Molecular Variability of Citrus tristeza virus Determined by
Assymmetric PCR-ELISA and SSCP
Silvija Černi1, Vera Martins2, Mladen Krajačić1, Dijana Škorić1 ,Gustavo Nolasco2
of Zagreb, Faculty of Science, Department of Biology, Marulićev trg
9A, Zagreb, 2Universidade do Algarve, Faculdade de Engenharia de Recursos
Naturais, Gampus de Gambelas, Faro, Portugal
1University
Dipl. ing. Silvija Černi, University of Zagreb, Faculty of Science, Department of
Biology, Marulićev trg 9A, 10000 Zagreb, Croatia
Tel. +(385)14843851
Fax. +(385)14844001
E-mail: scerni@botanic.hr
1
Citrus tristeza Closterovirus (CTV) is one of the most important viral
pathogens of citrus. CTV virions are phloem-limited flexuous filaments of
2000x11 nm in size. With its monopartite, single-stranded, positive-sense RNA
genome of 19.3 kb organized into 12 open reading frames (ORFs), CTV is
considered to be the largest single-stranded RNA plant virus characterized so
far. Aphids naturally spread the virus but it is also easily transmitted by grafting.
Depending on a virus strain, scion cultivar and rootstock, CTV can induce one of
the three main syndromes: “quick decline”, “stem pitting” and “seedling yellows”,
but some mild strains can go unnoticed because of the lack of visible symptoms.
In nature, CTV usually exists as a mixture of strains. Due to a low concentration
and seasonal variations of the virus in field trees, the detection is difficult and it is
necessary to use methods for sensitive and reliable CTV diagnosis and strain
typing.
Five CTV positive Croatian citrus samples have been strain-typed using
asymmetric PCR ELISA and single strand conformation polymorphism (SSCP)
analysis. CTV coat protein (CP) gene was amplified in an immunocapture
asymmetric RT-PCR (IC/RT-PCR) and amplicons were hybridized with the eight
CTV group-specific probes. In addition, SSCP analysis of CTV CP gene was
performed. According to the probe-typing results, all five analyzed samples were
mix-infected and showed homology to CTV phylogenetic groups 2 and M which
represent mild, asymptomatic, CTV strains. One sample also showed homology
to probe from group 4 and another to probe from group 3a. Groups 3a and 4
encompass very severe, “quick decline” strains and group 3a can additionally
induce “stem pitting” syndrome. Multiple band patterns in SSCP profiles
confirmed mixed-infectious in all analyzed samples. Probe-typing results were
further corroborated by cloning and sequencing of IC/RT-PCR amplicons.
Phylogenetic analysis was in accordance with the probe-typing groupclassification for all the samples.
Neither of the amplicons, even from the same virus strain, had the same
sequence. All the results obtained represent molecular confirmation of the
considerable biological diversity of CTV found in Croatian citrus samples.
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