The laurel wilt disease fungus, Raffaelea lauricola, is killing redbay trees, spreading rapidly in the U.S. southeastern coastal plain forest, and posing a serious threat to the avocado industry in Florida. A molecular tool is urgently required to facilitate detection of this pathogen. The 5′ region of the large ribosomal RNA (28S) gene is highly variable among Raffaelea spp. and ideal for this purpose but amplification of this sequence from R. lauricola has been difficult. Different amplification conditions were tested and a high-fidelity polymerase chain reaction (PCR) procedure utilizing a dNTP mix containing 7-deaza-dGTP was found to reliably amplify 28S sequences from R. lauricola. Sequencing the amplified products or cloned inserts also turned out to be difficult and required using a custom-blended sequencing mix containing 1 M betaine, 5% dimethyl sulfoxide, and dGTP-BigDye v3.1. Three GC-rich stem and loop or cruciform secondary structures were discovered, which may have interfered with amplification. This improved protocol made it possible to partially characterize the internal transcribed spacers sequence from R. lauricola, which also has interfering secondary structures. A TaqMan real-time PCR assay was designed using the species-specific 28S sequences and this allowed detection of R. lauricola from wood tissues or cultures. Wood tissues from symptomatic redbay, avocado, and sassafras trees in Florida were screened using this TaqMan assay and several were found to test positive for R. lauricola. Results were further confirmed by performing Koch's postulates for avocado specimens collected from commercial grooves.
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