Department of Molecular and Cell Biology, University of the Witwatersrand, Private Bag 3, PO Wits 2050, Johannesburg, South Africa1
Department of Microbiology, University of Cape Town, Private Bag, Rondebosch 7701, Cape Town, South Africa2
Author for correspondence: Leigh Berrie. Present address: Department of Microbiology, University of Cape Town, Private Bag, Rondebosch 7701, Cape Town, South Africa. Fax +27 21 689 7573. e-mail Leigh{at}molbiol.UCT.ac.za
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Healthy cassava plants and plants infected with SACMV were obtained from Mpumalanga Province in South Africa and maintained in a greenhouse at 25 °C with a day length of 16 h and a dark period of 8 h. Total DNA was extracted as previously described (Berrie et al., 1998 ) from young leaves of SACMV-infected cassava plants. Full-length DNA-A and DNA-B genomic clones of SACMV were generated by digestion of 30 µg of total DNA with SalI and EcoRI, respectively. Fragments of 2·8 kbp were gel purified, cloned into the SalI or EcoRI sites of pBluescript KS(+), respectively, and transformed into E. coli JM109 cells. Positive clones were selected by dot blot hybridizations using DIG-labelled DNA-A- or DNA-B-specific probes (DIG DNA labelling and detection kit, Roche): a 310 bp SACMV DNA-A-specific probe was generated using PCR primers PAV1978 and PAC496 (Rojas et al., 1993
) followed by digestion with PstI and Bam HI. The resulting 310 bp fragment consisted of sequences contained within the Rep gene of SACMV, being specific for SACMV DNA-A sequences only. A 687 bp DNA-B probe was generated using PCR primers PCRC2 and PBV2039 (Wyatt & Brown, 1996
). The resulting fragment consisted of 596 bp of the BC1 ORF, as well as 91 bp of the 3'-end of the IR, allowing the probe to hybridize to both SACMV DNA-A and DNA-B sequences. Genomic clones hybridizing to both DNA probes were accepted to contain DNA-A sequences, whereas those hybridizing to the DNA-B probe only were assumed to contain DNA-B sequences.
Complete sequences of infectious clones of SACMV DNA-A and -B (GenBank accession nos AF155806 and AF155807, respectively) were determined in both directions by automatic sequence analysis with the ALF Express system (Amersham Pharmacia), according to the manufacturers instructions. Sequence data were assembled and analysed, and both pairwise and multiple sequence alignments were generated using DNAMAN versions 2.6 and 3.0 for Windows (Lynnon Biosoft, Quebec, Canada). Phylogenetic trees were constructed from the multiple alignments by the neighbour-joining method using DNAMAN and drawn using Treeview (Page, 1996 ) (1000 bootstrap replicates).
Evidence of recombination in the evolutionary history of SACMV was detected using the pairwise scanning-based recombination analysis program, RDP (version 1.07; Martin & Rybicki, 2000 ), with the following settings: window size=10, highest acceptable probability=0·0001, internal reference sequences. The recombinant origin of regions of SACMV indicated by RDP was verified by constructing neighbour-joining trees using the aligned sequences in the putatively recombinant region. Over 60% bootstrap support for location of the putatively recombinant region of SACMV in a divergent branch of the neighbour-joining tree was accepted as confirmation that the region had been acquired through recombination. Geminivirus DNA sequences used for comparison and their database accession numbers are presented in Table 1
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Phylogenetic comparisons of SACMV DNA-A with other bipartite geminiviruses revealed a close relationship with geminiviruses from the Old World (Fig. 1a). SACMV is most closely related to EACMV type 2 isolates (-MH and -MK, 85%) (Zhou et al., 1998 a
), and less closely to EACMV type 1(-KE and -TZ, 79%) and EACMV-UG isolates (79%) (data not shown). Complete DNA-B sequence comparisons showed SACMV clustering with EACMV-UG-Mld and EACMV-UG-Svr (Fig. 1b
), with sequence similarities of 90%, meaning that SACMV could be considered a strain of EACMV. As SACMV DNA-A shares sequence similarities of <90% with other begomoviruses, we feel that SACMV should be considered a distinct virus, given that the A-component is the master sequence upon which replication depends (Padidam et al., 1995
).
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Agroinoculations of Nicotiana tabacum L. Samsun, Lycopersicon esculentum Mill. Poleboy Burpee, Capsicum annuum L. Anaheim, Phaseolus vulgaris L. Tenderpod green bush snap, Malva parviflora L., Solanum melongena L. Burpees black beauty, Datura stramonium L. and cassava (Manihot esculenta Crantz) were then carried out. Ten plants of each test species were agroinoculated with SACMV dimers as mentioned previously, and the experiment conducted in triplicate. Plants from the species Phaseolus vulgaris, Malva parviflora and cassava (Manihot esculenta Crantz) were successfully inoculated with SACMV, and displayed symptoms of leaf curling, chlorosis and stunting. These results are in contrast to the experimental host-range of ACMV-KE which is largely restricted to the Solanaceae, within which the virus is more or less readily transmitted to several species in the genera Nicotiana and Datura (Bock et al., 1978 ). The host-range of EACMV has not yet been defined. DNA extracted from plants showing symptoms tested positive in Southern blot hybridizations with an SACMV-specific probe. No symptoms were evident in Nicotiana tabacum, Lycopersicon esculentum, Capsicum annuum, Solanum melongena and Datura stramonium, and no viral DNA was detected in these plants by Southern blot hybridizations.
In cassava, whitefly-transmission experiments have proved to be restricted by the feeding preferences of the vector and due to biotype incompatibility with test plants. Mechanical inoculation experiments using SACMV-infected cassava plant sap have also been unsuccessful (Berrie et al., 1997 ). Recently, cassava has been successfully inoculated with full-length genomic clones of ACMV-NG using a hand-held biolistics device (Briddon et al., 1998
). This work demonstrated the first successful inoculation of a cloned geminivirus to cassava. Similar attempts have been made to infect cassava with full-length genomic clones of ACMV-CM and EACMV-CM, as well as inoculation by tissue wounding (V. Fondong, personal communication). Clones of both viruses were shown to be biolistically infectious; however, no success was obtained by tissue wounding. Here, we propose an alternative strategy for the inoculation of cassava with geminiviral clones by agroinoculation, and describe the first successful report of infection of cassava with a geminivirus using Agrobacterium tumefaciens. Successful infection of cassava with full-length genomic clones of SACMV, with appearance of characteristic symptoms, indicated that SACMV is almost certainly the causative agent of cassava mosaic disease in South Africa.
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References |
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Received 10 July 2000;
accepted 10 October 2000.