Rebecca Spanner

The genus Blumeria, comprised of obligate biotrophic fungi, includes some of the most economically significant pathogens of cereal crops worldwide (Glawe 2008). Most notable are the powdery mildews...

Barley leaf rust disease, caused by Puccinia hordei, leads to substantial yield losses and diminished malting quality of barley across temperate growing regions worldwide. To address the paucity of high-resolution genomic resources for this pathogen, we generated haplotype-phased, chromosome-scale assemblies for ten globally distributed isolates using PacBio HiFi and Hi-C sequencing. Phylogenomic analysis revealed seven distinct lineages of P. hordei, including evidence of nuclear exchange, with a shared nuclear haplotype detected between two US lineages. Nuclear genome sizes ranged from ∼140-147Mbp, with the exception of isolate 90ISR03 from Israel (∼163Mbp), which also harbored a 6.2Mbp extra scaffold in one nucleus exhibiting chromosomal characteristics. Consistent with its larger genome, P. hordei had a higher repeat content (∼70%) than related cereal rust fungi, driven primarily by the proliferation of LTR retroelements and DNA transposons. Across the global pan-genome of 13 unique nuclear haplotypes, approximately one-third of all protein orthogroups were conserved across all isolates. Only 18% of predicted effector orthogroups were conserved across all haplotypes, reflecting the highly dynamic and variable nature of the effector repertoire. The long-term propagation of clonal P. hordei lineages is apparent both within the US and globally, and nuclear exchange plays a role in generating novel diversity. Genome plasticity is evident in extensive structural variation, including large-scale translocations and inversions as well as an extra chromosome. These chromosome-level, haplotype-resolved genomes provide a foundational resource for exploring the evolution, diversity, and avirulence gene repertoire of P. hordei.

Wheat stem rust is a disease of global importance caused by the fungal pathogen Puccinia graminis f. sp. tritici ( Pgt ). Here we generate chromosome-level, nuclear-phased genome references for Pgt isolates ETH2013-1 and ITA2018-1, representing races TKTTF and TTRTF respectively, that have caused major epidemics in Africa and Europe. The nuclear haplotypes of ETH2013-1 and ITA2018-1 are unique and unrelated to those of Ug99 and Pgt21. Pgt nuclear haplotypes show extensive variation in sequence and copy number of six known Avr genes and AvrSr33 , which we identify through an effector gene library screen. Recognition properties of 22 novel Avr gene variants explain the race virulence phenotypes and the outbreak of TTRTF on durum cultivars containing Sr13b , since ITA2018-1 carries a homozygous deletion of AvrSr13 . This work establishes an Avr gene atlas for Pgt that can inform wheat breeding and enable development of sequence-based virulence diagnostic tools for pathogen surveillance.

Barley leaf rust disease, caused by Puccinia hordei , leads to substantial yield losses and diminished malting quality of barley across temperate growing regions worldwide. To address the paucity of high-resolution genomic resources for this pathogen, we generated haplotype-phased, chromosome-scale assemblies for ten globally distributed isolates using PacBio HiFi and Hi-C sequencing. Phylogenomic analysis revealed seven distinct lineages of P. hordei , including evidence of nuclear exchange, with a shared nuclear haplotype detected between two US lineages. Nuclear genome sizes ranged from ∼140-147Mbp, with the exception of isolate 90ISR03 from Israel (∼163Mbp), which also harbored a 6.2Mbp supernumerary scaffold in one nucleus exhibiting chromosomal characteristics. Consistent with its larger genome, P. hordei had a higher repeat content (∼70%) than related cereal rust fungi, driven primarily by the proliferation of LTR retroelements and DNA transposons. Across the global pan-genome of 13 unique nuclear haplotypes, approximately one-third of all protein orthogroups were conserved across all isolates. Only 18% of predicted effector orthogroups were shared between all haplotypes, reflecting the highly dynamic and variable nature of the effector repertoire. The long-term propagation of clonal P. hordei lineages is apparent both within the US and globally, and nuclear exchange is important for generating novel diversity and virulence profiles. The high degree of genome plasticity is evident in extensive structural variation, including large-scale translocations and inversions as well as a putative accessory chromosome. These chromosome-level, haplotype-resolved genomes provide a foundational resource for exploring the evolution, diversity, and avirulence gene repertoire of P. hordei .

Host resistance is a critical component of oat crown rust disease management. Pc94 is a qualitative resistance locus derived from diploid Avena strigosa with several independent introgressions into A. sativa that have been used in cultivar deployment. Quantitative trait locus (QTL) analysis combining previously published data for a historic A. strigosa population segregating for Pc94 revealed a large effect QTL on the distal end of A. strigosa chromosome 7A. Genome assembly of the parents identified a cluster of five nucleotide binding site leucine-rich repeat receptor (NLR) candidate genes within the QTL region. A single candidate NLR with an integrated zinc finger BED domain, AstNLR94 , was determined as necessary for Pc94 resistance based on map-based cloning and forward mutagenesis. A presence/absence allele specific PCR marker was designed in AstNLR94 and verified for accuracy and specificity in a diverse panel of A. strigosa and A. sativa . Pc94 introgressions in A. sativa ranged in size from 1.7-71 Mbp and two different introgression locations appear to have occurred. In A. sativa ‘Leggett’, a 6.3 Mbp Pc94 introgression is located at the end of chromosome 2A, and the same sized introgression was discovered in the OT3098 v2 genome. Finally, a QTL analysis identified an additional minor resistance locus on A . strigosa chromosome 4A, which has complicated previous efforts to characterize the Pc94 locus. This is the first report of an NLR gene underlying disease resistance in Avena spp. and delivers a Pc94 marker for marker assisted selection to produce disease resistant cultivars. Key message: We mapped a zfBED-NLR encoding gene necessary for Pc94 resistance, developed a diagnostic marker, and revealed diverse introgression sizes, clarifying Pc94 ’s history and utility for durable oat crown rust resistance.

To exploit allelic variation in Hordeum vulgare subsp. spontaneum, the Wild Barley Diversity Collection was subjected to paired-end Illumina sequencing at ∼9 × depth and evaluated for several agronomic traits. We discovered 240.2 million single nucleotide polymorphisms (SNPs) after alignment to the Morex V3 assembly and 24.4 million short (1 to 50 bp) insertions and deletions. A genome-wide association study of lemma color identified one marker-trait association (MTA) on chromosome 1H close to HvBlp, the cloned gene controlling black lemma. Four MTAs were identified for seedling stem rust resistance, including 2 novel loci on chromosomes 1H and 6H and one co-locating to the complex RMRL1-RMRL2 locus on 5H. The whole-genome sequence data described herein will facilitate the identification and utilization of new alleles for barley improvement.

Cercospora beticola, causal agent of Cercospora leaf spot of sugarbeet ( Beta vulgaris subsp. vulgaris), is the most economically damaging foliar pathogen inhibiting sugarbeet production. Despite the global importance of C. beticola, significant knowledge gaps exist pertaining to pathogen virulence and adaptation to management practices that include the use of resistant host varieties and the application of different fungicide chemistries. The lack of a high-quality reference genome for C. beticola has hindered research progression addressing these critical knowledge gaps. Here, we report the complete and gapless genome assembly and annotation of the reference C. beticola isolate Cb09-40. We identified misassembled regions in the previous reference genome and annotated previously missing protein-coding genes. This work establishes a high-quality reference genome that can serve as the foundation for future comparative genomic, population genetic, and evolutionary studies into the virulence and evolution of this important plant pathogen. [ ] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Cercospora leaf spot (CLS) is a globally important disease of sugar beet (Beta vulgaris) caused by the fungus Cercospora beticola. Long-distance movement of C. beticola has been indirectly evidenced in recent population genetic studies, suggesting potential dispersal via seed. Commercial sugar beet “seed” consists of the reproductive fruit (true seed surrounded by maternal pericarp tissue) coated in artificial pellet material. In this study, we confirmed the presence of viable C. beticola in sugar beet fruit for 10 of 37 tested seed lots. All isolates harbored the G143A mutation associated with quinone outside inhibitor resistance, and 32 of 38 isolates had reduced demethylation inhibitor sensitivity (EC₅₀> 1 µg/ml). Planting of commercial sugar beet seed demonstrated the ability of seedborne inoculum to initiate CLS in sugar beet. C. beticola DNA was detected in DNA isolated from xylem sap, suggesting the vascular system is used to systemically colonize the host. We established nuclear ribosomal internal transcribed spacer region amplicon sequencing using the MinION platform to detect fungi in sugar beet fruit. Fungal sequences from 19 different genera were identified from 11 different sugar beet seed lots, but Fusarium, Alternaria, and Cercospora were consistently the three most dominant taxa, comprising an average of 93% relative read abundance over 11 seed lots. We also present evidence that C. beticola resides in the pericarp of sugar beet fruit rather than the true seed. The presence of seedborne inoculum should be considered when implementing integrated disease management strategies for CLS of sugar beet in the future.

Cercospora beticola is a hemibiotrophic fungus that causes cercospora leaf spot disease of sugar beet (Beta vulgaris). After an initial symptomless biotrophic phase of colonization, necrotic lesions appear on host leaves as the fungus switches to a necrotrophic lifestyle. The phytotoxic secondary metabolite cercosporin has been shown to facilitate fungal virulence for several Cercospora spp. However, because cercosporin production and subsequent cercosporin‐initiated formation of reactive oxygen species is light‐dependent, cell death evocation by this toxin is only fully ensured during a period of light. Here, we report the discovery of the effector protein CbNip1 secreted by C. beticola that causes enhanced necrosis in the absence of light and, therefore, may complement light‐dependent necrosis formation by cercosporin. Infiltration of CbNip1 protein into sugar beet leaves revealed that darkness is essential for full CbNip1‐triggered necrosis, as light exposure delayed CbNip1‐triggered host cell death. Gene expression analysis during host infection shows that CbNip1 expression is correlated with symptom development in planta. Targeted gene replacement of CbNip1 leads to a significant reduction in virulence, indicating the importance of CbNip1 during colonization. Analysis of 89 C. beticola genomes revealed that CbNip1 resides in a region that recently underwent a selective sweep, suggesting selection pressure exists to maintain a beneficial variant of the gene. Taken together, CbNip1 is a crucial effector during the C. beticola–sugar beet disease process.

Cercospora leaf spot is the most important disease of sugar beet worldwide. The disease is caused by the fungus Cercospora beticola and is managed principally by timely application of fungicides including those of the sterol demethylation inhibitor (DMI) class. However, reliance on DMIs has caused an increase in resistance to this class of fungicides in multiple C. beticola populations. To better understand the genetic and evolutionary basis for resistance in C. beticola , a genome-wide association study (GWAS) and selective sweep analysis were conducted for the first time in this fungal plant pathogen. We performed whole genome resequencing of 190 C. beticola isolates predominantly from North Dakota and Minnesota that were phenotyped for sensitivity to tetraconazole, the most widely used DMI fungicide in this region. GWAS identified mutations in genes associated with DMI fungicide resistance including a Regulator of G-protein Signaling (RGS) protein, an ATP-binding cassette (ABC) pleiotropic drug resistance transporter, a dual-specificity tyrosine phosphorylation-regulated kinase (DYRK), and a gene annotated as a hypothetical protein. A SNP upstream of CbCYP51 , the gene encoding the target of DMI fungicides, was also identified via GWAS. Haplotype analysis of CbCYP51 identified a synonymous mutation (E170) in high linkage disequilibrium with the upstream SNP, and multiple non-synonymous mutations (L144F, I387M and Y464S) associated with DMI resistance. Additionally, a putative codon bias effect for the L144F substitution was identified that generated different resistance potentials. We also identified a CbCYP51 paralog in C. beticola , CbCYP51-like, with high protein homology to CYP51C found uniquely in Fusarium species but CbCYP51-like does not appear to influence DMI sensitivity. Genome-wide scans of selection showed that several of the GWAS mutations for fungicide resistance resided in regions that have recently undergone a selective sweep. Using radial plate growth on selected media as a fitness proxy, we did not find a trade-off associated with DMI fungicide resistance suggesting that resistance mutations can persist in C. beticola populations. Taken together, we show that population genomic data from a crop pathogen can allow the identification of mutations conferring fungicide resistance and inform about their origins in the pathogen population.