Download Isolation, identification and characterization of a tospovirus causing

Isolation, identification and characterization of a tospovirus causing
chlorotic necrosis1and ringspots on2 moth orchids
(Phalaenopsis
spp.)
1
1,3
You-Xiu Zheng , Ching-Chung Chen , Shyi-Dong Yeh , and Fuh-Jyh Jan
1
2
3
Department of Plant Pathology, National Chung Hsing University, Taichung 402, TAIWAN;
Taichung District Agricutural Improvement Station, Changhua 515, TAIWAN
Fax: +886-4-22854145; E-mail: [email protected]
Introduction
Phalaenopsis orchids plants bearing virus-like
symptoms of chlorotic spots with centric necrosis or
chlorotic ringspot on leaves have been observed in
Taiwan for several years. Although the causal agent of
this special disease was unclear, a so-called “Taiwan
virus” was postulated to cause this disease. The
objectives of this study were to isolate, identify and
characterize the causal agent of this orchid disease.
Our results indicate that the virus causing chlorotic
and necrotic spots on Phalaenopsis orchids in Taiwan
is a tospovirus with high nucleotide and amino acid
sequences identity of nucleocapsid (N) gene with those
of Capsicum chlorosis virus (CaCV), and therefore has
been designated as CaCV-Ph.
Results
Fig. 3. Serological relationships of CaCV-Ph and
Watermelon silver mottle virus (WSMoV) by western
blotting. Antisera against the nucleocapsid protein (NP)
of CaCV-Ph and WSMoV were used to react with the
crude saps from healthy Chenopodium
quinoa (H), and leaves infected with CaCV-Ph (C)
and WSMoV (W).
5’
NSs
N
N3101
FJJ2003-8
Fig. 1. Symptoms induced by Capsicum chlorosis
virus isolate CaCV-Ph on phalaenopsis orchids.
Symptoms of chlorotic spots with centric necrosis
(A) and chlorotic ringspot (B) on leaves of
Phalaenopsis orchids collected from field. Backinoculation of healthy Phalaenopsis orchids with
CaCV-Ph from infected Chenopodium quinoa
displaying similar symptoms (C and D).
Fig. 2. Isometric enveloped virions measuring
about 70-100 nm in diameter as observed in an
electron microscopic examination of ultra-thin
sections from naturally infected phalaenopsis
orchids showing chlorotic spots with centric
necrosis (A), CaCV-Ph infected Nicotiana
benthamiana (B) and Datura stramonium (C),
and back-inoculated phalaenopsis seedling (D).
Bar=200 nm.
3’
N3534c
FJJ2003-10
Fig. 6. Strategies of cloning genomic RNA L, M
and S of CaCV-Ph by RT-PCR. The overlapped
cDNA regions covering the entire genomic L, M
and S RNA of CaCV-Ph were obtained by the use
of primers based on the RNA sequences of
Gloxinia tospovirus HT, WSMoV and Peanut
bud necrosis virus (PBNV).
Conclusions
1. The virus causing chlorotic and necrotic spots
Fig. 4. The Nucleocapsid protein gene was cloned
by RT-PCR using the degenerated primers designed
for the WSMoV serogroup of tospoviruses.
on Phalaenopsis orchids was isolated and found
to be a tospovirus.
2. The sequence of N gene shares 73.6% and 96.1%
nucleotide identity and 83.7 and 97.5% amino
acid identity with WSMoV and Capsicum
chlorosis virus (CaCV).
3. Based on our results it was concluded that the
Phalaenopsis tospovirus is an isolate of CaCV
and was designated as CaCV-Ph.
4. We have also cloned and sequenced the entire
tripartite genome of the CaCV-Ph. The complete
genomic sequence of CaCV-Ph are 8916 nts of L
RNA, 4848 nts of M RNA and 3608 nts for S
RNA.
5. To our knowledge, this is the first investigation
of a tospovirus that can infect Phalaenopsis
orchids.
Fig. 5. Phylogenetic relationships of the NP amino
acid sequences of CaCV-Ph with those of other
tospoviruses.
References
1. F. H. Chu et al., Phytopathology 91, 361 (2001).
2. Y. H. Lin et al., Phytopathology 95, 1482 (2005).
3. L. A. McMichael et al., Australas. Plant Pathol. 31,
231 (2002).
4. Y. X. Zheng et al., Plant Pathol. Bull. 12, 293 (2003).