During the spring of 2008, a chrysanthemum plant showing mild mottle on young leaves was observed in a garden in Beijing, China. After the plant was moved into a greenhouse, symptoms became severe with obvious yellowing and complete chlorosis on new leaves. In addition, when a survey was conducted for chrysanthemum diseases in 2010, plants with mild chlorotic spots on leaves were also found occasionally in a commercial field in Hainan, China. These symptoms resembled symptoms induced by Chrysanthemum chlorotic mottle viroid (CChMVd). Therefore, total RNA of 13 samples collected from Beijing (cultivar unknown) and Hainan (cv. Golden) was extracted according to Li et al. (2) and tested for CChMVd by northern blot hybridization using DIG-labeled CChMVd cRNA probe (1). All samples were CChMVd positive, and the healthy control was negative. The viroid was further confirmed by reverse transcription (RT)-PCR using CChMVd specific primers (forward: 5′-AGGTCGTA(T)AAACTTCCCCTCTAAA(G)CG-3′, homologous to nucleotides 134 to 159; and reverse: 5′-TCCAGTCGAGACCTGAAGTGGGTTTC-3′, complementary to nucleotides 133 to 108) (1). Two amplified products of approximately 400 bp were cloned into the pGEM-T vector (Promega, Madison, WI) and transformed into E. coli DH5α competent cells. Two positive clones were obtained from each isolate and sequenced. Four sequences obtained have been submitted to GenBank (Accession Nos. HQ891014 to HQ891017). Sequence analysis revealed that the obtained sequences shared 96.49 to 96.99% similarity with the reference sequence CChMVd (GenBank Accession No. NC003540). All the clones are 399 nucleotides long and are thought to be the symptomatic type based on their UUUC sequence at positions 82 to 85 in the CChMVd tetraloop (1). In addition, both isolates were mechanically inoculated to three healthy chrysanthemum plants of the unknown cultivar from Beijing. All inoculated plants developed chlorosis after 5 weeks and CChMVd infections were confirmed by northern blot hybridization and RT-PCR. CChMVd is an important pathogen that may potentially cause losses to the chrysanthemum industry. It is necessary to survey for CChMVd infection in various chrysanthemums cultivated in China. To our knowledge, this is the first report of CChMVd in chrysanthemum in China. References: (1) P. M. De la Pena et al. Proc. Natl. Acad. Sci. USA. 96:9960, 1999. (2) S. F. Li et al. Ann. Phytopathol. Soc. Jpn. 61:381, 1995.
The slab gel electrophoretic assay for detection and investigation of chrysanthemum chlorotic mottle viroid (CChMV) in infected plants
