Jeyaprakash A.,Bureau of Entomology |
Davison D.A.,Bureau of Entomology |
Schubert T.S.,Bureau of Entomology
Plant Disease | Year: 2014
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. © 2014 The American Phytopathological Society.
Srinivasan R.,University of Georgia |
Guo F.,University of Georgia |
Riley D.,University of Georgia |
Diffie S.,University of Georgia |
And 3 more authors.
Journal of Entomological Science | Year: 2011
The onion thrips, Thrips tabaci (Lindeman), is the only known vector of Iris yellow spot virus (IYSV). IYSV was detected in Georgia for the first time in 2003. Phylogenetic analysis using nucleotide sequences of the IYSV capsid gene indicated that it may have been accidentally introduced from repackaging of imported Peruvian onions in the Vidalia onion-growing region. The tobacco thrips, Frankliniella fusca (Hinds), has been the dominant thrips species on onions in Georgia. However, in recent years the incidence of T. tabaci on onions has been consistently increasing. Laboratory competition studies indicated that T. tabaci outcompeted F. fusca on onion foliage. This led to speculation that a new biotype of T. tabaci may have been introduced along with IYSV through importation of Peruvian onions. This hypothesis was tested by analyzing variations in the mitochondrial cytochrome oxidase I gene and internal transcribed spacer region 2 of T. tabaci populations from Georgia and Peru. DNA was extracted from T. tabaci samples from Georgia and Peru and subjected to PCR using specific primers. The resulting amplicons were sequenced. Parsimony and Bayesian analysis of the COI sequences indicated that all the Peruvian taxa fell into a single clade along with one Georgia taxon. All the other Georgia taxa were in a separate clade. ITS2 sequence comparisons indicated that Georgia and Peru taxa were found in numerous clades. High variation among taxa from each region indicated that ITS2 may not be suitable to assess intraspecific variation among T. tabaci populations.