Oral: Pathogen Dispersal
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Detection and quantification of Bremia lactucae by spore trapping and quantitative PCR.
S. KUNJETI (1), A. Anchieta (2), F. Martin (3), Y. Choi (4), M. Thines (5), R. Mitchelmore (1), S. Koike (6), C. Tsuchida (1), W. Mahaffee (7), K. Subbarao (1), S. Klosterman (3) (1) University of California Davis, U.S.A.; (2) USDA Salinas, U.S.A.; (3) US
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Bremia lactucae 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 condition. This approach could lead 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 as nonspecific but a single sporangium could be detected at a Cq of 34. The correlation (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 is produced. The sensitivity of the B. lactucae-specific assay enabled detection of the pathogen within the two-week lettuce free period, and indicated that the assay can be useful for predicting inoculum load for timing of fungicide applications.