Citrus viroid (CVd) group II is comprised of hop stunt viroid (HSVd)-related variants of 295 to 302 nucleotides. Included in this group are the cachexia-inducing agents citrus cachexia viroid (or CVd-IIb), CVd-IIc, Ca-903, and Ca-909 as well as the non-cachexia-inducing variant CVd-IIa. The cachexia indexing hosts 'Parson's Special' mandarin and 'Orlando' tangelo as well as Citrus macrophylla responded with symptoms of gumming, discoloration, and stem pitting when infected by CVd-IIb, CVd-IIc, or Ca-903. However, 'Palestine' sweet lime, the indicator host used to describe the xyloporosis disease, displayed a distinctly different fine-pitting reaction and no discoloration or gumming when infected by the same viroids. Cachexia-inducing variants contain a number of nucleotide changes more similar to hop-type HSVd sequences than to the citrus-type HSVd sequences, as typified by CVd-IIa. The nucleotide sequence of CVd-IIc was identical to CVd group II isolates common to trees expressing xyloporosis. Experimental evidence indicates that either CVd-IIb or CVd-IIc can cause citrus diseases known as cachexia and xyloporosis and that the two disease designations reflect the distinct responses of different indexing hosts to the same viroids.
Vitis vinifera cultivars Zinfandel-1A and Mission were found to harbor different grapevine yellow speckle viroid-1 (GYSVd-1) variants and characterized to define the relationship to yellow speckle (YS) and vein-banding (VB) diseases. Products from the left terminal (T1), pathogenic (P), and a portion of the central (C) domains of Zinfandel-1A and Mission displayed distinct single-stranded conformation polymorphism (SSCP) patterns, presumably reflecting nucleotide changes in the P domain. The two selections were shown to contain homogeneous populations of type 1 and type 2 GYSVd-1 variants described in Australia. Symptoms of YS were induced only in vines containing the type 2 variant by treatment at a constant temperature of 32 degreesC in continuous light. SSCP of Pagadebit selections from Italy revealed the nonsymptomatic variant was essentially identical to Zinfandel-1A, whereas symptomatic selections were unlike any other previously described. Nucleotide sequence confirmed that nonsymptomatic selections from Italy contained the GYSVd-1 type 1 variant. A total of 43 changes were spread throughout the T1, C, V, and T2 domains from symptomatic selections. This study establishes the Australian type 1 variant as the non-symptom-inducing form of GYSVd-1 and type 2 as the symptom-inducing variant. The distinct symptom-inducing variant from Italy is proposed as a new type 3 variant of GYSVd-1.
Three isolates of sugarcane mosaic virus (SCMV) from sugarcane and maize in Thailand, were selected and used as viral sources for coat protein (CP) genes cloning by immunocapture reverse transcription polymerase chain reaction (IC-RT-PCR). The CP gene sequences and their deduced amino acids were determined. The sequences of three cloned CP genes, UT6TH-sgc and UD7TH-sgc from sugarcane, and SBC2TH-mz from maize were compared with other previously reported sequences of SCMV-CP gene and their phylogeny were studied. The analysis revealed that Thai SCMV CP gene contained 942 nucleotides encoded for coat protein MW of 33.65 kDa. The nucleotide sequence identity among cloned CPs from three isolates were 98-99% to each other. The N-terminus of the Thai SCMV CP contained a distinctive sequence, especially at nucleotide positions 28-46 of the N-terminal variable 70 amino acid residues which discriminated Thai SCMV from all previously reported SCMV isolates. Thai SCMV were placed in a separate branch of SCMV-MDB cluster which is closely related to most of SCMV isolates from maize, and distinct from the other cluster containing sugarcane-SCMV strains.
At the end of 2009, 60 leaf samples of tomato plants (Solanum lycopersicum) were collected from the field in Lampang province, Thailand. The plant samples were stunted and had leaf symptoms including necrosis, distortion and discoloration. After initial examination by electron microscopy, that revealed no virus particles, the samples were tested for viroids. Total RNA was extracted using a CTAB method (Tangkanchanapas et al., 2005) and then tested for viroids by reverse transcription-polymerase chain reaction (RT-PCR;Tangkanchanapas et al., 2005). Three different primer sets were employed (Table 1), with the PC2 primer set designed to detect six pospiviroids: Columnea latent viroid (CLVd), Tomato apical stunt viroid (TASVd), Tomato planta macho viroid (TPMVd), Tomato chlorotic dwarf viroid (TCDVd), Potato spindle tuber viroid (PSTVd) and Mexican papita viroid (MPVd). Of the 60 samples collected, three tested positive using the PC2 primer set. The amplified DNA bands from these three positive samples were purified, ligated to pGEM®-T Easy vector (Promega, Madison, USA) and sent for automated sequencing. Identical 348 bp sequences were obtained from all three isolates and these were deposited in GenBank (Accession Nos. JF446891, JF446892 and JF446893).These sequences shared 99% identity with a Pepper chat fruit viroid (PCFVd) isolate (FJ409044), previously reported from Netherlands (Verhoeven et al., 2009).One of the isolates (PCFVd-Thai; JF446893) was further characterised by mechanical transmission onto tomato seedlings (cv. Rutgers).Four weeks after inoculation, the infected plants showed necrosis on leaf veins, petioles and stems, leaf distortion and stunting (Fig. 1), closely matching the symptoms seen on the original tomato plants found in the field. Viroid diseases have previously been reported in Thailand: Citrus exocortis viroid (CEVd) in citrus and tomato, Hop stunt viroid (HSVd) and Grapevine yellow speckle viroid (GYSVd) in grapevine and CLVd in tomato. This is the first report of PCFVd occurring in the country.
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