Characterisation of an isolate of Narcissus degeneration virus from Chinese narcissus (Narcissus tazetta var. chinensis)


Chen, J.; Shi, Y.H.; Adams, M.J.; Zheng, H.Y.; Qin, B.X.; Chen, J.P.

Archives of Virology 152(2): 441-448

2006


A potyvirus from Chinese narcissus was transmitted mechanically to three species of Narcissus and to Lycoris radiata but not to 22 other test species. In western blot, the coat protein reacted strongly with Narcissus degeneration virus (UK isolate) antiserum. Antiserum raised to the Chinese virus did not react with eighteen other potyviruses. The complete nucleotide sequence ( 9816 nt) had the typical genome organisation for a member of the genus Potyvirus. Sequence comparisons and phylogenetic analysis showed that the Chinese virus was different from all previously sequenced potyviruses but distantly related to onion yellow dwarf and shallot yellow stripe viruses.

Arch
Virol
(2007)
152:
441-448
DOI
10.1007/s00705-006-0841-9
Printed
in
The
Netherlands
Archives
of
Virology
Characterisation
of
an
isolate
of
Narcissus
degeneration
virus
from
Chinese
narcissus
(Narcissus
tazetta
var.
chinensis)
Brief
Report
J.
Chen
l
,
Y.-H.
Shi
1
-
2
,
M.
J.
Adams
3
,
H.-Y.
Zheng
l
,
B.-X.
Qin
4
,
and
J.-P.
Chen
l
l
Institute
of
Virology
and
Biotechnology,
Zhejiang
Academy
of
Agricultural
Sciences,
Hangzhou,
People's
Republic
of
China
2
College
of
Life
Science,
Zhejiang
University,
Hangzhou,
People's
Republic
of
China
3
Wheat
Pathogenesis
Programme,
Plant-Pathogen
Interactions
Division,
Rothamsted
Research,
Harpenden,
Herts,
UK
4
Department
of
Plant
Protection,
Guangxi
Academy
of
Agricultural
Sciences,
Nanning
City,
People's
Republic
of
China
Received
May
8,
2006;
accepted
July
13,
2006
Published
online
August
24,
2006
©
Springer-Verlag
2006
Summary.
A
potyvirus
from
Chinese
narcissus
was
transmitted
mechanically
to
three
species
of
Narcissus
and
to
Lycoris
radiata
but
not
to
22
other
test
species.
In
western
blot,
the
coat
protein
reacted
strongly
with
Narcissus
degeneration
virus
(UK
isolate)
antiserum.
Antiserum
raised
to
the
Chinese
virus
did
not
react
with
eighteen
other
potyviruses.
The
complete
nucleotide
sequence
(9816
nt)
had
the
typical
genome
organisation
for
a
member
of
the
genus
Potyvirus.
Sequence
com-
parisons
and
phylogenetic
analysis
showed
that
the
Chinese
virus
was
different
from
all
previously
sequenced
potyviruses
but
distantly
related
to
onion
yellow
dwarf
and
shallot
yellow
stripe
viruses.
At
least
four
members
of
the
genus
Potyvirus
are
known
to
infect
narcissus,
causing
damaging
symptoms
on
the
leaves
and
sometimes
also
the
flowers
or
bulbs
[7].
These
are
Narcissus
yellow
stripe
virus
(NYSV;
causing
stunting,
leaf
distortion
and
chlorotic
streaks
early
in
the
season
and
some
flower
'breaking'
[5,
6]),
Narcissus
late
season
yellows
virus
(NLSYV;
causing
chlorotic
streaks
and
extensive
leaf
chlorosis
after
flowering
[13]),
Narcissus
degeneration
virus
(NDV;
causing
chlorotic
leaf
striping
but
only
reported
so
far
in
N.
tazetta
cv.
Grand
Soleil
d'
Or
[6])
and
Ornithogalum
mosaic
virus
(OrMV;
sometimes
found
in
mixed
infections
[10]).
Diagnosis
can
be
difficult
because
individual
plants
may
442
J.
Chen
et
al.
contain
mixtures
of
viruses,
which
accumulate
in
bulbs
because
of
the
vegetative
propagation
of
the
crop.
Chinese
narcissus
(Narcissus
tazetta
var.
chinensis)
is
an
economically
important
ornamental
plant
in
China,
and
virus
diseases
causes
loss
of
yield
and
poor
quality.
In
previous
work,
we
used
degenerate
primers
to
detect
and
then
to
sequence
the
3'-terminal
region
of
potyvirus
isolates
from
some
symptomatic
Chinese
and
European
narcissus
plants
and
attempted
to
identify
the
viruses
by
comparison
with
some
small
fragments
of
sequences
previously
described
from
Dutch
Narcissus
stocks
[10,
12].
NYSV
was
detected
in
both
Chinese
and
European
narcissus plants
and
a
virus
tentatively
identified
as
NLSYV
was
found
only
in
the
European
sample
together
with
OrMV.
Those
studies
also
indicated
the
presence
of
a
further
potyvirus
in
two
samples
of
Chinese
narcissus
(Zhangzhou,
AJ311373
and
Chongming
Island,
AJ311374).
We
have
now
done
some
more
detailed
studies
using
the
Chinese
Zhangzhou
isolate
and
report
here
the
characterization
of
this
virus,
including
its
complete
sequence.
These
new
data
suggest
that
it
is
an
isolate
of
NDV.
The
virus
isolate
used
was
that
previously
described
from
Zhangzhou
city,
Fujian
province
[10].
Subsequent
molecular
studies
with
degenerate
primers
for
potyviruses
and
specific
RT-PCR
tests
for
other
known
narcissus
viruses
suggested
that
there
was
one
virus
in
these
plants.
Crude sap
(1:100,
w/v)
from
leaf
extracts
of
six
virus-infected
plants
were
mechanically
inoculated
to
28
plant
species
from
six
families.
The
plants
used
(with
total
numbers
inoculated)
were:
Chenopodium
amaranticolor
(27),
C.
murale
(24),
C.
quinoa
(24),
Lycopersicon
esculentum
(12),
Physalis
floridana
(14),
Nicandra
physalodes
(15),
Datura
stramonium
(21),
Nicotiana
tabacum
(18),
N.
tabacum
Samsun
NN
(19),
N.
tabacum
Xanthi
nc
(15),
N.
tabacum
White
Burley
(15),
N.
benthamiana
(18),
Solanum
melongena
(8),
Phaseolus
vulgaris
(11),
P.
radiatus
(15),Glycine
max
(21),
Cucurbita
pepo
(12),
C.
moschata (12),
Cucumis
sativus
(9),
Luffa
cylindrica
(6),
Momordica
charantia
(6),
Brassica
campestris
(9),
Narcissus
tazetta
var.
chinensis
(8),
N.
pseudonarcissus
(8),
N.
poeticus
(6),
Leucojum
aestivum
L.
(5)
and
Lycoris
radiata
(8).
The
plants
were
dusted
with
Carborundum
(320
grit,
Fisher
Scientific)
prior
to
inoculation
and
then
maintained
in
a
glasshouse
and
observed
for
symptom
development.
Tests
for
virus
infection
were
also
made
by
ELISA
using
polyclonal
antiserum
(see
below)
15-25
days
post
inoculation.
Virus
was
purified
from
infected
leaves
using
a
method
described
previously
[14].
Purified
viral
particles
were
used
to
prepare
a
polyclonal
antiserum
in
mouse
[15]
and
this
antiserum
was
then
tested
by
Western
blot
[15]
against
the
purified
coat
proteins
of
12
potyviruses
(shallot
yellow
stripe
virus
(SYSV),
Thunberg
fritillary
mosaic
virus
(TFMV),
fritillary
virus
Y
(FVY),
sugarcane
mosaic
virus
(SCMV),
sorghum
mosaic
virus
(SrMV),
tuberose
mild
mottle
virus
(TuMMoV),
soybean
mosaic
virus
Pinellia
strain
(SMV),
onion
yellow
dwarf
virus
(OYDV),
leek
yellow
stripe
virus
(LYSV),
scallion
mosaic
virus
(ScaMV),
NLSYV
and
NYSV),
and
the
over-expressed
coat
proteins
of
6
other
potyviruses
(zucchini
yellow
mosaic
virus
(ZYMV),
lily
mottle
virus
(LMoV),
potato
virus
Y
(PVY),
turnip
mosaic
virus
(TuMV),
Zantedeschia
mosaic
virus
(ZaMV),
and
OrMV).
Experiments
were
done
three
times
for
each
virus.
A
rabbit
polyclonal
antiserum
Narcissus
degeneration
virus
443
raised
to
a
UK
isolate
of
Narcissus
degeneration
virus,
originally
prepared
by
Dr.
A. A.
Brunt,
was
kindly
provided
by
Dr.
John
Walsh.
RNA
was
isolated
from
purified
virions
using
the
RNeasy
Plant
Mini
Kit
(QIAGEN),
and
first-strand
cDNA
was
then
synthesised
using
M-MLV-reverse
a
rr
i;
4
40,
d
M
1
44.3
lommu
l
p
IOW
29.0
ism.
20.1
2
3
4
5
ma
Sop
Fig.
1.
a
Symptoms
of
the
NDV
Zhangzhou
isolate
on
Narcissus
tazetta
var.
chinensis,
(b)
purified
virus
particles
(scale
bar
200
nm),
(c)
electron
micrograph
of
thin
section
of
infected
leaf
cells
showing
typical
potyvirus
inclusion
bodies
(scale
bar
200
nm),
(d)
gradient
SDS-PAGE
analysis
of
purified
virus
particles
(1)
with
protein
size
markers
(kDa)
(M)
and
Western
blot
analysis
of
healthy
host
plant
sap
(2,
4)
and
purified
coat
protein
(3,
5)
probed
with
either
polyclonal
mouse
antiserum
raised
to
the
virus
3
or
NDV
antiserum
(5)
444
J.
Chen
et
al.
transcriptase
(Life
Technologies
Ltd)
according
to
the
manufacturer's
instructions
and
with
M4T
(5'-
arr
TTC
CCA
GTC
ACGACA
C
(T)16-3')
as
the
initial
primer.
Degenerated
PCR
amplifications
and
5'-RACE
were
done
to
amplify
fragments
of
the entire
genome
as
described
previously
[9].
The
LA
Taq
DNA
polymerase
system
(TaKaRa)
was
used
according
to
the
manufacturer's
protocols.
Successful
amplification
of
fragments
of
the
expected
size
was
confirmed
by
electrophoresis
through
1%
(w/v)
agarose
gels.
PCR
fragments
were
purified
using
the
Gel
Extraction
Kit
(QIAGEN)
and
cloned
into
the
pGEM-T
vector
(Promega)
following
the
manufacturers'
protocols.
Additional
primers
were
designed
to
internal
sequences
for
subcloning
and
sequencing.
Three
independent
clones
were
auto-sequenced
in
both
directions
by
the
ABI
PRISM
TM
3770
DNA
Sequencer.
Table
1.
Comparisons
were
made
using
GCG
GAP
between
NDV
Zhangzhou
isolate
and
other
fully-sequenced
potyviruses
(44
different
species)
in
different
regions
of
the
genome
Genome
region
%
nt
identity
%
as
identity
5'-UTR
41.6-57.9
Polyprotein
49.9-52.4
41.4-45.6
3'-UTR
21.0-68.2
P1
protein
37.7-42.9 10.7-28.6
HC-Pro
41.0-52.9
36.0-48.3
P3
protein
40.1-52.5
18.3-26.8
6K1
41.0-64.7
38.5-59.6
CI
51.4-57.3
48.7-54.3
6K2
41.5-56.4
20.8-40.4
VPg
47.7-54.5
33.2-42.4
Ma-Pro
47.7-56.4
36.3-50.6
Mb
53.8-62.8
53.0-65.8
Coat
protein
45.7-61.2
37.2-59.4
Fig.
2.
Phylogenetic
analysis
of
the
nucleotide
sequence
of
the
complete
ORFs
of
poty-
and
rymo-viruses
to
show
the
position
of
the
new
NDV
sequence.
The
values
at
the
forks
indicate
the
percentage
of
trees
in
which
this
grouping
occurred
after
bootstrapping
(10,000
replicates;
shown
only
when
>60%).
The
scale
bar
shows
the
number
of
substitutions
per
base.
Accession
numbers:
AgMV,
AY623626;
BCMNV,
AY138897;
BCMV,
U34972;
BtMV,
AY206394;
BYMV,
U47033;
CABMV,
AF348210;
ChiVMV,
AJ237843;
ClYVV,
AB011819;
CSV,
AF499738;
DsMV,
AJ298033;
EAPV,
AB246773;
FVY,
AM039800;
HoMV,
AY623627;
JGMV,
Z26920;
JYMV,
AB016500;
KoMV,
AB219545;
LMoV,
AJ564636;
LMV,
X97704;
LYSV,
AJ307057;
MDMV,
AJ001691;
NDV,
AM182028;
NYSV,
AM158908;
OYDV,
AJ510223;
PeMoV,
AF023848;
PenMV,
AY642590;
PepMoV,
AF501591;
PLDMV,
AB088221;
PPV,
X16415;
PRSV,
AY010722;
PSbMV,
D10930;
PTV,
AJ516010;
PVA,
AF543212;
PVV,
AJ243766;
PVY,
X12456;
RGMV,
Y09854;
ScaMV,
AJ316084;
SCMV,
AJ297628;
SMV,
D00507;
SPFMV,
D86371;
SrMV,
AJ310197;
SYSV,
AJ865076;
TEV,
M11458;
TFMV,
AJ851866;
TuMV,
AF169561;
TVMV,
X04083;
WMV,
AY437609;
WPMV,
AJ437279;
WVMV,
AY656816;
YMV,
U42596;
ZYMV,
AJ307036
Narcissus
degeneration
virus
445
Sequence
analysis
used
programs
from
the
Wisconsin
(GCG)
package
[3]
and,
in
particular,
pairwise
comparisons
were
done
with
GAP
(using
a
gap
creation
penalty
of
50
and
a
gap
extension
penalty
of
3
for
nucleotide
comparisons
and
100
100
PV.
Lis
I
84
Vial
KoMV
PPV
SPFMV
LMV
BYMV
ClYVV
EV
oV
FMV
LYSV
100
JYMV
TuMV
ScaMV
NYS
V
PVY
93
85
100
PepMoV
PTV
PVV
100
100
WPMV
PLDMV
ChiVMV
PRSV
67
87
99
86
100
100
89
100
65
98
100
100
PeMoV
BtMV
DsMV
ZYMV
CABMV
BCMV
BCMNV
EAPV
FVY
WVMV
SMV
WMV
PSbMV
CSV
9
JGMV
99
100
100
PenMV
SCMV
SrMV
MDMV
NDV
100
97
100
OYDV
SYSV
100
RGMV
100
0.1
100
AgMV
HoMV
446
J.
Chen
et
al.
values
of
8
and
2
respectively
for
amino
acids).
Phylogenetic
analysis
was
done
by
Neighbor-Joining
(Tamura-Nei
distances)
using
MEGA
3.1
[11].
Following
mechanical
inoculation,
the
virus
could
be
detected
by
ELISA
and
RT-PCR
in
Narcissus
tazetta
var.
chinensis
(6/8),
N.
pseudonarcissus
(4/8),
N.
poeticus
(4/6)
and
Lycoris
radiata
(2/8),
but
not
in
any
of
the
other
test
plants
used
(results
not
shown).
Within
2
wk,
the
typical
streak
mosaic
symptoms
that
had
been
seen
in
the
field
developed
on
the
infected
plants
(Fig.
la).
Purified
viral
particles
were
750-770
nm
long
and
slightly
flexuous
(Fig.
lb).
Characteristic
potyvirus
cylindrical
inclusion
bodies
(`pinwheels')
could
be
seen
in
sections
of
virus-infected
leaf
cells
(Fig.
lc).
When
the
purified
virus
was
run
in
SDS-PAGE,
a
single
protein
band
with
MW
of
nearly
30
kDa
was
detected,
and
the
antiserum
raised
to
the
purified
virus
particles
reacted
specifically
with
this
band
in
Western
blots
(Fig.
1d).
There
were
no
reactions
with
the
CPs
of
the
other
potyviruses
tested
(data
not
shown).
The
antiserum
raised
to
NDV
reacted
strongly
to
the
coat
protein
of
the
Chinese
narcissus
potyvirus
(Fig.
1d).
The
complete
nucleotide
sequence
was
9816
nt
long
excluding
the
polyA
tail
and
had
the
typical
genome
organisation
for
a
member
of
the
genus
Potyvirus.
The
first
AUG
codon
at
nt
114-116
appears
to
be
the
initiator
for
the
long
ORF,
which
terminates
with
an
UAG
codon
at
nt
9666-9668,
followed
by
a
3'-NCR
of
148
nt.
The
predicted
translation
product
of
this
ORF
contains
3184
amino
acids
(aa)
with
a
calculated
M
r
of
363.4
kDa
(polyprotein).
The
polypro-
tein
amino
acid
sequence
was
aligned
with
those
of
the
other
completely
se-
quenced
potyviruses,
and
the
characteristic
proteolytic
cleavages
sites
of
the
ten
mature
potyvirus
proteins
were
identified.
They
were
IKYY394/S
(P1/HC-
Pro),
YAVG852/G
(HC-Pro/P3),
VKFQ1236/Y
(P3/6K1),
VQFQ1288/S
(6K1/CI),
VQFQ1924/N
(Cl/6K2),
VQFQ19
77
/G
(6K2NPg),
VDFE2162/N
(VPg/Nla-Pro),
VGMQ2403/S
(NIa-Pro/Nlb)
and
VSFQ2923/S
(Nlb/CP).
Previous
analysis
demon-
strated
that
many
potyvirus
species
have
a
consistent
pattern
of
residues
at
position
P2
(the
second
residue
before
the
scissile
bond)
amongst
the
seven
sites
cleaved
by
the
Ma
cysteine
protease
[1].
The
new
narcissus
potyvirus
resembles
potato
virus
A
(PVA),
lettuce
mosaic
virus
(LMV),
tobacco
vein
mottling
virus
(TVMV),
Thunberg
fritillary
mosaic
virus
(TFMV),
bean
yellow
mosaic
virus
(BYMV)
and
clover
yellow
vein
virus
(C1YVV)
in
having
F
at
this
position.
The ten
proteins
all
contained
the
expected,
well-characterised
motifs
and
are
not
presented
in
detail
here.
The
deduced
CP
was
261
aa
long
and
had
a
predicted
molecular
weight
of
29.9
kDa,
which
was
similar
to
that
calculated
from
SDS-PAGE
(Fig.
1d).
Comparisons
were
made,
using
GCG
GAP,
between
the
new
narcissus
potyvirus
and
other
fully-sequenced
potyviruses
(44
different
species)
in
different
regions
of
the
genome.
The
complete
polyprotein
regions
of
the
new
narcissus
potyvirus
had
only
49.9-52.4%
nt
and
41.4-45.6%
aa
identity
to
that
of
other
potyviruses.
The
greatest
sequence
differences
were
in
the
P1
and
P3
region,
whereas
the
6K1,
CP,
Mb
and
CI
regions
were
relatively
conserved
(Table
1).
Comparisons
were
also
made
with
representative
coat
protein
gene
sequences
corresponding
to
each
species
(or
tentative
species)
in
the
genus
for
which
at
least
a
partial
sequence
was
available
(another
71
viruses).
The
narcissus
potyvirus
had
Narcissus
degeneration
virus
447
53.4-59.5%
nt
identity
(45.9-58.7%
aa
identity)
with
these
sequences,
with
the
exception
of
that
of
a
potyvirus
from
Lycoris
aurea
(golden
spider
lily)
in
Taiwan.
This
sequence
(AF511486,
[8])
consists
of
the
3'-terminal
1836
nt
and
has
95.1%
nt
(89.5%
aa)
identity
to
the
Zhangzhou
isolate
in
the
CP
cistron.
The
list
of
species
demarcation
criteria
given
by
ICTV
for
the
genus
Potyvirus
includes
CP
aa
identity
less
than
about
80%
and
complete
nt
identity
less
than
85%
[4].
Recent
studies
have
shown
that
a
value
of
76%
nt
identity
for
either
the
complete
ORF
or
the
CP
cistron
is
the
most
appropriate
for
separating
species
in
the
genus
[2].
Corresponding
values
for
amino
acid
comparisons
were
80-82%,
but
nucleotide
comparisons
were
preferred
for
discrimination
purposes.
The
new
narcissus
potyvirus,
together
with
the
Lycoris
potyvirus
from
Taiwan
therefore
clearly
satisfy
the
molecular
criteria
for
classification
as
a
separate
species
in
the
genus.
The
Zhangzhou
narcissus
virus
was
able
to
infect
a
species
of
Lycoris,
in
which
it
caused
symptoms
similar
to
those
on
Narcissus,
but
(unlike
the
report
for
the
Lycoris
potyvirus
[8])
it
was
unable
to
infect
Chenopodium
quinoa.
Although
the
type
strain
of
NDV
did
not
infect
N.
pseudonarcissus
(A.
A.
Brunt,
personal
communication),
the
strong
serological
relationship
suggests
that
the
Chinese
virus
(and
the
Lycoris
potyvirus)
should
be
classified
as
an
isolate
of
NDV.
Relatively
little
is
known
about
this
virus.
In
the
1960s
it was
very
prevalent
in
Narcissus
tazetta
cv.
Grand
Soleil
d'
Or,
an
important
commercial
variety
in
the
Isles
of
Scilly,
UK.
It
proved
to
be
serologically
unrelated
to
other
potyviruses,
but
after
virus-free
plants
had
been
produced
as
stock
for
propagation,
no
further
work
was
done
on
it
(A.
A.
Brunt,
personal
communication).
In
the
phylogenetic
analysis,
NDV
was
distantly
related
to
OYDV
and
SYSV
(Fig.
2).
In
GCG
gap,
the
nucleotide
identity
of
the
entire
genome
to
these
two
was
52.9
and
52.8%,
respectively.
The
sequence
described
here
was
deposited
in
the
EMBL/GenBank/DDBJ
databases
with
the
accession
number
AM182028.
Acknowledgements
We
thank
Alan
Brunt
for
his
advice
and
John
Walsh
(Warwick
HRI,
Wellesbourne,
UK)
for
kindly
providing
the
antiserum
to
Narcissus
degeneration
virus.
Yuhong
Shi
was
a
student
registered
in
Zhejiang
University
for
the
degree
of
Ph.D.
The
work
was
funded
by
the
Zhejiang
Provincial
Natural
Science
Foundation
of
China
(ZA0207)
and
the
National
Natural
Science
Foundation
of
China
(30200008,
30470080).
Rothamsted
Research
receives
grant-
aided
support
from
the
Biotechnology
and
Biological
Sciences
Research
Council
of
the
United
Kingdom.
References
1.
Adams
MJ,
Antoniw
JF,
Beaudoin
F
(2005)
Overview
and
analysis
of
the
polyprotein
cleavage
sites
in
the
family
Potyviridae.
Mol
Plant
Pathol
6:
471-487
2.
Adams
MJ,
Antoniw
JF,
Fauquet
CM
(2005)
Molecular
criteria
for
genus
and
species
discrimination
within
the
family
Potyviridae.
Arch
Virol
150:
459-479
3.
Anon.
(2001)
Wisconsin
Package
version
10.3.
Accelrys
Inc.,
San
Diego,
CA,
USA
448
J.
Chen
et
al.:
Narcissus
degeneration
virus
4.
Berger
PH,
Adams
MJ,
Barnett
OW,
Brunt
AA,
Hammond
J,
Hill
JH,
Jordan
RL,
Kashiwazaki
S,
Rybicki
E,
Spence
N,
Stenger
DC,
Ohki
ST,
Uyeda
I,
van
Zaayen
A,
Valkonen
J,
Vetten
HJ
(2005)
Potyviridae.
In:
Fauquet
CM,
Mayo
MA,
Maniloff
J,
Desselberger
U,
Ball
LA
(eds)
Virus
taxonomy:
Eighth
Report
of
the
International
Committee
on
Taxonomy
of
Viruses.
Elsevier/Academic
Press,
London,
pp
819-841
5.
Brunt
AA
(1971)
Narcissus
yellow
stripe
virus.
AAB
Descriptions
of
Plant
Viruses
No.
76
CMI/AAB,
Kew,
UK,
4
pp
6.
Brunt
AA,
Atkey
PT
(1967)
Rapid
detection
of
narcissus
yellow
stripe
and
two
other
filamentous
viruses
in
crude
negatively
stained
narcissus
sap.
Rep
Glasshouse
Res
Inst
1966,
pp
155-159
7.
Brunt
AA
(1995)
Narcissus.
In:
Loebenstein
G,
Lawson
RH,
Brunt
AA
(eds)
Virus
and
Virus-like
Diseases
of
Bulb
and
Flower
Crops.
John
Wiley
&
Sons
Ltd,
Chichester,
UK,
pp
322-334
8.
Chang
C-A,
Chen
C-C,
Hsu
HT
(2002)
Partial
characterization
of
two
potyviruses
associated
with
golden
spider
lily
severe
mosaic
disease.
Acta
Hort
568:
127-134
9.
Chen
J,
Chen
JP
(2002)
Determination
of
genome
sequence
of
potyviruses
by
degenerated
PCR
and
RACE
method.
Chinese
J
Virol
18:
371-374
10.
Chen
J,
Chen
JP,
Langeveld
SA,
Derks
AFLM,
Adams
MJ
(2003)
Molecular
char-
acterization
of
carla-
and
potyviruses
from
Narcissus
in
China.
J
Phytopathol
151:
1-4
11.
Kumar
S,
Tamura
K,
Nei
M
(2004)
MEGA3:
Integrated
software
for
Molecular
Evolutionary
Genetics
Analysis
and
sequence
alignment.
Brief
Bioinform
5:
150-163
12.
Langeveld
SA,
Derks
AFLM,
KonichevaV,
Muiloz
D,
Zhin-nan
C,
Denkova
ST,
Lemmers
MEC,
Boonekamp
PM
(1997)
Molecular
identification
of
potyviruses
in
Dutch
stocks
of
Narcissus.
Acta
Hort
430:
641-648
13.
Mowat
WP,
Dawson
S,
Duncan
GH
(1998)
Narcissus
late
season
yellows
virus.
AAB
Descriptions
of
Plant
Viruses
No.
367.
AAB,
Wellesbourne,
UK
14.
Philip
HB,
Patrick
JS
(1998)
Potyvirus
isolation
and
RNA
extraction.
In:
Foster
GD,
Taylor
SC (eds)
Plant
virology
protocols:
from
virus
isolation
to
transgenic
resistance.
Humana
Press
Inc,
Totowa,
New
Jersey,
pp
151-161
15.
Sambrook
J,
Fritsch
EF,
Maniatis
T
(1989)
Molecular
cloning:
a
laboratory
manual,
2nd
edn.
Cold
Spring
Harbor
Laboratory,
New
York
Author's
address:
Prof.
Jiong
Chen,
Institute
of
Virology
and
Biotechnology,
Zhejiang
Academy
of
Agricultural
Sciences, Hangzhou
310021,
People's
Republic
of
China;
e-mail:
jchen1975@zjip.com