Detection of citrus psorosis‐ringspot virus using RT‐PCR and DAS‐ELISA

Abstract

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.

Recent advances in Citrus psorosis virus

“…Portions of RNA1 have been cloned and a consensus sequence was obtained from which primers were designed for RT-PCR [20]. The CPG has been used to develop RT-PCR [5].…”

“…The CPG has been used to develop RT-PCR [5]. In both cases, the RT-PCR was able to detect CPsV at concentrations substantially lower than those required for detection by immunoassays and electron microscopy [20]. However, the sensitivity has not been sufficiently high to detect the heterologous psorosis isolates using primers designed from CPV-4.…”

“…The tools used to detect CPsV in citrus trees are mainly based on using indicator plants and on laboratory tests including enzyme-linked immunosorbent assay (ELISA) [20,33,80]. After CPsV sequences became available, molecular tests based on RT-PCR and dot blot or tissue-print hybridization were also developed for CPsV detection [5,20,26,28,32,55,57,67].…”

“…Availability of an improved antiserum [20] and of monoclonal antibodies (MAbs) to the coat protein (CP) [1,46], has allowed detection of CPsV by double and triple antibody sandwich (DAS-and TAS-) ELISA, in biologically characterized and non-characterized psorosis sources [2,12,32]. Monoclonal antibodies (24 MAbs) raised to the southern Italian CPsV isolate IAM-191Xa have been found to be useful to distinguish between CPsV isolates.…”

“…The original isolate CPV-4 produces necrotic local lesions when upon mechanical inoculation of the older, fully expanded leaves of C. quinoa [53,54]. Another factor is that CPV-4 can reach relatively high concentrations in infected tissues [20].…”