Psorosis, sometimes also associated with ringspot symptoms, is a widespread and damaging disease of citrus in many parts of the world including South America and the Mediterranean basin. We describe the application of RT‐PCR and DAS‐ELISA diagnostics to an isolate of citrus ringspot virus (CtRSV‐4) and other virus isolates associated with this disease. Fragments of cDNA from bottom‐component RNA of CtRSV‐4 were cloned and sequenced, and PCR primers were designed, 5′ACAATAAGCAAGACAAC upstream, and 5′CCATGTCACTTCTATTC downstream. RT‐PCR experiments using these primers allowed detection of CtRSV‐4 in infected citrus leaves down to a tissue dilution of 1/12 800 representing 2 μg of tissue, and less sensitive detection of the related citrus psorosis‐associated virus (CPsAV90‐1‐1) and four other psorosis isolates from Argentina and the USA. In addition, CtRSV‐4 particles were partially purified from local lesions in Chenopodium quinoa, and the preparations used to raise a rabbit antiserum. The antiserum was absorbed with extracts of healthy C. quinoa leaves, and a DAS‐ELISA kit was prepared and tested for detection of CtRSV‐4, CPsAV90–1‐1, and other psorosis isolates from Argentina, the USA, Italy and Spain. The ELISA detected CtRSV‐4 down to a tissue dilution of 1/1600, and most other psorosis isolates down to dilutions of 1/200–1/800. Three of a total of 20 heterologous isolates were consistently negative. Comparison of the PCR and ELISA results suggests that both methods can be used for detection of a range of psorosis isolates, but that variation of the viruses in the field might cause problems for any one diagnostic test.
Citrus psorosis virus (CPsV) causes a citrus disease occurring worldwide. Isolate CPV 4 has a genome with three single-stranded RNAs. The complete sequence of RNA 2 (1643 nucleotides) is reported here. Northern blot hybridization with strandspecific probes showed that most of the encapsidated RNA 2 is of negative polarity, although a small amount of the complementary strand may also be present in particles. The RNA 2 complementary strand contained a single open reading frame encoding a protein of 476 amino acids, which includes a motif resembling a nuclear localization signal. The sequence of this putative protein shows no significant similarity to any other in the databases. In the 3h-terminal untranslated region there is a putative polyadenylation signal. No subgenomic RNAs derived from RNA 2 were detected. Psorosis is a serious disease affecting citrus in many countries (Roistacher, 1993). In Argentina, it seems to be spread by an unknown vector (Ben4 atena & Portillo, 1984) and it causes important losses (Danos, 1990). The presumed causal agent, Citrus psorosis virus (CPsV), is the type member of the genus Ophiovirus (Milne et al., 2000) but the low concentration of virus particles in citrus tissues and their instability has so far impeded characterization of the genome. All species of the genus, Ranunculus white mottle virus (RWMV), Tulip mild mottle mosaic virus (TMMMV) and Mirafiori lettuce virus (MiLV), have similar morphology : circular, filamentous naked nucleocapside about 3 nm in diameter of different lengths. Limited information is available about their genomic structure : RWMV has at least three RNAs of approximately 7n5, 1n8 and 1n5 kb and a coat protein of 43 kDa (Vaira et al., 1997) ; TMMMV
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