Characterisation of Sri Lankan cassava mosaic virus and Indian cassava mosaic virus: evidence for acquisition of a DNA B component by a monopartite begomovirus

Two bipartite begomoviruses, Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus (SLCMV), have been isolated from mosaic-diseased cassava originating from central India and Sri Lanka, respectively. ICMV was transmitted with low efficiency from cassava to Nicotiana benthamiana by sap inoculation to give leaf curl symptoms. SLCMV was much more virulent in this host, producing severe stunting, leaf curl, and chlorosis. These symptoms were reproduced when their cloned genomic components (DNAs A and B) were introduced into N. benthamiana by either mechanical or Agrobacterium-mediated inoculation (agroinoculation). SLCMV is more closely related to ICMV (DNA A, 84%; DNA B, 94% nucleotide identity) than African cassava mosaic virus (ACMV) (DNA A, 74%; DNA B, 47% nucleotide identity). Sequence comparisons suggest that SLCMV DNA B originated from ICMV DNA B by a recombination event involving the SLCMV DNA A intergenic region. Pseudorecombinants produced by reassortment of the cloned components of ICMV and ACMV were not infectious in N. benthamiana, emphasising their status as distinct virus species. In contrast, a pseudorecombinant between ACMV DNA A and SLCMV DNA B was infectious. Consistent with these observations, iteron motifs located within the intergenic region that may be involved in the initiation of viral DNA replication are conserved between SLCMV and ACMV but not ICMV. When introduced into N. benthamiana by agroinoculation, SLCMV DNA A alone produced a severe upward leaf roll symptom, reminiscent of the phenotype associated with some monopartite begomoviruses. Furthermore, coinoculation of SLCMV DNA A and the satellite DNA beta associated with ageratum yellow vein virus (AYVV) produced severe downward leaf curl in N. glutinosa and yellow vein symptoms in Ageratum conyzoides, resembling the phenotypes associated with AYVV DNA A and DNA beta infection in these hosts. Thus, SLCMV DNA A has biological characteristics of a monopartite begomovirus, and the virus probably evolved by acquisition of a DNA B component from ICMV.     

Both Indian cassava mosaic virus and Sri Lankan cassava mosaic virus are found in India and exhibit high variability as assessed by PCR-RFLP

Summary. The biodiversity of geminiviruses associated with the Cassava Mosaic Disease (CMD) in India was investigated using PCR to specifically amplify the DNA of Indian cassava mosaic virus (ICMV) or Sri Lankan cassava mosaic virus (SLCMV) and also by using PCR to amplify specific viral genes, followed by digestion with different restriction endonucleases to obtain polymorphic patterns (PCR-RFLP). Results showed that both ICMV and SLCMV were present in mosaic-affected cassava; ICMV was geographically restricted to certain regions, whereas SLCMV was widespread. PCR-RFLP analysis showed that, in addition to ICMV-type and SLCMV-type patterns, a high proportion (40%) of the samples displayed novel patterns, some of which were localized in certain areas, whereas others were widely distributed.     

A novel cassava-infecting begomovirus from Madagascar: cassava mosaic Madagascar virus

Cassava mosaic geminiviruses (CMGs) are implicated in cassava mosaic disease (CMD), the main constraint to cassava production in Africa. Here, we report the complete nucleotide sequences of the DNA-A and DNA-B of a newly characterized CMG found infecting cassava in Madagascar, for which we propose the tentative name cassava mosaic Madagascar virus. With the exception of two recombinant regions that resembled a CMG, we determined that the non-recombinant part of the DNA-A component is distantly related to the other CMGs. Whereas the DNA-B component possesses one recombinant region originating from an unidentified virus, the rest of the genome was seen to be closely related to members of the species East African cassava mosaic Zanzibar virus (EACMZV). Phylogenetic analysis based on complete genome sequences demonstrated that DNA-A and DNA-B components are outliers related to the clade of EACMV-like viruses and that DNA-A is related to the monopartite tomato leaf curl begomoviruses described in islands in the south-west Indian Ocean.     

Host range and genetic diversity of croton yellow vein mosaic virus, a weed-infecting monopartite begomovirus causing leaf curl disease in tomato

Croton yellow vein mosaic virus (CYVMV) is a widely occurring begomovirus in Croton bonplandianum, a common weed in the Indian subcontinent. In this study, CYVMV (genus Begomovirus, family Geminiviridae) was transmitted by whiteflies (Bemisia tabaci) to as many as 35 plant species belonging to 11 families, including many vegetables, tobacco varieties, ornamentals and weeds. CYVMV produced bright yellow vein symptoms in croton, whereas in all the other host species, the virus produced leaf curl symptoms. CYVMV produced leaf curl in 13 tobacco species and 22 cultivars of Nicotiana tabacum and resembled tobacco leaf curl virus (TobLCV) in host reactions. However, CYVMV was distinguished from TobLCV in four differential hosts, Ageratum conyzoides, C. bonplandianum, Euphorbia geniculata and Sonchus bracyotis. The complete genome sequences of four isolates originating from northern, eastern and southern India revealed that a single species of DNA-A and a betasatellite, croton yellow vein mosaic betasatellite (CroYVMB) were associated with the yellow vein mosaic disease of croton. The sequence identity among the isolates of CYVMV DNA-A and CroYVMB occurring in diverse plant species was 91.8-97.9 % and 83.3-100 %, respectively. The CYVMV DNA-A and CroYVMB generated through rolling-circle amplification of the cloned DNAs produced typical symptoms of yellow vein mosaic and leaf curling in croton and tomato, respectively. The progeny virus from both the croton and tomato plants was transmitted successfully by B. tabaci. The present study establishes the etiology of yellow vein mosaic disease of C. bonplandianum and provides molecular evidence that a weed-infecting monopartite begomovirus causes leaf curl in tomato.     

Molecular identification of a new begomovirus associated with yellow mosaic disease of Jatropha gossypifolia in India

Yellow mosaic disease was observed on Jatropha gossypifolia plants growing in Kathaupahadi, Madhya Pradesh, India, and whiteflies (Bemisia tabaci) were found in the vicinity. Association of a new begomovirus with yellow mosaic disease of J. gossypifolia has been detected by PCR using begomovirus DNA-A-specific primers. The complete DNA-A genome (~2.7 kb) of this virus isolate was amplified by rolling-circle amplification (RCA) followed by digestion with Bam HI. The ~2.7-kb amplicons was cloned and sequenced, and the data obtained were submitted to GenBank under accession numbers FJ177030. The genome of the virus isolate consisted of six open reading frames (ORFs): V2 (pre-coat protein) and V1 (coat protein) in the virion sense and C3 (REn protein), C2 (TrAP protein), C1 (replication-associated protein) and C4 (C4 protein) in the complementary sense. BLASTn analysis of the nucleotide sequence (2757 nt) of the viral genome (FJ177030) showed 84–85% identity and a distinct phylogenetic relationship with DNA-A of tomato leaf curl virus-Bangalore II (U38239) and tomato leaf curl Karnataka virus (AY754812). Based on its 85% sequence identity to all other begomoviruses known to date and ICTV species demarcating criteria (< 88% identity), the name Jatropha yellow mosaic India virus (JYMIV) is proposed. JYMIV is considered to be monopartite, as neither DNA-B nor DNA-β components associated with begomoviruses were detected.     

Biological and molecular characterization of a begomovirus associated with yellow mosaic vein mosaic disease of pumpkin from Northern India

The biological and molecular properties of Squash leaf curl China virus from Varanasi, India (SLCCNV-IN[IN:Var:Pum]) were characterized. SLCCNV-IN[IN:Var:Pum] could be transmitted by grafting and through whitefly transmission. The complete DNA-A and DNA-B components were amplified through PCR using specific DNA-A and DNA-B primers. The DNA-A of the isolate was comprised of 2,738 nucleotides, encoding typical six open reading frames, and DNA-B of 2,704 nucleotides, encoding two ORFs. Genome organization of the isolate was typical of an old world bipartite begomovirus. Comparisons showed that DNA-A and its intergenic region have the highest sequence identity (97.6 and 97.4%, respectively) with the SLCCNV-IN[IN:Luc:Pum]; (DQ026296). This data suggested that the isolate is a same begomovirus species for which the name Squash leaf curl China virus-India[India:Varanasi:Pumpkin] is proposed. DNA-B showed maximum sequence identity (89.2%) with SLCCNV-IN[IN:Coi:Pum] (AY184488). Phylogenetic analysis of the present isolate showed close relationship to other cucurbit-infecting geminiviruses. This is the first evidence of occurrence of the bipartite Squash leaf curl China virus associated with severe yellow mosaic disease of pumpkin in northern India.     

Multiplex PCR for the detection of African cassava mosaic virus and East African cassava mosaic Cameroon virus in cassava

A multiplex PCR was developed for simultaneous detection of African cassava mosaic virus (ACMV) and East African cassava mosaic Cameroon virus (EACMCV) in cassava affected with cassava mosaic disease (CMD). One set of three primers consisting of an upstream primer common for both viruses and two down stream virus-specific primers were designed for simultaneous amplification of 368 base pair (bp) and 650 bp DNA fragments specific to the replicase gene of ACMV and EACMCV, respectively. Similarly, a second set of three primers were designed for simultaneous amplification of 540 bp and 655 bp fragments specific to the coat protein gene of EACMCV and ACMV, respectively. Primers that can amplify a 171 bp fragment of the large subunit of ribulose bisphosphate carboxylase oxygenase L were included as an internal control in these assays to determine the reliability of multiplex PCR. A simplified, cost-effective and rapid sample preparation method was adapted in place of the conventional plant DNA extraction procedure for multiplex PCR detection of ACMV and EACMCV. The method was validated using CMD-infected cassava samples obtained from farmers' fields in Nigeria. The multiplex PCR is useful for reliable assessment of the prevalence of CMBs in epidemiological studies and for crop improvement and phytosanitary programs in African countries.     

Complete genome sequence of a new begomovirus associated with yellow mosaic disease of Hemidesmus indicus in India

The complete DNA A genome of a virus isolate associated with yellow mosaic disease of a medicinal plant, Hemidesmus indicus, from India was cloned and sequenced. The length of DNA A was 2825 nucleotides, 35 nucleotides longer than the unit genome of monopartite begomoviruses. Comparison of the nucleotide sequence of DNA A of the virus isolate with those of other begomoviruses showed maximum sequence identity of 69 % to DNA A of ageratum yellow vein China virus (AYVCNV; AJ558120) and 68 % with tomato yellow leaf curl virus- LBa4 (TYLCV; EF185318), and it formed a distinct clade in phylogenetic analysis. The genome organization of the present virus isolate was found to be similar to that of Old World monopartite begomoviruses. The genome was considered to be monopartite, because association of DNA B and β satellite DNA components was not detected. Based on its sequence identity (<70 %) to all other begomoviruses known to date and ICTV (International Committee on Taxonomy of Viruses) species demarcating criteria (<89 % identity), it is considered a member of a novel begomovirus species, and the tentative name “Hemidesmus yellow mosaic virus” (HeYMV) is proposed.     

Alternate hosts of African cassava mosaic virus and East African cassava mosaic Cameroon virus in Nigeria

Cassava mosaic disease (CMD) caused by African cassava mosaic virus (ACMV) and East African cassava mosaic Cameroon virus (EACMCV) is the major constraint to cassava production in Nigeria. Sequences of the DNA-A component of ACMV and EACMCV isolates from leguminous plant species (Senna occidentalis, Leucana leucocephala and Glycine max), castor oil plant (Ricinus communis), a weed host (Combretum confertum) and a wild species of cassava (Manihot glaziovii) were determined. All ACMV isolates from these hosts showed 96-98% nucleotide sequence identity with cassava isolates from West Africa. EACMCV was found only in four hosts (S. occidentalis, L. leucocephala, C. confertum, M. glaziovii), and sequences of these isolates showed 96-99% identity with cassava isolates from West Africa. These results provide definitive evidence for the natural occurrence of ACMV and EACMCV in plant species besides cassava.     

Bhendi yellow vein mosaic disease in India is caused by association of a DNA Beta satellite with a begomovirus

Yellow vein mosaic disease is the major limitation in the production of bhendi or okra (Abelmoschus esculentus), an important vegetable crop of India. This disease is caused by a complex consisting of the monopartite begomovirus Bhendi yellow vein mosaic virus (BYVMV, family: Geminiviridae) and a small satellite DNA beta component. BYVMV can systemically infect bhendi upon agroinoculation but produces only mild leaf curling in this host. DNA beta induces typical symptoms of bhendi yellow vein mosaic disease (BYVMD) when co-agroinoculated with the begomovirus to bhendi. The DNA beta component associated with BYVMD has a number of features in common with those reported for ageratum yellow vein disease and cotton leaf curl disease. BYVMV represents a new member of the emerging group of monopartite begomoviruses requiring a satellite component for symptom induction.     

A new strain of Indian cassava mosaic virus causes a mosaic disease in the biodiesel crop Jatropha curcas

Jatropha curcas mosaic disease is a newly emerging disease that challenges the productivity of a prospective biofuel crop, J. curcas. The aetiology of this disease has not been resolved. Here, we report the complete nucleotide sequences of a Jatropha virus isolated from Dharwad, Southern India. Phylogenetic analysis of the virus genome suggests it is a new strain of Indian cassava mosaic virus. Agroinfiltration of the two cloned viral DNA components produced systemic infection and typical mosaic symptoms in J. curcas, thereby fulfilling Koch’s postulates. The availability of infectious clones will provide a valuable tool to screen J. curcas cultivars for disease resistance and facilitate the generation of virus-resistant J. curcas plants by transgenic technology.     

Complete nucleotide sequence of a monopartite begomovirus associated with yellow vein mosaic disease of mesta from north India

Yellow vein mosaic disease of mesta in northern India was found to be associated with a distinct begomovirus species. Except the AC1 gene, this begomovirus isolate shares low sequence identity with the Mesta yellow vein mosaic virus reported to be associated with a similar disease of mesta from eastern India.     

Infectivity of the cloned components of a begomovirus: DNA beta complex causing chilli leaf curl disease in India

The full-length genome of a begomovirus and its cognate DNA-β satellite component associated with chilli leaf curl disease (ChLCD), originating from Varanasi, India, were cloned. Sequence analysis revealed that the viral genome (EF190217) is 2,750 bp and the DNA-β satellite (EF190215) is 1,361 bp in length. Agroinoculation with partial tandem repeats of the viral genome along with the satellite induced symptoms typical of ChLCD in chilli and Nicotiana benthamiana. However, symptom expression was delayed and milder when the viral genome was agroinoculated alone in these hosts. Sequence comparisons revealed that the genome had the highest sequence identity (95%) with that of chilli leaf curl virus-PK[PK:Mul:98]. The DNA-β satellite shared maximum sequence identity (88%) with a DNA-β satellite associated with tomato leaf curl disease from Rajasthan (ToLCBDB-[IN:Raj:03]). These results demonstrate that ChLCD is caused by a complex consisting of the monopartite chilli leaf curl virus and a DNA-β satellite component. This is the first experimental demonstration of Koch’s postulates using cloned DNA molecules associated with chilli leaf curl disease. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The use of biolistic inoculation of cassava mosaic begomoviruses in screening cassava for resistance to cassava mosaic disease

Inoculation of cassava with infectious clones of cassava mosaic geminiviruses (Geminiviridae: Begomovirus) and total DNA extracts from plants infected with well-characterised viruses was evaluated using the Bio-Rad Helios Gene Gun System. Total DNA extracts from infected plants and cloned viruses were produced for coating gold particles and bombardment onto new cassava genotypes, 96/1089A, 96/1039, 96/0160, 96/0304 and three local landraces TME 117, TME 3 and TME 4. Cloned DNA of a Kenyan isolate of the recombinant variant of East African cassava mosaic virus (EACMV-UG2-[Ka]), was only infectious to TME 117 (7/10 plants), 3 weeks post-inoculation with mild infection symptoms in the newly developing leaves. Biolistic inoculation with a chimeric pseudorecombinant virus between DNA A and B components from EACMV-[Ke-Kilifi] and EACMV-UG2-[Ka], respectively, was infectious to TME 117, 96/1039 and 96/0304 and developed very severe and persistent symptoms. TME 3 and TME 4 also developed symptoms, 12 days post-inoculation (d.p.i.). Total DNA extracts of ACMV and EACMV-[Ke-Kilifi] resulted in serious infections with symptoms already evident, 10d.p.i. In general, biolistic inoculation trials with total DNA extracts resulted in a higher number of infected plants expressing symptoms at a much earlier stage (10-12d.p.i.) compared with trials inoculated with virus clones.     

Efficient replication of cloned African cassava mosaic virus in cassava leaf disks

A transient viral replication assay for cloned African cassava mosaic virus (ACMV) was developed using cassava leaf disks. TMS60444 leaf disks were transfected using biolistic-mediated inoculation with ACMV clones pKACMVA and pKACMVB, which originate from West Kenya ACMV isolate 844 (ACMV-KE). Viral DNA synthesized de novo was monitored by Southern hybridization with an AV1 DNA probe. By using the methylation-sensitive restriction enzymes DpnI and MboI, it was possible to distinguish between the input DNA (dam-methylated) and the de novo synthesized viral DNA (not methylated). Different media used for pre- and post-culture of inoculated leaf disks significantly affected the efficiency of viral DNA accumulation. Without pre-culture, replicated viral DNA was not detectable. Culture time in optimized medium also affected the accumulation of nascent viral DNA, and the best results were obtained after 4 days pre-culture on CIM medium followed by 4-6 days post-culture in SH medium. Time-course analysis showed that viral DNA replication can persist for 5-6 days post-inoculation. Our results also confirmed that DNA B of ACMV could assist the accumulation of viral DNA in the leaf disks. The novel protocol described here has also been used successfully with other cassava cultivars (MCol22, MCol1505, TME282 and TMS92/0326) and ACMV clones from the ACMV Nigeria isolate (ACMV-NOg).     

Molecular characterization and interviral relationships of a flexuous filamentous virus causing mosaic disease of sugarcane (Saccharum officinarum L.) in India

Summary.  A virus isolate causing mosaic disease of commercial sugarcane was purified to homogeneity. Electron microscopy revealed flexuous filamentous virus particles of ca 890 × 15 nm. The virus isolate reacted positively with heterologous antiserum to narcissus latent virus form UK, but failed to react with potyvirus group specific antiserum. N-terminal sequencing of the intact coat protein (CP) and the tryptic peptides indicated that the virus was probably a potyvirus but distinct from several reported potyviruses. Comparison of the 3′-terminal 1084 nucleotide sequence of the RNA genome of this virus revealed 93.6% sequence identity in the coat protein coding region with the recently described sugarcane streak mosaic virus (Pakistani isolate). The molecular weight of the coat protein (40 kDa) was higher than that deduced from the amino acid sequence (34 kDa). The apparent increase in size was shown to be due to glycosylation of the coat protein which has not been reported thus far in the family, Potyviridae. This is the first report on the molecular characterization of a virus causing mosaic disease of sugarcane in India and the results demonstrate that the virus is a strain of sugarcane streak mosaic virus, a member of the Tritimovirus genus of the Potyviridae. We have named it sugarcane streak mosaic virus – Andhra Pradesh isolate (SCSMV-AP). Received October 14, 1997 Accepted August 7, 1998     

Biolistic infection of cassava using cloned components of Indian cassava mosaic virus

Summary. Cassava mosaic disease (CMD) is a major constraint to cassava production in Africa and Asia. Of the two begomoviruses associated with CMD on the Indian subcontinent, Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus, only the latter has been successfully reintroduced into cassava to resolve the aetiology of the disease. Here, we report the complete nucleotide sequence of an ICMV isolate from Maharashtra (ICMV-[Mah2]), central India. Biolistic inoculation of the cloned components produced a systemic infection and typical mosaic symptoms in cassava, thereby fulfilling Koch’s postulates. The availability of infectious clones will provide a valuable tool to screen new cassava cultivars for disease resistance under defined conditions.     

Infectivity of African cassava mosaic virus clones to cassava by biolistic inoculation

Summary.  Clones of an African cassava mosaic virus isolate originating from Nigeria (ACMV-NOg) were shown to be infectious to cassava by biolistic inoculation. The production of pseudorecombinants between ACMV-NOg and clones of an ACMV isolate originating from Kenya (ACMV-K) indicated that the lack of infectivity of ACMV-K to cassava was due to defect(s) in the DNA B genomic component; this component encodes two proteins involved in cell-to-cell movement. This is the first demonstration of infectivity of a cloned geminivirus to cassava and conclusively proves that ACMV is the causative agent of cassava mosaic disease. The potential uses of infectious ACMV clones and the means by which to introduce them into cassava are discussed. Received January 18, 1998 Accepted May 27, 1998