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Detection of citrus psorosis‐ringspot virus using RT‐PCR and DAS‐ELISA
Author(s) -
GARCIA M. L.,
SANCHEZ DE LA TORRE M. E.,
DAL BO E.,
DJELOUAH K.,
ROUAG N.,
LUISONI E.,
MILNE R. G.,
GRAU O.
Publication year - 1997
Publication title -
plant pathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.928
H-Index - 85
eISSN - 1365-3059
pISSN - 0032-0862
DOI - 10.1046/j.1365-3059.1997.d01-81.x
Subject(s) - biology , serial dilution , antiserum , virology , virus , polymerase chain reaction , plant virus , chenopodium quinoa , heterologous , veterinary medicine , microbiology and biotechnology , horticulture , antibody , gene , genetics , medicine , alternative medicine , pathology
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.