Project Description
Delve into the heart of our initiative, understanding its objectives, scope, and the motivation behind it.
Engineering a Direct Detection Assay for Clubroot Pathogen in Canola Soil and Roots
Clubroot is a plant disease caused by the soil-borne protist Plasmodiophora brassicae. The disease is characterized by the development of abnormal galls on the roots of brassicae crops. Further manifestations of the disease result in swollen roots and hypocotyl deformation which produces clubbed roots. Clubbed root formation and disease progression depletes essential nutrients typically used for plant growth and survival, while infected plants demonstrate reduced aboveground growth, premature organ ripening, and shriveled seeds in severe infections. Moreover, clubbed roots impede nutrient and water uptake, resulting in leaf discolouration, wilting, and stunted growth (Javed et al., 2023). As a result, crop yield and quality are severely diminished (Strelkov et al., 2016). This issue is widespread in Alberta, affecting approximately 67% of counties and districts, with canola fields being particularly susceptible (Government of Alberta, 2022). Given the economic importance of canola in Alberta, it is imperative to develop effective detection strategies for Clubroot. Currently, there exists no treatment for Clubroot and limited mitigation strategies, including crop rotation, equipment sterilization and the development of genetically resistant crops, are unsustainable. Furthermore, current detection methods are costly and take a long time to obtain results as they utilize PCR amplification, which requires outsourcing to off-site laboratories.
The objective of our project is to create a reliable and inexpensive direct detection kit for Clubroot using a rapid and direct ELISA based testing system as proof of concept with a long term objective of engineering a qualitative Clubroot test strip. We have rationally designed and engineered model-based chimeric proteins fused with fluorescent probes (eGFP) that bind to P. brassicae-specific protein targets, Pro1 and PbEL04. The high resolution structures of these protein targets are still not yet published. Our end-goal is to offer farmers a user-friendly rapid-test that can be used on-site and provides results in hours. This system will enable farmers to identify the presence of pathogenic spores quickly, efficiently, and at a lower cost as compared to existing methods. The development of such a detection kit will greatly benefit farmers by providing timely information and facilitating the implementation of appropriate measures, such as longer crop rotations or equipment sanitization to limit spore spread. The successful creation of a reliable and effective detection kit represents a significant advancement in combating Clubroot.
Currently there are limited effective mitigation methods for Clubroot. Our team hopes to eventually develop a mitigation system with more refined chimeric proteins that can be applied to Clubroot infested fields. Through our research this year we have obtained an improved understanding of Clubroot and our chimeric proteins, and we plan to implement and test this new knowledge further in the coming year to design and engineer such a mitigation system.
By addressing the urgent need for reliable detection and mitigation strategies, our project will make substantial progress in the fight against Clubroot. Our technology will empower farmers with the knowledge to take appropriate action and establish proactive measures to limit the impact of Clubroot on their crops. Ultimately, the successful development and implementation of our detection and mitigation systems will contribute significantly to preserving the productivity and economic viability of Brassica crops, and particularly canola crops in Alberta.
Supplementary: Plasmodiophora brassicae Lifecycle
Resting spore germinate into zoospore
When the resting spores detect secretions from the roots of host plants during warmer, wetter weather, they germinate and transform into primary zoospores to start the infection process anew.
Zoospore
The primary zoospores created by resting spores are highly mobile, and capable of seeking out potential host plants. Though this phase is short-lived, it bypasses the need for the pathogen to rely on external distribution methods.
Initial Infection
The primary zoospores can be introduced to plants through root hairs or wounds on the plant. This zoospore produces a plasmodium, which is capable of producing and releasing secondary zoospores. These new zoospores infect the root cortex on either the initial host plant, or neighbooring plants. Once inside of the root cortex, these zoospores develop another plasmodium body.
Beginning of Gall Formation
The secondary plasmodium is able to manipulate plant metabolism and hormones to induce the characteristic galls in the hosts by promoting cell division. In different host plants, gall formation occurs on different places on the roots - Arabidopsis plants show infection in the upper roots, whereas other Brassicaceae crops show gall formation more on lateral roots.
Galls Formed
The galls of infected host plants are used as carbohydrate reservoirs to supply nutrients to future resting spores. Infected plants show that sugars from above-ground tissues are relocated to infected roots and galls, through manipulating and stunting vascular development in plants.
Plant Decay
After the secondary plasmodium in the roots mature, they divide into resting spores. The symptoms of this infection stunt the growth of the plant as the roots cease nutrient and water uptake. This eventually kills the plant, and the tissues no longer maintained by the host defence and immune response are degraded by soil microbes.
Resting Spore Released
After galls decay, the resting spores created by secondary plasmodium are released into the soil. These resting spores can survive in the soil for up to 10 years, though they do become inactive after two years without access to an appropriate host. This is a reason why P. brassicae is such a difficult pathogen to manage, and shows the importance of detection and mitigating it.
References
Botero, A., García, C., Gossen, B. D., Strelkov, S. E., Todd, C. D., Bonham‐Smith, P. C., & Pérez‐López, E. (2019). Clubroot disease in Latin America: Distribution and management strategies. Plant Pathology, 68(5), 827–833. https://doi.org/10.1111/ppa.13013
Clubroot Disease: Canola Encyclopedia. Canola Council of Canada. (2023, October 4). https://www.canolacouncil.org/canola-encyclopedia/diseases/clubroot/
Javed, M. A., Schwelm, A., Zamani‐Noor, N., Salih, R., Silvestre Vañó, M., Wu, J., González García, M., Heick, T. M., Luo, C., Prakash, P., & Pérez‐López, E. (2022). The clubroot pathogen plasmodiophora brassicae: A profile update. Molecular Plant Pathology, 24(2), 89–106. https://doi.org/10.1111/mpp.13283
Vañó, M. S., Nourimand, M., MacLean, A., & Pérez-López, E. (2023). Getting to the root of a club – understanding developmental manipulation by the Clubroot pathogen. Seminars in Cell & Developmental Biology, 148–149, 22–32. https://doi.org/10.1016/j.semcdb.2023.02.005
References
Javed, M. A., Schweim, A.,, Zamani-Noor, N., Salih, R., Vano, M. S., Wu, J., Gonzalez Garcia, M., Heick, T. M., Luo, C., Prakash, P. & Perez-Lopez, E (2023). The clubroot pathogen Plasmodiophora brassicae: A profile update. Molecular Plant Pathology, 24(2). 89–106. https://doi.org/10.1111%2Fmpp.13283
Strelkov, S. E., Hwang, S. F., Manolii, V. P., Cao, T., & Feindel, D. (2016). Emergence of new virulence phenotypes of Plasmodiophora brassicae on canola (brassica napus) in Alberta, Canada. European Journal of Plant Pathology, 145(3), 517–529. https://doi.org/10.1007/s10658-016-0888-8
Alberta Clubroot Management Plan. Alberta.ca. (2022). https://www.alberta.ca/alberta-clubroot-management-plan.aspx