Discrimination of Stem-Pitting from Other Isolates of Citrus tristeza virus

Abstract

Stem-pitting isolates of Citrus tristeza virus (CTV) are not thought to be widely distributed in commercial citrus in Florida, so prevention of their introduction and their detection is a regulatory priority. A test was needed as a supplement to the MCA13 monoclonal antibody test which could rapidly discriminate MCA13 reactive stem-pitting (SP) isolates from other MCA13 reactive, non-stem-pitting CTV isolates in field trees, and which also could replace or supplement biological indexing for stem-pitting symptoms. Three nucleic acid based and one serological technique were evaluated as diagnostic tools using isolates from the Florida and the USDAARS international CTV isolate collections that caused stem-pitting symptoms in citrus indicators. Sequence specific primers for amplifying CTV genome fragment PM33 and RF137 (from type II isolates of CTV) and genome fragment VT-1, and oligonucleotide probes (ONP) III, IV and V for hybridization studies gave positive results with many isolates that caused stem pitting in sweet orange, grapefruit or both. Enzyme linked immunosorbent assay (ELISA) designed for specific detection of sweet orange stem-pitting CTV and ONP II gave inconsistent results and were not tested further. For nucleic acid based assays, cDNA synthesized from total RNA extracts gave spurious results, whereas cDNA from immunocaptured virions produced clear, reproducible results. No one nucleic acid based technique was superior to the others and none could be used as a stand alone test. Therefore, test results from primers for Type II and VT-1 genome markers and probes II, IV, and V were used together to obtain stem-pitting profiles for isolates testing positive. False negatives were more common than false positives, and new primers are needed to detect isolates not identified by the current tests.