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The short-wave and highly energetic ultraviolet‑C (UV-C) radiation has a disinfectant effect on various microorganisms. It is also known that UV‑C radiation can have an effect on stimulating plant defense. In this study, we used the phytopathogen Phoma lingam as a model organism to examine UV‑C at a wavelength of 254 nm application as an alternative to control this pathogen in Brassica napus. The aim of the study was to determine direct effects on the pathogen and indirect effects of stimulating the plant defense. Mycelia of P. lingam were grown in vitro and treated with different doses of UV‑C (0.015–1.57 kJ/m2). Mycelia diameters were then measured 1, 2, and 7 days after UV‑C treatment. In the in planta tests, cotyledons were injured by a needle and inoculated with 106 spore suspension of P. lingam. To determine the direct effects the oilseed rape seedlings were treated with different UV‑C doses (0.2–1.5 kJ/m2) 1, 3, or 7 days after inoculation with P. lingam. To investigate the indirect effects the UV‑C treatments (0.2–1.5 kJ/m2) were applied 1, 3, or 7 days before inoculation with P. lingam. The in vitro experiments showed a significant reduction in mycelia growth on agar plates one and two days after UV‑C treatment. However, the fungal growth recovered; after 7 days no significant differences were detectable. The in planta results showed an effect on disease severity affected by the application time of UV‑C treatment and the applied UV‑C dose. UV‑C application 1 day before inoculation significantly increased the disease severity. Whereas, UV‑C treatment 7 days before inoculation with a UV‑C dose of 0.8 kJ/m2 reduced the disease severity by 44%. If the UV‑C application was done after inoculation, the disease severity could be reduced by approximately 68% at a dose of 0.8 kJ/m2 1 day after inoculation. The results of the study show, that UV‑C treatment can stimulate plant defense and damage the pathogen directly.

Extending the durability of plant resistance genes towards fungal pathogens is a major challenge. We identified and investigated the relationship between two avirulence genes of Leptosphaeria maculans, AvrLm3 and AvrLm4‐7. When an isolate possesses both genes, the Rlm3‐mediated resistance of oilseed rape (Brassica napus) is not expressed due to the presence of AvrLm4‐7 but virulent isolates toward Rlm7 recover the AvrLm3 phenotype.Combining genetic and genomic approaches (genetic mapping, RNA‐seq, BAC (bacterial artificial chromosome) clone sequencing and de novo assembly) we cloned AvrLm3, a telomeric avirulence gene of L. maculans. AvrLm3 is located in a gap of the L. maculans reference genome assembly, is surrounded by repeated elements, encodes for a small secreted cysteine‐rich protein and is highly expressed at early infection stages.Complementation and silencing assays validated the masking effect of AvrLm4‐7 on AvrLm3 recognition by Rlm3 and we showed that the presence of AvrLm4‐7 does not impede AvrLm3 expression in planta. Y2H assays suggest the absence of physical interaction between the two avirulence proteins.This unusual interaction is the basis for field experiments aiming to evaluate strategies that increase Rlm7 durability.

Identifying areas of forest loss is a fundamental aspect of sustainable forest management. Global Forest Change (GFC) datasets developed by Hansen et al. (in Science 342:850–853, 2013) are publicly available, but the accuracy of these datasets for small forest plots has not been assessed. We used a forest-wide polygon-based approach to assess the accuracy of using GFC data to identify areas of forest loss in an area containing numerous small forest plots. We evaluated the accuracy of detection of individual forest-loss polygons in the GFC dataset in terms of a “recall ratio”, the ratio of the spatial overlap of a forest-loss polygon determined from the GFC dataset to the area of a corresponding reference forest-loss polygon, which we determined by visual interpretation of aerial photographs. We analyzed the structural relationships of recall ratio with area of forest loss, tree species, and slope of the forest terrain by using linear non-Gaussian acyclic modelling. We showed that only 11.1% of forest-loss polygons in the reference dataset were successfully identified in the GFC dataset. The inferred structure indicated that recall ratio had the strongest relationships with area of forest loss, forest tree species, and height of the forest canopy. Our results indicate the need for careful consideration of structural relationships when using GFC datasets to identify areas of forest loss in regions where there are small forest plots. Moreover, further studies are required to examine the structural relationships for accuracy of land-use classification in forested areas in various regions and with different forest characteristics.

Fungi are of primary ecological, biotechnological and economic importance. Many fundamental biological processes that are shared by animals and fungi are studied in fungi due to their experimental tractability. Many fungi are pathogens or mutualists and are model systems to analyse effector genes and their mechanisms of diversification. In this study, we report the genome sequence of the phytopathogenic ascomycete Leptosphaeria maculans and characterize its repertoire of protein effectors. The L. maculans genome has an unusual bipartite structure with alternating distinct guanine and cytosine-equilibrated and adenine and thymine (AT)-rich blocks of homogenous nucleotide composition. The AT-rich blocks comprise one-third of the genome and contain effector genes and families of transposable elements, both of which are affected by repeat-induced point mutation, a fungal-specific genome defence mechanism. This genomic environment for effectors promotes rapid sequence diversification and underpins the evolutionary potential of the fungus to adapt rapidly to novel host-derived constraints.

Blackleg (also known as Phoma or stem canker) is a major, worldwide disease of Brassica crop species, notably B. napus (rapeseed, canola), caused by the ascomycete fungus Leptosphaeria maculans . The outbreak and severity of this disease depend on environmental conditions and management practices, as well as a complex interaction between the pathogen and its hosts. Genetic resistance is a major method to control the disease (and the only control method in some parts of the world, such as continental Europe), but efficient use of genetic resistance is faced with many difficulties: (i) the scarcity of germplasm/genetic resources available, (ii) the different history of use of resistance genes in different parts of the world and the different populations of the fungus the resistance genes are exposed to, (iii) the complexity of the interactions between the plant and the pathogen that expand beyond typical gene-for-gene interactions, (iv) the incredible evolutionary potential of the pathogen and the importance of knowing the molecular processes set up by the fungus to “breakdown’ resistances, so that we may design high-throughput diagnostic tools for population surveys, and (v) the different strategies and options to build up the best resistances and to manage them so that they are durable. In this paper, we aim to provide a comprehensive overview of these different points, stressing the differences between the different continents and the current prospects to generate new and durable resistances to blackleg disease.

Blackleg disease, caused by the fungal pathogen Leptosphaeria maculans, is a serious disease of Brassica napus. The disease is mainly controlled by genetic resistance and crop rotation. However, L. maculans has displayed a high evolutionary potential to overcome major resistance genes in B. napus. This study aimed to analyze the major-gene and adult-plant resistance (APR) of Canadian B. napus varieties/lines (accessions) and the avirulence allele frequency in L. maculans populations in western Canada. For resistance identification, a set of L. maculans isolates with known avirulence genes were used to characterize major resistance (R) genes in 104 Canadian B. napus accessions and 102 seed samples collected from growers' fields; with 104 B. napus accessions further evaluated for APR under controlled conditions. In addition, avirulence genes of 300 L. maculans isolates collected from infected canola stubbles in growers' fields were determined by cotyledon inoculation and gene-specific PCR assays. The results indicated that R genes were present in the majority of these B. napus accessions, with the Rlm3 gene being predominant while other R genes were rarely detected. APR was identified in more than 50 % of the accessions. Predominance of Rlm3 in 102 seed samples from growers' fields suggested Rlm3-carrying B. napus varieties were currently widely used in western Canada. Avirulence allele frequency identification of field L. maculans isolates revealed the scarcity of the avirulence allele towards Rlm3, AvrLm3. This indicated the breakdown of Rlm3 resistance, which could be due to the over use of this single resistance gene in Canadian B. napus germplasm.

• The control of stem canker disease of Brassica napus (rapeseed), caused by the fungus Leptosphaeria maculans is based largely on plant genetic resistance: single-gene specific resistance (Rlm genes) or quantitative, polygenic, adult-stage resistance. Our working hypothesis was that quantitative resistance partly obeys the gene-for-gene model, with resistance genes ‘recognizing’ fungal effectors expressed during late systemic colonization. • Five LmSTEE (stem-expressed effector) genes were selected and placed under the control of the AvrLm4-7 promoter, an effector gene highly expressed at the cotyledon stage of infection, for miniaturized cotyledon inoculation test screening of a gene pool of 204 rapeseed genotypes. • We identified a rapeseed genotype, ‘Yudal’, expressing hypersensitive response to LmSTEE98. The LmSTEE98–RlmSTEE98 interaction was further validated by inactivation of the LmSTEE98 gene with a CRISPR-Cas9 approach. Isolates with mutated versions of LmSTEE98 induced more severe stem symptoms than the wild-type isolate in ‘Yudal’. This single-gene resistance was mapped in a 0.6 cM interval of the ‘Darmor_bzh’ × ‘Yudal’ genetic map. • One typical gene-for-gene interaction contributes partly to quantitative resistance when L. maculans colonizes the stems of rapeseed. With numerous other effectors specific to stem colonization, our study provides a new route for resistance gene discovery, elucidation of quantitative resistance mechanisms and selection for durable resistance.

Phytopathogenic fungi frequently contain dispensable chromosomes, some of which contribute to host range or pathogenicity. In Leptosphaeria maculans, the stem canker agent of oilseed rape (Brassica napus), the minichromosome was previously suggested to be dispensable, without evidence for any role in pathogenicity. Using genetic and genomic approaches, we investigated the inheritance and molecular determinant of an L. maculans-Brassica rapa incompatible interaction.Single gene control of the resistance was found, while all markers located on the L. maculans minichromosome, absent in the virulent parental isolate, co-segregated with the avirulent phenotype. Only one candidate avirulence gene was identified on the minichromosome, validated by complementation experiments and termed AvrLm11. The minichromosome was frequently lost following meiosis, but the frequency of isolates lacking it remained stable in field populations sampled at a 10-yr time interval, despite a yearly sexual stage in the L. maculans life cycle.This work led to the cloning of a new 'lost in the middle of nowhere' avirulence gene of L. maculans, interacting with a B. rapa resistance gene termed Rlm11 and introgressed into B. napus. It demonstrated the dispensability of the L. maculans minichromosome and suggested that its loss generates a fitness deficit.

In agricultural systems, major (R) genes for resistance in plants exert strong selection pressure on cognate/ corresponding avirulence effector genes of phytopathogens. However, a complex interplay often exists between trade- offs linked to effector function and the need to escape R gene recognition. Here, using the Leptosphaeria maculans- oilseed rape pathosystem we review evolution of effectors submitted to multiple resistance gene selection. Characteristics of this pathosystem include a crop in which resistance genes have been deployed intensively resulting in ` boom and bust ' cycles; a fungal pathogen with a high adaptive potential in which seven avirulence genes are cloned and for which population surveys have been coupled with molecular analysis of events responsible for virulence. The mode of evolution of avirulence genes, all located in dispensable parts of the ' two-speed ' genome, is a highly dynamic gene- specific process. In some instances, avirulence genes are readily deleted under selection. However, others, even when located in the most plastic genome regions, undergo only limited point mutations or their avirulence phenotype is ` camouflaged ' by another avirulence gene. Thus, while hundreds of effector genes are present, some effectors are likely to have an important and nonredundant function, suggesting functional redundancy and dispensability of effectors might not be the rule.

LepR3, found in the Brassica napus cv ‘Surpass 400’, provides race-specific resistance to the fungal pathogen Leptosphaeria maculans, which was overcome after great devastation in Australia in 2004. We investigated the LepR3 locus to identify the genetic basis of this resistance interaction. We employed a map-based cloning strategy, exploiting collinearity with the Arabidopsis thaliana and Brassica rapa genomes to enrich the map and locate a candidate gene. We also investigated the interaction of LepR3 with the L. maculans avirulence gene AvrLm1 using transgenics. LepR3 was found to encode a receptor-like protein (RLP). We also demonstrated that aviru-lence towards LepR3 is conferred by AvrLm1, which is responsible for both the Rlm1 and LepR3-dependent resistance responses in B. napus. LepR3 is the first functional B. napus disease resistance gene to be cloned. AvrLm1's interaction with two independent resistance loci, Rlm1 and LepR3, highlights the need to consider redundant phenotypes in ‘gene-for-gene’ interactions and offers an explanation as to why LepR3 was overcome so rapidly in parts of Australia.