C. C. Chen

A chlorotic spot disease on calla lilies (Zantedeschia spp.) is caused by a tospovirus serologically but distantly related to Watermelon silver mottle virus

A new tospovirus, Calla lily chlorotic spot virus (CCSV), was isolated from calla lilies (Zantedeschia spp.) in Taiwan. Chlorotic spots, ranging from light green to yellow, appear on the middle leaves of the affected plants. Virions measuring 75 to 105 nm, similar in size to tospovirus particles, were present in crude extracts and ultrathin sections of diseased leaves. Of 35 plant species inoculated mechanically, 24, including wax gourd (Benincasa hispida) and zucchini squash (Cucurbita pepo), were susceptible to the virus. CCSV was transmitted from infected wax gourd by Thrips palmi to healthy wax gourd and zucchini squash. The virus was weakly related to Watermelon silver mottle virus (WSMoV) in enzyme-linked immunosorbent assay (ELISA) and western blot tests. WSMoV-specific N gene primers, however, failed to produce DNA fragments from total RNA extracts of CCSV-infected plants in reverse transcription-polymerase chain reaction (RT-PCR). Results of RT-PCR show that the conserved regions of the L genes of tospoviruses are present in CCSV.

Identification of Turnip mosaic virus Isolates Causing Yellow Stripe and Spot on Calla Lily

Two virus cultures, RC4 and YC5, were isolated in Taiwan from calla lily (Zantedeschia spp.) cv. Black magic displaying yellow spot and stripe on leaves. Both isolates were mechanically transmitted to various hybrids of Zantedeschia and induced systemic symptoms similar to those observed on diseased Black magic. In addition to Zantedeschia spp., the two virus isolates also infected several cruciferous species and induced mosaic symptoms. Electron microscopy revealed the presence of flexuous virus particles about 750 nm in length. The two isolates were propagated in and purified from mustard plants and were used as immunogens for production of antisera in rabbits. In enzyme-linked immunosorbent assay and sodium dodecyl sulfate-immunodiffusion tests, both antisera reacted strongly with their homologous antigens and with antigens of two Turnip mosaic virus (TuMV) isolates from radish (TuMV-R) and lisianthus (TuMV-L), but not with 21 other different potyviruses tested. In reciprocal tests, antisera against TuMV-R and TuMV-L also reacted strongly with RC4 and YC5 antigens, indicating that these two calla lily isolates are serologically indistinguishable from other known TuMV strains. Cloning and sequence analyses confirmed that both isolates shared 95 to 99% of deduced amino acid sequence identities in the coat protein genes with those of various known TuMV strains. This investigation represents the first record of the natural infection of TuMV in calla lily.

Characterization of a virus infecting lisianthus.

A virus was isolated in Taiwan from imported lisianthus (Eustoma russellianum) plants bearing viruslike symptoms and was biologically, physically, and serologically characterized. Purified virions are isometric and measure 32 to 33 nm in diameter. The virus from Taiwan is morphologically similar to and serologically related to Lisianthus necrosis necrovirus (LNV) reported in Japan. This lisianthus virus does not have the same host range that was reported for LNV from Japan, but inoculation studies show that it is a strain of LNV.

Identification of Carnation mottle virus from Lisianthus Plants in Taiwan

Lisianthus (Eustoma exaltatum (L.) Salisb. ex G. Don subsp. russellianum (Hook.) Kartesz) is an economically important ornamental crop in Taiwan. Over the past decade, nine viruses have been identified or detected in lisianthus including: Bean yellow mosaic virus (BYMV), Lisianthus necrosis virus (LNV) (2), Cucumber mosaic virus (CMV) (1), Turnip mosaic virus (TuMV), Tomato spotted wilt virus (TSWV), Broad bean wilt virus (BBWV), Tomato mosaic virus (ToMV), Pepper veinal mottle virus (PVMV), and Ageratum yellow vein virus (AYVV) (4). In May 2007 (late period of growing season) in central Taiwan, systemic necrotic spots, which are similar to that caused by LNV (2), were found on approximately 20% of the lisianthus plants. Spherical virus particles, approximately 32 nm in diameter, were found in the crude sap of infected lisianthus collected from the fields. However, the diseased samples did not react with antisera against domestic lisianthus-infecting spherical viruses, LNV (2) and CMV (1). A virus culture was isolated via mechanical inoculation on Chenopodium quinoa and serologically identified as Carnation mottle virus (CarMV) by ELISA, western blotting, and immunoelectron microscopy using antiserum against the CarMV zantedeschia strain (3). The virus induced necrotic local lesions on the inoculated leaves of C. quinoa, C. amaranticolor, Gomphrena globosa, Cucurbita moschata, Phaseolus angularis, P. vulgaris, and Vigna unguiculata. Lisianthus was previously reported as a local lesion host for CarMV (3). In current studies with 8 of 10 lisianthus plants, the newly isolated virus induced necrotic local lesions on inoculated leaves 20 days post inoculation (dpi). However, systemic necrotic lesions on noninoculated upper leaves, as were observed in the fields, appeared 120 dpi on inoculated plants, indicating that CarMV induces systemic infection in lisianthus during late growth stages. Noninoculated plants did not develop symptoms. Complementary DNA fragments of viral genomic RNA were amplified with a specific primer of the coat protein gene (3) and sets of degenerate primer for CarMV. The amplified cDNA fragments were cloned and sequenced. The full-length sequence was submitted as GenBank Accession No. FJ843021. The genomic RNA consists of 4,003 nucleotides and has an identical genome organization to that reported for members of the genus Carmovirus. The nucleotide sequence of the full-length genome shares more than 95% identity to isolates of CarMV (GenBank Accession Nos. AF192772, AJ304989, AJ811998, NC_001265, and X02986), and the nucleotide and deduced amino acid sequence of coat protein shares more than 98% identity with that of CarMV-TW (AY383566) (3), CarMV-FO25 (EF622206), CarMV-Italy-Ca1 (EF622207), and CarMV-Netherland Ca2 (EF622210). To our knowledge, this is the first report of natural infection of CarMV in lisianthus in Taiwan. References: (1) C. C. Chen and C. C. Hu, Plant Prot. Bull. 41:179, 1999. (2) C. C. Chen et al. Plant Dis. 84:506, 2000. (3) C. C. Chen et al. Plant Dis. 87:1539, 2003. (4) Y. H. Cheng et al. J. Taiwan Agric. Res. 58:196, 2009.

First report of Capsicum chlorosis virus infecting amaryllis and blood lily in Taiwan.

Capsicum chlorosis virus (CaCV), a thrips-transmitted, tentative species in the genus Tospovirus, family Bunyaviridae, was first identified in solanaceous crops, but also infects several ornamental crops such as orchid (4), gloxinia (3), and calla lily (1). From 2005 to 2007, virus-like yellow ringspots were observed on the leaves of amaryllis (Hippeastrum hybridum Hort.) and blood lily (Haemanthus multiflorus Martyn.) plants cultured in screenhouses and a private garden, respectively. Three of several hundred amaryllis plants in screenhouses from two places were observed as showing yellow ringspot symptoms and one of six blood lily plants was observed as showing similar yellow ringspot symptoms. Sap extracts from symptomatic leaves were inoculated to Chenopodium quinoa Willd. and the resulting local lesions were passaged three successive times to C. quinoa for virus isolation. Using the tospovirus genus-specific primers gL3637 and gL4435c designed from the conserved region in the L RNA (2), DNA fragments of the expected size of 800 bp were amplified by reverse transcription (RT)-PCR from field samples and local lesions from C. quinoa. Extracts from the diseased plants and local lesions of C. quinoa reacted strongly with antiserum against the nucleocapsid (N) protein of CaCV in ELISA and western blotting. To confirm the identity of this virus, we amplified the N gene from three amaryllis and one blood lily source using primer pair WN2328 and WN3534 designed from the S RNA of Watermelon silver mottle virus (1), and these products were cloned and sequenced. The sequence from each virus isolate was determined from three independent clones. The nucleotide and deduced amino acid sequences of N genes for the blood lily isolate (GenBank Accession No. EF101344) and three amaryllis isolates (GenBank Accession Nos. EF101343, EF137177, and FJ185170) had identities greater than 97% with that of a CaCV isolate infecting Capsicum spp. found in Australia (GenBank Accession No. AY036057). Phylogenetic analysis using maximum parsimony showed that these sequences clustered with CaCV. These results show that the virus identified from amaryllis and blood lily that were expressing yellow ringspot symptoms are isolates of CaCV. To our knowledge, this is the first report of CaCV naturally infecting amaryllis and blood lily and it could become an important threat to ornamental production in Taiwan. References: (1) C. C. Chen et al. Plant Dis. 91:1201, 2007. (2) F. H. Chu et al. Phytopathology 91:361, 2001. (3) H. T. Hsu et al. J. Gen. Plant Pathol. 66:167, 2000. (4) Y. X. Zheng et al. Eur. J. Plant Pathol. 120:199, 2008.

Occurrence of a Severe Strain of Lisianthus necrosis virus in Imported Carnation Seedlings in Taiwan

In the 1995 to 1996 season, severe viral disease symptoms were observed on carnations (Dianthus caryophyllus [hybrid Kooij Echo kgr]) propagated from imported seedlings on farms in central Taiwan. Disease symptoms began on upper leaves as numerous yellow spots that enlarged and fused into large chlorotic patches and expanded to cover entire leaves, which eventually became necrotic. Electron microscopy of crude extracts, purified preparations, and ultrathin sections of diseased tissues revealed the presence of isometric particles ≈32 to 33 nm in diameter. Earlier, in the 1994 to 1995 season, a strain of Lisianthus necrosis virus (LNV-L) was identified in lisianthus (Eustoma russellianum (Don.) Griseb) in a nearby nursery propagating seedlings (1). Both the lisianthus and carnations were imported from Europe. Chlorotic leaves from carnations reacted strongly with antiserum prepared against LNV-L in tissue blot immunoassay. Extracts of diseased leaves also reacted positively to LNV-L antiserum in both immunodiffusion and doubleantibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) tests. Mouse monoclonal antibodies prepared against LNV-L reacted positively based on indirect ELISA with extracts of chlorotic carnation leaves. The capsid protein of the carnation virus (LNV-D) was ≈38 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, similar to the LNV-L coat protein (1), and reacted with LNV-L antiserum in western blot analysis. LNV-D differs biologically from LNV-Japan and LNV-L isolates previously reported in Japan and Taiwan, respectively (1,2). In experiments, LNV-D has induced systemic infection in many hosts that are either nonhosts or local-lesion hosts for LNV-Japan or LNV-L. D. caryophyllus L. and D. chinensis L. are susceptible to systemic invasion by LNV-D but are nonhosts for LNV-Japan and LNV-L. D. barbatus L. is a systemic host for LNV-D but a nonhost for LNV-L and has not been tested as a host for LNV-Japan. Chenopodium amaranticolor Coste & Reyn. and C. quinoa Willd. are systemic hosts for LNV-D but are local-lesion hosts for both LNV-Japan and LNV-L. Capsicum annuum L. is a systemic host for LNV-D and LNV-L but is not susceptible to LNV-Japan. Lycopersicon esculentum Mill. is a systemic host for LNV-D, a local-lesion host for LNV-L, and a nonhost for LNV-Japan. All three isolates systemically infect E. russellianum, the only systemic host for all three isolates tested. The first reports of LNV in Japan and later in Taiwan were in lisianthus. To our knowledge, this is the first report of the natural occurrence of LNV in imported carnation seedlings in Taiwan. LNV infection in Taiwan was only noticed once in lisianthus (1994 to 1995 season) and once in carnation (1995 to 1996 season) in farms propagating imported seedlings. LNV is transmitted by Olpidium sp. (2). Olpidium-like structures were not observed in Taiwan in rootlets of diseased carnation and lisianthus nor were they isolated from soil around diseased plants. Surveys of LNV in the nurseries and nearby areas in subsequent years have not found a new case of infection. We believe that LNV disease is not endemic in Taiwan and that its occurrence in lisianthus and carnation are one-time incidents caused by the importation of infected seedlings or contaminated culture matrices associated with the seedlings. References: (1) C. C. Chen et al. Plant Dis. 84:506, 2000. (2) M. Iwaki et al. Phytopathology 77:867, 1987.