LIBRO-CONGRESO-CITRUS

S14
Conformation Polymorphism (SSCP) and samples with different electrophoretic pattern were cloned and their
nucleotide sequence compared by CLUSTALW. The results confirmed the presence of CLBV in Italy and three
new sequences were found and deposited in GenBank. No positive samples were found in varieties other
than Nagami kumquat, with 80% of infected trees, or calamondin, with 31% infected trees. This low CLBV
incidence in symptomatic calamondin trees could be due to a low virus titer in calamondin, or to bud union
crease being caused a different pathogen or by the interaction between biotic or abiotic factors.
S14P26
Citrus leaf blotch virus (CLBV) invades meristematic regions in Nicotiana benthamiana and citrus
Agüero J., Vives M.C., Velázquez K., Ruiz-Ruiz S., Juárez J., Navarro L., Moreno P., and Guerri J.
Instituto Valenciano de Investigaciones Agrarias (IVIA), Centro de Protección Vegetal y Biotecnología, Moncada, Valencia, Spain.
jguerri@ivia.es
Systemic infection by plant viruses involves long distance movement through the phloem sieve tubes.
Since the vascular system is not developed in the vicinity of apical meristems, most viruses are unable to
reach this region, a phenomenon that has been exploited to recover virus-free citrus plants by shoot-tip
grafting in vitro. However, Citrus leaf blotch virus (CLBV) is difficult to eliminate by this method and seed
transmission has been observed, suggesting that this virus does invade meristematic regions. To examine this
hypothesis we monitored CLBV movement and accumulation using and infectious cDNA clone of the CLBV
genome engineered to express the green fluorescent protein (GFP) gene. For this purpose, the promoter
inducing transcription of the ORF2 subgenomic RNA was conveniently reduced and duplicated in the 3’
terminal untranslated region to obtain the infectious clone clbv3’pr-GFP. This extra gene does not affect virus
functionality and produces GFP fluorescence in infected cells. Nicotiana benthamiana plants agroinoculated
with clbv3’pr-GFP showed fluorescence in all tissues including meristematic regions, but in citrus plants
infected with CLBV-GFP fluorescence was not visible and GFP had to be detected by western blot analysis. In
lime plants inoculated with Citrus tristeza virus (CTV), which cannot infect meristematic regions, CLBV or CLBV
plus CTV, hybridization and real time RT-PCR allowed detection of CLBV, but not of CTV, in shoot tips less than
0.2 mm long.
S14P27
Simultaneous detection of Citrus exocortis viroid and Hop stunt viroid in citrus plants by direct
tissue-print duplex real-time RT-PCR
Bertolini E. 1 , Martínez M.C. 1 , Serra P. 2 , Olmos A. 2 , Carbó C. 1 , Duran-Vila N. 1 , and Cambra M. 1
1 Instituto Valenciano de Investigaciones Agrarias (IVIA), Centro de Protección Vegetal y Biotecnología, Moncada, Valencia, Spain; and
2 Instituto de Biología Molecular y Celular de Plantas (IBMCP), Departamento de Biología del Estrés, Valencia, Spain.
ebertoli@ivia.es
Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd) are the causal agents of Exocortis and Cachexia,
respectively, the most important viroid diseases of citrus trees. Traditionally the detection of citrus viroids
has been performed by biological indexing and/or biochemical and molecular techniques. Biological indexing
(based on the use of indicator citrus species) is time consuming and requires expensive infrastructures.
Biochemical and molecular methods (based on sPAGE, tissue print-hybridization after graft-inoculation on
Citrus medica or conventional RT-PCR), require extract preparation, and they are generally laborious and
time consuming and present risk of contamination, often resulting inconvenient for routine diagnostics. The
usefulness of real-time RT-PCR to detect viroids has been reported previously. A real-time RT-PCR method
using two newly designed primer pairs and TaqMan probes was successfully developed and patented for the
simultaneous detection of both viroid species. A direct tissue-print sample preparation method was tested
and used to analyze samples from different citrus species and in different seasons. Results confirm that the
real-time RT-PCR approach is as reliable as biological indexing and more sensitive than conventional sPAGE
and molecular hybridization, enabling reliable detection of both citrus viroids at any season with the same
accuracy. The developed methods open new possibilities for sensitive, simple and accurate detection of citrus
viroids. A kit for the detection of both citrus pathogens has been validated and its use could facilitate the
sanitary controls required in certification programs.
248 - VALENCIA CONFERENCE CENTER, 18th-23rd NOVEMBER 2012