LIBRO-CONGRESO-CITRUS

S14
infected tree incites the psorosis A syndrome (PsA), including a shock reaction of the first flush and transient
chlorotic flecking in young leaves of the following flushes, whereas inoculation with scaled bark pieces incites
the psorosis B syndrome (PsB), that in addition to PsA includes gummy pustules in old leaves and branches.
Psorosis disease is caused by Citrus psorosis virus (CPsV), an ophiovirus with three negative-stranded genomic
RNAs. While comparison of RNA1 or RNA3 fragments by single-strand conformation polymorphism (SSCP)
analysis did not show any difference between plants affected by the PsA or the PsB syndromes, a fragment of
RNA2 enabled discriminating both syndromes by their SSCP profile. Some plants inoculated with scaled bark
did not show PsB at 6 months post-inoculation (mpi) and their RNA2 gave a PsA-type SSCP profile. At 12 mpi
these plants showed pustules in the trunk but not in the leaves. SSCP analysis of RNA2 showed only PsA-type
variants in the leaves, whereas the pustuled trunk areas contained both PsA and PsB variants, the latter being
predominant. These results suggest that psorosis-affected trees contain the two sequence variants and that
the PsB variant is associated with pustules and tends to accumulate in the trunk bark.
S14P21
Sensitivity to Citrus psorosis virus of species and hybrids of the genus Citrus and relatives
Velázquez K., Alba L., Zarza O., Vives M.C., Pina J.A., Juárez J., Navarro L., Moreno P., and Guerri J.
Instituto Valenciano de Investigaciones Agrarias (IVIA), Centro de Protección Vegetal y Biotecnología, Moncada, Valencia, Spain.
velazquez_kar@gva.es
Citrus psorosis virus (CPsV), genus Ophiovirus, causes an important disease in many citrus growing regions.
Virus damage can be controlled using resistant or tolerant cultivars, but sensitivity of many species and hybrids
of Citrus and related genera is unknown. To find potential sources of CPsV resístance we first propagated 61
species (or cultivars) and hybrids of Citrus and related genera [Citrus (37), Microcitrus (5), Fortunella (6),
Eremocitrus (1), Pleiospermium (1), Atalantia (1), Severinia (1), Clausena (1), Poncirus (1) and hybrids (7)] on
rough lemon seedlings inoculated with the CPsV isolate PB-143. Out of the 61 accessions tested 54 showed
symptoms and positive ELISA reaction, 2 (M. inodora y F. hindsii) were symptomless but gave high ELISA values,
suggesting tolerance to CPsV, 5 (C. depresa, C. reshni, C. excavata, Carrizo citrange (CC) and citrumelo CPB
4475) showed symptoms in the first flush but negative ELISA reaction, and 1 (P. trifoliata) was ELISA negative
and symptomless. We then examined infection by quantitative real time RT-PCR in C. reshni, P. trifoliata and
CC seedlings inoculated with CPsV isolates P-121, PB-102 and PB-143. While P-121 was detected in all C.
reshni, and CC, and in 75% of P. trifoliata plants, the other isolates infected only 32-82% (PB-143) or 20-91%
(PB-102) of the inoculated plants. These three accessions showed necrosis around the inoculum patch that
impaired or delayed infection, suggesting isolate-dependent partial resistance.
S14P22
Detection and quantitation of Citrus psorosis virus by real time RT-PCR
Velázquez K., Alba L., Guerri J., Moreno P., Navarro L., and Vives M.C.
Instituto Valenciano de Investigaciones Agrarias (IVIA), Centro de Protección Vegetal y Biotecnología, Moncada, Valencia, Spain.
cvives@ivia.es
Citrus Psorosis, an important disease in many citrus growing regions, is caused by Citrus psorosis virus (CPsV),
an ophiovirus transmitted through infected buds and naturally spread in areas of South America. The CPsV
genome consists of three single-stranded, negative-sense RNAs. Psorosis has been diagnosed by biological
indexing on indicator plants, and more recently by ELISA, tissue-print hybridization or RT-PCR. ELISA enables
fast and specific detection of CPsV, albeit with low sensitivity, and the other procedures do not allow virus
quantitation in different varieties, tissues and seasons. To develop a highly sensitive and reliable technique
for CPsV detection and quantitation we set up a real-time RT-PCR assay (qrt-RT-PCR) using SYBR Green and
the CPsV RNA3 as target. This protocol enables detection of 1000 CPsV RNA copies in plant extracts with a
linear response along a dynamic range of 7 log units of concentration. Sensitivity of CPsV detection by qrt-RT-
PCR is much higher than with ELISA or conventional RT-PCR, and it enables detection of distinct viral isolates
infecting field trees of different citrus species all year around. Maximum accumulation of CPsV was observed
in young leaves and bark, but it was also detected in old bark and leaves. The lowest CPsV accumulation was
246 - VALENCIA CONFERENCE CENTER, 18th-23rd NOVEMBER 2012