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Identification of Strains of Citrus tristeza virus by Subtraction Hybridization

Citrus sudden death (CSD) appears to be a new disease that is a serious problem in Brazil. Symptoms of CSD include yellow stain in the phloem of the rootstock. The cause is not known, but it appears to be infectious and may only affect trees budded on Rangpur lime. In a survey in Brazil, in addition to CSD, we observed numerous trees on Rangpur lime that were obviously declining but had remained in production for several years. Trees with this disease, referred to as Rangpur lime decline (RLD) were different from those with citrus blight (CB). They had near-normal size fruit compared with the small fruit associated with CB and were negative in the serological test for the CB-associated protein (p12). Moreover, they did not have the yellow stain symptom and obviously were declining much more slowly than was reported for CSD. To determine what viruses or virus strains might be associated with CSD, double-stranded (ds)RNAs from fibrous roots of a tree with CSD and stem bark from greenhouse trees infected with Citrus tristeza virus (CTV) isolates T30 and T36 were used to make random primed cDNAs. A Clontech PCR-Select cDNA Subtraction Kit was used to subtract the CSD cDNA with cDNA from an equal mixture of dsRNA from T30 and T36. Of 28 clones that were sequenced, five were found to be significantly different from published CTV sequences. One clone (SDA-1) was found to be only 48% similar to CTV T30 based on amino acid sequence. Using samples collected in October 2001, hybridization assays with a DIG probe of SDA-1 were positive for RNA from roots of declining trees from an area where CSD is reported to occur and from a second area where trees were declining with what had been thought to be CB and are now considered to be RLD. The SDA-1 probe reacted weakly or not at all with RNA from stem bark of trees with CSD, collected in October 2001, or RNA from roots of trees that were declining with CB. Using samples collected in March 2003 from trees with severe decline (nearly dead), the SDA-1 probe reacted with all preparations from both stems and roots. Reactions to the SDA-1 probe also were observed in many stem or root samples from trees with RLD, with early symptoms of CSD, and nonsymptomatic trees. The SDA-1 probe did not react with samples from roots or stems of healthy or CB trees from Florida.