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Title: Detection and quantification of Bremia lactucae by spore trapping and quantitative PCR

item KUNJETI, SRIDHARA - University Of California
item Anchieta, Amy
item Martin, Frank
item CHOI, YOUNG-JOON - Kunsan National University
item THINES, MARCO - Biodiversity And Climate Research Centre (BIK-F)
item MICHELMORE, RICHARD - University Of California
item KOIKE, STEVEN - University Of California - Cooperative Extension Service
item TSUCHIDA, CAYLA - University Of California
item Mahaffee, Walter - Walt
item SUBBARAO, KRISHNA - University Of California
item Klosterman, Steven

Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/24/2016
Publication Date: 8/29/2016
Citation: Kunjeti, S.G., Anchieta, A.G., Martin, F.N., Choi, Y.-J., Thines, M., Michelmore, R.W., Koike, S.T., Tsuchida, C., Mahaffee, W.F., Subbarao, K.V., Klosterman, S.J. 2016. Detection and quantification of Bremia lactucae by spore trapping and quantitative PCR. Phytopathology. 106:1426-1437.

Interpretive Summary: The lettuce downy mildew pathogen, Bremia lactucae, causes lettuce leaf lesions and discolorations that render the fresh product unmarketable. This pathogen is dispersed from field-to-field as airborne spores, and can quickly destroy entire lettuce fields after initial infection and sporulation. This study was undertaken to develop a quick and specific assay for detection and quantification of the DNA of B. lactucae in the environment, and particularly around commercial lettuce fields where it may be useful for disease predictions. The results indicate that the assay is species specific, only detecting DNA of B. lactucae and not the 14 other Bremia species examined. The assay was deployed to assess the relationship between the levels of detectable lettuce downy mildew pathogen and levels of actual downy mildew disease in fields. Using the assay, we detected the windborne pathogen at a low level in the Salinas Valley in the summer of 2015, but at higher levels near an infected lettuce field. Thus there are low levels of the pathogen present typically throughout the valley that complicate the usefulness of simple presence or absence reporting. However, increased pathogen detection was associated with increased disease incidence in a commercial field examined simultaneously. Taken together, the results suggest that the assay developed for airborne detection of B. lactucae may allow prediction of periods of higher airborne inoculum loads, enabling growers to better time spray applications for disease management, as well as fungicide resistance (in the pathogen) management.

Technical Abstract: Bremia lactucae causes the characteristic vein-delimited lesions, leaf chlorosis and necrosis and adversely affects marketability of lettuce. The disease has been managed with a combination of host resistance and fungicide applications with mixed success over the years. Fungicide applications are routinely applied under the assumption that inoculum is always present during favorable environmental conditions. This approach often leads to fungicide resistance in B. lactucae populations. Detection and quantification of airborne B. lactucae near lettuce crops may lead to a more judicious timing of fungicide applications. For specific detection of B. lactucae, we designed a qPCR assay based on a target sequence in B. lactucae mitochondrial DNA. PCR tests using DNA from 83 geographically diverse isolates, representing 14 Bremia spp., and amplicon sequencing confirmed that the primers developed for the TaqMan assays are species-specific and only amplify template from B. lactucae. Cq values >35 were considered nonspecific because a single sporangium could be detected at a Cq value of 34. The coefficient of determination (R2) for regression between sporangial density derived from flow cytometry and Cq values derived from the qPCR was 0.86. The assay was deployed using spore traps in the Salinas Valley, where nearly half the US lettuce crop is produced. The sensitivity of the B. lactucae-specific assay enabled detection of the pathogen during the two-week lettuce-free period as well as during the cropping season. These results suggest that the assay can be useful for predicting inoculum load for timing of fungicide applications.