Detection of mRNA by reverse-transcription PCR as an indicator of viability in Phytophthora ramorum

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Abstract:

In the last few decades, the use of molecular tools has greatly improved the efficiency of plant disease diagnosis. However, one of the major setbacks of most molecular diagnostic approaches is their inability to differentiate between dead and viable pathogens. We propose a new strategy for the detection of plant pathogens, based on the use of mRNA as a viability marker, on the basis that mRNA degradation in dead cells is significantly more rapid than that of DNA. A real-time reverse-transcription PCR (RT-PCR) assay targeting the mRNA of the subunit I of the cytochrome oxidase gene was designed for Phytophthora ramorum, the causal agent of sudden oak death and ramorum blight. In controlled laboratory tests, the developed RT-PCR assay did not detect the target mRNA a week after the pathogen had been killed by rapid lyophilization, while DNA of the pathogen could still be detected 3 months after the pathogen had died. The RT-PCR assay was then compared with a traditional culturing approach using PARP selective medium and two nested real-time PCR techniques on symptomatic California bay laurel leaves. Samples were either collected in three different sites in July, or in the same site but in three different seasons. Overall, RT-PCR results showed less positive samples than DNA-based nested PCR techniques (p < 0.0001), but more than culturing (p = 0.017). Nested PCR-positive but RT-PCR-negative samples may not be viable. On the other hand, RT-PCR-positive but culture-negative samples may be viable but dormant. A comparative analysis of the results indicated that RT-PCR and culturing provide comparable results when climatic conditions are optimal for the pathogen, but RT-PCR may be the most accurate approach to determine pathogen viability when climatic conditions are less than optimal for the pathogen.

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