Reassessment of the Columnea latent viroid (CLVd) Taxonomic Classification.

Columnea latent viroid (CLVd) is a member of the Pospiviroid family and its naked circular RNA genome typically forms native "rod-like" secondary structures. In this work, the CLVd taxonomy was reevaluated based on sequence similarity and phylogenetic analysis, as well as the evaluation of the symptom development and disease severity of four selected CLVd isolates in a range of host species. The phylogenetic analysis showed that all CLVd isolates were clustered into five distinct clades: (I) severe isolates originally found in tomato crops in Thailand, (II) ornamental isolates, (III) mild isolates originally found in tomato crops in Thailand, and two clades (IV and V) containing mild isolates originating mainly from tomato crops in European countries, with different virulence levels on several hosts. Our analysis demonstrated that some CLVd isolates have a sequence similarity of less than 90% within the species taxon, as well as distinct biological characteristics (symptom development and virulence), both of which are important ICTV criteria for viroid classification. For these reasons, we propose that CLVd should be re-classified into at least three main taxonomic lineages: a "CLVd-tomato Asian lineage" (I), a "CLVd-tomato European lineage" (IV) and a "CLVd-ornamental European lineage" (II), plus two minor lineages (III and V), fitting the ICTV criteria.

Whole-Genome Deep Sequencing Reveals Host-Driven in-planta Evolution of Columnea Latent Viroid (CLVd) Quasi-Species Populations.

Columnea latent viroid (CLVd) is one of the most serious tomato diseases. In general, viroids have high mutation rates. This generates a population of variants (so-called quasi-species) that co-exist in their host and exhibit a huge level of genetic diversity. To study the population of CLVd in individual host plants, we used amplicon sequencing using specific CLVd primers linked with a sample-specific index sequence to amplify libraries. An infectious clone of a CLVd isolate Chaipayon-1 was inoculated on different solanaceous host plants. Six replicates of the amplicon sequencing results showed very high reproducibility. On average, we obtained 133,449 CLVd reads per PCR-replicate and 79 to 561 viroid sequence variants, depending on the plant species. We identified 19 major variants (>1.0% mean relative abundance) in which a total of 16 single-nucleotide polymorphisms (SNPs) and two single nucleotide insertions were observed. All major variants contained a combination of 4 to 6 SNPs. Secondary structure prediction clustered all major variants into a tomato/bolo maka group with four loops (I, II, IV and V), and a chili pepper group with four loops (I, III, IV and V) at the terminal right domain, compared to the CLVd Chaipayon-1 which consists of five loops (I, II, III, IV and V).

First reported occurrence of citrus bent leaf viroid and citrus dwarfing viroid on imported oranges from China and lime fruits from Cambodia.

A number of viroids can cause serious damage on Citrus spp. ranging from stunting, bark scaling, yellowing and epinasty of leaves over stem pitting and gumming. However, so far, they have never been found in Thailand. In recent years, the import of orange and lime fruits from China and Cambodia, respectively, increased, and holds a substantial risk of viroid introduction and spread in Thailand. Orange and lime fruit samples in 2013 and 2014 were screened for the presence of citrus exocortis viroid (CEVd), hop stunt viroid (HSVd), citrus bent leaf viroid (CBLVd) and citrus dwarfing viroid (CDVd; CVd-IIId) by means of specific RT-PCR methods. Only CBLVd and CDVd were detected, clearly generating the expected amplification bands of around 320 and 300 base pairs, respectively. The presence of CBLVd and CDVd was confirmed by amplicon sequencing and RNA secondary structure analysis. About 34.2 and 19.5% of 41 samples (around 2300 fruits) of the imported lime fruit were infected with CBLVd and CDVd, respectively. CBLVd was detected in 62.3% of the 77 samples (around 2000 fruits) from imported oranges, while CDVd was found in 75.3%. This result indicates that the incidence of both CBLVd and CDVd in the imported citrus fruits is quite high. In addition, both viroid diseases have not been reported in Thailand. However, lack of information on the actual status of both viroids leads to difficulties in determining their impact on the Thai citrus industry.

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) designed for fast and sensitive on-site detection of Pepper chat fruit viroid (PCFVd).

Pepper chat fruit viroid (PCFVd) is one of the most important tomato and pepper diseases causing serious losses, affecting productivity, fruit quality and even international seed trade. Reverse transcription Loop-mediated isothermal amplification (RT-LAMP) is a fast and reliable RNA amplification assay, out competing conventional reverse transcription polymerase chain reaction (RT-PCR) in robustness, analytical sensitivity and specificity, and cost-effectiveness. In this work, a PCFVd specific RT-LAMP detection assay, based on a set of six primers was developed. Under the optimized conditions, PCFVd could be detected within 15 min, with a sensitivity of detecting PCFVd that was almost the same as a probe-based qRT-PCR and 10-100 times higher than the available RT-PCR methods. No cross-amplification with other viroids and tomato viruses was observed. The validated assay was also adapted for convenient on-site detection. Besides replacing the full RNA extraction with a simple lysis procedure, several visualization options, of which the use of SYTO9 was the most convenient, were presented to accommodate any in-field application of the method.