Adaptation of genetically monomorphic bacteria: evolution of copper resistance through multiple horizontal gene transfers of complex and versatile mobile genetic elements.

Copper-based antimicrobial compounds are widely used to control plant bacterial pathogens. Pathogens have adapted in response to this selective pressure. Xanthomonas citri pv. citri, a major citrus pathogen causing Asiatic citrus canker, was first reported to carry plasmid-encoded copper resistance in Argentina. This phenotype was conferred by the copLAB gene system. The emergence of resistant strains has since been reported in Réunion and Martinique. Using microsatellite-based genotyping and copLAB PCR, we demonstrated that the genetic structure of the copper-resistant strains from these three regions was made up of two distant clusters and varied for the detection of copLAB amplicons. In order to investigate this pattern more closely, we sequenced six copper-resistant X. citri pv. citri strains from Argentina, Martinique and Réunion, together with reference copper-resistant Xanthomonas and Stenotrophomonas strains using long-read sequencing technology. Genes involved in copper resistance were found to be strain dependent with the novel identification in X. citri pv. citri of copABCD and a cus heavy metal efflux resistance-nodulation-division system. The genes providing the adaptive trait were part of a mobile genetic element similar to Tn3-like transposons and included in a conjugative plasmid. This indicates the system's great versatility. The mining of all available bacterial genomes suggested that, within the bacterial community, the spread of copper resistance associated with mobile elements and their plasmid environments was primarily restricted to the Xanthomonadaceae family.

Early detection with the ß-LACTA™ test of extended-spectrum ß-lactamase-producing Enterobacteriaceae in blood cultures.

Effective antibiotic therapy is crucial for the outcome of septic patients and requires early diagnosis of ß-lactam resistance in cases of Gram-negative bacteremia. Here, we report high sensitivity of the ß-LACTA™ test in rapid detection of extended-spectrum ß-lactamase-producing Enterobacteriaceae in blood cultures positive for Gram-negative bacilli.

MultiLocus Sequence Analysis- and Amplified Fragment Length Polymorphism-based characterization of xanthomonads associated with bacterial spot of tomato and pepper and their relatedness to Xanthomonas species.

MultiLocus Sequence Analysis (MLSA) and Amplified Fragment Length Polymorphism (AFLP) were used to measure the genetic relatedness of a comprehensive collection of xanthomonads pathogenic to solaneous hosts to Xanthomonas species. The MLSA scheme was based on partial sequences of four housekeeping genes (atpD, dnaK, efp and gyrB). Globally, MLSA data unambiguously identified strains causing bacterial spot of tomato and pepper at the species level and was consistent with AFLP data. Genetic distances derived from both techniques showed a close relatedness of (i) X. euvesicatoria, X. perforans and X. alfalfae and (ii) X. gardneri and X. cynarae. Maximum likelihood tree topologies derived from each gene portion and the concatenated data set for species in the X. campestris 16S rRNA core (i.e. the species cluster comprising all strains causing bacterial spot of tomato and pepper) were not congruent, consistent with the detection of several putative recombination events in our data sets by several recombination search algorithms. One recombinant region in atpD was identified in most strains of X. euvesicatoria including the type strain.

Phylogeny and population structure of brown rot- and Moko disease-causing strains of Ralstonia solanacearum phylotype II.

The ancient soilborne plant vascular pathogen Ralstonia solanacearum has evolved and adapted to cause severe damage in an unusually wide range of plants. In order to better describe and understand these adaptations, strains with very similar lifestyles and host specializations are grouped into ecotypes. We used comparative genomic hybridization (CGH) to investigate three particular ecotypes in the American phylotype II group: (i) brown rot strains from phylotypes IIB-1 and IIB-2, historically known as race 3 biovar 2 and clonal; (ii) new pathogenic variants from phylotype IIB-4NPB that lack pathogenicity for banana but can infect many other plant species; and (iii) Moko disease-causing strains from phylotypes IIB-3, IIB-4, and IIA-6, historically known as race 2, that cause wilt on banana, plantain, and Heliconia spp. We compared the genomes of 72 R. solanacearum strains, mainly from the three major ecotypes of phylotype II, using a newly developed pangenomic microarray to decipher their population structure and gain clues about the epidemiology of these ecotypes. Strain phylogeny and population structure were reconstructed. The results revealed a phylogeographic structure within brown rot strains, allowing us to distinguish European outbreak strains of Andean and African origins. The pangenomic CGH data also demonstrated that Moko ecotype IIB-4 is phylogenetically distinct from the emerging IIB-4NPB strains. These findings improved our understanding of the epidemiology of important ecotypes in phylotype II and will be useful for evolutionary analyses and the development of new DNA-based diagnostic tools.

Differential disease phenotype of begomoviruses associated with tobacco leaf curl disease in Comoros.

In the 2000s, tobacco plantations on the Comoros Islands were afflicted with a previously unobserved tobacco leaf curl disease characterised by symptoms of severe leaf curling and deformation. Previous molecular characterization of potential viral pathogens revealed a complex of African monopartite tobacco leaf curl begomovirus (TbLCVs). Our molecular investigation allowed the characterization of a new monopartite virus involved in the disease: tomato leaf curl Namakely virus (ToLCNamV). Agroinoculation experiments indicated that TbLCVs and tomato leaf curl viruses (ToLCVs) can infect both tomato and tobacco but that infectivity and symptom expression fluctuate depending on the virus and the plant cultivar combination.

Genetic and Pathological Diversity Among Xanthomonas Strains Responsible for Bacterial Spot on Tomato and Pepper in the Southwest Indian Ocean Region.

Bacterial spot of tomato and pepper, a major problem in tropical climates, can be caused by several Xanthomonas genospecies. We examined the genetic and pathological diversity of a collection of 72 strains from the southwest Indian Ocean region as part of a regional research and development program to update inventories of agricultural pests and pathogens. Xanthomonas euvesicatoria, X. perforans, X. gardneri, and X. vesicatoria were identified in our strain collection. The identification of strains at the species level was consistently achieved by amplified fragment length polymorphism (AFLP) and multilocus sequence analysis (MLSA). Overall, X. euvesicatoria was the species recovered prevalently. MLSA data based on four housekeeping genes identified two to three sequence types per genospecies. It suggested that sequence variations primarily consisted of synonymous mutations, although a recombination event spanning several hundred nucleotides was detected for some strains of X. euvesicatoria on the atpD gene coding for the F1-F0-ATPase ß subunit. The pathogenicity of strains was consistent with data found in the literature. Some pathological variations were primarily observed among strains identified as X. euvesicatoria. This study provides the first ever comprehensive description of the status of Xanthomonas species that cause bacterial spot of tomato and pepper in the southwest Indian Ocean region.

Polyphasic characterization of xanthomonads pathogenic to members of the Anacardiaceae and their relatedness to species of Xanthomonas.

We have used amplified fragment length polymorphism (AFLP), multilocus sequence analysis (MLSA) and DNA-DNA hybridization for genotypic classification of Xanthomonas pathovars associated with the plant family Anacardiaceae. AFLP and MLSA results showed congruent phylogenetic relationships of the pathovar mangiferaeindicae (responsible for mango bacterial canker) with strains of Xanthomonas axonopodis subgroup 9.5. This subgroup includes X. axonopodis pv. citri (synonym Xanthomonas citri). Similarly, the pathovar anacardii, which causes cashew bacterial spot in Brazil, was included in X. axonopodis subgroup 9.6 (synonym Xanthomonas fuscans). Based on the thermal stability of DNA reassociation, consistent with the AFLP and MLSA data, the two pathovars share a level of similarity consistent with their being members of the same species. The recent proposal to elevate X. axonopodis pv. citri to species level as X. citri is supported by our data. Therefore, the causal agents of mango bacterial canker and cashew bacterial spot should be classified as pathovars of X. citri, namely X. citri pv. mangiferaeindicae (pathotype strain CFBP 1716) and X. citri pv. anacardii (pathotype strain CFBP 2913), respectively. Xanthomonas fuscans should be considered to be a later heterotypic synonym of Xanthomonas citri.

Widely conserved recombination patterns among single-stranded DNA viruses.

The combinatorial nature of genetic recombination can potentially provide organisms with immediate access to many more positions in sequence space than can be reached by mutation alone. Recombination features particularly prominently in the evolution of a diverse range of viruses. Despite rapid progress having been made in the characterization of discrete recombination events for many species, little is currently known about either gross patterns of recombination across related virus families or the underlying processes that determine genome-wide recombination breakpoint distributions observable in nature. It has been hypothesized that the networks of coevolved molecular interactions that define the epistatic architectures of virus genomes might be damaged by recombination and therefore that selection strongly influences observable recombination patterns. For recombinants to thrive in nature, it is probably important that the portions of their genomes that they have inherited from different parents work well together. Here we describe a comparative analysis of recombination breakpoint distributions within the genomes of diverse single-stranded DNA (ssDNA) virus families. We show that whereas nonrandom breakpoint distributions in ssDNA virus genomes are partially attributable to mechanistic aspects of the recombination process, there is also a significant tendency for recombination breakpoints to fall either outside or on the peripheries of genes. In particular, we found significantly fewer recombination breakpoints within structural protein genes than within other gene types. Collectively, these results imply that natural selection acting against viruses expressing recombinant proteins is a major determinant of nonrandom recombination breakpoint distributions observable in most ssDNA virus families.

Begomovirus 'melting pot' in the south-west Indian Ocean islands: molecular diversity and evolution through recombination.

During the last few decades, many virus species have emerged, often forming dynamic complexes within which viruses share common hosts and rampantly exchange genetic material through recombination. Begomovirus species complexes are common and represent serious agricultural threats. Characterization of species complex diversity has substantially contributed to our understanding of both begomovirus evolution, and the ecological and epidemiological processes involved in the emergence of new viral pathogens. To date, the only extensively studied emergent African begomovirus species complex is that responsible for cassava mosaic disease. Here we present a study of another emerging begomovirus species complex which is associated with serious disease outbreaks in bean, tobacco and tomato on the south-west Indian Ocean (SWIO) islands off the coast of Africa. On the basis of 14 new complete DNA-A sequences, we describe seven new island monopartite begomovirus species, suggesting the presence of an extraordinary diversity of begomovirus in the SWIO islands. Phylogenetic analyses of these sequences reveal a close relationship between monopartite and bipartite African begomoviruses, supporting the hypothesis that either bipartite African begomoviruses have captured B components from other bipartite viruses, or there have been multiple B-component losses amongst SWIO virus progenitors. Moreover, we present evidence that detectable recombination events amongst African, Mediterranean and SWIO begomoviruses, while substantially contributing to their diversity, have not occurred randomly throughout their genomes. We provide the first statistical support for three recombination hot-spots (V1/C3 interface, C1 centre and the entire IR) and two recombination cold-spots (the V2 and the third quarter of V1) in the genomes of begomoviruses.

A multiplex PCR method discriminating between the TYLCV and TYLCV-Mld clades of tomato yellow leaf curl virus.

Tomato yellow leaf curl virus (TYLCV) is one of the causal agents of tomato yellow leaf curl disease (TYLCD) and can cause up to 100% yield losses in tomato fields. As TYLCV continues to spread, many isolates have been described in different parts of the world. Recently two closely related but distinct TYLCV clades, called TYLCV and TYLCV-Mld, have been identified. Isolates from those two clades differ mainly in the nucleotide sequences of their replication associated protein genes but do not display significantly different symptomatology. In order to improve monitoring of the rapidly expanding worldwide TYLCD epidemic, a multiplex polymerase chain reaction assay (mPCR) was developed. A set of three primers were designed to detect and characterize the TYLCV and TYLCV-Mld clade isolates. The specificity and sensitivity of the mPCR were validated on TYLCV infected tomato plants and Bemisia tabaci whiteflies. Being cheap, fast and highly sensitive this new diagnostic tool should greatly simplify efforts to trace the global spread of TYLCV.

Duplication 9q34 syndrome.

Phenotypic, karyotypic, and developmental homology between affected children of carriers of an inverted insertion (9) (q22.1q34.3q34.1) led to recognition of a new chromosome syndrome: dup 9q34. Individuals with dup 9q34 have slight psychomotor retardation, understand simple directions, and acquire a limited vocabulary. In childhood, many are hyperactive. Clinical features include low birth weight, normal birth length, and initial poor feeding and thriving. Musculo-skeletal systems are affected: there are joint contractures, long thin limbs, and striking arachnodactyly. There is abnormal implantation of the thumb, increased space between the first and second fingers, and excess digital creases. Marfan syndrome was a provisional diagnosis for several cases prior to cytogenetic analysis. Cardiovascular and ocular systems are minimally affected, erythema and heart murmurs occur, and ptosis and strabismus are frequent, but lens dislocation is not observed. Features at birth include: dolichocephaly, facial asymmetry, narrow horizontal palpebral fissures, microphthalmia, prominent nasal bridge, small mouth, thin upper lip with down-turned corners, and slight retrognathia. In older children, retrognathia is diminished and the nose becomes long and narrow. The new culture and chromosome banding techniques enable sorting of cases with the distal dup 9q phenotype into two groups. The cases with a longer dup 9q are more likely to develop with life-threatening congenital anomalies. The cases with the shorter dup 9q34 have a less severe long-term prognosis and will benefit, together with their parents, from special education. Female carriers of the inv ins(9) (q22.1q34.3q34.1) have about a 31% risk in each pregnancy to conceive a fetus affected by the dup 9q34 syndrome. A comparable figure is not yet available for male carriers.