What is oak wilt?
Oak Wilt is a fungal disease caused by Bretziella fagacearum. It mainly affects Oak trees (Quercus spp.) by infiltrating into the vasculature and blocking the transportation of water and other nutrients, eventually leading to the death of the tree. It can devastate forests and urban landscapes as no treatment is available.
Current impact
Oak wilt is widespread across the Northeastern states and has already killed more than 500,000 oak trees in Michigan. On Belle Isle, a small island 600 meters from Windsor, Ontario, oak wilt has killed more than 100 trees. The cities of Windsor, Sarnia, London, and Hamilton, have all recently sounded the alarm on the potentially devastating impacts of oak wilt to Ontario’s parks and forests. Historically, vascular wilt tree diseases have had catastrophic consequences: Dutch elm disease killed more than 40 million elm trees in North America between 1930 and 1973; chestnut bark disease eliminated the chestnut tree population in the Eastern states (Campbell 2022).
Potential consequences of late detection
The cost to remove and replace infected oaks in Canada is estimated to be at least $266 million, with an estimated cost of $48.8 million to the City of Toronto alone. As well, the lumber industry is a critical sector of Ontario’s economy, with $47 million in revenue coming from oak wood exports. Oak trees are also important for biodiversity in Southern Ontario. They support over 450 species of caterpillars which make up an important food source for birds. Acorns from oaks also support blue jays and chipmunks (Cornelis 2021). The mass loss of oak trees will be devastating for our local ecosystem. Early detection of oak wilt can prevent the destruction of oak trees in Ontario and its damaging consequences to our local economy.
The traditional approach to diagnosing oak wilt is to ship a large amount of wood sample on ice to a nearby diagnostics lab for technicians to isolate and incubate the fungus. This process is time-consuming (takes 3-5 weeks) and is prone to false negatives. Notably, the difficulty in isolating the fungus from oak tree samples is responsible for delayed detection of oak wilt, especially in white oaks, leading some conservation authorities to mistakenly believe that white oaks are immune to oak wilt (Belter 2020). Diagnosis by visual inspection of wilting oak leaves is possible, but if a tree is infected during the summer, symptoms may only appear in the next year, leading to significant delays in a time-sensitive disease (Juzwik et al. 2008).
Our Approach
We designed LAMP primers targeting existing DNA regions known to be unique signatures of Bretziella fagacearum. To simplify and make more cost-effective the approaches of past iGEM teams using LAMP, we combined the DNA amplification and detection steps into one without the need for a separate downstream detection component such as a toehold switch or CRISPR/Cas12a. To achieve this, we used a pH-based detection method where a positive result can be easily detected by visible colour change of a pH indicator. A notorious problem with LAMP-based DNA detection methods is their potential for false positives which can be affected by the purity of the DNA extract. To account for potential contaminants and impurities from an on-site sample, we measured the sensitivity and specificity of our LAMP primers to Bretziella fagacearum DNA. Finally, we demonstrated that our one step DNA detection kit can be freeze-dried and thus shipped without relying on costly cold-chain transport.
Hardware
The current detection requires 7-10 days and $30-40 per test to obtain the result of infection and this process involves delivering the sample to a laboratory and culturing oak wilt fungus. Therefore, the hardware team developed a device to support on site fungus DNA extraction and the LAMP experiment. Our hardware designed a drill, a reagent mixing tool, a Dipstick DNA extraction tool, and a heater, and packaged them with the LAMP reagents into a single tool kit. This milestone is crucial as it complements the on-site detection of oak wilt using LAMP, helps the stakeholders to perform experiments on site with a lower cost, and reduces the amount of operational steps performed by human.
Integrated human practices
Though actively engaging with local stakeholders including forestry managers, plant pathologists, and the Invasive Species Centre, our team gained valuable insights into the limitations of current approaches to detect oak wilt. We understood the urgency of the issue and the devastating consequences of removing infected oak trees, ranging from a massive loss of carbon storage and the loss of ecosystems dependent on oak trees. Importantly, the stakeholders advised us to make the device as simple to use as possible while not sacrificing accuracy and would be of interest to government monitoring programs of oak wilt.
Relevant Literature
Campbell, F. (2022, May 16). Oak wilt. Don't Move Firewood. Retrieved June 21, 2022, from https://www.dontmovefirewood.org/pest_pathogen/oak-wilt/
CBC/Radio Canada. (2018, February 21). Erca joins oak wilt fight as deadly tree fungus threatens to spread from Michigan. CBCnews. Retrieved June 25, 2022, from https://www.cbc.ca/news/canada/windsor/erca-fights-oak-wilt-1.4544399
CBC/Radio Canada. (2021, December 5). Meet the latest threat to Ontario's forests - and it's lurking just off shore. CBC News. Retrieved June 21, 2022, from https://www.cbc.ca/news/canada/hamilton/oak-wilt-1.6271640
Cornelis, L. (2021, February 15). Majestic Oaks. Wallaceburg News. Retrieved June 21, 2022, from https://sydenhamcurrent.ca/2021/02/15/majestic-oaks/
Belter, K. (2020). PCR vs. Isolation and the War Against Oak Wilt. International Oaks, (31).
Juzwik, J., Harrington, T. C., MacDonald, W. L., & Appel, D. N. (2008). The origin of Ceratocystis fagacearum, the oak wilt fungus. Annual Review of Phytopathology. 46: 13-26., 46.
Wu, C.P., Chen, G.Y., Li, B., Su, H., An, Y.L., Zhen, S.Z. and Ye, J.R. (2011), Rapid and accurate detection of Ceratocystis fagacearum from stained wood and soil by nested and real-time PCR. Forest Pathology, 41: 15-21. >https://doi.org/10.1111/j.1439-0329.2009.00628.x
Singh, R., Feltmeyer, A., Saiapina, O., Juzwik, J., Arenz, B., & Abbas, A. (2017). Rapid and PCR-free DNA detection by nanoaggregation-enhanced chemiluminescence. Scientific Reports, 7(1). https://doi.org/10.1038/s41598-017-14580-w
Yang, A., & Juzwik, J. (2017). Use of nested and real-time PCR for the detection of ceratocystis fagacearum in the sapwood of diseased oak species in Minnesota. Plant Disease, 101(3), 480–486. https://doi.org/10.1094/pdis-07-16-0990-re
Moore, Melanie J.; Juzwik, Jennifer; Saiapina, Olga; Ahmed, Snober; Yang, Anna; Abbas, Abdennour. 2022. Use of Sodium Hydroxide DNA Extraction Methods for Nested PCR Detection of Bretziella fagacearum in the Sapwood of Oak Species in Minnesota . Plant Health Progress. 8 p. https://doi.org/10.1094/PHP-03-21-0057-RS.
Zhang, Y., Odiwuor, N., Xiong, J., Sun, L., Nyaruaba, R. O., Wei, H., & Tanner, N. A. (2020). Rapid molecular detection of SARS-CoV-2 (COVID-19) virus RNA using colorimetric LAMP. MedRxiv.
Becherer, Borst, N., Bakheit, M., Frischmann, S., Zengerle, R., & von Stetten, F. (2020). Loop-mediated isothermal amplification (LAMP) - review and classification of methods for sequence-specific detection. Analytical Methods, 12(6), 717–746. https://doi.org/10.1039/c9ay02246e
Valeri, J., Collins, K. M., Lepe, B. A., Lu, T. K., & Camacho, D. M. (2019). Sequence-to-function deep learning frameworks for synthetic biology. Biorxiv, 870055