Fnu Cherryl

Seminar Title: Harnessing isothermal amplification techniques with CRISPR/Cas systems for rapid and highly specific detection of plant diseases

Cherryl
PhD student

About the Presenter

Cherryl holds a BS (Hons.) in Agricultural Sciences from Punjab Agricultural University in Ludhiana, India, and an MS in Tropical Plant Pathology from the University of Hawaii at Manoa. She is a first-year PhD student in the Department of Plant Pathology at Washington State University, supervised by Dr. Youfu ‘Frank’ Zhao. Cherryl is based at the Irrigated Agriculture Research and Extension Center in Prosser, WA, where her research focuses on managing bacterial canker disease (BCD) of sweet cherries in Washington orchards. BCD is caused by members of diverse genetic lineages and pathotypes collectively assigned to the Pseudomonas syringae species complex. Cherryl is a member of the Plant Pathology Graduate Student Organization and serves as a Research Station Representative for IAREC, Prosser. Her hobbies are journalling, listening podcasts, and talking to her dad.

Abstract

Plant pathogens including bacteria, viruses, fungi, oomycetes, and nematodes pose serious agricultural threats to crops worldwide [1]. According to the Food and Agriculture Organization [2], plant pests and diseases result in a 40% yield decline for major economically important crops, which accounts for a loss of more than $220 billion USD (approx.) every year. Beyond severe economic losses, increasing cases of plant diseases across the world are an alarming concern for global food security, as the world population is estimated to rise to 9.7 billion by 2050 [3]. To combat this, diseases need to be managed at a much earlier stage, because early and timely intervention is a foundational approach for effective disease management and surveillance, thus reducing the socioeconomic burden on agricultural commodities. Conventional approaches such as microscopic analysis, biochemical assays, and morphological characterization are tedious and labor-intensive and provide only a moderate level of confidence [4]. More precisely, these methods cannot support the identification of many obligate pathogens, such as phytoplasmas and many phloem- or xylem-limited bacteria [5]. Among immunological assays, immunostrips are low-throughput testing tools, suitable for rapid on-site field diagnosis but insufficient for low-titer samples, while ELISA has improved throughput but is limited by cross-reactivity and contamination [1]. PCR provides more specific and faster responses than cultural methods [6], and qPCR adds quantification and higher throughput [7]. These methods are considered the gold standards. Although they are specific, sensitive, and reliable, they lack point-of-care capability, require sophisticated equipment, are prone to inhibitors, may miss the low titers found in asymptomatic tissue, and provide no visual readout suited to on-site diagnosis [8]. Isothermal amplification techniques such as RPA [9] and LAMP [10] can overcome these limitations. They operate at a constant temperature, RPA at 37 to 42 °C and LAMP at 65 °C and offer high sensitivity and rapid response without heavy equipment, making them efficient for on-field diagnosis using lateral flow dipstick assays [11]. The major weakness of these techniques is their heavy dependence on primer binding. At low stringency, primers can bind non-specifically, generating false amplicons and primer artifacts that give false positives [12]. Integrating isothermal amplification with CRISPR/Cas systems addresses this constraint by adding a step of sequence-dependent recognition. Guide RNA (gRNA), designed with a complementary sequence, pairs with the matching target site, and this binding activates the collateral cleavage activity of the Cas enzyme, which cleaves the target specifically and ssDNA reporters non-specifically [13]. Cas systems add an extra layer of specificity by identifying the amplicons generated upon isothermal amplification of the target pathogen by either RPA or LAMP. This combination eliminates false positives, as the signal is generated only when the amplicon binds the crRNA (gRNA). Coupling isothermal amplification with CRISPR can strengthen diagnostics by detecting the pathogen even in asymptomatic tissue, where it is present only in small traces [8]. Examples harnessing RPA with CRISPR/Cas systems for rapid pathogen detection include Guo et al. [14] and Xu et al. [15]. Hence, combining isothermal techniques with CRISPR/Cas systems is revolutionizing plant disease diagnostics through early, rapid, highly sensitive, and target-specific identification, disease monitoring, and surveillance.

Meeting Information

Mondays at 4:10 p.m. Clark Hall 151
Meeting ID: 874 8808 0602
Passcode: 3075

If you have any questions, please do not hesitate to contact Dmitri Mavrodi, instructor for PL_P 515 in Fall 2026 semester: dmavrodi@wsu.edu.

Literature Cited

1. Venbrux, M., Crauwels, S., & Rediers, H. (2023). Current and emerging trends in techniques for plant pathogen detection. Frontiers in Plant Science, 14, 1120968. https://doi.org/10.3389/fpls.2023.1120968
2. Food and Agriculture Organization of the United Nations. (2019). New standards to curb the global spread of plant pests and diseases. https://www.fao.org/newsroom/detail/New-standards-to-curb-the-global-spread-of-plant-pests-and-diseases/en 
3. Food and Agriculture Organization of the United Nations. (2017). The future of food and agriculture: Trends and challenges (pp. 11–16).
4. Wang, X., Liang, S., Gan, Q., Cai, B., & Liu, C. (2025). Current status and future perspectives of the diagnostic of plant bacterial pathogens. Frontiers in Plant Science, 16, 1547974. https://doi.org/10.3389/fpls.2025.1547974
5. Pardo, J. M., Suwannarach, N., Malichan, S., Cuellar, W. J., & Siriwan, W. (2026). Mixed-pathogen infections in vegetatively propagated crops: From biological synergism to integrated management. Plants, 15(9), Article 1332. https://doi.org/10.3390/plants15091332
6. Henson, J. M., & French, R. (1993). The polymerase chain reaction and plant disease diagnosis. Annual Review of Phytopathology, 31, 81–109.
7. Schaad, N. W., & Frederick, R. D. (2002). Real-time PCR and its application for rapid plant disease diagnostics. Canadian Journal of Plant Pathology, 24(3), 250–258. https://doi.org/10.1080/07060660209507006
8. Wheatley, M. S., Duan, Y. P., & Yang, Y. (2021). Highly sensitive and rapid detection of citrus huanglongbing pathogen (‘Candidatus Liberibacter asiaticus’) using Cas12a-based methods. Phytopathology, 111(12), 2375–2382. https://doi.org/10.1094/PHYTO-09-20-0443-R
9. Piepenburg, O., Williams, C. H., Stemple, D. L., & Armes, N. A. (2006). DNA detection using recombination proteins. PLoS Biology, 4(7), e204. https://doi.org/10.1371/journal.pbio.0040204
10. Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., & Hase, T. (2000). Loop-mediated isothermal amplification of DNA. Nucleic Acids Research, 28(12), e63. https://doi.org/10.1093/nar/28.12.e63
11. Dai, T., Yang, X., Hu, T., Jiao, B., Xu, Y., Zheng, X., & Shen, D. (2019). Comparative evaluation of a novel recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) assay, LAMP, conventional PCR, and leaf-disc baiting methods for detection of Phytophthora sojae. Frontiers in Microbiology, 10, 1884. https://doi.org/10.3389/fmicb.2019.01884
12. Anbazhagan, P., Parameswari, B., Anitha, K., Chaitra, G. V., Bajaru, B., Rajashree, A., Mangrauthia, S. K., Yousuf, F., Chalam, V. C., & Singh, G. P. (2024). Advances in plant pathogen detection: Integrating recombinase polymerase amplification with CRISPR/Cas systems. 3 Biotech, 14(9), 214. https://doi.org/10.1007/s13205-024-04055-x
13. Chen, J. S., Ma, E., Harrington, L. B., Da Costa, M., Tian, X., Palefsky, J. M., & Doudna, J. A. (2018). CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science, 360(6387), 436–439. https://doi.org/10.1126/science.aar6245
14. Guo, Y., Xia, H., Dai, T., & Liu, T. (2023). RPA-CRISPR/Cas12a mediated isothermal amplification for visual detection of Phytophthora sojae. Frontiers in Cellular and Infection Microbiology, 13, 1208837. https://doi.org/10.3389/fcimb.2023.1208837 
15. Xu, T., Cao, F., Dai, T., & Liu, T. (2024). RPA-CRISPR/Cas12a-mediated isothermal amplification for rapid detection of Phytopythium helicoides. Plant Disease, 108(12), 3463-3472. https://doi.org/10.1094/PDIS-06-24-1300-SR 
16.