Project : USDA ARS

ARS Home » Midwest Area » St. Paul, Minnesota » Cereal Disease Lab » Research » Research Project #441854

Research Project: Surveillance, Pathogen Biology, and Host Resistance of Cereal Rusts

2024 Annual Report

Objectives
Objective 1: Monitor, collect, and characterize U.S. cereal rust pathogen populations, and characterize foreign populations that threaten U.S. cereal production. • Sub-objective 1.A: Monitor, collect and characterize cereal rust pathogen populations in the U.S. for virulence that overcomes rust resistance genes in current cultivars. • Sub-objective 1.B: Characterize key exotic rust pathogen strains in advance of their introduction to the United States and contribute towards consortia for global pathogen surveillance through strategic partnerships and alliances. • Sub-objective 1.C: Development and/or improvement of molecular diagnostic tools for detection of cereal rust pathogen strains. Objective 2: Develop new genomic resources for cereal rust pathogens, identify links between pathogen phenotypes and genotypes, and improve understanding of the role of the sexual cycle in population dynamics. • Sub-objective 2.A: Develop genomic resources for population genetics and evolutionary studies of cereal rust pathogens. • Sub-objective 2.B: Identify linkages between phenotype and genotype of cereal rust fungi involved in pathogenicity and host resistance. • Sub-objective 2.C: Improve understanding of the roles of the sexual cycle in cereal rust fungal population dynamics. Objective 3: Improve host resistance in cereal crops to rust pathogens through investigations in sources and genetics of rust resistance, characterization of various germplasm, and incorporation into adapted germplasm. • Sub-objective 3.A: Evaluate wheat, oat and barley germplasm from U.S. breeding programs for rust resistance. • Sub-objective 3.B: Identify and characterize new sources of rust resistance in wheat, barley, and oat. • Sub-objective 3.C: Incorporate rust resistance into adapted germplasm.

Approach
Cereal rust fungi are dynamic leading to constant changes in the U.S. populations, which leads to the erosion of effective resistance in cereal crops. In addition, foreign isolates further threaten cereal production if they are introduced and established. Development of cereal cultivars with effective rust resistance will depend on the monitoring and characterization of virulence phenotypes of the rust pathogens with host differential lines containing single genes for rust resistance. Rust fungi have large, complex genomes and the uredinial stage is dikaryotic with two distinct haploid genomes. Genetic and genomic approaches will be used to (1) complete phased haploid genome assemblies for cereal rust fungi; (2) characterize population genetics of cereal rust pathogens; and (3) identify linkages between phenotype and genotype of cereal rust fungi involved in pathogenicity and host resistance. Surveys and identification of rust infections on Berberis and Mahonia species will be conducted to investigate the potential roles of alternate hosts in pathogen variations and disease epidemiology. Rust resistant cereal germplasm will be selected by testing wheat, oat, and barley lines from breeding programs throughout the U.S. for resistance to Pca, Pgt, P. hordei and P. triticina, using prevalent races, and races that have high virulence to rust resistance genes common in released cultivars and breeding lines. The identity of rust resistance genes in breeding lines will be postulated in seedling tests using specific races of these rust fungi. Adult plant resistance of breeding lines will be evaluated in field plots. Genetic loci that mediate rust resistance genes will be identified along with molecular markers that facilitate plant breeding via marker assisted selection. Advanced germplasm lines with combinations of rust resistance genes will be derived and distributed for use in cultivar development.

Progress Report
Objective 1: Monitor, collect, and characterize U.S. cereal rust pathogen populations, and characterize foreign populations that threaten U.S. cereal production. A total of 119 stem rust samples from wheat, barley, rye and oats were collected and analyzed in the 2023 crop season. Wheat stem rust pathogen (Puccinia graminis f. sp. tritici-Pgt) race QFCSC continues to be the dominant race across the country. Two races MCCDC and GBBBB were found in Louisiana and California, respectively. Race GBBBB has low virulence to wheat resistance genes but highly virulent on barley. Rye stem rust pathogen (P. graminis f. sp. secalis-Pgs) was identified from infected rye, barley and wild grasses and the alternate host, common barberry. Seven races of the oat stem rust pathogen (P. graminis f. sp. avenae-Pga) were identified, and race TGN continues to be the predominant race east of the Rocky Mountains. A total of 48 wheat leaf rust races were identified from 445 isolates derived from 183 wheat leaf rust samples collected in 2023. Races MNPSD, TCTNB and MCTNB with frequencies 15.3%, 10.8% and 7.5%, respectively, dominated the leaf rust population in 2023. A new race, MGPSB, with virulence to leaf rust resistance genes Lr16 and Lr23 was identified. There was a clear trend regional selection of races is driven by host resistance genes used in wheat cultivars. Survey data for the barley leaf rust pathogen (Puccinia hordei-Ph) included 519 isolates in the U.S. across the past 32 years. Virulence frequencies increased for resistance genes Rph3, Rph5, and Rph12 (Rph9.z) in this period. A total of 238 single pustule isolates of the oat crown rust pathogen (P. coronata f. sp. avenae-Pca) derived from 120 collections from 11 states, were tested on 40 oat varieties that carry different crown rust resistance genes. Like previous observations, high virulence diversity is present in the North American Pca population with greater than 88% of isolates being unique. The average number of ineffective resistance genes per isolate is 25, showing a continued trend for progressive increase of virulence. We analyzed 50 international wheat stem rust samples as part of an international surveillance program monitoring the evolution and spread of the pathogen. Results indicated the Ethiopian Pgt population mainly consisted of TTKTF and TKKTF of non-Ug99 races (68%) as well as the most virulent variant TTKTT of the Ug99 race group (26%). A single race TKKTF was identified from samples collected in Georgia. Diagnostic assays of Pgt were performed on 53 international Pgt isolates from Ethiopia and Kenya. Current populations in Ethiopia and Kenya were consistent with previous years suggesting a stable Pgt population that includes Ug99 lineages. In collaboration with USDA-APHIS, Pgt diagnostic assays for Ug99 lineages are being validated. Essential controls of Pgt diagnostic assays were disseminated to colleagues at North Dakota State University. ARS scientists at Saint Paul, Minnesota consulted on the development of USDA-APHIS New Pest Response Guidelines to emerging variants of Pgt. Objective 2: Develop new genomic resources for cereal rust pathogens, identify links between pathogen phenotypes and genotypes, and improve understanding of the role of the sexual cycle in population dynamics. A total of 188 Pgt isolates derived from a sexual population from Spain were tested for virulence on a panel of 50 wheat stem rust resistance gene stocks. The isolates were increased, DNA extracted, and are currently undergoing library construction and sequencing. To develop high quality annotated genomes, we performed RNA sequencing using several stages: spores and infected wheat. These resources will be used to identify causal variants in the Pgt genome that underlie recognition in wheat and barley. Infection of common barberry (Berberis vulgaris) have been observed and collected from southeastern Minnesota and Wisconsin in 2023. Rye stem rust was identified but not wheat stem rust. Rust samples on Berberis fendleri, a native barberry species known to be susceptible to stem and stripe rusts, were collected in Colorado and New Mexico and sequenced to investigate the potential role of this plant in pathogen variations and disease epidemiology. Long read sequencing was performed on seven international Pgt isolates and a unique Pgt isolate from California (23CA1107) with high virulence to barley but not wheat. Hi-C sequencing for chromosome level phasing of eight North American isolates was performed. In collaboration with others, we have developed several fully phased genome assemblies of Pca. Assemblies were resolved at the chromosome-level, fully phased nuclear haplotypes of isolates from across the globe. This data provides evidence of nuclear exchange and recombination as mechanisms that increase genetic diversity. The intercontinental migration of Pca provides further evidence for monitoring this pathogen at a global scale to develop means of effective plant resistance. In collaboration, we phenotyped and sequenced 217 world-wide Pca isolates. Data shows that the U.S. population is genetically diverse with contribution from the sexual cycle (alternate host, buckthorn). This diverse collection allowed genome-wide association analysis to localize 11 virulence-associated loci corresponding to 25 oat varieties that carry different crown rust resistance genes. This work facilitates generation of diagnostic markers for pathogen analysis. Objective 3: Improve host resistance in cereal crops to rust pathogens through investigations in sources and genetics of rust resistance, characterization of various germplasm, and incorporation into adapted germplasm. A total of 3,400 winter wheat, spring wheat and barley breeding lines from public and private breeding programs were tested for stem rust resistance with domestic and foreign stem rust races at the seedling stage, including Ug99 races, and resistance genes were postulated. Stem rust reaction at the adult plant stage is currently being evaluated. Data are distributed to breeders via regional nursery coordinators. Entries from the 2023 Northern and Southern Regional Performance, Uniform Southern and Eastern Soft Red Winter Wheat, and Uniform Southern Soft Red Winter Wheat nurseries were tested with 12 races of leaf rust in seedling tests. The leaf rust resistance genes were postulated based on infection types to different races and molecular marker data. The data and Lr gene postulations were distributed to the nursery organizers. In FY 2024, a total of 726 spring wheat and 374 spring barley lines from 11 collaborating breeders were assessed for response to virulent strains of the wheat stem rust pathogen (including Ug99) in the field in Kenya and Ethiopia. Lines with adult plant resistance to stem rust were identified. A hard red spring wheat line with consistent adult plant resistance in Kenya and Ethiopia was released and reported: MN-Torgy. A line previously identified with adult plant resistance to Ug99 in Kenya was released: MN-Rothsay. We evaluated 190 barley lines from ten breeding programs to identify and postulate resistance to Ph. Overall resistance to at least one isolate other than the moderately avirulent isolate, 17TX10B, was 69 percent. University of Minnesota and Anheuser-Busch had the highest number of resistant lines. Regarding gene postulation, Rph3 was the most common gene, especially in the University of California, Davis program, while Rph5 and Rph7 were postulated to a lesser extent. In collaborations with researchers in the University of Minnesota, we began to investigate the diversity and levels of oat stem rust resistance in oat breeding germplasm. A total of 2,000 cultivars and breeding lines from six U.S. oat breeding programs were tested with three races of Pga at the seedling stage and resistance genes were postulated. Selected resistant lines with potential useful resistance are being evaluated in the 2024 field stem rust nursery. Data has been distributed to breeders. The stem rust resistance gene SrTmp was fine-mapped to a 1.3 Mb region and diagnostic assays developed that can be used in marker-assisted selection. The adult plant resistance (APR) to Ug99 in Minnesota hard red spring wheat variety ‘Linkert’ was dissected and reported. A consistent QTL on chromosome arm 2BS was found and markers were validated for tracking this QTL. The sequence encoding the multi-allelic wheat stem rust resistance gene Sr9 was identified. Though various alleles confer resistance to different spectra of pathogen strains, the different variants of these genes only differed by as little as one amino acid. In 2023, over 2,300 oat breeding lines from various oat uniform nurseries and breeding programs in the U.S. and Canada were evaluated for crown rust resistance in the buckthorn nursery and nearby field plots. In 2024, over 3,400 lines have been planted in the buckthorn nursery and nearby field plots for evaluation. The 2023 lines included over 800 advanced oat breeding lines from the regional programs. Identification and molecular characterization of new/novel crown rust resistance genes is underway in approximately 300 diploid oat accessions from the U.S. and Canadian seed bank collections. Two oat lines carrying multiple APR genes to oat crown rust and linked molecular markers were released in 2024 for use by the oat breeding programs. These APR genes are postulated to be more durable than seedling resistance genes. We are now evaluating additional combination of APR genes in the buckthorn nursery for oat crown rust resistance. The barley stripe rust resistance gene Rps2 is inherited in an additive manner and was fine mapped on chromosome 2H. Three candidate resistance genes were identified, all belonging to the same gene family. Bioinformatic analysis of leaf transcriptome data of cv. Abed Binder 12 found one candidate gene expressed. Near-isogenic line development has advanced to the BC4 stage.

Accomplishments
1. Identification of a new reservoir of highly virulent stem rust isolates in the US. Stem rust, caused by the fungal pathogen Puccinia graminis, is a significant disease on wheat, barley, rye, and oats. A sexual population of stem rust is present in the Midwest by alternating between common barberry (Berberis vulgaris) and native grasses (Elymus spp. and Hordeum spp.). While this population is historically classified as rye stem rust pathogen (P. graminis f. sp. secalis) as it generally only infected rye, it remained unclear if this population could be a reservoir for new sources of virulence on other crops such as wheat and barley. ARS scientists in St. Paul, Minnesota developed a panel of new wheat lines that carry different resistance genes and discovered that isolates from this population can infect wheat cultivars carrying stem rust resistance genes, including Sr22, Sr27, Sr31, Sr50, Sr59, and the 1A.1R translocation. These resistance genes are valuable in current and future breeding for stem rust resistance in cereal crops. This research revealed the presence of new highly virulent population in the U.S. that can contribute to future wheat stem rust epidemics.

2. Three decades of rust surveys in the U.S. reveal drastic virulence changes in oat crown rust. Oat crown rust (Puccinia coronata f. sp. avenae, Pca) is a dynamic pathogen causing significant damage to oat production across the globe. To better understand how Pca has changed in the United States, ARS researchers in St. Paul, Minnesota analyzed 30 years of data from the USDA survey of isolates (n = 5,456) tested on 40 oat varieties that carry different crown rust resistance genes to understand trends in the ability of the pathogen to attack resistance oat varieties. Results showed an incredibly diverse pathogen population, that as a result of genetic exchange the majority of isolates each have a unique response to the panel of diverse oat cultivars. Over time, the pathogen undergoes drastic changes through sexual recombination and mutation that enables it to overcome the majority of sources of resistance available in oat (from 7 virulence per isolate in 1993 to 25 virulence per isolate in 2022). This research establishes empirical data on the increasing ability of oat crown rust to infect varieties and provides them motivation for oat pre-breeders to identify new sources of resistance that are more durable than current sources.

3. Identification of molecular markers for virulence in the oat crown rust fungus. Oat crown rust caused by the fungal pathogen Puccinia coronata f. sp. avenae (Pca) significantly limits US and international oat production. A major challenge for oat breeders is to develop oat cultivars that carry effective resistance to diverse Pca populations. To understand and identify the regions of the Pca genome that are predicted resistance/susceptibility, ARS researchers in St Paul MN, in collaboration with colleagues at other institutions, built upon previous partially phased genome assemblies and established a chromosome-level, and fully phased, reference genome of a historical isolate, Pca203. Disease incidence and sequence analysis of 217 Pca isolates from across the globe was completed. Genome wide association studies using single nucleotide polymorphisms (DNA sequence differences) and the disease incidence data for different fungal races were used to identify multiple virulence-associated genomic regions containing candidate effector genes. These results will be used to develop diagnostic markers to inform oat breeding to ensure that resistance genes are effectively deployed in oat cultivars.

4. Identifying new variants of a wheat stem rust resistance gene to tackle pathogen and environmental change. Wheat is protected from disease by resistance genes. While some resistance genes can function in diverse environments and diverse pathogen populations, others have more limited use. ARS scientists in St. Paul, Minnesota characterized the molecular genetics of stem rust resistance loci in wheat varieties. The DNA sequence in wheat was identified for stem rust resistance gene Sr9 including six different variants that confer resistance to different races of the stem rust pathogen. Based on diversity in wheat, the variants differed by as few as one amino acid. Similarly variation in the stem rust resistance gene Sr13 was also observed. Testing of six novel DNA variants of Sr13 in wheat plants indicated that each were effective and one may represent a new variant, effective to a unique set of races of the stem rust pathogen. Furthermore, wheat resistance genes Sr13 and Sr21 function in a temperature-dependent manner, being more effective at higher temperatures. This contrasts with the wheat resistance gene Sr6 that is more effective at lower temperatures. Together these studies elucidate the genetic and molecular pathways associated with stem rust race- and temperature specificity of stem rust resistance genes. This research will inform wheat breeding strategies using these resistance genes and identifies a method for engineering of genes adapted to appropriate (emerging) pathogen strains and target environments.

U.S. DEPARTMENT OF AGRICULTURE

Cereal Disease Lab: St. Paul, MN