Citrus tristeza virus (CTV) is the causal agent of various diseases with dramatic effects on citrus crops worldwide. Most Citrus species, grown on their own roots, are symptomless hosts for many CTV isolates. However, depending on different scion-rootstock combination, CTV infection should result in distinct syndromes, being 'tristeza' the more severe one, leading to a complete decline of the susceptible plants in a few weeks. Transcriptomic analyses revealed several genes involved either in defense response, or systemic acquired resistance, as well as transcription factors and components of the phosphorylation cascades, to be differentially regulated during CTV infection in Citrus aurantifolia species. To date little is known about the molecular mechanism of this host-pathogen interaction, and about the rootstock effect on citrus response to CTV infection. In this work, the response to CTV infection has been investigated in tolerant and susceptible scion-rootstock combinations by two-dimensional gel electrophoresis (2DE). A total of 125 protein spots have been found to be differently accumulated and/or phosphorylated between the two rootstock combinations. Downregulation in tolerant plants upon CTV infection was detected for proteins involved in reactive oxygen species (ROS) scavenging and defense response, suggesting a probable acclimation response able to minimize the systemic effects of virus infection. Some of these proteins resulted to be modulated also in absence of virus infection, revealing a rootstock effect on scion proteome modulation. Moreover, the phospho-modulation of proteins involved in ROS scavenging and defense response, further supports their involvement either in scion-rootstock crosstalk or in the establishment of tolerance/susceptibility to CTV infection.

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In the course of a survey to select superior old citrus lines in the area of Siracusa (Sicily, Italy), trees in several blocks of Fortune (Citrus reticulata Blanco), Nova (C. reticulata Blanco), Satsuma (C. unshiu (Macfad.) mandarins Marc.), and Marsh grapefruit (C. paradisi Macfad.) propagated on sour orange (C. aurantium L.) rootstock showed stunting, decline, dieback, and small-sized fruits. Stunting was particularly evident in grapefruit. Declined plants consistently showed pin-holing in the cambial face of sour orange bark below the bud union line, which is often associated with Citrus tristeza virus (CTV) infection. Young shoots from 600 Fortune, 300 Nova, 400 Satsuma, and 20 Marsh grapefruit plants showing decline were analyzed by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) (Loewe Phytodiagnostica Biochemica, Sauerlach, Germany) and by immunoprinting-ELISA (Agritest Srl Valenzano-Bari-Italy) using CTV specific polyclonal antibodies. All decline tree samples reacted positively with both techniques while healthy greenhouse controls were negative. Total RNA was extracted from 50 of those plants, 25 Fortune and 15 Nova mandarins, 5 Satsuma, and 5 Marsh grapefruit (Qiagen RNeasy Plant minikit, Qiagen S.P.A., Milan, Italy), and tested in reverse transcription-polymerase chain reaction (RT-PCR) using specific primers for genes p20 (forward 5′-CGA GCT TAC TTT AGT GTT A-3′ from CTV T36 genomic position 17767-17786 and reverse 5′-TAA TGT CAA ACT GAC CGC from CTV T36 position 18269-18286) and p23 (forward 5′-ACT AAC TTT AAT TCG AAC A-3′ from CTV T36 position 18347-18286 and reverse 5′-AAC TTA TTC CGT CCA CTT C-3′ from CTV T36 position 19026-19044) (2). In all cases, DNA fragments of the expected size were amplified. Equivalent samples from CTV-free greenhouse control plants did not react in ELISA and yielded no DNA after amplification with the same primers. When the history of the plants in the affected blocks was traced, it was found that all Fortune, Nova, satsuma and Marsh grapefruit trees had been propagated from budwood illegally imported from Spain 10 years before, suggesting the possibility that the imported buds were infected with CTV. The estimated number of infected plants in the area of Siracusa is approximately 10,000, and some evidence suggests that the virus might be spreading in the area (work in progress). Only scattered CTV-infected trees had been detected in Italy previously (1). To our knowledge, this is the first report of an important CTV outbreak in Italy. Additional surveys are being conducted to get a more accurate estimation of the CTV incidence, to determine if the virus is being dispersed by aphid vectors, and to biologically and molecularly characterize the virus strains present in the affected area. Presently, there are approximately 100,000 ha of citrus in Sicily, mostly grown on decline susceptible sour orange rootstock. The presence and potential spread of CTV is a major threat for this citrus industry. References: (1) M. Davino and G. Terranova. Frutticoltura 61:18, 1999. (2) A. Sambade et al. Plant Pathol. 51:257, 2002.

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