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).

Viroid-infected Tomato and Capsicum Seed Shipments to Australia

Pospiviroid species are transmitted through capsicum and tomato seeds. Trade in these seeds represents a route for the viroids to invade new regions, but the magnitude of this hazard has not been adequately investigated. Since 2012, tomato seed lots sent to Australia have been tested for pospiviroids before they are released from border quarantine, and capsicum seed lots have been similarly tested in quarantine since 2013. Altogether, more than 2000 seed lots have been tested. Pospiviroids were detected in more than 10% of the seed lots in the first years of mandatory testing, but the proportion of lots that were infected declined in subsequent years to less than 5%. Six pospiviroid species were detected: Citrus exocortis viroid, Columnea latent viroid, Pepper chat fruit viroid, Potato spindle tuber viroid, Tomato chlorotic dwarf viroid and Tomato apical stunt viroid. They were detected in seed lots exported from 18 countries from every production region. In many seed lots, the detectable fraction (prevalence) of infected seeds was estimated to be very small, as low as 6 × 10−5 (~1 in 16,000; CI 5 × 10−6 to 2.5 × 10−4) for some lots. These findings raise questions about seed production practices, and the study indicates the geographic distributions of these pathogens are uncertain, and there is a continuing threat of invasion.

First Report of Columnea latent viroid (CLVd) in Tomato in Mali

During surveys of tomato (Solanum lycopersicum) fields in Niono, Mali, conducted in March 2011, unusual disease symptoms, including stunted growth, epinasty, and chlorosis of leaves and necrosis of leaf veins and stems were observed in multiple fields. The incidence of these symptoms was low (~1 to 5%), but they were distinct from those associated with known diseases in the region. A representative leaf sample with these symptoms was applied to filter paper (FTA cards, Whatman), and DNA and RNA extracts were prepared according to manufacturer instructions. RT-PCR tests for Tomato spotted wilt virus, Tobacco streak virus, Tomato necrotic spot virus, Tobacco/tomato mosaic viruses, Cucumber mosaic virus, Alfalfa mosaic virus, torradoviruses, and potyviruses, and PCR tests for begomoviruses, phytoplasmas, and ‘Candidatus Liberibacter’ infection were also negative. However, virus-like symptoms developed in all 16 tomato seedlings (cv. Early Pak 7) 7 to 10 days after mechanical (sap) inoculation with inoculum prepared from the FTA sample. No symptoms developed in mock-inoculated control plants (n = 3). Symptoms induced included stunted growth and severe epinasty of leaves, followed by necrosis of leaf veins, petioles, and stems. These symptoms were similar to those observed in plants in Mali. When RNA extracts prepared from leaves of these symptomatic plants were mechanically inoculated onto 24 tomato seedlings, similar symptoms developed in all plants, suggesting the causal agent might be a viroid. RT-PCR tests with RNA from symptomatic tomato leaves and universal (3) and various specific Pospiviroid primer pairs were negative. However, equivalent RT-PCR tests conducted with the pCLV4/pCLVR4 primer pair specific for Columnea latent viroid (CLVd) (2) generated a DNA fragment of the expected size (~370 bp). The sequence of this DNA fragment (GenBank Accession No. JQ362419) was 99% identical with those of CLVd isolates from the Netherlands (AY373446 and AY372396). In host range studies, the CLVd isolate from Mali induced symptoms in all 48 mechanically-inoculated tomato plants, whereas no symptoms developed (up to 90 days after inoculation) in inoculated Chenopodium quinoa, C. amaranticolor, Nicotiana benthamiana, N. tabacum (cvs. Havana, Glurk and Turkish), N. glutinosa, Datura stramonium, common bean (cvs. Topcrop and Pinto bean), pumpkin (cv. Small Sugar), pepper (Capsicum annuum, cv. Yolo Wonder) and cucumber (cvs. Emparator and Poinsett 76) plants (results of three independent experiments with six plants per experiment). Symptomless infections were detected in pepper (24 of 30), N. benthamiana (25 of 25), and N. tabacum cv. Turkish (11 of 24) plants by RT-PCR with the pCLV4/pCLVR4 primer pair. To our knowledge, this is the first report of CLVd infecting tomato in Mali. RT-PCR tests of seeds collected from CLVd-infected tomato, pepper, and N. benthamiana plants also detected CLVd (1). Thus, it is possible that CLVd was introduced into Mali in association with seed. References: (1) O. Batuman and R. L. Gilbertson. Phytopathology 102:S4.9, 2012. (2) R. L. Spieker. Arch. Virol. 141:1823, 1996. (3) J. T. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004.

Columnea latent viroid. [Distribution map].

A new distribution map is provided for Columnea latent viroid. Pospiviroidae: Pospiviroid. Host: tomato (Solanum lycopersicum). Information is given on the geographical distribution in Europe (Belgium; Denmark; Germany; Italy; Netherlands; England and Wales, UK), North America (Canada, and Maryland, USA), and Central America and Caribbean (Costa Rica).