Details of DPV and References

DPV NO: 358 September 1998

Family: Geminiviridae

Genus: Begomovirus

Species: Squash leaf curl virus | Acronym: SLCV

Squash leaf curl virus

J. E. Duffus USDA Agricultural Research Service, 1636 E. Alisal St., Salinas, CA 93905, USA

D. C. Stenger Agricultural Research Service, 344 Keim Hall, University of Nebraska, Lincoln, NE 68583, USA

Contents

Introduction

Disease first described and virus whitefly-transmitted by Flock & Mayhew (1981). Virus first purified and characterized by Cohen et al. (1983).

Synonyms

Melon leaf curl virus (MLCV; Duffus et al., 1985)

Watermelon curly mottle virus (WMCMoV; Brown & Nelson, 1984; 1986)

Squash leaf curl virus (SLCV) is a virus with geminate particles, 22 x 38 nm. The circular ssDNA genome is bipartite and consists of two similar-sized species. Known hosts are in the Cucurbitaceae, Leguminosae, Solanaceae and Euphorbiaceae. The virus is transmitted by the whitefly, Bemisia tabaci, and by inoculation with sap. Squash leaf curl disease occurs in desert regions of southwestern USA and Mexico.

Main Diseases

SLCV causes severe stunting and leaf curling of squash (Cucurbita spp.) (Fig. 1). The disease may result in high rates of mortality, and yields may be reduced, particularly in autumn-planted crops when large populations of the whitefly vector are present (Flock & Mayhew, 1981; Dodds et al., 1982; Cohen et al., 1983). Wide host range isolates (Dodds et al., 1984; Duffus et al., 1985; Brown & Nelson, 1986) also cause severe leaf curling and stunting of Old World cucurbit species, including cantaloupe (Cucumis melo) and watermelon (Citrullus lanatus).

Geographical Distribution

The disease is common in cultivated low desert areas of California, Arizona and Texas in the USA, and also in adjacent desert regions of northern Mexico.

Host Range and Symptomatology

Transmissible by inoculation of sap to several species of Cucurbitaceae, and to Phaseolus vulgaris (Leguminosae) and Nicotiana benthamiana (Solanaceae) (Cohen et al., 1983; Dodds et al., 1984; Polston et al., 1989). At least one wide host range isolate is also capable of infecting Euphorbia heterophylla (Euphorbiaceae), Datura stramonium (Solanaceae) and Rhynchosia minima (Leguminosae) (Polston et al., 1989).

Diagnostic species

Cucurbita maxima (winter squash) (Fig. 3, Fig. 4). Severe stunting and leaf curl symptoms occur on new growth. Interveinal tissue may become mottled, and green vein-banding may be associated with leaf veins. Enations often form on the lower surface of symptom-bearing leaves. Occasionally, flowers fail to develop or set fruit, or fruits may be small and distorted. Similar symptoms develop on infected summer squash (C. moschata) and pumpkin (C. pepo) (Cohen et al., 1983).

Phaseolus vulgaris. Bean cultivars Tender Crop, Top Crop, Earliwax, Majestic and Greenpak develop a systemic green mosaic and veinal distortion, resulting in twisted, deformed leaves (Cohen et al., 1983) (Fig. 2).

Citrullus lanatus (watermelon), Cucumis melo (melon), Cucumis sativa (cucumber). The wide host range isolates SLCV-2 (Dodds et al., 1984), MLCV (Duffus et al., 1985) and WMCMoV (Brown & Nelson, 1986) induce leaf curling and stunting of these three Old World cucurbit species, whereas the narrow host range SLCV isolate of Cohen et al. (1983) does not.

Nicotiana benthamiana. Wide host range isolates induce a systemic leaf curling and distortion of N. benthamiana when cloned DNA is inoculated mechanically, or is delivered by Agrobacterium-mediated inoculation protocols (Lazarowitz & Lazdins, 1991). The SLCV isolate of Cohen et al. (1983) has not been tested on this species.

Propagation species

Squash-infecting isolates may be maintained by whitefly transmission to C. pepo. Wide host range isolates may be maintained also in P. vulgaris or N. benthamiana. Wide host range isolates have been maintained in P. vulgaris by mechanical transmission (Brown & Nelson, 1986; Polston et al., 1990).

Assay species

No local lesion hosts are known. Cohen et al. (1983) used infectivity assays based upon the proportion of C. pepo plants infected following whitefly inoculation. Infectivity of cloned DNA has been assayed by mechanical inoculation to N. benthamiana, or by Agrobacterium-mediated inoculation to N. benthamiana and P. vulgaris (Lazarowitz & Lazdins, 1991; Lazarowitz, 1991). Mechanical transmission assays with crude sap inocula have been reported for a wide host range isolate of SLCV (Brown & Nelson, 1986). SLCV was transmitted by B. tabaci that had acquired virus by feeding on inoculum through a membrane (Cohen et al., 1983).

Strains

Isolates of SLCV may be grouped into at least two strains based upon host range:

1. SLCV-2 (Dodds et al., 1984) and MLCV (Duffus et al., 1985) from the Imperial Valley, California, USA, and WMCMoV (Brown & Nelson, 1984; 1986) from Arizona, USA are able to infect watermelon, melon, and cucumber. MLCV and WMCMoV were initially described as distinct viruses, but may be considered, along with SLCV-2, as isolates of a wide host range strain of SLCV. They are closely related to a fourth isolate (SLCV-E, Polston et al., 1989) for which infective DNA clones have been characterized and completely sequenced (Lazarowitz & Lazdins, 1991; Lazarowitz, 1991).

2. The SLCV isolate of Cohen et al. (1983) has a narrow host range which does not include watermelon, melon or cucumber. An additional narrow host range isolate (SLCV-R) was cloned from a mixed culture also containing SLCV-E (Lazarowitz & Lazdins, 1991; Lazarowitz, 1991). Host range properties of SLCV-R are complex, with restriction in certain species being determined by variants of the DNA B component (Lazarowitz, 1991). Nucleic acid hybridization tests with component-specific probes (Polston et al., 1989) suggest that SLCV-R and the SLCV isolate of Cohen et al. (1983) are isolates of the same strain.

Transmission by Vectors

The results of vector transmission studies (Cohen et al., 1983; Polston et al., 1990) are consistent with a persistent, circulative mode of transmission by the whitefly B. tabaci. Minimum acquisition and inoculation access periods may be as brief as 30 min; however, a minimum latent period of 19-24 h is required (Cohen et al., 1983). Maximal transmission frequencies are achieved by an acquisition access period of 6-24 h, and an inoculation access period of 24-48 h (Cohen et al., 1983). The virus may be retained by individual adult whiteflies for as long as 26 days (Cohen et al., 1983), and there is a low frequency of passage through the moult (Polston et al., 1990). However, no transovarial passage was observed for 1000 progeny from viruliferous parents (Cohen et al., 1983). Nucleic acid probes and serological methods have been used to detect accumulations of SLCV DNA and antigen both in B. tabaci and in the non-vector whitefly species, Trialeurodes vaporariorum, suggesting that vector specificity is not determined simply by a lack of retention of virus by non-vectors (Polston et al., 1990). Although both the A and B biotypes of B. tabaci can acquire and transmit SLCV, the A biotype is a more efficient vector: serial transmission assays with individual whiteflies resulted in a transmission efficiency of 90% for the A biotype, compared to 30% for the B biotype (J. E. Duffus, unpublished data).

Transmission through Seed

No information.

Serology

Antiserum produced in a rabbit injected with purified virions yielded a titre of 1/128 in double diffusion tests, and a titre of 1/1028 in density gradient serological tests (Cohen et al., 1983). This same antiserum has been used in ELISA (1/800 dilution) to detect SLCV antigen in infected plant samples (Polston et al., 1989) and to discriminate among Begomovirus species (Cohen et al., 1983). ISEM has been used for detecting the virus and its relationships to other Begomovirus species (Roberts et al., 1984).

Relationships

SLCV shares biological and physical properties with geminiviruses in the genus Begomovirus (equivalent to subgroup III as defined by Matthews, 1991): it has a host range restricted to dicots (Cohen et al., 1983; Dodds et al., 1984; Brown & Nelson, 1986; Polston et al., 1989; Lazarowitz & Lazdins, 1991), is transmitted by the whitefly B. tabaci (Flock & Mayhew, 1981; Cohen et al., 1983; Dodds et al., 1984; Brown & Nelson, 1986), and possesses a bipartite genome (Lazarowitz & Lazdins, 1991) with an organization similar to those of other begomoviruses. Phylogenetic studies indicate that SLCV clusters within a clade consisting of other New World begomoviruses (Padidam et al., 1995). An ancestor of SLCV appears to have recombined with an ancestor of beet curly top virus to produce the genome of horseradish curly top virus (Klute et al., 1996).

SLCV may be distinguished from other begomoviruses by host responses within the Cucurbitaceae (Cohen et al., 1983; Lazarowitz, 1991), the nucleotide sequence of the Common Region (CR) conserved in both DNA species of a single virus (Lazarowitz, 1987; Lazarowitz & Lazdins, 1991), and the more divergent nucleotide sequence of DNA B (Lazarowitz & Lazdins, 1991).

Stability in Sap

No information concerning stability in sap is available, although most isolates have been transmitted mechanically. Purified virus preparations retained infectivity for at least 2 months at 4°C, as shown by assays in which whiteflies were allowed to acquire purified virus by feeding through Parafilm membranes (Cohen et al., 1983).

Purification

Purified virus particles (0.75-1.35 mg/kg fresh leaf; assuming A₂₆₀, 1 mg/ml, 1 cm light path = 7.7) may be recovered from squash by the following protocol (Cohen et al., 1983): Harvest infected leaves 4-7 days after symptom appearance and grind coarsely in a food grinder with 0.1 M phosphate buffer (Na₂HPO₄ - KH₂PO₄), pH 7.2, containing 0.002 M disodium EDTA and 0.01 M Na₂SO₃ at the rate of 1 ml/g leaf material. After further treatment with a homogenizer, filter the extract through muslin and stir with 1% (v/v) Triton-X100 for either 2 h at room temperature, or overnight at 4°C. Clarify by adding one-tenth volume of cold chloroform and then centrifuge at low speed (10,400 g, 4°C, 10 min). Precipitate the virus from the aqueous phase by stirring for 1.5-2.0 h at 4°C after the addition of polyethylene glycol (mol. wt 7000-9000) to 12% (w/v), and NaCl to a final concentration of 0.2 M. Recover the precipitate by centrifugation (10,400 g, 4°C, 15 min), and resuspend the pellet by stirring for at least 1 h at 4°C in one-tenth the original volume of 0.1 M phosphate buffer containing 0.002 M EDTA (resuspension buffer), and 1% Triton-X100 (v/v). Centrifuge at low speed (12,000 g, 4°C, 10 min), then subject the supernatant fluid to two cycles of differential centrifugation (high speed = 222,000 g, 4°C, 1 h; low speed = 12,000 g, 4°C, 10 min). Resuspend the purified virus in a minimal volume of resuspension buffer. The virus may be further purified by rate-zonal sedimentation in 10-30% sucrose gradients.

Properties of Particles

A₂₆₀/A₂₈₀: 1.5 (Cohen et al., 1983).

Particle Structure

Electron microscopy of purified particles prepared by negative staining in 2% uranyl acetate indicates that the majority of virions occur as paired geminate particles, 22 x 38 nm (Fig. 5). Unpaired ‘monomer’ particles measuring 22 nm are also present in purified preparations, and may be formed by disruption of geminate particles, or possibly may encapsidate half-unit length deletion derivatives of the viral DNA components. Although less abundant, ‘trimer’ (22 x 53 nm) and ‘tetramer’ (22 x 70 nm) particles also may be seen in purified preparations. The biological significance of monomer, trimer and tetramer particles is unknown.

Particle Composition

Nucleic acid: Two similar-sized species of circular, ssDNA (A and B, approximately 2600 nucleotides each) are present in virus particle preparations. Although each DNA species is thought to be encapsidated into separate, morphologically identical particles, this has not been established unequivocally.

Protein: The number and size of polypeptides present in the capsid of SLCV has not been determined by direct examination. However, determination of the nucleotide sequence of SLCV-E (Lazarowitz & Lazdins, 1991) indicates that the AR1 ORF encodes a 29.2 kDa protein that is highly conserved with the coat protein of other begomoviruses.

Genome Properties

Characterization of the biological and physical properties of infective DNA clones of two SLCV isolates (SLCV-E and SLCV-R) clearly demonstrates that the virus has a bipartite genome (Lazarowitz & Lazdins, 1991; Lazarowitz, 1991). Supercoiled, dsDNA forms of the virus genome are produced in vivo (Lazarowitz & Lazdins, 1991), and serve as replicative intermediates and as templates for transcription. Replication of SLCV DNA probably occurs through a rolling-circle mechanism (Stenger et al., 1991). The nucleotide sequences of the A (GenBank accession # M38183) and B (GenBank accession # M38182) DNA components of SLCV-E have been determined (Lazarowitz & Lazdins, 1991). The genome organization of SLCV (Fig. 6) is similar to those of other begomoviruses, with five ORFs encoded by DNA A, and two ORFs encoded by DNA B (Lazarowitz & Lazdins, 1991). A common region (CR) sequence of approximately 200 nt is present on both DNA components and contains cis-acting regulatory elements of the origin of replication (Lazarowitz, 1987; Lazarowitz et al., 1992).

All seven SLCV ORFs are homologous to ORFs of other begomoviruses, and perform similar functions. DNA A encodes the coat protein (ORF AR1), the replication initiator protein (ORF AL1), the transcriptional activating protein (ORF AL2), a replication enhancer protein (ORF AL3), and a potential protein of unknown function (ORF AL4). Specificity of replication is determined by AL1 protein-origin interactions (Lazarowitz et al., 1992), whereas the gene products of the AL2 and AL3 ORFs are functionally interchangeable among different begomoviruses (Sunter et al., 1994). DNA B encodes two proteins (BL1 and BR1) required for systemic movement. Both movement proteins are determinants of viral host range (Ingham et al., 1995). Mutational analysis of the BL1 and BR1 proteins (Ingham et al., 1995), and the production of disease symptoms produced in transgenic plants expressing the BL1 protein (Pascal et al., 1993) indicate that virulence is determined by the BL1 protein.

Relations with Cells and Tissues

Electron micrographs of vascular cells of infected squash leaves revealed the presence of virions (Hoefert, 1983; 1987) and fibrillar rings within nuclei (Hoefert, 1983). Hoefert (1983) also reported that vascular parenchyma cells contain cytoplasmic vesiculations not previously associated with other plant viruses that infect vascular tissue. The virus is also associated with maturing phloem sieve elements which may exhibit severe necrosis in as little as 9 days post-inoculation (Hoefert, 1987). The BR1 protein binds viral ssDNA and localizes to the cell nucleus (Pascal et al., 1994). The BL1 protein interacts with the BR1 protein and redirects the BR1 protein (and presumably SLCV DNA) to the cell periphery (Sanderfoot & Lazarowitz, 1995).

Notes

SLCV may be distinguished from other cucurbit-infecting viruses by mode of transmission, and by serological or nucleic acid hybridization assays with cloned DNA probes (Polston et al., 1990). Displacement of the B. tabaci A biotype by the B biotype may be responsible for the reduced occurrence of SLCV in southwestern USA.

Figures

References list for DPV: Squash leaf curl virus (358)

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