TREE DISEASE MITIGATION STRATEGIES USING A MOLECULAR APPROACH

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1013157
• Project Start Date: Sep 1, 2017
• Project End Date: Aug 31, 2022
• Program Code: [(N/A)]- (N/A)

Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824

Performing Department
Plant, Soil and Microbial Science

Non Technical Summary
DAD-PathWe will conduct focus group sessions and a statewide survey to systematically evaluate grower's (landscape, nursery, seedling, tree farm and Christmas tree growers) needs and concerns in relation to tree disease issues. The results of this survey will serve to drive the research focus of the Forest Pathology Lab at MSU initially in the development of specific, sensitive and robust molecular tools to rapidly and accurately detect pathogens and determine if they are dead or alive. This is critical to quickly screen plant material destined for export and also rapidly determine if a chemical treatment has been effective. Molecular tools will be developed and optimized in the Forest Pathology Lab at MSU and translated into operational tools in the MSU Diagnostic Services Lab. We will work closely with extension educators and industry professionals to ensure that the survey questions, tool development and application addresses industry needs and concerns. Research updates and findings will be communicated during grower meetings, extension bulletins, in peer reviewed journals, and on two websites: forestpathology.msu.edu and msue.anr.msu.edu.Oak WiltOur work will refine Michigan-specific Oak Wilt control and detection measures designed to reduce spread and improve detection confidence and speed. We will determine key high-risk periods of insect vector activity, fungal spore production and host tree susceptibility, and evaluate molecular methods for identification and source of spread. This project will serve to minimize OW spread and the loss of Michigan oak resources by evaluating and distributing clear, consistent and science-based messages to educate all industries involved with tree-related work, and for the public at large. Research updates and findings will be communicated during meetings, extension bulletins, in peer reviewed journals, and on two websites: forestpathology.msu.edu and msue.anr.msu.edu

Animal Health Component

25%

Research Effort Categories

Basic

50%

Applied

25%

Developmental

25%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
121	0699	1160	25%
123	0699	1160	25%
124	0699	1160	25%
125	0699	1160	25%

Knowledge Area
125 - Agroforestry; 124 - Urban Forestry; 123 - Management and Sustainability of Forest Resources; 121 - Management of Range Resources;

Subject Of Investigation
0699 - Trees, forests, and forest products, general;

Field Of Science
1160 - Pathology;

Keywords

tree pathogens

fungal diseases

fungal pathogens

oak

conifer

detection

population genetics

epidemiology

phytophthora diseases

phytophthora pathogens

survey

Goals / Objectives
Project 1 DAD-Path: Dead or Alive Detection of PathogensOur goal is to provide a rapid response to new disease incursions, a predictive focus to evaluate new disease risks and the means by which current detection and treatment methods of priority diseases can be improved and optimized. This will be achieved by the implementation of a streamlined approach to quantify disease issues across conifer tree crops and a tool development pipeline that can be used to develop and evaluate molecular assays designed to detect pathogens affecting tree crop industries in Michigan and specifically, provide a streamlined approach to link research and development directly to operational deployment of Dead and Alive Detection of Pathogens and chemical treatment evaluation. As a proof-of-concept we have initially targeted our assay development on a range of taxonomically diverse pathogens that target different tissue types in the plant to ensure our pipeline is robust and flexible enough to work on a wide range of disease issues. Additionally, we seek to facilitate systematic and open feedback and information sharing between researchers, educators, agencies and growers. Specific objectives include:1. Determine and quantify fungal disease issues affecting Michigan conifer growers.2. Determine communication pathways preferred by growers.3. Development of molecular detection pipeline and molecular tools that can detect pathogens.4. Development of molecular pipeline and molecular tools that can determine if a pathogen is dead or alive.Project 2. Epidemiology, biology and population genetics of Oak WiltWe seek to determine the key high-risk periods of insect vector activity, fungal spore production and host tree susceptibility in order to define the period during which oak related activities should be halted. We also seek to evaluate molecular methods for identification of OW and to determine the source of new infections. This will enable a rapid response to infection (removal of infected trees) and identify novel paths through which the pathogen is being spread, leading to recommendations to reduce spread rates.We will refine the key elements of the OW disease triangle (host, environment, pathogen and vector) within a MI context (Fig. 1). Specific Objectives include:1. Identification of potential insect vectors of C. fagacearum in MI, their phenology and peak activity periods (visits to oaks), and inoculum loads associated with key insect species.2. Determine key periods of oak susceptibility to infection and relation to xylem vessel development.3. Determine key periods that wounded trees are visited by insect vectors and assess likelihood of infection of wounded oaks by key insect vectors over time4. Determine the temporal duration of viable spore mat production.5. Determine the population structure of C. fagacearum in the US and within MI.6. Develop a SNPchip that can be used to assign new detections to source populations (if genetic structure correlates to geographical location).7. Evaluate whether molecular methods of detection can improve efficacy of positive C. fagacearum identification.

Project Methods
Project 1. DAD-Path: Dead or Alive Detection of PathogensActivity 1. Survey development will involve state-wide focus groups and one-on-one interviews of Agency and industry representatives. The survey will then be administered statewide in Michigan to all known nursery, seedling and Christmas tree growers to assess their current disease issues and treatment plans, their information needs, and their communication preferences. This survey is specifically designed to address both specific (we will list diseases & pathogens of concern) and general disease issues (allow growers to list disease issues not mentioned by specific questions). This will provide quantitative data on disease and pathogen issues which is critical to ensure our species-specific detection tools target the most destructive pathogens.Activity 2. For tool development, three key disease issues have been selected (spruce decline, Weir's cushion rust, and Phytophthora species), a literature review of current and available detection assays for these diseases will be conducted alongside building a DNA sequence database of barcoding genes linked to these pathogens. New sequencing of target genes will be conducted where appropriate, molecular assays will be designed using qPCR (quantitative PCR) and will be tested hierarchically using pure culture DNA mixed samples DNA (pure culture and plant DNA, pure culture DNA of target and sister species DNA) and finally environmental samples (either lab inoculated or from diseased samples collected from growers) (13). We will then be able to evaluate sensitivity, specificity and robustness of assays.Activity 3. RT-PCR (reverse-transcriptase PCR) assays will be designed to target constitutively expressed fungal and oomycete genes (14) in order to confirm if a target pathogen is dead or alive. These genes only express when a pathogen is alive. These assays will be tested initially on living cultures, then in a time series after heat shocking samples. Fungicide treatments of petri plates will then be tested before deployment in field conditions (fungicide treatment of inoculated and naturally infected plants). Extension educators will assist in finding already diseased sampling sites on which to test assays on.Activity 4. For technology transfer and data sharing assays will be developed in the MSU Forest Pathology Lab and tested in the MSU Diagnostic Lab under the guidance of Dr. Jan Byrne. Feedback will be provided to the MSU Forest Pathology Lab and assays modified where required. Research updates and findings will be communicated during grower meetings, extension bulletins, in peer-reviewed journals, and on our website forestpathology.msu.edu and on the MSU Extension website msue.anr.msu.edu. Meetings will be coordinated by the Extension educators. Presentations will be given by Dr. Sakalidis, Dr. Huff and extension educators.Project 2. Epidemiology, biology and population genetics of Oak WiltMolecular Assays. Compare detection by culturing (current method used for positive identification) with recent nested PCR and quantitative real time PCR (qPCR) tests (10 & 19) on samples sent to MSU Plant Diagnostic Clinic. Evaluate if molecular assays can detect pathogen in sub-optimal samples (old, kept at warm temperatures) as well as samples handled correctly. If current methods prove ineffective, target specific unique genes that can be retrieved from the C. fagacearum genome. Integration of molecular assays into current diagnostic practices at MSU Plant Diagnostic Clinic will increase rate, sensitivity and accuracy of OW detection.Population genetics. Obtain cultures of C. fagacearum from US states. Extract DNA and genotype using rad-seq or illumina resequencing (MSU RTSF Genomics Core). Genetic variation and structure will be identified (20 & 21). If genetic structure is sympatric with geographical location a representative set of SNPs will be extracted using DAPC (discriminant analysis of principle components) that can be used to rapidly genotype 96-384 new detections to source populations using a sequenome® array (SNPchip). Samples from study sites will be collected and included in the population study and the trace-back analysis. Trace-back analysis enables prevention strategy evaluation and can identify new pathways of spread.Spore mat production. As part of annual MDNR ground surveys for OW presence of spore mat producing trees will be recorded. In selected trees bark will be removed and any insects immediately present will be collected, identified and the viability and quantity of spores on insect bodies will be determined (22). This will enable monthly inoculum load to be quantified and correlated with insect visit activities.Red oak susceptibility. Trees will be inoculated once per month by applying a spore suspension to a small artificial wound (23) over a period of 6 months (May- October). We will place dendrometer bands on each tree to monitor timing and rates of radial growth. We will collect micro-core samples of bark and outer annual ring tissue from each tree every two to three weeks to assess timing of xylem development (24). Date of bud-break, shoot growth and leaf expansion will be tracked for individual trees to develop easily-observed indicators of tree phenology that can be linked to key events in xylem development (e.g., initiation or cessation of earlywood vessel development), as well as insect vector activity. Associations among tree susceptibility to infection, tree physiology, insect vector activity and climate variables will be determined.Pathogen infection. At OW positive sites, selected healthy trees will be wounded each month and trees will be checked for symptoms every two weeks over a period of 6 months (May- October) (23). Tree phenology as outlined above will be monitored. This will test the seasonal risk to natural infection from wounding and storm damage in OW positive areas.Insect Vectors. Monitor artificial tree wounds of healthy red oak near OW infection centers to identify and quantify and potential insect vectors of OW. Trap insects dispersing from OW infection centers over a period of 6 months (May - October). Insects will be collected from artificial wound sites (wounds will be made once per month) (25). Baited funnel traps (for nitidulid species) (26) will be placed in infection centers and non-baited flight traps (for bark beetle capture) will be placed in the crown of OW-killed trees to collect dispersing insects (27). Collected insects will be identified to species level. Presence of C. fagacearum and spore viability will be determined (26, 27). Insect phenological activity and spore viability will be related to cumulative degree-days (MSU Enviro-weather) to assess the onset, peak and end of vector activity and vector-pathogen-climate variables.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Endusers and stake holders: Invasive Species Centre, Ontario Sakalidis, M.L., Chahal, K. Morris, O., McCullough, D.G., Cregg, B. 2020. Oak wilt in Michigan. Invasive Species Centre, Oakville oak wilt forum, Queen Elizabeth Park Community Centre, Oakville, Ontario. Michigan Nursery and Landscape Association: Sakalidis, M.L. 2020. Pathology research updates on spruce decline and oak wilt. Michigan Nursery and Landscape Association's Great Lakes Trade Expo, January 27-29, Lansing, Michigan. Michigan State University Master Gardeners: Sakalidis, M.L. 2020. When Picnic Beetles Bring Along a Deadly Fungal Pathogen: Michigan oak wilt research updates and management recommendations. Master Gardener's Webinar Series. May 27th. General public: Chahal, K., Morris, O, McCullough, D.G., Cregg, B., Sakalidis, M.L. 2020. Insights into Oak Wilt Pathology in Michigan. ANR Week Forest Health. Addressing threats to Michigan's Forest Health. March 5th, East Lansing, Michigan. Morris, O, Chahal, K., McCullough, D.G., Sakalidis, M.L., Cregg, B., Poland, T. 2020. Insights into Oak Wilt Pathology in Michigan. ANR Week Forest Health. Addressing threats to Michigan's Forest Health. March 5th, East Lansing, Michigan. Changes/Problems:Due to covid my lab was shut down, and now back open due to physical distancing requirements, we operate slower then would typically be desired. What opportunities for training and professional development has the project provided?Includes previous cited oak wilt forum for industry and members of the public: Sakalidis, M.L., Chahal, K. Morris, O., McCullough, D.G., Cregg, B. 2020. Oak wilt in Michigan. Invasive Species Centre, Oakville oak wilt forum, Queen Elizabeth Park Community Centre, Oakville, Ontario. Includes previously cited factsheet distributed to the public: Sakalidis, M.L., Chahal, Morris, O., Cregg, B., McCullough, D.G., Accessed: May 12th 2020. How have the results been disseminated to communities of interest?Yes to industry, agencies and broader public. What do you plan to do during the next reporting period to accomplish the goals?Publish field part of oak wilt project. Publish dead and alive detection project Publish conifer growers survey project.

Impacts
What was accomplished under these goals? Project 1 1. Determine and quantify fungal disease issues affecting Michigan conifer growers. Critical disease concern information indicated that while Colorado blue spruce and Fraser fir account for the most commonly grown tree species, respondents avoided planting these trees due to disease concerns. Needle casts and blights (Rhizosphaeria Needle Blight of Spruce, Diplodia, and Rhabdocline Needlecast), Phytophthora root rot and spruce decline were considered the most common diseases encountered. Critical disease concern information indicated that while Colorado blue spruce and Fraser fir account for the most commonly grown tree species, respondents avoided planting these trees due to disease concerns. Needle casts and blights (Rhizosphaeria Needle Blight of Spruce, Diplodia, and Rhabdocline Needlecast), Phytophthora root rot and spruce decline were considered the most common diseases encountered. 2. Determine communication pathways preferred by growers. We distributed a survey to 587 conifer growers in Michigan, and received a response rate 16% (with 87 respondents) with respondents from both the lower and upper Peninsula of Michigan represented. Most respondents preferred online means of communication either through MSU Extension bulletins or websites. 3. Development of molecular detection pipeline and molecular tools that can detect pathogens. The mansucript for this work is in progress. 4. Development of molecular pipeline and molecular tools that can determine if a pathogen is dead or alive. We can detect if a pathogen is dead or alive using molecular tools. The manuscript is in progress. Project 2 1. Identification of potential insect vectors of C. fagacearum in MI, their phenology and peak activity periods (visits to oaks), and inoculum loads associated with key insect species. Peak periods of insect activity appear to be May and June; and second smaller peak occurs mid-August-mid-September. 2. Determine key periods of oak susceptibility to infection and relation to xylem vessel development. Trees appear susceptible late March-late September. 3. Determine key periods that wounded trees are visited by insect vectors and assess likelihood of infection of wounded oaks by key insect vectors over time Peak periods of insect activity appear to be May and June; and second smaller peak occurs mid-August-mid-September. 4. Determine the temporal duration of viable spore mat production. Mats can be produced any time of year except winter (although not surveyed) and in extremely hot and dry weather. Peak periods of imat production appear to be May and June; and second smaller peak occurs mid-August-mid-September. This overlaps well with insect vector phenology. 5. Determine the population structure of C. fagacearum in the US and within MI. 250 cultures have been obtained from 15 states. These cultures will form the basis of the population study. Reference culture genome has been released by JGI and is available on their website. 6. Develop a SNPchip that can be used to assign new detections to source populations (if genetic structure correlates to geographical location). This activity will not commence until Activity 5 is completed. 7. Evaluate whether molecular methods of detection can improve efficacy of positive C. fagacearum identification. Almost all enviromental samples have been collected for testing purposes.

Publications

• Type: Other Status: Published Year Published: 2020 Citation: Sakalidis, M.L., Chahal, Morris, O., Cregg, B., McCullough, D.G., 2020. Worried about oak wilt? Remember the no prune dates of April 15  July 15 to reduce the chance of oak wilt infection and spread (significant update). MSU Extension. Available: https://www.canr.msu.edu/news/worried_about_oak_wilt. Accessed: May 12th 2020.
• Type: Other Status: Published Year Published: 2020 Citation: Morris, O., McCullough, D.G., Chahal, K., Sakalidis, M.L. 2020. Oak wilt vectors: Activity and contamination rates of sap-feeding nitidulid beetles. Michigan Forest Health Highlights 2019. Page 16. Available: https://www.michigan.gov/documents/dnr/Forest_Health_Highlights_2019_FINAL_-_for_web_FINAL_683429_7.pdf Accessed: May 12th 2020.
• Type: Other Status: Published Year Published: 2020 Citation: Chahal, K., Sakalidis, M.L., McCullough, D.G., Morris, O. 2020. Sakalidis Lab report- Oak wilt research. Michigan Forest Health Highlights 2019. Page 32. Available: https://www.michigan.gov/documents/dnr/Forest_Health_Highlights_2019_FINAL_-_for_web_FINAL_683429_7.pdf Accessed: May 12th 2020.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Government agencies Affiliations: MDARD, MDNR Industry: Seedling and Christmas Tree Growers Academia: Conferences and meetings Oral presentations: Sakalidis, M.L. 2019. Forest Pathology in Michigan. Invited speaker, Department of Entomology and Plant Pathology, North Carolina State University. Chahal, K.*, Morris, O.R.*, McCullough, D.G., Cregg, B., Miles, T., Sakalidis, M.L. 2019. Biology and detection of Bretziella fagacearum, the causal agent of oak wilt, in Michigan. North Central Forest Pest Workshop, The Morton Arboretum, Lisle, Illinois, USA. Morris, O.R.*, McCullough, D.G., Cregg, B., Poland, T.B., Sakalidis, M.L., Chahal, K.* 2019. Oaks and nitidulids: small beetles, big threat by Michigan State University. North Central Forest Pest Workshop, The Morton Arboretum, Lisle, Illinois, USA. Sakalidis, M.L., Medina-Mora, C., Shin, K. 2019. DAD-Path: Dead and Alive Detection of Pathogens; a case study of the Phytophthora root rot-conifer pathosystem in Michigan. 14th International Christmas Tree Research and Extension Conference, Quebec City, Quebec, Canada. Chahal, K.*, Morris, O.R.*, McCullough, D.G., Cregg, B., Sakalidis, M.L. 2019. Preliminary biology and epidemiology of Oak Wilt in Michigan. Arboriculture Society of Michigan (Arborcon), February 12-13, Lansing, Michigan. Morris, O.R.*, McCullough, D.G., Cregg, B., Poland, T.M., Sakalidis, M.L., Chahal, K. 2019. Nitidulid activity and contamination rates compared to cumulative degree days and vascular tissue development in red oaks. Arboriculture Society of Michigan (Arborcon), February 12-13, Lansing, Michigan. Sakalidis, M.L. 2019 Research updates on spruce decline and oak wilt. Arboriculture Society of Michigan (Arborcon), February 12-13, Lansing, Michigan. Sakalidis, M.L. 2019. Oak Wilt Research Updates. FAP Oak Wilt training session, Michigan Department of Natural Resources. June 27th, Grayling, Michigan. Morris, O.R., McCullough, D.G., Cregg, B., Poland, T.M., Sakalidis, M.L., Chahal, K. 2019. Oak wilt: Nitidulid beetle activity, fungal contamination, and xylem development in red oaks. MSU Forestry Dept. Annual Graduate Student Symposium, February, East Lansing, Michigan. Poster presentations: Chahal, K., Morris, O.R., McCullough, D. G., Sakalidis, M.L. 2019. Biology, epidemiology and detection of oak wilt,American Phytopathological Society Meeting, Cleveland, Ohio, USA. Shin, K., Medina Mora, C., Sakalidis, M.L. 2019.Detection of viablePhytophthoraspp. associated with Christmas trees in Michigan using qPCR combined with propidium monoazide (PMA),American Phytopathological Society Meeting, Cleveland, Ohio, USA. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Includes previously cited factsheet and MSU extension article: Sakalidis, M.L. and Marcy, R.D. 2019. Smart Gardening to prevent oak wilt. MSU Extension. Available: https://www.canr.msu.edu/news/smart-gardening-to-prevent-oak-wilt Accessed: August 9th 2019. Sakalidis, M.L. 2019. Worried about oak wilt? Remember the no prune dates of April 15th- July 15th to reduce the chance of infection (updated). MSU Extension. Available: http://msue.anr.msu.edu/news/worried_about_oak_wilt Accessed: April 5th 2019. Includes previously cited training for the DNR Sakalidis, M.L. 2019. Oak Wilt Research Updates. FAP Oak Wilt training session, Michigan Department of Natural Resources. June 27th, Grayling, Michigan. How have the results been disseminated to communities of interest?Yes to industry, agencies and at academic conferences. What do you plan to do during the next reporting period to accomplish the goals?Finalize field part of oak wilt project. Publish dead and alive detection project Publish conifer growers survey project.

Impacts
What was accomplished under these goals? 1. Determine and quantify fungal disease issues affecting Michigan conifer growers. The survey data is now being analysed. 2. Determine communication pathways preferred by growers. This activity is dependent on activity 1 and will be determined once final data analysis has been completed. 3. Development of molecular detection pipeline and molecular tools that can detect pathogens. The mansucript for this work is in progress. 4. Development of molecular pipeline and molecular tools that can determine if a pathogen is dead or alive. We can detect if a pathogen is dead or alive using molecular tools. The manuscript is in progress. 1. Identification of potential insect vectors of C. fagacearum in MI, their phenology and peak activity periods (visits to oaks), and inoculum loads associated with key insect species. Peak periods of insect activity appear to be May and June. 2. Determine key periods of oak susceptibility to infection and relation to xylem vessel development. Trees appear susceptible late March-late September. 3. Determine key periods that wounded trees are visited by insect vectors and assess likelihood of infection of wounded oaks by key insect vectors over time May and June 4. Determine the temporal duration of viable spore mat production. Mats can be produced any time of year except winter (although not surveyed) and in extremely hot and dry weather. 5. Determine the population structure of C. fagacearum in the US and within MI. An additional 134 cultures have been obtained from over 14 states. These cultures will form the basis of the population study. DNA extraction methods have been optimized to ensure high quality and a high quantity of DNA is obtained. Reference culture has been submitted to JGI for genome sequencing. 6. Develop a SNPchip that can be used to assign new detections to source populations (if genetic structure correlates to geographical location). This activity will not commence until Activity 5 is completed. 7. Evaluate whether molecular methods of detection can improve efficacy of positive C. fagacearum identification. In progress as of October 2018.

Publications

• Type: Other Status: Published Year Published: 2019 Citation: Sakalidis, M.L. and Marcy, R.D. 2019. Smart Gardening to prevent oak wilt. MSU Extension. Available: https://www.canr.msu.edu/news/smart-gardening-to-prevent-oak-wilt Accessed: August 9th 2019.
• Type: Other Status: Published Year Published: 2019 Citation: Sakalidis, M.L. and Chahal, K. 2019. Oak wilt control. Forest Health Highlights. Page 13-14. Available: https://www.michigan.gov/documents/dnr/frsthlthhghlghts_513144_7.pdf?20160216094536 Accessed: March 27th 2019.
• Type: Other Status: Published Year Published: 2019 Citation: Sakalidis, M.L. 2019. Worried about oak wilt? Remember the no prune dates of April 15th- July 15th to reduce the chance of infection (updated). MSU Extension. Available: http://msue.anr.msu.edu/news/worried_about_oak_wilt Accessed: April 5th 2019.

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

Outputs
Target Audience:Interacted with other government agencies Affiliations: MDARD, MDNR Industry: Seedling and Christmas Tree Growers Fall Interagency Forest Invasives Committee Meeting. December 6th 2017. Lansing, Michigan Overview of Forest Pathology Lab Projects (15 min). Department of Natural Resources Professional Development Training "Leading Michigan in Conservation for the Common Good. January 25th 2018. Traverse City, Michigan. Oak Wilt, it's here, it's spreading- so what are we doing about it? (30 min talk) Winter Nursery Meeting. February 16th 2018. West Olive, Michigan. MSU Research Update on Spruce Decline" and "What are your concerns? Conifer Growers Disease Questionnaire" (20 min talk). ANR Week Forest Health. Addressing threats to Michigan's Forest Health. March 8th 2018. East Lansing, Michigan. Current Research on Oak Wilt? (40 min talk) What is causing spruce decline? (40 min talk). Sakalidis, M.L. (2018) Update on diseases that threaten conifer seedlings & other pathology research. Seedling Growers Meeting September 11th. Northern Pines Nursery, Lake City, Michigan. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Through various talks to conifer growers (citations in previous reponses). What do you plan to do during the next reporting period to accomplish the goals?1. Determine and quantify fungal disease issues affecting Michigan conifer growers. Analyze survey data and publish. 2. Determine communication pathways preferred by growers. This activity is dependent on activity 1 and will be determined 2019. 3. Development of molecular detection pipeline and molecular tools that can detect pathogens. Optimize Phythophthora detection and transfer to diagnostic lab. 4. Development of molecular pipeline and molecular tools that can determine if a pathogen is dead or alive. This activity is dependent on activity 3 and is in progress. 1. Identification of potential insect vectors of C. fagacearum in MI, their phenology and peak activity periods (visits to oaks), and inoculum loads associated with key insect species. Insects will continue to be collected during 2019. Data will be analysed 2020. 2. Determine key periods of oak susceptibility to infection and relation to xylem vessel development. Tree cores will continue to be collected and analyzed, trees will continue to be inoculated throughout 2017 -2019. Data will be analysed 2020. 3. Determine key periods that wounded trees are visited by insect vectors and assess likelihood of infection of wounded oaks by key insect vectors over time Insects will continue to be collected and identified and trees will continue to be wounded throughout 2017-2019. Data will be analysed 2020. 4. Determine the temporal duration of viable spore mat production. We expected further collections in 2019. 5. Determine the population structure of C. fagacearum in the US and within MI. DNA will be extracted from cultures and submitted for genome resequencing, data analysis will commence. Develop a SNPchip that can be used to assign new detections to source populations (if genetic structure correlates to geographical location). This activity will not commence until 2020 Evaluate whether molecular methods of detection can improve efficacy of positive C. fagacearum identification. This activity is in progress.

Impacts
What was accomplished under these goals? 1. Determine and quantify fungal disease issues affecting Michigan conifer growers. Survey was deployed and there were 90 useable respondants. The data is now being analysed. 2. Determine communication pathways preferred by growers. This activity is dependent on activity 1 and will be determined once final data analysis has been completed. 3. Development of molecular detection pipeline and molecular tools that can detect pathogens. Post doc has developed this on Phytophthora root rot and can detect Phytophthora from soil and root samples directly using q-PCR. 4. Development of molecular pipeline and molecular tools that can determine if a pathogen is dead or alive. This activity is dependent on activity and is in progress. We can detect if a pathogen is dead or alive using molecular tools. 1. Identification of potential insect vectors of C. fagacearum in MI, their phenology and peak activity periods (visits to oaks), and inoculum loads associated with key insect species. We screened 294 insects captured in traps at research sites between 29 August 2017 to 8 May 2018 for the presence of B. fagacearum. Based on morphological identification of the fungus, two beetles; Glischrochilus fasciatus and Colopterus truncates collected on 8 May from Nessen site were carrying viable B. fagacearum spores. 2. Determine key periods of oak susceptibility to infection and relation to xylem vessel development. Of the 30 trees infected with B. fagacearum only 7 remain healthy. Those infected in August and September 2018 and October 2017 are still healthy i.e. asymptomatic. 3. Determine key periods that wounded trees are visited by insect vectors and assess likelihood of infection of wounded oaks by key insect vectors over time Bi-weekly visits have resulted in over1109 nitidulid beetles representing six genera to species insects being collected. 4. Determine the temporal duration of viable spore mat production. 28 mats have been screened. 3 mats in March 2018, 1 mat in April, 3 mats in May and 1 mat in June contained spores. 5. Determine the population structure of C. fagacearum in the US and within MI. 80 cultures have been obtained from over 10 states. These cultures will form the basis of the population study. DNA extraction methods have been optimized to ensure high quality and a high quantity of DNA is obtained. 6. Develop a SNPchip that can be used to assign new detections to source populations (if genetic structure correlates to geographical location). This activity will not commence until Activity 5 is completed. 7. Evaluate whether molecular methods of detection can improve efficacy of positive C. fagacearum identification. In progress as of October 2018.

Publications

• Type: Other Status: Published Year Published: 2018 Citation: Sakalidis, M.L. 2018. Worried about oak wilt? Remember the no prune dates of April 15th- July 15th to reduce the chance of infection. MSU Extension. Available: http://msue.anr.msu.edu/news/worried_about_oak_wilt Accessed: March 30th 2018
• Type: Other Status: Published Year Published: 2018 Citation: Sakalidis, M.L. 2018. Oak Wilt. Forest Health Highlights. Page 11. Available: https://www.michigan.gov/documents/dnr/frsthlthhghlghts_513144_7.pdf?20160216094536 Accessed: May 28th 2018.

Progress 09/01/17 to 09/30/17

Outputs
Target Audience:Project Relavent Audiences (projects were discussed either formally with slides, or informally just spoken verbally to the group) Overview of oak wilt project and Introduction to the Sakalidis Lab. November 2nd 2016, Fall Interagency Forest Invasives Committee Meeting (Affiliations: MDARD, DNR, USDA FS, OMNRF). November 10th- attended Michigan Forest Association Board Meeting (Private landowners, foresters, MSU Extension (Georgia Petterson, Bill Cook) in Hardwick State Park, Michigan. November 17th- attended Arboriculture Society of Michigan board meeting (tree care professionals) in Lansing, Michigan. November 22nd- attended the "Oak Wilt Coalition" board meeting (attendees included representatives from Agencies (MDARD, DNR), tree care professionals (arborists), Utilities (Consumer Energy) and MSU extension in Lansing Michigan. Natural Resource Working group Zoom meeting. February 1st 2017. Talk (1 hour): Forest Pathology at MSU (MSU Extension specialists) Arborcon. February 15th 2017. Lansing Michigan. Talks: Sakalidis Lab Overview (15min) & A next generation approach to forest pathology (1 hour). Audience: Tree care professionals- arborists West Michigan Nursery Meeting. February 17th 2017. Grand Rapids, Michigan Talk (1 hour): Spruce Decline: past, present and future Michigan Christmas Tree Association Winter Meeting. March 2nd-3rd 2017. Traverse City, Michigan Talk (30min): Christmas Tree Disease Updates Sakalidis, M.L. 2017. A Next Gen Approach to Forest Pathology. March 7th. MDARD: Plant Industry Spring Training. Lansing, Michigan. Michigan Seedling Growers Meeting. August 24th 2017. Bosch's Countryview Nursery Choose and Cut Christmas Tree Barn 8075 Pierce St, Allendale, MI 49401. Talk Title:"Updates on MSUs Conifer Research Program" Changes/Problems:Key Personnel for this project (1 graduate student and 1 post doc) were not able to start until September 2017 (post doc) and October 2017 (graduate student) this has resulted in our activities not being as advanced as we had hoped. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?1. Determine and quantify fungal disease issues affecting Michigan conifer growers. Survey will be distributed to growers and results analysed 2. Determine communication pathways preferred by growers. This will be determined once results are known. 3. Development of molecular detection pipeline and molecular tools that can detect pathogens. This work will be commenced and completed by pathogen group. 4. Development of molecular pipeline and molecular tools that can determine if a pathogen is dead or alive. This work will be commenced in 2019. 1. Identification of potential insect vectors of C. fagacearum in MI, their phenology and peak activity periods (visits to oaks), and inoculum loads associated with key insect species. Insects will continue to be collected and identified throughout 2017 and 2018 (collection period). Data will be analysed 2019. 2. Determine key periods of oak susceptibility to infection and relation to xylem vessel development. Tree cores will continue to be collected and analyzed, trees will continue to be inoculated throughout 2017 and 2018 (collection period). Data will be analysed 2019. 3. Determine key periods that wounded trees are visited by insect vectors and assess likelihood of infection of wounded oaks by key insect vectors over time Insects will continue to be collected and identified and trees will continue to be wounded throughout 2017 and 2018. Data will be analysed 2019. 4. Determine the temporal duration of viable spore mat production. We expected further collections in 2018. 5. Determine the population structure of C. fagacearum in the US and within MI. DNA will be extracted from cultures and submitted for genome resequencing, data analysis will commence. 6. Develop a SNPchip that can be used to assign new detections to source populations (if genetic structure correlates to geographical location). This activity will not commence until 2019 7. Evaluate whether molecular methods of detection can improve efficacy of positive C. fagacearum identification. This activity will not commence until 2019

Impacts
What was accomplished under these goals? 1. Determine and quantify fungal disease issues affecting Michigan conifer growers. Survey has been drafted and questions are currently being reviewed prior to distribution to growers. 2. Determine communication pathways preferred by growers. This activity is dependent on activity 1 3. Development of molecular detection pipeline and molecular tools that can detect pathogens. Post doc has just started in the lab to work on this. We have collected cultures and tissue samples for testing purposes. 4. Development of molecular pipeline and molecular tools that can determine if a pathogen is dead or alive. This activity is dependent on activity 3. 1. Identification of potential insect vectors of C. fagacearum in MI, their phenology and peak activity periods (visits to oaks), and inoculum loads associated with key insect species. Traps were established at 4 sites in July. Bi-weekly visits have resulted in over 700 insects being collected. 2. Determine key periods of oak susceptibility to infection and relation to xylem vessel development. Tree cores have been collected bi-weekly since July. Cores are currently being analyzed. Trees have been infected starting in August. To date all 3 trees inoculated in August have become symptomatic for oak wilt infection and 1 tree inocuated in September has become symptomatic. 3. Determine key periods that wounded trees are visited by insect vectors and assess likelihood of infection of wounded oaks by key insect vectors over time Traps were established at 4 sites in July. Bi-weekly visits have resulted in over 700 insects being developed. 4. Determine the temporal duration of viable spore mat production. We have received a small number pressure pads but none were mature and therefore no spores were present. We expected further collections in 2018. 5. Determine the population structure of C. fagacearum in the US and within MI. 66 cultures have been obtained from 10 states. These cultures will form the basis of the population study. DNA extraction methods have also been tested to ensure high quality and a high quantity of DNA is obtained. 6. Develop a SNPchip that can be used to assign new detections to source populations (if genetic structure correlates to geographical location). This activity will not commence until Activity 5 is complted 7. Evaluate whether molecular methods of detection can improve efficacy of positive C. fagacearum identification. Not yet started

Publications

• Type: Other Status: Published Year Published: 2017 Citation: Sakalidis, M.L. 2017. Oak Wilt. Michigan Forest Association magazine Michigan Forests (non-peer reviewed). Volume 39, Issue 3, pages 14-15
• Type: Websites Status: Published Year Published: 2017 Citation: Sakalidis, M.L., Bhakta, B. 2017. National Invasive Species Awareness Week: Michigan Species and What You Should Know; Oak Wilt. URL: http://msue.anr.msu.edu/news/national_invasive_species_awareness_week_oak_wilt_msg17_bohling17 Accessed on Oct 3rd 2017(non-peer reviewed)