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Oilseed rape residues are a crucial determinant of stem canker epidemiology as they support the sexual reproduction of the fungal pathogen Leptosphaeria maculans. The aim of this study was to characterize the impact of a resistance gene against L. maculans infection on residue microbial communities and to identify microorganisms interacting with this pathogen during residue degradation. We used near‐isogenic lines to obtain healthy and infected host plants. The microbiome associated with the two types of plant residues was characterized by metabarcoding. A combination of linear discriminant analysis and ecological network analysis was used to compare the microbial communities and to identify microorganisms interacting with L. maculans. Fungal community structure differed between the two lines at harvest, but not subsequently, suggesting that the presence/absence of the resistance gene influences the microbiome at the base of the stem whilst the plant is alive, but that this does not necessarily lead to differential colonization of the residues by fungi. Direct interactions with other members of the community involved many fungal and bacterial amplicon sequence variants (ASVs). L. maculans appeared to play a minor role in networks, whereas one ASV affiliated to Plenodomus biglobosus (synonym Leptosphaeria biglobosa) from the Leptosphaeria species complex may be considered a keystone taxon in the networks at harvest. This approach could be used to identify and promote microorganisms with beneficial effects against residue‐borne pathogens and, more broadly, to decipher the complex interactions between multispecies pathosystems and other microbial components in crop residues., Characterizing the impact of a resistance gene against Leptosphaeria maculans on the microbiome of oilseed rape residues using near‐isogenic lines helps to identify microorganisms interacting with this pathogen.

Carrots with different Rhizoctonia-like symptoms were found in the main Swedish carrot production areas from 2001–2020. The most commonly observed symptoms were a greyish-white felt-like mycelium and black scurf, the latter often associated with Rhizoctonia solani anastomosis group (AG) 3-PT on potato. An overall increase in disease incidence in all studied fields over time was observed for both symptoms. The majority of Rhizoctonia isolates sampled from carrot in the period 2015–2020 were identified as AG 3 (45%) and AG 5 (24%), followed by AG 1-IB (13%), AG 11 (5%), AG-E (5%), AG BI (3%), AG-K (3%) and AG 4-HGII (2%). To our knowledge, this is the first report describing AG 5 in Sweden as well as AG 3, AG 11 and AG-E inducing Rhizoctonia-like symptoms on carrot. Secondly, we report for the first time that R. solani AG 3, and the less observed AGs: AG 1-IB and AG 5 can induce black scurf symptoms on the taproot of carrots. Due to a widely used carrot-potato crop rotation in Swedish areas, a possible cross-over from potato to carrot is suggested. This information is of high importance to reduce Rhizoctonia inoculum in soils, since avoiding carrot-potato crop rotations needs to be considered.

Studies were conducted to determine the role of 3-methylthioproprionic acid (MTPA) in the pathogenicity of potato stem canker, Rhizoctonia solani, and the concentrations required to inhibit growth of R. solani under laboratory and plant house-based conditions. The experiments were laid out in a completely randomized design with five treatments and five replications. The treatments were 0, 1, 2, 4, and 8 mM concentrations of MTPA. The purified toxin exhibited maximal activity at pH 2.5 and 30°C. MTPA at 1, 2, 4, and 8 mM levels reduced plant height, chlorophyll content, haulm fresh weight, number of stolons, canopy development, and tuber weight of potato plants, as compared to the control. MTPA significantly affected mycelial growth with 8 mM causing the highest infection. The potato seedlings treated with MTPA concentrations of 1.0-8.0 mM induced necrosis of up to 80% of root system area. Cankers were resulted from the injection of potato seedling stems with 8.0 mM MTPA. The results showed the disappearance of cell membrane, rough mitochondrial and cell walls, change of the shape of chloroplasts, and swollen endoplasmic reticulum. Seventy-six (76) hours after toxin treatment, cell contents were completely broken, cytoplasm dissolved, and more chromatin were seen in the nucleus. The results suggested that high levels of the toxin concentration caused cell membrane and cytoplasm fracture. The integrity of cellular structure was destroyed by the phytotoxin. The concentrations of the phytotoxin were significantly correlated with pathogenicity and caused damage to the cell membrane of potato stem base tissue.

Soil microorganisms and soil fauna may have a large impact on the tuber yield of potato crops. The interaction between root-lesion nematodes and the pathogenic fungus Rhizoctonia solani K&uuml, hn was studied on potato plants grown in pots under controlled conditions. In two similar experiments, different combinations of nematodes and fungal mycelium were added to the pots at three occasions, at planting, after 14 days, and after 28 days. The nematodes reduced root biomass and the combination of nematodes and R. solani resulted in reduced tuber yield in both experiments, but the interaction was not synergistic. In contrast, the number of stem canker lesions decreased in the presence of nematodes compared to treatments with R. solani only. The time of inoculation influenced the severity of both fungal and nematode damage. The nematode damage on tubers was less severe if the nematodes were added at 28 days, while the number of severe stem canker lesions increased if the fungus was added at 28 days. However, the time of nematode inoculation did not affect the incidence of fungal damage, hence the nematodes did not assist R. solani to infect the plant. Our results highlight the underestimated importance of root-lesion nematodes, not resulting in obvious above ground symptoms or misshaped tubers yet affecting the performance of other pathogens.

En el 2011 y 2012 se realizó una investigación de los agentes causales de las enfermedades asociadas al cultivo de Pitaya (Hylocereus costaricensis) en Costa Rica. Una de las más importantes durante el periodo de estudio, debido a su alta incidencia (100%), fue el cáncer en el tallo. Los síntomas observados fueron pequeñas manchas anaranjadas y rojizas irregulares y lesiones de coloración beige-grisáceas con el centro abultado. Para determinar el agente causal se recolectaron 85 muestras, cladodios completos con diferentes estados de desarrollo de la enfermedad, a partir de los cuales se realizaron 40 aislamientos. De estos creció un organismo en forma consistente, con el cual se llevó a cabo la identificación, caracterización morfológica y molecular así como la verificación de los postulados de Koch. De acuerdo con la forma de crecimiento, coloración del micelio y la morfología de los artroconidios y de las características moleculares (secuenciación de la región ITS), el patógeno asociado a los síntomas presentó un 99% de similitud con Neoscytalidium dimidiatum (Penz.) (no. de accesión KJ513460, del hongo aislado en Costa Rica). En las pruebas de los postulados de Koch se obtuvo un 92% de infección y se presentó una sintomatología similar a la observada en el campo, además, se recuperó el mismo organismo que fue inoculado. Este es el primer reporte de este hongo causando daños en el tallo de Hylocereus costaricensis en Costa Rica. In 2011 infected samples were examined to identify the causal agent of stem rot of Hylocereus costaricensis in Costa Rica. During the rainy season, symptoms associated with the disease included a small yellowish rot that typically started from the edge of the cladodium and extended inward towards the stem. Eventually the whole stem decayed. During the dry season, orange circular spots appeared on the stem, turning dark brown. For determine the causal agent, were collected 25 samples of each type of decay, complete cladodes with different stages of symptom development, from which 15 isolations of each disease were made. A bacterium was obtained consistently from each symptomatology, ith which the identification, characterization based on biochemical tests and molecular identification was carried out, as well as the verification of Koch’s postulates. Results indicated that the symptoms were caused by the same bacteria. Based on sequencing of the 16S region, the bacterium showed 99.2% similarity with Enterobacter hormaechei (KJ999997). According to Koch’s postulates, a similar symptomatology was obtained on plants from the field and on inoculated plants. This is the first report of this pathogen causing damage to the stem of Hylocereus costarricensis in American continent. Universidad de Costa Rica/[ED-313]/UCR/Costa Rica UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Agroalimentarias::Centro de Investigación en Protección de Cultivos (CIPROC) UCR::Vicerrectoría de Docencia::Ciencias Agroalimentarias::Facultad de Ciencias Agroalimentarias

Summary Background Teratosphaeria gauchensis and T. zuluensis are closely related fungi that cause Teratosphaeria (previously Coniothyrium) stem canker disease on Eucalyptus species propagated in plantations for commercial purposes. This disease is present in many countries in which Eucalyptus trees are planted, and continues to spread with the international trade of infected plant germplasm. Taxonomy Fungi, Ascomycota, Pezizomycotina, Dothideomycetes, Dothideomycetidae, Capnodiales, Teratosphaeriaceae, Teratosphaeria. Identification The causal agents form dark masses of pycnidia that are visible on the surface of distinct stem cankers that typically form on young green stem tissues. Accurate diagnosis of the causal agents requires DNA sequence data. Host range Nine species of Eucalyptus are known to be affected. Of these, E. grandis and its hybrids, which include some of the most important planting stock globally, appear to be particularly vulnerable. Disease symptoms Small necrotic lesions develop on young green stem tissue. These lesions coalesce to form large cankers that exude gum. Epicormic shoots develop below the girdling canker and, in severe cases, trees die. Useful websites Mycobank, https://www.mycobank.org; Publications of the Forestry and Agricultural Biotechnology Institute (FABI), https://www.fabinet.up.ac.za/index.php/journals.

The aim and tasks of the study. Theoretical justification of the methodology for forming a collection of sunflower lines by adaptability in resistance diseases to augment the collection fund of the NCPGRU.Results and discussion. The article presents the results of the theoretical justification and expediency of practical application of interval estimates as criteria of a dynamic system for evaluation of immunological characteristics of genotypes in terms of resistance to pathogens of downy mildew (Plasmopara helianthi Novot. f. helianthi), stem canker (Phomopsis / Diaporthe helianthi Munt. –Cvet. et al.) and gray mold (Botrytis cinerea Pers.) on the basis of the year-by-year prevalence of disease, taking into account the influence of weather conditions. A scheme for creating starting material of disease-resistant sunflower was developed as a result of practical application of this system in 2007–2013. Twenty-six sunflower lines - pollen fertility restorers with different combinations of resistance to major diseases were created. The type of adaptability of these lines was determined in relation to the stem canker development intensity. Fifteen lines combining homeostaticity or medium plasticity with a high or medium level of the genotypic effect with respect to stem canker were identified;5 of them (BI 54 V, BI 71 V, BI 77 V, BI 113 V, and BI 131 V) combine homeostaticity (biwithin -0.55 --0.39) and a high level of the genotypic effect (Еіwithin -11,13 - -5,22); 3 lines (BI 11 V, BI 66 V, and BI 127 V) have an medium plasticity (bi = 0,61–1,04) and a high level of the genotypic effect (Eiwithin -18,30--4,13); 6 lines (BI 43 V, BI 47 B, BI 55 V, BI 61 V, BI 72 V, and BI 78 V) are homeostatic (bi within -0,73 - 0,53) with a medium level of the genotypic effect (Eiwithin -2,30-1,22); and 1 line (BI 83 V) is mid-plastic (bi = 1.07) with a medium level of the genotypic effect (Ei = -0.47). Line- pollen fertility restorer BI 20 V is characterized by high resistance to downy mildew and gray mold, high oil content in seeds (31,86 %) and 1000-seed weight of 62,0 g; it is early ripening and multi-calathidium. Line- pollen fertility restorer BI131 V combines high resistance (0,0 % of affected plants) to downy mildew and gray mold, Septoria blight (8 points), and stem canker (22,0 % of affected stemarea), homeostaticity (bi = 0,37), a high genotypic effect (Ei = -9,13) manifested as weak intensity of stem canker development, a large calathidium (22,5 cm), high oil content in seeds (31,87 %) and 1000-seed weight of 87,5 g; it is early ripening and mono-calathidium.Conclusions. Thus, this paper presents the results of the theoretical justification of the methodology for forming of a collection of sunflower lines byadaptatbility in disease resistance. Starting material of sunflower that is resistant to the major diseases was created. Of the lines created, 15 were distinguished, since they combinehomeostaticity, a high genotypic effect manifested as weak intensity of stem canker development and a set of economically valuable traits.The lines were submitted for pre-registration examination at the NCPGRU for subsequent formation of a specific collection of sunflower lines by adaptability in resistance to Phomopsis helianthi., Цель. Теоретическое обоснование методологии формирования коллекции линий подсолнечника за адаптивностью по устойчивости к болезням для пополнения коллекционного фонда НЦГРРУ.Результаты и обсуждение. В статье изложены результаты теоретического обоснования и целесообразности практического применения интервальных оценок, как критериев динамической системы определения иммунологической характеристики генотипов по устойчивости к возбудителям ложной мучнистой росы (Plasmopara helianthi Novot. f. helianthi), фомопсиса (Phomopsis / Diaporthe helianthi Munt. - Cvet. Etal.) и серой гнили (Botrytis cinerea Pers.) на основе динамики распространенности болезней по годам с учетом влияния погодных условий. Разработана схема создания исходного материала подсолнечника, устойчивого к болезням, в результате практического применения которой в течение 2007–2013 гг. созданы 26 линий-восстановителей фертильности пыльцы подсолнечника, с различным сочетанием устойчивости к основным болезням. Определен тип адаптивности созданных линий относительно интенсивности развития фомопсиса. Выделены15 линий, сочетающих гомеостатичность или среднюю пластичность с высоким или средним уровнем проявления генотипического эффекта относительно устойчивости к фомопсису, из них пять (БИ 54 В, БИ 71 В, БИ 77 В, БИ 113 В, БИ 131 В) объединяют гомеостатичность (biот -0,55 до -0,39) и высокий уровень генотипического эффекта (Еіот -11,13 до -5,22), три линии (БИ 11 В, БИ 66 В, БИ 127 В) имеют среднюю пластичность (bi = 0,61–1,04) и высокий уровень генотипического эффекта (Еіот -18,30 до -4,13), шесть гомеостатических (biот -0,73 до 0,53) линий (БИ 43 В, БИ 47 В, БИ 55 В, БИ 61 В, БИ 72 В, БИ 78 В) со средним уровнем проявления генотипического эффекта (Еіот -2,30 до 1,22), одна (БИ 83 В) среднепластичная (bi = 1,07) линия со средним уровнем проявления генотипического эффекта (Еі = -0,47). Линия-восстановитель фертильности пыльцы БИ 20 В характеризуется высокой устойчивостью к ложной мучнистой росе и серой гнили, высокими содержанием масла в семенах (31,86 %) и массой 1000 семян (62,0 г), раннеспелая, многокорзиночная. Линия-восстановитель фертильности пыльцы БИ 131 В сочетает высокую устойчивость (0,0 % пораженных растений ) к ложной мучнистой росе и серой гнили, септориозу (8 баллов), фомопсису (22,0 % пораженной площади стебли), гомеостатичность (bi = 0,37), высокий генотипический эффект (Еі = -9,13) выраженный слабой интенсивностью развития фомопсиса, крупный размер корзинки (22,5 см), высокое содержание масла в семенах (31,87 %) и массу 1000 семян (87,5 г), раннеспелая, однокорзиночная.Выводы. Таким образом, в данной работе представлены результаты теоретического обоснования методологии формирования коллекции линий подсолнечника по адаптивности по устойчивости к болезням. Создан устойчивый к основным болезням исходный материал подсолнечника. Среди созданных линий выделены 15, сочетающих гомеостатичность, высокий генотипический эффект, выраженный слабой интенсивностью развития фомопсиса и комплекс ценных хозяйственных признаков. Линии переданы на предрегистрационную экспертизу в НЦГРРУ для последующего формирования специальной коллекции линий подсолнечника за адаптивностью по устойчивости к возбудителю фомопсиса., Викладено результати практичного застосування інтервальних оцінок як критеріїв системи визначення імунологічної характеристики генотипів за стійкістю до збудників несправжньої борошнистої роси (Plasmopara helianthi Novot. f. helianthi), фомопсису (Phomopsis / Diaporthe helianthi Munt. -Cvet. Etal.) та сірої гнилі (Botrytis cinerea Pers.) на основі динаміки хвороб за роками з урахуванням впливу погодних умов. Розроблено схему створення вихідного поширення хвороб за роками з урахуванням впливу погодних умов. Розроблено схему створення вихідного матеріалу соняшнику за стійкістю до хвороб, використання якої впродовж 2007–2013 рр. сприяло створенню 26 ліній-відновників фертильності пилку соняшнику з різним поєднанням стійкості до основних хвороб. Визначено тип адаптивності ліній відносно інтенсивності розвитку фомопсису. Виділено 15, які поєднують гомеостатичність чи середню пластичність з високим чи середнім рівнем прояву генотипового ефекту за стійкістю до фомопсису.

An experiment was conducted at Tuber Crops Research Centre, BARI, Bogra during 2008-2009 to evaluate the organic amendment for controlling the stem canker and black scurf of potato. The different kinds of organic amendments were: T 1 =Poultry refuse (PR) at 4 t ha -1 , T 2 =PR at 5 t ha -1 , T 3 =PR at 6 t ha -1 , T 4 =Mustard oil cake (MOC) at 300kg ha -1 , T 5 =MOC at 500kg ha -1 , T 6 =Neem oil cake (NOC) at 300kg ha -1 , T 7 =NOC at 500kg ha -1 , T 8 =Sawdust burning and T 9 = Control. The experiment was laid out in a randomized complete block design (RCBD) with four replications. The organic amendment significantly influenced the disease incidence, yield attributes and yield of potato. The lowest disease incidence (17.5%) and per cent disease index (9.99) were found in T 3 (poultry waste at 6 t ha -1 ). The minimum russet, deformed and Sclerotia infected tubers were also recorded in poultry waste at 6 t ha -1 . The highest healthy tubers and tuber yield were found in the same treatment. Therefore, poultry manure 6 t ha -1 along with recommended dose of chemical fertilizer can be recommended to produce healthy tubers and maximum tuber yield of potato.

In 2016, symptoms of stem canker with branches dieback were detected in several Cannabis sativa L. crops, located in the northeast part of Italy (Rovigo district). Morphological and molecular identifications of the fungal colonies isolated revealed the presence of Neofusicoccum parvum. The pathogenicity was verified with Koch’s postulates setup. To our knowledge, this is the first report of N. parvum as a pathogen of C. sativa worldwide.

During plant infection, pathogens secrete an arsenal of effectors, key elements of pathogenesis which modulate innate immunity of the plant and facilitate infection. Fungal effector genes typically encode small proteins, predicted to be secreted, with no homology in databases, and absence of known motif. As such their function or role in pathogenesis is mostly unknown. The phytopathogenic ascomycete Leptosphaeria maculans is the causal agent of stem canker of oilseed rape. More than 650 putative effector-encoding genes have been identified in its genome, 7 of them corresponding to avirulence proteins. We develop a project aiming at elucidating the involvement of L. maculans effectors in pathogenicity through the structural and functional characterization of a few major effector proteins and the determination of their interactants. Our strategy is illustrated here with AvrLm4-7, a 143 amino-acid long secreted protein important for fungal fitness and recognized by two oilseed rape resistance proteins, Rlm4 and Rlm7. One single amino-acid change is sufficient to lose recognition by Rlm4 while maintaining recognition by Rlm7. 3D-structure of an isoform of AvrLm4-7 only recognized by Rlm7 was previously determined, allowing us to define regions implicated in recognition by Rlm7 and translocation into plant cell. We recently determined the 3D-structure of another isoform of AvrLm4-7 recognized both by Rlm4 and Rlm7, showing that the amino-acid change allowing to escape Rlm4-recognition was located on an external loop and did not change the overall structure of the protein. AvrLm4-7 was also recently shown to suppress recognition of another L. maculans avirulence gene, AvrLm3, by its cognate resistance gene Rlm3, leading us to hypothesize a suppression of Effector-Triggered Immunity (ETI) by AvrLm4-7. In order to test that hypothesis, we transiently expressed AvrLm4-7 and several cell-death inducers in Nicotiana benthamiana epidermal cells: AvrLm4-7 was able to suppress cell death induced by BAX and AvrPto. We also generated transgenic lines of Arabidopsis thaliana constitutively expressing AvrLm4-7 and are currently characterizing the lines for their susceptibility to pathogens with contrasted lifestyles and for their ability to suppress recognition of Pseudomonas syringae avirulence proteins. A better understanding of the role of an effector implicated in the masking of another effector will allow us to develop alternative strategies to genetically control stem canker disease.

The stem canker disease, caused by Leptosphaeria maculans, is one of the most devastating diseases of oilseed rape (canola). It colonizes the plant in two stages: a short and early colonisation stage corresponding to cotyledon or leaf colonisation, and a late colonisation stage during which the fungus colonises systemically and symptomlessly the plant during several months before stem canker appears. To date, determinants of the late colonisation stage remain poorly understood. By a transcriptomic approach, we previously identified two waves of effector candidate expression during the early and late colonisation stages (Gervais et al, 2016). The late effector candidates are located in gene-rich genomic regions, whereas the early effector genes are located in gene-poor regions of the genome. Among the late effector candidates identified, we selected 6 genes for further characterization. We created mutants silenced for these effector candidates. For one of these genes, its expression level correlated negatively with the size of the necrosis observed in the stem. The identification of new effector genes would contribute to the identification of new resistance genes specific to these effectors. To easily identify matching resistance genes in oilseed rape, we created transgenic isolates expressing these 6 late effectors at the early steps of infection to provide medium-throughput strategies to screen more efficiently different cultivars. Preliminary results indicate that some cultivars with adult resistance were more resistant to these transgenic isolates in cotyledon assays. With this approach, we also identified a cultivar carrying a specific resistance to one these 6 effector candidates. Reference Gervais, J., Plissonneau, C., Linglin, J., Meyer, M., Labadie, K., Cruaud, C., Fudal, I., Rouxel, T. and Balesdent, M.H. (2016) Different waves of effector genes with contrasted genomic location are expressed by Leptosphaeria maculans during cotyledon and stem colonization of oilseed rape. Mol. Plant Pathol.

Leptosphaeria maculans is the fungus responsible for the stem canker disease of oilseed rape (Brassica napus). AvrLm3 and AvrLm7, two avirulence effector genes of L. maculans, are involved in an unusual relationship: the presence of AvrLm7 suppresses the Rlm3-mediated resistance of AvrLm3. Following the large-scale cropping of Rlm7 cultivars in Europe, the on-going breakdown of the Rlm7 resistance and the concomitant inactivation of AvrLm7 was accompanied by the resurgence of isolates expressing the AvrLm3 phenotype. Here, we evaluated adaptation mechanisms to escape Rlm3-mediated ETI by assessing AvrLm3 polymorphism in a collection of 236 L. maculans isolates. In isolates virulent towards both Rlm3 and Rlm7 (a3a7), the loss of the Rlm3-mediated resistance response was due to two distinct mechanisms. First, when AvrLm7 was inactivated (deletion or inactivating mutations), amino acid substitutions in AvrLm3 generated virulent isoforms of the protein. Secondly, point mutations in AvrLm7 could maintain the masking of the AvrLm3 phenotype while also allowing escape from the Rlm7 recognition. This virulence mechanism was found in 56.4% of the a3a7 isolates, and these contained an avirulent allele of AvrLm3. Signatures of positive selection were observed in AvrLm3 and, in spite of the telomeric location of AvrLm3, no a3a7 isolates exhibited deletion events or inactivating mutations in AvrLm3 as commonly observed for other avirulence genes of L. maculans. The complex evolutionary mechanisms enabling L. maculans to escape Rlm3-mediated resistance while preserving AvrLm3 sequence integrity, along with observed reduced aggressiveness of isolates silenced for AvrLm3, serves to emphasize importance of this effector in pathogenicity towards B. napus.

Species of the genus Vaccinium are commercially cultivated in Europe for their berries, which are highly valued for dietary and pharmaceutical properties. Cultivation is severely limited due to a range of fungal diseases, especially those caused by species of Diaporthe. A number of Diaporthe isolates have been collected from Vaccinium growing regions in Europe, and initially identified as D. vaccinii based on host association. Using DNA sequence inference of the combined ß-tubulin, calmodulin, translation elongation factor 1-alpha and the internal transcribed spacer region of the nuclear rDNA, along with morphological characteristics, six species were characterised. Diaporthe eres, D. vaccinii and D. viticola are known species and three novel taxa are described here as D. asheicola, D. baccae and D. sterilis. This study is the first confirmed report of D. vaccinii in Latvia and the Netherlands.

Stem canker (blackleg) is economically the most important disease of oilseed rape world­wide. This disease is caused by two species of pathogenic fungi of the genus Leptosphaeria: Leptosphaeria maculans (Desm.) ces. and de Not anamorf Phoma lingam (Tode. Fr.) Desmas, that causes stem canker and blackleg root and Leptospaeria biglobosa Shoem and Brun, that causes symptoms such as cancers of the upper part of the stem usually causing less damage, and can cause serious damage in countries with higher summer temperatures. Pathogenic fungi from one area to another are transmitted by ascospores and contaminated (infected) seed. In order to prove the modes of transmission of parasites, the test is done transmitting the parasite by seeds. Disinfected seeds of oilseed rape cultivars Quinta were submerged in the suspension of pycnospores. Submerged seeds were kept at 20 º C ± 1 º C and 12 h photoperiod for 48 h. After planting in plastic containers seeds were kept at 25 º C ± 1 º C and 12h photoperiod. In this experiment the following isolates were used: C-5, L-5, K-7, LJ-3, S-11, St-1, GS-3 and Lm as a reference isolate (L. maculans) fol­lowed by K-113, K-115 and Lb as a reference isolate (L. biglobosa). The symptoms were as­sessed after 7 and 14 days after emergence. Ratings were made with + which meant visible symptoms on cotyledons or hypocotyl and - what referred to healthy plants. After 7 days, the isolates (Lm, C-5, L-5, K-7, LJ-3, S-11, St-1, GS-3) were caused disease symptoms on plants of oilseed rape. This pathogenicity is reflected in the number of seedlings, leaf spots and stalk lodging cotyledons. At izolate Lb (reference isolate L. biglobosa) K-113 and K-115 after 7 and 14 days was not observed pathogenicity on rapeseed plants. The control variety seeds were soaked in distilled water. The trial was set in 6 reps, and pathogenicity of fungal isolates in relation to the control was tested using Dunette's test. After 14 days re-isolation of pathogen was done. Rak stabla (crna noga) je najznačajnije oboljenje uljane repice širom sveta. Bolest mogu prouzrokovati dve vrste patogenih gljiva iz roda Leptosphaeria: Leptosphaeria maculans (Desm.) ces. i de Not, anamorf Phoma lingam (Tode. Fr.) Desmas, koja prouzrokuje rak stabla i crnu nogu na korenu i Leptospaeria biglobosa Shoem and Brun, koja prouzrokuje simptome slične raku ali na gornjim delovima stabla obično prouzrokujući manje štete. Ozbiljnije štete mogu nastati u zemljama sa višim letnjim temperaturama. Patogene gljive se iz jedne oblasti u drugu prenose askosporama i zaraženim semenom. Da bi se utvrdili načini prenošenja parazita testirana je mogućnost prenošenja semenom. Dezinfikovano seme uljane repice, kultivara Quinta je potopljeno u suspenziju piknospora. Ovakvo seme je držano na 20ºC ± 1ºC i 12h fotoperiodu tokom 48 sati. Posle setve seme je preneto u plastične posude a temperatura čuvanja je bila 25ºC ± 1º C i fotoperiod od 12h. U ovom eksperimentu korišćeni su sledeći izolati: C-5, L-5, K-7, LJ-3, S-11, St-1, GS-3 i Lm kao referentni izolat (L. maculans) kao i K-113, K-115 i Lb kao referentni izolat (L. biglobosa). Ocena prisustva simptoma je rađena posle 7 i 14 dana od nicanja. Sa znakom + su se obeležavale biljke sa vidljivim simptomima na kotiledonima ili hipokotilu a znakom - biljke bez pojave simptoma. Posle 7 dana izolati Lm, C-5, L-5, K-7, LJ-3, S-11, St-1 i GS-3 su izazvali simptome bolesti na biljkama uljane repice. Patogenost se ogledala kroz broj oštećenih klijanaca, lisnih pega i povijenih kotiledona. Kod izolata Lb (referentni izolat L. biglobosa) K-113 i K-115 posle 7 i 14 dana nije primećena patogenost na biljkama uljane repice. Kontrolna varijanta semena je potapana u destilovanu vodu. Ogled je postavljen u 6 ponavljanja, i patogenost izolata gljive u odnosu na kontrolu je izražena Danetovim testom. Posle 14 dana izvedena je ponovna izolacija patogena.

Summary Leptosphaeria maculans is the fungus responsible for the stem canker disease of oilseed rape (Brassica napus). AvrLm3 and AvrLm4-7, two avirulence effector genes of L. maculans, are involved in an unusual relationship: AvrLm4-7 suppresses the Rlm3-mediated resistance. Here, we assessed AvrLm3 polymorphism in a collection of 235 L. maculans isolates. No field isolates exhibited deletion or inactivating mutations in AvrLm3, as observed for other L. maculans avirulence genes. Eleven isoforms of the AvrLm3 protein were found. In isolates virulent towards both Rlm3 and Rlm7 (a3a7), the loss of the Rlm3-mediated resistance response was due to two distinct mechanisms. First, when AvrLm4-7 was inactivated (deletion or inactivating mutations), amino acid substitutions in AvrLm3 generated virulent isoforms of the protein. Secondly, when only point mutations were observed in AvrLm4-7, a3a7 isolates still contained an avirulent allele of AvrLm3. Directed mutagenesis confirmed that some point mutations in AvrLm4-7 were sufficient for the fungus to escape Rlm7-mediated resistance while maintaining the suppression of the AvrLm3 phenotype. Signatures of positive selection were also identified in AvrLm3. The complex evolutionary mechanisms enabling L. maculans to escape Rlm3-mediated resistance while preserving AvrLm3 integrity, along with observed reduced aggressiveness of isolates silenced for AvrLm3, serves to emphasize the importance of this effector in pathogenicity towards B. napus. While the common response to resistance gene pressure is local selection of isolates depleted in the cognate avirulence gene, this example contributes to complexify the gene-for-gene concept of plant-pathogen evolution with a “camouflaged” model allowing retention of non-dispensable avirulence effectors. This article is protected by copyright. All rights reserved.

International audience; Leptosphaeria maculans, a hemibiotrophic fungus responsible of stem canker, colonises oilseed rape in two stages: an early stage of cotyledon or leaf colonisation, and a late colonisation stage during which the fungus colonises systemically without visible symptom the plant before stem canker appears. L. maculans presents a bipartite genome structure alternating gene-rich and transposable element (TE)-rich regions. TE-rich regions, which encompass one third of the genome, are enriched in putative effector-encoding genes that present the same expression pattern (no or a low expression level during in vitro growth and a strong over-expression during early infection in cotyledons and leaves; ‘early’ effectors). In contrast, gene-rich regions were recently reported to contain putative effector-encoding genes specifically expressed during the late stages of stem infection (‘late’ effectors). We have previously investigated the involvement of the chromatin structure of repeat-rich regions on the expression of ‘early’ effector genes: RNAi silencing of two genes encoding key players in heterochromatin assembly through histone modification H3K9me3, HP1 and KMT1, induced an over-expression of genes located in AT-isochores, particularly ‘early’ effector genes but no modification of ‘late’ effector genes expression. Here, we performed analysis of nucleosome positioning, chromatin structure and gene expression at the genome scale combining MAINE-seq, ChIP-seq and RNA-seq data during in vitro growth of L. maculans. We analysed in vitro ChIP-seq data targeting heterochromatin modifications, H3K9me3 and H3K27me3, and a euchromatin mark, H3K4me2 and found that gene-rich regions are associated with H3K4me2 and H3K27me3 while TE-rich regions are associated with H3K9me3. Effector genes are also associated with distinct heterochromatin marks according to their genomic location and expression kinetics: while ‘early’ effector genes located in TE-rich regions are associated with H3K9me3, ‘late’ effector genes located in gene-rich region are associated with H3K27me3. Genome-wide nuclesome positioning was analysed using MAINE-seq data, showing distinct nucleosome organization for genes located in TE-rich or gene-rich regions, and according to gene expression level. Finally, we recently investigated the role of another key player in heterochromatin assembly, KMT6, involved in the heterochromatic-associated histone modification H3K27me3, on the control of effector gene expression. RNAi silencing of KMT6 leads to a deregulation of genes not only associated with H3K27me3 in the wild type strain, suggesting a relocation of different histone modifications

National audience; Leptosphaeria maculans is responsible for the stem canker (phoma) disease of oilseed rape (Brassica napus). AvrLm3 and AvrLm4-7, two avirulence effector genes of L. maculans, are involved in an unusual relationship: the presence of AvrLm4-7 induces the Rlm7-mediated resistance but suppresses the Rlm3-mediated recognition (Plissonneau et al., 2015; doi: 10.1111/nph.13736). To investigate this relationship, we assessed the sequence diversity of AvrLm3 in L. maculans populations. The analysis of more than 200 isolates revealed a high level of allelic polymorphism for this gene, but no deletion event nor inactivating mutations were found. This observation contrasts with the presence/absence or RIP-(Repeat-induced-point-mutation) inactivation polymorphisms usually found for other L. maculans avirulence genes and questioned the role of AvrLm3 in fungal fitness. Two distinct mechanisms responsible for the «double virulent» phenotype (i.e. virulent toward both Rlm3 and Rlm7, «a3a7») were identified. In isolates displaying an inactivation of AvrLm4-7, amino acid changes in AvrLm3 were responsible for the virulent phenotype towards Rlm3. However, 56% of the a3a7 isolates displayed an avirulent allele of AvrLm3, combined with point mutations in AvrLm4-7. Such situations allow the fungus to escape Rlm7-mediated resistance while maintaining the suppression of the AvrLm3 phenotype effective. They also allow the fungus to escape to the recognition by two resistance genes while keeping the secretion of the two corresponding effector proteins. The complex evolutionary mechanisms displayed by L. maculans to escape Rlm3-mediated resistance while preserving AvrLm3 sequence integrity, along with the reduced virulence in isolates silenced for AvrLm3 confirmed the importance of this effector in pathogenicity towards B. napus. This work was funded by the INRA SMaCH metaprogram «K-masstec» and the CTPS project «ICOSCOP»

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.