Location: Cereal Disease Lab2021 Annual Report
Objective 1: Monitor, collect, and characterize U.S. cereal rust pathogens. Sub-objective 1.A. Monitor, collect, and characterize cereal rust pathogen populations in the U.S. for virulence to rust resistance genes in current cultivars. Sub-objective 1.B. Determine levels of genetic variation in Puccinia triticina, P. graminis and P. coronata populations. Sub-objective 1.C. Refine phylogenetics and systematics of P. graminis from Mahonia and other native Berberis spp. in North America. Objective 2: Further develop genomic resources of cereal rust pathogens and identify fungal genes involved in pathogenicity and development. Sub-objective 2.A. Identify effectors of P. graminis f. sp. tritici involved in fungal pathogenicity and host resistance. Sub-objective 2.B. Develop genomic resources and tools for more detailed analysis of P. coronata. 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; and incorporate into adapted germplasm.
Cereal rust fungi (Puccinia coronata, P. graminis, and P. triticina) are dynamic leading to constant changes in the U.S. population and erosion of effective rust resistance in cereal crops. In addition, introduction of foreign isolates, such as Ug99, further threaten cereal production. Development of cereal cultivars with effective rust resistance and management strategies of these diseases depend on monitoring, collection, virulence phenotyping, and genotypic characterization of cereal rust pathogen populations. Rust resistant cereal germplasm will be selected by testing wheat, oat, and barley lines from breeding programs throughout the U.S. and other sources for resistance to these pathogens using the prevalent races, and races that have high virulence to rust resistance genes common in released cultivars and breeding lines. Testing with selected isolates of the cereal rust pathogens and host genetics studies will identify the rust resistance genes in breeding lines and germplasm. Advanced germplasm lines with combinations of rust resistance genes will be selected and released for further use in cultivar development. Rust fungi produce a large arsenal of effector proteins in order to infect and colonize the plant host. Genetic and genomic approaches will be used to identify and characterize effector genes from P. graminis and P. coronata.
In the fourth full year we made progress in the following objectives: Objective1: Monitor, collect, and characterize U.S. cereal rust pathogens. In 2020 in the United States, 36 races of Puccinia triticina were identified in collections of leaf rust infected leaves that were sent to researchers at the Cereal Disease Laboratory (CDL) in Saint Paul, Minnesota. A total of 260 isolates were processed for race identification. Travel restrictions due to COVID-19 reduced the number of collections received in 2020. A total of 35 races were described in 2020. In Oklahoma, losses due to leaf rust were estimated to be 5%, with losses of 2% in Texas and 2.8% in Kansas. Losses in the other states were estimated to be 1% or less. Overall estimated losses in wheat in the United States due to leaf rust in 2020 were 15 million bushels. A total of 131 collections of Puccinia coronata from various regions of the United States were received by scientists in Saint Paul, Minnesota. From that sample, 85 individual pustules were purified and tested on the oat differential lines carrying various resistance genes. To date, we have received 14 collections of Puccinia coronata in 2021 from various regions of the United States (a substantial reduction relative to previous years) and are in process of characterizing the fungal population diversity. To date we have obtained 84 of Pca aecia from buckthorn for analysis of diversity in the alternate host. As expected oat crown rust collection displays a highly variable and virulent phenotype indicating the need for more diverse and durable plant resistance genes. A total of 17 isolates of Puccinia hordei, the barley leaf rust pathogen, were tested on barley leaf rust differential lines. No novel virulence was detected. A total of 324 isolates and 116 field samples of Puccinia graminis f. sp. tritici from seven countries were genotyped using single nucleotide polymorphism (SNP) markers. The majority of the isolates from Spain formed a separate phylogenetic clade that was genotypically diverse. A few isolates belonging to genotypes (clade III and IV) which are broadly distributed in southern Europe, Central Asia, Middle East, and Africa were also observed in this set of isolates from Spain. Genotypes observed in northeast Africa were the same as in previous years and included clades I (Ug99), III and IV. A total of 20 isolates derived from 15 stem rust samples from wheat and barley collected from the United States were analyzed, and three races were found in 2020. Three races of oat stem rust pathogen were identified from 10 samples collected across the United States. A total of 78 international stem rust samples from four countries were analyzed and eight races were detected from these samples. These races were identified previously. Rye stem rust pathogen was identified from aecial infections on common barberry collected from Minnesota, Wisconsin, and New England states. Twenty of the 24 resistance genes in the wheat stem rust differentials have reached the BC4F3 stage in backcrossing into Line E background. Objective 2: Whole genome sequencing and de novo assembly of three isolates of P. graminis f. sp. tritici and one isolate of P. graminis f. sp. avenae was completed. Annotation of the P. graminis f. sp. avenae genome has begun. A total of 60 isolates (30 isolates for 1990 and 30 isolates for 2015) of P. coronata from the United States were sequenced for analysis of genetic diversity, possible sources (i.e., the role of sexual vs. mutational derived variation) of pathogenicity and association of genomic segments with virulence. One hundred and twenty four isolates of P. triticina derived from a random mating population were tested for virulence to 24 lines of Thatcher wheat near isogenic for leaf rust resistance genes. DNA will be extracted from these isolates for genotyping by sequencing. The genotype and virulence data will be used in a genome wide association study (GWAS) in order to find SNPs that are tightly linked with loci that control avirulence/virulence in the leaf rust fungus. Objective 3: A total of 1790 spring wheat and barley lines were evaluated in stem rust field nurseries in Njoro, Kenya and Debre Zeit, Ethiopia. The lines were also tested at the seedling stage for response to four virulent isolates of the stem rust pathogen. In this reporting period, we described the genetics of adult plant resistance in spring wheat line CI 14275. This lines possessed three pyramiding quantitative trait loci (QTL) that were consistently detected across environments in the United States, Kenya, and Africa. One of the QTL was coincident with Sr12. Lines with the combination of the three QTL displayed significantly reduced stem rust disease compared to lines without the QTL. Over 375 oat breeding lines from different programs in the United States and Canada were evaluated for crown rust resistance in field plots. Additional 138 advanced oat breeding lines from the regional programs were analyzed for crown rust reaction. Nearly 487 lines from the Aberdeen USDA program were also evaluated for their adult plant resistance to oat crown rust in the buckthorn nursery. Analysis of four additional different oat recombinant inbred line populations has yielded several strong QTL loci explaining from 30% to 50% of phenotypic variance for adult plant crown rust resistance. Easy to use PCR based markers (Kompetitive Allele Specific PCR (KASP)) have been developed flanking these QTL regions. These markers were used to further validate and verify the significance of these genomic regions. A program to pyramid eight of these regions has continued and many lines carrying a combination of four QTL were evaluated in the buckthorn nursery to assess the additive effect of each region on crown rust resistance. Wheat lines with the combination of stem rust resistance gene Sr2 and Fusarium head blight resistance gene Fhb1 were backcrossed to the BC3F3 stage using recurrent parents contributed by collaborating wheat breeders at South Dakota State University, North Dakota State University, and Montana State University. A total of 2500 elite breeding lines and genetic stocks from public/private wheat breeding programs and research institutions in the United States were screened with nine domestic and several selected foreign stem rust races at the seedling stage and with five domestic races in field stem rust nursery. Resistance genes, especially those effective against Ug99 and other foreign races with significant virulence combinations, were postulated. Data were promptly distributed to collaborators. Entries from the 2021 Northern Regional Performance, Southern Regional Performance, Uniform Southern Soft Red Winter Wheat, Uniform Eastern Soft White Winter Wheat, and Uniform Southern Soft Red Winter Wheat nurseries were tested with 11 races of leaf rust in seedling tests. The leaf rust resistance genes were postulated on the basis of the infection types to the different races, and in conjunction with the molecular marker data. The data and Lr gene postulations were distributed to the organizers of each nursery.
1. Origin of the wheat stem rust race group Ug99. The wheat stem rust pathogen, Puccinia graminis f. sp. tritici, continues to evolve and generate new races that threaten wheat production worldwide. The origin of new race groups of the wheat stem rust pathogen is likely due to recombination events between different races but little is known about this process. Previous work by University of Minnesota scientists demonstrated that the Ug99 group was formed by the recombination of DNA between two races of P. graminis f. sp. tritici that did not involve the normal sexual cycle, which is called parasexual recombination, and identified one of the two races. ARS researchers at Saint Paul, Minnesota, identified the second race involved in the formation of the Ug99 group. A new method to characterize DNA sequence variation in individual genes was used to identify one race as the most likely progenitor of the Ug99 group. This was confirmed by comparing the total DNA sequence of the races in the Ug99 group with the progenitor race. This new method can be used for identifying parasexual recombination events in cereal rust fungi. Knowledge of how Ug99 originated and has evolved can be used to develop strategies to control and reduce yield losses in wheat due to stem rust.
2. Identification of a DNA region in the oat crown rust fungus Puccinia coronata f. sp. avenae (Pca) that is associated with overcoming a crown rust resistance gene in oat. Recently high-quality genome sequences of two Pca isolates were released by ARS scientists in Saint Paul, Minnesota. These two genomes were used to direct a population genomics-based study of two sets of Pca collections from 1990 and 2015 and analyze changes that may explain the most recent oat crown rust epidemics across the continental United States. This analysis indicated that the Pca population in 1990 was significantly different compared to the collections from 2015 for DNA sequence and for ability to overcome crown rust resistance genes in oat. We also found evidence for adaptation of the Pca collections to local oat cultivars and wild oats. The distribution of DNA sequence variation and virulence to crown rust resistance genes among the Pca isolates was consistent with both sexual and asexual reproduction occurring in the Pca population. Our work identifies genomic regions and genes, and linked DNA markers that may be involved in local host adaptation and the ability of Pca isolates to overcome crown rust resistance genes. This information will assist in development of crown rust resistant oat cultivars.
3. Identification of genetically distinct groups of Puccinia triticina in the United States. Leaf rust caused by Puccinia triticina is an important disease of wheat. New races of Puccinia triticina that can overcome leaf rust resistance genes in wheat cultivars constantly appear in the United States. ARS scientists at Saint Paul, Minnesota, tested a total of 159 isolates of P. triticina collected from 2011-2018 in the United States for ability to overcome leaf rust resistance genes on a set of 24 lines of wheat that differ by a single leaf rust resistance gene. The isolates were also genotyped for DNA sequence variation and eight genotype groups were identified. Over 80% of the isolates fall into two major groups that were widely distributed across the wheat growing regions of the Great Plains where hard red winter and spring cultivars are grown and the eastern and southern regions of the U.S. where soft red winter wheat cultivars are grown. Nineteen genomic regions were highly associated with overcoming the resistance of 11 leaf rust resistance genes. This data will provide insight on the evolution and increase of leaf rust races that can overcome the resistance in widely grown wheat cultivars in the United States.
4. Characterization of adult plant resistance to wheat stem rust. Stem rust is an important disease of wheat world-wide and new sources of resistance are needed for wheat breeding programs, since new races such as Ug99 have emerged that can overcome stem rust resistance in wheat cultivars. ARS scientists at Saint Paul, Minnesota, identified three genomic regions that confer adult plant resistance to stem rust in the wheat line CI 14275. The regions were located on chromosomes 2B, 3B, and 6A. The region on 2B was also found in a second study. The region on 3B was coincident with stem rust resistance gene Sr12, that was previously demonstrated to confer adult plant resistance to the Ug99 stem rust race group. Wheat lines with the three genomic regions had high levels of resistance that were consistent across environments. Molecular markers linked to the genomic regions can be used to select for wheat lines with adult plant resistance in wheat breeding programs. Adult plant resistance to stem rust in wheat may confer long lasting resistance to stem rust which is important since emerging races of the stem rust pathogen such as Ug99 can overcome frequently used stem rust resistance genes in wheat.
5. Developing oat germplasm with long lasting resistance to crown rust. Crown rust is an important disease of oat in the United States. Many new races of crown rust appear every year in the United States. The new races often overcome the resistance in oat cultivars. It is important for oat breeding programs to have sources of long lasting or durable crown rust resistance. ARS scientists at Saint Paul, Minnesota, conducted genetic studies over several years and at various locations with different oat germplasm lines that have durable crown rust resistance. A total of 11 genomic regions in oat were associated with the durable resistance. DNA markers for each genomic region were also developed. The regions that are associated with durable resistance are being combined in oat germplasm lines using the DNA markers. Oat germplasm with combinations of genomic regions associated with durable resistance can be used by oat breeding projects to develop cultivars with long lasting crown rust resistance.
6. Oat crown rust virulence in the United States has increased dramatically. Crown rust, caused by the fungus Puccinia coronata f. sp. avenae (Pca), is an important disease of oat in the United States. Many new races of crown rust appear every year in the United States. The new races often overcome the crown rust resistance genes in oat cultivars. ARS scientists at Saint Paul, Minnesota, have determined that the average crown rust isolate collected from the oat growing regions of the United States gained the ability to overcome nine crown rust resistance genes in oat between 1993 and 2017. The average isolate collected during the 2017 growing season has the ability to overcome 17 of the 26 oat resistance genes tested. This recent Pca population is capable of overcoming many more oat crown rust resistance genes than the populations from previous years. This result strongly undermines the strategy of how crown rust resistance genes are used in many oat breeding programs and suggests that any combination of previously deployed resistance genes will be quickly overcome by the Pca population.
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