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During the spring of 2006, nurserymen reported observations of the bud union disorder of 'Nagami' kumquat scions propagated on Troyer citrange rootstock to the CRA-Istituto Sperimentale per l'Agrumicoltura. These plants showed reduced canopy volume and new shoots below graft points 6 months after propagation; the bud union was brittle and broke down easily after 1 year. After tests excluded common citrus viruses and viroids that might cause the incompatibility (e.g., Citrus tristeza virus, Citrus psorosis virus, Citrus exocortis viroid, and Hop stunt viroid), we tested for Citrus leaf blotch virus (CLBV), a virus previously associated with a bud union crease in kumquat (2). Leaves were collected from 100 2-year-old kumquat plants from a nursery near Messina (Sicily [Italy]); 50 were grafted on sour orange rootstock (asymptomatic) and 50 were grafted on Troyer citrange rootstock (symptomatic). Total RNA was extracted using Qiagen RNeasy Plant Mini Kit (Qiagen S.P.A. Milan, Italy). Primers previously reported (1,2) and designed from a published CLBV sequence (Genbank Accession No. AJ318061) were used in reverse transcription (RT)-PCR assays to amplify the RNA-dependent RNA polymerase gene (sense primer KU 27, 5'-GATGCAAGCCAGGATGAATAC-3', genomic positions 5321-5340 and anti-sense primer KU 15, 5'-CAGACACTCCAAGACCTTTCC-3', genomic positions 5776-5756) and the coat protein gene (sense primer KU18, 5'-TTAAGATTACAGACACGAAGG-3' genomic positions 7686-7706 and anti-sense primer KU 19 5'-CTGTTTTTGAATTTTGCTCG-3', genomic positions 8123-8104). All kumquat samples yielded amplicons of the expected size (456 and 438 bp). No amplicons were obtained from healthy plants. Amplicons for each gene were cloned into the pGEM-T Easy Vector (Promega Italy, Milan), and four clones for each plasmid DNA were sequenced in both directions. Consensus sequences of the two genes (Genbank Accession Nos. EF203229 and EF203230) had 96 and 97% nucleotide sequence identity, respectively, and both had 99% amino acid identity with the previously reported CLBV sequence (Genbank Accession No. AJ318061). Approximately 400,000 ornamental kumquats are produced annually in Italy. CLBV infection can cause serious production losses because of the decline associated with bud union disorders grafted onto trifoliate orange and trifoliate-derived rootstocks. References: (1) L. Galipienso et al. Eur. J. Plant Pathol. 110:175, 2004. (2) M. C. Vives et al. Virology 287:225, 2001.

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.