Molecular and serological characterization of a distinct

potyvirus causing latent infection in calla lilies

Chin-Chih CHEN

1

, Hei-Ti HSU

2

, Ying-Huey CHENG

1

, Chun-Huei HUANG

1

, Jye-Yann LIAO,

Hei-Ting TSAI

1

, and Chin-An CHANG

1,

*

1

Plant Pathology Division, Taiwan Agricultural Research Institute, Wu-Feng, Taichung 413, TAIWAN

2

U.S. Department of Agriculture, Agricultural Research Service, Beltsville Agricultural Research Center, Beltsville, MD

20705, USA

(Received September 19, 2005; Accepted March 10, 2006)

ABSTRACT. A virus (isolate: Ca-M19) capable of inducing local lesions on Chenopodium quinoa Willd.

was isolated from calla lilies (Zantedeschia spp.). Subculture of Ca-M19 was easily maintained in C. quinoa,

but a back inoculation from single lesion of C. quinoa to calla lilies has so far not been successful. Typical

potyvirus-like flexuous particles were consistently detected in Ca-M19 infected plants, and a 1.3-kb DNA

fragment was amplified

from these plants by reverse-transcription polymerase chain reaction (RT-PCR)

using potyvirus degenerate primers. The PCR product was cloned and its sequence analyzed (AF469171).

The amplicon was revealed to correspond to the 3¡¦ terminal region of a potyviral genome. After comparing

this sequence with known potyvirus sequences in the GenBank, we considered the virus a new species

of Potyvirus based on the uniqueness in its coat protein gene (CP) and the 3¡¦ non-coding region (NCR).

Comparative studies showed that Soybean mosaic virus (SMV) and Watermelon mosaic virus 2 (WMV 2)

were the two most similar potyviruses with Ca-M19, but they shared only 80% of nucleotide identities in CP

and NCR with Ca-M19. Attempts to purify a sufficient quantity of Ca-M19 from C. quinoa for preparation

of antibodies were unsuccessful. Alternatively

,

Ca-M19 CP was expressed by the vector pET28b and puri-

fied from E. coli culture, and polyclonal antibodies were prepared in rabbits. The antibody was applied in

ELISA, Western blotting, SDS-immunodiffusion and immuno-specific electron microscopy for the detection

of Ca-M19 in calla lilies. It did not react with at least five calla lily infecting potyviruses, including Dasheen

mosaic virus, Bean yellow mosaic virus, Konjak mosaic virus, Turnip mosaic virus, and Zantedeschia mild

mosaic virus. Indirect ELISA and SDS-immunodiffusion tests showed that Ca-M19 was serologically related,

but distinct from Bean common mosaic virus (BCMV), Black cowpea mosaic virus (BlCMV), Melon vein

banding mosaic virus (MVbMV), Passionfruit mottle virus (PaMV), Passionfruit crinkle virus (PCV), Pas-

sionfruit woodness virus (PWV), Soybean mosaic virus (SMV), Watermelon mosaic virus 2 (WMV 2), and

Zucchini yellow mosaic virus (ZYMV). Besides serological techniques, a primer pair (M19u/M19d) and a

DNA probe were designed which could also specifically detect and differentiate Ca-M19 from other viruses.

By the use of specific antibodies in ELISA, Ca-M19 was frequently detected in calla lily plants collected

from several major calla lily production townships in Taiwan. Among 86 field samples positively reacting

to the antibody, 77 of them exhibited evident systemic mosaic symptoms, but these symptomatic plants were

confirmed to be infected simultaneously by other viruses. Nine plants were found to be infected by Ca-M19

alone. These plants were confirmed to have remained symptomless throughout a 6-month observation period.

Therefore, we propose naming this isolate Calla lily latent virus (CLLV) for its inability to develop any

visible symptoms on calla lily.

Keywords: Calla lily; Coat protein expression; Potyviruses; Sequence analysis; Serology.

Botanical Studies (2006) 47: 369-378.

*

Corresponding author: E-mail: cachang@wufeng.tari.gov.

tw; Fax: 886-4-23331089; Tel: 886-4-23302803.

mosaic virus (DsMV) is considered the most prevalent

and widespread in the family Araceae, including Zant-

edeschia spp. (Zettler et al., 1978; Zettler and Hartman,

1995). In addition to DsMV, several Potyvirus species¡X

including Bean yellow mosaic virus (BYMV) (Pham et al.,

2002), Konjak mosaic virus (KoMV) (Chang et al., 2001;

Hu, 2001; Kwon et al., 2002; Pham et al., 2002), Turnip

mosaic virus (TuMV) (Chen et al., 2003a)

,

and Zantede-

schia mild mosaic virus (ZaMMV) (Chen et al., 2003b;

MOLECULAR BIOLOGY

INTRODUCTION

Calla lily (Zantedeschia spp.), an aroid plant species, is

a popular ornamental crop in Taiwan and many parts of the

world. Viral diseases and bacterial soft rot are the major

factors limiting its production. Among viruses, Dasheen

370

Botanical Studies, Vol. 47, 2006

Huang and Chang, 2002,

2005)¡Xwere identified from cal-

la lilies. These potyviruses always induce systemic mottle

or mosaic symptoms with variable degree of severity on

leaves. Diagnosing the causal potyviruses simply based

on visual inspection of symptoms is difficult. Besides

potyviruses, spherical viruses such as Carnation mottle

virus (Chen et al., 2002b), Calla lily chlorotic ringspot vi-

rus (Lin et al., 2003), Cucumber mosaic virus (CMV)

,

and

Tomato spotted wilt tospovirus have also been reported to

occur naturally in calla lilies (Zettler and Hartman, 1995).

In general, these spherical viruses induce more severe

symptoms, such as chlorotic spotting, necrotic streaks,

ringspotting, and distortion of leaves, than those caused

by potyviruses (Chen et al., 2002b; Zettler and Hartman,

1995). Based on our observation, some calla lily cultivars

develop color breaking on their inflorescences when

infected by CMV (unpublished data).

In 2001, we isolated an unknown virus from a calla

lily plant dually infected with DsMV and KoMV. A

pure culture was established by single lesion isolation on

Chenopodium quinoa Willd. Although back inoculation

using the extract of the lesion to calla lily was not

successful, the virus was consistently detected from calla

lily plants in the field. The virus was later found to be

biologically, serologically and molecularly different from

previously described calla lily-infecting potyviruses. In

this paper, we provide evidence that this virus is unique.

Due to its inability to induce symptoms on calla lily, we

have christened this novel virus Calla lily latent virus

(CLLV) (Chen et al., 2004).

MATERIALS AND METHODS

Virus source and maintenance

In the beginning of our study, an unknown virus

designated Ca-M19 was isolated from a calla lily plant

doubly infected by DsMV and KoMV, as determined by

enzyme-linked immunosorbent assay (ELISA). Leaf ex-

tracts from this plant, however, induced local lesions on C.

quinoa which normally was not susceptible to DsMV or

KoMV. After three consecutive single lesion passages, a

pure culture of Ca-M19 in the infected leaves of C. quinoa

was preserved in 50% glycerol and stored in a freezer at

-20¢XC. The bulb of the original Ca-M19 infected calla lily

plant was grown in 7-inch pots under greenhouse condi-

tions for subsequent studies.

Host reaction tests

Ca-M19 induced lesions on C. quinoa were ground in

potassium phosphate buffer (50 mM, pH 7.5) at 1:10 dilu-

tion (g/ml) and used to mechanically inoculate tissue-cul-

ture derived calla lily plantlets cv. Black magic, Nicotiana

benthamiana Domin, Glycine max (L) Merrill., and C.

quinoa was used as control. Plants were kept in an insect-

proof greenhouse for 2 months for symptom development.

Ten plants each were inoculated, and inoculation tests

were repeated thrice.

Virus purification

Ca-M19

was mass propagated in C. quinoa, and the in-

oculated leaves were collected 7-10 days after inoculation

for virus purification. Virus was purified according to a

procedure described previously (Chen and Chang, 1998).

Yields of virions were determined by absorbance at 260

nm (HITACHI 220S spectrophotometer), and calculated

using an extinction coefficient of 2.4 for potyviruses with-

out light scattering correction (Holling and Brunt, 1981).

Purified virus samples were treated with an equal volume

of dissociation buffer (250 mM Tris-HCl, containing 2%

[w/v] of SDS, 4% [v/v] of 2-mercaptoethanol and 10% [w/

v] of sucrose) (Laemmli,

1970) before analysis by 12% of

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

(SDS-PAGE) (Sambrook and Russell, 2001).

Electron microscopy

Virus particles of Ca-M19 from leaf dips of C. quinoa

or purified samples were negatively stained with 2% PTA

(phosphotungstic acid) (Hall et al., 1945) and examined

with an electron microscope (HITACHI H-7000). Modal

length of

Ca-M19 virus particles was calculated by mea-

suring 100 virions. Immuno-sorbent electron microscopy

was used to determine the reaction of Ca-M19

with its

homologous antiserum. Grids were first coated with

Ca-M19

infected tissue extract, incubated with 1:1000

(v/v) diluted immunoglobulin G against

Ca-M19

CP, and

finally stained with 2% PTA before observation.

Cloning of 3¡¦-terminal region of

Ca-M19 and

nucleotide sequence analysis

A potyvirus-degenerate primer Hrp-5 (5¡¦-ATgATH-

gARKCNTgggg) designed by Pappu et al. (Pappu et al.,

1998) and an oligo d(T)

14

primer were used to amplify the

3¡¦-terminal region of

Ca-M19 by the reverse-transcription

polymerase chain reaction (RT-PCR). Extraction of viral

RNA and reverse transcription of the first strand cDNA

were conducted as described previously (Chen et al.,

2003a, Chen et al., 2002a). PCR amplification was car-

ried out by 26 repeated cycles (Perkin Elmer GeneAmp

system 2400): denaturing at 94¢XC for 1 min, annealing at

50¢XC for 45 s, and DNA synthesis at 72¢XC for 1 min 30

s. An elongation step at 72¢XC for 6 min was conducted at

the last cycle. DNA fragment amplified by RT-PCR was

cloned into a pCRII-TOPO vector (Invitrogen, California)

according to manufacturer¡¦s instructions. Nucleotides

were sequenced

by an automatic DNA sequencer (ABI

PRISM 377, Perkin-Elmer, CA). Three independent

clones were selected for alignment to determine the

complete nucleotide sequence. Sequence data were ana-

lyzed and compared with potyvirus species in GenBank

using the Vector NTI Suite (InforMax, Inc., Bethesda,

MD). In order to confirm the existence of

Ca-M19

in

infected calla lilies, a specific primer set was designed for

RT-PCR detection. A biotinylated DNA probe specific to

Ca-M19 prepared by a BrightStar

TM

kit (Psoralen-Biotin,

Ambion Inc. Texas) was further used to hybridize with

CHEN et al. ¡X Characterization of a distinct potyvirus infecting calla lily

371

RT-PCR products for confirmation. Hybridization were

determined by SEQ-Light Chemiluminscent Sequencing

system (Tropix Inc., MA, USA) and exposed to Kodak

intensifying screen (Eastman Kodak Co., New York). One

field isolate (Ca2) that positively reacted with

Ca-M19

specific primers was cloned, sequenced, and compared

with that of the type culture.

Expression of viral coat protein in Escherichia

coli

Based on the sequence data, a primer set was designed

for the amplification of Ca-M19 putative complete CP

gene. The recognition sites for restriction enzymes, NcoI

and XhoI, were created at the 5¡¦ end of the upstream

(M19CPu: 5¡¦ -TCACATACCATGGGCTCGGGAG

AAAAGACAGGT, NcoI site underlined) and down-

stream (M19CPd: 5¡¦-CCTGCCCTCGAGTTACTGCGGT

GGACCCATAC, XhoI site underlined) primers, respec-

tively. Viral RNAs from purified virions were used as a

template for RT-PCR amplification of

Ca-M19 CP gene,

which was then constructed into expression plasmid vector

pET28b (Novagen, Inc., Madison, WI) by

the

directional

cloning technique (Chen et al., 2002a). Constructed

pET plasmids were subsequently transformed into Esch-

erichia coli strain DH5£\ and later into E. coli strain BL21

(DE3) pLysS (Novagen) for protein expression. Protein

expression was induced by the addition of isopropyl £]-D-

thiogalactopyranoside (IPTG) as an inducer, and the

results were analyzed by SDS-PAGE

on 12% gels (Sam-

brook and Russell, 2001). The size and expression level of

Ca-M19

CP were determined by western blotting analysis

using the potyvirus-specific monoclonal antibody (Agdia

Inc., Elkhart, IN). Bacteria expressed

Ca-M19

CP was

eluted from the SDS-PAGE gel as described previously

(Chang et al., 1988).

Antiserum preparation and serological tests

Antiserum against bacterial expressed Ca-M19

CP

was prepared by immunizing New Zealand white rab-

bits. The immunization protocols were the same as those

described (Chang, 1992; Chen and Chang, 1998). Se-

rological relationships between

Ca-M19 and 20 known

potyviruses were analyzed by an SDS-immunodiffusion

test and ELISA as described previously (Chen and Chang,

1998; Purcifull and Batchlor, 1977). Antigens of four

calla lily-infecting potyviruses were used for comparison.

These included: S isolate of DsMV (Chen et al., 2003a),

R7 isolate of KoMV (Chen et al., 2003a), YC5 isolates of

TuMV (Chen et al., 2003a), and T17Q isolate of ZaMMV

(15) and the following potyviruses: Bean common mosaic

virus (BCMV) (Chang, 1993), Scott isolates of BYMV

(BYMV-Scott) (Chang, 1993), Blackeye cowpea mosaic

virus (BlCMV) (Chang, 1993), TP1 isolate of Lily mottle

virus (LiMV-TP1) (Chang et al., 1995), Lycoris mild

mottle virus (LyMMV) (Chang et al., 2002), Lycoris po-

tyvirus (LPV) (Chang et al., 2002), Melon vein banding

mosaic virus (MVbMV) (Liu et al., 1999), type W strain

of Papaya ringspot virus (PRV-W) (Purcifull and Hiebert,

1979), Passionfruit crinkle virus (PCV) (Chang and Lin,

1989), Passionfruit mottle virus (PaMV) (Chang, 1992),

Passionfruit woodness virus (PWV) (Chang, 1992), Ts

isolate of Peanut stripe virus (PStV-Ts) (Chang et al.,

1990), Pea seed-borne mosaic virus (PSbMV) (Chang et

al., 1989), Soybean mosaic virus (SMV) (Chang, 1993),

Tuberose mild mosaic virus (TMMV) (Chen and Chang,

1998), Watermelon mosaic virus 2 (WMV 2) (Purcifull

and Hiebert, 1979), and Zucchini yellow mosaic virus

(ZYMV) (Purcifull et al., 1984).

Surveys for the occurrence of

Ca-M19 in calla

lily fields

Symptomatic and asymptomatic plants were randomly

collected from various calla lily fields and indexed with

antisera against calla lily-infecting potyviruses by indirect

ELISA as described previously (Chen et al., 2003a).

RESULTS

Host reaction and symptomatology

Among the test plant species inoculated with Ca-M19

in the experiment, only C. quinoa responded consistently

with local lesion reactions. Nicotiana benthamiana, G.

max, and tissue-culture-derived calla lily plantlets, did

not show symptoms throughout the 2-month observation

period, nor did they react with antibody against Ca-M19

in ELISA within one month of mechanical inoculation.

Again, five different calla lily cultivars were subsequently

inoculated with

Ca-M19, but none were infected, as

confirmed by ELISA.

Virus purification and electron microscopy

Flexuous rod particles, typical of potyviruses, were

consistently observed in negatively stained leaf dips

of

Ca-M19 infected calla lilies and C. quinoa (Figure

1A). Inoculated leaves from C. quinoa 7-10 days af-

ter inoculation were used for virus purification,

and

approximately 0.6 mg Ca-M19 per 100 g of leaf tissue

was obtained. The UV absorption ratio (260/280) was

1.02, indicating that the purity of the sample was not

satisfactory (Stace-Smith and Tremaine, 1970). EM ex-

amination showed numerous flexuous rod particles similar

to those observed in negatively stained leaf dips, measured

about 755 nm in length as calculated from 100 purified

virus particles.

Particles either purified or from leaf dips

were readily decorated by the Ca-M19 CP antibodies (Fig-

ure 1B).

Nucleotide sequences and molecular

characterization

A 1.3-kb DNA product was consistently amplified

from the RNA templates of purified Ca-M19 virions or

total RNA extracted from Ca-M19 infected tissue by RT-

PCR using the potyvirus degenerate primer sets (data not

shown). The amplicon was cloned, sequenced, and filed

372

Botanical Studies, Vol. 47, 2006

to the GenBank with the accession number AF469171.

Disregarding the length of the poly A tail, the amplified

sequence comprises 1339 nucleotides (nts) corresponding

to the 3¡¦-terminal region of a potyvirus. The deduced ami-

no acid sequence contains 362 residues encoding part of

the 3¡¦-terminal region of the nuclear inclusion b gene (80

residues) and the complete sequence of the coat protein

(CP) gene (282 residues). A 253 nts non-coding region

(NCR) was found at the 3¡¦-terminal region of the DNA.

When the sequence was compared with known

potyviral sequences available in GenBank, the results

showed that Ca-M19 is unique in the coat protein gene

(CP) and 3¡¦ non-coding region (NCR). Comparisons also

showed that Soybean mosaic virus (SMV) and Watermelon

mosaic virus 2 (WMV 2) are most similar to Ca-M19 but

sharing only about 80% of nucleotide identities in CP and

NCR (Table 1). All other potyviruses share less than 72%

sequence identities with Ca-M19. Our analyses of a sec-

ond isolate of Ca-M19 (Ca2) from calla lily showed that it

was serologically and biologically indistinguishable from

Ca2 isolate, with a more than 98% sequence homology

(Table 1).

Based on the sequence data, we designed a primer set

(M19u: 5¡¦-ACAGGTGAGGATTTAGAT / M19d: 5¡¦-

AAATAAGTGCGACACAAT) that specifically amplified

a 850 bp product from the CP region of Ca-M19 ge-

nomic RNA. Using this primer set, Ca-M19 associated

calla lilies were identified from the field (Figure 2A).

This specific primer did not amplify four different calla

lily potyviruses including DsMV, KoMV, TuMV and

ZaMMV in RT-PCR (Figure 2A). Reciprocally, the

Ca-M19 specific DNA probe did not hybridize with RT-

PCR products of these potyviruses amplified by potyvirus-

degenerate primers (Figure 2C and 2D). The results

confirmed that Ca-M19 has a unique CP gene sequence

that is different from other known potyviruses infecting

calla lily.

Figure 1. Electron micrographs of virus particles of Calla

lily latent virus isolate M19 (Ca-M19). (A) Negative staining

of filamentous virus particles observed in leaf dip of Ca-M19

infected calla lily; (B) Virus particles decorated with antiserum

against bacteria expressed coat protein of CLLV.

Table 1. Comparisons of nucleotide and amino acid identities

of the coat protein (CP) genes and the 3

¡¦

-noncoding regions

(NCRs) of Calla lily latent virus isolate M19 (Ca-M19) with

those of known potyviruses species.

Percent identity

a

Potyvirus (Acc. No.)

b

CP nt CP aa 3

¡¦

-NCR

Ca-M19 (AF469171) 100 100 100

Ca-Ca2

99

99

98

AzMV (U60100)

70

71

67

BCMV (AJ293576)

69

72

68

BlCMV (AF395678)

69

72

68

BYMV (S77515)

60

56

40

CABMV (AF24123)

69

67

58

DeMV (U23564)

68

68

65

DsMV (AF511485)

59

61

47

KoMV (AF470620)

60

53

25

LiMV-TP1

56

52

45

LMV (Z78227)

61

60

45

LPV (AF511486)

52

40

31

LyMMV (AF399672) 59

47

47

MVbMV

71

71

65

PaMV-tw

68

68

71

PCV-tw

67

63

52

PSbMV (D10453)

57

52

39

PStV (NC001732)

70

72

65

PWV (AF208662)

69

69

67

SMV (NC002634)

77

81

81

SMV (S42280)

78

81

79

SMV (X96665)

77

81

83

TMMV (AF062926)

57

53

29

TuMV (AF530055)

59

54

45

WMV 2 (L22907)

80

82

81

WMV 2 (D13913)

80

82

79

WMV 2 (AB001994) 79

81

81

ZYMV (NC003224)

66

66

49

a

Percent identities of the respective nucleotide and amino acid

sequences were analyzed by the Vector NTI Suite program

(InforMax, Inc., Wisconsin).

b

The potyviruses listed are: Ca-Ca2 (Ca 2 isolate of Calla lily

latent virus), AzMV (Azukibean mosaic virus), BCMV (Bean

common mosaic virus), BYMV (Bean yellow mosaic virus),

BlCMV (Blackeye cowpea mosaic virus), CABMV (Cowpea

aphid-borne mosaic virus), DsMV (Dasheen mosaic virus),

DeMV (Dendrobium mosaic virus), KoMV (Konjak mosaic

virus), LMV (Lettuce mosaic virus), LiMV (Lily mottle virus),

LPV (Lycoris potyvirus), LyMMV (Lycoris mild mottle vi-

rus), MVbMV (Melon vein banding mosaic virus), PaMV

(Passionfruit mottle virus), PCV (Passionfruit crinkle virus),

PSbMV (Pea seedborne mosaic virus), PWV (Passionfruit

woodness virus), PStV (Peanut stripe virus), SMV (Soybean

mosaic virus), TMMV (Tuberose mild mosaic virus), TuMV

(Turnip mosaic virus ), WMV 2 (Watermelon mosaic virus 2 ),

and

ZYMV (Zucchini yellow mosaic virus).

CHEN et al. ¡X Characterization of a distinct potyvirus infecting calla lily

373

Expression of Ca-M19 CP in E. coli and

preparation of its antiserum

A 850-bp DNA product equivalent to the entire CP

gene of Ca-M19 was amplified by RT-PCR using the

aforementioned specific primer set. This product was

constructed directionally into expression vector pET28b

and transformed into E. coli strain BL21(DE3)pLys S for

protein expression. A protein about 35 kDa in size, similar

to that of purified Ca-M19 CP, was detected by SDS-

PAGE

i

n the lysate of bacteria transformed with BL21

clone, but not in the lysate of control bacteria (Figure 3A).

Using antiserum against a potyvirus-specific monoclonal

antibody in western blotting test, the potyviral CP nature

of the 35 kDa protein was confirmed (Figure 3B). The

protein was subsequently purified from the lysate of a

large volume culture of the transformed bacteria (Figure

3A). The estimated yield of expressed CP in 1000 ml of

bacteria culture was about 2 mg. A polyclonal antiserum

(#097) against the bacteria-expressed Ca-M19 CP was

thus prepared. It reacts strongly with both the CP from

purified virions and the bacteria expressed 35 kDa protein

(Figure 3C).

Serological tests

The Ca-M19 antiserum (#097) reacted strongly in

ELISA, SDS-immunodiffusion, and western blotting tests

against homologous antigens of Ca-M19 infected plants

but not with uninfected controls (Table 2, Figure 4). In

indirect ELISA, the antiserum cross reacted with some

potyviruses tested including BCMV, MVbMV, PaMV,

PCV, PStV, WMV 2, SMV, and ZYMV (Table 2). Among

them, SMV and WMV 2 showed ELISA readings as high

as those in homologous reactions. SDS-immunodiffusion

tests confirmed that these viruses were serologically

Figure 2. Molecular identification of Calla lily latent virus (CLLV) by reverse-transcription polymerase chain reaction (RT-PCR)

and DNA probe hybridization. (A) Amplification of CLLV and four different calla lily infecting potyviruses in RT-PCR using CLLV

specific primers; (B) Southern blot hybridization of the gel from figure (A) using biotin-labeled DNA probe specific to CLLV; (C)

Amplification of four different calla lily infecting potyviruses by RT-PCR using potyvirus-degenerate primer designed by Pappu et al.

(1998). (D) Southern blot hybridization of the gel from figure (C) by biotin-labeled DNA probe specific to CLLV. Lane M, kb DNA

ladder markers; lanes L and C are CLLV-infected tissues of Chenopodium quinoa and calla lily, respectively; lanes 1-6 are field col-

lected calla lily samples infected by CLLV; lanes H1 and H2 are uninfected control of C. quinoa and calla lily, respectively; lanes N, T,

D, K are four potyvirues infecting calla lily including Zantedeschia mild mottle virus, Turnip mosaic virus, Dasheen mosaic virus and

Konjak mosaic virus, respectively.

Figu re 3. Sodium dodecyl sulfate-polyacrylamide gel elec-

trophoresis (SDS-PAGE) and western blotting analyses of the

bacteria express ed coat protein (CP ) of M19 isolate of Calla

lily latent virus (Ca-M19). P roteins separated in SDS-PAGE

(A) were blotted onto nylon membrane and reacted with the

potyvirus-specific monoclonal antibody (Agdia Inc.) (B) and

antis erum against Ca-M19 (C); and subse quently detecte d

by alkaline phosphatase conjugated coat anti-rabbit antibody.

Lane M, protein markers; lane 1, IPTG-induced bacteria (E.

coli BL21(DE3)pLysS) lysate containing non-inserted expres-

sion vector pET28b; lane 2, IPTG-induced bacteria lysates of

pET28b inserted with Ca-M19 CP gene; lane 3, purified bacteria

expressed Ca-M19 CP; lane 4, purified Ca-M19 virions.

374

Botanical Studies, Vol. 47, 2006

Table 2. Reactions of antisera against bacteria expressed coat protein of M19 isolate of Calla lily latent virus (Ca-M19) with dif-

ferent potyviruses in enzyme linked immunosorbent assay

a

(ELISA)

and SDS-immunodiffusion tests

b

.

Antigens

c

Cross-reactivities

ELISA

d

SDS-immunodiffusion

e

25¡Ñ

50¡Ñ

100¡Ñ

1¡Ñ

Ca-M19 CP

3.08

2.47

1.11

I

e

Ca-M19

1.13

1.03

1.09

I

Ca-Ca2

1.27

1.14

1.12

I

BCMV

1.58

1.61

1.52

S

BlCMV

0.08

0.09

0.09

-

BYMV

0.18

0.19

0.15

-

DsMV

0.09

0.09

0.08

-

KoMV-R7

0.08

0.09

0.11

-

LPV

0.15

0.15

0.16

-

LyMMV

0.00

0.00

0.02

-

LiMV-TP1

0.14

0.16

0.15

-

MVbMV

0.74

0.37

0.33

S

PaMV

2.10

2.18

2.37

S

PSbMV

0.12

0.13

0.13

-

PCV

0.42

0.33

0.23

-

PRSV-W

0.15

0.14

0.16

-

PWV

2.57

2.62

2.58

S

PStV

1.62

1.46

1.29

-

SMV

2.74

2.51

2.23

S

TMMV

0.13

0.08

0.02

-

TuMV-YC5

0.20

0.19

0.19

-

WMV 2

2.46

2.43

2.25

S

ZYMV

0.86

0.75

0.62

S

a

Indirect enzyme-linked immunosorbent assay (ELISA), described previously (Chang et al., 1990), was conducted to determine

the serological relatedness between Ca-M19 and other potyviruses. Purified immunoglobulins of antiserum #097 were diluted to

1:1000 to react with antigens diluted to 1/25, 1/50, and 1/100.

b

SDS-immunodiffusion test, described previously (Chen et al., 2003a), was performed by the use of undiluted antiserum (#097)

against bacteria expressed Ca-M19 coat protein to react with undiluted SDS-treated antigens of different potyviruses as listed in

the table.

c

The antigens listed are: BCMV (Bean common mosaic virus), BlCMV (Blackeye cowpea mosaic virus), BYMV (Bean yellow

mosaic virus), DsMV (Dasheen mosaic virus), KoMV (Konjak mosaic virus), LPV (Lycoris potyvirus), LyMMV (Lycoris mild

mottle virus), LiMV (Lily mottle virus), MVbMV (Melon vein banding mosaic virus), PaMV (Passionfruit mottle virus), PSbMV

(Pea seedborne mosaic virus), PCV (Passionfruit crinkle virus), PRSV-W (Papaya ringspot virus type W), PWV (Passionfruit

woodiness virus), PStV (Peanut stripe virus), SMV (Soybean mosaic virus), TMMV (Tuberose mild mosaic virus), TuMV-YC5

(YC5 isolate of Turnip mosaic virus), WMV 2 (Watermelon mosaic virus 2), ZYMV (Zucchini yellow mosaic virus), Ca-M19 CP

(bacteria expressed coat protein of M19 isolate of Calla lily latent virus), Ca-M19 (infected tissue of M19 isolate of Calla lily

latent virus), and Ca-Ca2 (infected tissue of Ca2 isolate of Calla lily latent virus).

d

Reactivities in ELISA are shown as the absorbance readings (A

405nm

) taken about 40 min after addition of enzyme substrate

solution. The absorbance values are an average of two replicate wells. Readings lower than 0.2 were considered negative

reactions and are underlined.

e

Reactivities in SDS-immunodiffusion tests are shown as the reactions between homologous and heterologous antigens: I =

precipitation lines of homologous and heterologous reations fused without spur formation; S = homologous reactions spurred over

heterologous reactions; - = no reaction.

CHEN et al. ¡X Characterization of a distinct potyvirus infecting calla lily

375

related but distinct from Ca-M19 because the precipitin

lines formed spurs with those formed by homologous

reactions (Table 2). Furthermore, our data showed that

those calla lily-infecting potyviruses, including DsMV,

KoMV, TuMV and ZaMMV, were serologically unrelated

to Ca-M19 in ELISA and the SDS-immunodiffusion test

(Table 2, Figure 4), indicating the feasibility of using the

antiserum to specifically detect the presence of Ca-M19 in

calla lilies.

Surveys for the occurrence of Ca-M19 in calla

lily fields

A total of 245 calla lily samples were collected from

various fields in Taiwan. They were indexed by ELISA

using antisera against five different calla lily infecting

potyviruses including Ca-M19, DsMV, KoMV, TuMV

and ZaMMV. Ca-M19 was found in 86 of 245 sampled

calla lilies. Among these 86 Ca-M19-infected plants, 77

of them exhibited viral symptoms while the other nine

samples did not. Interestingly, all these symptomless

healthy looking plants were found to be infected only

by Ca-M19 while the other 77 symptomatic plants were

all found co-infected with other potyviruses. These as-

ymptomatic plants were transplanted from the field and

grown in a greenhouse for continuous observation. They

remained symptomless throughout a 6-month observation

period. During the 6-month observation, we routinely

checked these symptomless plants by ELISA and RT-

PCR (data not shown). They were confirmed consistently

infected by Ca-M19 only.

DISCUSSION

Based on the size and morphology of virus particles,

serological reactivities to potyvirus-specific monoclonal

antibodies, and the cross reactivities of the antiserum with

other potyviruses, Ca-M19 isolated from calla lilies was

identified as a member of the genus Potyvirus. Further-

more, the uniqueness in the nucleotide and the amino acid

sequences of coat protein gene and 3¡¦-NCR confirm that

the virus from calla lilies is a new species of Potyvirus

(Shukla et al., 1991; Ward et al., 1992). Thus, we conclude

that Ca-M19 isolate is a distinct potyvirus and designated

it Calla lily latent virus (CLLV). Like most potyviruses,

Ca-M19 has a very narrow host range. Besides calla

lilies, it only infects C. quinoa and induces local lesions

on the inoculated leaves in our experiment. Recovery

inoculation using extracts of an infected single lesion from

C. quinoa to calla lilies was not successful, making it diffi-

cult to conclude what specific symptoms on calla lilies are

induced by Ca-M19. Further studies showed that when

infected by Ca-M19 alone, calla lilies did not exhibit any

symptoms within a 6-month observation period. Our sur-

veys showed that symptomatic calla lilies observed in the

fields, when tested positive with Ca-M19 antiserum, were

always found co-infected by other viruses. The results

confirm our observation that Ca-M19 causes symptomless

infection in calla lilies. Therefore, we propose Calla lily

Figure 4. Serological comparison of M19 isolate of Calla lily

latent vir us (Ca-M19) with some potyviruses infecting calla

lily in sodium dodecyl sulfate (SDS) immunodiffusion tests.

The center wells were charged with antiserum (#097) against

bacteria-expressed coat protein of Ca-M19, and the peripheral

wells were filled with SDS-treated antigens of Dasheen mosaic

virus (D), Konjak mosaic virus (K), Ca-M19 (M), Turnip mosaic

virus (T), Zantedeschia mild mottle virus (N), healthy calla lily

(H), bacteria-expressed coat protein of Ca-M19 (Bep), and two

CLLV-infected calla lily antigens collected from fields (F1 and

F2).

latent virus (CLLV) as the name of this novel virus (Chen

et al., 2004). To our knowledge, CLLV is the first virus

reported to cause latent infection in calla lilies. CLLV-

associated symptomless calla lilies are indistinguishable

from uninfected plants in both growth vigor and flowering

potential, including yield and quality. However, possible

synergistic effects resulting from simultaneous infection

of CLLV with other viruses need to be clarified.

In this study, we developed DNA probes, specific

RT-PCR primers, and antiserum against bacteria-

expressed CP for the detection of CLLV. As in our

previous studies (Chen et al., 2002a), antiserum prepared

to bacteria-expressed CP was useful in ELISA, SDS-

immunodiffusion, immuno-specific electron microscopy,

and western blotting tests. Using these indexing tools,

CLLV was frequently detected in calla lily plants collected

from several major calla lily production townships in Tai-

wan. Although we did not carry out an insect transmission

test, we considered these infected plants to be vectored

by aphids. This is based on the fact that, as in most

potyviruses, a DAG triplet, the genetic code for aphid

transmissibility, is found in the N-terminus of CLLV CP.

CLLV is shown serologically related to at least eight

potyviruses, including BCMV, MVbMV, PaMV, PCV,

PStV, WMV 2, SMV, and ZYMV (Table 2). These

potyviruses were shown serologically to be closely related

to each other in our previous studies (Chang, 1992; Chang

and Lin, 1989; Chang et al., 1990). When the amino acid

sequence of CLLV CP gene is examined and aligned with

those of WMV 2 and SMV, the two most closely related

viruses based on ELISA readings, an extra 17-residues

were present at the N-terminus of CLLV CP (Figure

5). In the same region, an extra sequence is present in

WMV 2, but it is different from that of CLLV (Figure 5).

376

Botanical Studies, Vol. 47, 2006

Other than this variable N-terminal portion, the core and

C-terminal regions of CLLV CP are very similar to those

of SMV and WMV 2. This explains the close serological

relatedness among these three potyviruses.

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