Rapid detection of Cercospora beticola in sugar beet and mutations associated with fungicide resistance using LAMP or probe-based qPCR

Authors: SHRESTHA, SUBIDHYA - North Dakota State University; Neubauer, Jonathan; SPANNER, REBECCA - North Dakota State University; Natwick, Mari; RIOS, JOSHUA - North Dakota State University; METZ, NICHOLAS - North Dakota State University; SECOR, GARY - North Dakota State University; Bolton, Melvin

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/28/2020
Publication Date: 4/13/2020
Citation: Shrestha, S., Neubauer, J., Spanner, R., Natwick, M.B., Rios, J., Metz, N., Secor, G., Bolton, M.D. 2020. Rapid detection of Cercospora beticola in sugar beet and mutations associated with fungicide resistance using LAMP or probe-based qPCR. Plant Disease. 104:6. https://doi.org/10.1094/PDIS-09-19-2023-RE.
DOI: https://doi.org/10.1094/PDIS-09-19-2023-RE

Interpretive Summary: Cercospora leaf spot (CLS)is caused by the fungal pathogen Cercospora. It is the most devastating disease of sugar beet worldwide. Currently, disease management relies on timely application of fungicides. However, Cercospora populations are now resistant to many commonly used fungicides. Consequently, knowledge of the resistance levels that exist in a field is necessary in order to utilize fungicides that will manage the disease properly. In this work, we developed a visual assay that relies on a technology called loop-mediated isothermal amplification or LAMP to detect Cercospora in sugar beet leaves. Using a simple DNA isolation protocol, the LAMP assay can confirm the presence of Cercospora very early in the infection process. Once Cercospora has been confirmed, the grower will want to know what fungicides the identified Cercospora strain is resistant to. To that end, we developed LAMP and PCR assays to quickly and easily confirm whether the strains identified in the field are resistant to each of three commonly used fungicides for Cercospora disease management. With this knowledge in hand, sugar beet growers can make informed decisions on which fungicide chemistry to use, which will save growers both time and money.

Technical Abstract: Cercospora leaf spot (CLS), caused by the fungal pathogen Cercospora beticola, is the most destructive disease of sugar beet worldwide. Although growing CLS-tolerant varieties is helpful, disease management currently requires timely application of fungicides. However, over reliance on fungicides has led to the emergence of fungicide resistance in many C. beticola populations, resulting in multiple epidemics in recent years. Therefore, this study focused on developing a fungicide resistance management ‘toolbox’ for early detection of C. beticola in sugar beet leaves and mutations associated with different fungicides in the pathogen population. A loop-mediated isothermal amplification (LAMP) method was developed for rapid detection of C. beticola in infected sugar beet leaves. The Cb-LAMP assay was able to detect C. beticola in inoculated sugar beet leaves as early as 1 DPI. A QoI-LAMP assay was also developed to detect the G143A mutation in cytochrome b associated with QoI-resistance in C. beticola. The assay detected the mutation in C. beticola both in vitro and in planta with 100% accuracy. We also developed a probe-based qPCR assay for detecting a E198A mutation in ß-tubulin associated with benzimidazole-resistance and a probe-based qPCR assay for detection of mutations in cytochrome P450-dependent sterol 14a-demethylase (Cyp51) associated with resistance to sterol demethylation inhibitor (DMI) fungicides. The primers and probes used in the assay were highly efficient and precise in differentiating the corresponding fungicide resistant mutants from sensitive wild-type isolates.