Apple chlorotic leaf spot virus
Nobuyuki Yoshikawa
Faculty of Agriculture, Iwate University, Morioka 020-8550, Japan
Contents
Introduction
Described by
Cadman (1963),
Cropley (1963,
1964) and
Lister et al. (1965).
- Synonyms
- Pear ring pattern mosaic virus
(Van Katwijk, 1954)
- Apple latent virus Type 1
(Lister et al., 1965)
A virus with flexuous filamentous particles 680-780 nm long and 12 nm in width containing
linear positive-sense, ssRNA. Occurs in woody plants of the family Rosaceae including
apple,
peach, pear, plum, cherry, apricot and prune. Its natural mode of spread is unknown.
Transmissible
by mechanical inoculation to herbaceous plants.
Main Diseases
Though ACLSV infection is symptomless in most commercial apple varieties, the virus causes
topworking disease of apple trees grown on Maruba kaido (
Malus prunifolia var.
ringo)
rootstocks in Japan
(
Fig.1)
(
Yanase, 1974;
Yanase et al., 1979),
plum bark split
(
Dunez et al., 1972),
plum pseudopox
(
Jelkmann & Kunze, 1995),
pear ring pattern mosaic, and apricot pseudopox diseases. See
Nemeth (1986)
for an extensive review.
Geographical Distribution
Probably occurs wherever rosaceous fruit trees are cultivated.
Host Range and Symptomatology
The virus occurs in apple, peach, pear, plum, cherry, apricot, and prune
(
Lister, 1970;
Nemeth, 1986).
In addition, the virus infects experimentally species in a few families, including
Chenopodiaceae and
Leguminosae
(
Lister et al., 1965;
Saksena and Mink, 1969;
Yoshikawa et al., 1997).
Grafting is used for transmission between woody hosts. Transmissible from apple to
Chenopodium quinoa by mechanical inoculation of extracts from buds, young leaves or petals
ground in 0.05 M phosphate buffer (pH 7-8) containing 2% (v/v) nicotine base or 2% (w/v)
polyvinyl pyrrolidone
(
Lister, 1970).
Diagnostic species
Malus sylvestris cv. R12740-7A (Russian apple). Chlorotic blotches, usually
distributed asymmetrically in young leaves
(Fig.2),
asymmetric leaf distortion and puckering, stunting of shoot growth.
M. platycarpa. Irregular diffuse chlorotic ring and line patterns in leaves
(Fig.3),
which are smaller than normal, distorted, and often shed prematurely.
M. hupehensis. Red, necrotic spots and necrosis in young leaves
(Fig.4),
and malformed petals with necrotic ring spots and necrosis
(Fig.5)
(Machida, 1995).
M. hupehensis is the most sensitive indicator plant among the above three species.
However, the species also develops severe symptoms after infection with other apple viruses, e.g.,
Apple stem pitting virus (ASPV). Therefore, it is difficult to distinguish ACLSV infection from
ASPV infection when the plant is doubly infected with both viruses.
M. prunifolia var. ringo (MO-84a). Chlorotic spots, or flecks in young leaves,
accompanied by leaf distortion and inner bark necrosis
(Yanase, 1974;
Yanase et al., 1979).
MO-84a is useful for the detection of the type strain associated with the topworking disease,
though no symptoms result from inoculation of mild strains
(Yanase, 1974).
C. quinoa. Chlorotic and necrotic spots in inoculated leaves 3-4 days after
inoculation, followed by systemic symptoms consisting of chlorotic spots, mottling, ring and
line patterns in upper leaves
(Fig.6 & Fig.7).
C. amaranticolor. Small chlorotic spots in inoculated leaves and chlorotic spots,
vein clearing and mottling in upper leaves
(Fig.8).
Propagation species
C. quinoa is the most useful plant for maintaining cultures and propagating virus for
purification. To transmit the virus from fruit trees, Nicotiana occidentalis is useful
(Fig.9).
Assay species
C. quinoa is useful for assay.Strains
Many isolates are reported from apple, cherry, peach, plum, and prune, which can be differentiated
on symptomatology in indicator plants
(
Desvigens & Boye, 1988;
Chairez & Lister, 1973a;
German-Retana et al., 1997;
Yanase, 1974).
Some isolates from peach and apple trees comprise at least two or three variants that differ
considerably from each other in nucleotide sequence
(Candresse et al., 1995;
Yoshikawa, unpublished results), as found in Apple stem grooving and Apple stem pitting viruses
(Magome et al., 1997;
Yoshikawa et al., 2001).
Transmission by Vectors
No vectors are reported.
Transmission through Seed
Not reported.
Serology
Moderately antigenic, giving titers up to 1/1024. Polyclonal antisera have been prepared in
rabbits against purified virus
(
Saksena & Mink, 1969;
Chairez & Lister, 1973a;
Kerlan et al., 1981;
Yanase, 1974).
ELISA has been used to detect the virus in fruit trees
(
Flegg & Clark, 1979).
Monoclonal antibodies (MAbs) were produced and used to investigate the antigenic structure of the
virus. Epitope studies using these MAbs defined seven independent antigenic domains in ACLSV particles
(
Poul & Dunez, 1990).
MAbs were also used for sensitive and specific detection by ELISA
(
Poul & Dunez, 1989).
An immunocapture (IC)-RT-PCR was developed for the sensitive detection and the analysis of the
molecular variability of the virus
(
Candresse et al.,1995;
Nemchinov et al., 1995).
Relationships
Several strains can be differentiated serologically as well as by symptoms in indicator plants
(
Chairez & Lister, 1973a,
1973b;
Yanase, 1974).
Sequence comparisons among isolates indicate large molecular variability, i.e., sequence
conservation rates vary between 77.4% and 99.4%, with most of the isolates differing by 10-20% from
any other given isolates
(
German-Retana et al., 1997;
Candresse et al., 1995).
Serologically unrelated to all other known virus species in the genus Trichovirus.
Similarities exist between ACLSV and virus species in the genera Capillovirus and
Vitivirus in the amino acid sequences of the conserved polymerase motif, putative movement
protein and coat protein
(Yoshikawa et al., 1997).
Stability in Sap
In bentonite-clarified
C. quinoa sap, the thermal inactivation point (10 min) is between
52-55 °C, the half-life at 45 °C is 12±1 min, the dilution end-point is about 10
-4,
and infectivity is abolished in one day at 20 °C and about 10 days at 4 °C
(
Lister, 1970;
Saksena & Mink, 1969).
Infectivity was lost below pH 5.5 or above pH 9.5
(
Saksena & Mink, 1969).
Purification
The most satisfactory method depends on clarifying extracts with bentonite
(
Lister, 1970;
Lister & Hadidi, 1971;
Lister et al., 1965;
Saksena & Mink, 1969).
Purified from
C. quinoa by homogenizing 100g systemically infected leaves in 250 ml of 0.01 M
Tris-HCl buffer (pH 7.2-7.6) containing 0.01 M MgCl
2 or MgSO
4. Clarify the
extract by low speed centrifugation and squeeze through cheesecloth. Add a bentonite suspension
(30-40 mg/ml in 0.01 M phosphate buffer, pH 7-8) slowly and clarify by centrifuging at low speed.
Repeat this clarification step until the supernatant fluid is clear. Precipitate the virus from
the supernatant fluid by adding PEG (mol. wt 6,000) to 8% (w/v), allow to stand for 1 hr and centrifuge
at low speed. Resuspend the pellet in 0.05 M Tris-HCl buffer (pH 7.2) containing 0.01 M MgCl
2
and clarify by centrifuging at low speed. Concentrate the virus by ultracentrifugation and purify
further by sucrose density gradient centrifugation. Yields are 0.5-2 mg/100 g leaf tissue, depending
on the isolate.
Properties of Particles
Sedimentation coefficient:
s20,w (svedbergs) : about 96S (not corrected to
Infinite dilution). No accessory virus particles detected
(
Lister, 1970).
Buoyant density: 1.27 g/ml in Cs2SO4 gradients
(Bar-Joseph et al., 1974).
The particles degrade in CsCl gradients.
Electrophoretic mobility: -5.1 x 10-5 cm2. sec-1. V-1
in 0.05 M Tris + 0.005 M MgCl2 buffered to pH 7.5 in 3% sucrose (determined by
electrophoresis in a sucrose gradient)
(Lister, 1970).
A260/A280: 1.85-1.89 for very pure preparations (not corrected for
light-scattering)
(Lister, 1970;
Lister & Hadidi, 1971;
Yoshikawa & Takahashi, 1988).
Particle Structure
Particles are very flexuous filaments, 680-780 nm long and 12 nm in width
(
Fig.10),
with obvious cross-banding and helical symmetry; the pitch of the helix is about 3.8 nm
(
de Sequeira and Lister, 1969;
Lister, 1970,
Yoshikawa et al., 1997).
The particles require the presence of divalent cations for the integrity of the quaternary structure
(
Lister & Hadidi, 1971).
The best stains for electron microscopy are uranyl acetate or uranyl formate; particle breakage occurs
in phosphotungstate
(
Lister, 1970).
Particle Composition
Nucleic acid: Linear positive-sense ssRNA of M
r about 2.48 x 10
6 or 7,549
to 7,555 nucleotides excluding a polyA-tail
(
Bar-Joseph et al., 1974;
German et al., 1990;
German-Retana et al., 1997;
Sato et al., 1993;
Yoshikawa & Takahashi, 1988);
about 5% of particle weight. The 5' terminus of the RNA probably has a cap structure. Nucleotide
base ratios for isolate P863 from
Prunus domestica are: G 23.8%; A 31.5%; C 17.7%; U 27.0%
(
German et al., 1990).
Protein: Particles contain a single polypeptide species of Mr 21.4-21.5K
(Chairez & Lister, 1973b;
German et al., 1990;
Sato et al., 1993;
Yoshikawa & Takahashi, 1988).
Genome Properties
The complete nucleotide sequences of the single genomic RNA of four isolates have been determined.
They are isolates P863
(accession no.
M58152,
M31714)
from
Prunus domestica, P205
(
D14996)
from apple, Bal1
(
X99752) from Cherry, and PBM1
(
AJ243438) from plum
(
German et al., 1990;
German-Retana et al., 1997;
Sato et al., 1993).
Identities of the nucleotide sequences were 79.8% (P863/P205), 76.2% (P863/Bal1), 81.5% (P863/PBM1),
76.5% (P205/Bal1), 79.6% (P205/PBM1), and 76.5% (Bal1/PBM1). The genomic RNA has three ORFs in
the positive strand
(
Fig.11).
ORF1 encodes a 216 kDa protein containing the consensus motifs of methyltransferase,
papain-like protease, nucleotide triphosphate-binding helicase, and RNA polymerase
(
German et al., 1990;
German-Retana et al., 1997;
Sato et al., 1993).
The 50kDa protein encoded by ORF2 is localized on plasmodesmata in infected and transgenic plant leaves
(
Sato et al., 1995;
Yoshikawa et al., 1999).
The 50kDa protein fused to green fluorescent protein can move into adjacent cells from the cells
that produced it in the leaf epidermis
(
Satoh et al., 2000)
and transgenic plants expressing the protein can complement the systemic spread of mutants of
an infectious cDNA clone that are defective in ORF2
(
Satoh et al., 1999;
Yoshikawa et al., 2000),
indicating that the 50kDa protein is a movement protein. ORF3 encodes a 21-22 kDa coat protein
(
German et al., 1990;
German-Retana et al., 1997;
Sato et al., 1993).
Analysis of the dsRNAs of ACLSV-infected plants and in vitro translation of
ACLSV-RNA suggested that the 50 kDa protein and coat protein are expressed from 2.2 and
1.1 kb subgenomic RNAs, respectively
(Candresse et al., 1996;
German et al., 1992).
Relations with Cells and Tissues
In infected
C. quinoa leaves, the particles occur as aggregates in the cytoplasm of
vascular parenchyma and mesophyll cells, but not in the nucleus or vacuole. No virus-specific
inclusion bodies, such as pinwheels, viroplasmas or vesicles were observed
(
Ohki et al., 1989,
Yoshikawa et al., 1997).
Ecology and Control
Because the virus is thought to be transmitted in the field only by grafting, planting virus-free
plants is the best means of controlling decline problems in fruit trees due to the virus.
References
- Bar-Joseph, Hull & Lane, Virology 62: 563, 1974.
- Cadman, Plant Disease Reporter 47: 459, 1963.
- Candresse, Lanneau, Revers, Grasseau, Macquaire, German, Malinovsky & Dunez, Acta Horticulturae 386: 136, 1995.
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Apple topworking disease. Stem pitting of Malus prunifolia var. ringo rootstocks.
(Courtesy T. Ito).
Chlorotic spots in young leaves of M. sylvestris cv. R12740-7A (Courtesy T. Ito).
Chlorotic ring and line patterns in leaves of M. platycarpa (Courtesy T. Ito).
Necrotic spots and necrosis in leaves and top necrosis in a shoot of M. hupehensis
(Courtesy I. Machida).
Malformed petals with necrotic ring spots and necrosis on flowers of M. hupehensis
(Courtesy T. Ito).
Chlorotic spots, mottling and leaf distortion of a systemically infected
Chenopodium quinoa plant.
Necrotic spots in inoculated leaves of C. quinoa 7 days after inoculation.
Chlorotic spots and vein clearing in upper leaves of C. amaranticolor.
Chlorotic spots and vein clearing in leaves of Nicotiana occidentalis.
Virus particle stained with uranyl acetate. Bar represents 100nm.
Genome organization of Apple chlorotic leaf spot virus.
MET, methyltransferase; P-PRO, papain-like protease; HEL, helicase ; POL, RNA polymerase;
MP, putative movement protein; CP, coat protein.