A putative virus-induced disease showing chlorotic ringspots on leaves of Phalaenopsis orchids has been observed in Taiwan for several years. A virus culture, 91-orchid-1, isolated from a Phalaenopsis orchid bearing chlorotic ringspot symptoms was established in Chenopodium quinoa and Nicotiana benthamiana, and characterized serologically and biologically. The virus reacted slightly with the antiserum of Watermelon silver mottle virus (WSMoV) but not with those of Tomato spotted wilt virus (TSWV), Impatiens necrotic spot virus (INSV) and Groundnut ringspot virus (GRSV). Isometric particles measuring about 70-100 nm were observed. Inoculation with isolated virus was conducted to confirm that 91-orchid-1 is the causal agent of chlorotic ringspot disease of Phalaenopsis orchids. To determine the taxonomic relationships of the virus, the conserved region of L RNA and the complete nucleocapsid gene (N gene) were cloned and sequenced. The sequence of conserved region of L RNA shares 83.8, 82.5, 64.4 and 64.9% nucleotide identities and 96.5, 97.7, 67.3 and 67.6% amino acid identities with those of Peanut bud necrosis virus (PBNV), WSMoV, TSWV and INSV, respectively, indicating that 91-orchid-1 is a tospovirus related to WSMoV. The complete nucleotide sequence of the N gene determined from a cDNA clone was found to be 828 nucleotides long encoding 275 amino acids. Sequence analyses of the N gene showed that 91-orchid-1 is an isolate of Capsicum chlorosis virus (CaCV) which has been reported to infect tomato and capsicum plants in Australia and Thailand. 91-orchid-1 is therefore designated as CaCV-Ph. To our knowledge, this is the first formal report of a tospovirus infecting Phalaenopsis orchids
Begomoviruses of the Geminiviridae are usually transmitted by whiteflies and rarely by mechanical inoculation. We used tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus, to address this issue. Most ToLCNDV isolates are not mechanically transmissible to their natural hosts. The ToLCNDV‐OM isolate, originally identified from a diseased oriental melon plant, is mechanically transmissible, while the ToLCNDV‐CB isolate, from a diseased cucumber plant, is not. Genetic swapping and pathological tests were performed to identify the molecular determinants involved in mechanical transmission. Various viral infectious clones were constructed and successfully introduced into Nicotiana benthamiana, oriental melon, and cucumber plants by Agrobacterium‐mediated inoculation. Mechanical transmissibility was assessed via direct rub inoculation with sap prepared from infected N. benthamiana. The presence or absence of viral DNA in plants was validated by PCR, Southern blotting, and in situ hybridization. The results reveal that mechanical transmissibility is associated with the movement protein (MP) of viral DNA‐B in ToLCNDV‐OM. However, the nuclear shuttle protein of DNA‐B plays no role in mechanical transmission. Analyses of infectious clones carrying a single amino acid substitution reveal that the glutamate at amino acid position 19 of MP in ToLCNDV‐OM is critical for mechanical transmissibility. The substitution of glutamate with glycine at this position in the MP of ToLCNDV‐OM abolishes mechanical transmissibility. In contrast, the substitution of glycine with glutamate at the 19th amino acid position in the MP of ToLCNDV‐CB enables mechanical transmission. This is the first time that a specific geminiviral movement protein has been identified as a determinant of mechanical transmissibility.
Phalaenopsis orchids are popular ornamentals all over the world. A tospovirus, capsicum chlorosis virus (CaCV-Ph) had been identified as the cause of chlorotic ringspots on leaves of Phalaenopsis orchids in Taiwan. The tripartite genome of CaCV-Ph was found to contain 3608, 4848 and 8916 nt of S, M and L RNAs, respectively. Phylogenetic analysis of the nucleocapsid (N) protein confirmed that CaCV-Ph is a member of the watermelon silver mottle virus (WSMoV) serogroup in the genus Tospovirus. Based on the relations among the nonstructural protein (NSs), glycoprotein (GnGc), thrips genera, host and geographical distribution, tospoviruses and thrips could be classified into two major types: WSMoV-Thrips-Asian and Tomato spotted wilt virus (TSWV)-Frankliniella-EuroAmerican. The proline (P(459)) of all tospoviral Gn proteins was indispensable for thrips transmission, but the RGD motif, which is maintained by only six tospoviruses, may not be required for thrips transmission. An RdRp catalytic domain found in the conserved region of the L protein may recognize the typically conserved sequences on the 5' and 3' terminal regions (5' AGAGCAAU 3').
A new disorder exhibiting flower crinkle on Phalaenopsis orchids bearing white flowers has been observed in Taiwan, China and Japan for several years. This disorder decreased the flower longevity and was considered as a physiological syndrome. The objective of this study was to identify and characterize the real causal agent of this new Phalaenopsis disorder. Five plants of Phalaenopsis hybrids "V3" (Phal. Yukimai x Phal. Taisuco Kochdian) with flower crinkle symptoms were collected and tested by enzyme-linked immunosorbent assay with antisera against 18 viruses. The extract of leaves and flowers from one diseased plant (96-Ph-16) reacted positively only to antiserum against Odontoglossum ringspot virus (ORSV), while those from the other four plants (96-Ph-7, 96-Ph-17, 96-Ph-18 and 96-Ph-19) reacted positively to the antisera against ORSV and Cymbidium mosaic virus (CymMV). Five ORSV isolates, one each from flowers of those five diseased Phalaenopsis orchids, were established in Chenopodium quinoa. A CymMV culture was isolated from the flowers of one of the ORSV/CymMV mix-infected Phalaenopsis orchids (96-Ph-19). To determine the causal agent of the flower crinkle disease, healthy Phalaenopsis seedlings were singly or doubly inoculated with the isolated ORSV and/or CymMV. Results of back inoculation indicated that ORSV is the sole causal agent of the crinkle symptom on petals of Phalaenopsis orchid. The CP gene of the ORSV isolates from this study shared 97.3-100% nucleotide identity and 96.2-100% amino acid identity with those of 41 ORSV isolates available in GenBank. This is the first report demonstrating ORSV as the sole virus causing flower crinkle disease on Phalaenopsis orchids
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