Search Results (17)

Walnut canker is a common disease in the Xinjiang Uygur autonomous region of China, which is caused by Cytospora chrysosperma. To date, there is no effective control measure for this disease. Infection with mycoviruses has been widely proven to reduce the virulence of plant pathogenic fungi, with some mycoviruses even serving as potential biological control agents for plant diseases. In this study, mycoviruses associated with 31 strains of C. chrysosperma from Xinjiang Uygur autonomous region were identified by metatranscriptomic sequencing. Seven new mycoviruses were identified by BLAST and RT-PCR analysis, which were Botrytis cinerea partitivirus 5 (BcPV5), Gammapartitivirus sp-XJ1 (GVsp-XJ1), Botoulivirus sp-XJ2 (BVsp-XJ2), Luoyang Fusar tick virus 2 (LfTV2), Leptosphaeria biglobosa narnavirus 17 (LbNV17), Sclerotinia sclerotiorum narnavirus 6 (SsNV6), and Cytospora ribis mitovirus (CrMV3). Among these, BcPV5, GVsp-XJ1, BVsp-XJ2, CrMV3, and LfTV2 were found to co-infect C. chrysosperma strain WS-11 and significantly reduce both the colony growth rate and virulence of the host. After co-culturing the virus-free WS-FV strain with WS-11, the colony growth rate and virulence of the derivative strain were also decreased. These results provide potential biocontrol resources for the control of walnut canker. Full article

Alternative splicing (AS) is a prevalent post-transcriptional regulatory mechanism in eukaryotes and plays a crucial role in plant disease resistance. Here, we used the Illumina Novaseq sequencing platform to conduct transcriptome sequencing on canola (Brassica napus) leaves infected with the blackleg pathogen (Leptosphaeria biglobosa strain nm⁻¹) at 0 h, 72 h, 120 h, and 168 h post-inoculation to investigate the mechanism of AS coordination with transcriptional regulation in canola’s response to blackleg disease. The rMATS software (4.1.0) was employed to analyze different AS events in samples taken at 72 h, 120 h, and 168 h. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to elucidate the biological functions of differentially spliced genes at various time points, while Weighted Gene Co-expression Network Analysis (WGCNA) was used to identify key modules and hub genes. As a result, our analysis reveals 16908 AS events across three time points, with 221 being differently spliced. Intron retention (RI) was the most common AS event, accounting for approximately 55% of all events, while alternative 5′ splice site events were least common, comprising only 2%. Furthermore, a total of 213 significantly differentially spliced genes were identified, which were enriched in functions related to protein kinase activity, transferase activity, and pathways such as MAPK signaling pathway—plant and plant hormone signal transduction. WGCNA identified three key modules and ten hub genes, including calcium-binding transcription activator 1, LRR class receptor serine/threonine protein kinase FEI 2, PLATZ transcription factor family proteins, serine/threonine protein kinase PRP4, and E3 ubiquitin ligase SUD1, all of which are associated with canola resistance to L. biglobosa. Thus, this study provides a theoretical basis for identifying disease-resistance genes involved in AS and for exploring the functions of AS gene isoforms in canola. Full article

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. Full article

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. Full article

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. Full article

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%) (F_ST = 0.043, p = 0.042 < 0.05). Pairwise population differentiation (F_ST) 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. Full article

Mycoviruses widely exist in filamentous fungi and sometimes cause phenotypic changes in hosts. Trichoderma harzianum hypovirus 1 (ThHV1) and its defective RNA ThHV1-S were found in T. harzianum and exhibited high transmissibility. In our previous study, ThHV1 and ThHV1-S were transferred to an excellent biological control agent T. koningiopsis T-51 to form a derivative strain 51-13. In this study, we assessed the metabolic changes in strain 51-13 and antifungal activity of its culture filtrate (CF) and volatile organic compounds (VOCs). The antifungal activity of CF and VOCs of T-51 and 51-13 was different. Compared with the CF of T-51, that of 51-13 exhibited high inhibitory activity against B. cinerea, Sclerotinia sclerotiorum, and Stagonosporopsis cucurbitacearum but low inhibitory activity against Leptosphaeria biglobosa and Villosiclava virens. The VOCs of 51-13 exhibited high inhibitory activity against F. oxysporum but low inhibitory activity against B. cinerea. The transcriptomes of T-51 and 51-13 were compared; 5531 differentially expressed genes (DEGs) were identified in 51-13 with 2904 up- and 2627 downregulated genes. In KEGG enrichment analysis, 1127 DEGs related to metabolic pathways (57.53%) and 396 DEGs related to biosynthesis of secondary metabolites (20.21%) were clearly enriched. From the CF of T-51 and 51-13, 134 differential secondary metabolites (DSMs) were detected between T-51 and 51-13 with 39 up- and 95 downregulated metabolites. From these, 13 upregulated metabolites were selected to test their antifungal activity against B. cinerea. Among them, indole-3-lactic acid and p-coumaric acid methyl ester (MeCA) exhibited strong antifungal activity. The IC₅₀ of MeCA was 657.35 μM and four genes possibly related to the synthesis of MeCA exhibited higher expression in 51-13 than in T-51. This study revealed the mechanism underlying the increase in antifungal activity of T-51 because of the mycovirus and provided novel insights in fungal engineering to obtain bioactive metabolites via mycoviruses. Full article

A fungal strain (JC-1) of Simplicillium was isolated from a pod of oilseed rape (Brassica napus) infested with the blackleg pathogen Leptosphaeria biglobosa. This study was done to clarify its taxonomic identity using morphological and molecular approaches, to characterize its antifungal activity through bioassays and genome-based identification of antifungal metabolites, and to determine its efficacy in inducing systemic resistance (ISR) in oilseed rape. The results showed that JC-1 belongs to Simplicillium lamellicola. It displayed a strong antagonistic relationship with L. biglobosa, Botrytis cinerea (gray mold) and Sclerotinia sclerotiorum (stem rot). The cultural filtrates of JC-1 showed a high efficacy in suppressing infection by S. sclerotiorum on detached leaves of oilseed rape. Genome analysis indicated that JC-1 has the capability of producing multiple antifungal metabolites, including aureobasidin A1, squalestatin S1 and verlamelin. Inoculation of JC-1 on seeds of oilseed rape caused a suppressive effect on infection by L. biglobosa on the cotyledons of the resulting seedlings, suggesting that JC-1 can trigger ISR. Endophytic growth, accumulation of anthocyanins, up-regulated expression of CHI (for chalcone isomerase) and PR1 (for pathogenesis-related protein 1), and down-regulated expression of NECD3 (for 9-cis-epoxycarotenoid dioxygenase) were detected to be associated with the ISR. This study provided new insights into the biocontrol potential and modes of action of S. lamellicola. Full article

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. Full article

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. Full article

Winter oilseed rape (Brassica napus L.) is the main source of domestic oil in central and northern Europe, bringing profits to farmers, but the plants are often damaged by stem canker, caused by two fungal species belonging to the genus Leptosphaeria. Due to environmental concerns, the benefits of fungicide applications must outweigh disadvantages. The aim of this work was to determine the effect of stem canker on seed yield and its quality and find out the best timing of fungicide application. The multi-year field experiments were done at two sites in south-west Poland, where the disease is regarded as a serious problem. The fungicide treatments with the azole-containing preparation followed the same scheme each year; a single application was made at one-week intervals, starting in late September through mid-November for a total of eight treatments. Seed yield, oil and protein content, mass of thousand seeds as well as indole-and alkenyl-glucosinolate contents in seeds were statistically unrelated with the incidence and severity of phoma leaf spotting and stem canker symptoms. The significant decrease of the seed yield was observed in three (site × year combinations) of eight, in which phoma leaf spotting and stem canker were severe. Yield loss was noted only in years with warm and wet autumns, when cumulative mean temperatures between BBCH14 and BBCH19 plant growth stages exceeded 60 °C and precipitation in this period exceeded 110 mm of rain. Under these conditions, fungicide treatments were highly effective when they were done between BBCH15–BBC16 growth stages (5–6 true leaves). Full article

Background: Temperate agroforestry is regarded as a sustainable alternative to monoculture agriculture due to enhanced provisioning of ecosystem services. Plant health and food safety are crucial requirements for sustainable agriculture; however, studies of fungal diseases and mycotoxin contamination of crops grown under temperate agroforestry are lacking. This study therefore aimed to compare fungal colonization and mycotoxin contamination of crops grown in temperate agroforestry against conventional monoculture. Methods: The biomass of plant pathogenic fungi in oilseed rape plants and barley and wheat grain harvested in 2016 to 2018 at four paired agroforestry and monoculture sites was quantified using species-specific real-time PCR. Mycotoxin content of barley and wheat grain was determined by HPLC-MS/MS. Results: The colonization of oilseed rape plants with the vascular pathogen Verticillium longisporum and wheat grain with the head blight pathogen Fusarium tricinctum was lower in agroforestry than in conventional monoculture. Mycotoxin content of barley and wheat grain did not differ between agroforestry and monoculture systems and did not exceed the legal limits of the EU. Remarkably, fumonisin B₁ was detected in wheat grains at two sites in two years, yet the low levels found do not raise food safety concerns. No differences were found between the two production systems with regard to infection of wheat and barley grain with five Fusarium species (F. avenaceum, F. culmorum, F. graminearum, F. poae, and F. proliferatum) and oilseed rape with fungal pathogens Leptosphaeria biglobosa, Leptosphaeria maculans, and Sclerotinia sclerotiorum. Conclusions: Temperate agroforestry does not negatively affect the infection of wheat, barley and oilseed rape with major fungal pathogens though it may suppress the infection of oilseed rape with V. longisporum and wheat grain with F. tricinctum. Furthermore, temperate agroforestry does not increase mycotoxin contamination of barley and wheat. Therefore, temperate agroforestry does not negatively affect food safety. Full article

Blackleg (Phoma stem canker) of crucifers is a globally important disease caused by the ascomycete species complex comprising of Leptosphaeria maculans and Leptosphaeria biglobosa. Six blackleg isolates recovered from Brassica rapa cv. Mizspoona in the Willamette Valley of Oregon were characterized as L. biglobosa based on standard pathogenicity tests and molecular phylogenetic analysis. These isolates were compared to 88 characterized L. biglobosa isolates from western Canada, 22 isolates from Australia, and 6 L. maculans isolates from Idaho, USA using maximum parsimony and distance analysis of phylogenetic trees generated from the ITS rDNA (internal transcribed spacer rDNA) sequence, and the actin and β-tubulin gene sequences. The L. biglobosa isolates derived from B. rapa collected in Oregon formed a separate subclade based on concatenated gene sequences or a single gene sequence, regardless of the analyses. Pathogenicity tests showed that these isolates failed to infect either resistant or susceptible B. napus cultivars, but caused severe symptoms on three B. rapa cultivars (Accession number: UM1113, UM1112, and UM1161), a B. oleracea var. capitata (cabbage) cultivar (Copenhagen Market), and two B. juncea cultivars (CBM, a common brown Mustard, and Forge). These findings demonstrated that the L. biglobosa isolates derived from a B. rapa crop in Oregon were genetically distinct from existing species of L. biglobosa, and constitute a new subclade, herein proposed as L. biglobosa ‘americensis’. Full article

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. Full article

Here we report the molecular characterisation of a novel dsRNA virus isolated from the filamentous, plant pathogenic fungus Leptosphaeria biglobosa and known to cause significant alterations to fungal pigmentation and growth and to result in hypervirulence, as illustrated by comparisons between virus-infected and -cured isogenic fungal strains. The virus forms isometric particles approximately 40–45 nm in diameter and has a quadripartite dsRNA genome structure with size ranges of 4.9 to 4 kbp, each possessing a single ORF. Sequence analysis of the putative proteins encoded by dsRNAs 1–4, termed P1–P4, respectively, revealed modest similarities to the amino acid sequences of equivalent proteins predicted from the nucleotide sequences of known and suspected members of the family Quadriviridae and for that reason the virus was nominated Leptosphaeria biglobosa quadrivirus-1 (LbQV-1). Sequence and phylogenetic analysis using the P3 sequence, which encodes an RdRP, revealed that LbQV-1 was most closely related to known and suspected quadriviruses and monopartite totiviruses rather than other quadripartite mycoviruses including chrysoviruses and alternaviruses. Of the remaining encoded proteins, LbQV-1 P2 and P4 are structural proteins but the function of P1 is unknown. We propose that LbQV-1 is a novel member of the family Quadriviridae. Full article