First Report of Citrus leaf blotch virus on Kumquat in Italy.

Abstract

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.