Source: UNIV OF MASSACHUSETTS submitted to
FUNCTIONAL GENOMICS TO INVESTIGATE AND CHARACTERIZE GENETIC FACTORS GOVERNING DMI-SENSITIVITY IN SCLEROTINIA HOMOEOCARPA
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0230656
Grant No.
(N/A)
Project No.
MAS00436
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2012
Project End Date
Sep 30, 2014
Grant Year
(N/A)
Project Director
Jung, G.
Recipient Organization
UNIV OF MASSACHUSETTS
(N/A)
AMHERST,MA 01003
Performing Department
Stockbridge School of Agriculture
Non Technical Summary
The research described in this proposal involves utilizing genomics and molecular biology tools to understand the basis of DMI fungicide resistance in the most important disease of turf grasses for golf courses, dollar spot. For this, we will take advantage of cutting-edge research tools in genomics and molecular biology to shed light on how the dollar spot fungus is able to overcome fungicides at the molecular level. Benefits from this work include new targets for fungicide development, new targets for diagnostic assays to detect fungicide resistance, and a better scientific understanding of the molecular basis of DMI fungicide resistance.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2122130104010%
2124020104020%
2125220104020%
5112130104010%
5114020104020%
5115220104020%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants; 511 - New and Improved Non-Food Products and Processes;

Subject Of Investigation
2130 - Turf; 4020 - Fungi; 5220 - Pesticides;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
The planned research for this project employs a multidisciplinary approach to understanding the molecular basis of fungicide resistance in the most important of turf pathogens, Sclerotinia homoeocarpa, causal agent of the dollar spot disease of turf. Using cutting-edge tools in genomics and molecular biology, we plan to identify and characterize the genes involved in resistance to DMIs and additional fungicide classes. During the first year of this project, we aim to complete RNA-Seq experiments, so that we have a global picture of the total complement of genes, which show differential expression between highly DMI fungicide-resistant and fungicide-sensitive strains of S. homoeocarpa from New England. These results will be prepared for publication in a scientific journal and will be presented at the 2013 national conference for the American Phytopathological Society in Texas. During the second year of funding, we will begin to functionally characterize genes likely involved in fungicide resistance using quantitative PCR and yeast complementation experiments. These results will also be prepared for publication and presented at a national conference. We will also communicate relevant findings during the UMASS Turf Research Field Day events. Over the course of the project, we plan to train at least one PhD student, along with several motivated undergraduate students. In summary, the results from this work will provide the following to the agricultural science community: a better scientific understanding of fungicide resistance in dollar spot, new targets for the development of novel fungicides, and new targets for molecular diagnostic assays to detect fungicide resistance. This research will also benefit turf management for disease control of dollar spot, by providing grounds for more effective disease control strategies that maximize the efficacy of fungicide inputs, while minimizing the use of ineffective fungicides. Further, we plan to utilize results from this study to develop molecular diagnostic assays for fungicide resistance, which will complement the current development of fungicide resistance field kits in the Jung lab.
Project Methods
For this project, we are employing techniques in de novo fungal genomics, genetics, and molecular biology to Sclerotinia homoeocarpa. We have recently made publicly available the partial genome and transcriptome sequences of this pathogen, which will further stimulate research into mechanisms of it's evolution and pathogenicity. Our lab has undertaken genomics of this fungus for the express purpose of understanding the molecular basis of fungicide resistance, which is a novel approach for research into fungicide resistance of plant pathogens in general. More specifically, we are utilizing RNA-Seq technology, which entails a global analysis of gene expression data using Pearson Correlation Coefficients. Whereas most of previous published studies have investigated the role of single genes or gene families in fungicide resistance, we intend to look across gene families for global patterns of gene expression that underlie fungicide resistance phenotypes. We currently have a single biological replicate of RNA-Seq data, but the majority of the first year of the proposed work includes generating another biological replicate of data, which will allow us to compute robust statistical analyses and publish our work in a competitive scientific journal. The next phase of this research to begin in year two involves validating the RNA-Seq gene expression data using quantitative PCR and yeast complementation experiments. The quantitative PCR data will be gathered from isolates of the pathogen which have been recovered over the course of a two-year field efficacy study on DMI fungicides and S. homoeocarpa, and statistically analyzed for significant gene expression patterns in fungicide-resistant fungal isolates. In doing so, we are utilizing genomic data to better understand field resistance, which is a revolutionary approach for fungicide resistance research by bringing together field studies and genomics tools. Another research methodology we are employing, yeast complementation, will allow us to functionally characterize fungicide resistance genes. Briefly, this method involves expressing putative fungicide resistance genes in yeast mutants to determine if the putative fungicide-resistance transgenes can complement known mutations in the yeast strain, allowing the yeast to overcome fungicides in the growth medium. We plan to develop this method to not only screen DMI resistance determinants, but also genes potentially involved in resistance to multiple fungicide chemistries. Results from this portion of the study will provide a basis for DMI and multiple-resistance, and will also provide an experimental pipeline for screening additional genes of interest. Additionally, results from these experiments will be published and presented on, as they are novel for use in turf pathogens and fungicide resistance research in turf diseases. In summary, insight gained from our novel approach will be shared within the turf and agriculture scientific community at large and utilized to develop future services to benefit turf and agriculture.

Progress 10/01/12 to 09/30/14

Outputs
Target Audience: The target audiences are turfgrass pathologists, turfgrass chemical sale persons and turfgrass managers (golf courses, atheretic fields, lawn care companies). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? In terms of training, a graduate student, Hyunkyu Sang of the Plant & Soil Graduate Program, has been working under the guidance and training of Drs. Geunhwa Jung and Jon Hulvey, postdoctoral associate in the Jung lab. Dr. Jon Hulvey was trained under the project and has published a high impact journal publicaiton, Applied and Environmental Microbiology. Mr. Sang's research has led him to present at multiple national and regional conferences, and he has published a high impact paper, Molecular Plant Pathology. Several undergraduate students have been trained in the areas of bioinformatics, molecular biology/genetics, and genomics. These students worked on semester-long research projects as part of the Biology Undergraduate Research Assistantship program. Of these students was Mr. Dylan Kessler (Honor thesis), Mr. Jayson Stoner (Honor thesis) and Ms. Nicole Boisvert who presented research findings at a national and regional conference, as well as the Honors Undergraduate Research Symposium here at UMASS. Three other undergraduates, Soun Heang-Lee, Sean Gillis, and Robert Greens (Honor thesis) were also trained as part of this program. Sean Gillis and Jayson Stoner received a competitive honors college research fellowship through his work in my lab in 2013 and 2014. How have the results been disseminated to communities of interest? Results from this project have been presented at national conferences (American Phytopathological Society meeting in 2013 and 2014, and the Crop Science meeting in 2013 and 2014) and one regional conference, the Northeastern Division of the American Phytopathological Society in 2013 and 2014. Results have also been published as research abstracts for the American Phytopathological Society meeting and the Crop Sciences meeting. These abstracts are viewable by the greater research community. Finally, findings of the work have also been presented at the 2013 UMASS Turf Research Field Day and the Golf Course Superintendents Association meetings in 2013 and 2014. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The research outlined in this project has yielded the expected outcomes in terms of producing novel findings on fungicide resistance and training of two personnels (post-doctoral associate and Ph.D. student). This is evidenced by research presentations and publications inclduing a book chapter, extension publications, and extension talks. Additionally, seven undergraduate students have been trained over the course of this project. We have been working in characterizing novel genes (three were published in two reputable journals with high impacts) that were identified during genomic screens for their role in DMI fungicide resistance. With this information, we have been working to develop new molecular diagnostic tests for detecting fungicide resistance from infected plant material, which will be used as part of fee-based diagnostic services in future.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Development of molecular diagnostic assays for fungicide resistance in an important turfgrass pathogen, Sclerotinia homoeocarpa. J Hulvey, A Bousquet, H Sang, G Jung. PHYTOPATHOLOGY 103 (6), 63-63
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: De novo RNA-Seq and bioinformatic analyses uncover genetic determinants of fungicide detoxification in the turfgrass pathogen Sclerotinia homoeocarpa. J Hulvey, H Sang, A Berg, M Mullis, JT Popko, T Chang, G Jung. PHYTOPATHOLOGY 103 (6), 63-63
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Heterokaryons of Sclerotinia homoeocarpa exhibit enhanced adaptability to multiple fungicide pressures. D Kessler, J Hulvey, G Jung. PHYTOPATHOLOGY 103 (6), 73-73
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Hyunkyu Sang, Jon Hulvey, James T. Popko Jr., John Lopes, Aishwarya Swaminathan, Taehyun Chang, and Geunhwa Jung. 2015. A pleiotropic drug resistance transporter is involved in reduced sensitivity to 1 multiple fungicide classes in Sclerotinia homoeocarpa (F.T. Bennett). Molecular Plant Pathology: 16:251-261. DOI: 10.1111/mpp.12174.
  • Type: Book Chapters Status: Awaiting Publication Year Published: 2015 Citation: E. Allan, J. Hulvey, J. Popko, N. Mitkowski, J. Vargas, and G. Jung. 2014. Fungicide resistance in turfgrass pathogens. Fungicide Resistance In North America. American Phytopathological Society.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: High-resolution DNA melting analysis as a tool to screen for genetic polymorphism in fungicide resistance genes for two important fungal plant pathogens, Sclerotinia homoeocarpa and Colletotrichum cereale. J Hulvey, N Mitkowski, H Sang, G Jung. PHYTOPATHOLOGY 104 (3), 3-3
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: 2014 RNA-Seq and heterologous yeast expression provide evidence for novel fungicide resistance determinants with parallels to pesticide detoxification in plants and insects. J Hulvey, H Sang, T Chang, G Jung. PHYTOPATHOLOGY 104 (3), 2-2
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Investigation of the role of an ABC-G transporter from Sclerotinia homoeocarpa in resistance to multiple fungicide classes by a heterologous yeast expression system. H Sang, J Hulvey, G Jung PHYTOPATHOLOGY 104 (3), 6-6
  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Hulvey, J., Popko, J., Sang, H., and G. Jung. 2012. Overexpression of ShCYP51B and ShatrD in Sclerotinia homoeocarpa field isolates exhibiting practical field resistance to propiconazole. Applied and Environmental Microbiology 78:6674-6682.


Progress 10/01/12 to 09/30/13

Outputs
Target Audience: There are two targets audience for this work, research plant pathologists and managers of golf courses. Both audiences have been updated on research findings over the course of the past year through the publications (detailed in the Products section). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? In terms of training, a graduate student, Hyunkyu Sang of the Plant Biology Graduate Program, has been working under the guidance and training of Drs. Geunhwa Jung and Jon Hulvey, postdoctoral associate in the Jung lab. Mr. Sang's research has led him to present at two national conferences and one regional conference, and he is currently preparing his findings for publication. He and Dr. Hulvey are co-authors on a manuscript to describe new genes involved in fungicide resistance, which will be submitted to a competitive research journal in early 2014. Several undergraduate students have been trained in the areas of bioinformatics, molecular biology, and genomics by Dr. Hulvey, as well. These students worked on semester-long research projects as part of the Biology Undergraduate Research Assistantship program. Of these students was Mr. Dylan Kessler who presented research findings at a national and regional conference, as well as the Honors Undergraduate Research Symposium here at UMASS. Four other undergraduates, Martin Mullis, John Stevens, Soun Heang-Lee, and Sean Gillis were also trained as part of this program in 2013. Sean Gillis received a competitive honors college research fellowship through his work in our lab in 2013, as well. How have the results been disseminated to communities of interest? Results from this project have been presented at two national conferences (American Phytopathological Society 2013 meeting in Austin, TX, and the Crop Science meeting in Miami, FL) and one regional conference, the Northeastern Division of the American Phytopathological Society. Results have also been published as research abstracts for the American Phytopathological Society meeting. These abstracts are viewable by the greater research community. Finally, findings of the work have also been presented at the 2013 UMASS Turf Research Field Day. What do you plan to do during the next reporting period to accomplish the goals? We will continue to advance our research and present findings at conferences, while training graduate and undergraduate students along the way. We are in the midst of preparing a research publication for submission to a competitive plant pathology journal early this year. This will be the first report of a gene involved in reduced sensitivity to multiple fungicides in the dollar spot pathogen. Finally, we plan to refine our new molecular diagnostic tests developed in 2013 for detecting fungicide resistance in dollar spot samples collected from field sites.

Impacts
What was accomplished under these goals? The research outlined in this project has yielded the expected outcomes in terms of producing novel findings and training of personnel. This is evidenced by research presentations and publications, as well as informal extension talks. One graduate student has been trained, and has presented his findings at two national and one regional conference this year alone. This student is also involved in preparing a manuscript to describe a new gene involved in both DMI, and reduced sensitivity to multiple fungicides in the dollar spot fungus. Additionally, three undergraduate students have been trained over the course of the last year in this project. We are now working to characterize novel genes that were identified during genomic screens for their role in DMI fungicide resistance.With this information, we are also working to develop new molecular diagnostic tests for predicting fungicide resistance at golf course sites. Another new development is the development of a protocol for detecting resistance from infected plant material. These diagnostic assays are being refined for the purpose of using as a service to golf courses, in order to aid and promote proper fungicide usage. In total, our goals have been met and span research publications, training, developing tools to benefit the turf industry, and expanding the general knowledge base of DMI fungicide resistance.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Hulvey, J., H. Sang, A. Berg, M. Mullis, J. T. Popko, T. Chang, and G. Jung. 2013. "De novo RNA-Seq and bioinformatic analyses uncover genetic determinants of fungicide detoxification in the turfgrass pathogen Sclerotinia homoeocarpa." Phytopathology. 103:63.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Hulvey, J., A. Bousquet, H. Sang, and G. Jung. 2013. "Development of molecular diagnostic assays for fungicide resistance in an important turfgrass pathogen, Sclerotinia homoeocarpa." Phytopathology. 103: 63.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Sang, H., J. Hulvey, J. T. Popko, T. Chang, and G. Jung. 2013. "Investigating genetic mechanisms of decreased sensitivity to iprodione in field isolates of Sclerotinia homoeocarpa." Phytopathology. 103: 126.
  • Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2014 Citation: Hulvey, J., Mitkowski, N., and G. Jung. 2014. "High-resolution DNA melting analysis as a tool to screen for genetic polymorphism in fungicide resistance genes for two important fungal plant pathogens, Sclerotinia homoeocarpa and Colletotrichum cereale." Phytopathology (in press)
  • Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2014 Citation: Hulvey, J., Sang, H., and G. Jung. 2014. "RNA-Seq and heterologous yeast expression provide evidence for novel fungicide resistance determinants with parallels to pesticide detoxification in plants and insects." Phytopathology (in press)


Progress 09/30/11 to 09/30/12

Outputs
OUTPUTS: The first event to be considered a dissemination of our research results was an invited seminar to the Plant Biology Graduate Program Seminar Series, by postdoctoral researcher Dr. Jon Hulvey. Next, a layperson's summary of results from this project were presented to an audience of turf grass and landscape managers at the annual UMASS Turf Research Field Day on August 1, 2012. This talk was intended to share our findings with a general audience, so that they can better understand the practical applications of our genomic and molecular biology work. Finally, research results from this project have also been disseminated by means of research abstracts and presentations during the annual American Phytopathological Society meeting on August 4-8, 2012. The research of one graduate student, Mr. Hyunkyu Sang, as well as that of Dr. Hulvey, were presented in oral and poster formats. Finally, results from this research were most recently presented during the MassMyco meeting at Clark University in Worcester, MA. This was a regional meeting of fungal biologists in Massachusetts and surrounding states. Taken together, these presentations have been a catalyst for new collaborations and community interest in our unique approach to unraveling the mechanisms and implications of DMI fungicide resistance in Dollar Spot. PARTICIPANTS: During the course of this project, since September of 2011, Drs. Geunhwa Jung and Jon Hulvey have been involved in mentoring and training 2 PhD students (Ms. Elisha Allan and Mr. Hyukyu Sang) of the Plant Biology and Plant, Soil, and Insect Graduate Programs. Additionally, two undergraduate students majoring in Biology (Dylan Kessler and Martin Mullis) have completed Independent Study credits by assisting in research efforts directly related to the Hatch project. One student from the Plant Biology Graduate Program, Amanda Bousquet, is currently working to complete a lab rotation and has contributed to this project. Drs. Jung and Hulvey have recently published a recent research article that is the first demonstration of implementing the RNA-Seq technology to gain understanding of the genetic mechanisms of DMI fungicide resistance and field resistance. Drs. Jung and Hulvey also are working together on a book chapter on fungicide resistance in turf grasses for the 2nd Edition of Fungicide Resistance in North America. As mentioned in the Impacts/Outcome section, Drs. Jung and Hulvey have received funding from the USGA in 2013 for research and development of a molecular fungicide resistance assay, bringing the possibility that data from this Hatch project will have a direct application in the field for directing fungicide usage for control of Dollar Spot. Dr. Li-Jun Ma has provided technical support for the computational biology portion of this Hatch project. Dr. John Lopes has also provided research support for the development of experiments to functionally characterize the genetic determinants of fungicide resistance. TARGET AUDIENCES: There are two target audience for this work, research plant pathologists and managers of golf courses. Both audiences have been addressed with regards to our research findings over the course of the past year, through events detailed in the Outputs section, as well as in research and university extension publications. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
During the course of this work, the have developed techniques, which were not previously available, for functional genomics research of fungicide resistance in the Dollar Spot fungus. That is to say, we have put together an experimental pipeline for uncovering new genes and validating their role in practical field resistance to DMI fungicides. Another recent major outcome of this project has been an offshoot project aimed at the development of molecular fungicide resistance assays for Dollar Spot. The genetic data gathered for this Hatch grant has been used to develop a genetic-based fungicide resistance assay, which will save time and resources for golf course managers by giving them rapid diagnoses of fungicide resistance. The United States Golf Association in has committed to funding assay development for the year 2013. Ultimately the aim is to develop the assay so that it can be performed in the field, for a low cost, and provide dependable results onsite in a matter of hours. In turn, these results can be used to guide site-specific recommendations for fungicide usage.

Publications

  • Hulvey, J., Popko, J.T. Jr., Sang, H., and G. Jung. 2012. Overexpression of ShCYP51B and ShatrD in Sclerotinia homoeocarpa field isolates exhibiting practical field resistance to a demethylation inhibitor fungicide. Applied and Environmental Microbiology. 78: 6674- 6682.
  • UMASS Center for Agriculture, Research and Extension. 2012. "Turf Wars: How UMass Amherst Scientists Are Beating a Recalcitrant Fungus at Its Own Game". http://ag.umass.edu/news-events/highlights/turf-wars
  • Hulvey, J., Popko, J.T. Jr., Sang, H., and G. Jung. 2011. Overexpression of the BcatrD homolog from Sclerotinia homoeocarpa field isolates which exhibit practical field resistance to propiconazole. Phytopathology. 102: S1.1.