IMPROVING THE UNDERSTANDING AND GENETIC RESISTANCE TO FUSARIUM HEAD BLIGHT AND ROOT ROT DISEASES IN WHEAT

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1005871
• Project Start Date: Feb 1, 2015
• Project End Date: Sep 30, 2019
• Program Code: [(N/A)]- (N/A)

Recipient Organization
NORTH DAKOTA STATE UNIV
1310 BOLLEY DR
FARGO,ND 58105-5750

Performing Department
Plant Pathology

Non Technical Summary
Fusarium head blight (FHB) and Crown rot (CR) and common root rot (CRR) are among the most common and destructive diseases of wheat in North Dakota and other regions of North America. They are present in every growing season and can cause significant yield losses. FHB also reduces the quality of harvested grains due to contamination of mycotoxins (deoxynivalenol, nivalenol, or zearalenone) that are produced by the pathogens. These mycotoxins pose major threats to the health of humans and animals. Host resistance is considered as the most effective and economically sound method for controlling both FHB and root rot diseases. However, sources of resistance to these diseases are limited. Finding novel resources of resistance is essential for breeders to develop wheat varieties with improved resistance for growers to use. Furthermore, resistance to FHB and root rot is partial and often controlled by multiple quantitative trait loci (QTL) with expression significantly affected by environmental conditions. Therefore, selection of resistance to these diseases by traditional methods is very difficult. Identification of DNA markers associated with resistance QTL can help in development of resistant wheat varieties and thus reduce the impact of the diseases in North Dakota and the US. Disease resistance-associated DNA markers are powerful not only for the quick and effective transfer of the resistance loci into spring wheat varieties, but also for combining QTLs for FHB resistance from different sources to achieve a higher level of and/or more durable FHB resistance in wheat. An understanding of the genetic, biochemical, and molecular interactions of the pathogens with their hosts could ultimately lead to the development of novel strategies for controlling these serious cereal diseases. In this proposed project, we will screen and identify wheat germplasm and breeding lines for resistance to FHB and root rot diseases, map the genes (QTL) for resistance to FHB and DON accumulation using various mapping populations, and develop DNA markers associated with the resistance QTL for use in spring wheat breeding programs. Furthermore, the pathogenicity and virulence genes of the common root rot pathogen B. sorokiniana will be characterized using genomics and functional genomics approaches. The research project proposed will not only facilitate the development of new varieties with better resistance to FHB and root rot diseases, but also provide basic information about the biology of the common root rot pathogen B. sorokiniana so that we can develop novel approaches for disease control. All these research activities relate to the vision statement of NDAES: "to develop information, technology, and products that will allow North Dakotans to succeed in this new century."

Animal Health Component

30%

Research Effort Categories

Basic

60%

Applied

30%

Developmental

10%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1549	1160	100%

Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1549 - Wheat, general/other;

Field Of Science
1160 - Pathology;

Keywords

wheat

fusarium head blight

root rot diseases

resistance

molecular mapping

Goals / Objectives
The overall goal of this project is to develop a better understanding of biology and host resistance for the two diseases (Fusarium head blight and common root rot) in wheat. The specific objectives are:1. Identify novel sources of resistance to Fusarium head blight (FHB) and root rot diseases.2. Develop genetic markers associated with resistance to FHB and deoxynivalenol (DON) accumulation.3. Understand genetics and biology of the common root rot pathogen Bipolaris sorokiniana.

Project Methods
Objective 1. Identify novel sources of resistance to Fusarium head blight (FHB) and root rot diseases.1.1 Greenhouse Evaluation for FHB: Three replicates will be used for each wheat line/season in a randomized complete block design. Fifteen heads/line in each replicate at early anthesis stage will be inoculated near the center of each spike with l0 µl of 50,000 conidia/ml prepared from an equal contribution of four strains of pathogenic F. graminearum. Following the inoculation, the temperature will be kept at 22 to 25 ºC and the relative humidity will be kept above 90% by lightly misting the plants covered with a plastic tent for 72 hours. At 14 and 21 days post-inoculation, the blighted spikelets in each of the inoculated spikes will be counted and the total number of spikelets in each of the inoculated spikes will be used to calculate the disease severity based on the percentage of infected spikelets.1.2 Field Evaluation for FHB: Three replications (plots) will be planted for each line in a randomized complete block design. FHB-infected corn kernels will be spread in the field twice at the rate of 60 g per plot for inoculation. The field will be misted for 3 min (or 5 min when it is extremely dry) every 30 min for 12 h per day mostly during the night. FHB incidence (percentage of diseased spikes) and FHB severity (ratio of infected spikelets on diseased spikes) will be recorded 21 days after anthesis using a visual scale described by Stack and McMullen (1998). The grains will be harvested from each line and sent to a service lab for measurement of DON content.1.3 Greenhouse Evaluation for root rot diseases: The sand-cornmeal-inoculum method (Bilgi et al. 2008) will be used to evaluate the reactions of wheat lines to root rot diseases. We will first prepare the sand-cornmeal-inoculum. Fungal cultures are grown on PDA for 5 days in alternating 12 hr of light and 12 hr of dark. Sand, cornmeal and sterilized distilled water will be added to a 250ml flask in a 9:1:2 ratio (45g sand, 5g corn-meal and 10ml water). The mixture is autoclaved for 45 minutes at 1210C, cooled down to room temperature, and inoculated with eight 5mm2 plugs of fresh fungal culture. The inoculum is then incubated for 12 days. During the time of incubation, the mixture in the flask is swirled daily to ensure the even growth of the fungus. Then, vermiculite is autoclaved at 1210C for 45 minutes and cooled down to room temperature for use. Plastic cups are used as containers for planting. For each cup, five pin-sized holes are made at the bottom using a sterilized needle, and filled with 15g autoclaved vermiculite first, then 15g sand-cornmeal-inoculum, 5 seeds with embryo facing up and finally a layer of 8g of autoclaved vermiculite. To keep moisture, 80 ml of de-ionized water is added to each cup. The cups are kept in a humidifier chamber for 10 days with alternating 14 hr light and 10 hr dark and vaporizer turned on for 15 secs every 20 minutes. Seed germination, root growth, and browning of the root and base of seedlings will be examined and recorded at 10 to 12 days after planting.1.4 Field Evaluation for root rot diseases: Wheat lines and cultivars from private and public wheat breeding programs of the Northern Great Plains of United States will be evaluated at Fargo/Langdon, ND for reaction to CRR and CR. Cvs. Len and Amidon will be chosen as susceptible and moderately resistant checks, respectively, for CRR. Wheaton and Alsen will be used as susceptible and moderately resistant checks, respectively, for CR. A randomized complete block design is applied, with three replicates. Natural inocula of B. sorokiniana and Fusarium spp. are maintained by continuous cropping of cereals in the selected field and monitored by fungal isolation from the crown and subcrown parts of the cereal crops planted. Experimental units are two-row plots, 1.5 m long with 0.25 m spacing between rows. Planting, plot management, disease assessment and data analysis will be the same as described by Tobias et al. (2009).Objective 2. Develop genetic markers associated with resistance to FHB and deoxynivalenol (DON) accumulation.2.1 Greenhouse and Field Evaluation: Various mapping populations will be evaluated in greenhouse for Type II resistance using the point inoculation method as described by Stack (1989). Also, they will be evaluated in scab nurseries at Fargo and Langdon, ND, using the methods as described above (Objective 1).2.2 Genetic analysis and identification of QTL for FHB resistance: The analysis of variance, mean comparison and other statistic tests for FHB incidence, severity and DON content will be performed using the SAS program (SAS correlations and the Institute, Inc., 1990). For QTL mapping, a genetic linkage map will be developed for each mapping population using all DNA markers available as described by Chu et al. (2007) and SNP markers. The RILs and their parents will be genotyped using the wheat 90K-SNP arrays and polymorphic SNP markers will be identified and used for genetic map construction. Linkage analysis and map construction will be performed using MapDisto (Lorieux 2012) with a minimum LOD threshold of 3.0 and the Kosambi mapping function. For QTL analysis, composite interval mapping (CIM) will be performed using the software Windows QTL Cartographer Version 2.5 (Wang et al. 2012) to determine the genomic regions (marker intervals) associated with FHB resistance. A chromosome region identified to be associated with FHB resistance in one population will also be validated in other populations.Objective 3. Understand genetics and biology of the common root rot pathogen B. sorokiniana.3.1 Understand the infection process of B. sorokiniana during root disease development in wheat: Several strains of the root rot fungal pathogen B. sorokiniana have been transformed with the green fluorescent protein (GFP) gene. The transformants expressing the GFP protein will be used to inoculate the wheat cv. Len (susceptible to CRR) and Amidon (moderately resistant to CRR) using the sand-cornmeal-inoculum method as described above. Root samples will be taken at 12 hours, 24 hours, 3 days, 7 days and 14 days after inoculation, washed and examined under a confocal microscope. Fungal infection process will be observed. Photos will be taken and results from the susceptible and resistant wheat genotypes will be compared.3.2. Characterize fungal genes involved in root infection in wheat. A large-scale library of insertional mutants will be generated for the B. sorokiniana strain ND 4008 (highly virulent for causing CRR) through T-DNA tagging using the Agrobacterium-mediated transformation system developed for the fungus (Leng et al. 2009). The mutants will be characterized for growth, spore production, pathogenicity and virulence. We will particularly be interested in those mutants with normal growth but loss of pathogenicity or virulence on the susceptible wheat genotype. Flanking sequences of T-DNA insertions will be recovered by TAIL-PCR (Liu et al. 1995) and other methods to identify the disrupted genes. The function of the putative genes for virulence or pathogenicity will be further confirmed by gene knockout through gene deletion/replacement as described by Leng et al. (2012). Characterization of the insertional mutants will aid in the isolation and identification of genes related to virulence and pathogenicity in B. sorokiniana.

Progress 02/01/15 to 09/30/19

Outputs
Target Audience:1. Wheat breeders and geneticists; 2. Wheat growers; 3. Plant pathologists; 4. Extension agents; 5. Agricultural administrators/members. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Participants include Shaobin Zhong (PI), Yueqiang Leng (Research associate), Yan Liu (postdoctoral research associate), Rui Wang (Ph.D. graduate student), Mingxia Zhao (Ph.D. graduate student), Bikash Powdel (Ph.D. graduate student), and Anil Karmacharya (Ph.D. graduate student). The PI and other participants have attended 15 seminars and 18 conferences during the past five years. How have the results been disseminated to communities of interest?The results were presented in peer-reviewed journals and in conferences. FHB resistant wheat lines and DNA markers developed for FHB resistance are being used by wheat breeders and research geneticists for development of FHB resistant varieties or germplasm. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Both Fusarium head blight (FHB) and root rots significantly impact wheat production in North Dakota and many other regions of the world. FHB not only reduces yield of the crop but also contaminates the grains with mycotoxins produced by the pathogen. Root rot diseases cause annual yield loss ranging from 3-5%. Use of resistance is the most efficient way to manage the diseases and mitigate their impact on wheat production. In the meantime, understanding of biology of the pathogens causing the diseases is essential for deployment of disease resistance genes. We are evaluating various germplasm and varieties for resistance to these diseases, genetically map the quantitative trait loci (QTL) for disease resistance, developing DNA markers for the identified resistance genes or QTL, and functionally characterizing fungal genes related to virulence in the common root rot pathogen Bipolaris sorokiniana. To identify FHB resistance (Objective 1), we evaluated two mapping populations in durum wheat. One population contained 200 recombinant inbred lines (RILs) (F2:8) derived from the cross between Divide (durum wheat cultivar susceptible to FHB) and PI 277012 (hexaploid wheat line with a high level of FHB resistance) and the other population has 196 RILs (F2:7) derived from the cross between durum wheat cultivar Divide (susceptible to FHB) and emmer wheat line PI 254188 (resistant to FHB). A number of the RILs exhibited a high level of FHB resistance in greenhouse and field inoculation experiments. Some of the resistant durum wheat lines contains the major QTL (Qfhb.NDWP-5A) from 10Ae564 with resistance source coming from PI 277012 and minor QTL (Qfhb.NDWP-2A and Qfhb.NDWP-7A) existing in durum cultivars. Other RILs carry novel genes that are not detected in other known sources of FHB resistance. These RILs are useful sources of FHB resistance for developing FHB-resistant durum wheat germplasm. To develop DNA markers for FHB resistance in wheat (Objective 2), we phenotyped a mapping population consisting of 200 RILs derived from the Wheaton/PI 185843 cross for FHB reaction in greenhouse and field experiments. DON data were also collected from the greenhouse inoculation experiments. A total of 5370 SNP markers were identified from this mapping population using the genotyping by sequencing approach, and mapped to 21 linkage groups covering all 21 chromosomes. QTL analysis indicated that four QTL (Qfhb.ndwp-2AS, Qfhb.ndwp-2AL, Qfhb.ndwp-3BL, and Qfhb.ndwp-4D) control type II FHB resistance in PI 185843 (Surpressa). The QTL on chromosome 2A and 3B are likely novel based on physical locations of FHB resistance-linked markers. To clone the major QTL on 5AL for FHB resistance in the wheat line PI 277012, we developed additional PCR-based SNP markers, and constructed a non-gridded bacterial artificial chromosome (BAC) library using high molecular weight DNA of PI 277012. Sixteen BACs were identified to cover the 5AL genomic region containing the QTL. We also isolated and sequenced chromosome 5A-specific DNA from PI 277012 to identify the candidate gene for FHB resistance. The additional DNA markers are very useful not only for marker-assisted selection of the PI 277012 FHB resistance, but also for the map-based cloning of the FHB resistance QTL in this wheat line. The non-gridded BAC library constructed for PI 277012 and the chromosome-specific genome sequences are very useful for cloning and characterization of genes for FHB resistance. To better understand the genetics and biology of the fungal pathogen Bipolaris sorokiniana (Objective 3), we have sequenced genomes of four different pathotypes of Bipolaris sorokiniana and identified a number of genes that are involved in or regulates the virulence and secondary metabolite production of the fungus. The availability of fungal genome sequences and identification of fungal virulence genes will facilitate the study of host-pathogen interaction, which can provide important information for disease management.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Szabo-Hever, A., Zhang, Q., Friesen, T. L., Zhong, S., Elias, E. M., Cai, X., Jin, Y., Chao, S., and Xu, S. S. 2018. Genetic diversity and resistance to Fusarium head blight in synthetic hexaploid wheat derived from Aegilops tauschii and diverse Triticum turgidum subspecies. Front. Plant Sci. 9:1829. doi: 10.3389/fpls.2018.01829
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Wang, R., Leng, Y., Zhao, M., and *Zhong, S. 2019. Fine mapping of a single dominant gene for resistance to spot blotch caused by a new pathotype of Bipolaris sorokinianan in barley. Theor. Applied. Genet. 132:4151.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Wan, J., Zhong, S., Schwarz, P., Chen, B., and Rao, J. 2019. Physical properties, antifungal and mycotoxin inhibitory activities of five essential oil nanoemulsions: impact of oil compositions and processing parameters. Food Chemistry 291:199-206.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Wan, J., Zhong, S., Schwarz, P., Chen, B., and Rao, J. 2019. Enhancement of antifungal and mycotoxin inhibitory activities of food-grade thyme oil nanoemulsions with natural emulsifiers. Food Control 106: 106709.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Wu, D., Lu, J., Zhong, S., Schwarz, P., Chen, B., Rao, J. 2019. Physical stability, antifungal and mycotoxin inhibitory activities of lecithin stabilized cinnamon oil emulsions in the presence of chitosan. Food & Function 10:2817-2827.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Liu, Y., Evan, S., Fiedler, J. D., Hegstad, J. B., Green, A., Mergoum, M., Zhong, S., Li, X. 2019. Genetic mapping and prediction analysis of FHB resistance in a hard red spring wheat breeding population. Front. Plant Sci. 10:1007. https://doi.org/10.3389/fpls.2019.01007
• Type: Journal Articles Status: Awaiting Publication Year Published: 2019 Citation: Leng, Y., Zhao, M., Fiedler, J., Dreiseitl, A., Chao, S., Li, X., and *Zhong, S. 2019. Molecular mapping of loci conferring resistance to spot blotch and powdery mildew in barley using the genotyping by sequencing approach. Phytopathology (in press)
• Type: Journal Articles Status: Awaiting Publication Year Published: 2019 Citation: Wan, J., Jin, Z., Zhong, S., Schwarz, P., Chen, B., and Rao, J. 2019. Clove oil-in-water nanoemulsion mitigates growth of Fusarium graminearum and trichothecene mycotoxin production during the malting of Fusarium infected barley. Food Chemistry (in press)
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Poudel, B., Puri, K.D., Leng, Y., Mullins, J., Karmacharya A., Liu, Y., Hegstad, J., Li, X., and Zhong, S. 2019. Molecular mapping of quantitative trait loci for Fusarium head blight resistance in the Brazilian spring wheat cultivar Surpresa. Proceedings of the 2019 National Fusarium Head Blight Forum, Dec 8-10, 2019, Milwaukee, WI. P110-115.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Ren, S., Leng, Y., Zhang, W., Talukder, Z., Zhong, S., Fiedler, J., Qi, L., and Cai, X. 2019. Molecular mapping of hexaploid wheat-derived Fusarium head blight resistance in durum wheat. Proceedings of the 2019 National Fusarium Head Blight Forum, Dec 8-10, 2019, Milwaukee, WI. P117. (Poster #62)

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:The target audience includes: 1. Wheat breeders and geneticists; 2. Wheat growers; 3. Plant pathologists; 4. Extension agents; 5. Agricultural administrators/members. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Participants include Shaobin Zhong (PI), Yueqiang Leng (Research associate), Mingxia Zhao (Ph.D. graduate student), Bikash Powdel (Ph.D. graduate student), and Anil (Ph.D. graduate student). The PI and other participants have attended three seminars and three conferences. How have the results been disseminated to communities of interest?The results were presented in peer-reviewed journals and in conferences. FHB resistant wheat lines and DNA markers developed for FHB resistance are being used by wheat breeders and research geneticists for development of FHB resistant varieties or germplasm. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Fusarium head blight (FHB) and root rots are important diseases that threaten wheat production in North Dakota and many other regions of the world. Use of resistance is the most efficient way to manage the diseases and mitigate their impact on wheat production. We are evaluating various germplasm and varieties for resistance to these diseases, genetically map the quantitative trait loci (QTL) for disease resistance, and developing DNA markers for the identified resistance genes or QTL. To identify FHB resistance (Objective 1), we evaluated some of the selected recombinant inbred lines (RILs) (F2:6) derived from the cross between Divide (durum wheat cultivar susceptible to FHB) and PI 277012 (hexaploid wheat line with a high level of FHB resistance) in the Fargo FHB nursery in the summer of 2018. Four of the RILs exhibited a high level of FHB resistance in the nursery, with mean disease severity ranging from 7.54 to 24.40%. These durum wheat materials will be used as FHB resistance sources for durum wheat. We also selected some FHB resistant recombinant inbred lines (RILs) (F2:8) derived from the cross between durum wheat line 10Ae564 and cultivar Joppa and re-evaluated in the Fargo FHB nursery. Six of the RILs showed a high level of FHB resistance, with mean disease severity ranging from 17.6 to 31.6%. These durum wheat lines contains the major QTL (Qfhb.NDWP-5A) from 10Ae564 with resistance source coming from PI 277012 and minor QTL (Qfhb.NDWP-2A and Qfhb.NDWP-7A) existing in durum cultivars. These RILs have a combination of existing and new FHB resistance QTL and are useful sources of FHB resistance for developing FHB-resistant durum wheat germplasm. To develop DNA markers for FHB resistance in wheat (Objective 2), we phenotyped a mapping population consisting of 200 RILs derived from the Wheaton/PI 185843 cross for FHB reaction in two inoculation experiments, one in the 2018 Fargo FHB nursery and one in the greenhouse. DON data were also collected from the greenhouse inoculation experiment. The data will be used to map the QTL for FHB resistance in PI 185843. To clone the major QTL on 5AL for FHB resistance in the wheat line PI 277012, we developed additional PCR-based SNP markers, and constructed a non-gridded bacterial artificial chromosome (BAC) library using high molecular weight DNA of PI 277012 and made BAC pools in 12 X 96 well plates. Sixteen BACs were identified to cover the 5AL genomic region containing the QTL. The additional DNA markers are very useful not only for marker-assisted selection of the PI 277012 FHB resistance, but also for the map-based cloning of the FHB resistance QTL in this wheat line. The non-gridded BAC library constructed for PI 277012 are very useful for identification of BACs to cover the target region for candidate gene identification. To better understand the genetics and biology of the fungal pathogen Bipolaris sorokiniana (Objective 3), we identified a transcription factor gene (PacC), which regulates the virulence and secondary metabolite production of the fungus.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Ahmadpour, Castell-Miller, C., A, Javan-Nikkhah, A., Naghavi, M. R., Padasht Dehkaei, F., Leng, Y., Puri, K. D., and Zhong, S. 2018. Population structure, genetic diversity, and sexual state of the rice brown spot pathogen Bipolaris oryzae from three Asian countries. Plant Pathology 67: 181-192.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Zhao, M., Wang, G., Leng, Y., Wanjugi, H., Xi, P., Grosz, M. D., Pitkin, J., Mergoum, M., and Zhong, S. 2018. Molecular mapping of Fusarium head blight resistance in ND2710. Phytopathology 108: 972-979.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Wan, J., Zhong, S., Schwarz, P., Chen, B., and Rao, J. 2018. Influence of oil phase composition on antifungal and mycotoxin inhibitory activity of clove oil nanoemulsions. Food & Function 9: 2872-2882.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Leng, Y., Zhao, M., Wang, R., Steffenson, B. J., Bruggeman, R. S., and Zhong, S. 2018. The gene conferring susceptibility to spot blotch caused by Cochliobolus sativus is located at the Mla locus in barley cultivar Bowman. Theor. Applied. Genet. 131:1531-1539.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Zhao, M., Leng, Y., Chao, S., Xu, S. S., and Zhong, S. 2018. Molecular mapping of QTL for FHB resistance introgressed into durum wheat. Theor. Applied. Genet. 131:1939-1951.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Wu, D., Wan, J., Lu, J., Wang, X., Zhong, S., Schwarz, P., Chen, B., Rao, J. 2018. Chitosan coatings on lecithin stabilized emulsions inhibit mycotoxin production by Fusarium pathogens. Food Control 92: 276-285.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Wu, D., Lu, J., Zhong, S., Schwarz, P., Chen, B., and Rao, J. 2018. Physical Stability, Antifungal Properties, and Mycotoxin Inhibitory Activities of Chitosan Stabilized Cinnamon Oil-in-Water Emulsion. 2nd Mycokey International Conference - Integrated Solutions for Mycotoxin Management, September 16-18, 2018, Wuhan, China
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Leng, Y., Zhao, M., Fiedler, J., Dreiseitl, A., Chao, S., Li, X., and Zhong, S. 2018. Molecular mapping of loci conferring resistance to spot blotch and powdery mildew in barley using the genotyping by sequencing approach. 11th International Congress of Plant Pathology (ICPP), July 29-Agust 3, 2018, Boston, MA (Poster presentation)
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Leng, Y., Ahmadpour, A., Liu, Y., Shrestha, S., Poudel, B., and Zhong, S. 2018. The PacC transcription factor regulates pH-dependent fungal development and virulence in the barley pathogenic fungus Cochliobolus sativus. 11th International Congress of Plant Pathology (ICPP), July 29-Agust 3, 2018, Boston, MA (Poster presentation)
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Karmacharya, A., and Zhong, S. 2018. Establishment of a CRISPR-Cas9 system for targeting genes in wheat. 2018 North Central Division Meeting, Fargo, North Dakota. P37 (Poster#26)

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:The target audience includes: 1. Wheat breeders and geneticists; 2. Wheat growers; 3. Plant pathologists; 4. Extension agents; 5. Agricultural administrators/members. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Participants include Shaobin Zhong (PI), Yueqiang Leng (Research associate), Mingxia Zhao (Ph.D. graduate student), Subidhya Shrestha (Ph.D. graduate student), Bikash Powdel (Ph.D. graduate student), and Anil (Ph.D. graduate student). The PI and other participants have attended two seminars and two conferences. How have the results been disseminated to communities of interest?The results were presented in peer-reviewed journals and in conferences. FHB resistant wheat lines and DNA markers developed for FHB resistance are being used by wheat breeders and research geneticists for development of FHB resistant varieties or germplasm. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Fusarium head blight and root rot diseases continue to threaten wheat production by reducing yield and quality of the crop. Use of resistance is the most efficient way to manage the diseases and mitigate their impact on wheat production. We are screening various germplasm and varieties for resistance to these diseases, studying the genetics of the resistance in resistant materials, and developing DNA markers for the identified resistance genes. To identify FHB resistance (Objective 1), we re-evaluated the 164 TCAP wheat lines. Some of them consistently showed a high level of resistance to FHB in the 2017 summer disease nursery and will serve as resistant germplasm. We also phenotyped the mapping population consisting of 210 recombinant inbred lines (RILs) (F2:8) derived from the cross between 10Ae564 and Joppa in two more FHB inoculation experiments, including one in the greenhouse in the spring of 2016 and one in the Fargo FHB nursery in the summer of 2017. The results showed that disease severity varied among the population, as previously observed in 2016. To develop DNA markers for FHB resistance in wheat (Objective 2), we genotyped the 210 RILs derived from the cross between 10Ae564 and Joppa using DNA markers mapped on the 5AL QTL region associated with FHB resistance in PI 277012. The result confirmed that major QTL (Qfhb.NDWP-5A) from PI 277012 has been introgressed into durum wheat and FHB resistance of durum wheat can be improved by accumulation of existing and new FHB resistance QTL in durum wheat breeding program. In addition, we continued to develop PCR-based SNP for the 5AL QTL in PI 277012 using the 958 RILs derived from the cross between Grandin and PI 277012, and delimited the QTL in a 1.2 Mb genomic interval based on the reference genome sequence of Chinese Spring. This work not only provides more markers for effective selection of FHB resistance in wheat breeding programs, but also establishes a foundation for further cloning of the FHB resistance gene. To better understand the genetics and biology of the fungal pathogen Bipolaris sorokiniana (Objective 3), we were developing a CRISPR-Cas9-based genome editing approach for functional genomics study of Bipolaris sorokiniana. Plasmid vectors with CRISPR-Cas9 gene cassette have been constructed and will be used to target a reporter gene involved in melanin biosynthesis through PEG-mediated and Agrobacterium-mediated transformation. This tool should be useful for functional characterization of genes involved in fungal development and virulence in the pathogen.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Wang, R., Leng, Y., Ali, S., Wang, M., and Zhong, S. 2017. Genome-wide association mapping of seedling resistance to three different pathotypes of Cochliobolus sativus in a USDA barley core collection. Molecular Breeding 37:44
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Zhong. S., and Leng, Y. 2016. Molecular dissection of a complex gene locus conferring high virulence on barley cv. Bowman in the fungal cereal pathogen Cochliobolus sativus. O-29, APS Annual Meeting, July 30-August 3, 2016, Tampa, FL. (Oral)
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Leng, Y., Zhao, M., Wang, R., Steffenson, B. J., Bruggeman, R. S., *Zhong, S. 2016. The gene conferring susceptibility to spot blotch caused by Cochliobolus sativus is located at the Mla locus in barley cultivar Bowman. In: The 12th International Barley Genetics Symposium, June 26-30, 2016, Minneapolis-St. Paul, Minnesota United States of America (Poster 108).
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Wan, J., Zhong, S., Schwarz, P., and Rao, J. 2017. Application of nanoencapsulated clove oils to enhance antifungal activities and inhibit mycotoxin production in vitro in Fusarium graminearum. Proceedings of the 2017 National Fusarium Head Blight Forum, Dec 3-5, 2017, Milwaukee, WI. P39. (Poster #39)
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Zhang, Q., Faris, J. D., Chao, S., Friesen, T. L., Zhong, S., Cai, C., Elias, M., and Xu, S. S. 2017. Identification and Molecular Mapping of Quantitative Trait Loci for Resistance to Fusarium Head Blight in Cultivated Emmer PI 272527. Proceedings of the 2017 National Fusarium Head Blight Forum, Dec 3-5, 2017, Milwaukee, WI. P97. (Poster #55)
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Zhao, M., Leng, Y., Liu, Y., Xi, Xi., Li, J., Wang, R., Long, Y., Chao, S., Xu, S. S., and Zhong, S. 2017. Fine Mapping of a Novel Major QTL for Fusarium Head Blight Resistance in the Wheat Line PI 277012. Proceedings of the 2017 National Fusarium Head Blight Forum, Dec 3-5, 2017, Milwaukee, WI. P98. (Poster #56)

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:The target audience includes: 1. Wheat breeders andgeneticists; 2. Wheat growers; 3. Plant pathologists; 4. Extension agents; 5. Agricultural administrators/members. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Participants include Shaobin Zhong (PI), Yueqiang Leng (Research associate), Yan Liu (Postdoctoral associate), Mingxia Zhao (Ph.D. graduate student), Subidhya Shrestha (Ph.D. graduate student), Bikash Powdel (Ph.D. graduate student), and Anil (Ph.D. graduate student). The PI and other participants have attended four seminars and two conferences. How have the results been disseminated to communities of interest?The results were presented in peer-reviewed journals and in conferences. FHB resistant wheat lines and DNA markers developed for FHB resistance are being used bywheat breeders and research geneticists for development of FHB resistant varieties or germplasm. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? For FHB resistance identification (Objective 1), we re-evaluated 164 TCAP wheat lines which showed some levels of resistance to FHB in the previous year's screening in the field experiment.Sixof them were highly resistant to FHB in the 2016 summer disease nursery and will serve as resistant germplasm. Also, 108 spring wheat breeding lines from University of Minnesota and 120 spring wheat breeding lines from South Dakota State University have been evaluated for FHB resistance in the China nursery located in Nanjing, China. The disease severity ranged from 7.0% to 100% among the entries evaluated. Approximately 30% of these advanced breeding lines showed a disease severity below 15%, indicating they have a very good level of FHB resistance under natural infection conditions. To develop DNA markers for FHB resistance in wheat (Objective 2), 210 recombinant inbred lines (RIL) (F2:7) derived from the cross between 10Ae564 and Joppa were genotyped using the wheat 90K-SNP arrays and6323 SNP markers were identified in the mapping population. Excluding the co-segregated markers, 1676 SNP markers were used to construct a genetic linkage map which consisted of 34 linkage groups covering 626.42 cM in length. The 210 recombinant inbred lines were also phenotyped in two more FHB inoculation experiments, including one in the Fargo FHB nursery in the summer of 2015 and one in the greenhouse in the fall of 2015. QTL analysisindicated that one QTL for FHB resistance was detected on 5A and 6A from 10Ae564, and another QTL for FHB resistance from Joppa was identified on2A. For better use of the FHB resistance in PI 277012, thirty PCR-based SNP markers have been mapped to the two QTL regions on chromosome 5A, with 23 on the 5AL QTL region and 7 on the 5AS QTL region. The two closest flanking markers are 2.0 and 2.3 cM from the 5AL QTL and the two flanking markers are 0.8 and 0.9 cM from the 5AS QTL.To better understand the genetics and biology of the fungal pathogen Bipolaris sorokiniana (Objective 3), we characterized the functions of velvet genes which regulate fungal development and virulence. This information may be useful for development of new strategies for controlling the disease by targeting the virulence factors in the pathogen.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Zhu, X., Zhong, S., Chao, S., Gu, Y. Q., Kianian, S. F., Elias, E., and Cai, X. 2016. Toward a better understanding of the genomic region harboring Fusarium head blight resistance QTL Qfhs.ndsu-3AS in durum wheat. Theor. Appl. Genet. 129:31-43.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Zhu, X., Zhong, S., Xu, S. S., Jyoti, J., and Cai, X. 2016. Effects of D-genome chromosomes and their A/B-genome homoeologs on Fusarium head blight resistance in durum wheat. Crop Sci. 56:1049-1058.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Mergoum, M., Simsek, S., Zhong, S., Acevedo, M., Friesen, T. L., Xu, S. S., Liu, Z. 2016. Elgin-ND Spring Wheat: A Newly Adapted Cultivar to the North-Central Plains of the United States with High Agronomic and Quality Performance. J. Plant Reg. 10:130-134.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Wang, R., Leng, Y., Shrestha, S., and *Zhong, S. 2016. Coordinated and independent functions 1 of velvet-complex genes in fungal development and virulence of the fungal cereal pathogen Cochliobolus sativus. Fungal Biology 120:948-960.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Puri, K. D., Yan, C., Leng, Y., *Zhong, S. 2016. RNA-Seq revealed differences in transcriptomes between 3ADON and 15ADON populations of Fusarium graminearum in vitro and in planta. PLoS ONE 11(10): e0163803. doi:10.1371/journal.pone.0163803
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Shrestha, S., Poudel, R., Borowicz, P., and *Zhong, S. 2016. Histopathological study of compatible and incompatible interactions between barley and the spot blotch fungus Bipolaris sorokiniana. P7-211 In: IS-MPMI XVII Congress, July 17-21, 2016, Portland, OR. (Poster).
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Szabo-Hever, A., Zhang, Q., Zhong, S., Friesen, T. L., Elias, E. M., Xu, S. S., and Chao, S. 2016. Association mapping for Fusarium head blight resistance in synthetic hexaploid wheat. Proceedings of the 2016 National Fusarium Head Blight Forum, Dec 4-6, 2016, St. Louis, MO. P97. (Poster)
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Zhao, M., Leng, Y., Chao, S., Xu, S. S., and Zhong, S. 2016. Molecular mapping of QTL for FHB resistance introgressed into durum wheat. Proceedings of the 2016 National Fusarium Head Blight Forum, Dec 4-6, 2016, St. Louis, MO. P105. (Poster)

Progress 02/01/15 to 09/30/15

Outputs
Target Audience:The target audience includes: 1. Wheat and barley breeders; 2.Wheat and barley geneticists; 3. Wheat and barley growers; 4. Plant pathologists; 5. Extension agents; 6. Agricultural administrators/members. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Participants include Shaobin Zhong (PI), Yueqiang Leng (Research associate), Yan Liu (Postdoctoral associate), Rui Wang (Ph.D. graduate student), Mingxia Zhao (Ph.D. graduate student), Subidhya Shrestha (Ph.D. graduate student) and Bikash Powdel (M.S. graduate student). ThePI and other participants have attended four seminars and conferences. How have the results been disseminated to communities of interest?The results were presented in peer-reviewed journals and inconferences. DNA markers developed for FHB resistance have been sent to wheat breeders and research geneticists for selection of FHB resistant varieties or germplasm. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Use of disease resistant varietiesis the most effective and environmental friendly approach for disease management. However, sources of Fusarium head blight resistance are limited. To search for new sources of FHB resistance (Objective 1), we screened 2,186 wheat accessions collected by the National Small Grains Collection at USDA-ARS, Aberdeen, Idaho. Most of these wheat accessions were highly susceptible to FHB and only164 accessions showed FHB severity less than 50%. Among them, eleven accessions were highly resistant with a disease severity less than 21% and will be further used for genetic studies and as parents for wheat breeding programs.We also evaluated 297 advanced breeding lines from three wheat breeding programs (116 from South Dakota State University, 75 from North Dakota State University, and 106 from University of Minnesota) along with 83 diverse wheat accessions in a disease nursery located at Hangzhou, China. The disease severity ranged from 7.0% to 100% among the lines evaluated. Approximately 28% (107) of the materials showed a disease severity below 15%, indicating they have a very good level of FHB resistance under natural infection conditions. To develop DNA markers for FHB resistance inwheat (Objective 2), we genotyped a population consisting of 210 recombinant inbred lines (RILs) (F2:7) derived from the cross between durum line 10Ae564 and cultivarJoppa using the wheat 90K single nucleotide polymorphism (SNP) chips.A genetic map was constructed with the SNP markers. This population was also phenotyped for FHB resistance in two seasons of field experiments. The disease severity ranged from 9% to 97% among the RILs evaluated, suggesting that resistance to FHB segregated in the population.We collected grain samples from the inoculated plants for DON test. The data showed that DON contents ranged from 5.2 to 30.8 ppm among the RILs, indicating that the population segregated for DON accumulation in grains as well. To fine map the genes for FHB resistance in the wheat line PI 277012, we also evaluated 952 recombinant inbred lines (F2:7) from the cross between PI 277012 and the wheat cultivar Grandin for FHB resistance in two seasons of greenhouse experiments and two seasons of field experiments. We developed user-friendly assays for 10 DNA markers linked to the FHB resistance loci in PI 277012. These markers are being used for marker assisted selection of the FHB resistance transferred from PI 277012 into adapted wheat germplasm. To better understand the genetics and biology of the fungal pathogen Bipolaris sorokiniana (Objective 3),we characterized the functions of genes for non-ribosomal peptide synthetase (NRPS) unique in different pathotypes of the fungus. We have shown that these unique NRPS genes are involved in biosynthesis of virulence factors required fordisease development. This information may beuseful for development of new strategiesfor controlling the disease by targeting the virulence factors in the pathogen.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Szabo-Hever, A., Zhang, Q., Zhong, S., Friesen, T. L., Elias, E. M., Xu, S. S., and Chao, S. 2015. Characterization of new synthetic wheat germplasm for resistance to Fusarium head blight. Proceedings of the 2015 National Fusarium Head Blight Forum, Dec 6-8, 2015, St. Louis, MO. P107. (Poster)
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Zhao, M., Liu, Y., Leng, Y., Li, J., Wang, R., Long, Y., Chao, S., Xu, S. S., and Zhong, S. 2015 Development of User-Friendly DNA Markers for Fusarium Head Blight Resistance QTL in PI 277012. Proceedings of the 2015 National Fusarium Head Blight Forum, Dec 6-8, 2015, St. Louis, MO. P114. (Poster)
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Zhu, X., Zhong, S., and Cai, S. 2015. Cytogenetic Dissection of A, B, and D Genome Provides New Insights into Fusarium Head Blight Resistance in Durum Wheat. Proceedings of the 2015 National Fusarium Head Blight Forum, Dec 6-8, 2015, St. Louis, MO. P116. (Poster)