Life history parameters and predation capacities of Nesidiocoris volucer: a new biological control agent for tomato crop.

Whiteflies are one of the major pests of tomato under greenhouses, and their control partly relies on biocontrol strategies. Among those biocontrol agents, parasitoids or predators are widely used. However, the introduction of a biocontrol agent in a new area is not trivial. For that reason, we investigated the use of a tropical native mirid, Nesidiocoris volucer (Hemiptera: Miridae), for the biological control of whiteflies among other insect pests on tomato crops under greenhouses in the subtropical island of La Réunion, France. Nesidiocoris volucer life history traits and plant injury were examined. Nymphs developed and survived between 15 and 30°C and required on average 49.41 days at 15°C and on average 10.50 days at 30°C to develop (nymph survival >94%). At 25°C, each female produced on average 65 eggs. Nesidiocoris volucer was able to feed on several prey species, but performed better on whiteflies than on spider mites or thrips. No N. volucer feeding injury was observed on tomato. Nesidiocoris volucer has also been found in tropical countries of Africa, and we believe that the data presented on this natural enemy could be of great importance for the biocontrol of whiteflies in tropical areas.

Genome Sequence Resource of 'Candidatus Liberibacter asiaticus' from Diaphorina citri Kuwayama (Hemiptera: Liviidae) from La Réunion.

'Candidatus Liberibacter asiaticus' is an insect-transmitted, phloem-restricted α-proteobacterium associated with huanglongbing. Here, we provide the whole genome sequence of 'Ca. L. asiaticus' strain ReuSP1 from its insect vector Diaphorina citri (Hemiptera: Liviidae) collected in La Réunion. The genome is composed of 1,230,064 bp and has a 36.5% G+C content. This study reports the first 'Ca. L. asiaticus' genome sequence from La Réunion, which will add to 'Ca. L. asiaticus' genome resources and help elucidate our understanding of the introduction pathway into La Réunion.

Great Genetic Diversity but High Selfing Rates and Short-Distance Gene Flow Characterize Populations of a Tree (Foetidia; Lecythidaceae) in the Fragmented Tropical Dry Forest of the Mascarene Islands.

Following the global trend of deforestation and degradation, tropical dry forests in the Mascarenes archipelago on Reunion has undergone harsh reduction and fragmentation within 3 centuries of human occupation. We investigated the genetic diversity, mating system, and gene flow in fragmented populations of the native tree Foetidia mauritiana (Lecythidaceae) on Reunion, using microsatellite genotyping of adults (in- and ex situ) and seed progenies (in situ only). To test genetic isolation between the Mascarene islands, we also genotyped conspecific adults on Mauritius, and trees of Foetidia rodriguesiana on Rodrigues. We found a high genetic diversity among the trees on Reunion, but no population structure (G'ST: 0.039-0.090), and an increase of the fixation index (FIS) from adults to progenies. A subsequent analysis of mating systems from progeny arrays revealed selfing rates >50% in fragmented populations and close to 100% in lone trees. A paternity analysis revealed pollen flow ranging from 15.6 to 296.1 m within fragments. At broader scale, the populations of F. mauritiana on Reunion and Mauritius are genetically differentiated. The morphologically allied taxa F. rodriguesiana and F. mauritiana are clearly isolated. Therefore, this case study shows that genetic diversity may persist after deforestation, especially in long-lived tree species, but the reproductive features may be deeply altered during this process. This would explain the low seed production and the absence of recruitment in F. mauritiana. Restoration programs should take into account these features, as well as the importance that trees ex situ represent in restoring and conserving diversity.

Large-scale mitochondrial DNA analysis of native honey bee Apis mellifera populations reveals a new African subgroup private to the South West Indian Ocean islands.

BACKGROUND: The South West Indian Ocean (SWIO) archipelagos and Madagascar constitute a hotspot of biodiversity with a high rate of endemism. In this area, the endemic subspecies A. m. unicolor has been described in Madagascar. It belongs to the African lineage, one of the four described evolutionary lineages in honey bees. Despite a long beekeeping tradition and several recorded European introductions, few studies have been carried out on the diversity and proportion of honey bee subspecies. In order to identify and define which evolutionary lineages and potential sub-lineages are present in the SWIO, the COI-COII intergenic region and the ND2 gene of the mtDNA were sequenced in honey bee colonies from three archipelagos. An extensive sampling (n = 1184 colonies) was done in the Mascarene (La Réunion, Mauritius, Rodrigues), Seychelles (Mahé, Praslin, La Digue) and Comoros (Grande Comore, Mohéli, Anjouan, Mayotte) archipelagos. Islands genetic diversity was compared to newly sampled populations from Madagascar, continental African and European populations. RESULTS: African lineage haplotypes were found in all islands (except for Rodrigues). Madagascar, Comoros and Seychelles had 100% of A lineage, 95.5% in La Réunion and 56.1% in Mauritius. Among all African colonies detected in the SWIO, 98.1% (n = 633) of COI-COII haplotypes described the presence of the subspecies A. M. unicolor. Both genetic markers revealed i) a new private AI mitochondrial group shared by the SWIO archipelagos and Madagascar distant from continental populations; ii) the private African haplotypes for each island suggested diversity radiation in the archipelagos; iii) the detection of the Comoros archipelago as a possible contact area between insular and continental African populations. The exotic European C and M lineages were only detected in the Mascarene archipelago, but striking differences of proportion were observed among islands. Merely 4.6% of European colonies were found in La Réunion whereas Mauritius cumulated 44%. Here, among the 84 observed COI-COII haplotypes, 50 were newly described including 13 which were private to the SWIO archipelagos and Madagascar. Similarly, 24 of the 34 found ND2 haplotypes were novel which included six haplotypes particular to the SWIO populations. CONCLUSION: A new African subgroup was described in the SWIO region with mitochondrial genetic evidence that A. m. unicolor is the indigenous subspecies of the archipelagos surrounding Madagascar.

Divergent evolutionary and epidemiological dynamics of cassava mosaic geminiviruses in Madagascar.

BACKGROUND: Cassava mosaic disease (CMD) in Madagascar is caused by a complex of at least six African cassava mosaic geminivirus (CMG) species. This provides a rare opportunity for a comparative study of the evolutionary and epidemiological dynamics of distinct pathogenic crop-infecting viral species that coexist within the same environment. The genetic and spatial structure of CMG populations in Madagascar was studied and Bayesian phylogeographic modelling was applied to infer the origins of Madagascan CMG populations within the epidemiological context of related populations situated on mainland Africa and other south western Indian Ocean (SWIO) islands. RESULTS: The isolation and analysis of 279 DNA-A and 117 DNA-B sequences revealed the presence in Madagascar of four prevalent CMG species (South African cassava mosaic virus, SACMV; African cassava mosaic virus, ACMV; East African cassava mosaic Kenya virus, EACMKV; and East African cassava mosaic Cameroon virus, EACMCV), and of numerous CMG recombinants that have, to date, only ever been detected on this island. SACMV and ACMV, the two most prevalent viruses, displayed low degrees of genetic diversity and have most likely been introduced to the island only once. By contrast, EACMV-like CMG populations (consisting of East African cassava mosaic virus, EAMCKV, EACMCV and complex recombinants of these) were more diverse, more spatially structured, and displayed evidence of at least three independent introductions from mainland Africa. Although there were no statistically supported virus movement events between Madagascar and the other SWIO islands, at least one mainland African ACMV variant likely originated in Madagascar. CONCLUSIONS: Our study highlights both the complexity of CMD in Madagascar, and the distinct evolutionary and spatial dynamics of the different viral species that collectively are associated with this disease. Given that more distinct CMG species and recombinants have been found in Madagascar than any other similarly sized region of the world, the risks of recombinant CMG variants emerging on this island are likely to be higher than elsewhere. Evidence of an epidemiological link between Madagascan and mainland African CMGs suggests that the consequences of such emergence events could reach far beyond the shores of this island.

Asystasia mosaic Madagascar virus: a novel bipartite begomovirus infecting the weed Asystasia gangetica 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.

Description and phylogenetic placement of Beauveria hoplocheli sp. nov. used in the biological control of the sugarcane white grub, Hoplochelus marginalis, on Reunion Island.

On Reunion Island successful biological control of the sugarcane white grub Hoplochelus marginalis Fairmaire (Coleoptera: Melolonthidae) has been conducted for decades with strains from the entomopathogenic fungal genus Beauveria (Ascomycota: Hypocreales). A study based on morphological characters combined with a multisequence phylogenetic analysis of genes that encode the translation elongation factor 1-alpha (TEF1), RNA polymerase II largest subunit (RPB1), RNA polymerase II second largest subunit (RPB2) and the Bloc nuc intergenic region was carried out on Beauveria strains isolated on Reunion and Madagascar from H. marginalis. This study revealed that these strains, previously identified as Beauveria brongniartii, did not match that species and are closely related to but still distinct from B. malawiensis strains. Therefore we describe the Reunion Island fungus as the new species B. hoplocheli.

Frequency-dependent assistance as a way out of competitive exclusion between two strains of an emerging virus.

Biological invasions are the main causes of emerging viral diseases and they favour the co-occurrence of multiple species or strains in the same environment. Depending on the nature of the interaction, co-occurrence can lead to competitive exclusion or coexistence. The successive fortuitous introductions of two strains of Tomato yellow leaf curl virus (TYLCV-Mld and TYLCV-IL) in Réunion Island provided an ideal opportunity to study the invasion of, and competition between, these worldwide emerging pathogens. During a 7-year field survey, we observed a displacement of the resident TYLCV-Mld by the newcomer TYLCV-IL, with TYLCV-Mld remaining mostly in co-infected plants. To understand the factors associated with this partial displacement, biological traits related to fitness were measured. The better ecological aptitude of TYLCV-IL in single infections was demonstrated, which explains its rapid spread. However, we demonstrate that the relative fitness of virus strains can drastically change between single infections and co-infections. An epidemiological model parametrized with our experimental data predicts that the two strains will coexist in the long run through assistance by the fitter strain. This rare case of unilateral facilitation between two pathogens leads to frequency-dependent selection and maintenance of the less fit strain.

Complex recombination patterns arising during geminivirus coinfections preserve and demarcate biologically important intra-genome interaction networks.

Genetic recombination is an important process during the evolution of many virus species and occurs particularly frequently amongst begomoviruses in the single stranded DNA virus family, Geminiviridae. As in many other recombining viruses it is apparent that non-random recombination breakpoint distributions observable within begomovirus genomes sampled from nature are the product of variations both in basal recombination rates across genomes and in the over-all viability of different recombinant genomes. Whereas factors influencing basal recombination rates might include local degrees of sequence similarity between recombining genomes, nucleic acid secondary structures and genomic sensitivity to nuclease attack or breakage, the viability of recombinant genomes could be influenced by the degree to which their co-evolved protein-protein and protein-nucleotide and nucleotide-nucleotide interactions are disreputable by recombination. Here we investigate patterns of recombination that occur over 120 day long experimental infections of tomato plants with the begomoviruses Tomato yellow leaf curl virus and Tomato leaf curl Comoros virus. We show that patterns of sequence exchange between these viruses can be extraordinarily complex and present clear evidence that factors such as local degrees of sequence similarity but not genomic secondary structure strongly influence where recombination breakpoints occur. It is also apparent from our experiment that over-all patterns of recombination are strongly influenced by selection against individual recombinants displaying disrupted intra-genomic interactions such as those required for proper protein and nucleic acid folding. Crucially, we find that selection favoring the preservation of co-evolved longer-range protein-protein and protein DNA interactions is so strong that its imprint can even be used to identify the exact sequence tracts involved in these interactions.

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".

Spread and strain determination of Varroa destructor (Acari: Varroidae) in Madagascar since its first report in 2010.

Varroa destructor is a major pest in world beekeeping. It was first detected in Madagascar in 2010 on the endemic honeybee Apis mellifera unicolor. To evaluate V. destructor spread dynamics in Madagascar a global survey was conducted in 2011-2012. A total of 695 colonies from 30 districts were inspected for the presence of mites. 2 years after its introduction, nine districts were found infested. Varroa destructor spread was relatively slow compared to other countries with a maximum progression of 40 km per year, the five newly infested districts being located next to the first infested ones. The incidence of mite infestation was also investigated by monitoring 73 colonies from five apiaries during 1 year (2011-2012). Sixty percent of local colony mortality was recorded after 1 year of survey. Varroa destructor strain determination was done by partial sequencing of the cytochrome oxidase I gene of 13 phoretic mites sampled in five districts. A single V. destructor mitochondrial haplotype was detected, the Korean type, also present in the closest African countries. A global pathogen survey was also conducted on the colonies inspected for mite presence. The greater wax moth, Galleria mellonella has been found in all colonies all over the country. Two other pathogens and morphological abnormalities in workers, such as deformed wings, were found associated with only V. destructor presence. A prevention management plan must be implemented to delimit mite spread across the island.

Triple electroantennography captures the range and spatial arrangement of olfactory sensory neuron response on an insect antenna.

BACKGROUND: Electroantennography (EAG) is a basic neuroscientific tool that is widely used to measure olfactory responses in insects. It is particularly adapted to probing the olfactory systems of non-model insect species in chemical ecology and evolutionary biology. As currently practiced, EAG measures have varying degrees of correlation with olfactory responses, especially for insects whose olfactory sensory neurons (OSNs) are arranged in zones on the antennae. This limitation was shown to be partly due to the fact that only a single antennal position was recorded. NEW METHODS: We describe a setup using triple electroantennography (EAG3), whereby three antennal positions are recorded simultaneously. The spatial arrangement of the electrodes ensures the mechanical stability of the assembly. The EAG3 detector was coupled to a gas chromatograph (GC-EAD3), customized using a Dean's switch to improve the EAG signals by chopper modulation. EAG3 signals were analysed through a current point model to estimate olfactory responses across the antenna. RESULTS: Recordings were performed on Tephritidae and Drosophila species, which have antennae of different shapes and sizes. We confirmed that the spatio-temporal pattern of antennal activation was stimulus dependent and allowed us to quantify the antennal olfactory response. COMPARISONS WITH EXISTING METHOD: Compared to typical single-probe EAG, we show that EAG3 improves response quantification and increases the range of compounds for which a sensory response is detected. CONCLUSIONS: Our EAG3 setup is an original low-cost and easy-to-use method. It offers a useful bridge between comprehensive neurophysiological investigations and the broader themes explored in chemical ecology.

Bacterial plant pathogens affect the locomotor behavior of the insect vector: a case study of Citrus volkameriana-Triozae erytreae-Candidatus Liberibacter asiaticus system.

Plant pathogens can alter the behavior of their insect vectors as well as their survival and reproduction. The African psyllid, Trioza erytreae, is one of the vectors of Huanglongbing, a citrus disease caused mainly by "Candidatus Liberibacter asiaticus" (CLas). The purpose of this study was to characterize the effects of CLas on the psyllid, T. erytreae using Citrus volkamerina plants as the study system. The study focused more specifically on the CLas effects prior to and after its acquisition by the psyllid T. erytreae. Our results did not support the hypothesis that CLas effects psyllid probing behavior prior to acquisition; few differences were observed between uninfected T. erytrea feeding on CLas-infected versus control plants. On the other hand, compared to psyllids that had completed their development on control plants, the ones that had completed their development on a CLas-infected plant exhibited changes in their behavior (greater velocity), physiology (smaller mass) and biochemistry (lower water and lipid content). Altogether, our results confirm the existence of a marked postacquisition effect on the vector locomotor behavior and a minor preacquisition effect of CLas on the vector behavior, which can be partially explained by physiological and biochemical changes.

East African cassava mosaic-like viruses from Africa to Indian ocean islands: molecular diversity, evolutionary history and geographical dissemination of a bipartite begomovirus.

BACKGROUND: Cassava (Manihot esculenta) is a major food source for over 200 million sub-Saharan Africans. Unfortunately, its cultivation is severely hampered by cassava mosaic disease (CMD). Caused by a complex of bipartite cassava mosaic geminiviruses (CMG) species (Family: Geminivirideae; Genus: Begomovirus) CMD has been widely described throughout Africa and it is apparent that CMG's are expanding their geographical distribution. Determining where and when CMG movements have occurred could help curtail its spread and reveal the ecological and anthropic factors associated with similar viral invasions. We applied Bayesian phylogeographic inference and recombination analyses to available and newly described CMG sequences to reconstruct a plausible history of CMG diversification and migration between Africa and South West Indian Ocean (SWIO) islands. RESULTS: The isolation and analysis of 114 DNA-A and 41 DNA-B sequences demonstrated the presence of three CMG species circulating in the Comoros and Seychelles archipelagos (East African cassava mosaic virus, EACMV; East African cassava mosaic Kenya virus, EACMKV; and East African cassava mosaic Cameroon virus, EACMCV). Phylogeographic analyses suggest that CMG's presence on these SWIO islands is probably the result of at least four independent introduction events from mainland Africa occurring between 1988 and 2009. Amongst the islands of the Comoros archipelago, two major migration pathways were inferred: One from Grande Comore to Mohéli and the second from Mayotte to Anjouan. While only two recombination events characteristic of SWIO islands isolates were identified, numerous re-assortments events were detected between EACMV and EACMKV, which seem to almost freely interchange their genome components. CONCLUSIONS: Rapid and extensive virus spread within the SWIO islands was demonstrated for three CMG complex species. Strong evolutionary or ecological interaction between CMG species may explain both their propensity to exchange components and the absence of recombination with non-CMG begomoviruses. Our results suggest an important role of anthropic factors in CMGs spread as the principal axes of viral migration correspond with major routes of human movement and commercial trade. Finer-scale temporal analyses of CMGs to precisely scale the relative contributions of human and insect transmission to their movement dynamics will require further extensive sampling in the SWIO region.

Evidence of diversity and recombination in Arsenophonus symbionts of the Bemisia tabaci species complex.

BACKGROUND: Maternally inherited bacterial symbionts infecting arthropods have major implications on host ecology and evolution. Among them, the genus Arsenophonus is particularly characterized by a large host spectrum and a wide range of symbiotic relationships (from mutualism to parasitism), making it a good model to study the evolution of host-symbiont associations. However, few data are available on the diversity and distribution of Arsenophonus within host lineages. Here, we propose a survey on Arsenophonus diversity in whitefly species (Hemiptera), in particular the Bemisia tabaci species complex. This polyphagous insect pest is composed of genetic groups that differ in many ecological aspects. They harbor specific bacterial communities, among them several lineages of Arsenophonus, enabling a study of the evolutionary history of these bacteria at a fine host taxonomic level, in association to host geographical range and ecology. RESULTS: Among 152 individuals, our analysis identified 19 allelic profiles and 6 phylogenetic groups, demonstrating this bacterium's high diversity. These groups, based on Arsenophonus phylogeny, correlated with B. tabaci genetic groups with two exceptions reflecting horizontal transfers. None of three genes analyzed provided evidence of intragenic recombination, but intergenic recombination events were detected. A mutation inducing a STOP codon on one gene in a strain infecting one B. tabaci genetic group was also found. Phylogenetic analyses of the three concatenated loci revealed the existence of two clades of Arsenophonus. One, composed of strains found in other Hemiptera, could be the ancestral clade in whiteflies. The other, which regroups strains found in Hymenoptera and Diptera, may have been acquired more recently by whiteflies through lateral transfers. CONCLUSIONS: This analysis of the genus Arsenophonus revealed a diversity within the B. tabaci species complex which resembles that reported on the larger scale of insect taxonomy. We also provide evidence for recombination events within the Arsenophonus genome and horizontal transmission of strains among insect taxa. This work provides further insight into the evolution of the Arsenophonus genome, the infection dynamics of this bacterium and its influence on its insect host's ecology.

Evolution of African cassava mosaic virus by recombination between bipartite and monopartite begomoviruses.

BACKGROUND: Cassava mosaic disease (CMD) is a major constraint on cassava cultivation in Africa. The disease is endemic and is caused by seven distinct cassava mosaic geminiviruses (CMGs), some of them including several variants. FINDINGS: From cassava leaf samples presenting CMD symptoms collected in Burkina Faso, four DNA-A begomovirus components were cloned and sequenced, showing 99.9% nucleotide identity among them. These isolates are most closely related to African cassava mosaic virus (ACMV) but share less than 89% nucleotide identity (taxonomic threshold) with any previously described begomovirus. A DNA-B genomic component, sharing 93% nucleotide identity with DNA-B of ACMV, was also characterized. Since all genomic components have a typical genome organization of Old World bipartite begomoviruses, this new species was provisionally named African cassava mosaic Burkina Faso virus (ACMBFV). Recombination analysis of the new virus demonstrated an interspecies recombinant origin, with major parents related to West African isolates of ACMV, and minor parents related to Tomato leaf curl Cameroon virus and Cotton leaf curl Gezira virus. CONCLUSION: This is the first report of an ACMV-like recombinant begomovirus arisen by interspecific recombination between bipartite and monopartite African begomoviruses.

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.

The African citrus psyllid Trioza erytreae: An efficient vector of Candidatus Liberibacter asiaticus.

Introduction: Huanglonbing (HLB) is the most serious disease of citrus in the world, associated with three non-cultivable phloem-restricted bacteria Candidatus Liberibacter asiaticus (CLas), Ca L. africanus (CLaf) and Ca L. americanus (CLam). CLas is transmitted by the Asian citrus psyllid Diaphorina citri, and has spread to several countries. The African psyllid Trioza erytreae, the vector of CLaf occurs in Africa and neighbouring islands. Only two major citrus-growing regions - Australia/New Zealand and the Mediterranean Basin - are still HLB-free in the world. However, T. erytreae has recently been introduced into continental Europe (Portugal and Spain) and has become a potential threat to citrus production. The transmission of CLas by T. erytreae had been postulated but never tested. To evaluate the risk of T. erytreae transmitting CLas, comparative transmissions of CLas by T. erytreae and D. citri were assessed. Methods: Transmission tests were performed on excised leaves and seedlings of Citrus volkameriana with different inoculation access periods (in series) for both insect species. Quantifications of bacterial titers were made in excised leaves, seedlings three and six months after inoculation and on individual insects. Results: Our results showed that T. erytreae was able to efficiently acquire CLas. Furthermore, T. erytreae carried significantly higher bacterial titers than D. citri, and was able to efficiently transmit the bacteria to seedlings at a similar rate that D. citri highlighting the high risk of spread of the most aggressive variant of HLB (CLas) by T. erytreae in Europe. Discussion: Thus, extreme precautions to prevent any entry of CLas into Europe should be adopted.

A novel synthetic quantification standard including virus and internal report targets: application for the detection and quantification of emerging begomoviruses on tomato.

BACKGROUND: Begomovirus is a genus of phytopathogenic single-stranded DNA viruses, transmitted by the whitefly Bemisia tabaci. This genus includes emerging and economically significant viruses such as those associated with Tomato Yellow Leaf Curl Disease, for which diagnostic tools are needed to prevent dispersion and new introductions. Five real-time PCRs with an internal tomato reporter gene were developed for accurate detection and quantification of monopartite begomoviruses, including two strains of the Tomato yellow leaf curl virus (TYLCV; Mld and IL strains), the Tomato leaf curl Comoros virus-like viruses (ToLCKMV-like viruses) and the two molecules of the bipartite Potato yellow mosaic virus. These diagnostic tools have a unique standard quantification, comprising the targeted viral and internal report amplicons. These duplex real-time PCRs were applied to artificially inoculated plants to monitor and compare their viral development. RESULTS: Real-time PCRs were optimized for accurate detection and quantification over a range of 2 × 10(9) to 2 × 10(3) copies of genomic viral DNA/µL for TYLCV-Mld, TYLCV-IL and PYMV-B and 2 × 10(8) to 2 × 10(3) copies of genomic viral DNA/µL for PYMV-A and ToLCKMV-like viruses. These real-time PCRs were applied to artificially inoculated plants and viral loads were compared at 10, 20 and 30 days post-inoculation. Different patterns of viral accumulation were observed between the bipartite and the monopartite begomoviruses. Interestingly, PYMV accumulated more viral DNA at each date for both genomic components compared to all the monopartite viruses. Also, PYMV reached its highest viral load at 10 dpi contrary to the other viruses (20 dpi). The accumulation kinetics of the two strains of emergent TYLCV differed from the ToLCKMV-like viruses in the higher quantities of viral DNA produced in the early phase of the infection and in the shorter time to reach this peak viral load. CONCLUSIONS: To detect and quantify a wide range of begomoviruses, five duplex real-time PCRs were developed in association with a novel strategy for the quantification standard. These assays should be of a great interest for breeding programs and epidemiological surveys to monitor viral populations.

Molecular and biological characterization of pepper yellow vein Mali virus (PepYVMV) isolates associated with pepper yellow vein disease in Burkina Faso.

Yellow vein disease (YVD) is a major problem in pepper in West Africa. Despite the recent implication of a begomovirus in YVD in Mali and in Burkina Faso, the aetiology of the disease remains unclear. Using symptomatic samples from the main vegetable cultivation regions in Burkina Faso, 10 full-length DNA-A-like begomovirus sequences were obtained, each showing 98% nucleotide identity to pepper yellow vein Mali virus (PepYVMV). The host range was determined after construction of a viral clone for agroinfection. Severe symptoms developed in tomato and Nicotiana benthamiana. By contrast, no symptoms developed in either commercial or local pepper cultivars, demonstrating that the aetiology of YVD is not only associated with the presence of PepYVMV.