First Report of Chrysanthemum stunt viroid in Various Cultivars of Argyranthemum frutescens in France.

Described for the first time in Chrysanthemum indicum in the United States, Chrysanthemum stunt viroid (CSVd) was reported to naturally infect species in the Asteraceae family (1,3), as well a few hosts in other families. In May 2010 in a nursery in southwest France, the occurrence of stunted A. frutescens plantlets of cv. Butterfly showing yellow deformed leaves with terminal necrosis, which resembled the growth reduction, flower distortion or leaf necrosis symptoms reported for CSVd in Argyranthemum spp. (3), was reported. Mother plants from which the plantlets originated were asymptomatic. Reverse transcription (RT)-PCR with universal pospiviroid primers Pospi1-FW/RE (4) was performed on five symptomatic plants. A fragment of expected size (197 bp) was obtained in all cases. Viroid infection was confirmed by RT-PCR with two sets of primers: Vid-FW/RE using a 59°C annealing temperature instead of the recommended 62°C (4) and Vir-plus/minus that allows the amplification of the full-length viroid genome (2). Sequences of the three different uncloned amplicons were determined and a 355-nt contig was assembled (GenBank No. JF938538). A BLAST analysis of this full-length sequence revealed 99% identity with CSVd isolates from Chrysanthemum from Korea and Germany (GenBank Accession Nos. AF394452 and X16408). The Argyranthemum CSVd sequence differed from the Chrysanthemum ones by an A insertion at position 289 and substitutions (A to T) at positions 65 and 299. The insertion at position 289 is currently unique among CSVd sequences in GenBank. Thirty-five symptomless mother plants of A. frutescens cv. Butterfly were tested by PCR and all were shown to be infected. The difference in symptomatology observed between the mother plants and the commercial potting plants cannot be explained at this stage, but may reflect the different physiologies or growing conditions of the two kinds of plants, since these are known to affect CSVd symptoms in other hosts (1). To estimate the extent of CSVd contamination in A. frutescens, samples of 11 other cultivars originating from different nurseries were similarly analyzed. In addition to Butterfly, cvs. Sonnenstral, Maya Bofinger, Lili, Blanc Double, and Daisy Solenio were found to be infected by CSVd in the absence of clear symptomatology. The CSVd-free cultivars were Angelic Bordeaux, Dark Pink, Pink Delight, Angelic White, Dana, and Summer. The Pospi1-FW/RE amplicons from Blanc Double, Lili, and Daisy Solenio were identical to the Butterfly isolate sequence while the Maya Bofinger sequence showed one substitution (C to T) at position 256 and Sonnenstral had one substitution (T to A) at position 254. Although CSVd infection of Butterfly had been reported from Germany (3), to our knowledge, the results reported here represent the first report of CSVd in Argyranthemum for France and implicate a range of cultivars. CSVd being classified as a quarantine pest in Chrysanthemum spp. in the European Union, the finding of its significant prevalence in A. frutescens cultivars, frequently in the absence of clear symptomatology, raises the possibility that contaminated Argyranthemum may constitute a reservoir for future Chrysanthemum contamination. References: (1) I. Bouwnen and A. van Zaayen. Page 281 in: Viroids. Science Publishers, Enfield, NH, 2003. (2) T. Candresse et al. Plant Dis. 91:330, 2007. (3) W. Mentzel and E. Maiss. Z. Pflanzenk. Pfanzenschutz 107:548, 2000. (4) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004.