Development of a Greenhouse Assay to Screen Soybean Varieties for Resistance to Aerial Blight Caused by Rhizoctonia solani Anastomosis Group 1-IA.

Aerial blight, caused by the fungus Rhizoctonia solani anastomosis group (AG) 1-IA, is an economically important soybean disease in the mid-Southern United States. Management has relied on fungicide applications during the season, but there is an increasing prevalence of resistance to commonly used strobilurin fungicides and an urgent need to identify soybean varieties resistant to aerial blight. Because the patchy distribution of the pathogen complicates field variety screening, the present study aimed to develop a greenhouse screening protocol to identify soybean varieties resistant to aerial blight. For this, 88 pathogen isolates were collected from commercial fields and research farms across five Louisiana parishes, and 77% were confirmed to be R. solani AG1-IA. Three polymorphic codominant microsatellite markers were used to explore the genetic diversity of 43 R. solani AG1-IA isolates, which showed high genetic diversity, with 35 haplotypes in total and only two haplotypes common to two other locations. Six genetically diverse isolates were chosen and characterized for their virulence and fungicide sensitivity. The isolate AC2 was identified as the most virulent and was resistant to both active ingredients, azoxystrobin and pyraclostrobin, tested. The six isolates were used in greenhouse variety screening trials using a millet inoculation protocol. Of the 31 varieties screened, only Armor 48-D25 was classified as moderately resistant, and plant height to the first node influenced final disease severity. The study provides short-term solutions for growers to choose less susceptible varieties for planting and lays the foundation to characterize host resistance against this important soybean pathogen.

A Moroccan Pyrenophora teres f. teres Population Defeats Rpt5, the Broadly Effective Resistance on Barley Chromosome 6H.

Net form net blotch (NFNB), caused by Pyrenophora teres f. teres, is an important barley disease. The centromeric region of barley chromosome 6H has often been associated with resistance or susceptibility to NFNB, including the broadly effective dominant resistance gene Rpt5 derived from barley line CIho 5791. We characterized a population of Moroccan P. teres f. teres isolates that had overcome Rpt5 resistance and identified quantitative trait loci (QTL) that were effective against these isolates. Eight Moroccan P. teres f. teres isolates were phenotyped on barley lines CIho 5791 and Tifang. Six isolates were virulent on CIho 5791, and two were avirulent. A CIho 5791 × Tifang recombinant inbred line (RIL) population was phenotyped with all eight isolates and confirmed the defeat of the 6H resistance locus formerly mapped as Rpt5 in barley line CI9819. A major QTL on chromosome 3H with the resistance allele derived from Tifang, as well as minor QTL, was identified and provided resistance against these isolates. F2 segregation ratios supported dominant inheritance for both the 3H and 6H resistance. Furthermore, inoculation of progeny isolates derived from a cross of P. teres f. teres isolates 0-1 (virulent on Tifang/avirulent on CIho 5791) and MorSM 40-3 (avirulent on Tifang/virulent on CIho 5791) onto the RIL and F2 populations determined that recombination between isolates can generate novel genotypes that overcome both resistance genes. Markers linked to the QTL identified in this study can be used to incorporate both resistance loci into elite barley cultivars for durable resistance.

The Host Adapted Fungal Pathogens of Pneumocystis Genus Utilize Genic Regional Centromeres.

Centromeres are genomic regions that coordinate accurate chromosomal segregation during mitosis and meiosis. Yet, despite their essential function, centromeres evolve rapidly across eukaryotes. Centromeres are often the sites of chromosomal breaks which contribute to genome shuffling and promote speciation by inhibiting gene flow. How centromeres form in strongly host-adapted fungal pathogens has yet to be investigated. Here, we characterized the centromere structures in closely related species of mammalian-specific pathogens of the fungal phylum of Ascomycota. Methods allowing reliable continuous culture of Pneumocystis species do not currently exist, precluding genetic manipulation. CENP-A, a variant of histone H3, is the epigenetic marker that defines centromeres in most eukaryotes. Using heterologous complementation, we show that the Pneumocystis CENP-A ortholog is functionally equivalent to CENP-ACnp1 of Schizosaccharomyces pombe. Using organisms from a short-term in vitro culture or infected animal models and ChIP-seq, we identified centromeres in three Pneumocystis species that diverged ~100 million years ago. Each species has a unique short regional centromere (< 10kb) flanked by heterochromatin in 16-17 monocentric chromosomes. They span active genes and lack conserved DNA sequence motifs and repeats. CENP-C, a scaffold protein that links the inner centromere to the kinetochore appears dispensable in one species, suggesting a kinetochore rewiring. Despite the loss of DNA methyltransferases, 5-methylcytosine DNA methylation occurs in these species, though not related to centromere function. These features suggest an epigenetic specification of centromere function.

A High-Quality Genome Assembly for Cercospora janseana, Causal Agent of Narrow Brown Leaf Spot of Rice.

Cercospora janseana causes narrow brown leaf spot of rice. A nearly complete telomere-to-telomere reference genome was assembled with a combination of Oxford Nanopore and Illumina sequences. The genome assembly has a total length of 39,075,509 bp and consists of 15 chromosomes, 14 of which have telomeric repeats at both ends. The assembly N50 is 2.97 Mb and the L50 is five contigs. RNA-seq-mediated gene annotation identified 10,850 genes, including 955 predicted secreted proteins and 361 predicted effector proteins. This highly contiguous and almost complete C. janseana reference genome will be a vital resource for further investigation of host-pathogen interactions and genome evolution within this pathosystem. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Association mapping reveals a reciprocal virulence/avirulence locus within diverse US Pyrenophora teres f. maculata isolates.

BACKGROUND: Spot form net blotch (SFNB) caused by the necrotrophic fungal pathogen Pyrenophora teres f. maculata (Ptm) is an economically important disease of barley that also infects wheat. Using genetic analysis to characterize loci in Ptm genomes associated with virulence or avirulence is an important step to identify pathogen effectors that determine compatible (virulent) or incompatible (avirulent) interactions with cereal hosts. Association mapping (AM) is a powerful tool for detecting virulence loci utilizing phenotyping and genotyping data generated for natural populations of plant pathogenic fungi. RESULTS: Restriction-site associated DNA genotyping-by-sequencing (RAD-GBS) was used to generate 4,836 single nucleotide polymorphism (SNP) markers for a natural population of 103 Ptm isolates collected from Idaho, Montana and North Dakota. Association mapping analyses were performed utilizing the genotyping and infection type data generated for each isolate when challenged on barley seedlings of thirty SFNB differential barley lines. A total of 39 marker trait associations (MTAs) were detected across the 20 barley lines corresponding to 30 quantitative trait loci (QTL); 26 novel QTL and four that were previously mapped in Ptm biparental populations. These results using diverse US isolates and barley lines showed numerous barley-Ptm genetic interactions with seven of the 30 Ptm virulence/avirulence loci falling on chromosome 3, suggesting that it is a reservoir of diverse virulence effectors. One of the loci exhibited reciprocal virulence/avirulence with one haplotype predominantly present in isolates collected from Idaho increasing virulence on barley line MXB468 and the alternative haplotype predominantly present in isolates collected from North Dakota and Montana increasing virulence on barley line CI9819. CONCLUSIONS: Association mapping provided novel insight into the host pathogen genetic interactions occurring in the barley-Ptm pathosystem. The analysis suggests that chromosome 3 of Ptm serves as an effector reservoir in concordance with previous reports for Pyrenophora teres f. teres, the causal agent of the closely related disease net form net blotch. Additionally, these analyses identified the first reported case of a reciprocal pathogen virulence locus. However, further investigation of the pathosystem is required to determine if multiple genes or alleles of the same gene are responsible for this genetic phenomenon.

A triple threat: the Parastagonospora nodorum SnTox267 effector exploits three distinct host genetic factors to cause disease in wheat.

Parastagonospora nodorum is a fungal pathogen of wheat. As a necrotrophic specialist, it deploys effector proteins that target dominant host susceptibility genes to elicit programmed cell death (PCD). Here we identify and functionally validate the effector targeting the host susceptibility genes Snn2, Snn6 and Snn7. We utilized whole-genome sequencing, association mapping, gene-disrupted mutants, gain-of-function transformants, virulence assays, bioinformatics and quantitative PCR to characterize these interactions. A single proteinaceous effector, SnTox267, targeted Snn2, Snn6 and Snn7 to trigger PCD. Snn2 and Snn6 functioned cooperatively to trigger PCD in a light-dependent pathway, whereas Snn7-mediated PCD functioned in a light-independent pathway. Isolates harboring 20 SnTox267 protein isoforms quantitatively varied in virulence. The diversity and distribution of isoforms varied between populations, indicating adaptation to local selection pressures. SnTox267 deletion resulted in the upregulation of effector genes SnToxA, SnTox1 and SnTox3. We validated a novel effector operating in an inverse-gene-for-gene manner to target three genetically distinct host susceptibility genes and elicit PCD. The discovery of the complementary gene action of Snn2 and Snn6 indicates their potential function in a guard or decoy model. Additionally, differences in light dependency in the elicited pathways and upregulation of unlinked effectors sheds new light onto a complex fungal necrotroph-host interaction.

The Parastagonospora nodorum necrotrophic effector SnTox5 targets the wheat gene Snn5 and facilitates entry into the leaf mesophyll.

Parastagonospora nodorum is an economically important necrotrophic fungal pathogen of wheat. Parastagonospora nodorum secretes necrotrophic effectors that target wheat susceptibility genes to induce programmed cell death (PCD). In this study, we cloned and functionally validated SnTox5 and characterized its role in pathogenesis. We used whole genome sequencing, genome-wide association study (GWAS) mapping, CRISPR-Cas9-based gene disruption, gain-of-function transformation, quantitative trait locus (QTL) analysis, haplotype and isoform analysis, protein modeling, quantitative PCR, and laser confocal microscopy to validate SnTox5 and functionally characterize SnTox5. SnTox5 is a mature 16.26 kDa protein with high structural similarity to SnTox3. Wild-type and mutant P. nodorum strains and wheat genotypes of SnTox5 and Snn5, respectively, were used to show that SnTox5 not only targets Snn5 to induce PCD but also facilitates the colonization of the mesophyll layer even in the absence of Snn5. Here we show that SnTox5 facilitates the efficient colonization of the mesophyll tissue and elicits PCD specific to host lines carrying Snn5. The homology to SnTox3 and the ability of SnTox5 to facilitate the colonizing of the mesophyll also suggest a role in the suppression of host defense before PCD induction.

Identification and mapping of a novel resistance gene to the rice pathogen, Cercospora janseana.

KEY MESSAGE: The genetic architecture of resistance to Cercospora janseana was examined, and a single resistance locus was identified. A SNP marker was identified and validated for utilization in U.S. breeding germplasm Cercospora janseana (Racib.) is a fungal pathogen that causes narrow brown leaf spot (NBLS) in rice. Although NBLS is a major disease in the southern United States and variation in resistance among U.S. rice germplasm exists, little is known about the genetic architecture underlying the trait. In this study, a recombinant inbred line population was evaluated for NBLS resistance under natural disease infestation in the field across three years. A single, large-effect QTL, CRSP-2.1, was identified that explained 81.4% of the phenotypic variation. The QTL was defined to a 532 kb physical interval and 13 single nucleotide polymorphisms (SNPs) were identified across the region to characterize the haplotype diversity present in U.S. rice germplasm. A panel of 387 U.S. rice germplasm was genotyped with the 13 haplotype SNPs and phenotyped over two years for NBLS resistance. Fourteen haplotypes were identified, with six haplotypes accounting for 94% of the panel. The susceptible haplotype from the RIL population was the only susceptible haplotype observed in the U.S. germplasm. A single SNP was identified that distinguished the susceptible haplotype from all resistant haplotypes, explaining 52.7% of the phenotypic variation for NBLS resistance. Pedigree analysis and haplotype characterization of historical germplasm demonstrated that the susceptible haplotype was introduced into Southern U.S. germplasm through the California line L-202 into the Louisiana variety Cypress. Cypress was extensively used as a parent over the last 25 years, resulting in the susceptible CRSP-2.1 allele increasing in frequency from zero to 44% in the modern U.S. germplasm panel.

Botrytis spp.: A Contemporary Perspective and Synthesis of Recent Scientific Developments of a Widespread Genus that Threatens Global Food Security.

This perspective presents a synopsis of the topics contained in the Phytopathology Pathogen Spotlight on Botrytis spp. causing gray mold, including pathogen biology and systematics, genomic characterization of new species, perspectives on genome editing, and fungicide resistance. A timely breakthrough to engineer host plant resistance against the gray mold fungus has been demonstrated in planta and may augment chemical controls in the near future. While B. cinerea has garnered much of the research attention, other economically important Botrytis spp. have been identified and characterized via morphological and genome-based approaches. Gray mold control is achieved primarily through fungicide applications but resistance to various chemical classes is a major concern that threatens global plant health and food security. In this issue, new information on molecular mechanism(s) of fungicide resistance and ways to manage control failures are presented. Finally, a significant leap in fundamental pathogen biology has been achieved via development of CRISPR/Cas9 to assess gene function in the fungus which likely will spawn new control mechanisms and facilitate gene discovery studies.

The Barley HvWRKY6 Transcription Factor Is Required for Resistance Against Pyrenophora teres f. teres.

Barley is an important cereal crop worldwide because of its use in the brewing and distilling industry. However, adequate supplies of quality malting barley are threatened by global climate change due to drought in some regions and excess precipitation in others, which facilitates epidemics caused by fungal pathogens. The disease net form net blotch caused by the necrotrophic fungal pathogen Pyrenophora teres f. teres (Ptt) has emerged as a global threat to barley production and diverse populations of Ptt have shown a capacity to overcome deployed genetic resistances. The barley line CI5791 exhibits remarkably effective resistance to diverse Ptt isolates from around the world that maps to two major QTL on chromosomes 3H and 6H. To identify genes involved in this effective resistance, CI5791 seed were γ-irradiated and two mutants, designated CI5791-γ3 and CI5791-γ8, with compromised Ptt resistance were identified from an M2 population. Phenotyping of CI5791-γ3 and -γ8 × Heartland F2 populations showed three resistant to one susceptible segregation ratios and CI5791-γ3 × -γ8 F1 individuals were susceptible, thus these independent mutants are in a single allelic gene. Thirty-four homozygous mutant (susceptible) CI5791-γ3 × Heartland F2 individuals, representing 68 recombinant gametes, were genotyped via PCR genotype by sequencing. The data were used for single marker regression mapping placing the mutation on chromosome 3H within an approximate 75 cM interval encompassing the 3H CI5791 resistance QTL. Sequencing of the mutants and wild-type (WT) CI5791 genomic DNA following exome capture identified independent mutations of the HvWRKY6 transcription factor located on chromosome 3H at ∼50.7 cM, within the genetically delimited region. Post transcriptional gene silencing of HvWRKY6 in barley line CI5791 resulted in Ptt susceptibility, confirming that it functions in NFNB resistance, validating it as the gene underlying the mutant phenotypes. Allele analysis and transcript regulation of HvWRKY6 from resistant and susceptible lines revealed sequence identity and upregulation upon pathogen challenge in all genotypes analyzed, suggesting a conserved transcription factor is involved in the defense against the necrotrophic pathogen. We hypothesize that HvWRKY6 functions as a conserved signaling component of defense mechanisms that restricts Ptt growth in barley.

A Comparative Genomic Analysis of the Barley Pathogen Pyrenophora teres f. teres Identifies Subtelomeric Regions as Drivers of Virulence.

Pyrenophora teres f. teres causes net form net blotch of barley and is an economically important pathogen throughout the world. However, P. teres f. teres is lacking in the genomic resources necessary to characterize the mechanisms of virulence. Recently a high-quality reference genome was generated for P. teres f. teres isolate 0-1. Here, we present the reference quality sequence and annotation of four new isolates and we use the five available P. teres f. teres genomes for an in-depth comparison, resulting in the generation of hypotheses pertaining to the potential mechanisms and evolution of virulence. Comparative analyses were performed between all five P. teres f. teres genomes, examining genomic organization, structural variations, and core and accessory genomic content, specifically focusing on the genomic characterization of known virulence loci and the localization of genes predicted to encode secreted and effector proteins. We showed that 14 of 15 currently published virulence quantitative trait loci (QTL) span accessory genomic regions, consistent with these accessory regions being important drivers of host adaptation. Additionally, these accessory genomic regions were frequently found in subtelomeric regions of chromosomes, with 10 of the 14 accessory region QTL localizing to subtelomeric regions. Comparative analysis of the subtelomeric regions of P. teres f. teres chromosomes revealed translocation events in which homology was detected between nonhomologous chromosomes at a significantly higher rate than the rest of the genome. These results indicate that the subtelomeric accessory genomic compartments not only harbor most of the known virulence loci but, also, that these regions have the capacity to rapidly evolve.

Local adaptation drives the diversification of effectors in the fungal wheat pathogen Parastagonospora nodorum in the United States.

Filamentous fungi rapidly evolve in response to environmental selection pressures in part due to their genomic plasticity. Parastagonospora nodorum, a fungal pathogen of wheat and causal agent of septoria nodorum blotch, responds to selection pressure exerted by its host, influencing the gain, loss, or functional diversification of virulence determinants, known as effector genes. Whole genome resequencing of 197 P. nodorum isolates collected from spring, durum, and winter wheat production regions of the United States enabled the examination of effector diversity and genomic regions under selection specific to geographically discrete populations. 1,026,859 SNPs/InDels were used to identify novel loci, as well as SnToxA and SnTox3 as factors in disease. Genes displaying presence/absence variation, predicted effector genes, and genes localized on an accessory chromosome had significantly higher pN/pS ratios, indicating a higher rate of sequence evolution. Population structure analyses indicated two P. nodorum populations corresponding to the Upper Midwest (Population 1) and Southern/Eastern United States (Population 2). Prevalence of SnToxA varied greatly between the two populations which correlated with presence of the host sensitivity gene Tsn1 in the most prevalent cultivars in the corresponding regions. Additionally, 12 and 5 candidate effector genes were observed to be under diversifying selection among isolates from Population 1 and 2, respectively, but under purifying selection or neutrally evolving in the opposite population. Selective sweep analysis revealed 10 and 19 regions that had recently undergone positive selection in Population 1 and 2, respectively, involving 92 genes in total. When comparing genes with and without presence/absence variation, those genes exhibiting this variation were significantly closer to transposable elements. Taken together, these results indicate that P. nodorum is rapidly adapting to distinct selection pressures unique to spring and winter wheat production regions by rapid adaptive evolution and various routes of genomic diversification, potentially facilitated through transposable element activity.

Mapping of barley susceptibility/resistance QTL against spot form net blotch caused by Pyrenophora teres f. maculata using RIL populations.

Spot form net blotch (SFNB) caused by the necrotrophic fungal pathogen Pyrenophora teres f. maculata (Ptm) is an important disease of barley worldwide including the major barley production regions of North America. To characterize SFNB resistance/susceptibility quantitative trait loci (QTL), three recombinant inbred line (RIL) populations were developed from crosses between the malting barley cultivars, Tradition (six row) and Pinnacle (two row), and the two world barley core collection lines, PI67381 and PI84314. Tradition and Pinnacle were susceptible to many North American Ptm isolates, while PI67381 and PI84314 carry resistances to diverse Ptm isolates from across the globe. The RIL populations, Tradition/PI67381, Pinnacle/PI67381, and Pinnacle/PI84314 were genotyped using polymerase chain reaction-mediated genotype-by-sequencing single nucleotide polymorphism marker panels and phenotyped at the seedling stage with six geographically distinct Ptm isolates: FGOB10Ptm-1 (North Dakota, USA), Pin-A14 (Montana, USA), Cel-A17 (Montana, USA), SG1 (Australia), NZKF2 (New Zealand) and DEN2.6 (Denmark). The goal was to determine if the susceptible elite lines contained common susceptibility genes/QTL or if the resistant lines had common resistant genes/QTL effective against diverse Ptm isolates. The QTL analyses identified a total of 12 resistance and/or susceptibility loci on chromosomes 2H, 3H, 4H, 6H, and 7H of which three had not been previously reported. Common major QTL were detected on chromosome 2H (R2 = 14-40%) and 7H (R2 = 24-80%) in all three RIL populations, suggesting underlying genes with broad resistance specificity. The major 7H QTL was shown to be a dominant susceptibility gene in both susceptible malting barley varieties.

Genetics of Variable Disease Expression Conferred by Inverse Gene-For-Gene Interactions in the Wheat-Parastagonospora nodorum Pathosystem.

The wheat-Parastagonospora nodorum pathosystem involves the recognition of pathogen-secreted necrotrophic effectors (NEs) by corresponding wheat NE sensitivity genes. This inverse gene-for-gene recognition leads to necrotrophic effector-triggered susceptibility and ultimately septoria nodorum blotch disease. Here, we used multiple pathogen isolates to individually evaluate the effects of the host gene-NE interactions Tan spot necrosis1-Stagonospora nodorum ToxinA (Tsn1-SnToxA), Stagonospora nodorum necrosis1-Stagonospora nodorum Toxin1 (Snn1-SnTox1), and Stagonospora nodorum necrosis3-B genome homeolog1-Stagonospora nodorum Toxin3 (Snn3-B1-SnTox3), alone and in various combinations, to determine the relative importance of these interactions in causing disease. Genetic analysis of a recombinant inbred wheat population inoculated separately with three P. nodorum isolates, all of which produce all three NEs, indicated that the Tsn1-SnToxA and Snn3-B1-SnTox3 interactions contributed to disease caused by all four isolates, but their effects varied and ranged from epistatic to additive. The Snn1-SnTox1 interaction was associated with increased disease for one isolate, but for other isolates, there was evidence that this interaction inhibited the expression of other host gene-NE interactions. RNA sequencing analysis in planta showed that SnTox1 was differentially expressed between these three isolates after infection. Further analysis of NE gene-knockout isolates showed that the effect of some interactions could be masked or inhibited by other compatible interactions, and the regulation of this occurs at the level of NE gene transcription. Collectively, these results show that the inverse gene-for-gene interactions leading to necrotrophic effector-triggered susceptibility in the wheat-P. nodorum pathosystem vary in their effects depending on the genetic backgrounds of the pathogen and host, and interplay among the interactions is complex and intricately regulated.

Reference Quality Genome Assemblies of Three Parastagonospora nodorum Isolates Differing in Virulence on Wheat.

Parastagonospora nodorum, the causal agent of Septoria nodorum blotch in wheat, has emerged as a model necrotrophic fungal organism for the study of host-microbe interactions. To date, three necrotrophic effectors have been identified and characterized from this pathogen, including SnToxA, SnTox1, and SnTox3. Necrotrophic effector identification was greatly aided by the development of a draft genome of Australian isolate SN15 via Sanger sequencing, yet it remained largely fragmented. This research presents the development of nearly finished genomes of P. nodorum isolates Sn4, Sn2000, and Sn79-1087 using long-read sequencing technology. RNAseq analysis of isolate Sn4, consisting of eight time points covering various developmental and infection stages, mediated the annotation of 13,379 genes. Analysis of these genomes revealed large-scale polymorphism between the three isolates, including the complete absence of contig 23 from isolate Sn79-1087, and a region of genome expansion on contig 10 in isolates Sn4 and Sn2000. Additionally, these genomes exhibit the hallmark characteristics of a "two-speed" genome, being partitioned into two distinct GC-equilibrated and AT-rich compartments. Interestingly, isolate Sn79-1087 contains a lower proportion of AT-rich segments, indicating a potential lack of evolutionary hotspots. These newly sequenced genomes, consisting of telomere-to-telomere assemblies of nearly all 23 P. nodorum chromosomes, provide a robust foundation for the further examination of effector biology and genome evolution.

Reference Assembly and Annotation of the Pyrenophora teres f. teres Isolate 0-1.

Pyrenophora teres f. teres, the causal agent of net form net blotch (NFNB) of barley, is a destructive pathogen in barley-growing regions throughout the world. Typical yield losses due to NFNB range from 10 to 40%; however, complete loss has been observed on highly susceptible barley lines where environmental conditions favor the pathogen. Currently, genomic resources for this economically important pathogen are limited to a fragmented draft genome assembly and annotation, with limited RNA support of the P. teres f. teres isolate 0-1. This research presents an updated 0-1 reference assembly facilitated by long-read sequencing and scaffolding with the assistance of genetic linkage maps. Additionally, genome annotation was mediated by RNAseq analysis using three infection time points and a pure culture sample, resulting in 11,541 high-confidence gene models. The 0-1 genome assembly and annotation presented here now contains the majority of the repetitive content of the genome. Analysis of the 0-1 genome revealed classic characteristics of a "two-speed" genome, being compartmentalized into GC-equilibrated and AT-rich compartments. The assembly of repetitive AT-rich regions will be important for future investigation of genes known as effectors, which often reside in close proximity to repetitive regions. These effectors are responsible for manipulation of the host defense during infection. This updated P. teres f. teres isolate 0-1 reference genome assembly and annotation provides a robust resource for the examination of the barley-P. teres f. teres host-pathogen coevolution.

Genetic analysis of virulence in the Pyrenophora teres f. teres population BB25×FGOH04Ptt-21.

Pyrenophora teres f. teres is the causal agent of net form net blotch (NFNB) of barley. In order to map the genetics of avirulence/virulence in P. teres f. teres, a fungal population was developed using P. teres f. teres isolates BB25 (Denmark) and FGOH04Ptt-21 (North Dakota, USA) due to these two isolates differing in virulence on several common barley lines. 109 progeny isolates were obtained from the BB25 by FGOH04Ptt-21 cross that were then used for NFNB disease evaluation across eight barley lines, four of which have been used commonly as NFNB differential lines as well as four cultivars commonly used in barley production in the Northern Great Plains. BB25 was virulent on one of the barley lines and avirulent on seven of the barley lines whereas, FGOH04Ptt-21 was virulent on all eight barley lines evaluated. Genetic maps were generated with single nucleotide polymorphism (SNP) markers obtained using a restriction associated DNA genotyping by sequencing (RAD-GBS) approach. Sixteen linkage groups were formed and were used to identify quantitative trait loci (QTL) associated with avirulence/virulence. Nine unique QTL were identified on eight linkage groups out of which three QTL had major effects (R2≥45%) while the remaining six QTL were relatively minor (R2<20%). One or two major effect loci were identified for the lines commonly used as differentials. Conversely, variation in virulence on the local barley cultivars was mostly associated with small effect loci that contributed quantitatively to disease.

Characterizing the Pyrenophora teres f. maculata-Barley Interaction Using Pathogen Genetics.

Pyrenophora teres f. maculata is the cause of the foliar disease spot form net blotch (SFNB) on barley. To evaluate pathogen genetics underlying the P. teres f. maculata-barley interaction, we developed a 105-progeny population by crossing two globally diverse isolates, one from North Dakota and the other from Western Australia. Progeny were phenotyped on a set of four barley genotypes showing a differential reaction to the parental isolates, then genotyped using a restriction site-associated-genotype-by-sequencing (RAD-GBS) approach. Genetic maps were developed for use in quantitative trait locus (QTL) analysis to identify virulence-associated QTL. Six QTL were identified on five different linkage groups and individually accounted for 20-37% of the disease variation, with the number of significant QTL ranging from two to four for the barley genotypes evaluated. The data presented demonstrate the complexity of virulence involved in the P. teres f. maculata-barley pathosystem and begins to lay the foundation for understanding this important interaction.

Association mapping utilizing diverse barley lines reveals net form net blotch seedling resistance/susceptibility loci.

KEY MESSAGE: A diverse collection of barley lines was phenotyped with three North American Pyrenophora teres f. teres isolates and association analyses detected 78 significant marker-trait associations at 16 genomic loci. Pyrenophora teres f. teres is a necrotrophic fungal pathogen and the causal agent of the economically important foliar disease net form net blotch (NFNB) of barley. The deployment of effective and durable resistance against P. teres f. teres has been hindered by the complexity of quantitative resistance and susceptibility. Several bi-parental mapping populations have been used to identify QTL associated with NFNB disease on all seven barley chromosomes. Here, we report the first genome-wide association study (GWAS) to detect marker-trait associations for resistance or susceptibility to P. teres f. teres. Geographically diverse barley genotypes from a world barley core collection (957) were genotyped with the Illumina barley iSelect chip and phenotyped with three P. teres f. teres isolates collected in two geographical regions of the USA (15A, 6A and LDNH04Ptt19). The best of nine regression models tested were identified for each isolate and used for association analysis resulting in the identification of 78 significant marker-trait associations (MTA; -log10p value >3.0). The MTA identified corresponded to 16 unique genomic loci as determined by analysis of local linkage disequilibrium between markers that did not meet a correlation threshold of R 2 ≥ 0.1, indicating that the markers represented distinct loci. Five loci identified represent novel QTL and were designated QRptts-3HL, QRptts-4HS, QRptts-5HL.1, QRptts-5HL.2, and QRptts-7HL.1. In addition, 55 of the barley lines examined exhibited a high level of resistance to all three isolates and the SNP markers identified will provide useful genetic resources for barley breeding programs.