Martine Granier

A new silverleaf-inducing biotype Ms of Bemisia tabaci (Hemiptera: Aleyrodidae) indigenous to the islands of the south-west Indian Ocean.

Following the first detection of tomato yellow leaf curl virus (TYLCV) from Reunion (700 km east of Madagascar) in 1997 and the upsurge of Bemisia tabaci (Gennadius) on vegetable crops, two genetic types of B. tabaci were distinguished using RAPD-PCR and cytochrome oxidase I (COI) gene sequence comparisons. One type was assigned to biotype B and the other was genetically dissimilar to the populations described elsewhere and was named Ms, after the Mascarenes Archipelago. This new genetic type forms a distinct group that is sister to two other groups, one to which the B biotype is a member and one to which the Q biotype belongs. The Ms biotype is thought to be indigenous to the region as it was also detected in Mauritius, the Seychelles and Madagascar. Both B and Ms populations of B. tabaci induced silverleaf symptoms on Cucurbita sp., and were able to acquire and transmit TYLCV. Taken together these results indicate that the Ms genetic type should be considered a new biotype of B. tabaci.

Quasispecies nature of three maize streak virus isolates obtained through different modes of selection from a population used to assess response to infection of maize cultivars.

Three maize streak virus (MSV) isolates were derived from an MSV population used to assess the response to infection of maize cultivars. Isolate SP1 was obtained from this population through short acquisition and inoculation periods (1 and 5 min, respectively), using a single Cicadulina mbila vector. Isolate SP2 was derived from SP1 after transmission to a wild perennial host (Coix lacryma-jobi), on which it was maintained for about 4 years without insect transmission. Isolate N2A, the most pathogenic isolate, was obtained from the initial population after serial passages on almost completely resistant inbred maize lines. The complexity of each isolate was analysed by RFLP analysis and sequencing based on 120 SP1 clones, 36 SP2 clones and 40 N2A clones. All three isolates were composed of different but related clones, consistent with a quasispecies structure. The mutations were distributed throughout the genome. Mutation frequencies, based on all available sequences, were 3.8 x 10(-4) for SP1, 10.5 x 10(-4) for SP2 and 6.9 x 10(-4) for N2A. As expected from the bottleneck selection step, the intra-isolate variability of SP1 was relatively low. Comparison between SP1 and SP2 showed that SP1 heterogeneity increased during maintenance on the wild host. Furthermore, the consensus sequences of SP1 and SP2 differed by two non-synonymous substitutions in the complementary sense gene repA. N2A had a relatively low degree of heterogeneity, but was composed of several sub-populations. The results reflect the influence of the mode of selection of MSV isolates on their quasispecies organization, i.e. distribution of variants, and master sequence.

Genetic structure of the invasive pest Bemisia tabaci : evidence of limited but persistent genetic differentiation in glasshouse populations

The geographic range of plant pests can be modified by the use of glasshouses. Bemisia tabaci, originating from warm to hot climates, has been shown to be a complex of distinct genetic groups with very limited gene flow. The genetic structure of this pest was studied in glasshouses in southern France, a region beyond the northern limit of its open-field development area in Europe. Seven microsatellite loci were scored in 22 populations sampled from various regions over 3 years. Two genetic groups were distinguished using a Bayesian clustering method and were assigned to the so-called biotypes B and Q using the gene sequence of cytochrome oxidase 1 (CO1). All but one population corresponded to biotype Q, even though only biotype B was previously reported. Despite the enclosed environment of glasshouses and their expected isolation due to low outdoor survival during the winter, only limited differentiation among biotype Q glasshouses was observed. A single sample site was notable for a decrease in expected heterozygosity and the mean number of alleles over the years. The lack of spatial genetic structure among biotype Q populations was indicative of a recent colonization event combined with large dispersal at all spatial scales. This migration pattern of biotype Q populations was further supported by additional CO1 sequences, since individuals from France, Asia and America exhibited 100% nucleotide identity. The evolution of genetic diversity observed in glasshouses in France is part of the worldwide invasion of biotype Q, which is discussed in light of human activities.

Microsatellites reveal extensive geographical, ecological and genetic contacts between invasive and indigenous whitefly biotypes in an insular environment.

Human-mediated bioinvasions provide the opportunity to study the early stages of contact between formerly allopatric, divergent populations of a species. However, when invasive and resident populations are morphologically similar, it may be very difficult to assess their distribution in the field, as well as the extent of ecological overlap and genetic exchanges between invasive and resident populations. We here illustrate the use of data obtained from a set of eight microsatellite markers together with Bayesian clustering methods to document invasions in a group of major tropical pests, Bemisia tabaci, which comprises several morphologically indistinguishable biotypes with different agronomic impacts. We focus on the island of La Réunion, where an invasive biotype (B) has recently been introduced and now interacts with the resident biotype (Ms). The temporal and spatial distribution, host-plant range and genetic structure of both biotypes are investigated. We showed (i) that, without prior information, clustering methods separate two groups of individuals that can safely be identified as the B and Ms biotypes; (ii) that the B biotype has invaded all regions of the island, and showed no signs of genetic founder effect relative to the Ms biotype; (iii) that the B and Ms biotypes coexist in sympatry throughout most of their geographical ranges, although they tend to segregate into different host plants; and finally (iv) that asymmetrical and locus-specific introgression occurs between the two biotypes when they are in syntopy.

Spatial and Temporal Distribution of Geminiviruses in Leafhoppers of the Genus Cicadulina Monitored by Conventional and Quantitative Polymerase Chain Reaction

ABSTRACT Spatial and temporal distribution of Maize streak virus (MSV, family Geminiviridae, genus Mastrevirus) was monitored in the vector species Cicadulina mbila and the nonvector species C. chinaï using conventional and real-time quantitative polymerase chain reaction. Sustained feeding on MSV-infected plants showed that virus accumulation reaches a maximum in C. chinaï, but not in C. mbila. After a 3-day acquisition access feeding period (AAP), MSV was detected in the gut, the hemolymph, and the head of C. mbila, but only in the gut of C. chinaï. Similarly, Digitaria streak virus (genus Mastrevirus), which is not transmitted by either of the two species, was only detected in the gut. MSV was detected in the hemolymph of C. mbila 3 h after the beginning of the AAP. Although viral DNA progressively decreases in the vector and nonvector species after a 3-day AAP, MSV DNA remained stable in the salivary glands of C. mbila.

Nucleotide sequence evidence for three distinct sugarcane streak mastreviruses.

Summary The complete sequences of four clones of sugarcane streak virus (SSV) isolates from Egypt and one SSV clone from Reunion island were determined. The four Egyptian genomes were highly similar to one another (97–99% nt identity) and were considered as variants of the same virus. The Egyptian SSV was genetically different from all other mastreviruses, the closest virus being SSV from South-Africa (60% nt identity), and defined as a new mastrevirus species named SSEV. The SSV clone from Reunion was highly related to the SSV from Mauritius and SSV from Nigeria, for which only partial sequences were available, indicating that the three sugarcane streak isolates from Mauritius, Reunion and Nigeria were strains of the same virus tentatively named SSMV. This work further confirms that SSMV is a distinct viral species compared to other mastreviruses, including the SSEV (59% nt identity) and SSV (66% nt identity). By comparing two clones from the Mascarene islands, we correlated substitutions in the C-terminal end of the coat protein with a different response to a monoclonal antibody, providing data on the mapping of a specific epitope. Agroinoculations experiments demonstrated that an SSEV clone induced more severe symptoms on maize than two clones from the Mascarene. Inside the African streak virus cluster, the sugarcane mastrevirus isolates were gathered in a sub-cluster of three viruses, SSEV, SSV and SSMV. The diversity of the SSVs is discussed in relation to its host, sugarcane, an imported crop in Africa.

Infectivity and complete nucleotide sequence of the genome of a genetically distinct strain of maize streak virus from Reunion island

SummaryA complete infectious genome of an isolate of maize streak subgroup 1 geminivirus from Reunion Island (MSV-R) was cloned and sequenced. Using anAgrobacterium tumefaciens Ti plasmid delivery system, the cloned 2.7 kb circular DNA was shown to be infectious in maize. The agroinfected virus could be transmitted byCicadulina mbila, the most common vector species of MSV in Reunion. Analysis of open reading frames (ORFs) revealed seven potential coding regions including the 4 ORFs conserved in all geminiviruses infecting monocotyledonous plants, the 2 on the viral “+” strand (MP, CP), and the 2 on the complementary “−” strand (RepA, RepB). The nucleotide sequence of MSV-R was compared to previously determined sequence of three African clones from Nigeria (MSV-N), Kenya (MSV-K), and South Africa (MSV-S). More similarity was found between the African clones (97.0–97.3%) than between these and MSV-R (94.4–95.3%). Nucleotide substitutions were frequent in the large intergenic region, particularly in and around the most likely TATA box for the complementary sense genes, and in the 5′ end of ORF V1. The comparison of the predicted peptide sequences of the proteins encoded by ORFs MP, RepA and RepB confirmed the higher similarity between the African clones (97.8–99.3%) than between these and MSV-R (95.1–97.1%). However the amino acid sequences of the protein encoded by ORF CP (capsid protein) were very conserved among all the 4 clones, suggesting a high selection pressure on this ORF.

A novel cloning strategy for isolating, genotyping and phenotyping genetic variants of geminiviruses

BackgroundViruses of the genus Begomovirus (Geminiviridae) are emerging economically important plant viruses with a circular, single-stranded DNA genome. Previous studies have shown that geminiviruses and RNA viruses exhibit similar mutation frequencies, although geminiviruses are replicated by host DNA polymerases and RNA viruses by their own virus-encoded error-prone RNA-dependent RNA-polymerase. However, the phenotypic effects of naturally occurring mutations have never been extensively investigated in geminiviruses, particularly because, to be infectious, cloned viral genomes usually require sub-cloning as complete or partial tandem repeats into a binary vector from Agrobacterium tumefaciens.ResultsUsing Tomato yellow leaf curl virus (TYLCV), we show here that infectivity can be obtained when only a 41-nucleotide region containing a highly conserved stem-loop is repeated. A binary vector containing this 41-nt region and a unique restriction site was created, allowing direct cloning of infectious monomeric viral genomes provided that they harbour the same restriction site at the corresponding nucleotide position. This experimental system, which can be transferable to other geminiviruses, was validated by analysis of the phenotypic effect of mutations appearing in TYLCV genomes in a single tomato host plant originally inoculated with a unique viral sequence. Fourteen full-length infectious genomes extracted from this plant were directly cloned and sequenced. The mutation frequency was 1.38 × 10-4 mutation per nucleotide sequenced, similar to that found previously for another begomovirus by sequencing PCR-amplified partial sequences. Interestingly, even in this minimal pool of analysed genomes, mutants with altered properties were readily identified, one of them being fitter and reducing plant biomass more drastically than the parental clone.ConclusionThe cloning strategy presented here is useful for any extensive phenotyping of geminivirus variants and particularly of artificially generated mutants or recombinants.

Alfalfa Leaf Curl Virus: an Aphid-Transmitted Geminivirus

ABSTRACT The family Geminiviridae comprises seven genera differentiated by genome organization, sequence similarity, and insect vector. Capulavirus, an eighth genus, has been proposed to accommodate two newly discovered highly divergent geminiviruses that presently have no known vector. Alfalfa leaf curl virus, identified here as a third capulavirus, is shown to be transmitted by Aphis craccivora. This is the first report of an aphid-transmitted geminivirus.

Molecular characterization and prevalence of two capulaviruses: Alfalfa leaf curl virus from France and Euphorbia caput-medusae latent virus from South Africa

Little is known about the prevalence, diversity, evolutionary processes, genomic structures and population dynamics of viruses in the divergent geminivirus lineage known as the capulaviruses. We determined and analyzed full genome sequences of 13 Euphorbia caput-medusae latent virus (EcmLV) and 26 Alfalfa leaf curl virus (ALCV) isolates, and partial genome sequences of 23 EcmLV and 37 ALCV isolates. While EcmLV was asymptomatic in uncultivated southern African Euphorbia caput-medusae, severe alfalfa disease symptoms were associated with ALCV in southern France. The prevalence of both viruses exceeded 10% in their respective hosts. Besides using patterns of detectable negative selection to identify ORFs that are probably functionally expressed, we show that ALCV and EcmLV both display evidence of inter-species recombination and biologically functional genomic secondary structures. Finally, we show that whereas the EcmLV populations likely experience restricted geographical dispersion, ALCV is probably freely moving across the French Mediterranean region.