Tatsuya Kon

Characterization of a New World Monopartite Begomovirus Causing Leaf Curl Disease of Tomato in Ecuador and Peru Reveals a New Direction in Geminivirus Evolution

ABSTRACT All characterized whitefly-transmitted geminiviruses (begomoviruses) with origins in the New World (NW) have bipartite genomes composed of a DNA-A and DNA-B component. Recently, an NW begomovirus lacking a DNA-B component was associated with tomato leaf curl disease (ToLCD) in Peru, and it was named Tomato leaf deformation virus (ToLDeV). Here, we show that isolates of ToLDeV associated with ToLCD in Ecuador and Peru have a single, genetically diverse genomic DNA that is most closely related to DNA-A components of NW bipartite begomoviruses. Agroinoculation of multimeric clones of the genomic DNA of three ToLDeV genotypes (two variants and a strain) resulted in the development of tomato leaf curl symptoms indistinguishable from those of ToLCD in Ecuador and Peru. Biological properties of these ToLDeV genotypes were similar to those of Old World (OW) monopartite tomato-infecting begomoviruses, including lack of sap transmissibility, phloem limitation, a resistance phenotype in tomato germplasm with the Ty-1 gene, and functional properties of the V1 (capsid protein) and C4 genes. Differences in symptom phenotypes induced by the ToLDeV genotypes in tomato and Nicotiana benthamiana plants were associated with a highly divergent left intergenic region and C4 gene. Together, these results establish that ToLDeV is an emergent NW monopartite begomovirus that is causing ToLCD in Ecuador and Peru. This is the first report of an indigenous NW monopartite begomovirus, and evidence is presented that it emerged from the DNA-A component of a NW bipartite progenitor via convergent evolution and recombination.

Roles and interactions of begomoviruses and satellite DNAs associated with okra leaf curl disease in Mali, West Africa.

Okra leaf curl disease (OLCD) is a major constraint on okra (Abelmoschus esculentus) production in West Africa. Two monopartite begomoviruses (okra virus-1 and okra virus-2), a betasatellite and a DNA1 satellite are associated with OLCD in Mali. Okra virus-1 is an isolate of okra yellow crinkle virus (OYCrV), okra virus-2 is a recombinant isolate of cotton leaf curl Gezira virus (CLCuGV) and the betasatellite is a variant of cotton leaf curl Gezira betasatellite (CLCuGB). Cloned DNA of OYCrV and CLCuGV were infectious and induced leaf curl symptoms in Nicotiana benthamiana plants, but did not induce OLCD in okra. However, when these clones were individually co-inoculated with the cloned CLCuGB DNA, symptom severity and viral DNA levels were increased in N. benthamiana plants and typical OLCD symptoms were induced in okra. The CLCuGB was also replicated by, and increased symptom severity of, three monopartite tomato-infecting begomoviruses, including two from West Africa. The sequence of the DNA1 satellite was highly divergent, indicating that it represents a distinct West African lineage. DNA1 replicated autonomously, and replication required the DNA1-encoded Rep protein. Although DNA1 reduced helper begomovirus DNA levels, symptoms were not attenuated. In the presence of CLCuGB, DNA levels of the helper begomoviruses and DNA1 were substantially increased. Together, these findings establish that OLCD in Mali is caused by a complex of monopartite begomoviruses and a promiscuous betasatellite with an associated parasitic DNA1 satellite. These findings are discussed in terms of the aetiology of OLCD and the evolution of new begomovirus/satellite DNA complexes.

A severe symptom phenotype in tomato in Mali is caused by a reassortant between a novel recombinant begomovirus (Tomato yellow leaf curl Mali virus) and a betasatellite.

Tomato production in West Africa has been severely affected by begomovirus diseases, including yellow leaf curl and a severe symptom phenotype, characterized by extremely stunted and distorted growth and small deformed leaves. Here, a novel recombinant begomovirus from Mali, Tomato yellow leaf curl Mali virus (TYLCMLV), is described that, alone, causes tomato yellow leaf curl disease or, in combination with a betasatellite, causes the severe symptom phenotype. TYLCMLV is an Old World monopartite begomovirus with a hybrid genome composed of sequences from Tomato yellow leaf curl virus-Mild (TYLCV-Mld) and Hollyhock leaf crumple virus (HoLCrV). A TYLCMLV infectious clone induced leaf curl and yellowing in tomato, leaf curl, crumpling and yellowing in Nicotiana benthamiana and common bean, mild symptoms in N. glutinosa, and a symptomless infection in Datura stramonium. In a field-collected sample from a tomato plant showing the severe symptom phenotype in Mali, TYLCMLV was detected together with a betasatellite, identified as Cotton leaf curl Gezira betasatellite (CLCuGB). Tomato plants co-agroinoculated with TYLCMLV and CLCuGB developed severely stunted and distorted growth and small crumpled leaves. These symptoms were more severe than those induced by TYLCMLV alone, and were similar to the severe symptom phenotype observed in the field in Mali and in other West African countries. TYLCMLV and CLCuGB also induced more severe symptoms than TYLCMLV in the other solanaceous hosts, but not in common bean. The increased symptom severity was associated with hyperplasia of phloem-associated cells, but relatively little increase in TYLCMLV DNA levels. In surveys of tomato virus diseases in West Africa, TYLCMLV was commonly detected in plants with leaf curl and yellow leaf curl symptoms, whereas CLCuGB was infrequently detected and always in association with the severe symptom phenotype. Together, these results indicate that TYLCMLV causes tomato yellow leaf curl disease throughout West Africa, whereas TYLCMLV and CLCuGB represent a reassortant that causes the severe symptom phenotype in tomato.

Evidence of local evolution of tomato-infecting begomovirus species in West Africa: characterization of tomato leaf curl Mali virus and tomato yellow leaf crumple virus from Mali.

Tomato yellow leaf curl (TYLC) and tomato leaf curl (ToLC) diseases are serious constraints to tomato production in Mali and other countries in West Africa. In 2003 and 2004, samples of tomato showing virus-like symptoms were collected during a survey of tomato virus diseases in Mali. Three predominant symptom phenotypes were observed: (1) TYLC/ToLC (stunted upright growth and upcurled leaves with interveinal yellowing and vein purpling), (2) yellow leaf crumple and (3) broccoli or bonsai (severe stunting and distorted growth). Squash blot (SB) hybridization with a general begomovirus probe and/or SB/PCR analyses revealed begomovirus infection in plants with each of these symptom phenotypes and no evidence of phytoplasma infection. Sequence analysis of PCR-amplified begomovirus fragments revealed two putative new begomovirus species associated with the TYLC/ToLC and yellow leaf crumple symptom phenotypes, respectively. Full-length clones of these begomoviruses were obtained using PCR and overlapping primers. When introduced into N. benthamiana and tomato plants, these clones induced upward leaf curling and crumpling (the TYLC/ToLC-associated begomovirus) or downward leaf curl/yellow mottle (yellow leaf crumple-associated begomovirus) symptoms. Thus, these begomoviruses were named tomato leaf curl Mali virus (ToLCMLV) and tomato yellow leaf crumple virus (ToYLCrV). The genome organization of both viruses was similar to those of other monopartite begomoviruses. ToLCMLV and ToYLCrV were most closely related to each other and to tobacco leaf curl Zimbabwe virus (TbLCZV-[ZW]) and tomato curly stunt virus from South Africa (ToCSV-ZA). Thus, these likely represent tomato-infecting begomoviruses that evolved from indigenous begomoviruses on the African continent. Mixed infections of ToLCMLV and ToYLCrV in N. benthamiana and tomato plants resulted in more severe symptoms than in plants infected with either virus alone, suggesting a synergistic interaction. Agroinoculation experiments indicated that both viruses induced symptomatic infections in tomato and tobacco, whereas neither virus induced disease symptoms in pepper, common bean, small sugar pumpkin, African eggplant, or Arabidopsis. Virus-specific PCR primers were developed for detection of ToLCMLV and ToYLCrV and will be used to further investigate the distribution and host range of these viruses.

Recovery from Cucurbit leaf crumple virus (Family Geminiviridae, Genus Begomovirus) Infection Is an Adaptive Antiviral Response Associated with Changes in Viral Small RNAs

A strong recovery response occurs in cantaloupe (Cucumis melo) and watermelon (Citrullus lanatus) infected with the bipartite begomovirus Cucurbit leaf crumple virus (CuLCrV). This response is characterized by initially severe symptoms, which gradually become attenuated (almost symptomless). An inverse relationship was detected between viral DNA levels and recovery, indicating that recovered tissues had reduced viral titers. Recovered tissues also were resistant to reinfection with CuLCrV; i.e., recovered leaves reinoculated with the virus did not develop symptoms or have an increased level of viral DNA. In contrast, infection of CuLCrV-recovered leaves with the RNA virus, Cucumber mosaic virus (CMV), disrupted recovery, resulting in the development of severe disease symptoms (more severe than those induced by CMV or CuLCrV alone) and increased CuLCrV DNA levels. Small RNAs with homology to CuLCrV DNA were detected in recovered and nonrecovered tissues; as well as in phloem exudates from infected, but not uninfected plants. Levels of these small RNAs were positively correlated with viral titer; thus, recovered tissues had lower levels than symptomatic tissues. In addition, viral DNA from a host that undergoes strong recovery (watermelon) was more highly methylated compared with that from a host that undergoes limited recovery (zucchini). Furthermore, inoculation of CuLCrV-infected zucchini with a construct expressing an inverted repeat of the CuLCrV common region enhanced recovery and reduced viral symptoms and viral DNA levels in newly emerged leaves. Taken together, these results suggest that recovery from CuLCrV infection is an adaptive antiviral defense mechanism, most likely mediated by gene silencing.

Two genetically related begomoviruses causing tomato leaf curl disease in Togo and Nigeria differ in virulence and host range but do not require a betasatellite for induction of disease symptoms

Tomato leaf curl disease (ToLCD) has emerged as a major constraint on tomato production in some parts of West Africa. In this study, begomoviruses associated with ToLCD in Togo and Nigeria were characterized, as well as a betasatellite associated with the disease in Togo. The genome organization of both viruses is typical of Old World monopartite begomoviruses. Sequence analysis revealed that the begomovirus from Togo is a variant of tomato leaf curl Kumasi virus (ToLCKuV) from Ghana, and it is designated ToLCKuV-[Togo:Pagouda:2006] (ToLCKuV-[TG:Pag:06]). The begomovirus from Nigeria has a recombinant genome, composed of sequences of ToLCKuV (major parent) and a cotton leaf curl Gezira virus (CLCuGV)-like virus, and possesses an unusual non-reiterated replication-associated protein (Rep) binding site. Moreover, because the sequence has <89% identity with those of previously characterized begomoviruses, it is a new species and is designated tomato leaf curl Nigeria virus-[Nigeria:Odogbo:2006] (ToLCNGV-[NG:Odo:06]). The cloned DNAs of ToLCKuV-TG and ToLCNGV were infectious and induced leaf curl symptoms in tomato plants, but ToLCNGV was comparatively more virulent. Both viruses also induced stunted growth and leaf curl symptoms in other solanaceous species (various Nicotiana spp. and Datura stramonium), whereas ToLCNGV but not ToLCKuV-TG induced symptoms in common bean plants. The betasatellite associated with ToLCD in Togo is genetically distinct (i.e., <78% nucleotide sequence identity with previously identified betasatellites) and is designated tomato leaf curl Togo betasatellite-[Togo:Pagouda:2006] (ToLCTGB-[TG:Pag:06]). Replication and systemic spread of ToLCTGB in tomato was mediated by ToLCKuV-TG and ToLCNGV; however, the betasatellite had no effect on disease symptoms induced by either begomovirus. In contrast, ToLCTGB increased symptom severity induced by both viruses in Nicotiana spp. and D. stramonium. Thus, although ToLCTGB increased symptom severity in a host-dependent manner, it does not appear to play a role in ToLCD and may have been present with ToLCKuV-TG as a reassortant.

First report of the complete sequence of Sida golden yellow vein virus from Jamaica.

Begomoviruses are phytopathogens that threaten food security [18]. Sida spp. are ubiquitous weed species found in Jamaica. Sida samples were collected island-wide, DNA was extracted via a modified Dellaporta method, and the viral genome was amplified using degenerate and sequence-specific primers [2, 11]. The amplicons were cloned and sequenced. Sequence analysis revealed that a DNA-A molecule isolated from a plant in Liguanea, St. Andrew, was 90.9% similar to Sida golden yellow vein virus-[United States of America:Homestead:A11], making it a strain of SiGYVV. It was named Sida golden yellow vein virus-[Jamaica:Liguanea 2:2008] (SiGYVV-[JM:Lig2:08]). The cognate DNA-B, previously unreported, was successfully cloned and was most similar to that of Malvastrum yellow mosaic Jamaica virus (MaYMJV). Phylogenetic analysis suggested that this virus was most closely related to begomoviruses that infect malvaceous hosts in Jamaica, Cuba and Florida in the United States.

The molecular characterisation of a Sida-infecting begomovirus from Jamaica

The complete DNA sequence of both genome components of a new begomovirus (Sida golden mosaic Buckup virus-[Jamaica:St. Elizabeth:2004]; SiGMBuV-[JM:SE:04]) was determined from a field-infected Sida sp. sample from Buckup, St. Elizabeth, Jamaica. Phylogenetically, both genome components of SiGMBuV-[JM:SE:04] are most closely related to malvaceous weed-infecting Floridian and Mexican begomoviruses. Its DNA-B is a recombinant molecule, the majority of which was derived from a virus resembling Sida yellow mosaic Yucatan virus-[Mexico:Yucatan:2005] (SiYMYuV-[MX:Yuc:05]), while nucleotides 43-342 were derived from a virus resembling Sida golden mosaic virus-[United States of America:Florida] (SiGMV-[US:Flo]). Symptomatic infectivity of our cloned SiGMBuV-[JM:SE:04] components was confirmed in Nicotiana benthamiana.

Connecting smallholder tomato producers to improved seed in West Africa

AbstractBackgroundEnvironmental heterogeneity, emerging pathogens and limited access to financial and agricultural inputs prevent farmers from producing consistent crop yields in many developing countries. Yield instability impedes establishment of processing and export industries, thereby inhibiting economic development. Modern varieties offer significant increases in yield stability. ResultsA deeply collaborative, multi-national germplasm trialing network was established in West Africa to identify tomato varieties well adapted to each country and mobilize those varieties into local seed distribution networks alongside an integrated pest management program. Research partners in seven West African countries evaluated over 100 tomato varieties for resistance to tomato leaf curl disease (ToLCD). Using biotechnology, the identity and distribution of the key viruses (begomoviruses) causing ToLCD in these countries were identified, and a vector-independent inoculation method (agroinoculation) was developed. The trials identified a set of high-performing varieties with resistance. Agroinoculation with the three prevalent begomoviruses confirmed resistance. ConclusionsThese trial results fulfill the new Economic Community of West African States harmonized seed regulation policy, which requires at least 2 years of national performance trials prior to commercialization of a tomato variety. To compete with a rapidly expanding canned tomato import industry, West African growers need to increase productivity and processing capacity; therefore, we also assessed processing and export trade data for fresh and processed tomatoes from each of the seven countries.

Molecular and Biological Characterization of Distinct Strains of Jatropha mosaic virus from the Dominican Republic Reveal a Potential to Infect Crop Plants

In the Dominican Republic (DO), jatropha plants with yellow mosaic symptoms are commonly observed in and around fields of various crop plants. Complete nucleotide sequences of DNA-A and DNA-B components of four bipartite begomovirus isolates associated with symptomatic jatropha plants collected from three geographical locations in the DO were determined. Sequence comparisons revealed highest identities (91 to 92%) with the DNA-A component of an isolate of Jatropha mosaic virus (JMV) from Jamaica, indicating that the bipartite begomovirus isolates from the DO are strains of JMV. When introduced into jatropha seedlings by particle bombardment, the cloned components of the JMV strains from the DO induced stunting and yellow mosaic, indistinguishable from symptoms observed in the field, thereby fulfilling Kochs postulates for the disease. The JMV strains also induced disease symptoms in Nicotiana benthamiana, tobacco, and several cultivars of common bean from the Andean gene pool, including one locally grown in the DO. Asymmetry in the infectivity and symptomatology of pseudorecombinants provided further support for the strain designation of the JMV isolates from the DO. Thus, JMV in the DO is a complex of genetically distinct strains that have undergone local evolution and have the potential to cause disease in crop plants.