Investigation of quantitative real-time PCR as a mechanism for evaluating the efficacy of experimental fungicides against Septoria tritici
C. AVILA-ADAME (1), G. Gustafson (1). (1) Dow AgroSciences LLC, Indianapolis, IN, U.S.A.
The typical latent period for the wheat fungal pathogen <i>Septoria tritici</i> in greenhouse conditions is at least 20 days, leading to long cycle times when evaluating efficacy of fungicides. Quantitative real-time PCR was investigated as a technology that might significantly shorten the length of this evaluation period and reduce cycle time for testing experimental fungicides. Fluorescent oligonucleotide probes were designed that targeted the <i>Septoria</i> cytochrome <i>b</i> (cytb) and 14-α demethylase (<i>cyp51</i>) genes. Using these probes, fungal DNA could be detected in infected leaf tissue as early as 24 h after inoculation. DNA was extracted at 0, 5, 10, and 18 days after inoculation from leaves of wheat seedlings that eventually developed 5-100% disease severity. Overall, the cytb probe produced better correlations between <i>Septoria</i> DNA levels and disease severity than the cyp51 probe. There was a strong correlation between disease severity and fungal DNA levels in leaves with fully sporulating necrotic lesions with correlation coefficients of -0.89 and -0.85 for cytb and cyp51, respectively. However, DNA quantification at early stages of <i>S. tritici</i> development in leaf tissue was not a good predictor of final disease severity. Therefore, real-time PCR has a limited applicability for evaluating the efficacy of experimental fungicides against S. tritici at an early stage of development inside wheat leaf tissue.
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