Pyricularia oryzae: Lab star and field scourge.

Pyricularia oryzae (syn. Magnaporthe oryzae), is a filamentous ascomycete that causes a major disease called blast on cereal crops, as well as on a wide variety of wild and cultivated grasses. Blast diseases have a tremendous impact worldwide particularly on rice and on wheat, where the disease emerged in South America in the 1980s, before spreading to Asia and Africa. Its economic importance, coupled with its amenability to molecular and genetic manipulation, have inspired extensive research efforts aiming at understanding its biology and evolution. In the past 40 years, this plant-pathogenic fungus has emerged as a major model in molecular plant-microbe interactions. In this review, we focus on the clarification of the taxonomy and genetic structure of the species and its host range determinants. We also discuss recent molecular studies deciphering its lifecycle. TAXONOMY: Kingdom: Fungi, phylum: Ascomycota, sub-phylum: Pezizomycotina, class: Sordariomycetes, order: Magnaporthales, family: Pyriculariaceae, genus: Pyricularia. HOST RANGE: P. oryzae has the ability to infect a wide range of Poaceae. It is structured into different host-specialized lineages that are each associated with a few host plant genera. The fungus is best known to cause tremendous damage to rice crops, but it can also attack other economically important crops such as wheat, maize, barley, and finger millet. DISEASE SYMPTOMS: P. oryzae can cause necrotic lesions or bleaching on all aerial parts of its host plants, including leaf blades, sheaths, and inflorescences (panicles, spikes, and seeds). Characteristic symptoms on leaves are diamond-shaped silver lesions that often have a brown margin and whose appearance is influenced by numerous factors such as the plant genotype and environmental conditions. USEFUL WEBSITES Resources URL Genomic data repositories http://genome.jouy.inra.fr/gemo/ Genomic data repositories http://openriceblast.org/ Genomic data repositories http://openwheatblast.net/ Genome browser for fungi (including P. oryzae) http://fungi.ensembl.org/index.html Comparative genomics database https://mycocosm.jgi.doe.gov/mycocosm/home T-DNA mutant database http://atmt.snu.kr/ T-DNA mutant database http://www.phi-base.org/ SNP and expression data https://fungidb.org/fungidb/app/.

The bZIP transcription factor BIP1 of the rice blast fungus is essential for infection and regulates a specific set of appressorium genes.

The rice blast fungus Magnaporthe oryzae differentiates specialized cells called appressoria that are required for fungal penetration into host leaves. In this study, we identified the novel basic leucine zipper (bZIP) transcription factor BIP1 (B-ZIP Involved in Pathogenesis-1) that is essential for pathogenicity. BIP1 is required for the infection of plant leaves, even if they are wounded, but not for appressorium-mediated penetration of artificial cellophane membranes. This phenotype suggests that BIP1 is not implicated in the differentiation of the penetration peg but is necessary for the initial establishment of the fungus within plant cells. BIP1 expression was restricted to the appressorium by both transcriptional and post-transcriptional control. Genome-wide transcriptome analysis showed that 40 genes were down regulated in a BIP1 deletion mutant. Most of these genes were specifically expressed in the appressorium. They encode proteins with pathogenesis-related functions such as enzymes involved in secondary metabolism including those encoded by the ACE1 gene cluster, small secreted proteins such as SLP2, BAS2, BAS3, and AVR-Pi9 effectors, as well as plant cuticle and cell wall degrading enzymes. Interestingly, this BIP1 network is different from other known infection-related regulatory networks, highlighting the complexity of gene expression control during plant-fungal interactions. Promoters of BIP1-regulated genes shared a GCN4/bZIP-binding DNA motif (TGACTC) binding in vitro to BIP1. Mutation of this motif in the promoter of MGG_08381.7 from the ACE1 gene cluster abolished its appressorium-specific expression, showing that BIP1 behaves as a transcriptional activator. In summary, our findings demonstrate that BIP1 is critical for the expression of early invasion-related genes in appressoria. These genes are likely needed for biotrophic invasion of the first infected host cell, but not for the penetration process itself. Through these mechanisms, the blast fungus strategically anticipates the host plant environment and responses during appressorium-mediated penetration.

Whole-genome sequences of Bipolaris bicolor, Curvularia hawaiiensis, Curvularia spicifera, and Exserohilum rostratum isolated from rice in Burkina Faso, France, Mali, and Pakistan.

Different fungal species of the Pleosporaceae family infect rice, causing similar symptoms. Reference genomic sequences are useful tools to study the evolution of these species and to develop accurate molecular diagnostic tools. Here, we report the complete genome sequences of Bipolaris bicolor, Curvularia hawaiiensis, Curvularia spicifera, and Exserohilum rostratum.

A thousand-genome panel retraces the global spread and adaptation of a major fungal crop pathogen.

Human activity impacts the evolutionary trajectories of many species worldwide. Global trade of agricultural goods contributes to the dispersal of pathogens reshaping their genetic makeup and providing opportunities for virulence gains. Understanding how pathogens surmount control strategies and cope with new climates is crucial to predicting the future impact of crop pathogens. Here, we address this by assembling a global thousand-genome panel of Zymoseptoria tritici, a major fungal pathogen of wheat reported in all production areas worldwide. We identify the global invasion routes and ongoing genetic exchange of the pathogen among wheat-growing regions. We find that the global expansion was accompanied by increased activity of transposable elements and weakened genomic defenses. Finally, we find significant standing variation for adaptation to new climates encountered during the global spread. Our work shows how large population genomic panels enable deep insights into the evolutionary trajectory of a major crop pathogen.

The Combination of Both Heat and Water Stresses May Worsen Botryosphaeria Dieback Symptoms in Grapevine.

(1) Background: Grapevine trunk diseases (GTDs) have become a global threat to vineyards worldwide. These diseases share three main common features. First, they are caused by multiple pathogenic micro-organisms. Second, these pathogens often maintain a long latent phase, which makes any research in pathology and symptomatology challenging. Third, a consensus is raising to pinpoint combined abiotic stresses as a key factor contributing to disease symptom expression. (2) Methods: We analyzed the impact of combined abiotic stresses in grapevine cuttings artificially infected by two fungi involved in Botryosphaeria dieback (one of the major GTDs), Neofusicoccum parvum and Diplodia seriata. Fungal-infected and control plants were subjected to single or combined abiotic stresses (heat stress, drought stress or both). Disease intensity was monitored thanks to the measurement of necrosis area size. (3) Results and conclusions: Overall, our results suggest that combined stresses might have a stronger impact on disease intensity upon infection by the less virulent pathogen Diplodia seriata. This conclusion is discussed through the impact on plant physiology using metabolomic and transcriptomic analyses of leaves sampled for the different conditions.

CusProSe: a customizable protein annotation software with an application to the prediction of fungal secondary metabolism genes.

We report here a new application, CustomProteinSearch (CusProSe), whose purpose is to help users to search for proteins of interest based on their domain composition. The application is customizable. It consists of two independent tools, IterHMMBuild and ProSeCDA. IterHMMBuild allows the iterative construction of Hidden Markov Model (HMM) profiles for conserved domains of selected protein sequences, while ProSeCDA scans a proteome of interest against an HMM profile database, and annotates identified proteins using user-defined rules. CusProSe was successfully used to identify, in fungal genomes, genes encoding key enzyme families involved in secondary metabolism, such as polyketide synthases (PKS), non-ribosomal peptide synthetases (NRPS), hybrid PKS-NRPS and dimethylallyl tryptophan synthases (DMATS), as well as to characterize distinct terpene synthases (TS) sub-families. The highly configurable characteristics of this application makes it a generic tool, which allows the user to refine the function of predicted proteins, to extend detection to new enzymes families, and may also be applied to biological systems other than fungi and to other proteins than those involved in secondary metabolism.

Genetic diversity and structure of Bipolaris oryzae and Exserohilum rostratum populations causing brown spot of rice in Burkina Faso based on genotyping-by-sequencing.

In recent years, Brown spot disease of rice (BSR) has been observed on leaves and seeds of rice in all rice-growing areas of Burkina Faso. Bipolaris oryzae and Exserohilum rostratum are the main fungal species isolated from BSR infected tissues and they are frequently observed in the same field. However, we are lacking information on the genetic diversity and population structure of these fungi in Burkina Faso. The mode of reproduction is also unknown. The genetic diversity of isolates of B. oryzae (n=61) and E. rostratum (n=151), collected from major rice-growing areas of Burkina Faso, was estimated using genotyping-by-sequencing (GBS). The mean values for nucleotide diversity (π) were 1.9 x10-4 for B. oryzae and 4.8 x10-4 for E. rostratum. There is no genetic differentiation between the geographical populations of each species. The analysis of molecular variance revealed that 89% and 94% of the genetic variances were within the populations of B. oryzae and E. rostratum, respectively. For each species, four genetic clusters were identified by two clustering methods (DAPC and sNMF). The distribution of these genetic groups was independent of the geographical origin of the isolates. Evidence of recombination was detected in the populations of B. oryzae and E. rostratum. For B. oryzae balanced mating type ratios were supporting sexual reproduction. For E. rostratum overrepresentation of MAT1-2 isolates (79%) suggested a predominant asexual reproduction. This study provides important information on the biology and genetics of the two major fungi causing brown spot disease of rice in Burkina Faso.

Blocked at the Stomatal Gate, a Key Step of Wheat Stb16q-Mediated Resistance to Zymoseptoria tritici.

Septoria tritici blotch (STB), caused by the fungus Zymoseptoria tritici, is among the most threatening wheat diseases in Europe. Genetic resistance remains one of the main environmentally sustainable strategies to efficiently control STB. However, the molecular and physiological mechanisms underlying resistance are still unknown, limiting the implementation of knowledge-driven management strategies. Among the 22 known major resistance genes (Stb), the recently cloned Stb16q gene encodes a cysteine-rich receptor-like kinase conferring a full broad-spectrum resistance against Z. tritici. Here, we showed that an avirulent Z. tritici inoculated on Stb16q quasi near isogenic lines (NILs) either by infiltration into leaf tissues or by brush inoculation of wounded tissues partially bypasses Stb16q-mediated resistance. To understand this bypass, we monitored the infection of GFP-labeled avirulent and virulent isolates on Stb16q NILs, from germination to pycnidia formation. This quantitative cytological analysis revealed that 95% of the penetration attempts were unsuccessful in the Stb16q incompatible interaction, while almost all succeeded in compatible interactions. Infectious hyphae resulting from the few successful penetration events in the Stb16q incompatible interaction were arrested in the sub-stomatal cavity of the primary-infected stomata. These results indicate that Stb16q-mediated resistance mainly blocks the avirulent isolate during its stomatal penetration into wheat tissue. Analyses of stomatal aperture of the Stb16q NILs during infection revealed that Stb16q triggers a temporary stomatal closure in response to an avirulent isolate. Finally, we showed that infiltrating avirulent isolates into leaves of the Stb6 and Stb9 NILs also partially bypasses resistances, suggesting that arrest during stomatal penetration might be a common major mechanism for Stb-mediated resistances.

Diversity of Neofusicoccum parvum for the Production of the Phytotoxic Metabolites (-)-Terremutin and (R)-Mellein.

Two Neofusicoccumparvum isolates and a UV mutant were characterized for their phytotoxin production in vitro, their pathogenicity on grapevine, and their genome sequenced. The isolate Np-Bt67 produced high level of (-)-terremutin, but almost no (R)-mellein, and it was the most aggressive on grapevine, triggering apoplexy. Similar symptoms were not induced by purified (-)-terremutin. The isolate Bourgogne S-116 (Np-B) produced 3-fold less (-)-terremutin and high amounts of (R)-mellein, but it was less aggressive on grapevine than Np-Bt67. The UV9 mutant obtained from Np-B (NpB-UV9) no longer produced (-)-terremutin but overproduced (R)-mellein by 2.5-fold, and it was as pathogenic as its parent. NpB-UV9 differed from its parent by simple mutations in two genes (transcription factor UCR-NP2_6692, regulatory protein UCR-NP2_9007), not located neither near (R)-mellein, nor (-)-terremutin biosynthetic genes, but likely involved in the control of (-)-terremutin biosynthesis. Grapevine immunity was disturbed upon challenge with these pathogens or purified phytotoxins, leading to an upregulation of SA-dependent defenses, while (-)-terremutin interfered with host JA/ET-dependent defenses. Our results suggest that neither (-)-terremutin nor (R)-mellein alone is essential for the pathogenicity of N. parvum on grapevine, since isolate/mutant non-producing these toxins in vitro is pathogenic. However, these phytotoxins could play a quantitative role in the infection process.

Impacts of Sodium Arsenite on Wood Microbiota of Esca-Diseased Grapevines.

Although sodium arsenite was widely used in Europe until its ban in 2003, its effects on microorganisms is not clearly understood. To improve our understanding of sodium arsenite curative effect on GTDs, grapevines displaying esca-foliar symptoms from different French regions (Alsace, Champagne, Languedoc) were treated or not with sodium arsenite, and analyzed for their wood microbiota. Using metabarcoding, we identified the fungal and bacterial taxa composition of microbiota colonizing woody trunk tissues. Large differences in fungal microbiota composition between treated and untreated grapevines were observed while no major impacts were observed on bacteria microbiota. The main fungal species detected in untreated necrotic woody tissues was Fomitiporia mediterranea (63-94%), a fungal pathogen associated with esca. The relative abundance of this fungal species significantly decreased after sodium arsenite treatment in the three vineyards, in particular in white-rot necrotic tissues and their borders (-90%). F. mediterranea was the most sensitive to sodium arsenite among fungi from grapevine woody tissues. These results strongly suggest that the effect of sodium arsenite on GTDs is due to its ability to efficiently and almost specifically eliminate F. mediterranea from white-rot necrotic tissues, allowing saprobic fungi to colonize the tissues previously occupied by this pathogenic fungus.

Major changes in grapevine wood microbiota are associated with the onset of esca, a devastating trunk disease.

Esca, a major grapevine trunk disease in old grapevines, is associated with the colonization of woody tissues by a broad range of plant pathogenic fungi. To identify which fungal and bacterial species are involved in the onset of this disease, we analysed the microbiota from woody tissues of young (10-year-old) grapevines at an early stage of esca. Using meta-barcoding, 515 fungal and 403 bacterial operational taxonomic units (OTUs) were identified in woody tissues. In situ hybridization showed that these fungi and bacteria co-inhabited in grapevine woody tissues. In non-necrotic woody tissues, fungal and bacterial microbiota varied according to organs and seasons but not diseased plant status. Phaeomoniella chlamydospora, involved in the Grapevine trunk disease, was the most abundant species in non-necrotic tissues from healthy plants, suggesting a possible non-pathogenic endophytic behaviour. Most diseased plants (70%) displayed cordons, with their central white-rot necrosis colonized essentially by two plant pathogenic fungi (Fomitiporia mediterranea: 60%-90% and P. chlamydospora: 5%-15%) and by a few bacterial taxa (Sphingomonas spp. and Mycobacterium spp.). The occurrence of a specific association of fungal and bacterial species in cordons from young grapevines expressing esca-foliar symptoms strongly suggests that that microbiota is involved in the onset of this complex disease.

Infection cushions of Fusarium graminearum are fungal arsenals for wheat infection.

Fusarium graminearum is one of the most destructive plant pathogens worldwide, causing fusarium head blight (FHB) on cereals. F. graminearum colonizes wheat plant surfaces with specialized unbranched hyphae called runner hyphae (RH), which develop multicelled complex appressoria called infection cushions (IC). IC generate multiple penetration sites, allowing the fungus to enter the plant cuticle. Complex infection structures are typical for several economically important plant pathogens, yet with unknown molecular basis. In this study, RH and IC formed on the surface of wheat paleae were isolated by laser capture microdissection. RNA-Seq-based transcriptomic analyses were performed on RH and IC and compared to mycelium grown in complete medium (MY). Both RH and IC displayed a high number of infection up-regulated genes (982), encoding, among others, carbohydrate-active enzymes (CAZymes: 140), putative effectors (PE: 88), or secondary metabolism gene clusters (SMC: 12 of 67 clusters). RH specifically up-regulated one SMC corresponding to aurofusarin biosynthesis, a broad activity antibiotic. IC specifically up-regulated 248 genes encoding mostly putative virulence factors such as 7 SMC, including the mycotoxin deoxynivalenol and the newly identified fusaoctaxin A, 33 PE, and 42 CAZymes. Furthermore, we studied selected candidate virulence factors using cellular biology and reverse genetics. Hence, our results demonstrate that IC accumulate an arsenal of proven and putative virulence factors to facilitate the invasion of epidermal cells.

Nonproteinaceous effectors: the terra incognita of plant-fungal interactions.

Molecular plant-fungal interaction studies have mainly focused on small secreted protein effectors. However, accumulating evidence shows that numerous fungal secondary metabolites are produced at all stages of plant colonization, especially during early asymptomatic/biotrophic phases. The discovery of fungal small RNAs targeting plant transcripts has expanded the fungal repertoire of nonproteinaceous effectors even further. The challenge now is to develop specific functional methods to fully understand the biological roles of these effectors. Studies on fungal extracellular vesicles are also needed because they could be the universal carriers of all kinds of fungal effectors. With this review, we aim to stimulate the nonproteinaceous effector research field to move from descriptive to functional studies, which should bring a paradigm shift in plant-fungal interactions.

Broad-specificity GH131 ß-glucanases are a hallmark of fungi and oomycetes that colonize plants.

Plant-tissue-colonizing fungi fine-tune the deconstruction of plant-cell walls (PCW) using different sets of enzymes according to their lifestyle. However, some of these enzymes are conserved among fungi with dissimilar lifestyles. We identified genes from Glycoside Hydrolase family GH131 as commonly expressed during plant-tissue colonization by saprobic, pathogenic and symbiotic fungi. By searching all the publicly available genomes, we found that GH131-coding genes were widely distributed in the Dikarya subkingdom, except in Taphrinomycotina and Saccharomycotina, and in phytopathogenic Oomycetes, but neither other eukaryotes nor prokaryotes. The presence of GH131 in a species was correlated with its association with plants as symbiont, pathogen or saprobe. We propose that GH131-family expansions and horizontal-gene transfers contributed to this adaptation. We analysed the biochemical activities of GH131 enzymes whose genes were upregulated during plant-tissue colonization in a saprobe (Pycnoporus sanguineus), a plant symbiont (Laccaria bicolor) and three hemibiotrophic-plant pathogens (Colletotrichum higginsianum, C. graminicola, Zymoseptoria tritici). These enzymes were all active on substrates with ß-1,4, ß-1,3 and mixed ß-1,4/1,3 glucosidic linkages. Combined with a cellobiohydrolase, GH131 enzymes enhanced cellulose degradation. We propose that secreted GH131 enzymes unlock the PCW barrier and allow further deconstruction by other enzymes during plant tissue colonization by symbionts, pathogens and saprobes.

Low Amplitude Boom-and-Bust Cycles Define the Septoria Nodorum Blotch Interaction.

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.

Genome wide analysis of the transition to pathogenic lifestyles in Magnaporthales fungi.

The rice blast fungus Pyricularia oryzae (syn. Magnaporthe oryzae, Magnaporthe grisea), a member of the order Magnaporthales in the class Sordariomycetes, is an important plant pathogen and a model species for studying pathogen infection and plant-fungal interaction. In this study, we generated genome sequence data from five additional Magnaporthales fungi including non-pathogenic species, and performed comparative genome analysis of a total of 13 fungal species in the class Sordariomycetes to understand the evolutionary history of the Magnaporthales and of fungal pathogenesis. Our results suggest that the Magnaporthales diverged ca. 31 millon years ago from other Sordariomycetes, with the phytopathogenic blast clade diverging ca. 21 million years ago. Little evidence of inter-phylum horizontal gene transfer (HGT) was detected in Magnaporthales. In contrast, many genes underwent positive selection in this order and the majority of these sequences are clade-specific. The blast clade genomes contain more secretome and avirulence effector genes, which likely play key roles in the interaction between Pyricularia species and their plant hosts. Finally, analysis of transposable elements (TE) showed differing proportions of TE classes among Magnaporthales genomes, suggesting that species-specific patterns may hold clues to the history of host/environmental adaptation in these fungi.

Gene Flow between Divergent Cereal- and Grass-Specific Lineages of the Rice Blast Fungus Magnaporthe oryzae.

Delineating species and epidemic lineages in fungal plant pathogens is critical to our understanding of disease emergence and the structure of fungal biodiversity and also informs international regulatory decisions. Pyricularia oryzae (syn. Magnaporthe oryzae) is a multihost pathogen that infects multiple grasses and cereals, is responsible for the most damaging rice disease (rice blast), and is of growing concern due to the recent introduction of wheat blast to Bangladesh from South America. However, the genetic structure and evolutionary history of M. oryzae, including the possible existence of cryptic phylogenetic species, remain poorly defined. Here, we use whole-genome sequence information for 76 M. oryzae isolates sampled from 12 grass and cereal genera to infer the population structure of M. oryzae and to reassess the species status of wheat-infecting populations of the fungus. Species recognition based on genealogical concordance, using published data or extracting previously used loci from genome assemblies, failed to confirm a prior assignment of wheat blast isolates to a new species (Pyricularia graminis-tritici). Inference of population subdivisions revealed multiple divergent lineages within M. oryzae, each preferentially associated with one host genus, suggesting incipient speciation following host shift or host range expansion. Analyses of gene flow, taking into account the possibility of incomplete lineage sorting, revealed that genetic exchanges have contributed to the makeup of multiple lineages within M. oryzae These findings provide greater understanding of the ecoevolutionary factors that underlie the diversification of M. oryzae and highlight the practicality of genomic data for epidemiological surveillance in this important multihost pathogen.IMPORTANCE Infection of novel hosts is a major route for disease emergence by pathogenic microorganisms. Understanding the evolutionary history of multihost pathogens is therefore important to better predict the likely spread and emergence of new diseases. Magnaporthe oryzae is a multihost fungus that causes serious cereal diseases, including the devastating rice blast disease and wheat blast, a cause of growing concern due to its recent spread from South America to Asia. Using whole-genome analysis of 76 fungal strains from different hosts, we have documented the divergence of M. oryzae into numerous lineages, each infecting a limited number of host species. Our analyses provide evidence that interlineage gene flow has contributed to the genetic makeup of multiple M. oryzae lineages within the same species. Plant health surveillance is therefore warranted to safeguard against disease emergence in regions where multiple lineages of the fungus are in contact with one another.