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Title: Development and application of a hexaplex reverse transcription polymerase chain reaction for screening global citrus tristeza virus isolates

Author
item ROY, AVIJIT - University Of Florida
item ANANTHAKRISHNAN, G - University Of Florida
item Hartung, John
item BRLANSKY, RONALD - University Of Florida

Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/7/2010
Publication Date: 9/15/2010
Citation: Roy, A., Ananthakrishnan, G., Hartung, J.S., Brlansky, R.H. 2010. Development and application of a hexaplex reverse transcription polymerase chain reaction for screening global citrus tristeza virus isolates. Phytopathology. 100(10):1077-1088.

Interpretive Summary: Citrus tristeza virus (CTV) nearly destroyed the global citrus industry in the mid 20th century and it remains by far the most important virus of citrus. Previous research has demonstrated at least five biological types of this virus as measured by host range within citrus and the symptoms induced by various isolates of the virus, but the underlying basis for this variation is not understood. The University of Florida maintains a collection of isolates of CTV that originated in Florida, and the USDA ARS maintains a unique collection of CTV isolates that have originated around the world. In total, the diversity of 175 isolates of CTV that originated in Florida and 28 nations was characterized in this study. To do this, we used genetic information to design rapid laboratory assays that could distinguish each of the five types of CTV. These assays were validated singly against the 175 isolates and then were combined into a single assay that could for the first time simultaneously detect CTV and determine the type(s)of CTV present in individual trees. Our data show that mixed infections of different combinations of virus types are present in 72% of the trees sampled in Florida and around the world. Trees naturally infected with two, three, four and even all five biological and genetic types of the virus were observed. Two biological types of the virus were most common, infecting 55% of trees sampled. Other biological types were present in 43%, 40% and 14% of the trees sampled. Our experimental methods will be of interest to researchers who study CTV and will enable detailed studies of the interactions among isolates of the virus. Our results will be of interest to regulatory officials responsible for controlling the spread of CTV.

Technical Abstract: The discovery of the diversity of Citrus tristeza virus (CTV) genotypes has complicated detection and diagnostic measures. To simplify the identification and differentiation of CTV genotypes, an efficient multiplex reverse transcription polymerase chain reaction (M-RT-PCR) technique for the screening of CTV isolates was developed. Variable regions within the open reading frame (ORF) -1a of diverse CTV genotypes were identified to develop first a simplex (S) and then a hexaplex (H) RT-PCR. CTV isolates have been grouped into five genotypes namely, T3, T30, T36, VT and B165 based on nucleotide sequence comparisons and phylogenetic analyses. Nucleotide sequences from GenBank were used to design species- and genotype-specific primers (GSPs). The GSPs first were utilized for reliable detection of all CTV genotypes using S-RT-PCR. Furthermore, detection of all five recognized CTV genotypes was established using the H-RT-PCR. Six amplicons, one specific to all CTV isolates and one for each of the five recognized genotypes were identified on the basis of their molecular weights and were confirmed by sequence analysis. One hundred and seventy-five CTV isolates from 29 citrus growing countries were successfully analyzed by S- and H-RT-PCR. Ninety-seven isolates contained CTV-T36 genotypes, 95 contained CTV-T3 genotypes, 76 contained CTV-T30 genotypes, 71 contained CTV-VT genotypes and 24 contained CTV-B165 genotype isolates. A total of 126 isolates contained mixed infections of 2 to5 of the known CTV genotypes. Two of the CTV isolates could not be assigned to a known genotype. H-RT-PCR provides a sensitive, specific, reliable and high throughput way to screen for CTV genotypes as compared to other methods for CTV genotype detection. Efficient identification of CTV genotypes will facilitate a better understanding of CTV complexes, including the possible interaction of different genotypes in causing or preventing diseases. T he methods described can also be used in virus-free citrus propagation programs and in the development of CTV resistant cultivars.