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Title: The prevalence of the citrus tristeza virus trifoliate resistant breaking genotype among Puerto Rican isolates

item ROY, AVIJIT - University Of Florida
item CHOUDHARY, NANDLAL - University Of Florida
item Hartung, John
item BRLANSKY, R - University Of Florida

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/12/2013
Publication Date: 9/1/2013
Citation: Roy, A., Choudhary, N., Hartung, J.S., Brlansky, R.H. 2013. The prevalence of the citrus tristeza virus trifoliate resistant breaking genotype among Puerto Rican isolates. Plant Disease. 97:1227-1234.

Interpretive Summary: Citrus tristeza virus (CTV) causes the most important virus disease of citrus in the world. The disease has been managed by grafting sweet orange to resistant ‘trifoliate orange’ rootstocks. In 2008 a novel form of CTV was described in New Zealand, which is able to infect and multiply in ‘trifoliate orange’ rootstocks, although it does not produce any disease symptoms. We maintain a unique collection of 300 CTV isolates obtained from 40 countries. In this work we developed an assay for the specific strain of CTV from New Zealand that infects ‘trifoliate orange’. We found that a previously unclassified accession in our collection obtained from Puerto Rico, is in fact infected with a ‘trifoliate resistance breaking’ strain of CTV, and is very closely related to the strain from New Zealand. When we examined 8 other accessions of CTV from Puerto Rico we found that they also had the ‘trifoliate resistance breaking’ genotype, and were present as part of mixed infections with other previously characterized isolates. Thus CTV strains that can infect and multiply in ‘trifoliate orange’ are widespread in Puerto Rico, and are not limited to New Zealand. The strains from Puerto Rico also predate the strains from New Zealand. Such strains are likely to be present elsewhere. Producers and regulatory officials will want to use our assay to test citrus groves in Florida and elsewhere for the presence of these novel strains of CTV that can replicate in trifoliate orange, often in mixed infections. Although the trifoliate orange trees infected with these ‘new’ strains of CTV show minimal symptoms of disease, the presence of CTV in trifoliate orange is cause for concern.

Technical Abstract: Citrus tristeza virus (CTV) isolates have been grouped previously into five genotypes: T3, T30, T36, VT, B165 based on symptoms, host range and genomic sequence data. A sixth genotype has recently been identified with the novel property of replicating in trifoliate orange trees, a non host for the other five genotypes. CTV isolate B301 was obtained from the Exotic Pathogens of Citrus Collection (EPCC) at the USDA-ARS, Beltsville, MD, USA but was originally collected from Aguas Buenas, Puerto Rico in 1992. We were unable to place isolate B301 into one of the five traditional groups of CTV with our genotype specific multiplex (GSM) reverse transcription polymerase chain reaction (RT-PCR). The GSM RT-PCR can detect CTV-infected plants and identify all CTV groups except for the recently discovered resistance breaking genotype. CTV-B301 produced mild vein clearing symptoms in Mexican lime but did not induce seedling yellows or stem pitting reactions in appropriate indicator Citrus spp., as observed for a class of isolates typified by isolate T30. A primer pair for RT-PCR amplification of the CTV trifoliate resistance breaking genotype was designed based on the complete genomic sequences of trifoliate resistance breaking isolates from new Zealand deposited at NCBI. New Zealand resistance breaking isolates obtained from EPCC were used as positive controls. The CTV-trifoliate resistance breaking genotype specific primers (GSPs) successfully amplified isolate B301 and the amplicon sequence from this isolate was similar to that of other trifoliate resistance breaking genotypes. In addition, inoculation of B301 into a Carrizo citrange (a hybrid of Poncirus trifoliata and Washington navel sweet orange) cultivar did not induce any symptoms following successful inoculation judged by the presence of a RB-GSP amplicon obtained by RT-PCR. In addition, the complete genome sequence of CTV-B301 has 95-97% nucleotide sequence identity with New Zealand trifoliate resistance breaking isolates. Phylogenetic analysis of open reading frames (ORF) 1a and 1b from the 5' terminal half-region and ORFs 2-11 from 3' terminal half-region revealed that the isolate is part of the trifoliate resistance breaking clade with five other CTV trifoliate resistance breaking isolates from New Zealand. Additional CTV isolates obtained from Puerto Rico were tested with the RB-GSP and confirmed the presence of hidden trifoliate resistance breaking genotypes in mixed infection with other CTV genotypes. Although this is the first report of a CTV-trifoliate resistance breaking genotype from Puerto Rico, this genotype has been present there prior to 1992.