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ToxA is one of the most studied proteinaceous necrotrophic effectors produced by plant pathogens. It has been identified in four pathogens (Pyrenophora tritici-repentis, Parastagonospora nodorum, Parastagonospora pseudonodorum (formerly Parastagonospora avenaria f. sp. tritici) and Bipolaris sorokiniana) causing leaf spot diseases on cereals worldwide. To date, 24 different ToxA haplotypes have been identified. Some Py. tritici-repentis and related species also express ToxB, another small protein necrotrophic effector. We present here a revised and standardized nomenclature for these effectors, which could be extended to other poly-haplotypic (allelic) genes found across multiple species.

The fungus Parastagonospora nodorum is a narrow host range necrotrophic fungal pathogen that causes Septoria nodorum blotch (SNB) of cereals, most notably wheat (Triticum aestivum). Although commonly observed on wheat seedlings, P. nodorum infection has the greatest effect on the adult crop. It results in leaf blotch, which limits photosynthesis and thus crop growth and yield. It can also affect the wheat ear, resulting in glume blotch, which directly affects grain quality. Reports of P. nodorum fungicide resistance, the increasing use of reduced tillage agronomic practices, and high evolutionary potential of the pathogen, combined with changes in climate and agricultural environments, mean that genetic resistance to SNB remains a high priority in many regions of wheat cultivation. In this review, we summarize current information on P. nodorum population structure and its implication for improved SNB management. We then review recent advances in the genetics of host resistance to P. nodorum and the necrotrophic effectors it secretes during infection, integrating the genomic positions of these genetic loci by using the recently released wheat reference genome assembly. Finally, we discuss the genetic and genomic tools now available for SNB resistance breeding and consider future opportunities and challenges in crop health management by using the wheat–P. nodorum interaction as a model.

Plant‐pathogenic fungi are a significant threat to economic and food security worldwide. Novel protection strategies are required and therefore it is critical we understand the mechanisms by which these pathogens cause disease. Virulence factors and pathogenicity genes have been identified, but in many cases their roles remain elusive. It is becoming increasingly clear that gene regulation is vital to enable plant infection and transcription factors play an essential role. Efforts to determine their regulatory functions in plant‐pathogenic fungi have expanded since the annotation of fungal genomes revealed the ubiquity of transcription factors from a broad range of families. This review establishes the significance of transcription factors as regulatory elements in plant‐pathogenic fungi and provides a systematic overview of those that have been functionally characterized. Detailed analysis is provided on regulators from well‐characterized families controlling various aspects of fungal metabolism, development, stress tolerance, and the production of virulence factors such as effectors and secondary metabolites. This covers conserved transcription factors with either specialized or nonspecialized roles, as well as recently identified regulators targeting key virulence pathways. Fundamental knowledge of transcription factor regulation in plant‐pathogenic fungi provides avenues to identify novel virulence factors and improve our understanding of the regulatory networks linked to pathogen evolution, while transcription factors can themselves be specifically targeted for disease control. Areas requiring further insight regarding the molecular mechanisms and/or specific classes of transcription factors are identified, and direction for future investigation is presented., Transcription factors in plant‐pathogenic fungi are systematically reviewed. This includes their classification and a current state of the functional research into their diverse regulatory roles pertaining to fungal virulence.

Background The necrotrophic fungal pathogen Pyrenophora tritici-repentis (Ptr) causes tan (syn. yellow) spot of wheat and accounts for significant yield losses worldwide. Understanding the molecular mechanisms of this economically important crop disease is crucial to counteract the yield and quality losses of wheat globally. Substantial progress has been made to comprehend the race structure of this phytopathogen based on its production of necrotrophic effectors and genomic resources of Ptr. However, one limitation for studying Ptr in a laboratory environment is the difficulty to isolate high spore numbers from vegetative growth with mycelial contamination common. These limitations reduce the experimental tractability of Ptr. Results Here, we optimized a multitude of parameters and report a sporulation method for Ptr that yields robust, high quality and pure spores. Our methodology encompasses simple and reproducible plugging and harvesting techniques, resulting in spore yields up to 1500 fold more than the current sporulation methods and was tested on multiple isolates and races of Ptr as well as an additional seven modern Australian Ptr isolates. Moreover, this method also increased purity and spore harvest numbers for two closely related fungal pathogens (Pyrenophora teres f. maculata and f. teres) that cause net blotch diseases in barley (Hordeum vulgare), highlighting the usability of this optimized sporulation protocol for the wider research community. Conclusions Large-scale spore infection and virulence assays are essential for the screening of wheat and barley cultivars and combined with the genetic mapping of these populations allows pinpointing and exploiting sources of host genetic resistance. We anticipate that improvements in spore numbers and purity will further advance research to increase our understanding of the pathogenicity mechanisms of these important fungal pathogens.

BACKGROUNDPhytopathogenic oomycetes constitute some of the most devastating plant pathogens that cause significant crop and horticultural loss. Phytophthora cinnamomi is a phytopathogenic oomycete that causes dieback disease in native vegetation and a variety of crops. This pathogen can survive through harsh environmental conditions which gives it an advantage over its susceptible hosts. The only implemented chemical used to control P. cinnamomi is the oomyceticide phosphite. Despite its widespread use, the mode of action of phosphite is not well understood and it is unclear whether it works directly on the pathogen or through the host. Additionally, resistance to phosphite is emerging in P. cinnamomi isolates and other oomycete phytopathogens.RESULTSThe mode of action of phosphite on the pathogen and through a model host was investigated using label-free quantitative proteomics. In vitro treatment of P. cinnamomi with phosphite hinders growth by interfering with metabolism, signalling and gene expression, traits that are not observed in the tolerant isolate. When the model host L. angustifolius was treated with phosphite, enrichment of proteins that are associated with photosynthesis, carbon fixation and lipid metabolism in the host was observed. An increase in the production of a range of defence-related proteins was observed.CONCLUSIONWe hypothesise direct and indirect models of the multi-modal action of phosphite that directly targets the pathogen as well as alters plant metabolism and immune response.

Key message We identified allelic variation at two major loci, QSnb.nmbu-2A.1 and QSnb.nmbu-5A.1, showing consistent and additive effects on SNB field resistance. Validation of QSnb.nmbu-2A.1 across genetic backgrounds further highlights its usefulness for marker-assisted selection. Abstract Septoria nodorum blotch (SNB) is a disease of wheat (Triticum aestivum and T. durum) caused by the necrotrophic fungal pathogen Parastagonospora nodorum. SNB resistance is a typical quantitative trait, controlled by multiple quantitative trait loci (QTL) of minor effect. To achieve increased plant resistance, selection for resistance alleles and/or selection against susceptibility alleles must be undertaken. Here, we performed genetic analysis of SNB resistance using an eight-founder German Multiparent Advanced Generation Inter-Cross (MAGIC) population, termed BMWpop. Field trials and greenhouse testing were conducted over three seasons in Norway, with genetic analysis identifying ten SNB resistance QTL. Of these, two QTL were identified over two seasons: QSnb.nmbu-2A.1 on chromosome 2A and QSnb.nmbu-5A.1 on chromosome 5A. The chromosome 2A BMWpop QTL co-located with a robust SNB resistance QTL recently identified in an independent eight-founder MAGIC population constructed using varieties released in the United Kingdom (UK). The validation of this SNB resistance QTL in two independent multi-founder mapping populations, regardless of the differences in genetic background and agricultural environment, highlights the value of this locus in SNB resistance breeding. The second robust QTL identified in the BMWpop, QSnb.nmbu-5A.1, was not identified in the UK MAGIC population. Combining resistance alleles at both loci resulted in additive effects on SNB resistance. Therefore, using marker assisted selection to combine resistance alleles is a promising strategy for improving SNB resistance in wheat breeding. Indeed, the multi-locus haplotypes determined in this study provide markers for efficient tracking of these beneficial alleles in future wheat genetics and breeding activities.

The regulation of virulence in plant-pathogenic fungi has emerged as a key area of importance underlying host infections. Recent work has highlighted the role of transcription factors (TFs) that mediate the expression of virulence-associated genes. A prominent example is Pf2, a member of the Zn2Cys6 family of fungal TFs, where orthologues regulate the expression of genes linked to parasitism in several plant-pathogen lineages. These include PnPf2 which controls effector-gene expression in Parastagonospora nodorum, thereby determining the outcome of effector-triggered susceptibility on its host, wheat. PnPf2 is a promising target for disease suppression but the genomic targets, or whether other are regulators involved, remain unknown. This study used chromatin immunoprecipitation (ChIP-seq) and a mutagenesis analysis to investigate these components. Two distinct binding motifs connected to positive gene-regulation were characterised and genes directly targeted by PnPf2 were identified. These included genes encoding major effectors and other components associated with the P. nodorum pathogenic lifestyle, such as carbohydrate-active enzymes and nutrient assimilators. This supports a direct involvement of PnPf2 in coordinating virulence on wheat. Other TFs were also prominent PnPf2 targets, suggesting it also operates within a transcriptional network. Several TFs were therefore functionally investigated in connection to fungal virulence. Distinct metabolic and developmental roles were evident for the newly characterised PnPro1, PnAda1, PnEbr1 and the carbon-catabolite repressor PnCreA. Overall, the results uphold PnPf2 as the central transcriptional regulator orchestrating genes that contribute to virulence on wheat and provide mechanistic insight into how this occurs.ImportanceFungal pathogens cause large crop losses worldwide and consequently much attention has focused on improving host genetic resistance to diseases. These pathogens use effectors, which require coordinated expression at specific stages of the pathogenic lifecycle, to manipulate the host plant metabolism in favour of infection. However, our understanding of the underlying regulatory network in coordination with other genes involved in fungal pathogenicity is lacking. The Pf2 TF orthologues are key players underpinning virulence and effector gene expression in several fungal phytopathogens, including P. nodorum. This study provided significant insight into the DNA-binding regulatory mechanisms of P. nodorum PnPf2, as well as further evidence that it is central to the coordination of virulence. In the context of crop protection, the Pf2 taxonomic orthologues present opportune targets in major fungal pathogens that can be perturbed to reduce the impact of effector triggered-susceptibility and improve disease resistance.

Plant-pathogenic fungi span diverse taxonomic lineages. Their host-infection strategies are often specialised and require the coordinated regulation of molecular virulence factors. Transcription factors (TFs) are fundamental regulators of gene expression, controlling development and virulence in plant pathogenic fungi. Recent research has established regulatory roles for several taxonomically conserved fungal TFs, but the evolution of specific virulence regulators is not well understood. This study sought to explore the representation of TFs across a taxonomically-diverse range of fungi, with a focus on plant pathogens. A significant trend was observed among the obligate, host-associated pathogens, which possess a reduced overall TF repertoire, alluding to a lack of pressure for maintaining diversity. A novel orthology-based analysis is then presented that refined TF classifications, traditionally based on the nature of the DNA-binding domains. Using this analysis, cases of TF over/underrepresentation across fungal pathogen lineages are systematically highlighted. Specific examples are then explored and discussed that included the TF orthologues of Ste12, Pf2 and EBR1, plus phytotoxic secondary-metabolite cluster regulators, which all presented novel and distinct evolutionary insights. Ultimately, as the examples presented demonstrate, this resource can be interrogated to guide functional studies that seek to characterise virulence-specific regulators and shed light on the factors underpinning plant pathogenicity.

Phytopathogenic oomycetes pose a significant threat to global biodiversity and food security. The proteomes of these oomycetes likely contain important factors that contribute to their pathogenic success, making their discovery crucial for elucidating pathogenicity. Oomycetes secrete effector proteins that overcome or elicit a defence response in susceptible hosts. Phytophthora cinnamomi is a root pathogen that causes dieback in a wide variety of crops and a range of native vegetation world-wide. Virulence proteins produced by P. cinnamomi are not well defined and a large-scale approach to understand the biochemistry of this pathogen has not been documented. Here, soluble mycelial, zoospore and secreted proteomes were obtained and label-free quantitative proteomics was used to compare the composition of protein content of the three sub-proteomes by matching the MS/MS data to a sequenced P. cinnamomi genome. Mass spectra matched to a total of 4635 proteins, validating 17.7% of the predicted gene set of the P. cinnamomi genome. The mycelia were abundant in transporters for nutrient acquisition, metabolism and cellular proliferation. The zoospores had less metabolic related ontologies than the mycelia but were abundant in energy generating, motility and signalling associated proteins. Virulence-associated proteins were identified in the secretome such as candidate effector and effector-like proteins, which interfere with the host immune system. These include hydrolases, cell wall degrading enzymes, putative necrosis-inducing proteins and elicitins. The secretome elicited a hypersensitive response on the roots of a model host and thus suggests evidence of effector activity.SignificancePhytophthora cinnamomi is a phytopathogenic oomycete that causes dieback disease in native vegetation and several horticultural crops such as avocado, pineapple and macadamia. Whilst this pathogen has significance world-wide, its pathogenicity and virulence have not been described in depth. We carried out comparative label-free proteomics of the mycelia, zoospores and secretome of P. cinnamomi. This study highlights the differential metabolism and cellular processes between the sub-proteomes. Proteins associated with metabolism, nutrient transport and cellular proliferation were over represented in the mycelia. The zoospores have a specialised proteome showing increased energy generation geared towards motility. Candidate effectors and effector-like secreted proteins were also identified, which can be exploited for genetic resistance. This demonstrates a better understanding of the biology and pathogenicity of P. cinnamomi infection that can subsequently be used to develop effective methods of disease management

The wheat pathogen Parastagonospora nodorum has emerged as a model necrotrophic fungal species with growing genomic resources. Recent population-level pan-genome studies were leveraged to provide novel insights into pathogen evolution and effector-like gene contents relevant to local crop disease outbreaks. In this study, we examined 156 isolates representing a regional population from the Western Australian (WA) wheat-belt region, and 17 internationally sourced isolates. We observed a highly diverse local population, within which were numerous small and highly similar clusters of isolates from hotter and drier regions. Pan-genome assembly and orthologous gene datasets resulted in 3579 predicted effector candidates, 2291 of which exhibited presence-absence variation (PAV) across the population, and 1362 were specific to WA isolates. There was an abundance of mutations (including repeat-induced point mutation (RIP)), distributed in ‘hot-spots’ within the pan-genomic landscape that were rich in effector candidates. Three characterised effector loci (ToxA, Tox1 and Tox3) were located within sub- telomeric regions of lower diversity, but were nestled within larger high-diversity regions. RIP was widespread across the genome, but non-synonymous RIP-like mutations were strongly selected against. These improved bioinformatic resources for P. nodorum, represent progressive advancements in fungal pan-genomics, with a view towards supporting region- specific surveillance of host-pathogen interactions.

Wheat (Triticum aestivum L.) yields are commonly affected by foliar infection by fungal pathogens. Of these, three wheat leaf blotch fungal diseases, septoria nodorum blotch (SNB), tan spot (TS) and septoria tritici blotch (STB), caused by Parastagonospora nodorum (Pn), Pyrenophora tritici-repentis (Ptr) and Zymoseptoria tritici (Zt), respectively, induce major yield losses. Infection results in necrotic areas on the leaf, and it is often difficult to determine the underlying causative pathogen from visible symptoms alone, especially in mixed infections. Here, a regional survey of 330 wheat samples collected across three seasons (years 2015–2017) from four north-west European countries was undertaken. Using quantitative polymerase chain reaction (qPCR) assays specific for each pathogen, as well as disease assessment of leaf materials, distinct regional differences were identified. Two-thirds (65%) of all samples harbored at least two of the three pathogens. Norway had high SNB abundance, but also showed mixed infections of SNB, TS and STB. In Germany, TS was prevalent, with STB also common. Danish samples commonly possessed all three pathogens, with STB prevalent, followed by TS and SNB. The UK had a major prevalence of STB with minimal occurrence of TS and SNB. Across all samples, qPCR identified Zt, Pn and Ptr in 90%, 54% and 57% of samples, respectively. For each pathogen, average disease levels via visual assessment showed modest positive correlation with fungal DNA concentrations (R2 = 0.13–0.32). Overall, our study highlights that the occurrence of mixed infection is common and widespread, with important implications for wheat disease management and breeding strategies.

Phytophthora cinnamomi is a pathogenic oomycete that causes plant dieback disease across a range of natural ecosystems and in many agriculturally important crops on a global scale. An annotated draft genome sequence and annotation is publicly available (JGI Mycocosm) and suggests 26,131 gene models. In this study, soluble mycelial, extracellular (secretome) and zoospore proteins of P. cinnamomi were exploited to refine the genome by correcting gene annotations and discovering novel genes. By implementing the diverse set of sub-proteomes into a generated proteogenomics pipeline, we were able to improve the P. cinnamomi genome. Liquid chromatography mass spectrometry was used to obtain high confidence peptides with spectral matching to both the annotated genome and a generated 6-frame translation. 2,764 annotations from the draft genome were confirmed by spectral matching. Using a proteogenomic pipeline, mass spectra were used to edit the P. cinnamomi genome and allowed identification of 23 new gene models and 60 edited gene features using high confidence peptides obtained by mass spectrometry, suggesting a rate of incorrect annotations of 3% of the detectable proteome. The novel features were further validated by total peptide support, alongside functional analysis including the use of Gene Ontology and functional domain identification. We demonstrated the use of spectral data in combination with our proteogenomics pipeline can be used to improve the genome of important plant diseases and identify biologically relevant missed genes. This study presents the first use of spectral data to edit and manually annotate an oomycete pathogen.

Background The fungus Parastagonospora nodorum causes septoria nodorum blotch (SNB) of wheat (Triticum aestivum) and is a model species for necrotrophic plant pathogens. The genome assembly of reference isolate Sn15 was first reported in 2007. P. nodorum infection is promoted by its production of proteinaceous necrotrophic effectors, three of which are characterised – ToxA, Tox1 and Tox3. Results A chromosome-scale genome assembly of P. nodorum Australian reference isolate Sn15, which combined long read sequencing, optical mapping and manual curation, produced 23 chromosomes with 21 chromosomes possessing both telomeres. New transcriptome data were combined with fungal-specific gene prediction techniques and manual curation to produce a high-quality predicted gene annotation dataset, which comprises 13,869 high confidence genes, and an additional 2534 lower confidence genes retained to assist pathogenicity effector discovery. Comparison to a panel of 31 internationally-sourced isolates identified multiple hotspots within the Sn15 genome for mutation or presence-absence variation, which was used to enhance subsequent effector prediction. Effector prediction resulted in 257 candidates, of which 98 higher-ranked candidates were selected for in-depth analysis and revealed a wealth of functions related to pathogenicity. Additionally, 11 out of the 98 candidates also exhibited orthology conservation patterns that suggested lateral gene transfer with other cereal-pathogenic fungal species. Analysis of the pan-genome indicated the smallest chromosome of 0.4 Mbp length to be an accessory chromosome (AC23). AC23 was notably absent from an avirulent isolate and is predominated by mutation hotspots with an increase in non-synonymous mutations relative to other chromosomes. Surprisingly, AC23 was deficient in effector candidates, but contained several predicted genes with redundant pathogenicity-related functions. Conclusions We present an updated series of genomic resources for P. nodorum Sn15 – an important reference isolate and model necrotroph – with a comprehensive survey of its predicted pathogenicity content.

The fungus Parastagonospora nodorum is the causal agent of septoria nodorum leaf blotch (SNB) and glume blotch which are common in many wheat growing regions in the world. The disease is complex and could be explained by multiple interactions between necrotrophic effectors secreted by the pathogen and matching susceptibility genes in wheat. An Australian P. nodorum population was clustered into five groups with contrasting properties. This study was set to identify their pathogenicity profiles using a diverse wheat panel of 134 accessions which are insensitive to SnToxA and SnTox1 in both in vitro and in vivo conditions. SNB seedling resistance/susceptibility to five representative isolates from the five clusters, responses to crude culture-filtrates (CFs) of three isolates and sensitivity to SnTox3 semi-purified effector together with 11,455 SNP markers have been used for linkage disequilibrium (LD) and association analyses. While quantitative trait loci (QTL) on 1D, 2A, 2B, 4B, 5B, 6A, 6B, 7A, 7D chromosomes were consistently detected across isolates and conditions, distinct patterns and isolate specific QTL were also observed among these isolates. In this study, SnTox3–Snn3-B1 interaction for the first time in Australia and SnTox3–Snn3-D1 interaction for the first time in bread wheat were found active using wild-type isolates. These findings could be due to new SnTox3 haplotype/isoform and exotic CIMMYT/ICARDA and Vavilov germplasm used, respectively. This study could provide useful information for dissecting novel and different SNB disease components, helping to prioritise research targets and contributing valuable information on genetic loci/markers for marker-assisted selection in SNB resistance wheat breeding programme.

Phytophthora cinnamomi is a pathogenic oomycete that causes plant dieback disease across a range of natural ecosystems and in many agriculturally important crops on a global scale. An annotated draft genome sequence is publicly available (JGI Mycocosm) and suggests 26,131 gene models. In this study, soluble mycelial, extracellular (secretome), and zoospore proteins of P. cinnamomi were exploited to refine the genome by correcting gene annotations and discovering novel genes. By implementing the diverse set of sub-proteomes into a generated proteogenomics pipeline, we were able to improve the P. cinnamomi genome annotation. Liquid chromatography mass spectrometry was used to obtain high confidence peptides with spectral matching to both the annotated genome and a generated 6-frame translation. Two thousand seven hundred sixty-four annotations from the draft genome were confirmed by spectral matching. Using a proteogenomic pipeline, mass spectra were used to edit the P. cinnamomi genome and allowed identification of 23 new gene models and 60 edited gene features using high confidence peptides obtained by mass spectrometry, suggesting a rate of incorrect annotations of 3% of the detectable proteome. The novel features were further validated by total peptide support, alongside functional analysis including the use of Gene Ontology and functional domain identification. We demonstrated the use of spectral data in combination with our proteogenomics pipeline can be used to improve the genome annotation of important plant diseases and identify missed genes. This study presents the first use of spectral data to edit and manually annotate an oomycete pathogen.

Additional file 9: Supplementary Table 6. Properties of selected large regions of the Sn15 assembly exhibiting presence absence variation (PAV) across the Parastagonospora population.

Additional file 14: Supplementary Table 11. Summary of gene and effector candidate gene distances from nearest AT-rich regions in the P. nodorum Sn15 assembly.

Additional file 16: Supplementary Table 13. Summary of gene content and functional annotation for P. nodorum Sn15 accessory chromosome 23 (AC23).

Additional file 11: Supplementary Table 8. Summary of scaffold sequences from Syme et al. 2013 corresponding to chromosomes of new optical map-assisted long-read genome assembly for Parastagonospora nodorum Sn15

Additional file 4: Supplementary Table 1. Summary of draft (A) and high-quality (B) genome assemblies of Parastagonospora spp. alternate isolates used in this study for comparative genomics versus the Australian reference isolate Sn15.

Additional file 8: Supplementary Table 5. Summary of assembled sequence lengths in the new P. nodorum Sn15 genome assembly, and estimates of their potential to be unresolved by PFGE comparing a 1% size error range to the size difference with the next longest sequence.

Additional file 3: Supplementary Figure 3. Alignment of locally collinear blocks (LCBs) via Mauve, indicating large sections of similarity with structural rearrangements between Chromosome 4 of P. nodorum isolate Sn15 (top) and corresponding chromosomal sequences of isolates Sn4, Sn2000, and Sn79–1087, presented at the whole chromosome level (A) and within ~ 700–800 Kb of the telomere (B).

Additional file 2: Supplementary Figure 2. Comparison of non-repetitive regions of accessory chromosome 23 (AC23, black) of > 100 bp in length (grey arcs) to P. tritici-repentis BFP chromosomes 1,3 4, and 11 (red), P. tritici-repentis M4 chromosomes 1, 3, 4, 6 and 10 (green), Bipolaris maydis scaffold 16 (blue) and B. sorokiniana chromosomes 2, 4, and 9. This comparison indicated a trend of telomeric proximity in the relative matching regions of related species.

Additional file 12: Supplementary Table 9. Summary of genes and their functional annotations within the Chromosome 8 PAV region.

Additional file 17: Supplementary Text 1. Notes on the P. nodorum Sn15 genome assembly and the integration of optical mapping data.

Additional file 5: Supplementary Table 2. Comparison of repetitive sequence masking in the new P. nodorum Sn15 genome assembly using 3 different repeat libraries applied sequentially in 3 iterations.

Additional file 1: Supplementary Figure 1. Comparison of non-repetitive regions of accessory chromosome 23 (AC23, red) to other Sn15 chromosomes (black), indicating that it is not the product of duplication of a core, sister chromosome. The GC content of AC23 is indicated by the linear plot, and local repeat density is indicated in the heat map below (red). Nucleotide matches > 200 bp are indicated by grey arcs

Additional file 13: Supplementary Table 10. Summary of average SNP density and DN/DS selection metrics across the Parastagonospora spp. population, relative to the P. nodorum Sn15 reference genome assembly. )

We report a fungal pan-genome study involving Parastagonospora spp., including 21 isolates of the wheat (Triticum aestivum) pathogen Parastagonospora nodorum, 10 of the grass-infecting Parastagonospora avenae, and 2 of a closely related undefined sister species. We observed substantial variation in the distribution of polymorphisms across the pan-genome, including repeat-induced point mutations, diversifying selection and gene gains and losses. We also discovered chromosome-scale inter and intraspecific presence/absence variation of some sequences, suggesting the occurrence of one or more accessory chromosomes or regions that may play a role in host-pathogen interactions. The presence of known pathogenicity effector loci SnToxA, SnTox1, and SnTox3 varied substantially among isolates. Three P. nodorum isolates lacked functional versions for all three loci, whereas three P. avenae isolates carried one or both of the SnTox1 and SnTox3 genes, indicating previously unrecognized potential for discovering additional effectors in the P. nodorum-wheat pathosystem. We utilized the pan-genomic comparative analysis to improve the prediction of pathogenicity effector candidates, recovering the three confirmed effectors among our top-ranked candidates. We propose applying this pan-genomic approach to identify the effector repertoire involved in other host-microbe interactions involving necrotrophic pathogens in the Pezizomycotina.

Host genetic resistance is the most effective and sustainable means of managing tan spot or yellow spot of wheat. The disease is becoming increasingly problematic due to the adoption of minimum tillage practices, evolution of effector-mediated pathogenicity, and widespread cultivation of susceptible cultivars from a narrow genetic base. This highlights the importance of broadening the diversity of resistance factors in modern breeding germplasm. We explored 300 genetically diverse wheat accessions, originally sourced from the N. I. Vavilov Institute of Plant Genetic Resources (VIR), St. Petersburg, Russia. The collection was screened for resistance to tan spot at seedling and adult stage under controlled conditions, and in the field across two years. The phenotypic datasets, coupled with ToxA bioassay screening, identified a number of accessions with useful sources of resistance. Seedling disease response corresponded well with ToxA sensitivity (r = 0.49, P < 0.000), but not adult responses (r = -0.02–0.19, P < 0.002), and overall reactions to ToxA appeared to show poor correspondence with disease response at the adult stage. ToxA-insensitive accessions were generally found resistant across different growth stages (all-stage resistant, ASR) in all experiments (seedling and adult stage under controlled conditions and field). ToxA-sensitive accessions and susceptible at seedling stage, but resistant at both adult-plant stages, were deemed to carry adult-plant resistance (APR). We provide detailed information on the degree of tan spot resistance in the Vavilov wheat collection and discuss strategies to harness these sources to boost the diversity of resistance factors in modern wheat breeding germplasm. This article is protected by copyright. All rights reserved.

Key message The fungus Parastagonospora nodorum causes Septoria nodorum blotch (SNB) of wheat. A genetically diverse wheat panel was used to dissect the complexity of SNB and identify novel sources of resistance. Abstract The fungus Parastagonospora nodorum is the causal agent of Septoria nodorum blotch (SNB) of wheat. The pathosystem is mediated by multiple fungal necrotrophic effector–host sensitivity gene interactions that include SnToxA–Tsn1, SnTox1–Snn1, and SnTox3–Snn3. A P. nodorum strain lacking SnToxA, SnTox1, and SnTox3 (toxa13) retained wild-type-like ability to infect some modern wheat cultivars, suggesting evidence of other effector-mediated susceptibility gene interactions or the lack of host resistance genes. To identify genomic regions harbouring such loci, we examined a panel of 295 historic wheat accessions from the N. I. Vavilov Institute of Plant Genetic Resources in Russia, which is comprised of genetically diverse landraces and breeding lines registered from 1920 to 1990. The wheat panel was subjected to effector bioassays, infection with P. nodorum wild type (SN15) and toxa13. In general, SN15 was more virulent than toxa13. Insensitivity to all three effectors contributed significantly to resistance against SN15, but not toxa13. Genome-wide association studies using phenotypes from SN15 infection detected quantitative trait loci (QTL) on chromosomes 1BS (Snn1), 2DS, 5AS, 5BS (Snn3), 3AL, 4AL, 4BS, and 7AS. For toxa13 infection, a QTL was detected on 5AS (similar to SN15), plus two additional QTL on 2DL and 7DL. Analysis of resistance phenotypes indicated that plant breeders may have inadvertently selected for effector insensitivity from 1940 onwards. We identify accessions that can be used to develop bi-parental mapping populations to characterise resistance-associated alleles for subsequent introgression into modern bread wheat to minimise the impact of SNB. Electronic supplementary material The online version of this article (10.1007/s00122-018-3073-y) contains supplementary material, which is available to authorized users.

Key message A locus on wheat chromosome 2A was found to control field resistance to both leaf and glume blotch caused by the necrotrophic fungal pathogen Parastagonospora nodorum. Abstract The necrotrophic fungal pathogen Parastagonospora nodorum is the causal agent of Septoria nodorum leaf blotch and glume blotch, which are common wheat (Triticum aestivum L.) diseases in humid and temperate areas. Susceptibility to Septoria nodorum leaf blotch can partly be explained by sensitivity to corresponding P. nodorum necrotrophic effectors (NEs). Susceptibility to glume blotch is also quantitative; however, the underlying genetics have not been studied in detail. Here, we genetically map resistance/susceptibility loci to leaf and glume blotch using an eight-founder wheat multiparent advanced generation intercross population. The population was assessed in six field trials across two sites and 4 years. Seedling infiltration and inoculation assays using three P. nodorum isolates were also carried out, in order to compare quantitative trait loci (QTL) identified under controlled conditions with those identified in the field. Three significant field resistance QTL were identified on chromosomes 2A and 6A, while four significant seedling resistance QTL were detected on chromosomes 2D, 5B and 7D. Among these, QSnb.niab-2A.3 for field resistance to both leaf blotch and glume blotch was detected in Norway and the UK. Colocation with a QTL for seedling reactions against culture filtrate from a Norwegian P. nodorum isolate indicated the QTL could be caused by a novel NE sensitivity. The consistency of this QTL for leaf blotch at the seedling and adult plant stages and culture filtrate infiltration was confirmed by haplotype analysis. However, opposite effects for the leaf blotch and glume blotch reactions suggest that different genetic mechanisms may be involved.

OPEN ACCESS; International audience; Introduction: Septoria nodorum blotch (SNB) is a complex fungal disease of wheat caused by the Dothideomycete fungal pathogen Parastagonospora nodorum. The fungus infects through the use of necrotrophic effectors (NEs) that cause necrosis on hosts carrying matching dominant susceptibility genes. The Western Australia (WA) wheatbelt is a SNB “hot spot” and experiences significant under favorable conditions. Consequently, SNB has been a major target for breeders in WA for many years.Materials and Methods: In this study, we assembled a panel of 155 WA P. nodorum isolates collected over a 44-year period and compared them to 23 isolates from France and the USA using 28 SSR loci.Results: The WA P. nodorum population was clustered into five groups with contrasting properties. 80% of the studied isolates were assigned to two core groups found throughout the collection location and time. The other three non-core groups that encompassed transient and emergent populations were found in restricted locations and time. Changes in group genotypes occurred during periods that coincided with the mass adoption of a single or a small group of widely planted wheat cultivars. When introduced, these cultivars had high scores for SNB resistance. However, the field resistance of these new cultivars often declined over subsequent seasons prompting their replacement with new, more resistant varieties. Pathogenicity assays showed that newly emerged isolates non-core are more pathogenic than old isolates. It is likely that the non-core groups were repeatedly selected for increased virulence on the contemporary popular cultivars.Discussion: The low level of genetic diversity within the non-core groups, difference in virulence, low abundance, and restriction to limited locations suggest that these populations more vulnerable to a population crash when the cultivar was replaced by one that was genetically different and more resistant. We characterize the observed pattern as a low-amplitude boom-and-bust cycle in contrast with the classical high amplitude boom-and-bust cycles seen for biotrophic pathogens where the contrast between resistance and susceptibility is typically much greater. Implications of the results are discussed relating to breeding strategies for more sustainable SNB resistance and more generally for pathogens with NEs.

The fungus Parastagonospora nodorum infects wheat through the use of necrotrophic effector (NE) proteins that cause host-specific tissue necrosis. The Zn2Cys6 transcription factor PnPf2 positively regulates NE gene expression and is required for virulence on wheat. Little is known about other downstream targets of PnPf2. We compared the transcriptomes of the P. nodorum wildtype and a strain deleted in PnPf2 (pf2-69) during in vitro growth and host infection to further elucidate targets of PnPf2 signalling. Gene ontology enrichment analysis of the differentially expressed (DE) genes revealed that genes associated with plant cell wall degradation and proteolysis were enriched in down-regulated DE gene sets in pf2-69 compared to SN15. In contrast, genes associated with redox control, nutrient and ion transport were up-regulated in the mutant. Further analysis of the DE gene set revealed that PnPf2 positively regulates twelve genes that encode effector-like proteins. Two of these genes encode proteins with homology to previously characterised effectors in other fungal phytopathogens. In addition to modulating effector gene expression, PnPf2 may play a broader role in the establishment of a necrotrophic lifestyle by orchestrating the expression of genes associated with plant cell wall degradation and nutrient assimilation.

The fungus Parastagonospora nodorum is the causal agent of Septoria nodorum blotch of wheat (Triticum aestivum). The interaction is mediated by multiple fungal necrotrophic effector-dominant host sensitivity gene interactions. The three best-characterized effector-sensitivity gene systems are SnToxA-Tsn1, SnTox1-Snn1 and SnTox3-Snn3. These effector genes are highly expressed during early infection, but expression decreases as the infection progresses to tissue necrosis and sporulation. However, the mechanism of regulation is unknown. We have identified and functionally characterized a gene, referred to as PnPf2, which encodes a putative zinc finger transcription factor. PnPf2 deletion resulted in the down-regulation of SnToxA and SnTox3 expression. Virulence on Tsn1 and Snn3 wheat cultivars was strongly reduced. The SnTox1-Snn1 interaction remained unaffected. Furthermore, we have also identified and deleted an orthologous PtrPf2 from the tan spot fungus Pyrenophora tritici-repentis which possesses a near-identical ToxA that was acquired from P. nodorum via horizontal gene transfer. PtrPf2 deletion also resulted in the down-regulation of PtrToxA expression and a near-complete loss of virulence on Tsn1 wheat. We have demonstrated, for the first time, evidence for a functionally conserved signalling component that plays a role in the regulation of a common/horizontally transferred effector found in two major fungal pathogens of wheat.

This study aimed to compare the depth and reproducibility of total proteome and differentially expressed protein coverage in technical duplicates and triplicates using iTRAQ 4-plex, iTRAQ 8-plex, and TMT 6-plex reagents. The analysis was undertaken because comprehensive comparisons of isobaric mass tag reproducibility have not been widely reported in the literature. The highest number of proteins was identified with 4-plex, followed by 8-plex and then 6-plex reagents. Quantitative analyses revealed that more differentially expressed proteins were identified with 4-plex reagents than 8-plex reagents and 6-plex reagents. Replicate reproducibility was determined to be ≥69% for technical duplicates and ≥57% for technical triplicates. The results indicate that running an 8-plex or 6-plex experiment instead of a 4-plex experiment resulted in 26 or 39% fewer protein identifications, respectively. When 4-plex spectra were searched with three software tools-ProteinPilot, Mascot, and Proteome Discoverer-the highest number of protein identifications were obtained with Mascot. The analysis of negative controls demonstrated the importance of running experiments as replicates. Overall, this study demonstrates the advantages of using iTRAQ 4-plex reagents over iTRAQ 8-plex and TMT 6-plex reagents, provides estimates of technical duplicate and triplicate reproducibility, and emphasizes the value of running replicate samples.

National audience

This chapter compiled tabulated ultimate axial resistance, as well as axial and bending moment resistance (about major axis) under balanced failure for commonly used rectangular columns reinforced by up to 16 numbers of 16-mm-diameter rebar in all possible configurations. The column sizes ranged from 150 to 600 mm width with depth-to-width ratio from 1:1 to 4:1. For a compression member, concrete strength plays essential role. Therefore, the design data is tabulated based on concrete grade: C30/37, C40/50 and C50/60 to provide flexibility for designer. Ultimate axial resistance will aid the engineer to decide the size and reinforcement of an axially loaded column, whereas the axial and bending moment resistance under balanced failure provide insight in column capacity when it is subjected to bending moment.

This chapter compiled tabulated ultimate axial resistance, as well as axial and bending moment resistance (about major axis) under balanced failure for commonly used rectangular columns reinforced by up to 16 numbers of 12-mm diameter rebar in all possible configurations. The column sizes ranged from 150 to 600 mm width with depth-to-width ratio from 1:1 to 4:1. For a compression member, concrete strength plays essential role. Therefore, the design data is tabulated based on concrete grade: C30/37, C40/50 and C50/60 to provide flexibility for designer. Ultimate axial resistance will aid the engineer to decide the size and reinforcement of an axially loaded column, whereas the axial and bending moment resistance under balanced failure provide insight in column capacity when it is subjected to bending moment.

Core Ideas First genome‐wide association mapping of adult plant Septoria nodorum blotch resistance. Some adult plant resistance loci were shared with seedling resistance loci. Other adult plant resistance loci were significant across environments. Resistant haplotypes were identified, which can be used for breeding. Parastagonospora nodorum is the causal agent of Septoria nodorum leaf blotch (SNB) in wheat (Triticum aestivum L.). It is the most important leaf blotch pathogen in Norwegian spring wheat. Several quantitative trait loci (QTL) for SNB susceptibility have been identified. Some of these QTL are the result of underlying gene‐for‐gene interactions involving necrotrophic effectors (NEs) and corresponding sensitivity (Snn) genes. A collection of diverse spring wheat lines was evaluated for SNB resistance and susceptibility over seven growing seasons in the field. In addition, wheat seedlings were inoculated and infiltrated with culture filtrates (CFs) from four single spore isolates and infiltrated with semipurified NEs (SnToxA, SnTox1, and SnTox3) under greenhouse conditions. In adult plants, the most stable SNB resistance QTL were located on chromosomes 2B, 2D, 4A, 4B, 5A, 6B, 7A, and 7B. The QTL on chromosome 2D was effective most years in the field. At the seedling stage, the most significant QTL after inoculation were located on chromosomes 1A, 1B, 3A, 4B, 5B, 6B, 7A, and 7B. The QTL on chromosomes 3A and 6B were significant both after inoculation and CF infiltration, indicating the presence of novel NE–Snn interactions. The QTL on chromosomes 4B and 7A were significant in both seedlings and adult plants. Correlations between SnToxA sensitivity and disease severity in the field were significant. To our knowledge, this is the first genome‐wide association mapping study (GWAS) to investigate SNB resistance at the adult plant stage under field conditions.

This chapter compiled tabulated ultimate axial resistance, as well as axial and bending moment resistances (about major axis) under balanced failure for commonly used rectangular columns reinforced by up to 16 numbers of 20-mm-diameter rebar in all possible configurations. The column sizes ranged from 150 to 600 mm width with depth-to-width ratio from 1:1 to 4:1. For a compression member, concrete strength plays essential role. Therefore, the design data is tabulated based on concrete grade: C30/37, C40/50 and C50/60 to provide flexibility for designer. Ultimate axial resistance will aid the engineer to decide the size and reinforcement of an axially loaded column, whereas the axial and bending moment resistances under balanced failure provide insight in column capacity when it is subjected to bending moment.

This chapter compiled tabulated ultimate axial resistance, as well as axial and bending moment resistance (about major axis) under balanced failure for commonly used rectangular columns reinforced by up to 16 numbers of 32-mm-diameter rebar in all possible configurations. The column sizes ranged from 150 to 600 mm width with depth to width ratio from 1:1 to 4:1. For a compression member, concrete strength plays essential role. Therefore, the design data is tabulated based on concrete grade: C30/37, C40/50 and C50/60 to provide flexibility for designer. Ultimate axial resistance will aid the engineer to decide the size and reinforcement of an axially loaded column, whereas the axial and bending moment resistance under balanced failure provide insight in column capacity when it is subjected to bending moment.

This chapter compiled tabulated ultimate axial resistance, as well as axial and bending moment resistances (about major axis) under balanced failure for commonly used rectangular columns reinforced by up to 16 numbers of 25-mm-diameter rebar in all possible configurations. The column sizes ranged from 150 to 600 mm width with depth-to-width ratio from 1:1 to 4:1. For a compression member, concrete strength plays an essential role. Therefore, the design data is tabulated based on concrete grade C30/37, C40/50 and C50/60 to provide flexibility for designer. Ultimate axial resistance will aid the engineer to decide the size and reinforcement of an axially loaded column, whereas the axial and bending moment resistances under balanced failure provide insight into column capacity when it is subjected to bending moment.

Parastagonospora nodorum is a necrotrophic fungal pathogen of wheat (Triticum aestivum L.), one of the world’s most important crops. P. nodorum mediates host cell death using proteinaceous necrotrophic effectors, presumably liberating nutrients that allow the infection process to continue. The identification of pathogen effectors has allowed host genetic resistance mechanisms to be separated into their constituent parts. In P. nodorum, three proteinaceous effectors have been cloned: SnToxA, SnTox1, and SnTox3. Here, we survey sensitivity to all three effectors in a panel of 480 European wheat varieties, and fine-map the wheat SnTox3 sensitivity locus Snn3-B1 using genome-wide association scans (GWAS) and an eight-founder wheat multi-parent advanced generation inter-cross (MAGIC) population. Using a Bonferroni corrected P ≤ 0.05 significance threshold, GWAS identified 10 significant markers defining a single locus, Snn3-B1, located on the short arm of chromosome 5B explaining 32% of the phenotypic variation [peak single nucleotide polymorphisms (SNPs), Excalibur_c47452_183 and GENE-3324_338, -log10P = 20.44]. Single marker analysis of SnTox3 sensitivity in the MAGIC population located Snn3-B1 via five significant SNPs, defining a 6.2-kb region that included the two peak SNPs identified in the association mapping panel. Accordingly, SNP Excalibur_c47452_183 was converted to the KASP genotyping system, and validated by screening a subset of 95 wheat varieties, providing a valuable resource for marker assisted breeding and for further genetic investigation. In addition, composite interval mapping in the MAGIC population identified six minor SnTox3 sensitivity quantitative trait loci, on chromosomes 2A (QTox3.niab-2A.1, P-value = 9.17-7), 2B (QTox3.niab-2B.1, P = 0.018), 3B (QTox3.niab-3B.1, P = 48.51-4), 4D (QTox3.niab-4D.1, P = 0.028), 6A (QTox3.niab-6A.1, P = 8.51-4), and 7B (QTox3.niab-7B.1, P = 0.020), each accounting for between 3.1 and 6.0 % of the phenotypic variance. Collectively, the outcomes of this study provides breeders with knowledge and resources regarding the sensitivity of European wheat germplasm to P. nodorum effectors, as well as simple diagnostic markers for determining allelic state at Snn3-B1.

Fungal pathogen genomes can often be divided into core and accessory regions. Accessory regions ARs) may be comprised of either ARs (within core chromosomes (CCs) or wholly dispensable (accessory) chromosomes (ACs). Fungal ACs and ARs typically accumulate mutations and structural rearrangements more rapidly over time than CCs and many harbor genes relevant to host-pathogen interactions. These regions are of particular interest in plant pathology and include host-specific virulence factors and secondary metabolite synthesis gene clusters. This review outlines known ACs and ARs in fungal genomes, methods used for their detection, their common properties that differentiate them from the core genome, and what is currently known of their various roles in pathogenicity. Reports on the evolutionary processes generating and shaping AC and AR compartments are discussed, including repeat induced point mutation and breakage fusion bridge cycles. Previously ACs have been studied extensively within key genera, including Fusarium, Zymoseptoria, and Alternaria, but are growing in frequency of observation and perceived importance across a wider range of fungal species. Recent advances in sequencing technologies permit affordable genome assembly and resequencing of populations that will facilitate further discovery and routine screening of ACs.

The necrotrophic fungus Parastagonospora nodorum is an important pathogen of one of the world’s most economically important cereal crops, wheat (Triticum aestivum L.). P. nodorum produces necrotrophic protein effectors that mediate host cell death, providing nutrients for continuation of the infection process. The recent discovery of pathogen effectors has revolutionized disease resistance breeding for necrotrophic diseases in crop species, allowing often complex genetic resistance mechanisms to be broken down into constituent parts. To date, three effectors have been identified in P. nodorum. Here we use the effector, SnTox1, to screen 642 progeny from an eight-parent multiparent advanced generation inter-cross (i.e., MAGIC) population, genotyped with a 90,000-feature single-nucleotide polymorphism array. The MAGIC founders showed a range of sensitivity to SnTox1, with transgressive segregation evident in the progeny. SnTox1 sensitivity showed high heritability, with quantitative trait locus analyses fine-mapping the Snn1 locus to the short arm of chromosome 1B. In addition, a previously undescribed SnTox1 sensitivity locus was identified on the long arm of chromosome 5A, termed here QSnn.niab-5A.1. The peak single-nucleotide polymorphism for the Snn1 locus was converted to the KASP genotyping platform, providing breeders and researchers a simple and cheap diagnostic marker for allelic state at Snn1.