Research Project: Improving Control of Stripe Rusts of Wheat and Barley through Characterization of Pathogen Populations and Enhancement of Host Resistance

Location: Wheat Health, Genetics, and Quality Research

2020 Annual Report

Objectives
Stripe rust is one of the most important diseases of wheat throughout the U.S. and stripe rust of barley causes significant yield losses in western U.S. Significant progress has been made in understanding biology of the pathogen, virulence compositions of the pathogen population, identification of new sources of resistance, disease forecasting, and control of the disease using fungicides. However, research is needed to develop more effective strategies for sustainable control of stripe rusts. Therefore, this project has the following objectives for the next five years: Objective 1: Monitor and characterize stripe rust pathogen populations for providing essential information to growers for implementing appropriate measures to reduce damage on wheat and barley. Subobjective 1A: Identify virulent races of stripe rust pathogens to determine effectiveness of resistance genes in wheat and barley. Subobjective 1B: Develop molecular markers and characterize stripe rust pathogen populations to identify factors and mechanisms of pathogen dynamics for developing new management strategies. Objective 2: Enhance resistance in wheat and barley cultivars for sustainable control of stripe rusts. Subobjective 2A: Identify and map new genes for stripe rust resistance, and develop new germplasm for use in breeding programs. Subobjective 2B: Screen breeding lines for supporting breeding programs to develop wheat and barley cultivars with adequate and durable resistance to stripe rust. Accomplishment of these objectives will lead to improved knowledge of the disease epidemiology for developing more effective control strategies, more resistance genes and resistant germplasm to be used by breeders to develop stripe rust resistant wheat and barley cultivars, and more effective technology to be used by wheat and barley growers to achieve sustainable control of stripe rust.

Approach
Monitoring the prevalence, severity, and distribution of stripe rust and identify races of the pathogens, commercial fields, monitoring nurseries, trap plots, and experimental plots of wheat and barley, as well as wild grasses, will be surveyed during the plant growing-season. Recommendations will be made based on stripe rust forecasts and survey data to control stripe rust on a yearly basis. Rust samples will be collected by collaborators and ourselves during surveys and data-recording of commercial fields, experimental plots, and monitoring nurseries. Stripe rust samples will be tested in our laboratory for race identification. New races will be tested on genetic stocks, commercial cultivars, and breeding lines to determine their impact on germplasm and production of wheat and barley. Isolates of stripe rust pathogens will also be characterized using various molecular markers to determine population structure changes. To identify new genes for resistance to stripe rust in wheat, the mapping populations, which have been and will be developed in our laboratory, will be phenotyped for response to stripe rust in fields and/or greenhouse and genotyped using different approaches and marker techniques such as simple sequence repeat (SSR), single-nucleotide polymorphism (SNP), and /or genotyping by sequencing (GBS) markers. Linkage maps will be constructed with the genotype data using software Mapmaker and JoinMap. Genes conferring qualitative resistance will be mapped using the phenotypic and genotypic data using JoinMap or MapDisto, and quantitative trait locus (QTL) mapping will be conducted using the composite interval mapping program in the WinQTL Cartographer software. Relationships of genes or QTL to previous reported stripe rust genes will be determined based on chromosomal locations of tightly linked markers, type of resistance and race spectra, and origins of the resistance gene donors. Allelism tests will be conducted with crosses made between lines with potentially new gene with previously reported genes in the same chromosomal arms to confirm the new genes. A homozygous line carrying a identified new gene with improved plant type and agronomic traits will be selected from the progeny population using both rust testing and molecular markers. Lines with combinations of two genes in a same chromosome will be selected and confirmed using phenotypic and marker data. The new germplasm lines will be provided to breeding programs for developing resistant cultivars. To support breeding programs for developing cultivars with stripe rust resistance, wheat and barley nurseries will be evaluated in two locations: Pullman (eastern Washington) and Mount Vernon (western Washington) under natural infection of the stripe rust pathogens. Variety trials and uniform regional nurseries will also be tested in seedling and adult-plant stages with selected races of the stripe rust pathogen under controlled greenhouse conditions. The data will be provided to breeding programs for releasing new cultivars with adequate level and potentially durable stripe rust resistance.

Progress Report
Progress was made on the two objectives and four subobjectives, all of which fall under National Program 303 Plant Diseases. Under Sub-objective 1A, ARS scientists in Pullman, Washington, completed all tests for identifying races from the stripe rust collections in the United States in 2019, and reported the results to the cereal community of researchers, developers, and growers. During the 2020 growing season, field surveys were conducted and recommendations were made for control of stripe rust, and stripe rust samples were collected in the Pacific Northwest. Through collaborators in other regions, more than three hundred stripe rust samples were received from twelve states. These samples were used to inoculate susceptible wheat plants for recovering and increasing spores. Each isolate was tested on the set of wheat or barley differentials for identifying races of the wheat or barley stripe rust pathogen. From the 2019 samples, they identified 25 races (including two new races) of the wheat stripe rust pathogen and 10 races (including one new race) of the barley stripe rust pathogen and determined the distributions and frequencies of the races and virulence factors. From the 2020 collection, 10 races of the wheat stripe rust pathogen and three races of the barley stripe rust pathogen have been identified. Based on the virulence data of the races, resistance genes, such as Yr5, Yr15, and others, have been determined to be effective against all populations of the wheat stripe rust pathogen in the United States, and these genes can be used in breeding programs for developing stripe rust resistant wheat cultivars. Similar information has also been obtained for the barley stripe rust pathogen and barley resistance genes. Based on virulence patterns, distributions, and frequencies, they selected races for use in their wheat and barley germplasm screening research. This progress is on schedule. Under Sub-objective 1B, ARS scientists in Pullman, Washington, sequenced the genomes of a mutant population consisting of 30 isolates developed by chemical mutagenesis of an isolate representing the least avirulent race PSTv-18 and 117 isolates developed through selfing an isolate representing race PSTv-4 on alternate host barberry plants under controlled greenhouse conditions. Through analyzing the sequence data of the mutant population, they identified 62 genes highly associated to 16 avirulence genes, including 48 encoding secreted protein (SP) genes and 14 non-SP genes with high confidences based on various criteria. From the sexually reproduced population, they determined the modes of inheritance for 9 avirulence genes, constructed a genetic map consisting of 42 linkage groups and mapped 6 avirulence loci including a cluster containing four avirulence genes. The avirulence candidate genes identified in these studies will be used in further studies to clone the avirulence genes and used to develop avirulence specific markers for monitoring the stripe rust population and study the mechanisms of the plant-pathogen interactions. Using the core set of simple sequence repeat (SSR) markers, they completed the marker tests for the stripe rust collections up to 2017 and are currently working on the 2018 collection. Based on the molecular data, they identified several events of stripe rust pathogen introduction over the past 50 year and different population structures in various epidemiological regions. These studies improve the understanding of the pathogen biology and genetics and stripe rust epidemiology, and support the needs for developing wheat and barley varieties with non-race specific and durable resistance. Under Sub-objective 2A, ARS scientists in Pullman, Washington, have completed the genome-wide association studies (GWAS) for stripe rust resistance in a spring wheat panel consisting of 616 entries and a winter wheat panel consisting of 857 entries of commercial varieties, breeding lines, and genetic stocks developed or used in the United States. In these just published studies, they identified and mapped 37 genes in the spring wheat panel and 51 genes in the winter wheat panel, including a total of at least 20 genes that have not previously been reported, and determined the frequencies of these genes in various wheat regions. During the 2019-2020 crop season, 40 wheat mapping populations and three wheat germplasm panels originally from various countries were planted at Pullman and/or Mount Vernon, Washington, for the purpose of phenotyping stripe rust responses in order to map resistance genes in these crosses and collections. They have recorded the stripe rust responses. The rust response data will be used to identify and map new and effective resistance genes. Using the 2019 phenotypic data from the fields and the greenhouse tests with selected races of the wheat stripe rust pathogen, they selected 10 resistant and 10 susceptible lines, extracted DNA from each of the selected lines, and made the resistant and susceptible bulks for each cross. They genotyped the 80 resistant or susceptible bulks together with the 40 resistant parental varieties and the common susceptible parental variety and identified 233 SNP markers representing at least 78 different loci associated to stripe rust resistance in the 40 wheat germplasm varieties. The 2020 phenotypic data is being used to validate the marker-resistance relationships and using whole-population mapping of selected crosses further validates the mapped resistance loci. The three wheat germplasm panels with 420 to 520 entries were also tested with 7 races of the wheat stripe rust pathogen in the greenhouse. DNA is in the process of being extracted and genotyped using multiplexed sequencing for mapping genes for stripe rust resistance, especially genes conferring non-race specific high-temperature adult-plant resistance. Under Sub-objective 2B, ARS scientists in Pullman, Washington, planted more than 25,000 wheat and barley entries in the fields near Pullman and Mount Vernon, Washington for screening the materials for resistance to stripe rust. Some of the nurseries were also planted in three additional locations. They have completed stripe rust response data for the nurseries planted in different locations. They also have tested the wheat and barley variety trial nurseries and uniform nurseries from various wheat and barley production regions of the United States in both seedling and adult-plant stages with selected races of the stripe rust pathogen in the greenhouse. The thorough tests in both field and greenhouse allow for the identification of different types of resistance to stripe rust as well as different levels of resistance. Data to various breeding programs throughout the country has been provided for selecting resistant lines for releasing as new varieties. The test results of current varieties are used to update stripe rust reactions for the varieties listed in the Seed-Buyers’ Guide to be selected by growers to grow. Growing resistant varieties will reduce the potential risk of stripe rust damage.

Accomplishments
1. Large number of genes resistant to stripe rust in U.S. wheat varieties identified and mapped. Stripe rust is best controlled through growing resistant varieties, but stripe rust resistance genes in most U.S. wheat varieties are not clear. ARS scientists in Pullman, Washington, published two papers on identification and mapping of 37 genes in 616 spring wheat entries and 51 genes in 857 winter wheat entries, including commercial varieties, breeding lines, and genetic stocks developed or used in the United Statee for resistance to stripe rust using a genome-wide association analysis approach and molecular markers for previously reported genes. They determined the frequencies of these genes in varieties from different regions and the effect of the number of genes on the level of resistance. Their results point out the usefulness of individual genes and the importance of pyramiding different resistance genes. The resistant varieties, genes, and their markers identified in these studies are valuable for growers to select varieties with adequate levels of resistance to grow and breeders for selecting resistant varieties based on their genes to cross and markers to use for developing new wheat varieties.

2. Screened wheat and barley germplasm resistant to stripe rust. Developing resistant varieties is the most effective, easy-to use, and environmental-friendly approach to controlling stripe rust. ARS scientists in Pullman, Washington, screened more than 25,000 wheat and barley germplasm from breeding programs throughout the United States in various field locations and in the greenhouse for response to stripe rust in 2020. They provided the data to various breeding programs for releasing new resistant varieties and to growers for selecting released resistant varieties to grow. Based on their stripe rust data in the recent years, they collaborated with various breeding programs in releasing and registering 19 wheat varieties and onw barley variety which are resistant to stripe rust. Growing these new varieties will reduce potential risk of stripe rust.

3. Wheat and barley stripe rust pathogen races identified. The wheat and barley stripe rust pathogens evolve rapidly to produce new races that can overcome resistance in currently varieties, and the information of races with their virulence factors is essential for breeding programs to use effective genes for developing new varieties with adequate and durable resistance. ARS scientists in Pullman, Washington, identified 25 races, including 2 new races, of the wheat stripe rust pathogen and 10 races, including 1 new race, of the barley stripe rust pathogen, and determined the frequencies and distributions of these races and virulence factors in various epidemic regions in the United States. The results can be used by breeders to select effective resistance genes for developing new varieties and by pathologists to select important races for screening wheat and barley germplasm for releasing new varieties with adequate and durable resistance to stripe rust.

4. Genes associated to avirulence in the wheat stripe rust pathogen identified. Understanding the molecular mechanisms of virulence variation is important for control of stripe rust, but no avirulence/virulence genes in the wheat stripe rust fungus have been mapped and molecularly characterized. ARS scientists in Pullman, Washington, have recently published two papers on identifying genes associated to avirulence genes in the wheat stripe rust pathogen. Through genomic sequencing 30 mutant isolates developed by chemical mutagenesis and the wild-type isolate, 16 avirulence genes were mapped to the genome of the wheat stripe rust fungus and 62 candidate genes were identified, including 48 secreted protein (SP) genes and 14 non-SP genes, highly associated with the 16 avirulence loci. 117 progeny isolates developed by reproducing on alternate host barberry plants were sequenced and the parental isolate, mapped six avirulence loci to the pathogen linkage groups including four loci clustering in a small genome region (less than 200 kb), and 47 genes were identified as candidates for the four avirulence genes. These studies provide the genomic bases for understanding the molecular mechanisms of the pathogen evolution and resources for cloning avirulence genes for further study of the plant-pathogen interactions.

Review Publications
Muleta, K.T., Chen, X., Pumphrey, M. 2020. Genome-wide mapping of resistance to stripe rust caused by Puccinia striiformis f. sp. tritici in hexaploid winter wheat. Crop Science. 60(1):115-131. https://doi.org/10.1002/csc2.20058.
Tian, Y., Meng, Y., Zhao, X.C., Chen, X., Ma, H.B., Xu, S.D., Huang, L.L., Kang, Z.S., Zhan, G.M. 2019. Trade-off between triadimefon sensitivity and pathogenicity in a selfed sexual population of Puccinia striiformis f. sp. tritici. Frontiers in Microbiology. 10. https://doi.org/10.3389/fmicb.2019.02729.
Li, Y., Xia, C., Wang, M., Yin, C., Chen, X. 2020. Whole-genome sequencing of Puccinia striiformis f. sp. tritici mutant isolates identifies avirulence gene candidates. BMC Genomics. 21. https://doi.org/10.1186/s12864-020-6677-y.
Liu, Y., Qie, Y., Li, X., Wang, M., Chen, X. 2020. Genome-wide mapping of quantitative trait loci conferring all-stage and high-temperature adult-plant resistance to stripe rust in spring wheat landrace PI 181410. International Journal of Molecular Sciences. 21(2):478. https://doi.org/10.3390/ijms21020478.
Wang, J., Tian, W., Tao, F., Wang, J., Shang, H., Chen, X., Xu, X., Hu, X. 2020. TaRPM1 positively regulates wheat high-temperature seedling-plant resistance to Puccinia striiformis f. sp. tritici. Frontiers in Plant Science. 10(1). https://doi.org/10.3389/fpls.2019.01679.
Gill, K.S., Kumar, N., Randhawa, H.S., Carter, A.H., Yenish, J., Morris, C.F., Baik, B.V., Higginbotham, R.W., Guy, S.O., Engle, D.A., Chen, X., Murray, T.D., Lyon, D. 2020. Registration of 'Mela CL+' soft white winter wheat. Journal of Plant Registrations. 14(2):144:152. https://doi.org/10.1002/plr2.20006.
Sharma-Poudyal, S., Bai, Q., Wan, A., Wang, M., See, D.R., Chen, X. 2020. Molecular characterization of international collections of the wheat stripe rust pathogen Puccinia striiformis f. sp. tritici reveals high diversity and intercontinental migration. Phytopathology. 110(4):933-942. https://doi.org/10.1094/PHYTO-09-19-0355-R.
Chen, X. 2020. Pathogens which threaten food security: Puccinia striiformis, the wheat stripe rust pathogen. Food Security Journal. 12:239-251. https://doi.org/10.1007/s12571-020-01016-z.
Xia, C., Lei, Y., Wang, M., Chen, W., Chen, X. 2020. An avirulence gene cluster in the wheat stripe rust pathogen (Puccinia striiformis f. sp. tritici) identified through genetic mapping and whole-genome sequencing of a sexual population. mSphere. 5(3). https://doi.org/10.1128/mSphere.00128-20.
Wang, N., Fan, X., Zhang, S., He, M.Y., Chen, X., Tang, C.L., Kang, Z.S., Wang, X.J. 2020. Identification of a hyperparasitic Simplicillium obclavatum strain affecting the infection dynamics of Puccinia striiformis f. sp. tritici on wheat. Frontiers in Microbiology. 11. https://doi.org/10.3389/fmicb.2020.01277.
Tao, F., Hu, Y., Su, C., Li, J., Guo, L., Xu, X., Chen, X., Shang, H., Hu, X. 2020. Revealing differentially expressed genes and identifying effector proteins of Puccinia striiformis f. sp. tritici in response to high-temperature seedling-plant resistance of wheat based on RNA-seq. mSphere. 5(3). https://doi.org/10.1128/mSphere.00096-20.
Mu, J., Liu, L., Liu, Y., Wang, M.N., See, D.R., Han, D., Chen, X. 2020. Genome-wide association study and gene specific markers identified 51 genes or QTL for resistance to stripe rust in U.S. winter wheat cultivars and breeding lines. Frontiers in Plant Science. 11:998. https://doi.org/10.3389/fpls.2020.00998.
Liu, L., Wang, M.N., Zhang, Z.W., See, D.R., Chen, X. 2020. Identification of stripe rust resistance loci in U.S. spring wheat cultivars and breeding lines using genome-wide association mapping and Yr gene markers. Plant Disease. 104(8):2181-2192. https://doi.org/10.1094/PDIS-11-19-2402-RE.