Occurrence of Tomato chlorosis virus on tomato in Réunion Island

Pronounced yellowing symptoms on the lower and middle leaves of tomato plants, similar to those caused by magnesium deficiencies, were observed in 2004 and 2005 in farmer's greenhouses in Réunion Island; an island situated east of Madagascar in the Indian Ocean. Yellow leaf symptoms include irregular chlorotic mottling (Fig. 1) and the interveinal areas on leaves develop red or brown necrotic flecks. Furthermore, fruit sizes were reduced in most of the greenhouses having those symptomatic plants and yields were indirectly affected. The flame-like pattern of the discoloured leaves (Fig. 1) and the abundance of whiteflies on these plants suggested the possible involvement of Tomato chlorosis virus (ToCV) or Tomato infectious chlorosis virus (TICV) (Closteroviridae, Crinivirus) (Wisler et al., 1998).

Twenty symptomatic leaf samples were collected from tomato plants in March 2005, and total RNA was extracted from these samples using the Qiagen RNeasy Plant Mini kit. For the detection of a potential crinivirus, a nested PCR was performed (Dovas et al., 2002). The method consist of a one-step RT-PCR using primers HS-11 and HS-12, followed by the nested PCR with primers TIC-3/TIC-4 and ToC-5/ToC-6, for detecting TICV or ToCV respectively. These primers were designed to amplify the highly conserved region of the heat shock protein 70 gene. A PCR product at the expected size was observed with ToCV primers for 14 of the symptomatic leaf samples. No PCR product was observed for the PCR performed with the TICV primers. Three PCR products were cloned using pGEM-T easy vector system II (Promega, France) and sequenced (Genome Express, France) (Accession Nos AJ968394, AJ968395 and AJ968396). Sequences obtained from the three samples had 99.5% nucleotide identity when aligned (DNAMAN, Lynnon BioSoft, Quebec). The most significant sequence alignments (NCBI, BLASTn) were 98% with ToCV isolates from the USA (Accession No AF024630), Spain (Accession Nos AF233435, AF215818 and AF215817) and from Italy (Accession Nos AF234029 and AY048854). To our knowledge, this is the first report of the presence of ToCV in Réunion Island.