The Egyptian wheat cultivar Gemmeiza-12 is a source of resistance against the fungus Zymoseptoria tritici.

BACKGROUND: Wheat is one of the world's most important cereal crops. However, the fungal pathogen Zymoseptoria tritici can cause disease epidemics, leading to reduced yields. With climate change and development of new agricultural areas with suitable environments, Z. tritici may advance into geographical areas previously unaffected by this pathogen. It is currently unknown how Egyptian wheat will perform in the face of this incoming threat. This project aimed to assess the resistance of Egyptian wheat germplasm to Z. tritici, to identify cultivars with high levels of resistance and characterise the mechanism(s) of resistance present in these cultivars. RESULTS: Eighteen Egyptian wheat cultivars were screened against two Z. tritici model isolates and exhibited a wide spectrum of responses. This ranged from resistance to complete susceptibility to one or both isolates tested. The most highly resistant cultivars from the initial screen were then tested under two environmental conditions against modern UK field isolates. Disease levels under UK-like conditions were higher, however, symptom development on the cultivar Gemmeiza-12 was noticeably slower than on other Egyptian wheats. The robustness of the resistance shown by Gemmeiza-12 was confirmed in experiments mimicking Egyptian environmental conditions, where degree of Z. tritici infection was lower. The Kompetitive allele-specific PCR (KASP) diagnostic assay suggested the presence of an Stb6 resistant allele in several Egyptian wheats including Gemmeiza-12. Infection assays using the IPO323 WT and IPO323ΔAvrStb6 mutant confirmed the presence of Stb6 in several Egyptian cultivars including Gemmeiza-12. Confocal fluorescence microscopy demonstrated that growth of the IPO323 strain is blocked at the point of stomatal penetration on Gemmeiza-12, consistent with previous reports of Stb gene mediated resistance. In addition to this R-gene mediated resistance, IPO323 spores showed lower adherence to leaves of Gemmeiza-12 compared to UK wheat varieties, suggesting other aspects of leaf physiology may also contribute to the resistance phenotype of this cultivar. CONCLUSION: These results indicate that Gemmeiza-12 will be useful in future breeding programs where improved resistance to Z. tritici is a priority.

That's no moon, it's a Starship: Giant transposons driving fungal horizontal gene transfer.

To date, most reports of horizontal gene transfer (HGT) in fungi rely on genome sequence data and are therefore an indirect measure of HGT after the event has occurred. However, a novel group of class II-like transposons known as Starships may soon alter this status quo. Starships are giant transposable elements that carry dozens of genes, some of which are host-beneficial, and are linked to many recent HGT events in the fungal kingdom. These transposons remain active and mobile in many fungal genomes and their transposition has recently been shown to be driven by a conserved tyrosine-recombinase called 'Captain'. This perspective explores some of the remaining unanswered questions about how these Starship transposons move, both within a genome and between different species. We seek to outline several experimental approaches that can be used to identify the genes essential for Starship-mediated HGT and draw links to other recently discovered giant transposons outside of the fungal kingdom.

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.

Multi-stage resistance to Zymoseptoria tritici revealed by GWAS in an Australian bread wheat diversity panel.

Septoria tritici blotch (STB) has been ranked the third most important wheat disease in the world, threatening a large area of wheat production. Although major genes play an important role in the protection against Zymoseptoria tritici infection, the lifespan of their resistance unfortunately is very short in modern wheat production systems. Combinations of quantitative resistance with minor effects, therefore, are believed to have prolonged and more durable resistance to Z. tritici. In this study, new quantitative trait loci (QTLs) were identified that are responsible for seedling-stage resistance and adult-plant stage resistance (APR). More importantly was the characterisation of a previously unidentified QTL that can provide resistance during different stages of plant growth or multi-stage resistance (MSR). At the seedling stage, we discovered a new isolate-specific QTL, QSt.wai.1A.1. At the adult-plant stage, the new QTL QStb.wai.6A.2 provided stable and consistent APR in multiple sites and years, while the QTL QStb.wai.7A.2 was highlighted to have MSR. The stacking of multiple favourable MSR alleles was found to improve resistance to Z. tritici by up to 40%.

Scaling-up to understand tree-pathogen interactions: A steep, tough climb or a walk in the park?

Plants have proficient tools that allow them to survive interactions with pathogens. Upon attack, they respond with specific countermeasures, which are controlled by the immune system. However, defences can fail and this failure exposes plants to fast-spreading devastation. Trees face similar challenges to other plants and their immune system allows them to mount defences against pathogens. However, their slow growth, longevity, woodiness, and size can make trees a challenging system to study. Here, we review scientific successes in plant systems, highlight the key challenges and describe the enormous opportunities for pathology research in trees. We discuss the benefits that scaling-up our understanding on tree-pathogen interactions can provide in the fight against plant pathogenic threats.

The identification of a transposon affecting the asexual reproduction of the wheat pathogen Zymoseptoria tritici.

Zymoseptoria tritici, the causal agent of Septoria tritici blotch, is a fungal wheat pathogen that causes significant global yield losses. Within Z. tritici populations, quantitative differences in virulence among different isolates are commonly observed; however, the genetic components that underpin these differences remain elusive. In this study, intraspecific comparative transcriptomic analysis was used to identify candidate genes that contribute to differences in virulence on the wheat cultivar WW2449. This led to the identification of a multicopy gene that was not expressed in the high-virulence isolate when compared to the medium- and low-virulence isolates. Further investigation suggested this gene resides in a 7.9-kb transposon. Subsequent long-read sequencing of the isolates used in the transcriptomic analysis confirmed that this gene did reside in an active Class II transposon, which is composed of four genes named REP9-1 to -4. Silencing and overexpression of REP9-1 in two distinct genetic backgrounds demonstrated that its expression alone reduces the number of pycnidia produced by Z. tritici during infection. The REP9-1 gene identified within a Class II transposon is the first discovery of a gene in a transposable element that influences the virulence of Z. tritici. This discovery adds further complexity to genetic loci that contribute to quantitative virulence in this important pathogen.

The complex genomic basis of rapid convergent adaptation to pesticides across continents in a fungal plant pathogen.

Convergent evolution leads to identical phenotypic traits in different species or populations. Convergence can be driven by standing variation allowing selection to favour identical alleles in parallel or the same mutations can arise independently. However, the molecular basis of such convergent adaptation remains often poorly resolved. Pesticide resistance in agricultural ecosystems is a hallmark of convergence in phenotypic traits. Here, we analyse the major fungal pathogen Zymoseptoria tritici causing serious losses on wheat and with fungicide resistance emergence across several continents. We sampled three population pairs each from a different continent spanning periods early and late in the application of fungicides. To identify causal loci for resistance, we combined knowledge from molecular genetics work and performed genome-wide association studies (GWAS) on a global set of isolates. We discovered yet unknown factors in azole resistance including a gene encoding membrane associated functions. We found strong support for the "hotspot" model of resistance evolution with convergent changes in a small set of loci but additional loci showed more population-specific allele frequency changes. Genome-wide scans of selection showed that half of all known resistance loci were overlapping a selective sweep region. Hence, the application of fungicides was one of the major selective agents acting on the pathogen over the past decades. Furthermore, loci identified through GWAS showed the highest overlap with selective sweep regions underlining the importance to map phenotypic trait variation in evolving populations. Our population genomic analyses highlighted that both de novo mutations and gene flow contributed to convergent pesticide adaptation.

Victorin, the host-selective cyclic peptide toxin from the oat pathogen Cochliobolus victoriae, is ribosomally encoded.

The necrotrophic fungal pathogen Cochliobolus victoriae produces victorin, a host-selective toxin (HST) essential for pathogenicity to certain oat cultivars with resistance against crown rust. Victorin is a mixture of highly modified heterodetic cyclic hexapeptides, previously assumed to be synthesized by a nonribosomal peptide synthetase. Herein, we demonstrate that victorin is a member of the ribosomally synthesized and posttranslationally modified peptide (RiPP) family of natural products. Analysis of a newly generated long-read assembly of the C. victoriae genome revealed three copies of precursor peptide genes (vicA1-3) with variable numbers of "GLKLAF" core peptide repeats corresponding to the victorin peptide backbone. vicA1-3 are located in repeat-rich gene-sparse regions of the genome and are loosely clustered with putative victorin biosynthetic genes, which are supported by the discovery of compact gene clusters harboring corresponding homologs in two distantly related plant-associated Sordariomycete fungi. Deletion of at least one copy of vicA resulted in strongly diminished victorin production. Deletion of a gene encoding a DUF3328 protein (VicYb) abolished the production altogether, supporting its predicted role in oxidative cyclization of the core peptide. In addition, we uncovered a copper amine oxidase (CAO) encoded by vicK, in which its deletion led to the accumulation of new glycine-containing victorin derivatives. The role of VicK in oxidative deamination of the N-terminal glycyl moiety of the hexapeptides to the active glyoxylate forms was confirmed in vitro. This study finally unraveled the genetic and molecular bases for biosynthesis of one of the first discovered HSTs and expanded our understanding of underexplored fungal RiPPs.

Assessing the efficacy of CRISPR/Cas9 genome editing in the wheat pathogen Parastagonspora nodorum.

BACKGROUND: The genome-editing tool CRISPR/Cas9 has revolutionized gene manipulation by providing an efficient method to generate targeted mutations. This technique deploys the Cas9 endonuclease and a guide RNA (sgRNA) which interact to form a Cas9-sgRNA complex that initiates gene editing through the introduction of double stranded DNA breaks. We tested the efficacy of the CRISPR/Cas9 approach as a means of facilitating a variety of reverse genetic approaches in the wheat pathogenic fungus Parastagonospora nodorum. RESULTS: Parastagonospora nodorum protoplasts were transformed with the Cas9 protein and sgRNA in the form of a preassembled ribonuclear protein (RNP) complex targeting the Tox3 effector gene. Subsequent screening of the P. nodorum transformants revealed 100% editing of those mutants screened. We further tested the efficacy of RNP complex when co-transformed with a Tox3-Homology Directed Repair cassette harbouring 1 kb of homologous flanking DNA. Subsequent screening of resulting transformants demonstrated homologous recombination efficiencies exceeding 70%. A further transformation with a Tox3-Homology Directed Repair cassette harbouring a selectable marker with 50 bp micro-homology flanks was also achieved with 25% homologous recombination efficiency. The success of these homology directed repair approaches demonstrate that CRISPR/Cas9 is amenable to other in vivo DNA manipulation approaches such as the insertion of DNA and generating point mutations. CONCLUSION: These data highlight the significant potential that CRISPR/Cas9 has in expediting transgene-free gene knockouts in Parastagonospora nodorum and also in facilitating other gene manipulation approaches. Access to these tools will significantly decrease the time required to assess the requirement of gene for disease and to undertake functional studies to determine its role.

Transposon-Mediated Horizontal Transfer of the Host-Specific Virulence Protein ToxA between Three Fungal Wheat Pathogens.

Most known examples of horizontal gene transfer (HGT) between eukaryotes are ancient. These events are identified primarily using phylogenetic methods on coding regions alone. Only rarely are there examples of HGT where noncoding DNA is also reported. The gene encoding the wheat virulence protein ToxA and the surrounding 14 kb is one of these rare examples. ToxA has been horizontally transferred between three fungal wheat pathogens (Parastagonospora nodorum, Pyrenophora tritici-repentis, and Bipolaris sorokiniana) as part of a conserved ∼14 kb element which contains coding and noncoding regions. Here we used long-read sequencing to define the extent of HGT between these three fungal species. Construction of near-chromosomal-level assemblies enabled identification of terminal inverted repeats on either end of the 14 kb region, typical of a type II DNA transposon. This is the first description of ToxA with complete transposon features, which we call ToxhAT. In all three species, ToxhAT resides in a large (140-to-250 kb) transposon-rich genomic island which is absent in isolates that do not carry the gene (annotated here as toxa- ). We demonstrate that the horizontal transfer of ToxhAT between P. tritici-repentis and P. nodorum occurred as part of a large (∼80 kb) HGT which is now undergoing extensive decay. In B. sorokiniana, in contrast, ToxhAT and its resident genomic island are mobile within the genome. Together, these data provide insight into the noncoding regions that facilitate HGT between eukaryotes and into the genomic processes which mask the extent of HGT between these species.IMPORTANCE This work dissects the tripartite horizontal transfer of ToxA, a gene that has a direct negative impact on global wheat yields. Defining the extent of horizontally transferred DNA is important because it can provide clues to the mechanisms that facilitate HGT. Our analysis of ToxA and its surrounding 14 kb suggests that this gene was horizontally transferred in two independent events, with one event likely facilitated by a type II DNA transposon. These horizontal transfer events are now in various processes of decay in each species due to the repeated insertion of new transposons and subsequent rounds of targeted mutation by a fungal genome defense mechanism known as repeat induced point mutation. This work highlights the role that HGT plays in the evolution of host adaptation in eukaryotic pathogens. It also increases the growing body of evidence indicating that transposons facilitate adaptive HGT events between fungi present in similar environments and hosts.

Acquisition and Loss of Secondary Metabolites Shaped the Evolutionary Path of Three Emerging Phytopathogens of Wheat.

White grain disorder is a recently emerged wheat disease in Australia, caused by Eutiarosporella darliae, E. pseudodarliae, and E. tritici-australis. The disease cycle of these pathogens and the molecular basis of their interaction with wheat are poorly understood. To address this knowledge gap, we undertook a comparative genomics analysis focused on the secondary metabolite gene repertoire among these three species. This analysis revealed a diverse array of secondary metabolite gene clusters in these pathogens, including modular polyketide synthase genes. These genes have only been previously associated with bacteria and this is the first report of such genes in fungi. Subsequent phylogenetic analyses provided strong evidence that the modular PKS genes were horizontally acquired from a bacterial or a protist species. We also uncovered a secondary metabolite gene cluster with three polyketide/nonribosomal peptide synthase genes (Hybrid-1, -2, and -3) in E. darliae and E. pseudodarliae. In contrast, only remnant and partial genes homologous to this cluster were identified in E. tritici-australis, suggesting loss of this cluster. Homologues of Hybrid-2 in other fungi have been proposed to facilitate disease in woody plants, suggesting a possible alternative host range for E. darliae and E. pseudodarliae. Subsequent assays confirmed that E. darliae and E. pseudodarliae were both pathogenic on woody plants, but E. tritici-australis was not, implicating woody plants as potential host reservoirs for the fungi. Combined, these data have advanced our understanding of the lifestyle and potential host-range of these recently emerged wheat pathogens and shed new light on fungal secondary metabolism.

Rapid Parallel Evolution of Azole Fungicide Resistance in Australian Populations of the Wheat Pathogen Zymoseptoria tritici.

Zymoseptoria tritici is a globally distributed fungal pathogen which causes Septoria tritici blotch on wheat. Management of the disease is attempted through the deployment of resistant wheat cultivars and the application of fungicides. However, fungicide resistance is commonly observed in Z. tritici populations, and continuous monitoring is required to detect breakdowns in fungicide efficacy. We recently reported azole-resistant isolates in Australia; however, it remained unknown whether resistance was brought into the continent through gene flow or whether resistance emerged independently. To address this question, we screened 43 isolates across five Australian locations for azole sensitivity and performed whole-genome sequencing on 58 isolates from seven locations to determine the genetic basis of resistance. Population genomic analyses showed extremely strong differentiation between the Australian population recovered after azoles began to be used and both Australian populations recovered before azoles began to be used and populations on different continents. The apparent absence of recent gene flow between Australia and other continents suggests that azole fungicide resistance has evolved de novo and subsequently spread within Tasmania. Despite the isolates being distinct at the whole-genome level, we observed combinations of nonsynonymous substitutions at the CYP51 locus identical to those observed elsewhere in the world. We observed nine previously reported nonsynonymous mutations as well as isolates that carried a combination of the previously reported L50S, S188N, A379G, I381V, Y459DEL, G460DEL, and N513K substitutions. Assays for the 50% effective concentration against a subset of isolates exposed to the tebuconazole and epoxiconazole fungicides showed high levels of azole resistance. The rapid, parallel evolution of a complex CYP51 haplotype that matches a dominant European haplotype demonstrates the enormous potential for de novo resistance emergence in pathogenic fungi.IMPORTANCE Fungicides are essential to control diseases in agriculture because many crops are highly susceptible to pathogens. However, many pathogens rapidly evolve resistance to fungicides. A large body of studies have described specific mutations conferring resistance and have often made inferences about the origins of resistance based on sequencing data from the target gene alone. Here, we show the de novo acquisition of resistance to the ubiquitously used azole fungicides in genetically isolated populations of the wheat pathogen Zymoseptoria tritici in Tasmania, Australia. We confirm evidence for parallel evolution through genome-scale analyses of representative worldwide populations. The emergence of complex resistance haplotypes following a well-documented recent introduction of azoles into Australian farming practices demonstrates how rapidly chemical resistance evolves in agricultural ecosystems.

Genetic mapping of Stb19, a new resistance gene to Zymoseptoria tritici in wheat.

KEY MESSAGE: A new and dominant R gene Stb19 is identified from a soft wheat cultivar 'Lorikeet' and was mapped on the distal region of chromosome 1DS. Two tightly linked KASP markers were also discovered and validated for molecular-assisted breeding programs. A new R gene, designated as Stb19, provides resistance to Zymoseptoria tritici in wheat. This new dominant gene resides on the short arm of chromosome 1D, exhibiting complete resistance to three Z. tritici isolates, WAI332, WAI251, and WAI161, at the seedling stage. A genetic linkage map, based on an F2:3 population of 'Lorikeet' and 'Summit,' found the Stb19 gene at a 9.3 cM region on 1DS, closely linked with two Kompetitive Allele-Specific PCR markers, snp_4909967 and snp_1218021. Further, the two markers were tested and validated in another F2:3 population and 266 different wheat accessions, which gave over 95% accuracy of resistance/susceptibility prediction. Combined with the physical location of the identified SNPs and the previous evidence of gene order on chromosome 1DS (centromere-Sr45-Sr33-Lr21-telomere), Stb19 is proposed to be located between Sr33 and Lr21. Thus, the newly discovered Stb19 along with the KASP markers represents an increase in genetic resources available for wheat breeding resistance to Z. tritici.

Just the surface: advances in the discovery and characterization of necrotrophic wheat effectors.

For many years pathogens of wheat have remained poorly understood. Hindered by an inaccessible host and the obligate nature of many of the pathogens, our understanding of these interactions has been limited compared to other more amenable pathosystems. However, breakthroughs over recent years have shed new light on diseases of wheat, particularly those caused by the genetically tractable necrotrophic pathogens. We now understand that many of the necrotrophic fungal pathogens do interact with wheat in a strict gene-for-gene relationship, and that pathogen and host partners in these interactions have now been identified. This improved understanding of necrotrophic effector biology has fundamentally changed the way we consider these important wheat diseases.

The discovery of the virulence gene ToxA in the wheat and barley pathogen Bipolaris sorokiniana.

Bipolaris sorokiniana is the causal agent of multiple diseases on wheat and barley and is the primary constraint to cereal production throughout South Asia. Despite its significance, the molecular basis of disease is poorly understood. To address this, the genomes of three Australian isolates of B. sorokiniana were sequenced and screened for known pathogenicity genes. Sequence analysis revealed that the isolate BRIP10943 harboured the ToxA gene, which has been associated previously with disease in the wheat pathogens Parastagonospora nodorum and Pyrenophora tritici-repentis. Analysis of the regions flanking ToxA within B. sorokiniana revealed that it was embedded within a 12-kb genomic element nearly identical to the corresponding regions in P. nodorum and P. tritici-repentis. A screen of 35 Australian B. sorokiniana isolates confirmed that ToxA was present in 12 isolates. Sequencing of the ToxA genes within these isolates revealed two haplotypes, which differed by a single non-synonymous nucleotide substitution. Pathogenicity assays showed that a B. sorokiniana isolate harbouring ToxA was more virulent on wheat lines that contained the sensitivity gene when compared with a non-ToxA isolate. This work demonstrates that proteins that confer host-specific virulence can be horizontally acquired across multiple species. This acquisition can dramatically increase the virulence of pathogenic strains on susceptible cultivars, which, in an agricultural setting, can have devastating economic and social impacts.

Transition from heterothallism to homothallism is hypothesised to have facilitated speciation among emerging Botryosphaeriaceae wheat-pathogens.

White grain disorder (WGD) is a recently emerged wheat disease in Australia caused by three Botryosphaeriaceae fungi, from the genus Eutiarosporella. These species are E. tritici-australis, E. darliae, and E. pseudodarliae. Characterisation of the mating type genes for the WGD-species show that the genome sequence of a single E. darliae and E. pseudodarliae isolate both harbour MAT1-2-1 and MAT1-1-1, which suggests that these species are homothallic. However, unlike most other characterised mating-type loci from other homothallic Dothideomycetes, these species' MAT1-1-1 are located at a separate locus, inserted within the coding region of another gene. The sequenced strain of E. tritici-australis analysed did not harbour MAT1-1-1. Including the sequenced strain, we screened the mating type genes present in 16 E. tritici-australis individuals isolated from infected grain from fields in South Australia. Of these 16, 11 harbour MAT1-1-1 and the other five harbour MAT1-2-1. The genome of a MAT1-1-1 harbouring isolate was re-sequenced, which demonstrated that MAT1-1-1 was present at the MAT locus. We examined non-coding DNA surrounding the MAT1-1-1 gene in E. pseudodarliae and observed fragments of the MAT locus both up and downstream. These fragments and their orientation around MAT1-1-1 is similar to characterised heterothallic Botryosphaeriaceae. Based on these gene arrangements, we conclude that the new MAT1-1-1 containing locus likely originated from a cryptic DNA integration event between two heterothallic individuals. We hypothesise that this integration event led to the formation of a homothallic lineage, which is the common ancestor of E. darliae and E. pseudodarliae.

Fungal phytopathogens encode functional homologues of plant rapid alkalinization factor (RALF) peptides.

In this article, we describe the presence of genes encoding close homologues of an endogenous plant peptide, rapid alkalinization factor (RALF), within the genomes of 26 species of phytopathogenic fungi. Members of the RALF family are key growth factors in plants, and the sequence of the RALF active region is well conserved between plant and fungal proteins. RALF1-like sequences were observed in most cases; however, RALF27-like sequences were present in the Sphaerulina musiva and Septoria populicola genomes. These two species are pathogens of poplar and, interestingly, the closest relative to their respective RALF genes is a poplar RALF27-like sequence. RALF peptides control cellular expansion during plant development, but were originally defined on the basis of their ability to induce rapid alkalinization in tobacco cell cultures. To test whether the fungal RALF peptides were biologically active in plants, we synthesized RALF peptides corresponding to those encoded by two sequenced genomes of the tomato pathogen Fusarium oxysporum f. sp. lycopersici. One of these peptides inhibited the growth of tomato seedlings and elicited responses in tomato and Nicotiana benthamiana typical of endogenous plant RALF peptides (reactive oxygen species burst, induced alkalinization and mitogen-activated protein kinase activation). Gene expression analysis confirmed that a RALF-encoding gene in F. oxysporum f. sp. lycopersici was expressed during infection on tomato. However, a subsequent reverse genetics approach revealed that the RALF peptide was not required by F. oxysporum f. sp. lycopersici for infection on tomato roots. This study has demonstrated the presence of functionally active RALF peptides encoded within phytopathogens that harbour an as yet undetermined role in plant-pathogen interactions.

Utilizing Gene Tree Variation to Identify Candidate Effector Genes in Zymoseptoria tritici.

Zymoseptoria tritici is a host-specific, necrotrophic pathogen of wheat. Infection by Z. tritici is characterized by its extended latent period, which typically lasts 2 wks, and is followed by extensive host cell death, and rapid proliferation of fungal biomass. This work characterizes the level of genomic variation in 13 isolates, for which we have measured virulence on 11 wheat cultivars with differential resistance genes. Between the reference isolate, IPO323, and the 13 Australian isolates we identified over 800,000 single nucleotide polymorphisms, of which ∼10% had an effect on the coding regions of the genome. Furthermore, we identified over 1700 probable presence/absence polymorphisms in genes across the Australian isolates using de novo assembly. Finally, we developed a gene tree sorting method that quickly identifies groups of isolates within a single gene alignment whose sequence haplotypes correspond with virulence scores on a single wheat cultivar. Using this method, we have identified < 100 candidate effector genes whose gene sequence correlates with virulence toward a wheat cultivar carrying a major resistance gene.

Is Zymoseptoria tritici a hemibiotroph?

The growth of microorganisms in planta is often categorized based on their methods of nutrient acquisition and the physical appearance of symptoms on the host. For example, biotrophs thrive on living tissue while necrotrophic pathogens often quickly lyse cells to access nutrients. Hemibiotrophs are pathogens that initially feed on living host tissue without causing visible symptoms prior to switching to necrotrophy. During infection of wheat, the pathogen Zymoseptoria tritici undergoes a prolonged and asymptomatic phase during which it grows slowly and protects itself from host defenses prior to eliciting a strong necrotic response. However careful analyses of the asymptomatic phase indicate that the pathogen does not alter host growth, casting doubt on the biotrophic nature of this asymptomatic period. Consequently, we question whether Z. tritici is correctly defined as a hemibiotroph.

Next-generation re-sequencing as a tool for rapid bioinformatic screening of presence and absence of genes and accessory chromosomes across isolates of Zymoseptoria tritici.

The wheat pathogen Zymoseptoria tritici possesses a large number of accessory chromosomes that may be present or absent in its genome. The genome of the reference isolate IPO323 has been assembled to a very high standard and contains 21 full length chromosome sequences, 8 of which represent accessory chromosomes. The IPO323 reference, when combined with low-cost next-generation sequencing and bioinformatics, can be used as a powerful tool to assess the presence or absence of accessory chromosomes. We present an outline of a range of bioinformatics techniques that can be applied to the analysis of presence-absence variation among accessory chromosomes across 13 novel isolates of Z. tritici.