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ARS Home » Midwest Area » St. Paul, Minnesota » Cereal Disease Lab » Research » Research Project #423040

Research Project: CEREAL RUST FUNGI: GENETICS, POPULATION BIOLOGY, AND HOST-PATHOGEN INTERACTIONS

Location: Cereal Disease Lab

2017 Annual Report


Objectives
Objective 1: Monitor, collect, and characterize U.S. cereal rust pathogen populations. Sub-objective 1.A. Monitor, collect and characterize cereal rust pathogen populations in the U.S. for virulence phenotypes to rust resistance genes in cereal cultivars. Sub-objective 1.B. Determine levels of genetic variation in P. triticina and P. graminis populations. Sub-objective 1.C. Refine phylogenetics and systematics of P. graminis and P. triticina. Objective 2: Discover and characterize fungal genes that are involved in pathogenesis and the obligate biotrophic interactions of cereal rust pathogens and their hosts. Objective 3: Identify and characterize rust resistance genes in novel and elite germplasm to assist in the development of resistant cereal cultivars. 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. The proposed research objectives are central to the mission of the USDA ARS Cereal Disease Laboratory (CDL): to reduce losses in wheat, oat, and barley to major diseases using host resistance. Research is focused on genetic variation in both the host cereals and their rust pathogens that determine the resistance/susceptible phenotype of the interaction. Isolates of rust fungi obtained from annual surveys of the wheat, barley, and oat crops are used to inform the breeding process. Successful control of cereal rusts with host resistance cannot be achieved without knowledge of variation in cereal rust populations. Studies of virulence and molecular variation in cereal rust populations can answer questions that range from the applied, such as which host resistance genes are effective against the current rust population and what resistance genes are in current cereal cultivars, to more basic questions like what are the origins of new races and how do they spread. Discovery of the molecular determinants of pathogenesis and obligate biotrophy in cereal rust fungi via genomic approaches offers intriguing leads in the development of novel resistance mechanisms. Identification, characterization, and introgression of new host resistance to cereal rusts are key to increasing the diversity of resistance genes in our cereals and staying ahead of these "shifty" pathogens.


Approach
Cereal rust fungi are dynamic leading to constant changes in the U.S. population which leads to the erosion of effective rust resistance in cereal crops. In addition, the 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 will depend on the monitoring and characterization, virulence phenotypes and molecular genotypes, of cereal rust pathogen populations. Rust resistant cereal germplasm will be selected by testing wheat, oat, and barley lines from breeding programs throughout the United States for resistance to Puccinia coronata, P. graminis, and P. triticina, 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. 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.


Progress Report
Significant progress was made on all objectives. Objective 1: Monitor, collect, and characterize U.S. cereal rust pathogen populations. Annual surveys of wheat stem and leaf rust, oat stem and crown rust, and barley leaf rust in the United States were completed and 10 bi-weekly bulletins posted on the ARS website. Collections of rust from surveys were processed for race identity and results distributed through an interactive map hosted at the ARS website. Progress was also made in characterizing and mapping new sources of rust resistance in wheat, barley and oat. From 2013-2017, over 360 isolates of oat crown rust were processed for race identification, and 240 isolates of barley leaf rust tested for virulence. Differential responses were observed to barley leaf rust resistance genes Rph3, Rph5, Rph7, Rph9, Rph10, Rph11, Rph13, and Rph14. Virulence was not detected to Rph15 or resistance from barley line Tunisia 34. Also during this timeframe, 50 to 71 races of P. triticina were found each year in the United States. In the Great Plains from Texas to Minnesota, most common races had virulence to Lr39 present in the hard red winter wheat cultivars. Races with virulence to Lr21 were at highest frequency in the spring wheat region of Minnesota and North Dakota, where many cultivars have Lr21. In southeastern states, most common races had virulence to Lr11, Lr26, and Lr18 present in soft red winter wheat cultivars. Leading cultivars of the Great Plains and Eastern United States are generally susceptible to leaf rust and can suffer significant losses. In 2013, a leaf rust race with high virulence to durum wheat cultivars was found in a wheat field in Kansas. This virulent isolate may eventually spread to North Dakota where majority of the United States durum wheat is grown. A total of 831 isolates of wheat leaf rust were genotyped using 23 SSR markers. The leaf rust collections are from North America, South America, Central Asia, Russia, China, Pakistan, the Middle East, and East Africa. A total of 425 SSR genotypes were described. Each continental region has distinct SSR genotype groups with significant differentiation between regions. However, isolates with identical SSR genotypes and identical or similar virulence were described among different continental regions, indicating migration of the leaf rust fungus between different regions has occurred in recent years. Stem rust samples from wheat, barley, oats, and barberry obtained across the United States were processed. Race QFCSC was the dominant race east of the Rocky Mountains, with races such as MCCDC rarely detected. More than 60 races of stem rust were detected in the Pacific Northwest in 2012. Five races of P. graminis f. sp. avenae were found across the United States. Races with Pga virulence dominated the population. An isolate of P. graminis f. sp. secalis was identified from an infected barley nursery in California. Stem rust sentinel plots were established and monitored in southern Texas, California, and Arizona to detect potential Ug99 spread into North America. Rust infections on Barberry were surveyed and collected from Minnesota and Wisconsin, and P. graminis f. sp. secalis was identified and isolated. Native B. fendleri was surveyed in southern Colorado. Berberis x ottawensis, a naturally occurring hybrid in the New England area, was investigated for susceptibility to stem rust through inoculation experiments of 150 plants. Investigations in the role of barberry for stem rust variation in eastern Africa established that B. holstii is a functional alternate host for P. graminis in Ethiopia. Two custom high-throughput SNP genotyping arrays were developed for P. graminis f. sp. tritici. United States and foreign samples were genotyped. These represent current and historical collections. North American samples formed 3 distinct genetic groups, and are different from the 4 main genetic groups found in Africa, Asia, Europe, and the Middle East. Wide distribution of genotypes indicated broad movement of wheat stem rust between continental regions. Evidence for diverse populations was observed indicating P. graminis f. sp. tritici is going through sexual cycle on the alternate host in South Western Asia. These populations are likely sources of new genotypes and races. To date all of the genotypic data indicates that the variability detected in the P. graminis f. sp. tritici Ug99 race group is due to mutation, rather than sexual recombination. A molecular diagnostic assay was developed for P. graminis f. sp. tritici Ug99 race group. This assay is being used as a key component of the international wheat stem rust monitoring program as part of the Borlaug Global Rust Initiative. Over a five year period, more than 2,500 samples were analyzed. With the discovery of "new" critical wheat stem rust races in Africa and Europe a second diagnostic assay was developed to detect different genetic types. This second assay is now used as part of the international wheat stem rust monitoring program. Objective 2: Discover and characterize fungal genes that are involved in pathogenesis and the obligate biotrophic interactions of cereal rust pathogens and their hosts. Research continued on a genetic map for P. graminis f. sp. tritici and identification of candidate effector genes involved in race-specific resistance in wheat. A refined genetic map of P. graminis f.sp. tritici was developed using high-density SNP data derived from high throughput sequencing data of a mapping population. A few segments of the map have not been resolved, indicating that genomic rearrangements may have occurred. To resolve this issue, populations derived from the parents are being developed. Association mapping using the high-density SNP data was used to map the location of an effector gene. Confirmation of a candidate effector gene has been hampered by the lack of a robust functional assay for P. graminis f. sp. tritici. DNA sequence analysis of 2 isolates of the crown rust fungus, P. coronata continued. The genome assembly for theses isolates is almost complete and transcriptome analysis is being used to annotate the genomes. A diversity study has been initiated to identify nucleotide variants for further use in characterization of oat crown rust populations. Objective 3: Identify and characterize rust resistance genes in novel and elite germplasm to assist in the development of resistant cereal cultivars. A total of 933 oat breeding lines from different programs in the United States and Canada were evaluated for crown rust resistance in field plots. Additional oat lines from regional breeding programs were analyzed for crown rust reactions. In addition, 1600 wheat breeding lines were tested annually, with multiple leaf and stem rust races in greenhouses at the seedling stage and in the field. Data was provided to nursery coordinators and posted at the ARS website. Advanced and preliminary wheat breeding lines from the University of Minnesota were evaluated for leaf rust resistance in field plots. Hard red spring wheat entries in the Uniform spring wheat nursery were evaluated for seedling resistance with 10 leaf rust races and the leaf rust resistance genes were postulated for each entry. Eight populations of oat RILS were characterized for crown rust resistance. Analysis of one population identified 2 loci contributing significantly to adult plant resistance tagged with SNP markers. Another population carried a single genetic locus tagged with DNA markers responsible for resistance. This locus has been introgressed into various breeding material and tracked with linked molecular markers. Three promising populations were further phenotyped in the buckthorn nursery for adult plant resistance, their seeds increased and distributed to collaborators for analysis of other segregating agronomic traits, and tissue collected for marker analysis. In 2016, four F6 RIL populations of wheat lines (approximately 120 lines each) were evaluated for leaf rust resistance in field plots. Leaf rust resistance derived from the hard red winter wheat cultivars Duster and Santa Fe were mapped to chromosome arm 3BL. Leaf rust resistance derived from the soft red winter wheat Caldwell was mapped to chromosome arm 3BS. Resistance derived from AC Taber mapped to numerous chromosome locations. RIL populations of Tc*2/Bill Brown F3 and Tc*2/Deliver F3 were advanced to F6 generations. Adult plant wheat leaf rust resistance was mapped and indicated Lr23, Lr46 and Lr68 were likely present in variety Ulen. From 2013 to 2017, screening of 670 spring barley and 2155 spring wheat breeding lines respectively, were coordinated at Ug99 nurseries in Ethiopia and Kenya. Lines were submitted by 16 public and private breeders. Data was communicated to breeders to allow for selection of United States wheat and barley cultivars with Ug99 resistance. Seven mapping RIL populations that segregate for resistance to Ug99 were evaluated in Africa for 3 seasons. Germplasm combining 2 Ug99 resistance genes (Sr9h and Sr28) in coupling on the same chromosome arm were deposited in the National Small Grains Collection as CDL001, PI 670015. Work continued on characterizing over 80 wheat alloplasmic lines for resistance to stem and leaf rust, and S. nodorum blotch. The lines were evaluated in the field using a precision phenotyping approach with aerial imaging to measure agronomic traits. Progress has been made on genome sequencing of organellar DNA from alloplasmic wheat lines. Progress has been made on developing an imaging analysis function to calculate the percentage of rust pustule coverage.


Accomplishments
1. Ug99 resistance in United States durum wheat. The disease wheat stem rust threatens United States wheat production because a new strain called Ug99 is virulent to the majority of wheat varieties worldwide and few genetic resources are available that confer resistance, especially in durum wheat. ARS scientists in St. Paul, Minnesota discovered a unique stem rust resistance gene called Sr8155B1 present in durum wheat that is effective to most isolates of the Ug99 race group. Molecular markers linked to Sr8155B1 can be used to select for Ug99-resistance in durum wheat breeding. Selection and release of durum wheat varieties that possess both Sr8155B1 and previously characterized Ug99-effective Sr13 will provide a strategy for maximum immediate protection of United States durum wheat from the threat of Ug99.

2. A new gene for durable leaf rust resistance in wheat. The wheat cultivar Santa Fe has had long lasting resistance to leaf rust. Long lasting leaf rust resistance in wheat has been difficult to maintain since the leaf rust pathogen is highly variable for virulence to leaf rust resistance genes in wheat, and populations of this rust fungus can adapt quickly to the resistance in the released cultivars. ARS scientists in St. Paul, Minnesota determined that Santa Fe has a major gene for adult plant leaf rust resistance on chromosome arm 3BL. This is a new gene for adult plant leaf rust resistance and has been designated as Lr77 in the wheat gene catalogue. The cultivar Duster was also determined to have Lr77. KASP markers on 3BL can be used to select wheat germplasm for the new durable leaf rust resistance gene.

3. Leaf rust resistance in soft red winter wheat. The cultivar Caldwell has had long lasting resistance to leaf rust. Long lasting leaf rust resistance in wheat has been difficult to achieve since the leaf rust pathogen is highly variable for virulence to leaf rust resistance genes in wheat, and populations of this rust fungus can adapt quickly to the resistance in the released cultivars. ARS scientists in St. Paul, Minnesota discovered that Caldwell has a major QTL on chromosome arm 3BS for adult plant leaf rust resistance. This QTL mapped close to the region of Lr74, and Sr2. KASP markers on 3BS can be used to select for the adult plant leaf rust resistance on chromosome arm 3BS.

4. Virulence and molecular genotypes of Puccinia triticina (wheat leaf rust) from Pakistan. Wheat leaf rust, an important disease in Pakistan was studied by ARS scientists in St. Paul, Minnesota and collaborators to determine molecular genotypes of the representative leaf rust races in 2008, 2011, 2013, and 2014. Twenty-four races were described. The most common races had virulence to leaf rust resistance genes Lr16, Lr17, and Lr26. Twenty-seven SSR genotypes were found in three groups of SSR genotypes. Isolates in each SSR group had identical or similar SSR genotype and also similar virulence. The SSR genotypes from Pakistan were highly related to isolates from Turkey, Europe, Central Asia, the Middle East, North America, and South America, which indicated migration of the leaf rust fungus between continents.

5. A new adult plant resistance gene to crown rust in oat. A single effective adult plant resistance gene in oat with the associated KASP/SNP markers has been released to various oat breeding programs from across the United States for incorporation into adapted germplasm. The markers have proven effective in the breeding program for selection of the gene in their cultivar development. This new gene and associated markers are critical in developing long-term solutions to the persistent oat crown rust problem in the United States. ARS scientists in St. Paul, Minnesota observed that over the past two years of release no crown rust pustules have been identified on lines carrying this gene. Additional genes are being identified for pyramiding and development of an effective package against this highly variable pathogen.

6. Population structure of wheat stem rust pathogen in Kenya. Stem rust continues to be a threat to wheat production in the United States and worldwide. ARS scientists in St. Paul, Minnesota lead an effort with scientists in Kenya to characterize the population structure of wheat stem rust pathogen in Kenya during 2011, reporting the largest diversity of wheat stem rust races within the Ug99 lineage in Kenya. In 2017 extensive sampling across the four main wheat growing regions in Kenya was performed and 421 samples were genotyped using DNA markers. A total of 79 different genotypes were identified, all of them belonging to the Ug99 lineage. Analysis indicated that a single asexual population of the wheat stem rust pathogen exists across Kenya and that the diversity observed in the population is due to mutation rather sexual recombination. This is a significant finding in terms of developing strategies to address this disease problem.


Review Publications
Nirmala, J.H., Chao, S., Olivera, P., Babiker, E.M., Abeyo, B., Tadesse, Z., Imtiaz, M., Talbert, L., Blake, N., Akhunov, E., Pumphrey, M.O., Jin, Y., Rouse, M.N. 2016. Markers linked to wheat stem rust resistance gene Sr11 effective to Puccinia graminis f. sp. tritici race TKTTF. Phytopathology. 106(11):1352-1358.
Edae, E.A., Olivera, P., Jin, Y., Poland, J.A., Rouse, M.N. 2016. Genotype-by-sequencing facilitates genetic mapping of a stem rust resistance locus in Aegilops umbellulata, a wild relative of cultivated wheat. Biomed Central (BMC) Genomics. 17:1039. doi 10.1186/s12864-016-3370-2.
Kolmer, J.A., Jin, Y., Hughes, M.E., Gale, S.W. 2016. Wheat rusts in the United States in 2015. Wheat Newsletter. 62:73-78.
Kolmer, J.A., Mirza, J.I., Imtiaz, M., Shah, J.A. 2017. Genetic differentiation of the wheat leaf rust fungus Puccinia triticina in Pakistan and genetic relationship to other worldwide populations. Phytopathology. 107(6):786-790.
Turner, M.K., Kolmer, J.A., Pumphrey, M.O., Bulli, P., Chao, S., Anderson, J. 2016. Association mapping of leaf rust resistance loci in a spring wheat core collection. Theoretical and Applied Genetics. 130(2):345-361. doi: 10.1007/s00122-016-2815-y.
Li, G., Xu, X., Bai, G., Carver, B.F., Hunger, R., Bonman, J.M., Kolmer, J.A., Dong, H. 2016. Genome-wide association mapping reveals novel QTL for seedling leaf rust resistance in a worldwide collection of winter wheat. The Plant Genome. 9(3):1-12. doi:10.3835/plantgenome2016.06.0051.
Aoun, M., Breiland, M., Turner, M.K., Loladze, A., Chao, S., Xu, S.S., Ammar, K., Anderson, J.A., Kolmer, J.A., Acevedo, M. 2016. Genome-wide association mapping of leaf rust response in a durum wheat worldwide germplasm collection. The Plant Genome. 9(3). doi:10.3835/plantgenome2016.01.0008.
Johnson, J.W., Chen, Z., Buck, J.W., Buntin, G.D., Babar, M.A., Mason, R.E., Harrison, S.A., Murphy, J.P., Ibrahim, A.H., Sutton, R.L., Simoneaux, B.E., Bockelman, H.E., Baik, B.-K., Marshall, D.S., Cowger, C., Brown Guedira, G.L., Kolmer, J.A., Jin, Y., Chen, X., Cambron, S.E., Mergoum, M. 2017. ‘GA 03564-12E6’: A high-yielding soft red winter wheat cultivar adapted to Georgia and the southeastern regions of the United States. Journal of Plant Registrations. 11:159-164.
Kianian, P., Kianian, S. 2016. Methods in molecular biology: Plant cytogenetics. In: Kianian, S.F., Penny, M.A. Plant Cytogenics. New York, NY: Springer Publishers. https://doi.org/10.1007/978-1-4939-3622-9.
Kumar, J., Singh, S.P., Kianian, S. 2016. Engineering resistance to plant viruses: Present status and future prospects. In: Kumar S.D., Pandey, A., Sangwan, R.S. Current Developments in Biotechnology and Bioengineering. Amsterdam, Denmark: Elsevier Science Ltd. pp. 75-101.
Liu, H., Lu, L., Zhao, Y., Pan, Y., Sun, X., Hwang, C., Wu, V. 2014. Antibacterial activity of the essential oils extracted from cassia bark, bay fruits and cloves against Vibrio parahaemolyticus and Listeria spp. Natural Product Communications. 9(12):1-5.
Jia, S., Li, A., Avoles-Kianian, P., Kianian, S., Zhang, C., Holding, D. 2016. A population of deletion mutants and an integrated mapping and Exome-seq pipeline for gene discovery in maize. G3, Genes/Genomes/Genetics. doi: 10.1534/g3.116.030528.
Dukowic-Schulze, S., Sundararajan, A., Thiruvarangan, R., Kianian, S., Pawlowski, W., Mudge, J., Chen, C. 2016. Novel meiotic miRNAs and indications for a role of phasiRNAs in meiosis. Frontiers in Plant Science. 7:762. doi: 10.3389/fpls.2016.00762.
Kianian, P., Liberatore, K.L., Miller, M., Hegstad, J., Kianian, S. 2016. Dissecting plant chromosomes by the use of ionizing radiation. In: Kianian, S.K., Kianian, P.M.A. Methods in Molecular Biology. New York, NY: Springer Publishing. p. 91-102.
Abuhammad, W.A., Mamidi, S., Kumar, A., Pirseyedi, S., Kianian, S., Alamri, M., Mergoum, M., Elias, E. 2016. Identification and validation of a major cadmium accumulation locus and closely associated SNP markers in North Dakota durum wheat cultivars. Molecular Breeding. 36:112. doi: 10.1007/S11032-016-0536-1.
Yin, C.T., Downey, S.I., Klasges-Mundt, N.L., Ramachandran, S., Chen, X., Szabo, L.J., Pumphrey, M., Hulbert, S.H. 2015. Identification of promising host-induced silencing targets among genes preferentially transcribed in haustoria of Puccinia. Biomed Central (BMC) Genomics. 16:579.
Patpour, M., Hovmoller, M., Justesen, A.F., Newcomb, M., Olivera, P., Jin, Y., Szabo, L.J., Hodson, D., Shahin, A., Wanyera, R., Habarurema, I., Wobibi, S. 2016. Emergence of virulence to SrTmp in the Ug99 race group of wheat stem rust, Puccinia graminis f.sp. tritici in Africa. Plant Disease. 106:729-736.
Cuomo, C.A., Bakkeren, G., Khalil, H., Panwar, V., Joly, D., Linning, R., Sakthikumar, S., Song, X., Goldberg, J., Young, S., Zeng, Q., Bruce, M.A., McCallum, B., Szabo, L.J., Hulbert, S., Chen, X., Fellers, J.P. 2016. Comparative analysis highlights variable genome content of wheat rusts and divergence of the mating loci. Genes, Genomes, Genetics. doi:10.1534/g3.116.032797.
Zhang, X., Rouse, M.N., Nava, I., Jin, Y., Anderson, J. 2016. Development and verification of wheat germplasm containing both Sr2 and Fhb1. Molecular Breeding. 36(85):1-14. doi: 10.1007/s11032-016-0502-y.
Klindworth, D.L., Saini, J., Long, Y., Rouse, M.N., Faris, J.D., Jin, Y., Xu, S.S. 2017. Physical mapping of DNA markers linked to stem rust resistance gene Sr47 in durum wheat. Theoretical and Applied Genetics. 130:1135-1154. doi: 10.1007/s00122-017-2875-7.
Babiker, E.M., Gordon, T.C., Chao, S., Rouse, M.N., Brown Guedira, G.L., Pretorius, Z.A., Wanyera, R., Newcomb, M., Bonman, J.M. 2016. Genetic mapping of resistance to the Ug99 race group of Puccinia graminis f. sp tritici in a spring wheat landrace CItr 4311. Theoretical and Applied Genetics. 129(11):2161-2170.
Nirmala, J.H., Chao, S., Olivera, P., Babiker, E.M., Abeyo, B., Tadesse, Z., Imtiaz, M., Talbert, L., Blake, N., Akhunov, E., Pumphrey, M., Rouse, M.N., Jin, Y. 2017. Markers linked to wheat stem rust resistance gene Sr11 effective to Puccinia graminis f. sp. tritici race TKTTF. Phytopathology. 106(11):1352-1358.
Edae, E.A., Olivera, P., Jin, Y., Rouse, M.N. 2017. Genotyping-by-sequencing facilitates a high resolution consensus linkage map for Aegilops umbellulata, a wild relative of cultivated wheat. Genes, Genomes, and Genomics. 7:1551-1561.
Babiker, E.M., Gordon, T.C., Chao, S., Rouse, M.N., Jin, Y., Bhavani, S., Wanyera, R., Newcomb, M., Bonman, J.M. 2017. Genetic loci conditioning adult plant resistance to the Ug99 race group and seedling resistance to races TRTTF and TTTTF of the stem rust pathogen in wheat landrace CItr 15026. Plant Disease. 101(3):496-501.
Babiker, E.M., Gordon, T.C., Chao, S., Rouse, M.N., Acevedo, M., Wanyera, R., Brown Guedira, G.L., Bonman, J.M. 2017. Molecular mapping of stem rust resistance loci effective against the Ug99 race group of the stem rust pathogen and identification of SNP marker linked to stem rust resistance gene Sr28. Phytopathology. 107(2):208-215.
Mihalyov, P.D., Nichols, V.A., Bulli, P., Rouse, M.N., Pumphrey, M.O. 2017. Multi-locus mixed model analysis of stem rust resistance in a worldwide collection of winter wheat. The Plant Genome. 10(2):1-12.