Identification of a second begomovirus,Sri Lankan cassava mosaic virus,causing cassava mosaic disease in India

Summary. The DNA A and DNA B components of a begomovirus associated with cassava mosaic disease (CMD) originating from Kerala, India, were cloned. Biolistically inoculated clones induced symptoms typical of CMD in cassava. Sequence comparisons showed the virus to be an isolate of Sri Lankan cassava mosaic virus (SLCMV). This is the first time this begomovirus species has been identified in India and only the second species shown to cause CMD in the country. The implication of these findings on our understanding of the diversity and geographic distribution of CMD-associated begomoviruses in the region and on efforts to obtain resistance to CMD are discussed.     

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.     

Asystasia mosaic Madagascar virus: a novel bipartite begomovirus infecting the weed <Emphasis Type="BoldItalic">Asystasia gangetica</Emphasis> in Madagascar

Here, we describe for the first time the complete genome sequence of a new bipartite begomovirus in Madagascar isolated from the weed Asystasia gangetica (Acanthaceae), for which we propose the tentative name asystasia mosaic Madagascar virus (AMMGV). DNA-A and -B nucleotide sequences of AMMGV were only distantly related to known begomovirus sequence and shared highest nucleotide sequence identity of 72.9 % (DNA-A) and 66.9 % (DNA-B) with a recently described bipartite begomovirus infecting Asystasia sp. in West Africa. Phylogenetic analysis demonstrated that this novel virus from Madagascar belongs to a new lineage of Old World bipartite begomoviruses.     

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.     

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.     

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     

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.     

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 of a new alphasatellite associated with a cassava mosaic geminivirus in Madagascar

Two complete nucleotide sequences of an alphasatellite isolated from a cassava plant with mosaic disease symptoms in Madagascar are described and analyzed. While the helper begomovirus was identified as an isolate of East African cassava mosaic Kenya virus (EACMKV), its associated alphasatellite was most closely related (80 % nucleotide sequence identity) to cotton leaf curl Gezira alphasatellite. These satellite molecules have typical features of alphasatellites, with a single gene in the virion sense, an A-rich region and a stem-loop structure. According to the proposed species demarcation threshold of alphasatellites (83 % nucleotide identity), they are isolates of a new species for which we propose the name “Cassava mosaic alphasatellite”.     

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.     

Inhibition of African cassava mosaic virus systemic infection by a movement protein from the related geminivirus tomato golden mosaic virus.

Plant viruses encode proteins that mediate their movement through the host plant leading to the establishment of a systemic infection. We have analyzed the effect of tomato golden mosaic virus (TGMV) genes BL1 and BR1, which are thought to be involved in the process of virus movement, on the infectivity of African cassava mosaic virus (ACMV) in Nicotiana benthamiana. Recombinant genomes were constructed by replacing the ACMV coat protein coding sequence with those of either BL1 or BR1. Replication of recombinants containing BL1 and BR1 coding sequences in the sense orientation with respect to the coat protein promoter was detected in the inoculated leaves only when the constructs were co-inoculated, suggesting that both genes are being expressed and act in a cooperative manner. Co-inoculated recombinants induced localized symptoms on inoculated leaves but did not spread systematically, either because of a defect in BL1 and/or BR1 expression or due to the inability of the TGMV gene products to functionally complement their ACMV counterparts. Systemic spread of ACMV was inhibited when the recombinant containing the BL1 coding sequence in the sense, but not in the antisense, orientation was co-inoculated with ACMV DNA B. Disruption of the BL1 coding sequence by a frameshift mutation restored the ability of the recombinant to spread systemically, suggesting that the gene product is responsible for the inhibitory effect. The inhibitory phenotype was mimicked by a chimera containing amino-terminal sequences of TGMV BL1 and carboxy-terminal sequences of its ACMV homologue, BC1. The chimera has characteristics of a dominant negative mutant. We suggest that dominant negative mutants of virus movement genes may provide a novel source for virus resistance genes.     

Complete genome sequence of Jacquemontia yellow mosaic virus,a novel begomovirus from Venezuela related to other New World bipartite begomoviruses infecting Convolvulaceae

The complete genome of a bipartite begomovirus (genus Begomovirus, family Geminiviridae) infecting Jacquemontia sp. (Convolvulaceae) in Venezuela has been cloned and sequenced. Sequence comparison and phylogenetic analysis have shown that it represents an isolate of a novel species with closest relatives being two New World bipartite begomoviruses that infect Convolvulaceae, Jacquemontia mosaic Yucatan virus and Merremia mosaic virus. The DNA-As of these begomoviruses, however, share only 77.0-78.4 % nucleotide sequence identity with the DNA-A of the isolate described here, for which a recombinant origin is suggested. Based on the symptoms observed in the field, the name Jacquemontia yellow mosaic virus (JacYMV) is proposed for this novel bipartite begomovirus.     

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.     

Stachytarpheta leaf curl virus is a novel monopartite begomovirus species

Summary. Begomovirus isolates were obtained from Stachytarpheta jamaicensis plants showing leaf curl and chlorosis symptoms collected in the Hainan province of China. The complete sequences of isolates Hn5-4, Hn6-1, Hn30 and Hn34 were determined to be 2748, 2751, 2748 and 2748 nucleotides long, respectively. The complete sequences of the four isolates share more than 94.9% nucleotide sequence identity, but all of them have less than 86% nucleotide sequence identity with other reported begomoviruses. The molecular data show that Hn5-4, Hn6-1, Hn30 and Hn34 are isolates of a distinct begomovirus species, for which the name Stachytarpheta leaf curl virus (StaLCV) is proposed. PCR and Southern blot analyses demonstrate that all the collected field samples are not associated with DNAβ or DNA-B components. An infectious clone of StaLCV isolate Hn5-4 was constructed, and could efficiently infect Nicotiana benthamiana, N. tabacum Samsun, N. glutinosa, Lycopersicon esculentum and Petunia hybrida plants, inducing upward leaf roll and vein swelling symptoms. In addition, we illustrate that StaLCV can functionally interact with distinct DNAβ molecules in plants. These authors contributed equally to this work.     

Comparing the regional epidemiology of the cassava mosaic and cassava brown streak virus pandemics in Africa

The rapid geographical expansion of the cassava mosaic disease (CMD) pandemic, caused by cassava mosaic geminiviruses, has devastated cassava crops in 12 countries of East and Central Africa since the late 1980s. Region-level surveys have revealed a continuing pattern of annual spread westward and southward along a contiguous 'front'. More recently, outbreaks of cassava brown streak disease (CBSD) were reported from Uganda and other parts of East Africa that had been hitherto unaffected by the disease. Recent survey data reveal several significant contrasts between the regional epidemiology of these two pandemics: (i) severe CMD radiates out from an initial centre of origin, whilst CBSD seems to be spreading from independent 'hot-spots'; (ii) the severe CMD pandemic has arisen from recombination and synergy between virus species, whilst the CBSD pandemic seems to be a 'new encounter' situation between host and pathogen; (iii) CMD pandemic spread has been tightly linked with the appearance of super-abundant Bemisia tabaci whitefly vector populations, in contrast to CBSD, where outbreaks have occurred 3-12 years after whitefly population increases; (iv) the CMGs causing CMD are transmitted in a persistent manner, whilst the two cassava brown streak viruses appear to be semi-persistently transmitted; and (v) different patterns of symptom expression mean that phytosanitary measures could be implemented easily for CMD but have limited effectiveness, whereas similar measures are difficult to apply for CBSD but are potentially very effective. An important similarity between the pandemics is that the viruses occurring in pandemic-affected areas are also found elsewhere, indicating that contrary to earlier published conclusions, the viruses per se are unlikely to be the key factors driving the two pandemics. A diagrammatic representation illustrates the temporal relationship between B. tabaci abundance and changing incidences of both CMD and CBSD in the Great Lakes region. This emphasizes the pivotal role played by the vector in both pandemics and the urgent need to identify effective and sustainable strategies for controlling whiteflies on cassava.     

Synergism of a DNA and an RNA virus: enhanced tissue infiltration of the begomovirus Abutilon mosaic virus (AbMV) mediated by Cucumber mosaic virus (CMV)

Replication of the begomovirus Abutilon mosaic virus (AbMV) is restricted to phloem nuclei, generating moderate levels of virus DNA. Co-infection with Cucumber mosaic virus (CMV) evidently increased AbMV titers in Nicotiana benthamiana, tobacco, and tomato, resulting in synergistic symptom enhancement. In situ hybridization revealed that in double-infected leaves an increased number of nuclei contained elevated amounts of AbMV. Additionally, the begomoviral phloem-limitation was broken. Whereas CMV 3a movement protein-expressing tobacco plants did not exert any similar influence, the presence of CMV 2b silencing suppressor protein lead to enhanced AbMV titers and numbers of infected vascular cells. The findings prove that AbMV can replicate in nonvascular cells and represent the first report on a true synergism of an RNA/ssDNA virus combination in plants, in which CMV 2b protein plays a role. They indicate considerable consequences of mixed infections between begomo- and cucumoviruses on virus epidemiology and agriculture.     

Soybean chlorotic spot virus, a novel begomovirus infecting soybean in Brazil

A novel soybean-infecting begomovirus from Brazil was identified in Jaíba, in the state of Minas Gerais, and molecularly characterized. By using rolling-circle amplification-based cloning of viral DNAs, three DNA-A variants and a cognate DNA-B were isolated from infected samples. The DNA variants share more than 98 % sequence identity but have less than 89 % identity to other reported begomovirus, the limit for demarcation of new species. In a phylogenetic analysis, both DNA-A and DNA-B clustered with other Brazilian begomoviruses. Infectious cloned DNA-A and DNA-B components induced distinct symptoms in Solanaceae and Fabaceae species by biolistic inoculation. In soybean, the virus induced mild symptoms, i.e., chlorotic spots on the leaves, from which the name soybean chlorotic spot virus (SoCSV) was proposed. The most severe symptoms were displayed by common beans, which exhibited leaf distortion, blistering, interveinal chlorosis, mosaic and golden mosaic. The possibility that SoCSV may become a threat to bean production in Brazil is discussed.