Pyrenophora tritici-repentis (Ptr) is a necrotrophic fungal pathogen that causes the major wheat disease, tan spot. We set out to provide essential genomics-based resources in order to better understand the pathogenicity mechanisms of this important pathogen. Here, we present eight new Ptr isolate genomes, assembled and annotated; representing races 1, 2 and 5, and a new race. We report a high quality Ptr reference genome, sequenced by PacBio technology with Illumina paired-end data support and optical mapping. An estimated 98% of the genome coverage was mapped to 10 chromosomal groups, using a two-enzyme hybrid approach. The final reference genome was 40.9 Mb and contained a total of 13,797 annotated genes, supported by transcriptomic and proteogenomics data sets. Whole genome comparative analysis revealed major chromosomal segmental rearrangements and fusions, highlighting intraspecific genome plasticity in this species. Furthermore, the Ptr race classification was not supported at the whole genome level, as phylogenetic analysis did not cluster the ToxA producing isolates. This expansion of available Ptr genomics resources will directly facilitate research aimed at controlling tan spot disease.

The adaptive potential of plant fungal pathogens is largely governed by the gene content of a species, consisting of core and accessory genes across the pathogen isolate repertoire. To approximate the complete gene repertoire of a globally significant crop fungal pathogen, a pan genomic analysis was undertaken for Pyrenophora tritici-repentis (Ptr), the causal agent of tan (or yellow) spot disease in wheat. In this study, 15 new Ptr genomes were sequenced, assembled and annotated, including isolates from three races not previously sequenced. Together with 11 previously published Ptr genomes, a pangenome for 26 Ptr isolates from Australia, Europe, North Africa and America, representing nearly all known races, revealed a conserved core-gene content of 57 % and presents a new Ptr resource for searching natural homologues (orthologues not acquired by horizontal transfer from another species) using remote protein structural homology. Here, we identify for the first time a non-synonymous mutation in the Ptr necrotrophic effector gene ToxB, multiple copies of the inactive toxb within an isolate, a distant natural Pyrenophora homologue of a known Parastagonopora nodorum necrotrophic effector (SnTox3), and clear genomic break points for the ToxA effector horizontal transfer region. This comprehensive genomic analysis of Ptr races includes nine isolates sequenced via long read technologies. Accordingly, these resources provide a more complete representation of the species, and serve as a resource to monitor variations potentially involved in pathogenicity.

The ToxA effector is a major virulence gene of Pyrenophora tritici-repentis (Ptr), a necrotrophic fungus and the causal agent of tan spot disease of wheat. ToxA and co-located genes are believed to be the result of a recent horizontally transferred highly conserved 14kb region a major pathogenic event for Ptr. Since this event, monitoring isolates for pathogenic changes has become important to help understand the underlying mechanisms in play. Here we examined ToxA in 100 Ptr isolates from Australia, Europe, North and South America and the Middle East, and uncovered in isolates from Denmark, Germany and New Zealand a new variation, a novel 166 bp insertion element (PtrHp1) which can form a perfectly matched 59 bp inverted repeat hairpin structure located downstream of the ToxA coding sequence in the 3’ UTR exon. A wider examination revealed PtrHp1 elements to be distributed throughout the genome. Analysis of genomes from Australia and North America had 50–112 perfect copies that often overlap other genes. The hairpin element appears to be unique to Ptr and the lack of ancient origins in other species suggests that PtrHp1 emerged after Ptr speciation. Furthermore, the ToxA UTR insertion site is identical for different isolates, which suggests a single insertion event occurred after the ToxA horizontal transfer. In vitro and in planta-detached leaf assays found that the PtrHp1 element insertion had no effect on ToxA expression. However, variation in the expression of ToxA was detected between the Ptr isolates from different demographic locations, which appears to be unrelated to the presence of the element. We envision that this discovery may contribute towards future understanding of the possible role of hairpin elements in Ptr.

Pyrenophora is a fungal genus responsible for a number of major cereal diseases. Although fungi produce many specialised or secondary metabolites for defence and interacting with the surrounding environment, the repertoire of specialised metabolites (SM) within Pyrenophora pathogenic species remains mostly uncharted. In this study, an in-depth comparative analysis of the P. teres f. teres, P teres f. maculata and P. tritici-repentis potential to produce SMs, based on in silico predicted biosynthetic gene clusters (BGCs), was conducted using genome assemblies from PacBio DNA reads. Conservation of BGCs between the Pyrenophora species included type I polyketide synthases, terpene synthases and the first reporting of a type III polyketide synthase in P teres f. maculata. P. teres isolates exhibited substantial expansion of non-ribosomal peptide synthases relative to P. tritici-repentis, hallmarked by the presence of tailoring cis-acting nitrogen methyltransferase domains. P. teres isolates also possessed unique non-ribosomal peptide synthase (NRPS)-indole and indole BGCs, while a P. tritici-repentis phytotoxin BGC for triticone production was absent in P. teres. These differences highlight diversification between the pathogens that reflects their different evolutionary histories, host adaption and lifestyles.

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 P. 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 genes found across multiple species.

The incidence of wheat head infection by Pyrenophora tritici-repentis (Ptr), the etiological agent of tan spot disease, was evaluated during grain development in a glasshouse experiment. Heads artificially inoculated with a Ptr spore suspension developed widespread brown spots across the spikelets, and mycelia and conidophores were observed on glumes and awns. Seeds of heavily infected heads were darkened and shrivelled, but no red smudge symptoms were apparent. The recovery rate of Ptr isolates from the inoculated wheat heads was low, and colonies that were re-isolated displayed an irregular morphology with reddish mycelia when grown on agar plates. The presence of Ptr on inoculated wheat heads was assessed directly via PCR detection, and a limitation of Ptr hyphae to proliferate beyond the point of contact of spore inoculum on floret tissues was observed. The systemic transmission of Ptr from infected seeds was minimal; however, saprophytic growth of the pathogen occurred on the senescing leaves of wheat plants grown from inoculated seeds. Thus, even though Ptr seed infection is not as common as foliar infection, infected seeds are still a source of disease inoculum and screening for pathogen contamination is advisable.