Source: KANSAS STATE UNIV submitted to
NOVEL STRATEGIES FOR MANAGING BLAST DISEASES ON RICE AND WHEAT
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0231543
Grant No.
2013-68004-20378
Project No.
KS602582
Proposal No.
2012-02345
Multistate No.
(N/A)
Program Code
A5122
Project Start Date
Jan 1, 2013
Project End Date
Dec 31, 2018
Grant Year
2017
Project Director
Valent, B.
Recipient Organization
KANSAS STATE UNIV
(N/A)
MANHATTAN,KS 66506
Performing Department
Plant Pathology
Non Technical Summary
Rice and wheat are the top two sources of calories in the human diet, and both are important export crops for the U.S. Rice blast disease, caused by the fungus Magnaporthe oryzae remains an explosive threat to rice production in the U.S. and globally. Since 1985 when M. oryzae first appeared on wheat in Brazil, blast has become a major constraint to wheat production in several South American countries. It is critical to prevent establishment of wheat blast in the U.S. Disease control is complicated by the lack of a clear understanding of how the disease develops and spreads in the field, by extreme variability shown by the fungus and by unknowns associated with climate change. We will apply knowledge gained through previous research and cloned blast resistance genes for disease control, including developing methods for choosing effective resistance genes and rapidly deploying them, methods for forecasting disease risk and informing farmers of the best control options, and novel methods to control disease by turning off fungal genes needed for disease. Although genetic resistance is the most effective way to control blast diseases, high-yielding rice varieties preferred by U.S. farmers generally lack resistance. These varieties require expensive fungicide applications when weather conditions favor disease. There is urgent need for efficient strategies to move new resistance (R) genes into elite rice varieties while maintaining yield and quality. We will develop cisgenic technology, in which only rice DNA sequences are introduced, to precisely incorporate resistance genes into elite rice varieties. Adoption of cisgenic rice by U.S. farmers requires consumer acceptance in the U.S. as well as in Europe and other import markets. We will assess consumer attitudes in the U.S. and Europe toward cisgenic rice, as compared to transgenic rice in which some non-rice DNA sequences are also introduced. Strategies for promoting consumer acceptance will be developed based on this assessment. In contrast, few wheat blast resistance genes have been identified. We address this with strategies to identify wheat resistance genes through field tests of U.S. wheat varieties against native pathogen populations in South America. We will also test if moving rice resistance genes into wheat will provide effective wheat blast resistance. Development and use of a blast forecasting model will inform farmers if and when they need to apply fungicides, and will warn first responders when the weather is right for wheat blast disease to occur. Understanding wheat blast biology and training a first-responder network will enable rapid detection and response to any wheat blast outbreaks in the U.S. We will also develop novel strategies for blast control based on designing plants that block the fungus from expressing genes it needs to cause disease. Our extension programs will produce publications and workshops to inform extension specialists, farmers and other first responders about wheat blast and the novel disease management strategies. We will design and implement educational activities to attract undergraduate students to careers in plant pathology and in plant biosecurity.
Animal Health Component
(N/A)
Research Effort Categories
Basic
30%
Applied
70%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2121530116030%
2121540116010%
2121541116010%
2121542116010%
2121543116010%
2121544116010%
2121545116010%
2121549116010%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1549 - Wheat, general/other; 1540 - Hard red winter wheat; 1542 - Soft red wheat; 1541 - Hard red spring wheat; 1530 - Rice; 1545 - Durum wheat; 1543 - Soft white wheat; 1544 - Hard white wheat;

Field Of Science
1160 - Pathology;
Goals / Objectives
Rice blast, caused by the Magnaporthe oryzae Oryza pathotype remains an explosive threat to U.S. rice. High-yielding rice varieties preferred by farmers are often classified as "susceptible" or "highly susceptible" to blast, adding costs ranging from $4.75 to $20.87 per acre. Host resistance is one of the most effective means of control, but there is a critical need for efficient strategies to move resistance genes into elite rice varieties while maintaining yield and quality. In contrast for the newly-emerged wheat blast disease in South America (S.A.) caused by the M. oryzae Triticum pathotype, few resistance genes have been identified in wheat and fungicide treatments are unreliable. It is paramount to prepare for incursion and prevent establishment of the S.A. fungus on U.S. wheat. It is also essential to determine the potential for blast to emerge and establish in the U.S. wheat crop from native strains of the Lolium pathotype, which currently cause gray leaf spot disease on turf grasses. Turf grass strains infect wheat grown under greenhouse conditions and a native Lolium strain was isolated from a single severely-blasted wheat head in Kentucky in 2011. Therefore, wheat blast could establish in the U.S. by introduction of aggressive strains endemic to S.A., or by emergence of a native Lolium strain shifting to become an aggressive wheat pathogen in the field. The major premise for this proposal is that various diseases caused by host-differentiated M. oryzae populations can be viewed as a single system for developing novel control strategies, and that extensive research into disease on one host can aid in control on others. For example, pathogenicity factors and avirulence (Avr) effectors have been extensively characterized from rice pathogens, and 18 blast resistance genes have been cloned from rice. Our goal is to apply this knowledge to develop new genetic tools for controlling rice blast and preventing wheat blast in the U.S. Specific objectives are to: (1) enhance blast resistance in elite US rice varieties; (2) develop a novel strategy using host-induced gene silencing (HIGS) for controlling blast; (3) control wheat blast through the understanding of wheat blast genetics and genomics, pathology, ecology and epidemiology; (4) incorporate blast resistance into wheat; (5) develop and validate forecasting models for rice and wheat blast; (6) assess consumer attitudes and economics of cisgenic blast-resistant rice; (7) disseminate results through educational resources and programs for stakeholders; and (8) attract a new generation of plant pathologists to work on plant biosecurity. Outputs from this project will include efficient cisgenic strategies for incorporating resistance genes into high-yielding U.S. varieties, and pathogen population analysis to inform breeders' resistance gene choices. Understanding wheat blast field biology together with effective diagnostics, workshops and training resources will underpin a first-responder network for preventing establishment of wheat blast in the U.S. Deploying forecasting models and assessing economic and social impacts of new technologies will benefit both rice and wheat production.
Project Methods
This project involves collaborators with expertise in pathogen population dynamics, epidemiology, genetics, genomics, plant transformation, forecast modeling, extension, education and economics. Aspects of this project continue from previous NIFA support (grant # 2009-55605-05201). Major methods for each objective are summarized. (1) Enhance blast resistance in elite U.S. rice varieties. To simplify the regulatory process, cisgenic approaches (only rice sequences are retained in the new variety) will be developed to introduce cloned rice resistance genes into preferred rice varieties. To guide resistance gene deployment, Avr-effector compositions in extant blast populations will be assessed through sequencing of pooled, barcoded PCR samples. (2) Develop host-induced gene silencing (HIGS) for controlling blast. HIGS would control blast disease by silencing fungal genes needed for disease, through expressing siRNAs targeted against these fungal genes directly in rice. If effective, HIGS has the potential to exploit the wealth of fungal pathogenicity genes previously identified through foundational research. (3) Wheat blast control through understanding wheat blast genetics, pathology, ecology and epidemiology. We will focus on understanding the population structure, ecology and epidemiology of the wheat blast fungus in S.A., on determining the potential for native Lolium strains to establish on U.S. wheat, and on validating, refining and deploying PCR-based diagnostic protocol(s). (4) Incorporate blast resistance into wheat. We previously identified some U.S. wheat varieties with high levels of resistance to some wheat blast strains in growth chamber and greenhouse studies. Broadly useful resistance genes will be confirmed through field tests in S.A. Molecular markers will be developed for incorporating at least one effective resistance gene into wheat. We will also test if cloned rice resistance genes function to control blast after transformation into wheat. (5) Develop and test forecasting models for rice and wheat blast. Models will be developed based on both regional-scale, climate indices and local-scale weather, and validated for rice in the U.S. and for wheat in S.A. (6) Assess consumer attitudes and economics of cisgenic rice by implementing "willingness to pay (WTP)" surveys with participants in the U.S. and Belgium. Results will be broadly communicated in order to promote acceptance of cisgenics throughout the rice production and marketing chain. Analysis of the economic impact of cisgenic resistance and blast forecasting will also promote acceptance. (7) Disseminate results through educational resources and programs for stakeholders through rice blast training workshops in the U.S., wheat blast training workshops in the U.S. and in South America, extension publications and apps for mobile devices, and international educational outreach. (8) Attract a new generation of plant pathologists to work on international plant biosecurity. We will engage undergraduates in workshops focused on wheat production, policy and pathogens, on plant biosecurity and containment research, and on rice and the global importance of food security.

Progress 01/01/13 to 12/31/18

Outputs
Target Audience:Target audiences for the Blast Integrated Project (BIP) are the professional plant pathology community, rice and wheat production specialists, USDA-APHIS scientists, rice and wheat stakeholders in government and industry, and students with an interest in plant pathology and plant biosecurity. The target audience also includes scientists, students, farmers and other wheat stakeholders in South America and in Bangladesh, where wheat blast currently limits wheat production, as well as world-wide in countries wishing to block wheat blast from occurring. Our distinguished Scientific Advisory Board (SAB) includes James Correll, University of Arkansas, Fayetteville, Arkansas; Justin Faris, USDA-ARS Cereal Crops Unit, Fargo, North Dakota; Anna McClung, Dale Bumpers National Rice Research Center, Stuttgart, Arkansas; Tim Murray, Washington State University, Pullman, WA; Boyd Padgett, LSU Ag Center, Alexandria, LA; Pawan Singh, CIMMYT, El Batan, Mexico; and Didier Tharreau, CIRAD, France. The 2017 BIP Project Team and SAB meeting (December 2, 2017) was held in association with the 2017 National Fusarium Head Blight Forum in Milwaukee, Wisconsin. Team members Paul, Cruz and DeWolf organized a Wheat Blast Symposium (2017) that was held Dec. 3 immediately before the FHB Forum, and it was attended by many wheat stakeholders attending the Forum. Additionally, Cruz served as an instructor for the Regional Training Course and Wheat Blast Surveillance, organized in Bangladesh by the 'Delivering Genetic Gain in Wheat Project,' Feb 4-14, 2017. His lecture "Wheat Blast Pathology and Disease Identification" is available on YouTube (https://www.youtube.com/watch?v=CZGOSxypHKQ ). A Wheat Blast Training Workshop for South Asia (July 17-21, 2017) was conducted at the USDA ARS FDWSRU at Fort Detrick, MD by ARS, CIMMYT and KSU. The training course was attended by 10 visiting scientists from ARS, CIMMYT, Bangladesh Agricultural Research Institute, Uttar Banga Krishi Viswavidyalaya University and Indian Council of Agricultural Research, India. The workshop focused on greenhouse evaluation of wheat germplasm for blast resistance and development and application of molecular markers. In 2018, Co-PI Fernandes organized a Wheat Blast and Head Blight Workshop at the 12th Meeting of the Brazilian Wheat and Triticale Research Commission, July 2-5, Passo Fundo, Brazil. Valent discussed wheat blast to a large audience of Brazilian scientists, farmers and other wheat stakeholders. The final BIP project meeting was held on August 1, 2018, in association with the 11th International Congress of Plant Pathology in Boston, Massachusetts. Co-PI Cruz was involved in co-organizing a Wheat Blast Session at this meeting, entitled "Wheat Blast - Developing Strategies for Assessing and Managing a Global Threat on the Move" on August 3, 2018, and several BIP team members spoke at this session. Our second Extension Publication 'Wheat Disease Identification,' a popular wheat disease identification booklet, was updated to include wheat blast and republished. This publication was originally developed by the multi-state extension and research committees for small grain diseases, NCERA-184 and WERA-97. The updated books were distributed in 32 states that are most at risk for wheat blast.More than 18,000 copies were distributed at part of this effort. Multiple states liked the publication so much they elected to purchase additional copies to share with their growers.Publication authors: Erick DeWolf, and James P. Shroyer, Kansas State University (https://www.bookstore.ksre.ksu.edu/pubs/MF2994.pdf). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Plant Biosecurity in Theory and Practice, the highly successful 5-day short course developed in 2015 by CoPI Stack. For the duration of this grant, the course was presented at K-State's Biosecurity Research Institute May 18-22, 2015 (36 participants from 14 countries), May 23-27, 2016 (40 participants from 16 countries) and May 15-19, 2017 (38 participants from 12 countries on 6 continents), and May 14-18, 2018 (29 participants from at least 5 countries). In total, 143 participants from at least 16 countries included undergraduates, graduate students, postdoctoral researchers, faculty and other researchers from government agencies, industry and academia. An international panel of instructors, including project members Stack, Valent, Bockus, Cruz, and Cruppe, provided perspectives on plant biosecurity through a combination of lectures, hands-on biocontainment laboratory training, case studies, and open discussions Courses were recorded and are available on DVD. Project members organized 6 NATIONAL WORKSHOPS focused on rice blast and/or wheat blast and participated as organizers and/or participants in 11 INTERNATIONAL MEETINGS OR COURSES. Visitors attended the 7 BIP project meetings including APHIS scientists, who were involved in decisions on how to regulate import of wheat seed from blast-endemic areas of South America and needed to understand wheat blast disease. APHIS scientists attended the BIP Project Meetings in Manhattan, KS in June 2013; in Minneapolis, MN, on August 9; the Wheat Blast Workshop in St. Louis on December 7, 2014; the BIP/SAB meeting and workshop on Dec. 5-6, 2015; and the BIP/SAB meeting on Nov. 14-15, 2016 and the BIP/SAB meetings on Dec 2-3, 2017. Valent and Cruz are serving as reviewers for the USDA-APHIS New Pest Response Guideline (NPRG) for wheat blast. Valent is leading the team to update the Recovery Plan for Wheat Blast published on the National Plant Disease Recovery System web page in 2013 (http://www.ars.usda.gov/research/docs.htm?docid=14271 ). STUDENTS AND POSTDOCS WORKING ON THIS PROJECT: High School Student -- Holly Herald (summer intern, Arkansas Rice Research and Extension Center, AR, with Jia). UNDERGRADUATES -- Lindsey Ashmore (KSU, Wheat Blast Web Site, Facebook and Twitter; Wheat blast videos; Helping with Biosecurity Short Course); Payton DeLong (KSU, BSL-3 studies of wheat blast); Mia Hodges (UARK, Rice blast population biology). GRADUATE STUDENTS -- Jorge Bavaresco (University of Passo Fundo RS BR, MS student in the Applied Computing Graduate Program at University of Passo Fundo); Zhiyi Bian (Purdue, HIGS in rice); Li 'Lily' Chen (UK, population analyses of US Lolium pathotype); Giovana Cruppe (KSU, Wheat blast resistance, wheat blast field biology); Taylor Fischer (KSU, graduated with M.S. degree, blast forecast modeling); Javier Kiyuna (KSU, diagnostic protocols for detecting MoT fungus in wheat seed); Yang Li (Purdue, HIGS in rice); Karasi Mills (OSU; biology and epidemiology of wheat blast incited by MoT and MoL); Monica Navia Urrutia (KSU; Rice R gene function in wheat); Kerri Neugebauer (KSU; Rice R gene function in wheat); Carlos Augusto Pizolotto (University of Passo Fundo, Brazil, MoT occurrence and survival in the field); Aaron M. Shew (Univ. of AR, cisgenic survey in India); Stuart Sprague (KSU, Molecular analysis of transgenic rice); Tej Man Tamang (KSU, rice cisgenics); Francis Tisboe (UARK, Cisgenic survey in Ghana); Mengying Wang (NCSU, HIGS in rice); Xue Zhang (Purdue, HIGS in rice). POST-DOCTORAL ASSOCIATES -- Christian Cruz (KSU, wheat blast biology, resistance, Studies in BSL-3 containment and the field in SA; Currently Assistant Professor, Dept. Botany and Plant Pathology, Purdue University); Jorge David Salgado Moncada (OSU, wheat blast field studies in Bolivia); Shefali Dobhal (KSU, population biology of Bolivian wheat blast fungus); Ely Oliveira Garcia (Wheat blast genomics and cell biology; currently Assistant Professor, Dept. Plant Pathology and Crop Physiology, LSU AgCenter/College of Agriculture); Jianbing Ma (USDA/Dale Bumpers National Rice Research Institute, Arkansas, rice blast avirulence gene analysis); Mike Pieck (USDA-ARS-FSWSRU, molecular diagnostics sequence discovery); Prasanna Kankanala (KSU, wheat resistance, molecular markers); Denis Shah (Cornell,with DeWolf, blast forecasting models); Baohua Wang (UK, population analyses of US Lolium pathotype); Xueyan Wang (University of Arkansas Rice Research and Extension Center, Arkansas, rice blast population biology). LAB TECHNICIANS - Melinda Dalby (KSU, Research Assistant: Rice Blast Research) Edwin Arley Navia-Rodriguez (KSU, Lab Technician: Wheat Blast Research); Jung-Eun Kim Park (KSU, Transformation Specialist: Rice Genetic Transformation); Trevor Steiner (KSU, Lab Technician: Rice plant management and seed harvest in Greenhouse); Maria Bellizzi (OSU, Rice Transformation); Kelli Thomas (USDA-ARS FDWSRU Maryland, Wheat Blast Research). PERSONNEL/STUDENTS TRAINED IN BOLIVIA -- Javier Kiyuna (Fundación CETABOL, Wheat blast field tests); Juan Vasquez (Fundación CETABOL, Wheat blast laboratory tests); Santiago Rivera (Universidad Gabriel René Moreno, Wheat blast field work); Jorge Andrés Cuellar, Ing. (Tropical Agricultural Research Center - CIAT Bolivia, wheat blast field testing); Lidia Calderon, Jorge Cuéllar, and Darwin Coimbra, wheat blast field testing); Andres Cruz (KSU, wheat field studies). PROJECT COORDINATOR-- Forrest G. Chumley (KSU). COLLABORATORS -- On rice blast, we collaborated with rice disease specialists Rick Cartwright (AR), Don Groth (LA), Shane Zhou (TX), and Chris Greer (CA) to compile historical weather data for AR, CA, LA, and TX. We have also collaborated with Bo Zhou (IRRI, the Philippines). Rice Breeder Xueyan Sha (UA RREC) and Klaus Scheuermann (EPAGRI, Brazil) have collaborated on identification of US rice germplasm with Pi-9. For wheat blast, Brian Mills and his team at Penn State (including Paul Knight and Doug Miller) have collaborated on gathering weather data, implementing models for wheat blast, and summarizing results in a map-based format. For wheat blast, we are collaborating on molecular markers and GBS with Eduard Akhunov, Jesse Poland and Allan Fritz at KSU, and Mohammad Asif of Heartland Plant Innovations, Inc, Manhattan, Kansas. We are collaborating with Sanzhen Liu (KSU) on genomics of the wheat blast pathogen. We are working with Jorge Dubcovsky (Triticum CAP Project Director, UC Davis). International collaborators are listed according to location. Europe: Wim Verbeke, Professor at the University of Gent, and a leading European Ag Economist and E.U. advisor on GMO issues; Didier Tharreau (CIRAD-UMR BGPI, Montpellier, France); Pierre Gladieux (INRA-UMR BGPI, Montpellier, France); Elisabeth Fournier (INRA-UMR BGPI, Montpellier, France); Nick Talbot (University of Exeter, UK). Brazil: Alfredo Urashima at Universidade Federal de São Carlos, Brazil; Andre Rosa and Paulo Khunem (Biotrigo Genetica). Bolivia: Field work is being performed with Diego Baldelomar, (ANAPO), Marcia Gabriela Rivadeneira Caballero, Centro de Investigación Agrícola Tropical (CIAT Bolivia), and Takashi Bravo (Fundación CETABOL). Paraguay: Mohan Kohli (Cámara Paraguaya de Exportadores de Cereales y Oleaginosas, CAPECO). Australia: Evans Lagudah, CSIRO; Peter Solomon, Australian National University; International Maize and Wheat Improvement Center (CIMMYT): Etienne Duveiller, CIMMYT, India; Pawan Singh, CIMMYT, Mexico. Bangladesh: Md. Muzahid-E-Rahman, Paritosh Kumar Malaker, and Naresh Chandra Deb Barma, Bangladesh Wheat and Maize Research Institute, BARI. Japan: Yukio Tosa (Kobe University, Japan); Ryohei Terauchi (Iwate Biotechnology Research Center and Kyoto University). How have the results been disseminated to communities of interest?Many of the students and post-docs participating in the Plant Biosecurity in Theory and Practice short course were new to the field of Plant Biosecurity. We communicate with colleagues and directly with the general public through a webpage (http://www.k-state.edu/wheatblast/ ), Facebook (https://www.facebook.com/ksuwheatblast/ ) and Twitter (https://twitter.com/KSUWheatBlast ). Project members regularly participate in radio interviews, popular news articles and postings on You-Tube. We produced an interesting and informative video entitled "Wheat Blast, An Emerging Threat to Wheat" in order to educate the general public on issues associated with wheat blast and plant biosecurity (Stack, Cruz). The video is available on our web page and on youtube in English (https://www.youtube.com/watch?v=z_pCMbaY2io), in Spanish https://www.youtube.com/watch?v=FshZQZ-MgaA, and in Portuguese https://www.youtube.com/watch?v=VV-9c3FIYxs). Two additional videos are available in English, produced by Christian D. Cruz for the Regional Training Course and Wheat Blast Surveillance, Bangladesh, February 4-14, 2017. These are: "Wheat Blast Pathology and Disease Identification," (https://www.youtube.com/watch?v=CZGOSxypHKQ) and "Wheat Blast: Epidemiology and Management of an Emerging Global Threat," (https://www.youtube.com/watch?v=gf1UFz926og). International grain/soybean buyers, government officials and others responsible for the purchase, shipment and handling of U.S. grains and soybeans had an opportunity to understand the science, risks and benefits of genetically engineered crops at the annual grain purchasing workshop hosted by the KSU International Grains Program (Trick). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Project continued through 2018 with a 1-year no-cost extension. RICE BLAST: (OBJECTIVE 1) We developed new understanding of the complexity of the U.S. rice blast population. (Wang et al., 2017), and identified useful resistance (R) genes for controlling that population. We molecularly identified a novel broad-spectrum R gene Ptr, which will be valuable for controlling blast in the U.S. and worldwide (Zhao et al., 2018). We determined that a previously identified R gene, Pi9, is broadly effective against the U.S. rice population, and developed molecular markers and identified useful germplasm for U.S. breeders to use for new varieties (Scheuermann and Jia, 2016). A major take-home is that the blast population is so dynamic over time and that frequent monitoring of the population is required for informed deployment of effective R gene combinations. Our next manuscript will describe relationships between AVR gene alleles and efficacy of corresponding R genes. (Xueyan Wang, Yulin Jia, et al., in preparation). Although we have achieved cisgenic transformation (GMO with only rice genes, no foreign genes) of elite U.S. rice varieties with the Pi9 R gene, both efficiency of expression and efficacy of Pi9 are low in these elite rice varieties (Co-PIs Wang, Park and Valent). The Yinong Yang lab has developed efficient methods for CRISPR/Cas9-mediated genome editing in plants which facilitated the functional validation of the rice Ptr gene (Zhao et al., 2018). The Yang lab has also developed a method to improve the efficiency of multiplex genome editing in rice blast fungus for functional analyses. (OBJECTIVE 2) With some pathogen genes, Host-Induced Gene Silencing (HIGS) can provide up to 80% disease control under laboratory conditions (Mengying Wang, PhD thesis, 2019). However, field tests so far appear less promising. WHEAT BLAST: (OBJECTIVE 3) We sequenced and assembled genomes of >35 M. oryzae/M.grisea strains from rice, wheat, ryegrass and other hosts, and developed software tools for BLAST searches, SNP analyses and repeat masking in single and batch formats. By pooling our genome data with that of European collaborators, we developed a comprehensive phylogenetic analysis of host-specific forms, including understanding which host-specific populations are most closely related to the MoT population (Malaker et al. 2016; Farman et al. 2017, 2018; Gladieux et al. 2018). With collaborator Yukio Tosa, we identified Lolium strains as the likely source for the wheat blast outbreak in Brazil (Inoue et al, 2018). Our analyses clearly refute the recent assignment of wheat blast strains to a separate species (Gladieux et al. 2018; Valent et al. 2019). We produced a telomere-to-telomere reference genome for the aggressive wheat pathogen B71, and identified pathogen genomic features, such as supernumerary mini-chromosomes, with potentially significant impacts on pathogen evolution and variability (Peng et al. 2018; Yadav et al. 2019). These genome analyses underpin our development of Wheat Blast Specific-MoT3 Diagnostic Assays: An end-point and real-time PCR assay using the MoT3 locus of the retinol dehydrogenase gene for detection of M. oryzae Triticum (Pieck et al. 2017) was delivered to APHIS for work-plan development and deployment through the National Plant Diagnostic Network. Additionally, the PoT2 transposon and MoT3 locus were used to develop loop-mediated isothermal amplification (LAMP) assays for the specific detection of M. oryzae Triticum in-lab and in the field (Yasuhara-Bell et al. 2018 and 2019). Experiments are underway to optimize a protocol for detecting seed-borne MoT in seed/grain shipments (Kiyuna, M.S. thesis). We also focused on pathogen ecology and epidemiology. We quantified temporal and spatial progress of wheat blast, showing that the disease does not just show-up and affect 100% of the spikes. It increases in intensity over time, and the rate at which this occurs is influenced by cultivar resistance, in-field inoculum, inoculum density, temperature, relative humidity, and interactions among these factors (Mills, Ph.D. thesis; Mills, Salgado, Paul, Madden, unpublished). We have provided evidence that MoT inoculum produced on basal leaves can contribute to wheat head blast development (Cruz et al, 2015; Cruppe Ph.D. thesis), which highlights the critical importance of seed fungicide treatments and suggests utility for seedling applications. We have worked with multiple collaborators in Brazil and Bolivia to evaluate the performance of fungicides against wheat head blast under various environments and to determine the best scenarios for fungicide applications (Cruz et al, 2019, TPP). Although dependent on country and disease pressure, chemical control can be effective even under environmental conditions that favor disease. (OBJECTIVE 4) We have developed an efficient wheat blast field-testing platform in Bolivia. Working with the Bolivian Wheat Growers Association (ANAPO) and with CIAT Bolivia, we have trained personnel and developed effective methods for trial inoculations allowing for two rounds of field tests per year, even when the weather is not conducive to blast disease. Our analyses show good correlation between results in the field in Bolivia and our tests in controlled Biosafety Level-3 growth facilities at KSU and Fort Detrick. We showed that the 2NS/2AS translocation from Aegilops ventricosa confers useful, although partial, head blast resistance (Cruz et al, Crop Science, 2016). This wild wheat translocation fragment has already been widely deployed in wheat varieties planted world-wide because it carries several genes conferring rust and nematode resistance, and we have shown that the most blast resistant wheat varieties identified so far contain the 2NS resistance. 2NS-mediated partial resistance is stronger in some genetic backgrounds. In order to identify non-2NS sources of resistance, we have screened over 780 accessions in field and controlled environments and tested each for the presence of the 2NS molecular marker (Cruppe et al, Plant Disease, accepted). Four non-2NS spring wheat CIMMYT breeding lines (CM22, CM49, CM52, and CM61) and four wheat wild-relatives, Ae. tauschii (TA10142, TA1624, TA1667, and TA10140) were identified as resistant (<5% severity) or moderately resistant (5% to <25% severity) to head blast. We provided field trials in Bolivia and greenhouse tests at Ft. Detrick which led to the release of the first blast resistant, biofortified wheat variety released by the National Seed Board in Bangladesh. Called "BARI Gom 33", the variety was developed by the Wheat Research Centre in Bangladesh using a breeding line from CIMMYT. (OBJECTIVE 5) The wheat blast forecasting model developed by CoPI Fernandes for Brazil (Fernandes et al, 2017) has been adapted for the U.S., and is incorporated into the BIP risk assessment project. A climate suitability study for the U.S., performed using weather data from over a decade ago (Cruz et al., Plant Disease, 2016), showed most soft red winter wheat and over half of the hard red winter wheat is at risk. More accurate models needed, taking into account climate change. Using historical data, variables associated with rice blast epidemics (cooler temperatures in April; more precipitation in June), were incorporated into preliminary risk assessment models for rice blast (Taylor, M.S. thesis, 2016; DeWolf, Paul, Madden, Jia, Wamishe). (OBJECTIVE 6) We assessed the potential economic impact of cisgenically-resistant rice in the U.S. (Nalley et al, 2016). Assessment of consumer attitudes towards cisgenically resistant rice showed that consumers in developing countries are more accepting of GMO solutions (Shew et al, 2015; Tsiboe et al, 2017) than consumers from developed countries (Delwaide et al, 2015). (OBJECTIVE 7) Extension publications and workshops described in Outputs. (OBJECTIVE 8) Educational activities described in Opportunities for Training.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Cruppe, Giovana, Christian D. Cruz, Gary Peterson, Mohammad Asif, Allan Fritz, Lidia Calderon, Cristiano Lemes da Silva, Tim Todd, Paulo Kuhnem, Pawan K. Singh, Ravi P. Singh, Hans-Joachim Braun, and Barbara Valent. (201X) Novel sources of wheat head blast resistance in modern breeding lines and wheat wild relatives. Plant Disease. Accepted
  • Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Gongora-Canul, C., Salgado, J.D., Singh, D., Cruz, A.P., Valent, B., Paul, P.A., Madden, L.V., and Cruz, C.D. (201X) Temporal dynamics of wheat spike blast epidemics and disease quantification using multispectral aerial imagery. Under review.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Yadav, V., Yang, F., Reza, M. H., Liu, S., Valent, B., Sanyal, K., and Naqvi, N. I. (2019) Cellular dynamics and genomic identity of centromeres in the cereal blast fungus. mBio 10:e01581-19. https://doi.org/10.1128/mBio.01581-19./475574.
  • Type: Other Status: Published Year Published: 2019 Citation: Valent, B., Farman, M., Tosa, Y., Begerow, D., Fournier, E., Gladieux, P., Islam, M. T., Kamoun, S., Kemler, M., Kohn, L. M., Lebrun, M.-H., Stajich, J. E., Talbot, N. J., Terauchi, R., Tharreau, D., and Zhang, N. (2019) Pyricularia graminis-tritici is not the correct species name for the wheat blast fungus: response to Ceresini et al. (MPP 20:2). Molecular Plant Pathology 20:173-179.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Cruz, Christian D., Fl�vio M. Santana, Timothy C. Todd, Jo�o L. N. Maciel, Javier Kiyuna, Diego F. Baldelomar, Douglas Lau, Claudine S. Seixas, Augusto P. Goulart, Angelo A. Sussel, Carlos A. Schipanski, D�bora F. Chagas, Maur�cio Coelho, Tatiane Dalla Nora Montecelli, Carlos Utiamada, Adriano A. Cust�dio, Javier Toledo, William W. Bockus, and Barbara Valent. (2019) Multi-environment assessment of fungicide performance for managing wheat head blast (WHB) in Brazil and Bolivia. Tropical Plant Pathology, 44: 183-191, https://doi.org/10.1007/s40858-018-0262-9.
  • Type: Book Chapters Status: Awaiting Publication Year Published: 2019 Citation: Valent, Barbara, Pawan Kumar Singh, Xinyao He, Mark Farman, Yukio Tosa and Hans Joachim Braun. (2019) Blast Diseases: Evolution and challenges of a staple food crop fungal pathogen. In Emerging Plant Diseases and Global Food Security (Edited by Angela Records and Jean Ristaino) APS Press. In Press.
  • Type: Other Status: Published Year Published: 2019 Citation: Yasuhara-Bell, J., Pieck, M. L., Ruck, A., Farman, M., Peterson, G. L., Stack, J. P., Valent, B., and Pedley, K. F. 2019. A response to Gupta et al. (2019) regarding the MoT3 wheat blast diagnostic assay. Phytopathology 109:509-511, doi: 10.1094/PHYTO-10-18-0397-LE.
  • Type: Book Chapters Status: Published Year Published: 2019 Citation: Valent, Barbara and Jan E. Leach. (2019) Manipulating molecular interactions between hosts and pathogens for enhancing resistance and disease management. In Creating a sustainable global food supply. Editor: Robert S. Zeigler. CSIRO Publishing, Victoria, Australia with Oxford University Press, Oxford, England, U.K.
  • Type: Theses/Dissertations Status: Published Year Published: 2019 Citation: Wang, Mengying. 2019. Novel Strategies for Disease Control of Rice and Examination of the Associated Microbiome. Ph.D. Thesis, North Carolina State University, Raleigh, North Carolina (Under the direction of Dr. Ralph A. Dean).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Farman, Mark, Bradford Condon, Li Chen, Neil Moore, Jerzy Jaromczyk, Mike Pieck, Kerry Pedley, Jarred Yasuhara-Bell, Barbara Valent, James P. Stack. (2018) Genome-Enabled Molecular Diagnostics for Blast Diseases. In: Proceedings of the VIII Symposium on Current Phytopathology: Molecular Tools Applied to Phytopathology, Federal University of Vi�osa, Vi�osa, Minas Gerais, Brazil
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Yasuhara-Bell, Jarred, Kerry F. Pedley, Mark Farman, Barbara Valent and James P. Stack. (2018) Specific detection of the wheat blast pathogen (Magnaporthe oryzae Triticum) by loop-mediated isothermal amplification. Plant Disease 102:2550-2559.
  • Type: Other Status: Awaiting Publication Year Published: 2019 Citation: Peng, Zhao, Ely Oliveira Garcia, Guifang Lin, Ying Hu, Melinda Dalby, Pierre Migeon, Haibao Tang, Mark Farman, David Cook, Frank F. White, Barbara Valent and Sanzhen Liu. (2019) Effector gene reshuffing involves dispensable mini-chromosomes in the wheat blast fungus. (First publication in 2018, bioRxiv doi: https://doi.org/10.1101/359455.) PLoS Genetics, in Press.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Zhao, Haijun, Xueyan Wang, Yulin Jia, Bastian Minkenberg, Matthew Wheatley, Jiangbo Fan, Melissa H. Jia, Adam Famoso, Jeremy D. Edwards, Yeshi Wamishe, Barbara Valent, Guo-Liang Wang, Yinong Yang. (2018) The rice blast resistance gene Ptr encodes an atypical protein required for broad-spectrum disease resistance. Nature Communications 9:2039, doi:2010.1038/s41467-41018-04369-41464.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Gladieux, Pierre, Bradford Condon, Sebastien Ravel, Darren Soanes, Joao Leodato Nunes Maciel, Antonio Nhani, Ryohei Terauchi, Marc-Henri Lebrun, Didier Tharreau, Thomas Mitchell, Barbara Valent, Nicholas J. Talbot, Mark Farman, Elisabeth Fournier (2018) Gene flow between divergent cereal- and grass-specific lineages of the rice blast fungus Magnaporthe oryzae. mBio 9:e01219-17. https://doi.org/10.1128/mBio.01219-17.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Jiang, C., Zhang, X., Liu, H. Q., and Xu, J. -R. (2018) Mitogen-activated protein kinase signaling in plant pathogenic fungi. PLoS Pathogens 14(3): e1006875.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Inoue, Yoshihiro, Trinh T. P. Vy, Kentaro Yoshida, Hokuto Asano, Chikako Mitsuoka, Soichiro Asuke, Vu L. Anh, Christian Joseph R. Cumagun, Izumi Chuma, Ryohei Terauchi, Kenji Kato, Thomas Mitchell, Barbara Valent, Mark Farman, Yukio Tosa (2017) Evolution of the wheat blast fungus through functional losses in a host specificity determinant. Science 357:80-83.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Wang, X., Jia, Y., Wamishe, Y., H. Jia, M.H., Valent, B. (2017) Dynamic changes in the rice blast population in the USA through decades. Molecular Plant-Microbe Interactions 30:803-812, doi: 10.1094/MPMI-04-17-0101-R.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Hu, Ying, Qingyu Wu, Zhao Peng, Stuart A. Sprague, Wei Wang, Jungeun-Kim Park, Eduard Akhunov, K.S.V. Jagadish, Paul Nakata, Ninghui Cheng, Kendal D. Hirschi, Frank F. White, and Sunghun Park (2017) Silencing of OsGRXS17 in rice improves drought stress tolerance by modulating ROS accumulation and stomatal closure. Scientific Reports 7:15950 doi:10.1038/s41598-017-16230-7.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Zhang, X., Liu, W. D., Li, G. T., and Xu, J. -R. (2017) Expression of HopAI interferes with MAP kinase signaling in Magnaporthe oryzae. Environmental Microbiology. 10:4190-4204. doi: 10.1111/1462-2920.13884.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Li, Y., Zhang, X., Xu, Y., and Xu, J. -R. (2017) PKA activities are essential for appressorium formation and relieving the suppression of hyphal growth by MoSfl1 in Magnaporthe oryzae. PLoS Genetics. doi.org/10.1371/journal.pgen.1006954.
  • Type: Book Chapters Status: Published Year Published: 2017 Citation: Minkenberg, B., Wheatley, M., and Yang, Y. (2017) CRISPR/Cas9-enabled multiplex genome editing and its application. Progress in Molecular Biology and Translational Science 149: 111-132. Genome Editing in Plants (Eds. Donald Weeks and Bing Yang), Elsevier.
  • Type: Other Status: Published Year Published: 2017 Citation: Wang G.-L. and B. Valent. (2017) Durable resistance to rice blast. Science 355:906-907.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Tsiboe, Francis, Lawton L. Nalley, Bruce L. Dixon, Diana Danforth, Anne-C�cile Delwaide and Rodolfo M. Nayga (2017) Ghanaian consumers attitudes toward cisgenic rice: are all genetically modified rice the same? Ghana Journal of Development Studies Vol. 14, No. 1, May, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Del Ponte, Emerson M, Barbara Valent and Gary C. Bergstrom (2017). A special issue on Fusarium head blight and wheat blast. Tropical Plant Pathology 42:143-145, doi:10.1007/s40858-017-0166-0.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Fernandes, Jos� Maur�cio Cunha, M�rcio Nicolau, Willingthon Pavan, Carlos Amaral H�lbig, Maur�cio Karrei, Felipe de Vargas, Jorge Luis Boeira Bavaresco, Alexandre Tagliari Lazzaretti , Rodrigo Y. Tsukahara (2017) A weather-based model for predicting early season inoculum build-up and spike infection by the wheat blast pathogen. Tropical Plant Pathology 42:230-237, doi:10.1007/s40858-017-0164-2.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Cruz, C.D. and B. Valent (2017) Wheat Blast Disease: Danger on the Move. Tropical Plant Pathology 42:210-222, doi:10.1007/s40858-017-0159-z.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Villari C, Mahaffee WF, Mitchell TK, Pedley KF, Pieck ML and Hand FP. (2017) Early detection of airborne inoculum of Magnaporthe oryzae in turfgrass fields using a quantitative LAMP assay. Plant Disease 101:170-177.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Farman, M., Gary Peterson, Li Chen, Barbara Valent, Paul Bachi, Lloyd Murdock, Don Hershman, Kerry Pedley, J. Mauricio Fernandes and Jorge Bavaresco. (2017) The Lolium Pathotype of Magnaporthe oryzae recovered from a single blasted wheat plant in the United States. Plant Disease 101:684-692. doi: http://dx.doi.org/10.1094/PDIS-05-16-0700-RE.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Pieck, Michael L., Amy Ruck, Mark L. Farman, Gary L. Peterson; James P. Stack, Barbara Valent and Kerry F. Pedley. (2017) Genomics-Based Marker Discovery and Diagnostic Assay Development for Wheat Blast. Plant Disease 101:103-109. doi: http://dx.doi.org/10.1094/PDIS-04-16-0500-RE.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Minkenberg, B., Xie, K., and Yang, Y. 2017. Discovery of rice essential genes by characterizing CRISPR-edited mutation of closely related rice MAP kinase genes. Plant Journal 89:636-648. (doi/10.1111/tpj.13399/full).
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Nalley L, Tsiboe F, Durand-Morat A, Shew A, Thoma G. 2016. Economic and environmental impact of rice blast pathogen (Magnaporthe oryzae) alleviation in the United States. PLOS ONE 11(12): e0167295. doi: 10.1371/journal.pone.0167295.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Helliwell, E.E., Wang, Q., and Yang, Y. 2016. Ethylene biosynthesis and signaling is required for rice immune response and basal resistance against Magnaporthe oryzae infection. Mol. Plant-Microbe Interact. 29: 831-843 (dx.doi.org/10.1094/MPMI-06-16-0121-R).
  • Type: Other Status: Published Year Published: 2017 Citation: EXTENSION PUBLICATION: Wheat Disease Identification  A popular wheat disease identification booklet was updated to include wheat blast and republished in 2017. This publication was originally developed by the multi-state extension and research committees for small grain diseases, NCERA-184 and WERA-97. The updated books were distributed in 32 states that are most at risk for wheat blast. More than 18,000 copies were distributed at part of this effort. Multiple states liked the publication so much they elected to purchase additional copies to share with their growers. Publication authors: Erick De Wolf, and James P. Shroyer, Kansas State University. (https://www.bookstore.ksre.ksu.edu/pubs/MF2994.pdf).
  • Type: Theses/Dissertations Status: Other Year Published: 2019 Citation: Mills, Karasi. Anticipated 2019. Epidemiology of wheat blast incited by Triticum and Lolium pathotypes of Magnaporthe oryzae: Quantifying environmental effects and cultivar reactions. Ph.D. Thesis, The Ohio State University, Wooster, Ohio (Under direction of Pierce Paul and Laurence Madden).
  • Type: Theses/Dissertations Status: Other Year Published: 2019 Citation: Cruppe, Giovana. Anticipated 2019. Identifying new sources associated with wheat blast resistance, understanding the background effect of 2NS-based resistance, and characterizing the basic ecology of Magnaporthe oryzae Titicum. Ph.D. Thesis. Kansas State University, Manhattan, Kansas (Under the direction of Barbara Valent and Christian D. Cruz).
  • Type: Theses/Dissertations Status: Other Year Published: 2019 Citation: Kiyuna, Javier. Anticipated 2019. Detection of wheat blast within and on wheat seeds and preliminary risk evaluation tool for the detection of MoT infected seeds. M.S. Thesis, Kansas State University, Manhattan, Kansas (Under the direction of James Stack).
  • Type: Theses/Dissertations Status: Other Year Published: 2019 Citation: Navia-Urrutia, Monica. Anticipated 2019. Use of biotechnological tools in the incorporation of disease resistance in common wheat. Ph.D. Thesis. Kansas State University, Manhattan, Kansas (Under direction of Harold Trick).


Progress 01/01/16 to 12/31/16

Outputs
Target Audience:Target audiences for the Blast Integrated Project (BIP) continue to be the professional plant pathology community, rice and wheat production specialists, USDA-APHIS scientists, rice and wheat stakeholders in government and industry, and students with an interest in plant pathology and plant biosecurity. Due to the devastating occurrence of wheat blast in Bangladesh in February, our target audience has expanded to include many new international scientists and stakeholders involved in wheat production in Bangladesh and globally. Our distinguished Scientific Advisory Board (SAB), includes James Correll, University of Arkansas, Fayetteville, Arkansas; Justin Faris, USDA-ARS Cereal Crops Unit, Fargo, North Dakota; Anna McClung, Dale Bumpers National Rice Research Center, Stuttgart, Arkansas; Tim Murray, Washington State University, Pullman, WA; Boyd Padgett, LSU Ag Center, Alexandria, LA; Pawan Singh, CIMMYT, El Batan, Mexico; and Didier Tharreau, CIRAD, France. SAB member Correll visited our experimental sites in Bolivia June 16-30 to observe and to consult on ongoing field experiments. The 2016 BIP Project meeting (November 14) and SAB meeting (November 15) will be hosted by Reid Frederick (Supervisory Research Biologist, USDA ARS Foreign Disease and Weed Science Research Unit) and Co-PIs Peterson and Kedley at the Fort Detrick, MD. Our Extension Publication 'Identifying Wheat Diseases Affecting Heads and Grain' was distributed in 19 additional states, to bring the total distribution to 29 states and Canada (De Wolf and colleagues). This publication, the first such publication to include Wheat Blast, was individualized for distribution from each institution. Team members Wamishe and Jia organized the 'Rice and Wheat Blast Symposium' held preceding the 36th Rice Technical Working Group Meeting, March 1, 2016, Galveston, Texas. 57 attendees signed our list and responded positively to the workshop assessment questions. Team members Fernandes and Valent organized the 2nd International Workshop on Wheat Blast (IWWB), which was held April 6-10 at Costão do Santinho, Florianópolis, Brazil (http://scabandblastofwheat2016.org/). On July 26?27, team members Cruz and Fernandes represented the BIP project at a regional consultation workshop in Kathmandu, Nepal, which was organized by BARC/BARI/WRC and CIMMYT with support from USAID and Australian Centre for International Agricultural Research (ACIAR). Workshop participants discussed recommendations from the emergency task force to be implemented in Bangladesh and developed a collective set of mid?term and long term actions to combat and manage the disease. BIP team members gave numerous presentations, including 4 lectures and 2 posters at the 2nd International Workshop on Wheat Blast (April 6-10, Florianópolis, Brazil). Other presentation were giving at the Rice and Wheat Blast Symposium, 36th Rice Technical Working Group (March 1-4, Galveston, Texas); Rutgers University; the IS-MPMI meeting (July 17-21, Portland Oregon); the 7th International Rice Blast Conference (October 9-13, Manila, Philippines) and a specialty 2016 Durable Wheat Resistance Meeting, (November 2-3, Minneapolis, MN, https://www.ag.ndsu.edu/wheatresistance ). Changes/Problems:In August, Dr. Christian Cruz assumed the role of Objective 4 Team Leader due to the retirement of the previous Team Leader, Dr. William Bockus. No problems to report. What opportunities for training and professional development has the project provided?'Plant Biosecurity in Theory and Practice', the highly successful 5 day short course developed by CoPI Stack last year was again delivered at K-State's Biosecurity Research Institute May 23-27, 2016. Forty participants from 16 countries included undergraduates, graduate students, postdoctoral researchers, faculty and other research professionals. An international panel of instructors, including project members Stack, Bockus, Cruz, and Valent, provided a diverse array of perspectives on plant biosecurity through a combination of lectures, hands-on biocontainment laboratory training, case studies, and open discussions. Topics included plant biosecurity concepts, case studies, diagnostic technologies and applications, strategies to achieve biosecurity, and forensic analysis in a plant biosecurity context. Participants engaged in biosecurity problem solving while developing biosecurity plans for three defined scenarios. A limited number of participants (priority to students) gained an in-depth experience in handling high consequence pathogens in a biocontainment laboratory. The entire course was recorded and will be available on DVD. A unique hands-on course entitled 'Wheat Blast Diagnosis: From symptoms to molecular markers,' was held April 4, 2016 at the University of Passo Fundo, Brazil, immediately preceding the joint 5th International Symposium on Fusarium Head Blight / 2nd International Workshop on Wheat Blast (Instructors: Fernandes, Stack, Cruz). The course included: Introduction to Wheat and Wheat Blast; Chemical Management; Host Diagnosis in the field; Wheat Blast Epidemiology and Forecasted Models (lecture and lab); Disease Diagnosis and Pathogen Identification (microscopy, PCR - demonstration). STUDENTS AND POSTDOCS WORKING ON THIS PROJECT: High School Student -- Holly Herald (summer intern, Arkansas Rice Research and Extension Center, AR, with Jia). UNDERGRADUATES -- Lindsey Ashmore (KSU, Wheat Blast Web Site, Facebook and Twitter; Wheat blast videos; Helping with Biosecurity Short Course); Payton DeLong (KSU, BSL-3 studies of wheat blast); Mia Hodges (UARK, Rice blast population biology). GRADUATE STUDENTS -- Jorge Bavaresco (University of Passo Fundo RS BR, MS student in the Applied Computing Graduate Program at University of Passo Fundo); Zhiyi Bian (Purdue, HIGS in rice); Li 'Lily' Chen (UK, population analyses of US Lolium pathotype); Giovana Cruppe (KSU, Wheat blast resistance, wheat blast field biology); Taylor Fischer (KSU, graduated with M.S. degree, blast forecast modeling); Javier Kiyuna (KSU, diagnostic protocols for detecting MoT fungus in wheat seed); Yang Li (Purdue, HIGS in rice); Karasi Mills (OSU; wheat blast biology and field studies in Bolivia); Monica Navia (KSU; Rice resistance gene function in wheat); Kerri Neugebauer (KSU; Rice resistance gene function in wheat); Aaron M. Shew (Univ. of AR, cisgenic survey in India); Stuart Sprague (KSU, Molecular analysis of transgenic rice); Francis Tisboe (UARK, Cisgenic survey in Ghana); Mengying Wang (NCSU, HIGS in rice); Xue Zhang (Purdue, HIGS in rice). POST-DOCTORAL ASSOCIATES -- Christian Cruz (KSU, wheat blast biology, resistance, Studies in BSL-3 containment and the field in SA; Recently promoted to Research Assistant Professor); Jorge David Salgado Moncada (OSU, wheat blast field studies in Bolivia); Shefali Dobhal (KSU, population biology of Bolivian wheat blast fungus); Ely Oliveira Garcia (Wheat blast genomics and cell biology); Jianbing Ma (USDA/Dale Bumpers National Rice Research Institute, Arkansas, rice blast avirulence gene analysis); Mike Pieck (USDA-ARS-FSWSRU, molecular diagnostics sequence discovery); Prasanna Kankanala (KSU, wheat resistance, molecular markers); Denis Shah (Cornell,with DeWolf, blast forecasting models); Baohua Wang (UK, population analyses of US Lolium pathotype); Xueyan Wang (University of Arkansas Rice Research and Extension Center, Arkansas, rice blast population biology). LAB TECHNICIANS -- Jung-Eun Kim Park (KSU, Transformation Specialist: Rice Genetic Transformation); Trevor Steiner (KSU, Lab Technician: Rice plant management and seed harvest in Greenhouse); Maria Bellizzi (OSU, Rice Transformation); Melinda Dalby (KSU, rice blast research); Kelli Thomas (USDA-ARS FDWSRU Maryland, wheat blast research). PERSONNEL/STUDENTS TRAINED IN BOLIVIA -- Javier Kiyuna (Fundación CETABOL, Bolivia, Wheat blast field tests); Juan Vasquez (Fundación CETABOL, Bolivia, Wheat blast laboratory tests); Santiago Rivera (Universidad Gabriel René Moreno, Bolivia, Wheat blast field work); Jorge Andrés Cuellar, Ing. (Tropical Agricultural Research Center - CIAT Bolivia, wheat blast field testing); Lidia Calderon (ANAPO, Bolivia, wheat blast field testing); Andres Cruz (KSU, wheat field studies). COLLABORATORS -- On rice blast, we collaborated with rice disease specialists Rick Cartwright (AR), Don Groth (LA), Shane Zhou (TX), and Chris Greer (CA) to compile historical weather data for AR, CA, LA, and TX. We have also collaborated with Bo Zhou (IRRI, the Philippines). Rice Breeder Xueyan Sha (UA RREC) and Klaus Scheuermann from EPAGRI, Brazil have collaborated on identification of US rice germplasm with Pi-9 for immediate use by breeders. For wheat blast, Brian Mills and his team at Penn State (including Paul Knight and Doug Miller) have collaborated on gathering needed weather data, implementing models for wheat blast, and summarizing the results in a map-based format. We are collaborating on molecular markers and GBS with Eduard Akhunov, Jesse Poland and Allan Fritz at KSU. We have begun collaborating with Sanzhen Liu (KSU) to produce a high quality assembled genome sequence for a race 2 MoT strain, B71. Field work in Bolivia is being performed together with Diego Baldelomar (ANAPO, Bolivia) and Marcia Gabriela Rivadeneira Caballero (CIAT, Bolivia). We are also working with Dr. Jorge Dubcovsky (Triticum CAP Project Director, UC Davis); Dr. Peter Solomon (The Australian National University, Australia); Dr. Evans Lagudah, CSIRO, Australia; wheat blast expert Prof. Dr. Alfredo Urashima at Universidade Federal de São Carlos, Brazil; Dr. Pawan Singh at the International Maize and Wheat Improvement Center, CIMMYT, Dr. Etienne Duveiller, CIMMYT, India, and Dr. Paritosh Malaker, BARI, Bangladesh. Dr. Nalley has collaborated with Dr. Wim Verbeke, Professor at the University of Gent, and a leading European Ag Economist and E.U. advisor on GMO issues. How have the results been disseminated to communities of interest?We communicate with colleagues and directly with the general public through a webpage (http://www.k-state.edu/wheatblast/ ), Facebook (https://www.facebook.com/ksuwheatblast/ ) and Twitter (https://twitter.com/KSUWheatBlast ). Project members regularly participate in radio interviews, popular news articles and postings on You-Tube. We produced an interesting and informative video entitled "Wheat Blast, An Emerging Threat to Wheat" in order to educate the general public on issues associated with wheat blast and plant biosecurity (Stack, Cruz). The video is available on our web page and on youtube in English (https://www.youtube.com/watch?v=z_pCMbaY2io), in Spanish https://www.youtube.com/watch?v=FshZQZ-MgaA, and in Portuguese https://www.youtube.com/watch?v=VV-9c3FIYxs). International grain/soybean buyers, government officials and others responsible for the purchase, shipment and handling of U.S. grains and soybeans had an opportunity to understand the science, risks and benefits of genetically engineered crops at the annual grain purchasing workshop hosted by the KSU International Grains Program (Trick). What do you plan to do during the next reporting period to accomplish the goals?A major event during this reporting period was the occurrence of wheat blast in Bangladesh in February. Project members worked together with BARI and CIMMYT colleagues to show that the fungus infecting wheat in Bangladesh was derived from the MoT population in South America (Malaker et al, Plant Disease, 2016), and the strains resemble the highly aggressive race 2 strains currently coming from the field in SA. The large scale incidence of wheat blast on this first report of the disease outside South America has underscored a concern about the potential spread of wheat blast to other wheat producing areas in Bangladesh, South Asia and beyond. CIMMYT will now be making a major commitment to wheat blast research and we expect to expand our collaboration with them and with others. For Obj 4, development of mapping populations and discovery of resistance genes/QTL is a critical goal. We are currently working with CIMMYT, with Biotrigo Genetica Ltda (Passo Fundo, Brazil), the Bolivian Wheat Growers Association, and with others in SA to develop a comprehensive list of currently known blast resistant wheat lines. We have been testing these resistant lines for the presence of the 2NS translocation, and so far, most of them do contain the 2NS translocation. We have also shown that the efficacy of the 2NS translocation resistance varies depending on the wheat genetic background. Therefore, our search for additional resistance will include the strategy of identifying QTLs that complement the 2NS resistance.

Impacts
What was accomplished under these goals? (OBJECTIVE 1) (1.0) CRISPR-Cas9 constructs for genome editing of rice resistance genes Pita and Ptr were constructed and transformed into rice. Preliminary tests have been performed (Yang). (1.1) Pi9 cisgenic transformants of elite US varieties LaGrue and Juniper are being analyzed (Wang, Park). (1.2) Analyses of US rice pathogens were expanded to include infection assays with the IRRI monogenic blast differentials and with 13 polymorphic SSR markers. The population structure of M. oryzae rice pathogens in the US is complex (Jia, Wamishe). Two new races IB17 and IB1 were verified and 20 blast isolates were selected for breeding and genetics. (1.3) We developed a Pi-9 perfect DNA marker from 5' leader region including a portion of exon of the gene and used this marker to screen rice germplasm that is relevant for US rice breeders. A line with Pi-9 has been provided to U.S. rice breeders (Scheuermann and Jia, 2016, Phytopathology). (Objective 2) Stable T0 rice transformants have been produced containing HIGS constructs for 29 M. oryzae genes. Preliminary infection results with these transformants showed reduced pathogenicity for 3 of these genes. Seed is being produced and analyses continue. (Dean, Xu) (Objective 3) Significant findings in selected tasks are: (3.0) We worked with scientists at CIMMYT (Mexico, India) and BARI (Bangladesh) to identify fungal strains causing wheat blast disease in Bangladesh as members of the MoT population from South America, indicating that the disease has now moved beyond SA (Malaker et al, 2016, Plant Disease). The result is based on genome sequencing of 5 fungal strains from diverse locations in Bangladesh. Genomes of these strains appear identical, and they are nearly identical to the most highly aggressive MoT strains from SA. (3.2) We have sequenced and assembled genomes of >25 M. oryzae/M.grisea strains from rice, wheat, perennial and annual ryegrass and other hosts, and we have developed software tools for BLAST searches, SNP analyses and repeat masking in single and batch formats. Whole genome phylogenetic analyses confirm that (3.2.a) MoT and MoL isolates are closely related to each other and some SA isolates from wheat belong to the MoL subgroup; (3.2.b) the Kentucky isolate from wheat is a native MoL strain (Farman et al, accepted); (3.2.c) native MoL isolates belong to two distinct subpopulations; (3.2.d) MoT isolates from SA show a surprising degree of SNP variability. We are collaborating with European researchers who are also sequencing and analyzing genomes from various M. oryzae host-specific isolates. By pooling genome data, we are developing a comprehensive phylogenetic analysis of host-specific forms, including understanding which host-specific populations are most closely related to the MoT population, and potential for populations to shift to infecting new hosts. (Farman, Valent, Maciel, Mitchell) (3.3) MoT-specific diagnostic assay development is underway using the MoT3 marker (Pieck et al, 2016, Plant Disease, accepted and online). Experiments are underway to optimize a protocol for detecting seed-borne MoT in seed/grain shipments (Stack, Pedley). (3.4) Based on preliminary analysis of field and lab data, there is evidence that suggests an association between MoT inoculum produced on basal leaves and wheat head blast development (Cruz et al, 2015, Plant Pathology; Cruz and Salgado et al., in preparation). Data are being analyzed from fungicide field trials in Bolivia and Brazil (Cruz et al, in preparation). So far, it appears Mancozeb and some fungicide combinations provided significant reduction of head blast severity. New data on spatial and temporal development of leaf and spike blast within the wheat canopy have been generated in Bolivia. The first set of formal statistical analyses of the spatio-temporal data collected from fields in Bolivia suggest the importance of in-field hotspots for both the temporal and spatial spread of wheat blast (Pierce, Madden, Mills, Cruz). (Objective 4) (4.0) Based on discussions at the Bangladesh wheat blast workshop in Nepal, we published a standardized inoculation protocol to test wheat cultivars for reaction to head blast caused by MoT (Cruz et al, Plant Health Progress, 2016). Joao Maciel (Embrapa Trigo) spent a year working at KSU (ending Aug, 2016) and helped determine that recent MoT isolates from Bolivia and Brazil belong to a new highly aggressive race 2. (4.1) We showed that the 2NS/2AS translocation from Aegilops ventricosa confers wheat blast resistance (Cruz et al, Crop Science, 2016). The CIMMYT variety Milan tests positive for the 2NS marker. Wide-spread use of Milan as a source of resistance in breeding programs in South America suggested that the 2NS resistance locus accounts for much of the currently deployed resistance, and we have confirmed this by analyses of resistant germplasm using 2NS-molecular markers. 2NS-mediated resistance is stronger in some genetic backgrounds, raising the possibility that more resistant varieties contain wheat blast QTLs. (4.3) Additional spring, winter, landraces, and wild-type wheat relatives are being tested for resistance in the US and Bolivia. Cultivars showing good levels of resistance are screened for presence of the 2NS segment; selected candidates have been chosen to develop population(s) for QTL mapping. One Ae.tauschii core collection entry and one non-2NS SRPN winter wheat have shown significant resistance to recent more aggressive MoT strains. Through association analysis, a large panel of phenotypic and genotypic data will identify novel regions associated with wheat blast resistance. Avocet and Thatcher isolines containing the adult rust and mildew resistance genes Lr34, Lr46 or Lr67 (obtained from Evans Lagudah) are highly susceptible to head blast in growth chamber inoculations. Emmer wheats as well as collections of T. turgidum ssp. and X. Triticosecale are also being tested (Peterson, Bockus, Cruz, Maciel). (4.8) Transgenic wheat lines expressing the rice Piz-t resistance gene were tested for rice gene function in controlling wheat leaf and/or head blast resistance using MoT strain T25, which contains a functional avirulence allele of AvrPiz-t (Trick, Farman). However, Piz-t confers little, if any, head or leaf resistance to MoT stain. (Objective 5) (5.1) The wheat blast forecasting model (http://dev.sisalert.com.br/webinar/wheat_blast/ ) developed by CoPI Fernandes (Embrapa Wheat, Brazil) has been adapted for use in the US, and is incorporated into the BIP risk assessment project. A climate suitability study for the US (Cruz et al Plant Disease, 2016) predicted the climate was adequate for maintaining MoT populations in 40% of the US winter wheat production areas. Disease outbreak threshold levels were reached in 25% of the country. Probability of years suitable for outbreaks was greater than 70% in Louisiana, Mississippi, and Florida. Freezing and thawing of the wheat blast fungus on moist residue significantly affected its ability to produce viable conidia (Fischer M.S. thesis, KSU). Additionally, historical data was used to identify variables associated with rice blast epidemics, which are favored by cooler temperatures in April and higher levels of precipitation in June. Preliminary models based on these variables correctly classified epidemic years with >75% accuracy. These results will be used as part of a risk assessment for wheat blast and may serve as part of a rice blast forecast system in the Southern US (DeWolf, Paul, Madden, Jia, Wamishe). (Objective 6) An analysis of the economic impact of rice blast alleviation in the mid?south of the United States has been completed and submitted for publication. (Objective 7) An extension publication and workshops (DeWolf, Bockus, Pierce), including the 2nd International Workshop on Wheat Blast (IWWB) in Brazil are described under Outputs. (OBJECTIVE 8) Educational activities are described under Opportunities for Training.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Cruz, C. D., G. L. Peterson, W. W. Bockus, P. Kankanala, J. Dubcovsky, K. W. Jordan, E. Akhunov, F.G. Chumley, D. F. Baldelomar and B. Valent. 2016. The 2NS translocation from Aegilops ventricosa confers resistance to the Triticum Pathotype of Magnaporthe oryzae, Crop Science, 56:990-1000, doi: 10.2135/cropsci2015.07.0410.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Malaker, P.K., N.C.D. Barma, T.P. Tiwari, W.J. Collis, E. Duveiller, P.K. Singh, A.K. Joshi, R.P. Singh, H.-J. Braun, G.L. Peterson, K.F. Pedley, M.L. Farman and B. Valent. 2016. First report of wheat blast caused by Magnaporthe oryzae pathotype triticum in Bangladesh. Plant Disease Plant Disease 100:2330, doi: 10.1094/PDIS-05-16-0666-PDN.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Cruz, C. D., Magarey, R. D., Christie, D. N., Fowler, G. A., Fernandes, J. M., Bockus, W. W., Valent, B., and Stack, J. P. 2016. Climate suitability for Magnaporthe oryzae Triticum pathotype in the United States. Plant Disease 100:1979-1987, doi: 10.1094/PDIS-09-15-1006-RE.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Scheuermann, Klaus and Yulin Jia. 2016. Identification of a Pi9 containing rice germplasm with a newly developed robust marker. Phytopathology, 106:871-876 http://dx.doi.org.er.lib.k-state.edu/10.1094/PHYTO-02-16-0091-R.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Cruz, C. D., W. W. Bockus, J. P. Stack, B. Valent, J. N. Maciel and G.L. Peterson. 2016. A Standardized Inoculation Protocol to Test Wheat Cultivars for Reaction to Head Blast Caused by Magnaporthe oryzae (Triticum pathotype) Plant Health Progress 17:186-187, doi: 10.1094/PHP-BR-16-0041.
  • Type: Theses/Dissertations Status: Published Year Published: 2016 Citation: Fischer, Taylor Dawn. 2016. Influence of freezing on the survival of Magnaporthe oryzae and weather conditions that favor blast epidemics in rice. M.S. thesis. Kansas State University, Manhattan, Kansas.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Liu, Wende and Wang, Guo-Liang (2016) Plant innate immunity in rice: a defense against pathogen infection. National Science Review 3:295-308, doi:10.1093/nsr/nww015.
  • Type: Other Status: Published Year Published: 2016 Citation: EXTENSION PUBLICATION IDENTIFYING WHEAT DISEASES AFFECTING HEADS AND GRAIN was distributed in 19 ADDITIONAL STATES, to bring the total distribution to 29 states and Canada. This is the first US Extension publication to include Wheat Blast.
  • Type: Websites Status: Published Year Published: 2016 Citation: Wheat Blast: Web Site (http://www.k-state.edu/wheatblast/ ) with links to videos in English, Spanish and Portuguese; Facebook (https://www.facebook.com/ksuwheatblast/ ) and Twitter (https://twitter.com/KSUWheatBlast )


Progress 01/01/15 to 12/31/15

Outputs
Target Audience:Target audiences for the Blast Integrated Project (BIP) include the professional plant pathology community, rice and wheat production specialists, USDA-APHIS scientists, rice and wheat stakeholders in government and industry, and students with an interest in plant pathology and plant biosecurity. In addition to our interdisciplinary project team, we meet annually with a distinguished Scientific Advisory Board (SAB), with members James Correll, University of Arkansas, Fayetteville, Arkansas; Justin Faris, USDA-ARS Cereal Crops Unit, Fargo, North Dakota; Anna McClung, Dale Bumpers National Rice Research Center, Stuttgart, Arkansas; Tim Murray, Washington State University, Pullman, WA; Boyd Padgett, LSU Ag Center, Alexandria, LA; Pawan Singh, CIMMYT, El Batan, Mexico; and Didier Tharreau, CIRAD, France. The next SAB meeting is scheduled for December 5, together with a second US Wheat Blast Workshop (De Wolf and Bockus) on December 6, preceding the 2015 Fusarium Head Blight Forum in St. Louis, MO. An Extension publication entitled Identifying Wheat Diseases Affecting Heads and Grain was completed and distributed in 10 states and Canada (DeWolf and colleagues). This is the first US extension publication to include wheat blast. USDA-APHIS scientists supporting their agency's regulatory responsibilities for wheat blast disease have been attending the BIP Project Team meetings and the BIP Scientific Advisory Board meetings in order to learn about recent research findings. The 2015 meetings are scheduled for December 4 and 5 in St. Louis. Additionally, Valent and Cruz serve as reviewers and communicate project learnings relevant for drafting the USDA-APHIS New Pest Response Guideline (NPRG) for wheat blast. Following on from the success of our first wheat blast stakeholders workshop in St. Louis on Dec 7, 2014 (which attracted 64 attendees, including scientists from universities, USDA-ARS, USDA-APHIS and industry) DeWolf and Bockus organized a second workshop for Dec 6, preceding the 2015 Fusarium Head Blight Forum in St. Louis. Team members gave numerous presentations, including lectures at the American Phytopathological Society Annual Meeting in Pasadena, CA; the 2015 Rice Breeders Conference in Stoneville, Mississippi; Embrapa Wheat in Passo Fundo, Brazil; the Universidad Gabriel René Moreno - Instituto de Investigaciones Agrícolas El Vallecito - Santa Cruz de la Sierra, Bolivia; The Ohio State University - Ohio Agricultural Research and Development Center; and at CIMMYT (Mexico). Co-PI Fernandes presented his work on a wheat blast risk analysis tool in the Workshop for Advancing Pest and Disease Modeling held in Gainesville, Florida February 23-25, 2015. Changes/Problems:No problems to report. What opportunities for training and professional development has the project provided?FOR A UNIQUE EXPERIENCE IN PLANT BIOSECURITY EDUCATION, Co-PI Stack designed and delivered a 5-day short course entitled Plant Biosecurity in Theory and Practice at Kansas State University. The 36 course participants (from 14 countries) included undergraduates, graduate students, postdoctoral researchers, faculty and other research professionals. This course was convened May 18-22 at the KSU Biosecurity Research Institute (BRI) and offered a broad overview of the theory and practice of plant biosecurity with a focus on wheat blast as the specific example of an emerging disease. An international panel of instructors provided a diverse array of perspectives on plant biosecurity through a combination of lectures, hands-on biocontainment laboratory training, case studies, and open discussions. Topics included plant biosecurity concepts, case studies, diagnostic technologies and applications, strategies to achieve biosecurity, and forensic analysis in a plant biosecurity context. Participants engaged in biosecurity problem solving while developing biosecurity plans for three defined scenarios. A limited number of participants (priority to students) gained an in-depth experience in handling high consequence pathogens in a biocontainment laboratory. The entire course was recorded and will be available on DVD. IN A SEPARATE EDUCATIONAL EXPERIENCE, students, post-docs and faculty learned about genomics and biotech-based solutions to disease control at an International Genomics Workshop, a 2-day mini-symposium with >50 participants at Northwest Agricultural University in Yangling, China (CoPIs Dean and Xu). STUDENTS AND POSTDOCS WORKING ON THIS PROJECT: UNDERGRADUATES --Lindsey Ashmore (KSU, Wheat Blast Web Site, Facebook and Twitter; Wheat blast videos; Helping with Biosecurity Short Course); Payton DeLong (KSU, BSL-3 studies of wheat blast); Mia Hodges (UARK, Rice blast population biology); Javier Kiyuna (Fundación CETABOL, Bolivia, Wheat blast field tests); Juan Vasquez (Fundación CETABOL, Bolivia, Wheat blast laboratory tests); Santiago Rivera (Universidad Gabriel René Moreno, Bolivia, Wheat blast field work). GRADUATE STUDENTS -- Li Chen (UK, population analyses of US Lolium pathotype); Giovana Cruppe (KSU, Wheat blast resistance); Taylor Fischer (KSU, blast forecast modeling); Yang Li (Purdue, HIGS in rice); Karasi Mills (OSU; wheat blast biology and field studies in Bolivia); Monica Navia (KSU; Rice resistance gene function in wheat); Kerri Neugebauer (KSU; Rice resistance gene function in wheat); Aaron M. Shew (UARK, cisgenic survey in India); Francis Tisboe (UARK, Cisgenic survey in Ghana); Mengying Wang (NCSU, HIGS in rice). POST-DOCS -- Christian Cruz (KSU, wheat blast biology, resistance, Studies in BSL-3 containment and the field in SA; Recently promoted to Research Assistant Professor); Jorge Salgado (OSU, wheat blast field studies in Bolivia); Shefali Dobhal (KSU, population biology of Bolivian wheat blast fungus); Ely Oliveira Garcia (Wheat blast genomics and cell biology); Mike Pieck (USDA-ARS-FSWSRU, molecular diagnostics sequence discovery); Denis Shah (Cornell,with DeWolf, blast forecasting models); Baohua Wang (UK, population analyses of US Lolium pathotype). How have the results been disseminated to communities of interest?Project members have participated in radio interviews, postings on You-Tube (for example https://www.youtube.com/watch?v=z_pCMbaY2io in English and https://www.youtube.com/watch?v=FshZQZ-MgaA in Spanish) and popular news articles. International grain/soybean buyers, government officials and others responsible for the purchase, shipment and handling of U.S. grains and soybeans had an opportunity to understand the science, risks and benefits of genetically engineered crops at the annual grain purchasing workshop hosted by the KSU International Grains Program (7 attendees from private industry; June 6; CoPI Trick). What do you plan to do during the next reporting period to accomplish the goals?OBJECTIVE 1, we have begun collaborating with Dr. Yinong Yang of Pennsylvania State University on CRISPR-CAS genome editing for developing blast resistant rice. OBJECTIVE 3, we have begun collaborating with European researchers who are also sequencing and analyzing genomes from various M. oryzae host-specific isolates. By pooling genome data, we will be able to develop a comprehensive phylogenetic analysis of host-specific forms of M. oryzae, including understanding which host-specific populations are most closely related to the MoT population, and potential for populations to shift to infecting new hosts. In collaboration with the Cereal Disease Epidemiology Laboratory at the Ohio State University, statistical analysis is currently being performed to identify and characterize important source(s) of MoT inoculum in the development of wheat blast epidemics. OBJECTIVE 4, development of mapping populations and discovery of resistance genes/QTL is a critical goal. We had already begun developing possible mapping populations using susceptible and resistant winter wheat cultivars. However, we have now demonstrated that the 2NS segment confers wheat head blast resistance and that all of the resistant parents we used in our populations contain the 2NS segment. Therefore, they apparently all have the same source of resistance. Additionally, we have shown that the CIMMYT wheat variety Milan also carries the 2NS segment. Milan is currently the resistant parent for many CIMMYT populations developed for blast resistance, and it is being widely used by breeders in South America (SA). Therefore, it appears much of the resistant germplasm currently being developed and deployed in SA contains the same 2NS-resistance locus. Through biocontainment inoculations, we originally identified 11 other winter wheat cultivars that do not contain 2NS but showed high levels of resistance to head blast (<10%). However, at least 10 of these are highly susceptible when inoculating with other wheat blast strains under controlled environment conditions or in field tests in SA. We are currently lacking additional R loci. Based on the known ability of the rice blast fungus to quickly overcome single deployed R genes, it is critical to identify new sources of resistance to wheat blast disease. We have begun working with CIMMYT, with Biotrigo Genetica Ltda (Passo Fundo, Brazil), the Bolivian Wheat Growers Association, and with others in SA to develop a comprehensive list of currently known blast resistant wheat lines. We will test all of these resistant lines for the presence of the 2NS translocation. We will then focus on resistant lines that lack the 2NS chromosome segment. Additionally, we are testing two "Core" collections of Ae. tauschii (21 and 41 accessions from the KSU Wheat Genetics Resources Center) for reaction to head blast.

Impacts
What was accomplished under these goals? (OBJECTIVE 1) (1.1) Cisgenic transformation of the elite US rice varieties LaGrue and Juniper with the Pi9 gene is underway (Wang, Park). (1.2) Analyses of avirulence (AVR) gene compositions in US blast populations continue. Using our established pipeline, sequence variation in 5 cloned AVR genes was assessed in 480 recently collected field isolates. Seventy-four percent, 65%, 82%, 20% and 76% of these isolates contain AVR-Pi9, AVR-Pita1, AVR-Pizt, AVR-Pik and ACE1, respectively. Therefore, the AVR-Pi9 gene is widely present in the US population and Pi9 will be a valuable gene for deploying together with AVR-Pita1, AVR-Piz-t and/or ACE1. (Jia, Wamishe) (1.3) Screening of rice germplasm with our Pi9 molecular marker is underway. (OBJECTIVE 2) Progress is being made on development of HIGS strategies for rice blast control, and more will be reported next time. (Dean, Xu) (OBJECTIVE 3) The most significant findings are: (3.1) Native Lolium (MoL) isolates pose a significant risk to US wheat production. A subset of US MoL isolates are already 75-80% as aggressive on wheat as MoT strains from SA. This finding has been further confirmed by extensive assays on Soft Red Winter Wheat varieties (Paul, Peterson). (3.2) Our whole genome phylogenetic analysis of M. oryzae/M. grisea strains from rice, wheat, perennial and annual ryegrass and other hosts confirms that SA MoT and US MoL isolates are closely related to each other and relatively distant from other host specific populations; the Kentucky isolate from wheat is a native MoL strain (Farman et al, in preparation) ; some SA isolates from wheat belong to the MoL subgroup; and MoT isolates from SA show a surprising degree of SNP variability relative to isolates from other hosts. (Farman, Valent, Maciel, Mitchell) (3.3) We selected the MoT3 marker (Pedley) for development of a MoT-specific diagnostic assay; protocol development is underway. We will deploy the diagnostic protocol together with USDA-APHIS and NPDN scientists (Stack). (3.4) New insights on wheat blast field biology gained through project studies in Bolivia have been published (Cruz et al 2015 Plant Pathology). Specifically, we showed that MoT can be transmitted from spike to seed and from seed to seedlings; that MoT grows saprophytically on basal senescent wheat leaves and can be seen sporulating at the time head blast occurs, suggesting this saprophytic growth is a potential inoculum source; that certain cultivar and isolate combinations showed more disease on old wheat leaves than on young expanding leaves, which is the opposite situation from rice blast; and that foliar fungicide applications reduced the amount of conidia on basal leaves by 62-77%. (3.5) Seed treatments can provide significant wheat blast control, with Benomyl showing the best efficacy (Bockus et al, 2015, PDMR). (3.6) Fungicides applied at the head phase reduced MoT seed-borne inoculum and blast intensity and increased yield. Through a new collaboration with Fundacion CETABOL in Bolivia, we performed fungicide field trials in six locations. Several combinations of fungicides significantly reduced head blast severity on a highly susceptible cultivar, even at high disease pressure. Data analyzed from 18 locations in Brazil showed that Mancozeb provided a considerable reduction of head blast severity across environments. (OBJECTIVE 4) (4.1) Using BSL-3 containment inoculations and field tests, we have shown that the 2NS/2AS translocation from Aegilops ventricosa confers wheat blast resistance (Cruz et al, accepted for publication in Crop Science). This wild wheat chromosome segment has been deployed by breeders because it carries the Lr37, Yr17 and Sr38 rust resistance genes as well as a nematode resistance gene. We used a PCR marker for the 2NS/2AS translocation segment to test for this fragment in 418 of our phenotyped varieties. We confirmed the presence of the 2NS fragment in 14 out of 22 of the highly resistant wheat varieties identified at KSU. Of the 175 spring wheat entries tested at Fort Detrick, 6 showed less than 10% infection and 5 of these contained the 2NS fragment. Field tests of paired 2NS-isogenic lines (provided by Dubcovsky, UC Davis, and Pumphrey, WSU) showed an average of 85% less head blast than their corresponding non-2NS parents. However, the 2NS fragment confers less resistance to some fungal populations under field conditions, and additional sources of resistance must to be identified (Cruz et al, accepted for publication in Crop Science). (4.2) The CIMMYT variety Milan tests positive for the 2NS marker. This variety has been widely used as a source of resistance in breeding programs in South America, suggesting that the 2NS resistance locus accounts for much of the currently deployed resistance. (4.3) Inoculation of 2 Ae. tauschii core collections is underway in our BSL-3 facilities. Collections of T. turgidum ssp. and X. Triticosecale have been planted for testing. Forty entries with resistance to Ug99 were tested and related lines are being obtained from CIMMYT in Hungary and Turkey. (Bockus, Peterson) (4.4) Although soft red winter wheats showed a continuum of reaction to head blast incited by MoL and MoT strains (similar to HRWW and Spring wheats), the range of mean disease severity was much narrower for MoT (70 to 100%) when compared to MoL (2-78%) (Paul, Peterson). (4.8) Characterization of transgenic wheat lines expressing the rice Piz-t blast resistance gene continues (Trick). We have now shown that MoT strains typically have 2 AvrPiz-t alleles, one functional and the other non-functional in conferring avirulence on Piz-t rice. This confirms that the rice Piz-t gene, if indeed it functions in wheat, should control that portion of the pathogen population with the functional AVR gene (Farman). (OBJECTIVE 5) (5.1) Working with rice disease specialists Rick Cartwright (AR), Don Groth (LA), Shane Zhou (TX), and Chris Greer (CA), we have compiled historical weather data for AR, CA, LA, and TX and checked for missing observations and errors. Currently for rice blast, multiple candidate models have been developed based on variables from hourly records of temp, RH and rainfall, and these models are showing more than 70% accuracy. Refinement and validation are underway (DeWolf, Paul, Madden, Jia, Wamishe). (5.2) For wheat blast, Brian Mills and his team at Penn State (including Paul Knight and Doug Miller) have collaborated on gathering needed weather data, implementing models for wheat blast, and summarizing the results in a map-based format. J.M. Fernandes (Embrapa Wheat) visited KSU from December 2014 through February 2015 and shared his expertise and current wheat blast model from Brazil (http://dev.sisalert.com.br/webinar/wheat_blast/) with other BIP team members. (OBJECTIVE 6) The European Cisgenic Survey has been completed by MS student Anne-Céclie Delwaide and published (Delwaide, 2014, Delwaide et al, 2015). Graduate student Aaron Shew (UArk) has completed and published a similar survey in India, the second largest rice consumer (Shew et al, 2015). Results from a survey in Ghana, a major importer of US rice, are being prepared for publication. A major finding is that consumers in food-insecure countries such as India and Ghana are much more open to acceptance of transgenic solutions to crop production problems than are consumers in Europe (Nalley). (OBJECTIVE 7) As described in Target Audiences, we distributed the first extension publication that compares wheat blast symptoms to those of other head diseases of wheat, and we have organized the second US Wheat Blast Workshop for Dec 6, in St. Louis (DeWolf, Bockus). We have organized the 2nd International Workshop on Wheat Blast (IWWB) to be held April 6-10, 2016, at Costão do Santinho, Florianópolis, Brazil (http://scabandblastofwheat2016.org/). (Fernandes, Valent) (OBJECTIVE 8) Educational courses delivered by BIP team members are described under Opportunities for Training.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Delwaide, A.-C., Nalley, L.L., Dixon, B.L., Danforth, D.M., Nayga, R.M., Jr., Van Loo, E.J., and Verbeke, W. (2015). Revisiting GMOs: are there differences in European consumers acceptance and valuation for cisgenically vs transgenically bred rice? PLoS ONE 10, e0126060
  • Type: Other Status: Published Year Published: 2015 Citation: Bockus, W. W., Cruz, C. C., Stack, J. P., and Valent, B. 2015. Effect of seed-treatment fungicides on sporulation of Magnaporthe oryzae from wheat seed, 2014. (online) Plant Disease Management Reports 9:ST0004. DOI:10.1094/PDMR09. The American Phytopathological Society, St. Paul, MN
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Cruz, C. D., J. Kiyuna, W.W. Bockus, T.C. Todd, J.P. Stack and B. Valent, 2015. Magnaporthe oryzae conidia on basal wheat leaves as a potential source of wheat blast inoculum. Plant Pathology, doi: 10.1111/ppa.12414
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Shew, A.M., L.L. Nalley, D.M. Danforth, B.L. Dixon, R.M. Nayga, Jr., A.-C. Delwaide and B. Valent, 2015. Are all GMOs the same? Consumer acceptance of cisgenic rice in India. Plant Biotechnology Journal, doi: 10.1111/pbi.12442
  • Type: Other Status: Published Year Published: 2015 Citation: Extension Publication, Identifying Wheat Diseases Affecting Heads and Grain, completed and distributed in 10 states and Canada


Progress 01/01/14 to 12/31/14

Outputs
Target Audience: Our target audience includes the professional plant pathology community and rice and wheat production specialists. We organized and hosted a workshop entitled ‘Special Symposium: Blast control - a moving target’ for the rice stakeholder audience. The workshop was presented on February 18, 2014, directly before the 35th Rice Technical Working Group Meeting in New Orleans, which was convenient for many rice stakeholders. On December 7, 2014, we held a Wheat Blast Workshop for wheat stakeholders. The workshop was held directly before the Annual Fusarium Head Blight Meeting in St. Louis, MO, which is already attended by many interested individuals. Our audience also includes professionals involved in regulatory issues. USDA-APHIS scientists with a need to understand wheat blast disease attended the Blast Integrated Project Meeting in Minneapolis, MN, on August 9, and the Wheat Blast Workshop in St. Louis on December 7. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Objective 1. Objective Team Leader (OTL) Yulin Jia is advising postdoctoral fellow Xueyan Wang and Biological Science Lab Technicians Tracy Bianco, Michael Lin and Tiffany Sookaerm, all working on the population biology of the rice blast fungus. Co-PI Yeshi Wameshe, Univ of Arkansas (UArk) extension specialist has coordinated field observations and collection of disease specimens, with support from field technician Tebebu Gebremariam. Wameshe and Jia are working with rice extension specialists Rick Cartwright (AR), Don Groth (LA), Shane Zhou (TX), and Chris Greer (CA), all of whom have supported field collection of fungal specimens. Mia Hodges, an undergraduate student from UARK, is a student research assistant. Co-PI Guoliang Wang (OSU) and his technician Maria Bellizzi have produced and analyzed rice transgenics carrying R genes of interest. Objective 2. OTL R. Dean (NCSU) is advising graduate student Mengying Wang on the HIGS project. Co-PI J. Xu will advise graduate student Yang Li as she begins work on HIGS objectives in Year 3. Co-PI Sunghun Park (KSU), with support from technician Jung-Eun Kim Park, will produce and analyze rice transgenics. Objective 3. OTL P. Paul and Co-PI L. Madden (OSU) are advising graduate student K. McLean Mills in research on epidemiology of Lolium and Triticum pathotype strains and disease modeling and forecasting. Co-PI M. Farman (UK) is advising post-doc Baohua Wang and graduate student Lily Chen, both working on population analyses of the M. oryzae Lolium (Mol) pathotype in the US. Co-PI G. Peterson (USDA-ARS Ft. Detrick) is supported by Biological Science Technician Kelly Thomas. Co-PI K. Pedley is advising postdoc Mike Pieck on comparative genomics of M. oryzae pathotypes with the goal of developing high-specificity diagnostic tools for wheat blast. Objective 4. OTL Bockus and Co-PI Stack advised Dr. Christian Cruz when he was a graduate student supported by our previous wheat blast grant. Cruz continues as a post-doctoral researcher on this project. He competed for and won a Rotary Humanitarian Study Grant which provided additional funding during Year 2 for wheat blast research in SA. This funding provided living expenses for Dr. Cruz to work in Bolivia and Brazil to better study wheat blast disease in the field. He established working relationships with project collaborators J. M. Fernandes (Embrapa Trigo) and D. Baldelomar (ANAPO, Bolivia), as well as with wheat blast expert Prof. Dr. Alfredo Urashima at Universidade Federal de São Carlos, Brazil. Payton DeLong is an undergraduate research assistant supporting Dr. Bockus’s work on this Objective. In collaboration with Dr. Evans Lagudah, CSIRO, Australia, the team tested Avocet and Thatcher isolines containing the adult rust and mildew R genes Lr34, Lr46 or Lr67. Co-PI Trick (KSU) has advised Kerri Neugebauer (PhD student) and Hyeonju Lee (research technician) on wheat tissue culture, transformation, selection, identification and characterization of transgenic wheat lines containing the rice Piz-t resistance gene. PD Valent (KSU) with collaborator Eduard Akhunov (KSU) guided postdoc P. Kankanala who contributed GBS genotypes for wheat varieties that have varying levels of resistance to wheat blast. Objective 5. OTL DeWolf (KSU) is advising graduate student Taylor Fisher on modeling, risk evaluation and forecasting objectives and he will advise postdoc Denis Shah (Cornell) on modeling, risk evaluation and disease forecasting in 2015. P. Knight, D. Miller and B. Mills (all at Penn State) will collaborate on modeling, risk assessment and disease forecasting in 2015. Co-PI J.M. Fernandes (Embrapa Trigo, Brazil) will complete a 3-month residency at K-State early in 2015. He will continue collaborating with OTL DeWolf and Co-PI Stack when he returns to Brazil, closely coordinating with Willingthon Pavan and Jorge Bavaresco, researchers at the University of Passo Fundo, RS BR. Objective 6. With OTL Nalley (UArk) as her advisor, Anne-Céclie Delwaide completed a double M.S. degree in agricultural economics, one from the University of Arkansas and one from the University of Gent, Belgium. She administered and analyzed the European Cisgenic Survey in Europe and the U.S. Nalley and Delwaide collaborated with Dr. Wim Verbeke, Professor at the University of Gent, and a leading European Ag Economist and E.U. advisor on GMO issues. A new graduate student will be recruited to join the project in 2015. Nalley and the new student will collaborate with economists in Africa to conduct the Cisgenics Survey in selected African countries. Objective 7: OTL Stack (KSU) advised Lindsey Ashmore, a Junior in Agricultural Communications on creation of wheat blast videos and the Wheat Blast Web Site: (http://www.k-state.edu/wheatblast/). Other contributors on outreach include Co-PI Wameshe, OTL Jia, Co-PI Trick, postdoc Cruz, OTL DeWolf, Co-PI J.M. Fernandes, and PD Valent, all of whom are mentioned earlier in this section, and all of whom are contributing to achieving unique outcomes linked to Objective 7. Objective 8: OTL Mitchell (OSU) has been joined by OTL Stack in planning and conducting educational programs linke to the Blast Integrated Project. How have the results been disseminated to communities of interest? The outreach program created through this project has impacted the lives of the undergraduate students in 2013 through a Study Abroad trip to China covering the topics of “Food Security and Food Safety” as reported in detail in last year’s report. This trip will run again, through support of this project, in May of 2015. As part of the year 2 outreach efforts, a week-long workshop on real-time PCR on M. oryzae was conducted by Tom Mitchell at the University of Puerto Rico Mayaguez in June. For the workshop, 27 attendees performed hands-on experiments on rice blast samples to test hypotheses using real-time PCR technologies. They attended daily sessions taught by Tom on plant pathology, genomics, and bioinformatics. At the end of the workshop, students gave a presentation on their results and an interpretation of their meaning and possible biological impacts. In addition to exposing a new cohort of students to these topics, two were inspired to apply to graduate school in Plant Pathology. Additionally, Tom’s graduate student Nikki Tate assisted with all aspects of workshop training, preparation, and execution in Puerto Rico. This was an experience of a lifetime for her and helped train her to be the next generation of scientists that can communicate their work effectively to the public. A press release about the workshop can be found at http://www.uprm.edu/portada/article.php?id=2941. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Project members attended a BIP meeting in Minneapolis on Aug 9 and an External Scientific Advisory Board (SAB) meeting in St. Louis on Dec 6. Progress is summarized. (OBJECTIVE 1) (1.1) Cisgenic approaches to introduce cloned rice resistance (R) genes into elite US rice varieties focused on producing Pi9 marker-free transgenic rice using the dual vector system developed last year. Transformation conditions were optimized for US varieties LaGrue and Juniper. (1.2) Understanding avirulence (AVR) gene compositions in field populations will guide R gene deployment. Isolates being analyzed include 629 archival isolates (collected 1950 to 2005), and 880 isolates from 2012 to 2014. We shortened our AVR analysis pipeline, in which purified strains are assigned to lineages using Rep-PCR fingerprinting and analyzed for AVR gene composition, by isolating DNA for PCR analysis from fungus which is still in the storage filter papers (Jia et al. 2014. Crop Journal). We are focusing on AVR genes corresponding to 4 cloned rice R genes, Pi9, Pita, Pizt, and Pik. All archival isolates contain AVR-Pi9 and 83% contain AVR-Pita1. Among 480 recent isolates, 74% contain AVR-Pi9 and 65% contain AVR-Pita1. (1.3) We developed a perfect DNA molecular marker for Pi9 and are screening germplasm used by US rice breeders. (OBJECTIVE 2) The Dean and Xu labs have prepared a list of 20 target genes for the HIGS analysis, an appropriate HIGS vector, immature suspension culture cells, and actively growing callus cultures for rice transformation. (OBJECTIVE 3) (3.1) Native Lolium (MoL) isolates pose a significant risk to US wheat. The MoL strain isolated from a wheat head in Kentucky in 2011, and 2 other Kentucky isolates are 75, 57, and 81% as aggressive as Bolivian MoT strain B2 in causing head blast under controlled environment conditions. (3.2) We sequenced and assembled genomes of 25 fungal strains from rice, wheat, ryegrass and other hosts, and we developed software for BLAST searches, SNP analyses and repeat masking. We confirmed that M. oryzae isolates are distinct from M. grisea Digitaria isolates and that MoT and MoL isolates are closely related and relatively distant from other populations; and we showed that MoT isolates show a surprising degree of SNP variability relative to isolates from other hosts. (3.3) We are applying a Seq-to-SSR approach to develop diagnostics for differentiating MoT and MoL strains. Marker2 appears specific to MoT, but the final assay may require more than one marker. 20 additional unique MoT loci are being evaluated. (3.4) Experiments in Bolivia confirm that MoT is seed-borne and can be transmitted from spike to seed and from seed to seedling. MoT sporulation on basal senescent wheat leaves might provide an inoculum source in the field. New MoT isolates from Bolivia and Paraguay show a lower temperature optimum than previous isolates. (3.5) Seed treatments can provide significant wheat blast control and fungicides applied at the head phase reduced MoT seed-borne inoculum and blast intensity and increased yield. (OBJECTIVE 4) (4.1) Successful field tests were performed in Quirusillas and Okinawa in Bolivia. Some putatively-resistant cultivars identified in controlled environment studies with single isolates were susceptible in field studies, suggesting there are races in the field. Field tests of isogenic lines from Dubcovsky and Pumphrey show that the 2NS/2AS translocation from T. ventricosum, containing the Lr37, Yr17 and Sr38 rust R genes, also confers wheat blast resistance in some genetic backgrounds and to some pathogen field populations. (4.2) Soft Red Winter Wheat (SRWW) varieties assayed at OSU show a continuum of reactions to MoL isolate PL2-1 similar to HRWWs inoculated with MoT strains. Differential reactions to point and spray inoculations suggest that SRWW may possess different types of resistance to MoL-resistance to infection and resistance to spread within the spike. Thatcher, reported in the literature to contain 2 leaf blast resistance genes, is moderately susceptible to head blast and may not be a good candidate for follow-up. Avocet and Thatcher isolines containing the adult rust and mildew R genes Lr34, Lr46 or Lr67, from the CSIRO, Australia, are highly susceptible to head blast in growth chamber inoculations. A list of resistant cultivars was produced and uploaded onto the wheat blast website (http://www.k-state.edu/wheatblast/). (4.3) DNA was isolated from about 450 wheat cultivars of known reaction to blast and screened for the presence of the 2NS translocation segment using published PCR markers. The 2NS fragment was confirmed in 14 out of 22 highly resistant wheat varieties identified at KSU. Other varieties (Hatcher, Overland, RonL, TAM 112) may contain different resistance genes. Of the 175 spring wheat entries tested at ARS, 6 appeared highly resistant and 5 of these contained the 2NS fragment. We initiated Genotyping by Sequencing (GBS) studies for wheat blast resistance. (OBJECTIVE 5) Activities for rice blast modeling focused on identifying cooperators and compiling historical observations of blast outbreaks and associated weather data for rice producing regions in AR, LA, TX and CA. Quality assessment and verification of weather data are completed from AR and LA and in process for TX and CA. We calculated potential model variables from the hourly records of temp, RH and rainfall, and developed preliminary models for rice blast. We have also identified rice blast models based on research in Asia and will take advantage of these where possible. J.M. Fernandes (Embrapa, Brazil) is working at KSU for 3 months to incorporate his expertise and his current wheat blast model into the project modeling effort. (OBJECTIVE 6) The European Cisgenic Survey has been administered in 5 European countries, Belgium, England, France, Holland, and Spain. A total of 3002 surveys were completed and the data was cleaned and analyzed in SAS using a survivor model. The “willingness to pay (WTP)” survey was designed to assess consumers’ attitudes towards cisgenic rice and to estimate which factors influence consumers’ willingness to accept cisgenic rice. There were important differences among countries and consumers do have a more positive attitude towards cisgenic rice than transgenic rice. Many consumers report that they need more information, which represents opportunity for education to have an impact. (OBJECTIVE 7) (7-1) Our workshop entitled: ‘Special Symposium: Blast control-a moving target’ on Feb 18, before the 35th Rice Technical Working Group Meeting in New Orleans had 75 rice stakeholder attendees. After the workshop, Jia and Wamishe responded to many requests for information, DNA marker protocols, and field inspections and 200 samples of blast resistant germplasm were distributed to public and private institutions from the GSOR of DB NRRC. (7-2) A draft wheat blast extension publication was made available for comment at the wheat blast stakeholders workshop Dec 7. (7.3) We had 15 attendees from private industry at a presentation entitled Science, Risks and Benefits of GMOs at a grain purchasing workshop (KSU International Grains Program) on Mar 31. (7.4) A Wheat Blast Workshop in Bolivia provided disease survey and management information to wheat producers, diagnosticians, extension specialists, breeders, research plant pathologists, and students (52 attendees). (7.5) Our Wheat Blast Stakeholders Workshop at the annual Fusarium Head Blight Meeting in St. Louis on Dec 7 attracted 64 attendees, including scientists from universities, USDA-ARS, USDA-APHIS and industry. (OBJECTIVE 8) We presented a 1-week Genomics and Bioinformatics Workshop for undergraduates, graduate students, post-docs and faculty at the University of Puerto Rico Mayaguez in June. The 27 attendees performed hands-on real-time PCR on rice blast samples, and learned about blast, fungal diseases, genomics and bioinformatics.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Jia, Y., Wamishe, Y.A., and Zhou, B. 2014. An expedited method for isolaton of DNA for PCR from Magnaporthe oryzae stored on filter paper. The Crop Journal 2: 267-271. Doi: 10.1016/j.cj.2014.06.003.
  • Type: Theses/Dissertations Status: Published Year Published: 2014 Citation: Delwaide, Anne-C�cile. 2014. European consumers attitudes towards cisgenic rice. M.S. thesis, University of Arkansas, Fayetteville, AR


Progress 01/01/13 to 12/31/13

Outputs
Target Audience: The professional plant pathology community and wheat production specialists: We organized and hosted a Wheat Blast Symposium associated with the annual meeting of the North Central Division of the American Phytopathological Society (NC-APS) held June 13-15 in Manhattan, Kansas (http://www.apsnet.org/members/divisions/nc/Pages/default.aspx). Professionals involved in regulatory issues: APHIS scientists with a need to learn about wheat blast disease attended the BLAST Project Meeting in Manhattan, KS, on June 15. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Jia Lab (Dale Bumpers National Rice Research Center, Arkansas) - Biological Science Lab Technicians Tracy Bianco and Michael Lin are working on the population biology of the rice blast fungus. Farman Lab (University of Kentucky) - post-doc Baohua Wang and graduate student Li Chen are working on population analyses of the M. oryzae Lolium (Mol) pathotype in the US. Wang Lab (OSU) - Maria Bellizza is performing cisgenic rice transformation. Paul/Madden labs (OSU) - Kara McLean, a new graduate student has begun working on the comparative epidemiology of the Lolium and Triticum pathotype strains and other objective goals. Bockus Lab (KSU) - Dr. Christian Cruz was a graduate student supported by our previous wheat blast grant and he continues as a post-doctoral researcher on this project. He competed for and won a Rotary Humanitarian Study Grant which will provide additional funding for wheat blast research in SA. This funding will provide living expenses for Dr. Cruz to have extended stays in Brazil and Bolivia in order to better study wheat blast disease in the field. He will be working with project collaborators Fernandes and Baldelomar, as well as with wheat blast expert Prof. Dr. Alfredo Urashima at Universidade Federal de São Carlos, Brazil. Stack Lab (KSU) - Lindsey Ashmore, a Junior majoring in Agricultural Communications at KSU is contributing to the Extension/Outreach objective by working on videos and the Wheat Blast Web Site: (http://www.k-state.edu/wheatblast/). Trick Lab (KSU) – Kerri Neugebauer (PhD student) and Hyeonju Lee (research technician) performed the wheat tissue culture, transformation, selection, identification and characterization of transgenic wheat lines containing the rice Piz-t resistance gene. Nalley Lab (University of Arkansas) – Anne-Céclie Delwaide is obtaining a double Master’s degree in agricultural economics, one from the University of Arkansas and one from the University of Gent. She is performing and analyzing the Cisgenics surveys in Europe and the U.S. How have the results been disseminated to communities of interest? The outreach program created through this project has impacted the lives of the undergraduate students that participated in the study abroad program in China as well as a student that spent the summer working in a lab in Lyon France studying rice blast. Participating students represented a variety of majors from Agricultural Engineering to Microbiology. As a result of the summer experience, 92% of the students reported a heightened understanding of the importance of rice in and other staple crops on worldwide food security and social rest. Post trip evaluations also showed that students shared their new insights with friends and family members, thus increasing the reach of the program. As a direct result of the summer experience, three students selected Plant Pathology as a minor and one student changed their major to Plant Health Management. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Major activities completed: The project team, including collaborators from South America (SA) met together with members of the External Scientific Advisory Board (SAB) on June 15 in Manhattan, KS, to discuss project priorities and objectives. The SAB provided valuable feedback and suggestions. The project meeting was coordinated with the annual meeting of the North Central Division of the American Phytopathological Society, and project members organized and presented a well-attended symposium on Wheat Blast Disease at this meeting. (Objective 1) Cisgenic approaches (only rice sequences are retained in the new variety) are being developed to introduce cloned rice resistance (R) genes into elite rice varieties. The cloned rice blast R gene Pi9 does not occur in US germplasm and our previous results showed that it confers a high level of resistance to US pathogen populations. We are performing cisgenic transformation of the Pi9 gene into two elite US rice cultivars. We have developed a novel co-transformation vector system consisting of a cargo vector (pLJ42) carrying the R gene together with marker gene vector (pRB01) carrying genes for Hygromycin resistance and the Green Fluorescent Protein (GFP). Expression of GFP provides visual indication on whether particular T1 plants carry the marker gene vector. In the initial experiments, T1 plants that fail to express GFP were shown by PCR analysis to contain Pi9 without the GFP and selection marker genes, validating this strategy for producing cisgenic rice. Avirulence (AVR) gene compositions in extant blast populations are underway to guide R gene deployment. We have collected >300 rice blast isolates in Arkansas, Louisiana, Texas and Missouri in 2013, and we are analyzing these strains together with 100 isolates from 2012. We have established a pipeline in which strains are purified, assigned to lineages using Rep-PCR fingerprinting, and analyzed for sequence variation in 5 cloned AVR genes relevant to US rice. Selected strains will be inoculated on monogenic lines harboring 24 major blast R genes. (Objective 2) We have prepared a prioritized list of fungal target genes for analyses. Funding for this objective begins in Year 2. (Objective 3) We have collected >100 isolates of SA wheat strains (M. oryzae Triticum pathotype, Mot) as well as SA strains from other grasses that might serve as inoculum sources for wheat. We also collected >150 strains from annual and perennial ryegrass (M. oryzae Lolium pathotype, Mol) and other grasses in the US. Analyses of Mot and Mol isolates continue to show their close genetic relatedness and confirm the ability of some Mol strains to infect wheat in BSL-3 containment experiments. Genomic DNA is being extracted for DNA fingerprinting and genome sequencing, and AVR gene content and structure are being monitored. Experiments have been initiated to compare the host specificity, ecology and epidemiology of the Mot and Mol populations. A major goal of this objective is to deploy PCR-based diagnostics for identification of exotic wheat blast introductions and detection of wheat blast in seed lots and asymptomatic tissues. An assay that differentiates Mot and Mol strains is important for monitoring and regulatory activities as well as for epidemiology and disease management. We have identified markers that appear unique to the Mot isolates from genome sequencing. We are testing the diagnostics assay on multiple isolates to determine specificity, and we are measuring sensitivity on infected wheat heads, seeds, and leaves. We are planning fungicide tests for wheat seed treatment and for head blast control. (Objective 4) One goal is validation of our BSL-3 containment results by field tests in SA. Two field tests were performed in 2013, one in Brazil (with Dr. Andre Rosa, Biotrigo Genetica) and one in Bolivia (with Diego Baldelomar, ANAPO). Unfortunately, neither provided excellent data due to low disease pressure during heading of our winter wheat varieties. Most field tests are performed under natural infection conditions. Therefore, we are establishing new testing sites in Paraguay and Brazil in order to maximize field testing potential. A second goal is to choose broadly resistant germplasm for developing molecular markers. We previously made crosses between resistant and susceptible cultivars, and we have now produced a set of F2-derived, F3 families from Jagalene (R) by Everest (S) for phenotype testing. Many of the wheat lines that appear immune to the Mot isolates we use contain the 2NS fragment from Triticum ventricosum that was translocated onto the 2AS wheat chromosome. Inoculations of sets of paired isogenic lines with and without the 2NS fragment are confirming the associated blast resistance. Reports from SA support the 2NS fragment as an important R gene source. These results point to a promising R locus for molecular marker analysis and wheat blast control. The third goal is to test if cloned blast R genes from rice can control blast in wheat. We have transformed the rice Piz-t blast R gene into wheat varieties Fielder and Bobwhite. The plasmid pPi-zt-C1305 expressing Piz-t was co-transformed with pACH20 (carrying the bar gene for selection). T1 progeny seed have been harvested from 4 Liberty resistant events. At the same time, we are confirming that the fungal AVR gene AvrPiz-t, which is required for Pizt resistance, is broadly present in the Mot population. (Objective 5) We have gathered more than a decade of historical data for wheat blast in Brazil, as a first step to preliminary model development. Preliminary analysis of the information from Brazil indicates possible patterns in temperature and relative humidity that are correlated with wheat blast epidemics. We are assessing potential sources of historic data for wheat blast in Bolivia and for rice blast in the US. (Objective 6) We are constructing a “willingness to pay (WTP)” survey to be administered to participants in the US and Europe. We have decided to move quickly on administration of this survey in Europe, because the European Union has not yet decided how to regulate cisgenic material, and we want to act in time to potentially impact consumer acceptance and regulatory decisions. We will administer the survey in Belgium, Holland, France, Spain and England in the coming months. A top Ag Economist in Europe, Dr. Wim Verbeke (University of Gent), will be helping with the experiments. (Objective 7) We have planned the first rice blast workshop, entitled “Blast control: a moving target”, to coincide with the 35th Rice Technical Working Group Meeting (RTWG) in New Orleans in February 2014. The RTWG provides a multi-state rice-working-group audience for introducing project objectives, including wheat blast and cisgenic routes to blast resistant rice. We are working on a wheat blast extension publication and a mobile device app. (Objective 8) In May, we sponsored participation for 20 undergraduate students in OSU’s China Study Abroad Program, which is focused on Food Security and Production. This was a one month experience where the students also had experiences with food policy as well as meat and dairy production. These students experienced first-hand the challenges of rice production and impact of diseases such as rice blast. We are currently designing a week-long Wheat Production and Policy Workshop (Plant Biosecurity Research Experience) to be held in BSL-3 facilities at KSU and OSU.

Publications