Australasian Plant Pathology, 2002, 31, 345–350
Host range and symptomatology of Subterranean clover mottle virus in alternative pasture, forage and crop legumes J. Fosu-NyarkoA, R. A. C. JonesB,C,E, L. J. SmithB,C, M. G. K. JonesA,C and G. I. DwyerA,C,D A
Plant Biotechnology Research Group, State Agricultural Biotechnology Centre, Division of Science and Engineering, Murdoch University, WA 6150, Australia. B Crop Improvement Institute, Department of Agriculture, Locked Bag No. 4, Bentley Delivery Centre, WA 6983, Australia. C Centre for Legumes in Mediterranean Agriculture, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia. D Centre for Bioinformatics and Biological Computing, Division of Business, Information Technology and Law, Murdoch University, WA 6150, Australia. E Corresponding author; email:
[email protected]
Abstract. Fifty one plant species from 25 different genera in seven families were challenged with Subterranean clover mottle virus (SCMoV) by inoculation with infective sap. These included 39 legume species and 12 species belonging to the families Amaranthaceae, Apiaceae, Chenopodiaceae, Cruciferae, Cucurbitaceae and Solanaceae. Twenty one new host species belonging to eight different legume genera were found for SCMoV, nine of which were alternative annual pasture or forage species and 12 were crop legumes. Of these, seven pasture or forage species and six crop species were infected systemically. Two Chenopodium spp. were also hosts. The host status of eight previously tested legume species was also re-examined. The implications of these results for paddocks sown with alternative pasture or forage crop legumes, and for plant improvement programs evaluating such species for commercial use, are discussed. AP02038 .JHeFotaslur. -aNgnyearknodsymptomatol gy ofCS MoV
Additional keywords: grain legumes, pulse crops, risk assessment, sensitivity, susceptibility, vulnerability.
Introduction The most widely grown annual pasture legume species in Australia are Trifolium subterraneum (subterranean clover) and annual Medicago spp. (annual medics) (Puckridge and French 1983). However, there are many situations where neither persists well (Cocks et al. 1980). Also, ley farming systems based around annual Medicago spp. and T. subterraneum are being replaced by phase farming systems to which these pasture species are poorly adapted, and there is a growing need for more forage species, to which neither T. subterraneum nor annual Medicago spp. are well suited (Ewing 1998). The pasture and forage legume program in Western Australia (WA) is evaluating a wide range of alternative legume species and recent releases include cultivars of Biserrula pelecinus (biserrula) for permanent annual pastures, Ornithopus compressus (yellow serradella) for ley farming, O. sativum (French serradella) for phase farming systems with extended rotations, T. incarnatum (crimson clover) for forage, and T. vesiculosum (arrowleaf clover) for sites with perched water tables (e.g. © Australasian Plant Pathology Society 2002
Ewing 1998). Similarly, employing appropriate crop legume species in cropping rotations increases production and aids development of sustainable farming systems. The most widely grown crop legume in southern Australia is Lupinus angustifolius (narrow-leafed lupin) but this species is poorly adapted to neutral to alkaline fine-textured or shallow soils (Gladstones et al. 1998). The cool season crop legume program in WA is evaluating a number of crop species better adapted to such soils. Locally adapted cultivars of Cicer arietinum (chickpea), Lens culinaris (lentil), Pisum sativum (field pea), Vicia ervilia (bitter vetch) and Lathyrus cicera (dwarf chickling) have been released (e.g. Siddique and Sykes 1997; Siddique et al. 1999). Virus diseases pose a major constraint to production of legume crops and pastures worldwide (Bos et al. 1988; Edwardson and Christie 1991). The most economically important viral pathogen infecting T. subterraneum pastures in Australia is Subterranean clover mottle virus (SCMoV), which causes economic losses of ca. AUD$31 million per year to the Australian dairy industry and also causes
10.1071/AP02038
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substantial losses to national wool and meat industries (Ferris and Jones 1994, 1995; Jones 1996). SCMoV is transmitted by trampling and grazing by stock, on mower blades, on the wheels of vehicles and through seed (Wroth and Jones 1992b; Jones 1996; McKirdy et al. 1998; Jones et al. 2001). Infection is common in pastures in WA, Tasmania, South Australia, New South Wales and Victoria. In addition to infecting T. subterraneum, it also occurs in other cultivated annual clovers, such as T. vesiculosum, and wild annual clovers, such as T. glomeratum (cluster clover) (Francki et al. 1983; Johnstone and McLean 1987; Wroth and Jones 1992b; Helms et al. 1993; Ferris and Jones 1994; Jones et al. 2001). Diseased T. subterraneum plants develop leaf mottling and distortion, decreased leaf size and plant stunting (Wroth and Jones 1992a). Infection decreases herbage and seed production, diminishing feed for stock and ability of pastures to regenerate annually from seed (Wroth and Jones 1992b; Ferris and Jones 1994, 1995; Barbetti et al. 1996; Barbetti and Jones 1999). Recent ‘risk assessment’ studies established the probable consequences of introducing alternative commercial or potentially commercial legume species into agricultural systems in which they are likely to encounter widespread infection with four common aphid-borne viruses, Alfalfa mosaic virus, Bean yellow mosaic virus, Cucumber mosaic virus and Pea seed-borne mosaic virus (McKirdy et al. 2000; Latham and Jones 2001; Latham et al. 2001). Although the reactions of T. subterraneum and some annual Medicago spp. to infection with SCMoV are known (Wroth and Jones 1992a; Ferris et al. 1996), host range studies so far provide rather incomplete information on whether infection with SCMoV might pose a threat to pastures sown with the alternative annual pasture and forage legumes currently under evaluation for their commercial potential or already being sown commercially. Similarly, although P. sativum is known to become infected (Francki et al. 1983; Wroth and Jones 1992a), there is no information on the host status of other cool season crop legumes. Francki et al. (1983) inoculated 16 species within the Papilionaceae, Chenopodiaceae and Solanaceae with SCMoV but only T. subterraneum, P. sativum and Chenopodium murale became infected and in the latter two species, infection was restricted to the inoculated leaves. Wroth and Jones (1992a) examined a wider range of species and infected P. sativum, T. subterraneum, six annual Medicago spp., five wild annual Trifolium spp., two perennial Trifolium spp. and nine alternative annual Trifolium spp. with SCMoV. All but four of the species infected were systemic hosts. In this paper we report the results of more extensive host range studies involving 51 species belonging to 25 different genera. The extent of the threat posed by infection with SCMoV to the viability of pastures sown with alternative pasture or forage legumes is discussed along with the need to screen advanced selections
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for their vulnerability to this virus before the release of new cultivars and species for sowing in pastures. Methods Origins and maintenance of virus cultures, and virus inoculations Three SCMoV isolates were obtained from infected T. subterraneum pastures in WA: severe isolate P23 and very severe isolate MJ were originally collected from Manjimup (Wroth and Jones 1992a, 1992b; Ferris and Jones 1995) and moderate isolate MB was collected from Mt Barker. Very severe isolate AL was collected from a T. vesiculosum plant in a pasture at North Dandalup in WA. All isolates were maintained in T. subterraneum cv. Woogenellup. Inoculations were done by grinding infected leaves in 0.2 M sodium phosphate buffer, pH 7.2, mixing the sap with diatomaceous earth and rubbing it gently onto leaves. Plants Lupinus spp. (lupin) and Vicia faba (faba bean) were grown in washed river sand, otherwise all plant species were grown in a peat, soil and sand mix. Plants were kept in an air-conditioned glasshouse at 18–20°C. Legume species were inoculated with appropriate Rhizobium spp. The plant species inoculated with SCMoV are shown in Table 1. Five plants of each genotype (Table 1) were inoculated with isolate P23 and an additional five plants kept as uninoculated controls. In addition, five plants each of Medicago truncatula and P. sativum were inoculated with SCMoV isolates MJ, MB and AL and five left uninoculated. Symptom observations and virus detection All plants were inspected weekly over 6–9 weeks for viral symptoms in inoculated and uninoculated leaves. Samples were collected from inoculated leaves 2–3 weeks after inoculation and tested by RT-PCR. Depending on the length of time it took for systemic symptoms to develop, newly emerged leaves were sampled and tested by RT-PCR 3–9 weeks after inoculation. In a few instances, samples from inoculated and newly emerged leaves were tested by ELISA instead of RT-PCR. Nucleic acid extraction Leaf samples (50–100 mg fresh weight) were macerated in 500 μL of extraction buffer (50 mM Tris-HCl pH 8.5, 10 mM EDTA and 200 mM NaCl) and total nucleic acid extracted in an equal volume of TE (10 mM Tris-HCl and 1 mM EDTA, pH 8.0)-buffered phenol:chloroform:isoamyl alcohol (50:49:1), followed by chloroform:isoamyl alcohol (24:1). The aqueous phase was then transferred to a fresh tube containing 0.7 volumes of ice-cold isopropanol, mixed by inversion and stored at –80°C for 30 min. Total nucleic acid was pelleted at 20 000 g for 30 min, washed with 70% ethanol, the pellet dried under vacuum and resuspended in 50 μL of TE. RT-PCR RT-PCR was carried out in a two-step reaction; all RT-PCR reagents were supplied by Perkin Elmer Corp. RT-PCR primers were designed from the cDNA sequence of SCMoV isolate P23 (Dwyer et al. 2003). Reverse transcription was done in a 10 μL reaction volume containing 1.5 μL of total nucleic acid from a single leaf, 5 mM MgCl2, 1 × PCR buffer (500 mM KCl, 100 mM Tris-HCl, pH 8.3), 0.5 μM scm37 reverse primer (5´ TGCCTCAGGAGAGAGCAGT 3´), 1 mM dNTPs, 1 U/μL of RNase inhibitor, and 1.25 units of M-MuLV reverse transcriptase. The reaction mixture was then incubated at 42°C for 30 min followed by 95°C for 15 min. For PCR, the volume was increased to 50 μL with the addition of 0.1 μM upstream scm34 primer (5´ TTCTACCGAGTCGTCGC), 1 × PCR buffer, 2 mM of MgCl2 and
Host range and symptomatology of SCMoV
1.25 units of AmpliTaq DNA polymerase. PCR was carried out in a GeneAmp PCR System 2400 (Perkin Elmer). PCR mixtures were initially incubated at 94°C for 3 min followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 1 min, with a final extension step of 72°C for 7 min. RT-PCR products were analysed by electrophoresis in a 1.0% agarose gel buffered in Tris-acetate-EDTA (pH 8.0) containing 0.3 μg/mL ethidium bromide as described by Sambrook et al. (1989). ELISA Leaf samples were extracted (1 g/20 mL) in phosphate-buffered saline solution (10 mM potassium phosphate, 150 mM sodium chloride) using a leaf press (Pollahne, Germany). The extracts were tested for SCMoV by double antibody sandwich ELISA as described by Clark and Adams (1977). The polyclonal antiserum to SCMoV was prepared by Dr J. I. Cooper and Ms D. Li at the State Agricultural Biotechnology Centre, WA. Each sample was tested in duplicate wells in microtitre plates with SCMoV-infective and healthy leaf sap included in paired wells as controls. The substrate used was 0.6 mg/mL p-nitrophenyl phosphate in 100 mL/L of diethanolamine, pH 9.8. Absorbance values (A405) were measured in a Multiskan plate reader (Labsystems, Finland). A sample was considered positive when the A405 value was greater than three times that of healthy leaf sap.
Results Twenty-one new host species of SCMoV were found belonging to eight different legume genera (Table 1). Of the new hosts, seven pasture or forage species and six crop species were systemically infected, but infection remained restricted to inoculated leaves in B. pelecinus, T. glanduliferum (gland clover) and six other crop legume species. When infection became systemic, this developed in all inoculated plants of most species. The exceptions were T. isthmocarpum (Moroccan clover) and T. vesiculosum, in which 3/5 and 1/5 inoculated plants, respectively, developed systemic infection. Inoculated leaves developed obvious necrotic spots in Trigonella balansae (Trigonella) (Fig. 1a), faint necrotic spots or rings in L. clymenum, and faint necrotic or chlorotic spots in T. spumosum 24BP (bladder clover). In all other legume hosts, inoculated leaf infection was asymptomatic (Table 1). Severity of systemic symptoms varied between species and the different types found included vein clearing (mostly an initial transient symptom), chlorosis, mottle, vein banding, systemic necrotic spotting, streaking and line patterns, leaf distortion/deformation, leaf curling, epinasty, decreased leaf size and plant stunting. Trigonella balansae and T. clypeatum (helmet clover) developed severe systemic symptoms 10–14 days post-inoculation and are potentially useful as diagnostic hosts (Fig. 1b, c, d). Additional hosts that developed distinct systemic symptoms included three crop legumes, C. arietinum, Lathyrus ochrus (grass pea) and Vicia narbonensis (narbon bean). Epinasty in C. arietinum was a symptom unique to this species. In general, in T. incarnatum, T. michelianum (balansa clover), T. purpureum (purple clover) and T. resupinatum (Persian clover), the symptoms caused by SCMoV resembled those reported previously by Wroth and Jones
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(1992a), except that initial vein clearing developed and symptoms were milder in T. incarnatum, T. michelianum and T. resupinatum (Table 1). Unlike the genotypes of M. truncatula and T. hirtum (rose clover) tested by Wroth and Jones (1992a), the genotypes of these species tested by us were not infected by SCMoV. In M. polymorpha (burr medic) cv. Circle Valley, the infection was asymptomatic, as reported previously (Wroth and Jones 1992a), but in our inoculations, infection was always restricted to the inoculated leaves. The reaction of P. sativum cv. Greenfeast to inoculation with SCMoV was as reported previously (Francki et al. 1983; Wroth and Jones 1992a), but for cvv. Dundale, Laura and Wirrega, infection in the inoculated leaves was asymptomatic. When P. sativum cv. Greenfeast and M. truncatula cv. Caliph were inoculated with SCMoV isolates MB, MJ and AL, the reactions in cv. Greenfeast were the same as those caused by isolate P23 and, as with P23, no infection was detected in cv. Caliph. Chenopodium quinoa developed chlorotic spot lesions in inoculated leaves 7–14 days after inoculation and systemic mottling in young leaves after 3 weeks (Table 1). C. amaranticolor was symptomlessly infected in inoculated leaves without systemic infection. No virus was detected in inoculated or tip leaves of the ten species inoculated with SCMoV from five other families. Discussion This study extends the known host range of SCMoV within the Papilionaceae. In addition to the 25 host species reported previously (Francki et al. 1983; Wroth and Jones 1992a), a further 21 were identified. However, outside this family the host range of SCMoV was narrow, and included only two further species from the Chenopodiaceae. With eight of the 21 new legume host species and one of the Chenopodium spp., infection was restricted to inoculated leaves. Moreover, like Wroth and Jones (1992a), we found that in a small number of systemically infected host species, systemic invasion occurred only in a proportion of the plants inoculated. Within T. subterraneum, this response to sap inoculation is genotype dependent (Wroth and Jones 1992a, 1992b; Ferris et al. 1996) and is due to impaired cell-to-cell movement of the virus rather than to diminished virus multiplication (Njeru et al. 1995). Except with P. sativum and M. truncatula, we used only one isolate in our host range studies. Possibly, had additional isolates been used, they might have infected more species. SCMoV isolates differ in the severity of the symptoms they cause when infecting T. subterraneum plants (Wroth and Jones 1992b), but no serologically distinct strains are reported (Jones et al. 2001). As isolates differ in severity, others would presumably have tended to induce symptoms of different intensity from those caused by the relatively severe isolate we used. Ferris et al. (1996) evaluated the performance of different genotypes of T. subterraneum inoculated with
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Table 1. Family
Amaranthaceae Apiaceae Chenopodiaceae Cruciferae Cucurbitaceae Papilionaceae
Solanaceae
Reactions of different species of pasture, forage and crop legumes to inoculation with SCMoV isolate P23 SpeciesA
Gomphrena globosa Daucus carota Chenopodium amaranticolor C. quinoa Brassica juncea B. napus Cucumis sativus Biserrula pelecinus (PL) Cicer arietinum (CL) C. arietinum (CL) Dorycnium hirsutum (PL) Hedysarum coronarium (PL) Lathyrus cicera (CL) L. clymenum (CL) L. ochrus (CL) L. ochrus (CL) L. sativus (CL) L. sativus (CL) Lens culinaris (CL) Lupinus albus (CL) L. angustifolius (CL) L. luteus (CL) Medicago murex (PL) M. polymorphaC (PL) M. sativa (PL) M. truncatulaC (PL) Melilotus alba (PL) Ornithopus compressus (PL) O. sativus (PL) Phaseolus vulgaris (CL) Pisum sativumC (CL) P. sativum (CL) Trifolium cherleri (PL) T. clypeatum (PL) T. dasyurum (PL) T. dasyurum (PL) T. glanduliferum (PL) T. hirtumC (PL) T. incarnatumC (PL) T. isthmocarpumD (PL) T. michelianumC (PL) [= T. balansae] T. purpureumC (PL) T. resupinatumC (PL) T. spumosum (PL) T. spumosum (PL) T. vesiculosumD (PL) Trigonella balansae (PL) Vicia benghalensis (CL) V. faba (CL) V. narbonensis (CL) V. sativa (CL) Vigna unguiculata (CL) Capsicum annuum Lycopersicon esculentum Nicotiana benthamiana N. glutinosa Physalis floridana
Common name
None Carrot None None Mustard Canola Cucumber Biserrula Chickpea ‘kabuli’ Chickpea ‘desi’ Hairy canary clover Sulla Dwarf chickling None Grass pea Grass pea Grass pea Grass pea Lentil White lupin Narrow-leafed lupin Yellow lupin Murex medic Burr medic Lucerne, Alfalfa Barrel medic White sweet clover Yellow serradella Pink serradella Climbing bean Field pea Field pea Cupped clover Helmet clover Eastern Star clover Eastern Star clover Gland clover Rose clover Crimson clover Moroccan clover Balansa clover Purple clover Persian clover Bladder clover Bladder clover Arrowleaf clover Trigonella Purple vetch Faba bean Narbon bean Common vetch Cowpea Capsicum, Bell pepper Tomato None None None
Genotype (Accession/breeding line/cultivar) — cv. Stefano — — cv. Tendergreen cv. Pinnacle — cv. Casbah cv. Kaniva cv. Sona SA1111 cv. Grimaldi ATC 80521 C7022 ATC 80532 ATC 80123 ATC 80488 ATC 80517 cv. Matilda cv. Kiev mutant cv. Gungurru cv. Wodjil cv. Zodiac cv. Circle Valley cv. Aquarius cv. Caliph 35635 cv. Santorini cv. Cadiz cv. Stringless blue cv. Greenfeast cvv. Dundale, Laura, Wirrega cv. Lisare CFD13 24GCN39 42BT ATC87181+ATC87182 GCN39 cv. Caprera MAR14.10.1 Paradana 136780 Persian Prolific 87144 24BP cv. Seelu SA5054 cv. Popany cvv. Ascot, Fiesta SA26554 cv. Namoi – cv. Rialto cv. Grosse Lisse – – –
SymptomsB Inoculated Non-inoculated leaves leaves and shoots NI NI SI LCS NI NI NI SI SI SI NI NI SI LNR, LNS SI SI SI SI SI SI SI SI NI SI NI NI NI NI NI NI LNS, LVN SI SI SI SI SI SI NI SI SI SI SI SI SI LNS, LCS SI LNS SI NI SI SI NI NI NI NI NI NI
NI NI NI M NI NI NI NI VC, C, RLS, TB, SSt VC, C, R, RLS, TB, TD, SSt NI NI NI C C, MM, VB, LD, SNS, RLS, St C, M, LD, SNS, RLS, SSt SS NI M, RLS NI NI NI NI NI NI NI NI NI NI NI NI NI VC, RLS, St SVC, C, SM, SNS, RLS, St VC, C, SM, LD, SNS, RLS, SSt VC, C, SM, VB, LD, SNS, RLS, SSt NI NI VC, MM, LD, RLS, St VC, SM, LC, LD, St VC, C, M, LD, RLS, St SVC, M, LD, RLS, St VC, C, MM, MLD, RLS, St VC, MM, SNS, RLS, St VC, MM, LC, RLS, MSt VC, C, MM, St VC, SM, SLD, NL, RLS, SSt NI NI VC, C, M, LD, RLS, St NI NI NI NI NI NI NI
A
PL = pasture or forage legume; CL = crop legume. Symptom codes: LCS = local chlorotic spots; LNS = local necrotic spot lesions; LNR = local necrotic rings; LVN = local veinal necrosis; SI = symptomless infection in inoculated leaves; VC = vein clearing; SVC = severe vein clearing; C = chlorosis; R = reddening; MM = mild mottle; M = mottle; SM = severe mottle; VB = vein banding; LC = leaf curling; LD = leaf deformation/distortion; SLD = severe leaf deformation/distortion; SNS = systemic necrotic spotting and/or streaking; NL = systemic necrotic line patterns; RLS = reduced leaf size; TB = shoot tip bending (epinasty); TD = shoot tip death; St = stunting; MSt = mild stunting; SSt = severe stunting; SS = symptomless systemic infection; NI = no infection detected in non-inoculated leaves. Where the code NI is used, the leaf sample tested negative for SCMoV by either RT-PCR or ELISA. C Species previously tested by Wroth and Jones (1992a). D Only a proportion of the plants inoculated became infected systemically. B
SCMoV in the field using grazing animals as virus vectors. Due to the expense involved, we were unable to repeat such experiments using the new hosts identified here. However, the information obtained by Wroth and Jones (1992a) and by us on the reactions of alternative pasture and forage
legume species to sap inoculation with SCMoV provides an indication of the risk that SCMoV-induced losses may occur upon the release of these species into pastures in different regions of Australia. Previous extensive surveys of pastures in southern Australia revealed that species at
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Fig 1b
Fig 1a
Fig 1d
Fig 1c
Fig. 1. (a) Spreading necrotic spots in SCMoV-inoculated leaves of Trigonella balansae. (b) Severe mottle and leaf deformation in leaves of Trigonella balansae systemically infected with SCMoV. (c) Persistent severe vein clearing in a leaf of Trifolium clypeatum infected systemically with SCMoV. (d) Obvious mottle and necrotic streaking in the leaf of a Trifolium clypeatum plant infected systemically with SCMoV.
greatest risk of damage from SCMoV are those sown within the highest rainfall zones (Wroth and Jones 1992b; Helms et al. 1993; Jones 1996). Whether it is appropriate to deploy SCMoV-vulnerable pasture species [e.g. T. clypeatum, T. dasyurum (eastern star clover), T. isthmocarpum and Trigonella balansae] should be considered carefully before sowing them in such zones. Instead, deploying species that do not become infected systemically, like B. pelecinus and T. glanduliferum, or non-hosts, like H. coronarium, O. compressus and O. sativus, may be more suitable. In the future, it would seem prudent that all new selections of alternative pasture and forage legumes be tested by inoculation with SCMoV prior to release so that vulnerable species and genotypes can be identified. Depending upon the relative importance of any beneficial traits they possess versus their vulnerability to damage caused by SCMoV, it may then be appropriate for such species or genotypes to be targeted only for sowing in lower SCMoV-risk regions.
Of the 13 crop legume species that were hosts of SCMoV, only six became infected systemically, namely C. arietinum, L. clymenum, L. ochrus, L. sativus (grass pea), Lens culinaris and V. narbonensis. No surveys have been made to determine whether SCMoV naturally infects crops of these six species. However, even though the virus is readily contact transmissible and could sometimes be introduced on contaminated farm machinery, it seems unlikely that incidences of infection would become high enough to cause economic losses in grain yield, especially as there is no known insect vector. Therefore, unlike the situation with pasture and forage legumes within pastures, SCMoV does not seem to pose much of a threat to the productivity of legume crops. Acknowledgements Seeds were supplied by K. Foster, C. Hanbury, R. Snowball and L. Latham. We thank G. Burchell for glasshouse assistance. Funding for the first author was
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provided by a Murdoch University Postgraduate Research Scholarship.
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International
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Received 2 January 2002, accepted 10 April 2002
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