Apple chlorotic leaf spot virus

Nobuyuki Yoshikawa
Faculty of Agriculture, Iwate University, Morioka 020-8550, Japan

Contents

Introduction Main Diseases Geographical Distribution Host Range and Symptomatology Strains Transmission by Vectors Transmission through Seed Transmission by Grafting Transmission by Dodder Serology Nucleic Acid Hybridization Relationships Stability in Sap

Purification Properties of Particles Particle Structure Particle Composition Properties of Infective Nucleic Acid Molecular Structure Genome Properties Satellites Relations with Cells and Tissues Ecology and Control Notes References Acknowledgements Figures

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 MgCl₂ or MgSO₄. 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 MgCl₂ 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: s_20,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 10⁶ 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 M_r 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

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Figure 1

Apple topworking disease. Stem pitting of Malus prunifolia var. ringo rootstocks. (Courtesy T. Ito).

Figure 2

Chlorotic spots in young leaves of M. sylvestris cv. R12740-7A (Courtesy T. Ito).

Figure 3

Chlorotic ring and line patterns in leaves of M. platycarpa (Courtesy T. Ito).

Figure 4

Necrotic spots and necrosis in leaves and top necrosis in a shoot of M. hupehensis (Courtesy I. Machida).

Figure 5

Malformed petals with necrotic ring spots and necrosis on flowers of M. hupehensis (Courtesy T. Ito).

Figure 6

Chlorotic spots, mottling and leaf distortion of a systemically infected Chenopodium quinoa plant.

Figure 7

Necrotic spots in inoculated leaves of C. quinoa 7 days after inoculation.

Figure 8

Chlorotic spots and vein clearing in upper leaves of C. amaranticolor.

Figure 9

Chlorotic spots and vein clearing in leaves of Nicotiana occidentalis.

Figure 10

Virus particle stained with uranyl acetate. Bar represents 100nm.

Figure 11

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.