systems generated similar results as in singleplex qPCR. The duplex qPCR system, along with the sample preparation and DNA extraction specified in this study, constituted a first-reported duplex qPCR protocol for detection and quantification of the two blackleg pathogens from field samples., Competing Interests: The author(s) declare no conflict of interest.

Copyright of Chinese Journal of Oil Crop Sciences is the property of Oil Crops Research Institute of Chinese Academy of Agricultural Sciences and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Stem canker of brassicas, also known as blackleg is the most damaging disease of many Brassicaceae. The disease is caused by Leptosphaeria maculans (Desm.) Ces et de Not. and L. biglobosa sp. nov., Shoemaker & Brun, which coexist in plants and resulting in disease symptoms and decreased yield, quantity and quality of cultivated vegetables and oilseed rape. The paper presents taxonomic relationships between these coexisting pathogen species, describes particular stages of their life cycles, summarizes the differences between the species, and reviews methods for their identification.

Stem canker of brassicas is a severe disease of oilseed rape in Australia, Canada and Europe, including Poland. The disease is caused by Leptosphaeria maculans and L. biglobosa - two pathogens belonging to the class Dothideomycetes. The species differ in pathogenicity, but they have identical shape and size of fruiting bodies and spores of the generative and vegetative stages. Both pathogens are often found together in infected tissues of oilseed rape plants. The main goal of the experiments was to measure the rate of pseudothecial maturation and to monitor ascospore concentration of L. maculans and L. biglobosa in air samples. The paper is the first investigation on the generative stage development of these two species in south-east Poland. The studies were done for three consecutive years (2005-2007), for six most important months in pathogen development and plant infection, including 3 months in the spring (March - May) and 3 months in the autumn (September - November). The stage of pseudothecial maturation was assessed visually, based on the development of asci and ascospores. Monitoring of spore concentration in the air was performed using a Hirst-type 7-day volumetric trap. It was proved that differences in pseudothecial maturation rate in south-east Poland, encompassing the climatic regions of the Carpathian Foothills and Cracow, do not exceed two weeks within one season. The first and the highest ascospore concentration dates depended on weather conditions in a particular season. The total number of spores during the studied seasons varied from 9 to 12 spores/m3, which was from 70 to 90 times lower than the average from five other monitoring sites around Poland. The short exposition to spore showers and very small concentrations of L. maculans and L. biglobosa ascospores in air samples were the most probable reasons for relatively small damage of oilseed rape crops by stem canker in the south-east part of Poland.

Recent advances in molecular detection of living organisms facilitate the introduction of novel methods to studies of the transport of fungal spores over large distances. Monitoring the migration of airborne fungi using microscope based spore identification is limited when different species produce very similar spores. In our study, DNA-based monitoring with the use of species-specific probes allowed us to track the aerial movements of two important fungal pathogens of oilseed rape ( Brassica napus L.), i.e. , Leptosphaeria maculans and Leptosphaeria biglobosa , which have identical spore shape and size. The fungi were identified using dual-labelled fluorescent probes that were targeted to a β -tubulin gene fragment of either Leptosphaeria species. Spore identification by Real-Time PCR techniques capable of detecting minute amounts of DNA of selected fungal species was combined with back-trajectory analysis, allowing the tracking of past movements of air masses using the Hybrid Single Particle Lagrangian Integrated Trajectory model. Over a study period spanning the previous decade (2006–2015) we investigated two specific events relating to the long distance transport of Leptosphaeria spp. spores to Szczecin in North-West Poland. Based on the above mentioned methods and the results obtained with the additional spore sampler located in nearby Szczecin, and operating at the ground level in an oilseed rape field, we have demonstrated that on both occasions the L. biglobosa spores originated from the Jutland Peninsula . This is the first successful attempt to combine analysis of back-trajectories of air masses with DNA-based identification of economically important pathogens of oilseed rape in Europe. In our studies, the timing of L. biglobosa ascospore dispersal in the air was unlikely to result in the infection of winter oilseed rape grown as a crop plant. However, the fungus could infect other host plants, such as vegetable brassicas, cruciferous weeds, spring rapeseed and winter rapeseed growing as a volunteer plant. [ABSTRACT FROM AUTHOR]

Copyright of Progress in Plant Protection is the property of Institute of Plant Protection and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

In October 2010 the occurrence of the stem canker symptoms in rapeseed was observed at the locality Rimski Šančevi in Serbia. Several strains of fungi were isolated from the stem. Morphological characteristics of the isolates were studied on a PDA medium at 25±1°C: growth rate, colour, shape and appearance of colonies and the edge of the colony, the pigment, size, colour and shape of pycnidiospores and pycnidia in 10 isolates (isolated in Serbia K-111, K-112, K-113, K-114, K-115, K-116, K-117, K-118, K-119, and K-120). All tested strains had fast growth, the regular form of colonies and poor sporulation on nutrient medium. Pycnidiospores are unicellular, hyaline, and mostly straight, with or without a drop of oil. Molecular identification was performed by the application the PCR technique using primers PN3/PN10. In addition to these 10 isolates, two reference strains obtained from the Centre for Agricultural Studies, Rothamsted, UK, which are marked with L. m (Leptosphaeria maculans), L. b (Leptosphaeria biglobosa) and 7 reference isolates originating from Serbia, which are marked K-7, St-16, GS-25, L-5, C-3, LJ-2, S-1 were used. On the basis of DNA amplification with primers PN3 and PN10 tape length was about 580 bp for isolates (L.b, K- 111, K-112, K-113, K-114, K-115, K-116, K-117, K-118, K-119, K-120) while that was 560 bp for other group of isolates (L.m, K-7, St-16, GS-25, L-5, C-3, LJ- 2, S-1).This study showed that 10 isolates (K-111, K-112, K-113, K- 114, K-115, K-116, K-117, K-118, K-119, K-120) isolated from the stem of rapeseed belong to the species Leptosphaeria biglobosa. [ABSTRACT FROM AUTHOR]

Juncea canola ( Brassica juncea L.) is being developed throughout the worlds canola growing countries as a drought tolerant, shatter resistant and highly blackleg resistant option to canola ( Brassica napus L.). Juncea canola was grown commercially in Australia for the first time in 2007. This study determined the incidence and severity of blackleg infection in juncea canola prior to commercial release throughout south-eastern Australia in 2006 and 2007, and then again 5 years after commercialisation (2010-2013) to determine if blackleg severity had increased. Blackleg was found at all 127 sites surveyed throughout Victoria, New South Wales, South Australia and Western Australia. The severity of blackleg infection differed among sites and among the juncea canola cultivars and breeding lines suggesting that differences in resistance may be present. This is the first report that L. maculans isolates virulent on B. juncea are already widespread throughout the Australian canola growing regions and contradicts the widespread opinion that B. juncea is immune to blackleg. This also demonstrates that blackleg infection was already occurring in juncea canola prior to commercialisation of this crop in Australia and that disease management strategies similar to those used in canola cultivation will need to be implemented. [ABSTRACT FROM AUTHOR]

Blackleg (Phoma) disease, caused by the ascomycete fungi Plenodomus biglobosus and P. lingam , threatens oilseed rape (OSR; Brassica napus ) crops internationally. In many parts of the world, both species co-occur, but in China only P. biglobosus has so far been reported. Plenodomus biglobosus reproduces asexually (pycnidiospores), but also sexually (pseudothecia-yielding ascospores), via a heterothallic mating system requiring MAT1-1 and MAT1-2 genotypes. However, the roles of airborne ascospore inoculum in driving blackleg disease outbreaks in China are less well understood compared to elsewhere in the world. This is despite the very different agronomic cropping practices in parts of China, in which paddy rice and OSR are often grown in rotation; OSR stubble is often submerged under water for long periods potentially affecting pseudothecial development. Here, we indirectly investigate the potential role of sexual reproduction by developing new polymerase chain reaction (PCR) -based mating-type diagnostics for P. biglobosus and subsequently screening an international collection of 59 European and 157 Chinese isolates. Overall, in both Europe and China, P. biglobosus mating types did not deviate from a 1:1 ratio, such as is generally thought to occur under frequency-dependent selection in sexually reproducing pathogen populations. Both mating types were balanced in all the individual European countries tested (Austria, France, Poland, UK). Conversely, in China, mating types were only balanced in the eastern region; in the northern and southwestern regions there were skewed ratios, more typical of predominantly asexual reproduction, towards MAT1-1 and MAT1-2 , respectively. The implications of these findings and future research directions for improved understanding of P. biglobosus epidemiology on OSR, particularly in China, are considered.

Blackleg, caused by Leptosphaeria maculans, is a major threat to canola production in Canada. With the exception of China, L. maculans is present in areas around the world where cruciferous crops are grown. The pathogen can cause trade barriers in international canola seed export due to its potential risk as a seed contaminant. The most recent example is China restricting canola seeds imported from Canada and Australia in 2009. Therefore, it is important to assess the level of Blackleg infection in Canadian canola seed lots and dockage (seeds and admixture). In this study, canola seed lots and dockage samples collected from Western Canada were tested for the presence of the aggressive L. maculans and the less aggressive L. biglobosa. Results showed that both L. maculans and L. biglobosa were present in seed lots and dockage samples, with L. biglobosa being predominant in infected seeds. Admixture separated from dockage had higher levels of L. maculans and L. biglobosa infection than samples from seed lots. Admixture appears to harbour higher levels of L. maculans infection compared to seeds and is more likely to be a major source of inoculum for the spread of the disease than infected seeds.

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]

When airborne propagules of Leptosphaeria maculans and L. biglobosa were collected in Poland at three ecologically different sites from 1 September to 30 November in 2004 to 2008, using a Hirst-type seven-day volumetric spore trap, there were fluctuations in timing of ascospore release and diverse ratios between airborne propagules of both species depending on season, field location and weather conditions. The detection was done using the microscope as well as quantitative PCR with species-specific primers targeted against fragments of β- tubulin genes and quantified with a dual-labelled fluorescent probe approach. This detection chemistry is described for the first time for L. maculans and L. biglobosa. Its advantage over the previous ITS-based SYBR-Green chemistry resides in improved sensitivity and the virtual absence of false positives in the detection of these fungi. There were significant, positive correlations between data obtained using visual assessment of ascospore numbers and DNA concentrations that were measured by qPCR. Climatic differences between the oilseed rape growing regions could have significantly affected the biological processes of pseudothecial maturation and ascospore development of the pathogens. The data suggest that regular rain events of intermediate intensity recorded in the Maritime region favoured the maturation of the pathogen more than the drier weather recorded in the Silesia or Pomerania regions. It was observed that the number of rainy days was of greater importance than the cumulative rainfall to obtain the generative sporulation of the pathogen. Accurate detection of airborne inoculum of pathogenic Leptosphaeria spp. facilitates improved targeting of disease management decisions for oilseed rape protection against phoma stem canker and stem necrosis diseases. [ABSTRACT FROM AUTHOR]

Blackleg (causal agents Leptosphaeria maculans and L. biglobosa) is the most significant disease of canola (Brassica napus) worldwide. This was the first survey of Leptosphaeria isolates recovered from Brassica juncea stubble in Australia. Sixty-four L. maculans isolates and 88 L. biglobosa 'canadensis' isolates were collected from B. juncea stubble over two years (2005-2006). These isolates were screened over three canola quality B. juncea and three B. napus differentials. As expected, L. biglobosa 'canadensis' isolates caused lower disease severity than the L. maculans isolates. However, 13 L. biglobosa 'canadensis' isolates were capable of causing ≥50% mean internal infection on at least one of the six differentials. Although these isolates were recovered from B. juncea stubble, disease severity was low when canola quality B. juncea differentials were inoculated. The same differential lines were also screened over two field sites by placing pots containing each of the differential lines onto B. napus and B. juncea stubble from commercial fields. At one site, inoculum in B. napus stubble caused higher internal infection severity than the B. juncea stubble in all differentials except B. napus differential 'ATR-Beacon'. At the other site, however, inoculum from B. juncea stubble produced more internal infection in one B. juncea differential in 2007 and in four differentials in 2008. This investigation suggests that when B. juncea is widely grown, increased blackleg symptoms could occur and that L. biglobosa 'canadensis' isolates can contribute to disease severity. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Plant Protection Research is the property of Institute of Plant Protection and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The article cites a study describing the application of pathogenicity testing in the evaluation of virulence variability in L. maculans populations to control blackleg disease in canola. The researchers made use of samples that were collected from North America, Canada, and North Dakota from 2002-2004. Results indicated that new strains have emerged and is causing a significant threat to the canola industry in Canada and the U.S.