Ian D. Bedford

A phylogeographical analysis of the Bemisia tabaci species complex based on mitochondrial DNA markers

Mitochondrial 16S (~550 bp) and cytochrome oxidase I (COI) (~700 bp) sequences were utilized as markers to reconstruct a phylogeography for representative populations or biotypes of Bemisia tabaci. 16S sequences exhibited less divergence than COI sequences. Of the 429 characters examined for COI sequences, 185 sites were invariant, 244 were variable and 108 were informative. COI sequence identities yielded distances ranging from less than 1% to greater than 17%. Whitefly 16S sequences of 456 characters were analysed which consisted of 298 invariant sites, 158 variable sites and 53 informative sites. Phylogenetic analyses conducted by maximum parsimony, maximum‐likelihood and neighbour‐joining methods yielded almost identical phylogenetic reconstructions of trees that separated whiteflies based on geographical origin. The 16S and COI sequence data indicate that the B‐biotype originated in the Old World (Europe, Asia and Africa) and is most closely related to B‐like variants from Israel and Yemen, with the next closest relative being a biotype from Sudan. These data confirm the biochemical, genetic and behavioural polymorphisms described previously for B. tabaci. The consideration of all global variants of B. tabaci as a highly cryptic group of sibling species is argued.

Diversity of DNA β, a satellite molecule associated with some monopartite begomoviruses

DNA beta molecules are symptom-modulating, single-stranded DNA satellites associated with monopartite begomoviruses (family Geminiviridae). Such molecules have thus far been shown to be associated with Ageratum yellow vein virus from Singapore and Cotton leaf curl Multan virus from Pakistan. Here, 26 additional DNA beta molecules, associated with diverse plant species obtained from different geographical locations, were cloned and sequenced. These molecules were shown to be widespread in the Old World, where monopartite begomoviruses are known to occur. Analysis of the sequences revealed a highly conserved organization for DNA beta molecules consisting of a single conserved open reading frame, an adenine-rich region, and a region of high sequence conservation [the satellite conserved region (SCR)]. The SCR contains a potential hairpin structure with the loop sequence TAA/GTATTAC; similar to the origins of replication of geminiviruses and nanoviruses. Two major groups of DNA beta satellites were resolved by phylogenetic analyses. One group originated from hosts within the Malvaceae and the second from a more diverse group of plants within the Solanaceae and Compositae. Within the two clusters, DNA beta molecules showed relatedness based both on host and geographic origin. These findings strongly support coadaptation of DNA beta molecules with their respective helper begomoviruses.

Cotton leaf curl disease is associated with multiple monopartite begomoviruses supported by single DNA β

Summary For bipartite begomoviruses (family Geminiviridae) trans-replication of the DNA B component by the DNA A-encoded replication-associated protein (Rep) is achieved by virtue of a shared sequence, the “common region”, which contains repeated motifs (iterons) which are sequence-specific Rep binding sites and form part of the origin of replication. Recently cotton leaf curl disease (CLCuD), a major constraint to cotton production on the Indian subcontinent, has been shown to be caused by a monopartite begomovirus (Cotton leaf curl Multan virus [CLCuMV]) and a novel single-stranded DNA satellite molecule termed CLCuD DNA β. The satellite molecule is trans-replicated by CLCuMV but does not possess the iteron sequences of this virus. We have investigated the ability of CLCuD DNA β to interact with three further clones of monopartite begomoviruses, isolated from cotton, that have distinct Rep binding specificities. All three cloned viruses were capable of trans-replicating the satellite molecule and inducing CLCuD symptoms in cotton, indicating that the interaction between begomovirus and DNA β is relaxed in comparison to the interaction between DNA A and DNA B components. Field surveys across all the cotton growing regions of Pakistan indicate that dual and multiple infections are the norm for CLCuD with no evidence of synergism. Despite the diversity of begomoviruses associated with CLCuD, only a single class of DNA β has been detected, suggesting that this satellite has the capacity to be recruited by unrelated begomoviruses.

Characterization and distribution of esterase electromorphs in the whitefly,Bemisia tabaci (Genn.) (Homoptera: Aleyrodidae)

Esterase profiles were examined for over 40 populations of the whitefly,Bemisia tabaci, obtained from native and cultivated plant hosts worldwide. Twelve unique electromorphs were identified from distinct populations concentrated largely in Central America, Africa, and India. One electromorph, type B, has recently been proposed as a separate species,Bemisia argentifolii, and has recently spread throughout much of the world. When considered with evidence from mating studies and the ability to induce phytotoxic disorders (squash silverleaf disorder), our data suggest that the single taxonBemisia tabaci may actually represent a species complex.

Biotype status and genetic polymorphism of the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) in Greece: mitochondrial DNA and microsatellites

The genetic polymorphism and the biotype identity of the tobacco whitefly Bemisia tabaci (Gennadius) have been studied in population samples taken from different localities within Greece from cultivated plants growing in greenhouses or in open environments and from non-cultivated plants. Two different approaches were used: sequencing of the mitochondrial cytochrome oxidase I (mtCOI) gene and genotyping using microsatellite markers. Analyses of the mtCOI sequences revealed a high homogeneity between the Greek samples which clustered together with Q biotype samples that had been collected from other countries. When genetic polymorphism was examined using six microsatellite markers, the Greek samples, which were all characterized as Q biotype were significantly differentiated from each other and clustered into at least two distinct genetic populations. Moreover, based on the fixed differences revealed by the mtCOI comparison of known B. tabaci biotype sequences, two diagnostic tests for discriminating between Q and B and non-Q/non-B biotypes were developed. Implementation of these diagnostic tools allowed an absence of the B biotype and presence of the Q biotype in the Greek samples to be determined.

Genome organization of ageratum yellow vein virus, a monopartite whitefly-transmitted geminivirus isolated from a common weed.

A full-length copy of a single genomic component of the whitefly-transmitted geminivirus ageratum yellow vein virus (AYVV) has been cloned from an extract of infected Ageratum conyzoides originating from Singapore. Sequence analysis shows that the genomic component encodes two virion-sense (V1 and V2) and four complementary-sense open reading frames (C1-C4), typical of DNA A of whitefly-transmitted geminiviruses from the Eastern hemisphere. A genomic component equivalent to DNA B was not detected in extracts of infected A. conyzoides. The cloned genomic component produced a systemic infection in Nicotiana benthamiana, Phaseolus vulgaris and Lycopersicon esculentum when introduced into plants by agroinoculation, and symptoms were identical to those produced by wild-type virus introduced into these hosts using viruliferous whiteflies. However, attempts to re-establish a systemic infection in A. conyzoides either by agroinoculation or by whitefly transmission of the cloned progeny were unsuccessful, suggesting that additional factors are required for infection of the natural host. The significance of A. conyzoides as a reservoir host for the economically important geminivirus diseases is discussed.

Clones of cotton leaf curl geminivirus induce symptoms atypical of cotton leaf curl disease.

The causative agent of cotton leaf curl disease has previously been shown to be transmissible by the whitefly Bemisia tabaci (Gennadius) and a begomovirus (Geminiviridae) was shown to be associated with the disease. This virus was provisionally called cotton leaf curl virus (CLCuV) although no causal relationship between virus and disease was shown. In the present study full-length clones of CLCuV, equivalent to the DNA A component of bipartite begomoviruses, were obtained. The clones of CLCuV were systemically infectious to both Nicotiana benthamiana and cotton. Infected plants did not exhibit symptoms characteristic of cotton leaf curl disease, producing mild leaf curling, yellowing and some stunting. Efforts to identify a second genomic component were not successful. These findings suggest that the begomovirus, CLCuV, is not or not the sole cause of cotton leaf curl disease. The transmission of cotton leaf curl disease by B. tabaci, however, may indicate that the begomovirus plays a part in the transmission of the disease. The implications of these findings are discussed.

Efficient whitefly transmission of African cassava mosaic geminivirus requires sequences from both genomic components

Clones of two subgroup III geminiviruses, the common strain of tomato golden mosaic virus (csTGMV) and African cassava mosaic virus originating from Kenya (ACMV-K), were shown to be non-transmissible by whitefiles. Lack of transmissibility of cloned ACMV-K was investigated by exchanging genomic components with a whitefly-transmissible ACMV isolate from Nigeria (ACMV-NOg). Neither pseudorecombinant was transmissible, indicating that defects in both genomic components contributed to the lack of transmissibility. Analysis of the acquisition of the pseudorecombinats by Bemisia tabaci indicated that accumulation of virus within the insect was DNA B dependent. Return of virus to plants was determined by DNA A, although the coat protein was essential for acquisition. Repeated passaging of both the wild strain of ACMV-NOg and the cloned virus led to loss of insect transmissibility of the wild isolate but not the cloned virus. Products encoded on both genomic components are required for transmission of bipartite geminiviruses by B. tabaci.

Genetic Analysis of Bemisia (Hemiptera: Aleyrodidae) Populations by Isoelectric Focusing Electrophoresis

Twenty-one whitefly populations in the genus Bemisia were evaluated for genetic variation at 3 allozyme loci. Nine of the 22 populations that exhibited polymorphic loci were subjected to allozyme analysis using a minimum of 10 enzymes, representing 10 to 14 distinct loci. Among those nine variants examined, calculated genetic distances ranged between 0.03 and 0.52, with three main groups emerging from the analysis. One group comprised two closely related Western Hemisphere variants of B. tabaci: type A from California, United States and a geographically proximal population from Culiacan, Mexico. A second cluster contained five collections previously identified as B. tabaci type B and Bemisia argentifolii, while a third group contained a single population from Benin, Africa. The latter two groups were grouped separately from New World populations and are thought to have a recent origin in the Eastern Hemisphere.

Comparison of a beet curly top virus isolate originating from the old world with those from the new world

The complete nucleotide sequence of an infectious, insect-transmissible clone of a beet curly top virus isolate originating from Iran (BCTV-I) has been determined. The nucleotide sequence of BCTV-I shows high levels of similarity to the sequences of BCTV strains isolated from North America, and is nearly identical to the CFH strain of BCTV. The symptoms produced by BCTV-I in Nicotiana benthamiana and Beta vulgaris most closely resemble those of the CFH strain and are distinct from the other isolates. The significance of these findings with respect to the possible geographic origins and evolution of BCTV are discussed.