Source: VIRGINIA POLYTECHNIC INSTITUTE submitted to NRP
INTEGRATED MANAGEMENT OF OOMYCETE DISEASES OF SOYBEAN AND OTHER CROP PLANTS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0224426
Grant No.
2011-68004-30104
Cumulative Award Amt.
$9,425,000.00
Proposal No.
2015-03267
Multistate No.
(N/A)
Project Start Date
Apr 1, 2011
Project End Date
Mar 31, 2017
Grant Year
2015
Program Code
[A5121]- Global Food Security: Oomycete Pathosystems in Crop Plants to Minimize Disease
Recipient Organization
VIRGINIA POLYTECHNIC INSTITUTE
(N/A)
BLACKSBURG,VA 24061
Performing Department
Virginia Bioinformatics Institute
Non Technical Summary
Oomycetes are fungal-like microbes that cause highly destructive plant diseases that affect agriculture, horticulture, and forestry. Soybean is a high value food and feed crop in the US that is heavily impacted by root and stem rot diseases caused by oomycetes. Losses to oomycetes have increased by four-fold in the last nine years. Over the last ten years, knowledge of the biology and genomics of oomycete pathogens has expanded dramatically. The goal of this project is to exploit this knowledge to create new disease management technologies that integrate with current practices to improve the sustainability of soybean production. The research activities will focus on three complementary approaches to management of oomycete diseases of soybean: improved molecular diagnostics, new natural resistance genes, and novel transgenes. To ensure that the new technologies meet the needs of producers, and are well-integrated with current farming practices, a network of extension specialists from the 12 major soybean producing states will communicate between stakeholders and the project team. A detailed economic assessment will measure the potential value of the new technologies to the soybean growing industry, from farmers to seed companies to soybean processors. A network of undergraduate institutions will create opportunities for students to participate in oomycete bioinformatics research and contribute to devising new disease resistance strategies. School students will be introduced to sustainable agriculture in collaboration with 4-H programs.
Animal Health Component
35%
Research Effort Categories
Basic
30%
Applied
35%
Developmental
35%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2121820104020%
2121820116020%
2121820302010%
2124099104020%
2124099116020%
2124099302010%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
4099 - Microorganisms, general/other; 1820 - Soybean;

Field Of Science
1040 - Molecular biology; 1160 - Pathology; 3020 - Education;
Goals / Objectives
Oomycetes cause highly destructive plant diseases that affect agriculture, horticulture, and forestry. Over the last ten years, knowledge of the biology of oomycete species has expanded dramatically. Soybean is a high value crop that is heavily impacted by oomycete diseases. Losses have increased by four-fold in the last nine years as resistance genes have been losing their effectiveness. Moreover, increasing adoption of early planting and no-till farming have increased soybean losses due to soil-borne oomycete diseases. The goal of this transdisciplinary project is to exploit new knowledge of the biology, pathology, genetics, biochemistry and genomics of oomycete pathogens of soybean, especially PHYTOPHTHORA SOJAE, to create new disease management technologies that integrate with current practices to improve the sustainability of soybean production. The project will emphasize technologies that can readily be deployed against oomycete and fungal diseases in other crops, and will actively foster early transfer of promising technologies to other crops. Detailed economic and social analyses will assess the potential value of each new technology to farmers and the farm economy. The principal objectives are: 1. Develop DNA-based diagnostic tools for PYTHIUM species and PHYTOPHTHORA SOJAE to improve the ability of farmers to select successful combinations of seed treatments and soybean varieties. 2. Use essential P. SOJAE virulence proteins to rapidly screen germplasm of soybean and other legumes for new resistance genes against P. SOJAE that can be introduced by molecular breeding or soybean transformation. 3. Evaluate novel transgene-based technologies for disrupting oomycete infection, including blocking oomycete virulence proteins, and delivery of novel proteins and RNAs into oomycete cells to inhibit their growth. 4. Create and mentor a network of faculty at predominantly undergraduate institutions, including minority-serving institutions, who will utilize oomycete bioinformatics and functional genomics in their teaching and undergraduate research programs. Engage those faculty and students in the research goals of the project. 5. Using input from farmers, crop professionals, industry stakeholders and extension personnel, assess the economic, social and regulatory impacts of the technologies developed by this project. 6. Facilitate communication and collaboration among stakeholders, extension personnel, plant scientists interested in oomycete disease control, and oomycete molecular genetics researchers by supporting annual conferences, training workshops and mini-sabbaticals. Particular emphasis will be placed on disseminating project outcomes. 7. Implement an Extension program for soybean growers and crop professionals that promotes awareness of oomycete diseases and their impact on soybean production, and enhances their understanding of genetic and GMO technologies. 8. Collaborate with the 4-H program to expand a highly successful program, Kid's Tech, that introduces elementary and middle school children to STEM disciplines through university-style lectures, hands-on and on-line activities. Create new modules on sustainable agriculture.
Project Methods
The approaches to be employed to achieve each principal objective are as follows. 1. DNA-based diagnostics tools will leverage information gained by surveying pathogen prevalence across the 12 principal soybean-growing states, and will employ state-of-the-art polymerase chain reaction (PCR) technologies including isothermal PCR. 2. Essential virulence (i.e. effector) proteins, will be identified by silencing strongly-expressed, monomorphic effector genes. Essential effector genes will be inserted into a bacterial delivery system to create a high throughput screening system. New resistance genes will be isolated by genetic mapping and/or association mapping, followed by testing of candidate genes. 3. Oomycete virulence proteins will be blocked by seeking plant-delivered proteins that can bind to the machinery they use to enter host cells. Potential oomycete resistance proteins will be created by joining proteins that can enter oomycete hyphae to proteins that interfere with oomycete growth. Potential oomycete resistance RNAs will consist of small silencing RNAs produced by the plant and targeted against oomycete genes required for growth. 4. At each participating undergraduate institution, PIs will recruit 2-3 students each year to participate in a research project over a two year period. A web portal, SPACES, will form a forum of faculty and students to exchange resources and experiences. Virginia Tech will host a common bioinformatics resource and will provide an annual training workshop in its use. 5. An ex ante economic impact assessment will performed to estimate the benefits of new technologies developed by the project. National surveys of US soybean producers and industry experts will be used to estimate potential farm-level economic impact, potential adoption of the new technologies, and potential aggregate economic impact and distribution 6. Communication and collaboration among stakeholders and researchers will be facilitated by supporting annual conferences, training workshops and mini-sabbaticals. Particular emphasis will be placed on disseminating project outcomes. 7. To inform soybean growers and crop professionals, we will create Powerpoint presentations, a trifold publication, several on-line educational modules, and open access webcasts. 8. We will establish a new Kid's Tech program at Bowling Green State University, in collaboration with the local 4-H program, by adapting the successful program at Virginia Tech.

Progress 04/01/11 to 03/31/16

Outputs
Target Audience:Soybean producers, extension personnel, and certified crop advisors are the first target audience of this project. A final webinar titled "Diagnostic tools for identification of soybean root rot diseases" is available within the Focus on Soybean site of the Plant Management Network (plantmanagementnetwork.org). Three other webinars are also available on this site. The second target audience is students interested in careers in plant pathology, genomics, and bioinformatics. In the first five years, 93 undergraduate students (4 from under-represented minority groups) were involved in research at the five cooperating institutions. In the final year, undergraduate research projects were continued at all five institutions. Finally, children aged 9-12 were exposed to a University setting and STEM (Science, Technology, Engineering, and Mathematic) disciplines at the Kids' Tech University at Bowling Green State University. In the past four years, over 460 registrants and their parents participated in 4 separate events each spring semester. The final year there were 75 registrants. The third target audience is researchers and industry personnel interested in soybean production and oomycete diseases. This audience was targeted through presentations at various society meetings and invited talks. Students, postdoctoral associates, and faculty have presented their research at many meetings including: the American Phytopathological Society Annual Meeting, July 30-Aug 3, 2016, Tampa, FL; Plant and Animal Genome Conference, San Diego, CA, January 14-18, 2017; International Society for Molecular Plant Microbe Interactions (IS-MPMI), July 17-21, Portland, OR, 2016; and the 16th Oomycete Molecular Genetics Network (OMGN) Annual Meeting, Asilomar, CA March 11-14, 2017, immediately preceding the 29th Fungal Genetics Conference. The OMGN Annual Meeting brings together oomycete molecular geneticists, pathologists, and evolutionary and population biologists within the U.S. and world-wide, particularly Europe and China. Finally, this project was highlighted as a success story within a report prepared by the SoAR foundation (Supporters of Agricultural Research, supportagresearch.org). The report, called "Retaking the Field: Strengthening the Science of Food and Farm Production, summarized AFRI-funded projects from 11 universities. This report was targeted to legislators as part of SoAR's strategic priority of advocating for increased funding for NIFA. The release of the report was coordinated with a visit to Washington DC, in which representatives of each project (e.g., PI McDowell), testified before the House and Senate Subcommittees on Agriculture, and other audiences. The report and visit were reported through several news outlets (see the SoAR web site for examples). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project supported travel and subsistence to the annual Oomycete Molecular Genetics Network Conference at the Asilomar Conference Grounds, Pacific Grove, CA with approximately 82 attendees. There were 27 travel fellowships awarded, ranging from $900-$1600 each depending upon geographic location, and totaling close to $36K. Two awards were given to individuals from Brazil and Spain. U.S. institutions represented included: College of Wooster, Wooster OH; Iowa State University, Ames IA; North Carolina State University, Raleigh NC; Nova Southeastern University, Miami FL; Oregon State University, Corvallis OR; UC-Davis, Davis CA; UC-Riverside, Riverside, CA; University of Delaware, Newark DE; University of Florida, Gainesville FL; University of Hawaii-Manoa, Honolulu HI; Virginia Tech, Blacksburg VA. These awards provided attendees an important opportunity to present their work, collaborate with other labs, and exchange techniques and ideas. Young scientists and awardees are encouraged to present their work as oral presentations. For this meeting, out of 32 total presentations, 11 were given by awardees and 20 presentations were given by graduate students or post-docs. Six oral presentations were given by individuals receiving travel awards to prior OMGN meetings. The project also supported travel for several undergraduate and graduate students in the education network to attend and present their work at state, regional, and national meetings. Students presented work at national/international meetings such as; the Annual Phytopathology Meeting; the Society for Invertebrate Pathology Annual Meeting; the American Society for Biochemistry and Molecular Biology; and several state/regional meetings such as the Phytobiomes Meeting, Washingdon, D.C., Northwest Ohio Undergraduate Research Symposium, the Undergraduate Bioinformatics Education Conference, St. Vincent's College, Latrobe, PA, and the Pennsylvania Academy of Science Meeting, Bradford, PA. How have the results been disseminated to communities of interest?Extension project results were disseminated to soybean producers and industry stakeholders via powerpoints, trifold publications, on-line learning environments, and other extension materials at extension meetings and conferences, newsletters and bulletins, and workshops. Molecular and genetic project results have been disseminated to academic, government, and industry researchers at several professional meetings including but not limited to: Plant and Animal Genome Conference XXIV, January 8-13, 2016, San Diego, CA. The International Society for Molecular Plant-Microbe Interactions XVII, July 17-21, 2016, Portland, OR. The Annual Phytopathology Meeting, July 30-Aug 3, 2016, Tampa, FL. Oomycete Molecular Genetics Network Meeting, March 11-14, 2017, Pacific Grove, CA. The 29th Fungal Genetics Conference, Pacific Grove, CA, March 14-19, 2017. Project results and technology were also presented in many invited seminars. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1. Molecular Diagnostics. In previous years, diagnostic assays were developed using isolates collected during previous field surveys. In the final year, plant disease diagnosticians were trained on the proper use of these diagnostic assays at an MSU training workshop. A high-throughput screen for sensitivity to mefenoxam and ethaboxam fungicide was also developed. This screen was compared to a standard amended media method using EC50 values. Out of 190 isolates tested, only 1 Phytophthora isolate had an EC50 < 1 ug/mL and a number of Pythium species within clades A, B, and E had EC50 values ³ 20 ug/mL. These results indicate that both chemistries are necessary for management of oomycete communities and suggest that insensitivity to ethaboxam is phylogenetically related. 2. New resistance. In previous years, we screened 1100 Glycine max and G. soja accessions and identified a large number of Phytophthora-resistant accessions: 83 G. max and 60 G. soja accessions were made publically available through a publication (Matthiesen et al. 2016, Plant Dis.). A subset of the resistant accessions including 31 G. max and 17 G. soja, which contained Rps8 or new resistance genes, were challenged with ten P. sojae effectors via a Pseudomonas Type III secretion system. In order to map resistance loci, we developed segregating populations by crossing resistant accessions with the P. sojae susceptible 'Williams'. Results from analysis of two crosses at F2:3 generation suggest that effector recognition genes are segregating as single loci. During the final year of the project, over 1200 Recombinant Inbred Lines from six key resistant by susceptible crosses were planted in the field for generation advancement. Seed from F7, F8 or F9 generations of these populations were harvested for disease screening. DNA samples from all population were genotyped with SOY6KSNP chip and chromosomal linkage maps were constructed. Currently, resistant breeding lines are being selected in the field from advanced generations of the five populations that contain the most promising resistance response. Three additional manuscripts from this work will be submitted by the end of the calendar year. 3. Novel resistance strategies. In previous years, six constructs for expression of secreted phosphatidylinositol 3-phospate (or 4-phosphate) binding PIP-binding proteins and controls have been transformed into soybean and 110 events have been confirmed to date by PCR. To date, several lines from multiple independent events have been found to exhibit resistance to P. sojae that is comparable to major R gene resistance in controlled environments. Work is on-going with delivering inhibitory proteins into the oomycete pathogen with the intent to either eliminate or reduce pathogenicity on soybean. Six proteins or peptides have been identified that mediate entry into Phytophthora hyphae. Nuclear localization signals for targeting of nucleases to P. sojae nuclei have been identified in three proteins. Ta-si RNAs directed against 9 essential P. sojae genes have been designed and constructs were introduced into soybean transgenics. More than 40 putative ta-si RNA expressing lines are undergoing evaluation. For this final project year, four lines of transgenic soybean expressing PIP-binding proteins that show very promising levels of resistance to P. sojae along with appropriate controls were evaluated in the field in Iowa and in Michigan during the summer of 2016. Disease resistance and agronomic characteristics were evaluated throughout the year. These trials will be harvested in the next several weeks. 4. Education network. In the previous five years, 118 undergraduates (36) from under-represented minority groups) were involved in research at the 5 cooperating institutions. Many have continued on with either graduate studies or medical/dental school. Oomycete genomic analysis has also been integrated into several undergraduate curricula. For the final year, progress continues on multiple oomycete bioinformatic projects. At Nova SE, work continues on three novel glycoside hydrolase genes that play a role in mosquito-oomycete pathogenesis. At BGSU, a high throughput assay was developed to determine if Pseudomonas species were capable of inhibiting growth of P. sojae hyphae. Work at Lafayette College, PA continues on characterizing Phytophthora pectin methylesterases and at College of Wooster with ochemically characterizing oomycete phosphate kinases and analyzing a panel of RXLR genes using the yeast model system. As a result of this project, a proposal was awarded by NSF for the project "Exploitation of genetic and epigenic variation in the regulation of tomato fruit quality traits" van Der Knaap, OSU and Fraga, College of Wooster. In the first 5 project years, 18 papers were published by the education network with extensive student contributions. In the final year, three more papers have been published. 5. Economic analysis. Once the transgenic field trial is harvested from aim 3, data will be statistically analyzed and the results will be used to construct detailed scenarios that characterize the technologies in the context of the partial equilibrium model already developed. Finally, a book titled "The economics of soybean disease" has been proposed and accepted by CABI Press. This book will cover the technical aspects of plant pathology and epidemiology and will place disease management decisions into an economic framework. Although technical in nature and aimed predominantly at academics, the book will be approachable by non-economists and should become a resource for a range of disciplines including farmers and others in the crop production industry. 6. Communication & collaboration. For the final year, 27 travel awards were funded to the 18th Annual OMGN Meeting March 11-14, 2017, Pacific Grove, CA. A number of awardees were able to attend and present their research at the 29th Fungal Genetics Conference immediately following the OMGN. 7. Extension network. In previous years, an on-line learning resource in oomycete biology was developed with three lessons and four webinars were developed for the Plant Management Network. A final webinar was developed by Alejandro Rojas titled "Diagnostic Tools for Identification of Soybean Root Rot Diseases". Two trifolds were also produced over the course of the project and distributed to over 1000 growers and CCAs in each state of the Midwest. 8. Kids' Tech University. The intent of this program is to increase interest in STEM careers for children aged 9-12. It was established at BGSU in 2012 and well received in the first four years with 470 registrants. In the final year of the project, the program was a continued success with 75 participants. Topics were: "Two reasons why coral reef systems exist: microbes and sunlight", LaJeunesse; "Nature's navigators: the remarkable migratory behavior of birds", Bingman; "How do you measure a growing plant?", Spalding; and "Blue water or green muck: solving one of Ohio's most urgent environmental and economic challenges - harmful algal bloom", Midden. The program has been self-sustaining for the past few years through a registration fee, solicited funds, and in-kind support from Bowling Green State University.

Publications

  • Type: Theses/Dissertations Status: Published Year Published: 2016 Citation: Fedkenheuer, Michael. Understanding plant pathosystems in wild species of crop plants.
  • Type: Journal Articles Status: Submitted Year Published: 2017 Citation: Noel, Z.A., Rojas, A.J., Jacobs, J.L., Chilvers, M.I. Development and evaluation of a high-throughput fungicide sensitivity assay for oomycetes. Phytopathology. Submitted with revisions Sep 08, 2017.
  • Type: Theses/Dissertations Status: Published Year Published: 2016 Citation: Rojas, J. Alejandro. Diversity of oomycetes associated with soybean seedling diseases.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Fang Y, Jang HS, Watson GW, Wellappili DP, Tyler BM. (2017). Distinctive nuclear localization signals in the oomycete Phytophthora sojae. Font. Microbiol. doi: 10.3389/fmicb.2017.00010
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Helliwell, E.E., Vega-Arregu�n, J., Shi, Z., Bailey, B., Xiao, S.Y., Maximova, S.N., Tyler, B.M. and Mark J. Guiltinan, M.J. (2016). Enhanced resistance in Theobroma cacao against oomycete and fungal pathogens by secretion of phosphatidylinositol-3-phosphate-binding proteins Plant Biotechnology J. 14(3)875-886.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Panda A, Sen D, Ghosh A, Gupta A, Malar MC, Mishra GP, Singh D, Ye W, Tyler BM, Tripathy S. (2016). EumicrobDBLite: a lightweight genomic resource and analytic platform for draft oomycete genomes. Mol Plant Pathol. 2016 Oct 27. doi: 10.1111/mpp.12505.
  • Type: Books Status: Published Year Published: 2013 Citation: Mueller D, Wise K, Dufalt N, Bradley C, and Chilvers MI. (Edited) 2013. Fungicides for Field Crops. APS Press. 120 pages.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Hinkel, L. & Ospina-Giraldo, M.D. (2017).Structural characterization of a putative chitin synthase gene in Phytophthora spp. and analysis of its transcriptional activity during pathogenesis on potato and soybean plants. Curr Genet (2017) 63: 909. https://doi.org/10.1007/s00294-017-0687-6.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Ahmed S, M Ariyaratne, J. Patel, A. Howard, A.Kalinoski, V. Phuntumart and P.F. Morris. (2017). Altered expression of polyamine transporters reveals a role for spermidine in the timing of flowering and other developmental response pathways. Plant Science 258:146-155.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Kong, P., McDowell, J. M., & Hong, C. (2017). Zoospore Exudates from Phytophthora nicotianae Affect Salicylic Acid Independent Defense in Arabidopsis. PLOS One, doi.org/10.1371/journal.pone.0180523.
  • Type: Journal Articles Status: Other Year Published: 2017 Citation: Michelmore, R., G. Coaker, R. Bart, G. Beattie, A. Bent, T. Bruce, D. Cameron, J. Dangl, S. Dinesh-Kumar, R. Edwards, S. Eves-van den Akker, W. Gassmann, J. T. Greenberg, L. Hanley-Bowdoin, R. J. Harrison, J. Harvey, P. He, A. Huffaker, S. Hulbert, R. Innes, J. D. G. Jones, I. Kaloshian, S. Kamoun, F. Katagiri, J. Leach, W. Ma, J.M. McDowell, J. Medford, B. Meyers, R. Nelson, R. Oliver, Y. Qi, D. Saunders, M. Shaw, C. Smart, P. Subudhi, L. Torrance, B. Tyler, B. Valent and J. Walsh 2017. Foundational and Translational Research Opportunities to Improve Plant Health. Molecular Plant Microbe Interactions, 30:515-516.
  • Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Dalio, R.J.D., Herlihy, J., Oliveira, T.S., McDowell, J.M., Machado, M. 2017 Effector biology in focus: a primer for computational prediction and functional characterization Molecular Plant-Microbe Interactions, In press.
  • Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Deb, D., Anderson, R. G., Kim, T., Tyler, B., and McDowell, J. M., 2017,�Conserved RxLR effectors from oomycetes Hyaloperonospora arabidopsidis and Phytophthora sojae suppress PAMP- and effector-triggered immunity in diverse plants, Molecular Plant-Microbe Interactions, Accepted with revision.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Coffua LS, Veterano ST, Clipman SJ, Mena-Ali JI, Blair JE. 2016. Characterization of Pythium spp. associated with asymptomatic soybeans in southeastern Pennsylvania. Plant Dis. 100(9):1870-1879.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Dorrance, A. E., Kurle, J., Robertson, A. E., Bradley, C. A., Giesler, L., Wise, K. and Concibido, V. C. 2016. Pathotype diversity of Phytophthora sojae in eleven states in the United States. Plant Dis. 100:1429-1437.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Fang Y, Cui L, Gu B, Arredondo F, Tyler BM. 2017. Efficient genome editing in the oomycete Phytophthora sojae using CRISPR/Cas9. Curr. Protoc. Microbiol. 4:21A.1.1-21A.1.26. doi: 10.1002/cpmc.25.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Fang, Y. and Tyler, B. M. (2017), Nuclear localization of a putative Phytophthora sojae bZIP1 transcription factor is mediated by multiple targeting motifs. Molecular Microbiology, 104: 621635. doi:10.1111/mmi.13652.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Matthiesen, R. L., Abeysekara, N. S., Ruiz-Rojas, J. J., Biyashev, R. M., Saghai Maroof, M. A., and Robertson, A. E. 2016. A method for combining isolates of Phytophthora sojae to screen for novel sources of resistance to Phytophthora stem and root rot in soybean. Plant Dis. 100:1424-1428.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Matthiesen, R., Ahmad, A. and Robertson, A.E. 2016. Temperature affects aggressiveness and fungicide sensitivity of four Pythium spp. that cause soybean and corn damping off in Iowa. Plant Dis. 100(3):583-591.
  • Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Noel, Z.A., Wang, J. Chilvers, M.I. Guidelines for accurate EC50 estimation for fungal and oomycete plant pathogens. Plant Disease.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Rojas A, Miles TD, Coffey, MD, Martin, MI. 2017. Development and application of qPCR and RPA genus- and species-specific detection of Phytophthora sojae and P. sansomeana root rot pathogens of soybean. Plant Dis. 101:1171-1181.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Rojas AJ, Jacobs JL, Napieralski S, Karaj B, Bradley CA, Chase T, Esker PD, Giesler LJ, Jardine DJ, Malvick DK, Markell SG, Nelson BD, Robertson AE, Rupe JC, Smith DL, Sweets LE, Tenuta AU, Wise KA, and Chilvers MI. 2017. Oomycete species associated with soybean seedlings in North AmericaPart II: Diversity and ecology in relation to environmental and edaphic factors. Phytopathology 107:293-304.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Stewart, S., Robertson, A.E. Wickramasinghe, D, Michel, A. and Dorrance A.E. 2016. Population structure among and within Iowa, Missouri, Ohio and South Dakota populations of Phytophthora sojae. Plant Dis. 100(2)367-379.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Zitnick-Anderson, K.K., Norland, J. E., del Rio, L., Fortuna, A. M., and Nelson, B. 2017. Probability models based on soil properties for predicting presence-absence of Pythium in soybean roots. Microb. Ecol. 74(3):550-560
  • Type: Theses/Dissertations Status: Published Year Published: 2016 Citation: Fang, Yufeng. Nuclear localization of proteins and genome editing in the oomycete Phytophthora sojae.
  • Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: Davis, Colin. Identification, validation, and mapping of Phytophthora sojae and soybean mosaic virus resistance genes in soybean.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Edgar, R.E., Morris, P.F., Rozmarynowycz, M.J., D'souza, N.A., Moniruzzaman, M., Bourbonniere, R.A., Bullerjahn, G.S, Phuntumart, V., Wilhelm, S.W., McKay, R.M.L. �(2016) Adaptations to photoautotrophy associated with seasonal ice cover in a large lake revealed by metatranscriptome analysis of a diatom bloom. Journal Great Lakes Research 42(5):1007-1015.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Ahmed S, M Ariyaratne, J. Patel, A. Howard, A.Kalinoski, V. Phuntumart and P.F. Morris 2017. Altered expression of polyamine transporters reveals a role for spermidine in the timing of flowering and other developmental response pathways. Plant Science 258:146-155.
  • Type: Theses/Dissertations Status: Published Year Published: 2016 Citation: Fedkenheuer, Kevin. Molecular analysis of oomycete pathogens to identify and translate novel resistance mechanisms to crops.


Progress 04/01/14 to 03/31/15

Outputs
Target Audience:Soybean producers, pathologists and extension personnel, and certified crop advisors throughout the United States were the first targeted audience through more than 100 presentations that impacted 11,000+ attendees by providing information about soybean seedling diseases caused by oomycetes and strategies for management of these diseases. In the first 3 years of the project, an on-line oomycete learning environment (passel.unl.edu/communities/oomycete) was established with two lessons on oomycete biology and soybean interaction. The third lesson "Transgenic strategies for oomycete disease resistance" was developed in year 4 to target the advanced extension and academic learner who needs to understand the role of molecular pathology and genetic engineering to produce oomycete resistant soybeans. This lesson was tested on a pilot group of eight seed industry professionals throughout the U.S. and Brazil. A project-developed master slide set with project logos and a tri-fold publication detailing Pythium seed blight have been used extensively in many outreach activities and presentations. A second trifold on Phytophthora root and stem rot is in the final stages of preparation. To date, three webinars have been developed for the Plant Management Network focus on Soybean Webcasts (https://www.plantmanagementnetwork.org/infocenter/topic/focusonsoybean/). The latest webinar discusses "A Role for Oomycete Biology in the Development of Disease Resistant Soybean" and is authored by Kevin Fedkenheuer and co-authors Mike Fedkenheuer and John McDowell, Virginia Tech. Several more webinars are planned for the final year including: "Genetic Future: Diagnostic refinements, Transgene deployment, and Regulatoryprocesses" by Wayne Parrott, University of Georgia and "Diagnostics as a Component of Crop Management" by Martin Chilvers, Michigan State University. Researchers and industry personnel interested in soybean root rot were brought together in the 4th reinstatement of the Annual Soybean Root Rot Workshop (rootrot.org) in November 2014 in Duck Key, FL. This workshop provides a forum for informal exchange of information and protocols. There were 17 participants (one Chinese). Eight travel fellowships were awarded to attend and present research. The second target audience is students interested in careers in plant pathology, genomics, and bioinformatics. In the first three years, 73 undergraduate students were involved in research at the five cooperating institutions - many have continued in graduate studies. In the 4th year, this project supported 20 undergraduate students (4 from under-represented minority groups) in oomycete research. Some of the representative projects include: Nova Southeastern University - a transcriptome analysis of an oomycete pathogen (Lagenidium giganteum) that infects insects and characterization of a cellulose synthase 3 gene and a glycoside hydrolase gene; Franklin & Marshall University - primer design, sequence alignment, and phylogenetics of oomycete genomes in FungiDB; College of Wooster- identified yeast mutants that were hypersensitive to the P. sojae effector PsAvh172; and Bowling Green State University - characterizing ooomycete polyamine transporters. Many of these students attended the Annual Summer Oomycete Bioinformatics Training Workshop that is supported by this project and held at the SCALE-Up classroom in Newman Library, Virginia Tech June 3-5, 2015. Finally, children aged 9-12 were exposed to a University setting and STEM (Science, Technology, Engineering, and Mathematic) disciplines at the newly established Kids' Tech University at Bowling Green State University. In year 4, there were 70 registrants who participated in 4 separate themed events. Lastly, the third target audience is researchers and industry personnel interested in soybean production and oomycete diseases. This audience was reached through presentations at various Society meetings and invited talks. Students, postdoctoral associates, and faculty have presented their research at many meetings including: the American Phytopathological Society Annual Meeting, Minneapolis, MN, Aug 2014; Plant and Animal Genome Conference, San Diego, CA, January 2015; Molecular Plant-Microbe Interactions, Rhodes, Greece, July 2014; and the 15th Oomycete Molecular Genetics Network (OMGN) Annual Meeting, Norwich, U.K. July 2014. The OMGN Annual Meeting brings together oomycete molecular geneticists, pathologists, and evolutionary and population biologists. This conference continues to grow and was the largest to date in 2014 with approximately 140 attendees. The project awarded 16 travel awards of up to $1500 each to attend. The latest conference was held March 14-17, 2015 at the Asilomar Conference Grounds, immediately preceding the 28th Fungal Genetics Conference. The project awarded 19 travel awards totaling $20,100. There were 118 attendees (87 US, 16 UK, 3 China, 2 Brazil, 1 Australia, 1 Peru, remainder from Europe). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The Summer Bioinformatics Training Workshop was held at the SCALE-Up classroom, Newman Library at Virginia Tech on June 3-5, 2014. The workshop included brief introductions on genome and genome sequencing, gene predictions, and using the FungiDB database and suite of bioinformatics tools. Eighteen travel fellowships were awarded with four foreign awardees (Brazil, India, the U.K., and Colombia) totaling $19,200. There were 42 participants (14 undergraduates, 13 graduates, and 15 faculty or academic professionals) from throughout the U.S. including OH, NY, PA, MI, NC, FL, OR, MD, and VA. Workshop expenses (facility rental, refreshments, lodging, local transportation) and attendee travel and subsistence were supported by this project. The project also supported travel and subsistence to the annual Oomycete Molecular Genetics Network Conference in Norwich, UK with approximately 140 attendees. There were 16 travel fellowships awarded for up to $1500 each and totaling $22,500. Institutions represented included: Bowling Green State Univ., North Carolina State University, UC-Davis, Lafayette College, University of Delaware, Penn State, Oklahoma State University, Iowa State University, UC-Riverside, Michigan State University, and Cornell University. This project will support at least 12 travel fellowships to the next annual meeting in Norwich, UK July 2-4, 2014. The latest conference was held March 14-17, 2015 at the Asilomar Conference Grounds. The project awarded 19 travel awards totaling $20,100. These awards provide attendees an important opportunity to present their work, collaborate with other labs, and exchange ideas. For example, 18 of the 35 oral presentations were given by graduate students or post-docs. The project also supported travel for several undergraduate and graduate students in the education network to attend and present their work at state, regional, and national meetings. Students presented work at national/international meetings such as; the Annual Phytopathology Meeting August 2014, Minneapolis, MN; the Society for Invertebrate Pathology Annual Meeting, Pittsburgh, Aug 2014; and several state/regional meetings such as the Ohio Branch American Microbiology Conference, Ashland University, OH, and the Science, Engineering & Technology Gateway, Bowling Green, OH and the Pennsylvania Academy of Science Meeting, Bradford, PA. This project also provided 8 travel fellowship awards to the Soybean Root Rot Workshop held November 2014 in Duck Key, FL. The workshop provided an opportunity for students to present their work, solicit advice, and interact with soybean researchers and industry personnel in a communal, and informal atmosphere. Students and senior project members have also been accorded opportunities for oral and poster presentations and networking at the Annual Project Meetings in Amicolala State Park, Dawsonville, GA March 19-21, 2014 and the most recent one in Chicago, IL, May 20-21, 2015. In the 2014 meeting, Emily Helliwell (Post-doc, Penn State) presented "Expression of apoplast-targeted PI3P-binding proteins confers oomycete and fungal resistance in cacao and rice" and Leah Sandall (Instructor, Univ. Nebraska-Lincoln) presented "Oomycete learning environment". In the 2015 meeting, Alejandro Rojas and Zach Noel (PhD Candidates, Michigan State University) presented "Molecular diagnostic and population genetics"; Kevin Fedkenheuer (PhD Candidate, VA Tech) presented "Effector-directed breeding"; Emily Helliwell (Post-doc, Oregon State University) presented "Transgenic soybean response to P. sojae"; and Alex Howard (Undergraduate, Bowling Green State University) presented "FungiDB, a tool for DNA methylation research in P. sojae". How have the results been disseminated to communities of interest?Extension project results have been disseminated to soybean producers and industry stakeholders via powerpoints, trifold publications, on-line learning environments, and other extension materials at extension meetings and conferences (e.g. Great Lakes Crop Summit, Iowa Soybean Association Research Conference, Minnesota Ag Expo, North Dakota Soybean Council Annual Expo, and Wisconsin Crop Conference), newsletters and bulletins, and workshops. Molecular and genetic project results have been disseminated to academic, government, and industry researchers at several professional meetings including but not limited to: the 15th Biennial Conference on the Molecular and Cellular Biology of the Soybean, Minneapolis, MN, August 3-5, 2014; the Annual Phytopathology Meeting, August 9-13, Minneapolis, MN Oomycete Molecular Genetics Network Meeting, Pacific Grove, CA, Norwich, UK, July 2-4, 2014 and Pacific Grove, CA March 15-17, 2015; the Gordon Research Conference on Cellular and Molecular Biology, Holderness, NH, June 18, 2014 Plant and Animal Genome Conference XXII, San Diego, CA, January 11-15, 2014. The 28th Fungal Genetics Conference, Pacific Grove, CA, March 17-22, 2015. Project results were also presented in many invited seminars, including but not limited to: McDowell, J.M. 2014, "Translating functional genomics of plant-oomycete interactions into better tools for disease control". Ohio State University Practical Summer Workship in Plant Functional Genomics, Columbus Ohio. McDowell, J.M. 2014, "Translating functional genomics of plant-oomycete interactions into better tools for disease control". Department of Plant Pathology, Texas A & M University, College Station, Texas. McDowell, J.M., "Why are the foods that you love the most in danger from rusts, blights, molds and mildews?" Kids Tech, Bowling Green State University, April 2015. Robertson, A. 2014. Plant pathology and disease resistance. Research experience for teachers summer program. Iowa State University Tyler BM, "From genomics to effectors: new ways to protect crop plants from disease". College of Plant Protection. Northwest A & F University, Yangling, China. April 24, 2014. Tyler BM. "Collaborative Research in Oomycete Molecular Biology" Shandong Agricultural University, Tai'an, China. April 28, 2014. Tyler BM. "From Genomics to Effectors: How Oomycete Plant Pathogens Cause Disease" Ohio Agricultural Research and Development Center. Wooster, Ohio. February 13, 2015. What do you plan to do during the next reporting period to accomplish the goals?1. Molecular Diagnostics. Submit a manuscript describing diversity of oomycete species that were collected and identified in the Midwest during the first two years of the project. Develop a genus specific marker for Pythium. Complete a manuscript describing the molecular diagnostic assays. Train and implement diagnostic assays across the project extension and National Plant Diagnostic Networks. Use the developed high-throughput fungicide screen to assay resistance of oomycete isolates to fungicides including Mefenoxam and the new chemistry Ethaboxam from Valent. 2. New resistance. Analyze results of genotyping F2:3 populations with molecular markers to validate type of resistance, inheritance studies, and genetic mapping of resistance loci. Continue developing the segregating populations from several crosses (Phytophthora sojae susceptible 'Williams' x resistant G. max and G. soja lines, Pythium resistant 'Archer' x resistant lines, and intercrosses among the resistant lines) - over 100 F2 populations. Produce F5, F6, and F7 seed from 16 of 30 crosses of susceptible mapping populations X resistant soybean accessions in winter nurseries - these 16 were chosen because they respond to one or more of the effectors tested to date. These recombinant inbred lines (RILs) will be screened for resistance using effector-based and inoculum-based assays and for linkage mapping. DNA from these RILs will be used for genotyping with the Illumina 6K SNP Infinium Chip. Finish screening soybean accessions with additional effectors (9) identified as monomorphic and essential to moderately needed for virulence. Evaluate 32 Phytopthora resistant G. max lines for resistance to Pythium, facilitating developing of breeding lines resistant to both oomycete species. 3. Novel resistance strategies. Continue evaluating transgenic soybean plants expressing phosphatidyl-inositol binding proteins for disease resistance and normality through resistance assays and expression data. Promising plants will be advanced to field tests for normality and disease resistance. Based on results from a parallel study in cacao, evaluate transgenic soybeans to determine if they are exceptionally response to chemical stimulants of the plant immune response (e.g. beta-amino-butyric acid). Use new biosensors for PI3P and other lipids to identify the location of PI3P in soybean membranes. Evaluate a bZIP transcription factor that is strongly down-regulated during infection as a possible protein that could be delivered into oomycete hyphae to reduce infection. Determine if two small protein domains that mediate strong nuclear localization can be used to deliver toxic proteins to the nucleus of oomycetes. Continue evaluating ta-si RNA- expressing lines for disease resistance and normality in the laboratory and greenhouse. Silence Hypotaurocyamine kinase (enzyme involved in cellular energy metabolism specific to oomycetes) and other potential targets in P. sojae to determine effect on infection. 4. Education network. Train undergraduates in oomycete biology and bioinformatics at the five institutions. Obtain pre- and post-participation data on students and develop a manuscript detailing the model, assessment strategy, and impact on student participants for the past five years. Submit several manuscripts in preparation with student contributions. Continue refining the pipeline for uploading and annotating genomes to FungiDB. Upload 3 more genomes. Refine a method to re-annotate hypothetical genes using the InterPro database. The work will focus particularly on effectors. 5. Economic analysis. Finalize analysis of the third CCA survey with Iowa State University. Complete the final producer survey instrument for distribution in summer/fall 2015. Continue estimating the global impact of oomycete mitigation technologies and the project more broadly. Estimate farm level impact and adoption for the economic analysis models. Finish both economic analysis models; a partical equilibrium model that will estimate the net impact of project-developed technologies and an input-output model will contextualize impacts in terms of sectoral impact. Finish and submit several technical manuscripts detailing the methods, analyses, and key results of the CCA and producer surveys. Develop 6. Communication & collaboration. The soybean oomycete root rot workshop will continue to be organized and supported by this project and eight travel fellowships will be awarded. In this year, the workshop will be combined with the 1st International Soilborne Oomycete Conference December 8-10, 2015 (oomyceteconference.org/). The 17th Annual Oomycete Molecular Genetics Conference in Malmo, Sweden will be supported. This project will also sponsor six to nine training internships for project participants, international collaborators, and other researchers interested in furthering a project aim or implementing project outcomes toward the control of other oomycete diseases. 7. Extension network. In collaboration with the University of Missouri, analyze the third CCA survey. Develop powerpoint presentation summarizing project findings for use at annual grower and agricultural professional meetings. Finish development of the second trifold publication on Phytophthora root and stem rot. This trifold will include recent data on pathotype diversity of P. sojae isolates in the Midwest. Phytophthora sojae trifold publication similar to the completed Pythium trifold. Develop two more webinars for the Plant Management Network on "Genetic Future: Diagnostic refinements, Transgene deployment, and Regulatoryprocesses" and "Diagnostics as a Component of Crop Management". Finish interactive on-line educational modules that educate stakeholders about breeding for disease resistance and the strategies used. This will qualify for Continuing Education Credits. 8. Kids' Tech University. Continue to hold and expand four KTU events at Bowling Green State University and become financially self-sufficient through registration fees, in-kind donations, and solicited contributions.

Impacts
What was accomplished under these goals? 1. Molecular Diagnostics. Isolates collected from the first two years of field surveys were used to develop diagnostic assays for identification of oomycete species. A multiplex qPCR assay can be used to confirm the oomycete belongs to the genus Phytopthora and can also correctly identify Phytopthora sojae and sansomeana. A duplex isothermal assay has been developed for markers at the genus level for Pythium. A diagnostician training is planned in June for these assays. We have also initiated amplicon-based approaches to determine the composition of oomycete species in plant and soil samples. A high-throughput fungicide sensitive screen has been developed to screen the collection of isolates. 2. New resistance. In previous years, we screened 1100 accessions and identified 31 Glycine max and 17 Glycine soja lines that contained either a new resistant gene or Rps8. These are currently being re-screened with an isolate diagnostic for Rps8. These resistant lines were also challenged with three essential pathogen effectors using a system optimized to deliver effectors into soybean leaves via a Pseudomonas Type III secretion system. 13 out of 31 G. max lines and 3 out of 16 G. soja lines responded to one or more of the three effectors. Challenging these lines with an additional set of seven effectors yielded five putative resistance genes. In order to map resistance loci, we developed mapping populations that segregate for these putative resistance genes in classic genetic ratios. Resistant lines were crossed with both the Phytophthora sojae susceptible 'Williams' and the Pythium resistant 'Archer'. Mapping populations have been developed for 30 G. max lines. Of these, sixteen have been advanced to recombinant-inbred lines that are at either F5, F6 or F7 generations. In order to map the five putative R genes, F2 populations were genotyped with 1536 single nucleotide polymorphism (SNP) markers. Screening of F3 families from F2 progeny from crosses of two resistant lines X Williams is underway. Preliminary results from analysis of two crosses suggest that effector recognition genes are segregating as single loci. Screens of 20 P. vulgaris cultivars with Avh16, Avh240, and Avh180 were negative. 3. Novel resistance strategies. Six constructs for expression of secreted phosphatidylinositol 3-phospate (or 4-phosphate) binding PIP-binding proteins and controls have been transformed into soybean and 110 events have been confirmed to date by PCR. Among these transgenic lines, 38 lines from 23 indendent events are homozygous for expression of these proteins. They are currently being independently evaluated at Virginia Tech and Oregon State for resistance to P. sojae. So far, 11 of these lines, representing 7 independent events, have been found to exhibit resistance to P. sojae that is comparable to major R gene resistance. More lines will be evaluated in both locations and with several assays. Work continues with delivering inhibitory proteins into the oomycete pathogen that will either eliminate or reduce pathogenicity on soybean. Six proteins or peptides that mediate entry into Phytopthora hyphae have been identified. Nuclear localization signals for targeting of nucleases to P. sojae nuclei have been identified in three proteins. Ta-si RNAs directed against 9 essential P. sojae genes have been designed and constructs introduced into soybean transgenics. 41 putative ta-si RNA expressing lines are currently undergoing evaluation. 4. Education network. In the 4th year, 20 students (4 from under-represented groups) were engaged in education and research projects supported by the education network. Progress continues on multiple oomycete bioinformatic projects at each location. Training includes: DNA methylation in oomycete genomes; data mining and primer design; sequence alignment and phylogenetics; PCR and gel electrophoresis; qRT-PCR; microscopy; flow cytrometry; and microscopy . A paper was published describing the transcriptome of Lagenidium giganteum, an oomycete that infects mosquito larvae. Network members presented their work at several annual meetings. A genome re-annotation effort was undertaken for a subset of the genomes available from FungiDB. Close to ten thousand hypothetical genes could be re-annotated with defined products. Eight more oomycete sequences have been uploaded to FungiDB. Three more are planned for the upcoming year. 5. Economic analysis. A third survey of certified crop advisors (CCAs) was undertaken in year 4 to further assess: 1) use and value of seed treatments, 2) how new oomycete testing technologies might be perceived, valued, and adopted, and 3) how CCAs value seed attributes and employ them with respect to oomycete diseases. The survey was completed in early May 2015. Preliminary analysis indicates that results are consistent with the two prior surveys and suggest that a CCA's perception of indicidence, severy, and impact of the disease have remained stable over time. They have also estimated larger yield losses from oomycete diseases than reported previously. However, these experts continue to place relatively modest value on what growers are willing to pay for resistant soybean varieties. A complementary grower survey instrument has been developed and will be executed in the summer of 2015. This survey will use refined estimates of grow valuation and potential adoption of resistant traits and the results will feed into the on-going economic impact assessment modeling work. 6. Communication & collaboration. The soybean oomycete root rot workshop (rootrot.org) was reinstated in north-central WI and held annually for 4 years. For the 4th year, there were 17 participants and 8 travel awards. The Summer Oomycete Bioinformatics Training Workshop was held June 3-5, 2014 at Virginia Tech. The Oomycete Molecular Genetics Network Meeting was held July 2014 in Norwich, UK. The project awarded 16 travel awards of up to $1500 each to attend this meeting. The latest conference was held March 2015 at the Asilomar Conference Grounds, Pacific Grove, CA. The project awarded 19 travel awards totaling $20,100. 7. Extension network. In previous years, an on-line learning resource in oomycete biology (passel.unl.edu/communities/oomycete) was developed with two lessons. A third lesson was developed in year 4 entitled "Transgenic strategies for oomycete disease resistance". A second webinar was developed for the plant management network entitled "A role for oomycete biology in the development of disease resistant soybean". A second trifold publication detailing Phytophthora root and stem rot was developed and in the final stages of preparation. It will be printed and distributed to the Extension Network (EN) for use in winter meetings in fall 2014/spring 2015. More than 15 on-line extension newsletter articles were written. Over 100 presentations and workshops on soybean seedling diseases were given to more than 11,000 stakeholders across the Midwest from October 2014 through January 2015. 8. Kids' Tech University. This program (KTU), originally established at the Virginia Bioinformatics Institute at Virginia Tech to increase the interest of children ages 9-12 in STEM careers, was initiated at Bowling Green State University. It was well received in the first three years with 325 registrants. The 4th year was a continued success with 70 participants. Topics ranged from fossils, forensic DNA analysis of oak pathogens, chemistry and the best way to grow crystals, and why the foods we love the most are the most attacked by rusts, blights, and mildews.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Matthiesen R, Ahmad A, Ellis M. and Robertson AE, 2014. First report of Pythium schmitthenneri causing maize seedling blight in Iowa. Plant Dis. 98(7):994.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Hyde KD, Nilsson RH, Alias SA, Ariyawansa HA, Blair JE et al. 2014. One stop shop: backbone trees for important phytopathogenic genera: I (2014). Fungal Diversity 67:21-125.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Kamoun, S., Furzer, O., Jones, J. D., Judelson, H. S., Ali, G. S., Dalio, R. J., Roy, S. G., Schena, L., Zambounis, A., Panabieres, F., Cahill, D., Ruocco, M., Figueiredo, A., Chen, X. R., Hulvey, J., Stam, R., Lamour, K., Gijzen, M., Tyler, B. M., Grunwald, N. J., Mukhtar, M. S., Tome, D. F., Tor, M., Van den Ackerveken, G., McDowell, J., Daayf, F., Fry, W. E., Lindqvist-Kreuze, H., Meijer, H. J., Petre, B., Ristaino, J., Yoshida, K., Birch, P. R., and Govers, F. (2014) The Top 10 oomycete pathogens in molecular plant pathology. Mol Plant Pathol Sep 1. doi: 10.1111/mpp.12190.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Martin FM, Blair JE, Coffey MD. 2014. A combined mitochondrial and nuclear multilocus phylogeny of the genus Phytophthora. Fungal Genetics & Biology 66: 19-32
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Matari NH, Blair JE. 2014. A multilocus timescale for oomycete evolution estimated under three distinct molecular clock models. BMC Evolutionary Biology 14:101.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Matthiesen, R. and Robertson, A.E. 2014. Pathotype diversity in single zoospore isolates from a race 1 isolate of Phytophthora sojae. Phytopathology 103(Suppl.)
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Matthiesen, R., Ahmad, A., Ellis, M. and Robertson, A.E. 2014. First report of Pythium schmitthenneri causing maize seedling blight in Iowa. Plant Dis. 98:994
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: McDowell J., J. Carr, M. Loretto, 2014. Focus on Translational Research, Molecular Plant-Microbe Interactions. 27:195.
  • Type: Book Chapters Status: Published Year Published: 2014 Citation: McDowell JM, 2014, Hyaloperonospora arabidopsidis, in "Genomics of plant associated fungi and oomcyetes: Dicot pathogens", eds. Ralph A. Dean, Ann Lichens-Park, and Chittaranjan Cole, Springer, New York.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Mingora, C., Ewer, J., Ospina-Giraldo, M. 2014. Comparative structural and functional analysis of genes encoding pectin methylesterases in Phytophthora spp. Gene 538: 7483.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Quiroz Velasquez PF, Abiff SK, Fins KC, Conway QB, Salazar NC, Delgado AP, Dawes JK, Douma LG, Tartar A. 2014. Transcriptome Analysis of the Entomopathogenic oomycete Lagenidium giganteum Reveals Putative Virulence Factors. Appl Environ. Microbiol. Oct 15;80(20):6427-36. doi: 10.1128/AEM.02060-14.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Stewart, S., Abeysekara, N. and Robertson, A.E. 2014. Pathotype and genetic shifts in population of Phytophthora sojae under soybean cultivar rotation. Plant Disease 98:614-621.
  • Type: Book Chapters Status: Published Year Published: 2014 Citation: Tyler, B.M. and Gijzen, M.J. (2014) The Phytophthora sojae genome sequence: foundation for a revolution In: Genomics of Plant-Associated Fungi and Oomycetes: Dicot Pathogens. Dean, R.A., Lichens-Park, A., and Kole, Chittaranjan (Eds). Chapter 7, pp 133-157. Springer-Verlag, Berlin, Heidelberg.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Zitnick-Anderson, KK and Nelson, BD, Jr. 2014. Identification and Pathogenicity of Pythium on soybean in North Dakota. Plant Dis. 99(1):31-38.


Progress 04/01/13 to 03/31/14

Outputs
Target Audience: The first target audience is soybean growers, soybean pathologists and extension personnel, and certified crop advisors throughout the United States. There were more than 40 presentations that impacted 6000+ attendees by providing information about soybean seedling diseases caused by oomycetes and strategies for management of these diseases. An on-line oomycete learning environment (passel.unl.edu/communities/oomycete) has been established and two lessons developed to date: 1) Oomycete Biology and 2) Photophthora sojae and Soybean Interaction. The environment was tested at participating project extension network locations including South Dakota State University and Iowa State University. The environment is currently awating approval by the American Society of Agronomy staff for Continuing Education Units (CEU) approval. A master slide set with project logos was developed and has been used in outreach activities. A webinar developed for the Plant Management Network in the 2nd project year was launched. Two more webinars are being developed for completion by end of the 4th project year. A tri-fold publication detailing Pythium seed blight was developed and distributed to the project extension network. Researchers and industry personnel interested in soybean root rot were brought together in the 3rd reinstatement of the Annual Soybean Root Rot Workshop (rootrot.org) in September 2013 in North-Central Wisconsin. This workshop provides a forum for informal exchange of information and protocols. Eighteen participants attended from seven research institutions (Iowa State Univ., North Dakota State Univ., Virginia Tech, Kansas State Univ., Univ. Minnesota, Oregon State. Univ., and Univ. Arkansas). Six travel fellowships were awarded to attend and present research. A second target audience is students interested in careers in plant pathology, genomics, and bioinformatics. In the 3rd project year, this project supported 26 undergraduate students (10 from under-represented minority groups) in oomycete research. Some of the representative projects include: Nova Southeastern University - sequencing of potential virulence factors from an oomycete pathogen (Lagenidium giganteum) that infects insects; Franklin & Marshall University - determining diversity of oomycete species in water and agriculture using baiting techniques and ribosomal sequencing; and College of Wooster/Bowling Green State University - determining if decreasing expression of an enzyme (phosphagen kinase) involved in regulation of cellular energy (adenosine tri-phosphate) molecules in the oomycete causing root rot of soybean (Phytophthora sojae) can reduce disease expression. Many of these students attended the Annual Summer Oomycete Bioinformatics Training Workshop that is supported by this project and held at the Graduate Life Center in Donaldson Brown, Virginia Tech June 24-26, 2013. The workshop included brief introductions on genome and genome sequencing, gene predictions, and using the FungiDB database and suite of bioinformatics tools. There were 45 attendees including 17 undergraduates, 19 graduate, and 18 faculty or academic professionals. Thirty-two travel awards were equally supported by this project and an NSF Research Coordinate Network grant. Finally, children aged 9-12 were exposed to a University setting and STEM (Science, Technology, Engineering, and Mathematic) disciplines at the newly established Kids' Tech University at Bowling Green State University. In year 3, there were 115 registrants who participated in 4 separate themed events. Lastly, the third target audience is researchers and industry personnel interested in soybean production and oomycete diseases. This audience was reached through presentations at various Society meetings and invited talks. Students, postdoctoral associates, and faculty have presented their research at many meetings including: the American Phytopathological Society Annual Meeting, Austin TX, Aug 2013; Plant and Animal Genome Conference, Sandiego, CA, January 2014; Molecular Plant-Microbe Interactions, Taipei, Taiwan, October 2013; and the Oomycete Molecular Genetics Network (OMGN) Annual Meeting, Pacific Grove, CA, March 2013; and the 27th Fungal Genetics Conference, Pacific Grove, CA, March 2013. The OMGN Annual Meeting brings together oomycete molecular geneticists, pathologists, and evolutionary and population biologists. This conference continues to grow and was the largest to date in 2013with 117 attendees (66 US, 21 UK, 37 Spain/China/Africa/Other): ~ 32 are involved in this project as project personnel, collaborators, or members of the advisory board. This project supported travel awards for 38 participants equally split between this project and the NSF RCN. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The Summer Bioinformatics Training Workshop was held at the Graduate Life Center in Donaldson Brown at Virginia Tech on June 24-26, 2013. There were 45 participants with 32 receiving travel awards. This workshop provides introductory lectures on genomic and bioinformatics topics such as sequencing, transcriptomics, and annotation. Attendees included students from the five Education Network institutions (Bowling Green State Univ., College of Wooster, Lafayette College, Franklin & Marshall College, and Nova Southeastern Univ. Other institutions represented included North Carolina State University, Cornell University, Pennsylania State University, the University of Vermont, Oregon State University, and the Ohio State University. There were a few international participants including Brazil, UK, France, India, and Colombia. Workshop expenses (facility rental, refreshments, lodging, local transportation) and attendee travel and subsistence were supported by this project and an NSF Research Coordination Network grant (Training, communication and resources for the oomycete genomics community, Award No. MCB-0639226). The NSF RCN also supported travel and subsistence to the annual Oomycete Molecular Genetics Network Conference in Pacific Grove, CA March 10-12, 2013. There were 27 travel fellowships awarded to travel to and attend/present at the meeting. Institutions represented included: Bowling Green State Univ., Iowa State, Michigan State, Nova Southeastern, Oklahoma State, Oregon State, Penn State, UC-Riverside, Univ. Rhod Island, Univ. Tennessee, and Virginia Tech. This project will support at least 12 travel fellowships to the next annual meeting in Norwich, UK July 2-4, 2014. The project also supported travel for several undergraduate and graduate students in the education network to attend and present their work at state, regional, and national meetings. Students presented work at national/international meetings such as the U.S. Department of Energy (DOE) Joint Genome Institute (JGI) User Meetings, March 20-22, 2012, Walnut Creek, CA; the Annual Phytopathology Meeting August 2013, Austin, TX; the Society for Invertebrate Pathology Annual Meeting, Pittsburgh, Aug 2013; and several state/regional meetings such as the Ohio Branch American Microbiology Conference, Ashland University, OH, and the Science, Engineering & Technology Gateway, Bowling Green, OH and the Pennsylvania Academy of Science Meeting, Bradford, PA. This project also provided 6 travel fellowship awards to the Soybean Root Rot Workshop held September 16-17, 2013 at the Kemp Natural Resources Station Woodruff, WI. The workshop provided an opportunity for students to present their work, solicit advice, and interact with soybean researchers and industry personnel in a communal, rustic, and informal atmosphere. Shared meal preparation, lodging, and no television ensured optimal interaction. The project awarded two training internships in 2013. The first was to Shahin Ali at USDA/ARS Sustainable Perennial Crops Lab, Beltsville, MD to learn how to transform Phytophthora palmivora and P. megakarya. The award was for up to $2500 for travel and subsistence to Dr. Brett Tyler's lab at Oregon State University between July 7-26, 2013. The second award was to Junli Quan at Northwest A&F University, Shanghai, P.R. China to learn how to transform Phytophthora sojae. The award was for up to $2800 for travel and subsistence to Dr. Brett Tyler's lab at Oregon State University between March 22-April 25, 2013. Students and senior project members have also been accorded opportunities for oral and poster presentations and networking at Project Meetings. One oral presentation was presented by a senior project member in Chicago, IL May23-24, 2014: Peter Lafayette, "Soybean transgenics and commercialization". Five students also presented project-related work: J. Alejandro Rojas, "Oomycete ID and diversity in soybean"; Kevin Fedkenheuer, "High through-put screening of soybean resistance"; Qunqing Wang, "PI3P-mediated entry of effectors"; Tom Jacobs, "Short, simple, silencing using the trans-acting siRNA pathway"; and Brittany Begress, "Characterization of a hypotaurocyamine kinase from Phytophthora sojae". How have the results been disseminated to communities of interest? Extension project results have been disseminated to soybean producers and industry stakeholders via powerpoints, trifold publications, on-line learning environments, and other extension materials at extension meetings and conferences (e.g. Great Lakes Crop Summit and Minnesota Ag Expo), newsletters and bulletins, and workshops. Molecular and genetic project results have been disseminated to academic, government, and industry researchers at several professional meetings including: Gordon Conference on Plant Lipids: Structure, Function, and Metabolism, Galveston, TX Jan 27-Feb 1, 2013; Oomycete Molecular Genetics Network Meeting, Pacific Grove, CA, March 10-12, 2013; the American Chemical Society Annual Meeting, April 2013, New Orleans, LA; the Annual Phytopathology Meeting, August 10-14, 2013, Austin, TX; the International Congress of Plant Pathology, August 24-25, 2013 Beijing, China; and the Academia Sinica Symposium on Molecular Plant-Microbe Interactions. Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan October 3-4, 2013. Project results were also presented in many invited seminars, including: Yangzhou University, October 2013; Shandong Agricultural university, September 2013; College of Plant Protection, Northwest A&F University, April 2013; and College of Tropical Agriculture and Human Resources, University of Hawaii, Feb. 15, 2013. Project results are also posted to the public website: oscap.org. What do you plan to do during the next reporting period to accomplish the goals? 1. Molecular Diagnostics. Complete a manuscript describing diversity of oomycete species that were collected and identified in the Midwest during the first two years of the project. Initiate screening of oomycete species for fungicide sensitive using both existing fungicides such as Mefenoxam and the new chemistry Ethaboxam. Complete validation of molecular diagnostic assays at the Phytophthora and Pythium genus level and at the P. sojae species level using TaqMan markers and isothermal amplification assays. 2. New resistance. Continue optimizing the transient effector delivery system for efficiency in Glycine max, G. soja, and other Phaseolus species. This will be done by optimizing an ion-leakage assay as a way to quantitively measure cell death, stably integrating effector transgenes into the bacterial chromosome and improving assay response, and determining if a "helper" bacterial strain is necessary for other Phaseolus species. Re-screen the 32 Glycine max lines identified to contain either a new resistance gene or Rps8 with another isolate that should consistently break resistance. Finish genotyping of F2:3 populations with molecular markers to validate resistance, inheritance studies, and genetic mapping of resistance loci. Continue developing the segregating populations from several crosses (Phytophthora sojae susceptible 'Williams' x resistant G. max and G. soja lines, Pythium resistant 'Archer' x resistant lines, and intercrosses among the resistant lines). Screen soybean accessions with additional effectors (9) identified as monomorphic and essential to moderately needed for virulence. Continue advancing the F2:3 mapping populations and mapping resistance genes through genotyping with 1536 single nucleotide polymorphism (SNP) markers. Screen progeny of populations using the effector-based screen system and inoculum-based assays. Continue screening soybean lines for additional putative resistance genes. 3. Novel resistance strategies. Evaluate transgenic soybean plants expressing phosphatidyl-inositol binding proteins for disease resistance and normality through resistance assays and expression data. Optimize targeting of TAL effector nucleases to deliver toxic proteins to P. sojae nuclei. Evaluate ta-si RNA- expressing lines for disease resistance and normality. Silence Hypotaurocyamine kinase (enzyme involved in cellular energy metabolism specific to oomycetes) and other potential targets in P. sojae to determine effect on infection. 4. Education network. Continue to recruit and train undergraduates in oomycete biology and bioinformatics at the five institutions. Continue to obtain pre- and post-participation data on students to develop a manuscript detailing the model, assessment strategy, and impact on student participants. Each institution will submit manuscripts resulting from student participation. The bioinformatics team will continue to develop tools for use by the education network and other oomycete researchers. The network will prepare a manuscript detailing the model, assessment strategy, and impact on student participation. Publish several manuscripts in preparation. Continue refining the pipeline for uploading and annotating genomes to FungiDB. Upload 7 genomes. Develop a method to fix genome annotation of several oomycete species, particularly with respect to effecor genes. A separate, lightweight database called EumicrobeDB will continue to be developed and serve as a repository and toolbox for "un-polished" oomycete genomes. 5. Economic analysis. The database of soybean varies will be finished and used to evaluate market share, pricing, and resistance traits over time and states. A component/attribute pricing analysis is also planned. A second survey of soybean growers is planned to measure their valuation and willingness to pay for various attributes of soybean seeds, including soybean root rot resistance. Results from analyses of the various surveys (grower, CCA, major ag-bio companies) will be synthesized to develop the expected adoption paths of the new technologies developed in the project across classes of soybean farms/types of growers. The impacts of various factors that might condition such adoption will be quantified. A technical report that details such results will be developed. 6. Communication & collaboration. The soybean oomycete root rot workshop will continue to be organized and supported by this project along with eight travel fellowships each year. The annual Oomycete Molecular Genetics Conference will also be supported through twelve travel fellowships. This project will also sponsor six to nine training internships for project participants, international collaborators, and other researchers interested in furthering a project aim or implementing project outcomes toward the control of other oomycete diseases. 7. Extension network. Develop powerpoint presentation summarizing project findings for use at annual grower and agricultural professional meetings. Finish development of a Phytophthora sojae trifold publication similar to the completed Pythium trifold. Develop a second webcast covering molecular diagnostics in association with the Plant Management Network. Finish interactive on-line educational modules that educate stakeholders about breeding for disease resistance and the strategies used. This will qualify for Continuing Education Credits. 8. Kids' Tech University. Continue to hold four KTU events at Bowling Green State Univ and become financially self-sufficient through registration fees, in-kind donations, and solicited contributions.

Impacts
What was accomplished under these goals? 1. Molecular Diagnostics. Isolates collected from the first two years of field surveys were cleaned up, entered into a database, and identified (82 different oomycete species) using the ITS region of ribosomal DNA. Representative isolates were screened in pathogenicity and fungicide assays. Diagnostic assays have been developed with initial validation screens conducted for markers at the genus level for Phytophthora and species level for Phytophthora sojae. 2. New resistance. Among 1100 accessions screened, 31 Glycine max and 17 Glycine soja lines were identified that contained either a new resistance gene or Rps8. They are being screened with an isolate diagnostic for Rps8. To enable us to map resistance genes, crosses were made between these resistant lines with both the susceptible 'Williams' and Pythium resistant 'Archer'. We have developed F2:3 mapping populations of 22 resistant Glycine max lines. We have also screened the resistant lines using a modified Pseudomonas system that delivers 3 essential avirulence effectors into soybean. Among the 31 soybean lines: 13 responded to 1 or more effectors; 3 lines responded to 2 effectors; and 1 responded to all 3 effectors (Avh16, Avh180, and Avh240). These results have been corroborated with a particle bombardment assay in a different lab. We also used the same effector-based system to screen 20 Phaseolus vulgaris (common bean) and 31 Medicago truncatulata (barrel clover) accessions. To date, resistance has not been identified in either species: effectors appear to be suppressing plant triggered immunity (PTI) in Phaseolus and all the Pseudomonas strains tested to deliver effectors appear to be strongly pathogenic in Medicago. 3. Novel resistance strategies. Six constructs for expression of secreted PIP-binding proteins and controls have been transformed into soybean and 110 events have been confirmed. We have confirmed more than ten events for each binding protein by amplifying DNA via polymerase-chain reaction (PCR) and visualizing/quantifying expression level through an attached green fluorescent protein (GFP). The majority are now in the greenhouse for seed production. Since seed is currently limiting, we have developed a detached trifoliate leaf assay to determine if enhanced expression of these binding proteins in soybean leaves reduces disease. Entry of these binding proteins into Phytophthora hyphae has also been confirmed, providing a basis for delivering inhibitory proteins into P. sojae and killing cells. We hope to target pathogen DNA sequences such as ribosomal RNA genes. Targeting is done through Transcription-Activator-Like-Effector Nucleases (TALENS). As a first step, we have been validating nuclear localization signals (NLSs) for delivery of these TALENS into the pathogen nucleus. We have screened a large number of pathogen proteins with nuclear locations and found two small protein domains that mediate nuclear localization. Nucleases that target P. sojae HIS4 have been confirmed and are being prepared for E. coli expression. Ta-si RNAs directed against two essential P. sojae genes have been designed and constructs introduced into soybean transgenics. Hypotaurocyamine kinase, an enzyme involved in cellular energy metabolism, is being tested as a way to reduce pathogenicity. 4. Education network. In the 3rd year, 26 students (10 from under-represented groups) were engaged in research project. Progress continues on multiple oomycete bioinformatic projects at each institution. Two papers with significant student contributions were recently published. The Oomycete Bioinformatics Training Workshop at VT was well attended with 45 participants in year 3. The workshop was also extended to three full days to enhance the training experience, enabling bioinformatics trainers to give hands-on demonstrations of expressed RNA data in normal compared to diseased soybeans. Network members presented their work at several annual meetings including the Society of Invertebrate Pathology (Pittsburg), American Phytopathology Society (Austin), Ohio Plant Biotechnology Meeting (Columbus), and the Oomycete Molecular Genetics Network (Monterey). 5. Economic analysis. Survey results from the first two years were statistically analyzed for grower willingness to pay and implied demand for various oomycete control strategies, including any developed by this project. These surveys clearly indicate that there is significant uncertainty about the incidence and potential impact of seedling disease. These analyses attempt to quantify the perceived economic benefits of technologies developed by this project and are expected to drive adoption when technologies are commercialized. However, other stakeholders in the agricultural supply chain (e.g. seed producers, synthetic pesticide producers, crop consultants, retailers of agricultural inputs) can also influence the adoption and use of the new technologies as well as their ultimate economic value. Similarly, competitive or complementary technologies can condition their adoption. 6. Communication & collaboration. The soybean oomycete root rot workshop (rootrot.org) was reinstated in north-central WI and held annually for 3 years. For the 3rd year, there were 14 participants and 6 travel awards. Other conferences, workshops, internships, and mini-sabbaticals that are currently supported by the NSF RCN (ended August 31, 2013) will be supported by this project in 2013-2015. The Summer Oomycete Bioinformatics Training Workshop was held June 24-26, 2013 at Virginia Tech had 45 participants with 32 receiving travel awards. 7. Extension network. An on-line learning resource in oomycete biology was developed in the 2nd year of the project. In year 3, a second module was developed and tested at an Extension meeting. The first of four webinars for the Plant Management Network was launched. A trifold publication detailing Pythium seedling blight was printed and distributed to the Extension Network (EN) for 2014 meetings. A trifold publication on Phytophthora root and stem rot was drafted and will be edited by the EN, printed and distributed in summer 2014. Several on-line extension newsletter articles were written. Over 40 presentations and workshops on soybean seedling diseases were given to more than 6000 stakeholders across the Midwest from October 2013 through January 2014. 8. Kids' Tech University. This program (KTU), originally established at the Virginia Bioinformatics Institute at Virginia Tech to increase the interest of children ages 9-12 in STEM careers, was initiated at Bowling Green State University. It was well received in the first two years with 245 registrants. In order for KTU to be self-sustaining, an enrollment fee has been initiated for the 3rd year. Registration opened October 28, 2013. For the 3rd year, two events have been completed [Jan 25, Dr. Doug Kane, "What is this green slime in Lake Erie, why is it there, and what can be done to get ride of it?; Feb 22, Dr. Victor Raboy, 'The Genetic Code: Is it the piano that's important, or the music played on it?"]. Two more events are planned.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Heyman F, Blair JE, Persson L, Wikstrom M. 2013. Root rot of pea and faba bean in southern Sweden caused by Phytophthora pisi, sp. nov. Plant Disease 97(4): 462-471.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Matthiesen, R and Robertson AE. 2013. Fungicide sensitivity of Pythium species affecting corn and soybean in Iowa at three temperatures. Phytopathology 103 (Suppl. 3):S3.6
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Matthiesen, R and Robertson AE. 2013. Pathogenicity of Pythium species affecting corn and soybean in Iowa at three temperatures using two assay methods. Phytopathology 103 (Suppl. 2):S2.92
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: McDowell JM and Meyers BC, 2013, A transposable element is domesticated for service in the plant immune system, Proc. Natl. Acad. Sci. USA, 110:14821-14822.
  • Type: Other Status: Published Year Published: 2013 Citation: Mueller D, Wise K, Dufalt N, Bradley C, and Chilvers MI. (Edited) 2013. Fungicides for Field Crops. APS Press. 120 pages.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Palmer, A*, Begres BN*, Van Houten JM, Snider MJ, and Fraga D. 2013. Characterization of a putative oomycete taurocyamine kinase: implications for the evolution of the phosphagen kinase family. Comparative Biochemistry and Physiology: Part B Biochemistry and Molecular Biology. Nov-Dec;166(3-4):173-81. doi: 10.1016/j.cbpb.2013.08.003. Epub 2013 Aug 23.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Shtarkman YM, Ko�er ZA, Edgar R, Veerapaneni RS, DElia T, Morris PF, Rogers SO. 2013. Subglacial Lake Vostok (Antarctica) Accretion Ice Contains a Diverse Set of Sequences from Aquatic, Marine and Sediment-Inhabiting Bacteria and Eukarya. PLoS ONE 8(7): e67221. doi:10.1371/journal.pone.0067221.


Progress 04/01/12 to 03/31/13

Outputs
Target Audience: The first target audience is soybean growers, soybean pathologists and extension personnel, certified crop advisors, and industry stakeholders, throughout the United States. This project supported more than 25 presentations to 3000+ attendees providing information about soybean seedling diseases caused by oomycetes and strategies for management of these diseases. An on-line learning resource in oomycete biology targeting the extension learning market has been developed and is currently under revision. A master slide set with project logos was developed for use in all webinars. The first of four webinars was developed for the Plant Management Network and the Continuing Education Unit (CEU) audience. Researchers and industry personnel interested in soybean root rot were brought together in the Annual Soybean Root Rot Workshop (rootrot.org) that provides a forum for informal exchange of information and protocols. Eighteen participants attended from seven research institutions (Iowa State Univ., Univ. Illinois, Michigan State Univ., North Dakota State Univ., Virginia Tech, Kansas State Univ., Univ. Minnesota). Seven travel fellowships were awarded. A second target audience is students interested in careers in plant pathology, genomics, and bioinformatics. In the 2nd project year, this project supported 28 undergraduate students in oomycete research. Many of these students attended the Oomycete Bioinformatics Summer Training Workshop supported by this project, held at the Virginia Bioinformatics Institute at Virginia Tech July 6-7, 2012. There were 54 attendees including 17 undergraduates, 19 graduate, and 18 faculty or academic professionals. The Annual Oomycete Molecular Genetics Network Meeting was held March 10-12, 2013 in Pacific Grove CA. This project supported travel and expenses for eight students among the 118 participants. Finally, children aged 9-12 were exposed to a University setting and STEM (Science, Technology, Engineering, and Mathematic) disciplines at the newly established Kids' Tech University at Bowling Green State University. In year 2, there were 115 registrants who participated in 4 separate themed events. Lastly, the third target audience is researchers interested in soybean pathology, particularly diseases caused by oomycetes. This audience was reached through presentations at various Society meetings and invited talks. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The Summer Bioinformatics Training Workshop was held at the Virginia Bioinformatics Institute at Virginia Tech on July 6-7, 2012. This workshop provides introductory lectures on genomic and bioinformatics topics such as sequencing, transcriptomics, and annotation. There were 54 attendees, including 17 undergraduate and 19 graduate students from the five Education Network institutions (Bowling Green State Univ., College of Wooster, Lafayette College, Franklin & Marshall College, and Nova Southeastern Univ. The remainder came from oomycete research labs throughout the country with a few international participants including the UK, France, and Colombia. Workshop expenses (facility rental, refreshments, lodging, local transportation) and attendee travel and subsistence were supported by this project and an NSF Research Coordination Network grant (Training, communication and resources for the oomycete genomics community, Award No. MCB-0639226). Both projects will equally support the next workshop scheduled for June 24-26, 2013. The NSF RCN also supported travel and subsistence to the annual Oomycete Molecular Genetics Network Conference in Asilomar, CA March 10-12, 2013. This project will support 12 travel fellowships to the next annual meeting in Norwich, UK July 2-4, 2014. The project also supported travel for several undergraduate and graduate students in the education network to attend and present their work at state, regional, and national meetings. Students presented work at national/international meetings such as the U.S. Department of Energy (DOE) Joint Genome Institute (JGI) User Meetings, March 20-22, 2012, Walnut Creek, CA; the Annual Phytopathology Meeting August 4-8, 2012, Providence, RI; the Society for Invertebrate Pathology Annual Meeting, Pittsburgh, Aug 11-15, 2012; and several state/regional meetings such as the Ohio Plant Biotechnology Consortium, Fall 2012; the Pennsylvania Academy of Science Meeting, Bradford, PA. This project also provided 7 travel fellowship awards to the Soybean Root Rot Workshop held September 23-25, 2012 at the Kemp Natural Resources Station Woodruff, WI. The workshop provided an opportunity for students to present their work, solicit advice, and interact with soybean researchers and industry personnel in a communal, rustic, and informal atmosphere. Shared meal preparation, lodging, and no television ensured optimal interaction. Students and senior project members have also been accorded opportunities for oral and poster presentations and networking at Project Meetings. One oral presentation was presented by a senior project member in Helen, GA March 16018, 2013. One oral presentation by a senior member and 5 by students are scheduled for the upcoming meeting in Chicago, IL on May 22-24, 2013. How have the results been disseminated to communities of interest? Extension project results have been disseminated to soybean producers and industry stakeholders via powerpoints, trifold publications, on-line learning environments, and other extension materials at extension meetings and conferences, newsletters and bulletins, and workshops. Molecular and genetic project results have been disseminated to academic, government, and industry researchers at several professional meetings including: Gordon Conference on Plant Lipids: Structure, Function, and Metabolism, Galveston, TX, Jan 27-Feb 1, 2013; International Society for Molecular Plant-Microbe Interactions, Kyoto, Japan, May 22-24, 2012; Oomycete Molecular Genetics Network Meeting, Nanjing, China, May 26-28, 2012 and March 10-12, 2013; the American Chemical Society Annual Meeting, April 2013, New Orleans, LA; and Annual Phytopathology Meeting, August 4-8, 2012, Providence, RI. Project results were also presented in many invited seminars including: Canadian Dept. of Agriculture and Agri-Foods Canada, Ottawa, Canada; the Conference of Soil-borne Plant Pathogens at Oregon State University, March 27-28, 2013; Dept. Plant Pathology, Nanjing Agricultural University, Aug 3, 2012; and College of Tropical Agriculture and Human Resources, University of Hawaii, Feb. 15, 2013. Project results are also posted to the public website: oscap.org. What do you plan to do during the next reporting period to accomplish the goals? 1. Molecular Diagnostics. Complete fungicide sensitivity screens to determine which oomycetes are most destructive. Develop diagnostic markers for these oomycetes. Identify P. sojae pathotype distribution among the twelve cooperating states. 2. New resistance. Screen 400 soybean lines and 10 each Medicago and Phaseolus lines for resistance to P. sojae using available effectors. Cross lines containing putative new resistance genes with each other and with ‘Archer’ which contains resistance to Pythium and Phytophthora. Genetically characterize resistance genes in segregating populations. 3. Novel resistance strategies. Evaluate transgenic soybean plants expressing PIP binding proteins for disease resistance and normality. Continue optimizing targeting of TAL effector nucleases to deliver toxic proteins to P. sojae nuclei. Evaluate ta-si RNA- expressing lines for disease resistance and normality. Silence Hypotaurocyamine kinase (enzyme involved in cellular energy metabolism specific to oomycetes) and other potential targets in P. sojae to determine effect on infection. 4. Education network. Continue to recruit and train undergraduates in oomycete biology and bioinformatics at the five institutions. Continue to obtain pre- and post-participation data on students to develop a manuscript detailing the model, assessment strategy, and impact on student participants. Each institution will submit manuscripts resulting from student participation. The bioinformatics team will continue to develop tools for use by the education network and other oomycete researchers. Once the recently hired bioinformatician is up to speed, they will continue to refine the pipeline for uploading and annotating genomes to FungiDB. A separate, lightweight database called EumicroDB will continue to be developed and serve as a repository and toolbox for “un-polished” oomycete genomes. 5. Economic analysis. The supply chain for industry stakeholders will be analyzed to determine potential commercialization strategies of new resistance strategies, their likelihood of success, and opportunities/constraints to adoption. 6. Communication & collaboration. The soybean oomycete root rot workshop will continue to be organized and supported by this project along with eight travel fellowships each year. The annual Oomycete Molecular Genetics Conference will also be supported through twelve travel fellowships. This project will also sponsor six to nine training internships for project participants, international collaborators, and other researchers interested in furthering a project aim or implementing project outcomes toward the control of other oomycete diseases. 7. Extension network. Paper-based "clicker" surveys of participants at extension meetings and field days to increase awareness, improved understanding of oomycete-soybean pathosystem, medium term actions, and recommendations adopted. Finish and distribute first tri-fold publication on Pythium root rot. Continue development of second tri-fold on Phytophthora root and stem rot of soybean. Finish open-access webcast covering molecular diagnostics as a component of crop management. Using current review, refine interactive on-line educational module with continuing education credits (CEU) for CCAs, to educate stakeholders strategies used to reduce losses to soybean root rot diseases.

Impacts
What was accomplished under these goals? 1. Molecular Diagnostics. Over 4500 (3239 yr 1, 1287 yr2) isolates of oomycete species were obtained from the cooperating institutions in IL, IN, IA, KS, MI, MN, MO, NE, ND, WI. From these, 82 different oomycete species were identified. Pathogenicity assays have been developed. Results of the assay will inform the development of molecular diagnostic tools. 2. New resistance. Over 1100 accessions of cultivated and wild soybean were screened with Phytophthora sojae isolates diagnostic for known Rps genes: of these, 31 contained either a new resistance gene or Rps8. These resistant lines were also screened with three essential effectors using a modified Pseudomonas effector-detector system. Several resistant accessions were identified with the effector-based screen. 3. Novel resistance strategies. Six constructs for expression of secreted PIP-binding proteins and controls have been transformed into soybean and 133 events have been confirmed. To date, we have PCR- and GFP-confirmed more than ten events for each binding protein construct with the majority at the greenhouse for seed production. Entry of PI3P- and PI4P-binding proteins into Phytophthora hyphae have been confirmed, providing a basis for delivering inhibitory proteins into P. sojae. TAL effector nuclease constructs were validated in yeast and introduced into P. sojae transformants, but expression in P. sojae was poor. Nuclear localization signals for targeting of nucleases to P. sojae nuclei are being evaluated. Ta-si RNAs directed against two essential P. sojae genes have been designed and constructs introduced into soybean transgenics. The ta-si RNA- expressing lines are currently undergoing hygromycin selection. Hypotaurocyamine kinase, a member of the phosphagen kinase family with key roles in cellular energy metabolism, has been suggested as a target. 4. Education network. Twenty-eight undergraduate students (eleven either Hispanic or African American) were involved in multiple oomycete projects at the five institutions comprising the network. Resources for students to purse career options in oomycetes, bioinformatics, and genomics were loaded into the SPACES collaborative environment hosted at Lafayette College. The first cohort of students in year 1 have undergone pre- and post-test surveys while the students in the 2nd project year have undergone pre-test surveys. Six oomycete genomes were migrated into FungiDB. A lightweight genome database called EumicrobDB was established to host “rough draft” genomes and provide data and tools for small labs who do not have genome sequence hosting and analysis capabilities. 5. Economic analysis. A national producer survey was designed in year 1 and completed in year 2 to assess understanding of the incidence and severity of seedling disease and mid-season root rots. The survey indicates that producers highly value an easy and inexpensive diagnostic test and place an even higher value on resistant varieties (a 28% premium relative to the average). Moreover, producers value the economic benefits of early planting and no/reduced tillage and are unwilling to alter those practices to control oomycetes. As such, they are even more willing to pay for resistant varieties rather than plant after soils have warmed and soil moisture content reduced. A Monte Carlo farm-level simulation model was further developed to calculate implied values for testing technologies and resistant soybean varieties based their performance, cropping practices, insurance, and other factors. 6. Communication & collaboration. The reinstated soybean oomycete root rot workshop (rootrot.org) was held for the 2nd time September 23-25, 2012. There were 18 participants with 7 receiving travel awards. The Annual Oomycete Molecular Genetics Network Meeting was held March 10-12, 2013 in Asilomar, CA. There were 118 attendees (66 US, 21 UK, 37 Spain/China/Africa/Other): ~ 32 are involved in this project as project personnel, collaborators, or members of the advisory board. The Summer Bioinformatics Training Workshop will be held June 23-26, 2013 and supported by travel fellowships. 7. Extension network. Certified crop advisors (CCAs) were surveyed for a second year to evaluate their experience, management strategies, and value of controlling seedling and mid-season diseases caused by oomycetes. The response rate doubled from 6% to 14% in year 2. Almost all CCAs reported moderate to severe stand establishment issues associated with Pythium (77%), Phytophthora (76%), Rhizoctonia (54%), and Fusarium (39%). Similar to producers, CCAs also believe that identification of the pathogen causing disease is extremely important and would use a simple, quick, and low-cost diagnostic test. An on-line lesson was developed to introduce oomycete biology. Powerpoint presentations highlighting oomycete biology, soybean seedling diseases, implications for management of crop diseases, and summaries of the soybean seedling disease survey were also developed. An on-line learning environment targeting the extension learning market has been developed and is currently under peer review (passel.unl.edu/communities/oomycete). The environment has been developed specifically for CCAs who want more in-depth knowledge about oomycetes and targets outcomes such as: define and describe oomycetes, identify the symptoms of Phytophthora and Pythium in soybean, and predict the best management steps for a given scenario. Two trifold publications are also undergoing development. 8. Kids’ Tech University. This educational initiative was held for a second year with 115 registrants in four events. Feb 9, 2012, Jennifer Kay, National Center for Climate Research, Boulder, CO, “Bright blankets and boating bonanzas: How do clouds and ice affect our planet? Feb 16, Craig Zirbel, Mathematics and Statistics, Bowling Green, OH, “Practical math for the digital age”. Apr 6, Brett Tyler, Center for Genome Research and Biocomputing, Oregon State Univ., OR, “Why doesn’t my banana get the flu”. Apr 20, Ron Woodruff, Biological Sciences, Bowling Green, OH, “What can fly genetics tell us about human health and evolution”. Students in the Middle School Science Education program have been recruited to help develop science, technology, engineering, and mathematic activities, particularly experiential ones. Facilities, marketing support, personnel, and in-kind/matching support have been solicited from Bowling Green State University, 4-H, and other organization to support future programs and expansion of Kids’ Tech.

Publications

  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Blair JE, Coffey MD, and Martin FN. 2012. Species tree estimation for the late blight pathogen, Phytophthora infestans, and close relatives. PLoS ONE 7(5): e37003
  • Type: Journal Articles Status: Published Year Published: 2011 Citation: Bienapfl JC, Malvick DK, Percich JA. 2011. Specific molecular detection of Phytophthora sojae using conventional and real-time PCR. Fungal Biology. 115 733-740.
  • Type: Other Status: Published Year Published: 2012 Citation: Chilvers MI. 2012. Molecular diagnostics in plant disease diagnostic clinics&whats the status? (Editorial) Fungal Genomics and Biology. 2:e102. doi:10.4172/2165-8056.1000e102.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Heyman F, Blair JE, Persson L, Wikstrom M. 2013. Root rot of pea and faba bean in southern Sweden caused by Phytophthora pisi, sp. nov. Plant Disease 97(4): 462-471.
  • Type: Books Status: Published Year Published: 2013 Citation: Mueller D, Wise K, Dufalt N, Bradley C, and Chilvers MI. (Edited) 2013. Fungicides for Field Crops. APS Press. 120 pages.
  • Type: Journal Articles Status: Submitted Year Published: 2012 Citation: Shtarkman YM, Zeynep A, Ko�er ZA, Edgar R, Veerapaneni RS, DElia T, Morris PM, Rogers SO. 2012 Subglacial Lake Vostok (Antarctica) accretion ice contains a diverse set of sequences from aquatic and marine Bacteria and Eukarya. Submitted to Plos ONE.
  • Type: Journal Articles Status: Under Review Year Published: 2013 Citation: Blair JE, Matari NH, and Mena-Ali JI. 2013 A Timescale for Oomycete Evolution Estimated from Conserved Regulators of Gene Expression. Mol. Bio. Evol. (in review).


Progress 04/01/11 to 03/31/12

Outputs
OUTPUTS: To determine which oomycete pathogens are responsible for soybean losses, diseased seedlings or soil samples were collected from 10 major soybean producing states: IL, IN, IA, KS, MI, MN, MO, NE, ND, WI. More than 3000 oomycete cultures were isolated and are undergoing identification via rDNA sequencing of the ITS region. Thirty-two soybean lines were identified that contain resistance to known Rps genes. An effector-detector system that uses PSEUDOMONAS SYRINGAE bacteria to deliver known PHYTOPHTHORA SOJAE (PS) effectors to soybean has been modified and validated. Eight PS effectors were transiently silenced and 7 of 8 appear to measurably contribute to virulence. Efforts are underway to re-validate entry of plant proteins into PS hyphae. Six constructs for expression of PI-3-P or PI-4-P binding proteins were created and biolistically transformed into soybean. More than 10 transformation events have been confirmed for each of the 6 constructs via PCR and GFP analysis. Similar constructs are being used to transform rice, switchgrass, cacao, and BRACHYPODIUM. Transcription activator-like effector nucleases constructs are being assembled to target essential PS genes. A partnership has been established with FungiDB to upload oomycete genomes to their servers and leverage their powerful interface and tools for project analysis and annotation purposes. Several oomycete genomes including P. SOJAE, P. INFESTANS, HYALOPERONOSPORA ARABIDOPSIS, and PYTHIUM ULTIMUM have been uploaded to a beta site of FungiDB and will become public once initial testing is completed. A partnership with DOE JGI allows the seamless transfer of data from FungiDB to their Mycocosm database and leverages their suite of bioinformatic tools. The agronomic and economic impacts of oomycetes in soybean production were reviewed and informed two nationally representative surveys of soybean industry stakeholders: certified crop advisors (CCA) and soybean producers. CCAs were surveyed on-line in March 2011 about their experience, management strategies, and value of controlling soybean seedling diseases. A second survey was done March 2012. A phone survey of soybean producers was developed and fine-tuned. A Monte Carlo farm-level simulation model was developed to calculate implied values for project outcomes including new testing technologies and new resistance genes or strategies against Phytophthora root rot. On-line lessons of oomycete biology including sampling activities and pathogen isolation were developed for the CEU audience. The extension network has also developed powerpoint presentations introducing oomycete biology and implications for disease management that have been incorporated into extension meetings in each participating state. An instance of the collaborative environment called SPACES at Lafayette College was developed for members of the oomycete undergraduate network to communicate, collaborate, and upload useful bioinformatics content. Kids' Tech University at VBI, emphasizing STEM disciplines, was expanded to Bowling Green State University. Four events were held in spring 2012 with university-style lectures given by well-established authorities. PARTICIPANTS: The project is managed by the PD, Brett Tyler and project manager, Joel Shuman (Virginia Tech). Other co-PDs are John McDowell, M.A. Saghai Maroof, Joao Setubal, Christopher Lawrence, and Reinhard Laubenbacher (Virginia Tech) and Wayne Parrott (Univ. Georgia), Martin Chilvers (Michigan State Univ.), Paul Morris, Vipa Phuntumart, and Karen Sirum (Bowling Green State Univ.), Nicholas Kalaitzandonakes (Univ. Missouri), William Morgan and Dean Fraga (College of Wooster), Manuel Ospina-Giraldo (Lafayette College), Jaime Blair (Franklin & Marshall College), Aurelian Tartar (Nova Southeastern Univ.), Donald Lee and Loren Giesler (Univ. Nebraska), Alison Robertson (Iowa State Univ.), Carl Bradley (Univ. Illinois), Dean Malvik (Univ. Minnesota), Doug Jardine (Kansas State Univ.), Thomas Chase (South Dakota State Univ.), Kiersten Wise (Purdue Univ.), Samuel Markell and Berlin Nelson (North Dakota State Univ.), and Paul Esker (Univ. Wisconsin). Senior research or project personnl include: Sucheta Tripathy (VBI, Bioinformatician), Kristy Collins (VBI, KTU), Julio Vega-Arreguin (VBI), Peter Lafayette (UGA), James Kaufman (Univ. Missouri). Postdoctoral associates include: Qunqing Wang (Oregon State), Hua Wise (VT), Nilwala Abeysekara (ISU), Ehsan Shakiba (VT), and Ryan Anderson (VT). Graduate students included: Yufeng Fang, Alejandro Rojas, Thomas Jacobs, Rashelle Matthieson-Anderson, Devdutta Deb, Technicians/Other Professional staff were: Janette Jakobs, Noah Lawler, Lisa Sutton, Kyle Broaderick, and Dianne Pederson. Undergraduates included: Kenyetta Robinson, Nathan Steele, Chrispin Otondi, Jason Ewer, Jhanelle Dawes, Amy Chabitnoy, Ana Paula Delgado, Corey Mark Howe, Eric Budge, Josie Hug, Patrick Wolfkiel, Rebecca Cull, Robin Edgar, Jason van Houten, Andrew Lamade, Meyesa Mansour, Kilian Lafreniere, Crystal Floyd, Kim Chapman, and Justin Bailies. There were at least 83 people (31 female, 52 male, 20 under-represented races and ethnicities) involved in the project. Undergraduates, graduate students, and postdoctoral scholars were given a variety of research, extension, and teaching experiences in their respective laboratories ranging from molecular diagnostics and pathology,DNA sequencing, DNA cloning, soybean transformation, bioinformatic analysis, and extension pathology. The oomycete bioinformatics training workshop was held June 29-July 1, 2011 at the VBI and attended by 26 participants. Topics included phylogenetic analysis, structural annotation, gene annotation and biological ontology, and FungiDB and hands-on bioinformatics exercises. Results of the oomycete isolate collection and their molecular ID were presented at the North Central Division American Phytopathological Society (APS) meeting. Oomycete diversity in PA soybean fields using baiting and rRNA sequencing was presented at the Northeast Division APS meeting. TARGET AUDIENCES: The main target audience are soybean growers throughout the United States, soybean pathologists and extension, and certified crop advisors. They were reached through more than 39 meetings of 5000 attendees on oomycete biology and implications for disease management. A tri-fold publication was developed for dissemination. On-line lessons on oomycete isolation and biology were developed for the CEU audience. A project website (oscap.org) was also developed to provide stakeholders with information about the project, meeting and workshop dates, links to information on managing and detecting oomycete pathogens on soybean and other crop plants, and other project-developed materials and resources. Soybean industry stakeholders and soybean pathologists are also targeted in the Annual Root Rot Workshop (rootrot.org) that provides a forum for informal exchange of information and protocols. A second audience are the undergraduates recruited by the oomycete undergraduate network or involved in extension and research activities related to the project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Isolation and identification of oomycete species in soybean will enable project stakeholders to develop management solutions and identify which oomycete species are causing disease in each represented state or region. At least 52 different PYTHIUM species were ID'd in year 1, highlighting the wide range of oomycete species in soybean fields across the U.S. These isolates are being used to develop a molecular diagnostic assay. The identification of 32 soybean accessions with resistance to known Rps genes will be used in future soybean breeding and to develop molecular markers. The identification of at least two RXLR effectors that appear to substantially contribute to virulence provides two targets for new resistance genes. The optimized and validated high-throughput effector-detector system will allow the rapid screening of resistant soybeans for this project and a newly-established collaboration with Chinese colleagues to screen an estimated 2000 Chinese soybean. The first on-line CCA survey highlights the need for more extension information/education and more definitive diagnostic tools. To this end, the extension network has educated close to 5000 participants in more than 39 meetings on oomycete biology and implications on disease management in soybean through project-created presentations, videos, webcasts, and on-line extension newsletter articles. The first cohort of students in the oomycete undergraduate network contributed to network bioinformatics projects and advanced their academic careers. At least 13 posters were developed and presented by members of the oomycete undergraduate network at their respective institutions or at various regional and national meetings. A Careers in SPACES assisted oomycete undergraduates in writing personal statements and developing career options. Students will continue graduate studies at Wake Forest Univ. (Chytrid genomics), Laval University-Quebec (Arctic diatoms), and masters at their respective institutions. The newly established Kids' Tech University at BGSU had 130 registrants with support from a partnership with the 4-H program at the Ohio State University. Four travel awards were awarded to the Soybean Root Rot workshop that brought together industry, extension, and research stakeholders with a free exchange of information and protocols. An initial project meeting was held May 12, 2011 in Chicago, IL to initiate the project and obtain feedback from the Scientific Advisory Board (SAB). A project year-end meeting was held March 16-18, 2012 to update project members and the SAB of progress for the nine project aims through oral and poster presentations and to provide feedback and suggestions for improving or modifying project goals. Based upon discussion and feedback, the sampling protocol was modified to include a second medium that should increase our recovery of typically slower-growing PHYTOPHTHORA and PHYTOPYTHIUM species of which relatively few were isolated and ID'd in year 1 samples.

Publications

  • Arbuckle, J. and Robertson, A. (2011). Oomycete diseases in the North Central Region: A survey of certified crop advisors. Iowa State University Extension and Outreach: Sociology Technical Report 2011. 12pp.
  • Anderson, R.G., Casady, M.S., Fee, R.A., Vaughan, M.M., Deb, D., Fedkenheuer, K., Huffaker, A., Schmelz, E.A., Tyler, B.M. and McDowell, J.M. (2012). Homologous RXLR effectors from Hyaloperonospora arabidopsidis and Phytophthora sojae suppress immunity in distantly related plants. The Plant Journal, Accepted manuscript online: 18 JUN 2012 11:32AM EST DOI: 10.1111/j.1365-313X.2012.05079.x
  • Andersen, K., Ospina-Giraldo, M.D. (2011). Relative disease susceptibility of cultivated varieties of potato to different isolates of Phytophthora infestans. J. PA Acad. Sci. 85:140-146.
  • Andersen, K., Ospina-Giraldo, M.D. (2011). Assessment of the effect of temperature on the late blight disease cycle using a detached leaf assay. J. PA Acad. Sci. 85:165-173.
  • Chilvers, M.I. (2012). Molecular diagnostics in plant disease diagnostic clinic. What is the status (Ed.). Fungal Genomics and Biology. doi: 10.4172/2165-8056.1000e102.
  • McDowell, J.M., (2011). Beleaguered Immunity. Science, 334:1354.
  • McDowell JM, 2011. Genomes of obligate plant pathogens reveal adaptations for obligate parasitism. Proc Natl Acad Sci USA, 108:8921-2.
  • Tripathy, S., Deo, T., Tyler, B.M. (2012). Oomycete transcriptomics database: A resource for oomycete transcriptomes, BMC Genomics, doi:10.1186/1471-2164-13-303.