Multiplex quantitative PCR assay for detection of spinach and lettuce downy mildews using spore trapping

Authors: Clark, Kelley; Anchieta, Amy; CAVANAUGH, KERI - University Of California; Martin, Frank; CORRELL, JAMES - University Of Arkansas; MONTAZAR, ALIASGHAR - University Of California Agriculture And Natural Resources (UCANR); PUTMAN, ALEXANDER - University Of California; Klosterman, Steven

Submitted to: PhytoFrontiers
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/21/2023
Publication Date: 7/24/2023
Citation: Clark, K.J., Anchieta, A.G., Cavanaugh, K., Martin, F.N., Correll, J.C., Montazar, A., Putman, A.I., Klosterman, S.J. 2023. Multiplex quantitative PCR assay for detection of spinach and lettuce downy mildews using spore trapping. PhytoFrontiers. 4(1):72-80. https://doi.org/10.1094/PHYTOFR-04-23-0045-FI.
DOI: https://doi.org/10.1094/PHYTOFR-04-23-0045-FI

Interpretive Summary: Spinach and lettuce downy mildews continue to cause crop loss for the leafy greens industry. Here we developed a method for dual amplification and detection of the airborne spores of both of the pathogens causing these diseases. We paired this method with two different spore trapping platforms and found cyclone spore traps, when compared to impaction spore trap samplers, collect significantly more spores, and provide more ease-of-use. This detection tool can be used to increase awareness of spore presence and abundance at a given field site, which in return may reduce fungicide usage or improve their efficiency.

Technical Abstract: Downy mildews are major constraints on spinach and lettuce production globally. Disease management can be achieved with fungicides, but routine applications are costly and can lead to pathogen resistance. Detection of airborne spores could guide sustainable fungicide application and considering spinach and lettuce are often grown in the same cycles simultaneous detection of both pathogens is practical. Here, a multiplex hydrolysis probe quantitative PCR assay was designed using single copy mitochondrial DNA (mtDNA) targets for spinach downy mildew (Peronospora effusa) and lettuce downy mildew (Bremia lactucae). To quantify P. effusa and B. lactucae sporangia, a standard curve was developed for each pathogen using the multiplex qPCR to amplify DNA obtained from known dilutions of sporangia. Analysis of these curves revealed a greater sensitivity for B. lactucae, indicating that the sporangia of B. lactucae may harbor more mitochondria than those of P. effusa, providing insight into the biology of these pathogens. The multiplex qPCR assay was partnered with two different spore trap types: a cyclone spore trap and an impaction spore trap. Results from air sampling revealed that the cyclone spore traps collect significantly more sporangia compared to impaction traps. Exposure of P. effusa sporangia to desiccation was performed to assess environmental impact on the assay and while detection levels were reduced, they were still apparent. This detection and quantification tool will be useful in efforts to improve the accuracy of downy mildew forecasting, which in return may reduce fungicide usage or improve their efficiency.