Population structure and recombination in environmental isolates of Legionella pneumophila

Legionella pneumophila is a water-borne bacteria responsible for most cases of legionellosis, an emerging disease with an increasing incidence in industrialized countries. Although early analysis based on multilocus enzyme electrophoresis (MLEE) described the population structure of this species as clonal, more recent reports have suggested that recombination also contributes to shaping variation across its genome. We report here the results of analysing the nucleotide sequences of 19 loci in 31 environmental samples of L. pneumophila from a small Spanish region (near Alcoi, province of Alicante) where legionellosis has become almost endemic. We analysed the six loci currently incorporated to the sequence-based typing scheme developed by European Working Group for Legionella Infections (EWGLI) for L. pneumophila and 13 intergenic regions, for which we developed primers anchored in flanking, conserved genes. Our results show that recombination among natural isolates of this species is a common phenomenon, as 20 of the 31 isolates contained at least one locus in which recombination was revealed by at least three different methods. The mapping of the recombination events on the maximum likelihood tree of the concatenate sequence of the 19 loci indicated that at least nine independent recombination events might explain the observed distribution of recombinant loci among isolates. In consequence, we have shown that recombination in L. pneumophila is much more frequent than previously considered and that it does not seem to be restricted to already described pathogenicity islands or other genome constituents which provide it with a high plasticity.     

Reappearance of Cucumber mosaic virus Isolates Belonging to Subgroup IB in Tomato Plants in North-eastern Spain

A disease characterized by symptomless leaves and fruit decolouration, loss of consistency and mild deformation on ripening was detected in tomato fields in north‐eastern Spain during 2003 and 2004. DAS‐ELISA analysis revealed the presence of the Cucumber mosaic virus (CMV) in all diseased plants. CMV isolates were characterized by polyacrylamide gel electrophoresis (PAGE) analysis of double‐stranded RNAs (dsRNAs) and nucleotide sequence analysis, and compared with some CMV isolates belonging to different subgroups used as controls. A total of 12 isolates obtained from the infected tomato plants selected for this study gave the same electrophoresis pattern for the three genomic dsRNAs, which was different to the patterns showed by the CMV isolates collected in the same region a few years ago. The identity of the complete nucleotide sequence of one of these CMV isolates and the partial sequence of other five isolates compared to the Tfn strain from Italy and the BAR92/1 isolate from tomato collected in Barcelona in 1992 was higher than 99%, both belonging to subgroup IB of CMV. The CMV isolates of this subgroup found in eastern Spain in previous studies were not detected after 1996. The nucleotide sequences of two isolates that were chosen as representatives of the CMV isolates more frequently detected in previous years revealed that they belonged to the CMV subgroups IA. The origin and the possible causes of reappearance of CMV IB isolates in north‐eastern Spain are discussed.     

Differentiation of Indian,East Timorese,Papuan and Floridian ‘Candidatus Liberibacter asiaticus’ isolates on the basis of simple sequence repeat and single nucleotide polymorphism profiles at 25 loci

Japanese isolates of ‘Candidatus Liberibacter asiaticus’ have been shown to be clearly differentiated by simple sequence repeat (SSR) profiles at four loci. In this study, 25 SSR loci, including these four loci, were selected from the whole‐genome sequence and were used to differentiate non‐Japanese samples of ‘Ca. Liberibacter asiaticus’ (13 Indian, 3 East Timorese, 1 Papuan and 8 Floridian samples). Out of the 25 SSR loci, 13 were polymorphic. Dendrogram analysis using SSR loci showed that the clusters were mostly consistent with the geographical origins of the isolates. When single nucleotide polymorphisms (SNPs) were searched around these 25 loci, only the upstream region of locus 091 exhibited polymorphism. Phylogenetic tree analysis of the SNPs in the upstream region of locus 091 showed that Floridian samples were clustered into one group as shown by dendrogram analysis using SSR loci. The differences in nucleotide sequences were not associated with differences in the citrus hosts (lime, mandarin, lemon and sour orange) from which the isolates were originally derived.     

Lateral gene transfer of O1 serogroup encoding genes of Vibrio cholerae

In Gram-negative bacteria, the O-antigen-encoding genes may be transferred between lineages, although mechanisms are not fully understood. To assess possible lateral gene transfer (LGT), 21 Argentinean Vibrio cholerae O-group 1 (O1) isolates were examined using multilocus sequence typing (MLST) to determine the genetic relatedness of housekeeping genes and genes from the O1 gene cluster. MSLT analysis revealed that 4.4% of the nucleotides in the seven housekeeping loci were variable, with six distinct genetic lineages identified among O1 isolates. In contrast, MLST analysis of the eight loci from the O1 serogroup region revealed that 0.24% of the 4943 nucleotides were variable. A putative breakpoint was identified in the JUMPstart sequence. Nine conserved nucleotides differed by a single nucleotide from a DNA uptake signal sequence (USS) also found in Pastuerellaceae . Our data indicate that genes in the O1 biogenesis region are closely related even in distinct genetic lineages, indicative of LGT, with a putative DNA USS identified at the defined boundary for the DNA exchange.     

Multiple polymorphic sites at the ITS and ATAN loci in cultured isolates of Perkinsus marinus

Sequence analysis of genomic DNA from the protozoan parasite Perkinsus marinus at two loci revealed genetic polymorphisms within and among different cultured isolates. Genomic DNA from 12 Perkinsus marinus isolates was amplified at the internal transcribed spacer region and at an anonymous locus previously identified to contain polymorphisms by restriction fragment length polymorphism analysis. Fourteen polymorphic nucleotide positions were identified at the internal transcribed spacer region; eight in internal transcribed spacer 1 and six in internal transcribed spacer 2. Thirteen polymorphic nucleotide sites were identified within the anonymous locus. In some instances, more than three different sequences were observed at both the internal transcribed spacer region and at the anonymous locus from a single clonal isolate, suggesting the possibility of recombination in cultured cells and/or strand jumping during the polymerase chain reaction. Intra-isolate sequence variation (3.46% for the anonymous locus and 3.08% for internal transcribed spacer 1) was in several cases as high as inter-isolate sequence variation, even in one isolate where recombination was not evident. High intra- and inter-isolate variation detected at both loci demonstrates the importance of determining the genetic variation of each locus prior to development of sequence-based molecular diagnostics.     

Development of Genomic SSR Markers and Molecular Characterization of Magnaporthe oryzae Isolates from Wheat in Brazil

Magnaporthe oryzae, the causal agent of wheat blast, was characterized on a molecular level with 38 newly isolated genomic SSR loci. Among the 31 wheat isolates analyzed, 15 polymorphic loci were detected, with an average of 1.7 alleles per locus, 28.9% of them being highly or reasonably informative. The number of polymorphic loci was higher in isolates from Londrina in the Brazilian state of Paraná and Coromandel in Minas Gerais compared with Goiânia in Goiás and São Borja in Rio Grande do Sul. The rice isolate was clearly different from the wheat isolates, and the size difference in polymorphic SSR loci between one isolate from wheat and one isolate from rice was associated with the number of repeats. Some isolates collected from different states and in different years demonstrated similarities of 100%. The markers developed here are useful for the genetic analysis of M. oryzae isolated from wheat, and isolates representing the variability detected in the field can be used to search for better wheat blast resistance.     

Genetic variations in Lycoris radiata var. radiata in Japan

The genetic variations of Lycoris radiata var. radiata, a completely sterile triploid from Japan, were examined by comparing the nucleotide sequences of genomic DNA regions in 11 triploid strains sampled from Japan and four triploid strains sampled from China, and in two diploid strains of Lycoris radiata var. pumila, which is endemic to China and fertile. For this purpose, two genes were analyzed, the lectin gene in the nuclear genome and the maturase gene in the chloroplast genome. A clear genetic constancy was observed in their DNA nucleotide sequences. For both genes, completely identical nucleotide sequences were detected in the 11 Japanese and four Chinese triploid strains and also between the two Chinese diploid strains. However, some genetic variations were observed between the Japanese and Chinese triploid strains, and between the triploid and diploid strains. These results are consistent with the findings obtained from previous chromosome karyotype analyses and allozyme analyses. In addition, in our preliminary FISH analysis of the physical mapping of the rRNA gene family, the 18S-5.8S-26S rRNA and 5S rRNA loci were localized on six and four chromosomes, respectively. Regarding the 18S-5.8S-26S rRNA loci, two were associated with two SAT chromosomes. The remaining four were distinguished by having no secondary constriction. Localization of 5S rRNA loci to chromosome spreads revealed three sites on the proximal part of the long arm of three acrocentric chromosomes and one site on the distal part of the long arm of the SAT chromosome; the latter site was juxtaposed to the 18S-5.8S-26S rRNA loci. These findings indicate that L. radiata var. radiata is not a typical autotriploid. The present paper discusses the possible origin of L. radiata var. radiata from a diploid variety of L. radiata var. pumila, based on the molecular cytogenetic analysis and DNA sequence analysis.     

Biogeography of the ubiquitous marine bacterium Alteromonas macleodii determined by multilocus sequence analysis

Twenty‐three isolates of the widely distributed marine bacteria Alteromonas macleodii have been analysed by multilocus sequence analysis combined with phylogenetic and multivariate statistical analyses. The strains originated from the Pacific Ocean, Mediterranean Sea, English Channel, Black Sea and Thailand. Using the nucleotide sequences of nine loci for each of the 23 isolates, a robust identification was achieved of different clades within the single species. Strains generally clustered with the depth in the water column from which the isolate originated. Strains also showed more recombination with isolates from the same vicinity, suggesting that genetic exchange plays a role in diversification of planktonic marine prokaryotes. This study thus shows for the first time for a large set of isolates of a species of planktonic marine prokaryotes that multilocus sequence analysis overcomes the problems associated with the analysis of individual marker genes or presence of extensive recombination events. It can thus achieve intraspecific identification to the level of genotypes and, by comparison with relevant environmental data, ecotypes.     

Population structure of the fish-pathogenic bacterium Flavobacterium psychrophilum

Flavobacterium psychrophilum is currently one of the main bacterial pathogens hampering the productivity of salmonid farming worldwide, and its control mainly relies on antibiotic treatments. To better understand the population structure of this bacterium and its mode of evolution, we have examined the nucleotide polymorphisms at 11 protein-coding loci of the core genome in a set of 50 isolates. These isolates were selected to represent the broadest possible diversity, originating from 10 different host fish species and four continents. The nucleotide diversity between pairs of sequences amounted to fewer than four differences per kilobase on average, corresponding to a particularly low level of diversity, possibly indicative of a small effective-population size. The recombination rate, however, seemed remarkably high, and as a consequence, most of the isolates harbored unique combinations of alleles (33 distinct sequence types were resolved). The analysis also showed the existence of several clonal complexes with worldwide geographic distribution but marked association with particular fish species. Such an association could reflect preferential routes of transmission and/or adaptive niche specialization. The analysis provided no clues that the initial range of the bacterium was originally limited to North America. Instead, the historical record of the expansion of the pathogen may reflect the spread of a few clonal complexes. As a resource for future epidemiological surveys, a multilocus sequence typing website based on seven highly informative loci is available.     

Sexual Reproduction Plays a Major Role in the Genetic Structure of Populations of the Fungus Mycosphaerella Graminicola

The relative contributions of sexual and asexual reproduction to the genetic structure of populations can be difficult to determine for fungi that use a mixture of both types of propagation. Nuclear RFLPs and DNA fingerprints were used to make indirect and direct measures of departures from random mating in a population of the plant pathogenic fungus Mycosphaerella graminicola during the course of an epidemic cycle. DNA fingerprints resolved 617 different genotypes among 673 isolates sampled from a single field over a 3-month period. Only 7% of the isolates represented asexual clones that were found more than once in the sample. The most common clone was found four times. Genotypic diversity averaged 85% of its maximum possible value during the course of the epidemic. Analyses of multilocus structure showed that allelic distributions among RFLP loci were independent. Pairwise comparisons between individual RFLP loci showed that the majority of alleles at these loci were in gametic equilibrium. Though this fungus has the capacity for a significant level of asexual reproduction, each analysis suggested that M. graminicola populations maintain a genetic structure more consistent with random-mating over the course of an epidemic cycle.     

Experimental herpes-like viral infections in marine bivalves: demonstration of interspecies transmission

The sequences of gene segments 2 and 8 from 10 different isolates of infectious salmon anaemia virus (ISAV) sampled in Norway, Canada and Scotland between 1987 and 1999 were determined and compared. Pairwise comparisons revealed a high degree of homology between the European isolates, with identities of 98 to 100% for both genes examined. The Canadian isolate showed identities of 84 and 87 to 88% with the European isolates for the nucleotide sequence of segments 2 and 8, respectively. Phylogenetic analyses were performed to establish the interrelationship between the European virus isolates. The evolutionary rate based on 4 Norwegian isolates clustered together in the analysis of segment 2 was calculated to be 0.96 x 10(-3) nucleotides site(-1) yr(-1). On the basis of this mutation rate it was estimated that the Norwegian Glesvaer 90 and Canadian Bay of Fundy 97 isolates diverged around 1900, which coincides with transportation of salmonids between Europe and North America starting in the late nineteenth century.     

Development of novel microsatellites from Moniliophthora perniciosa, causal agent of the witches' broom disease of Theobroma cacao

Moniliophthora perniciosa is the causal agent of the witches' broom disease of cacao. Based on available genomic sequences, we identified 30 new microsatellite loci, which were analysed using 50 isolates from four populations sampled over a wide geographical area in Brazil, including three populations from the Amazon, the fungal putative centre of diversity, plus one from Bahia. Nine loci were polymorphic, with an average of 2.9 alleles per locus. The level of polymorphism observed was low, but these markers may allow the evaluation of pathogen diversity and the establishment of molecular standards for isolate fingerprinting to support cacao breeding.     

Mitochondrial DNA sequence variation among geographical isolates of Opisthorchis viverrini in Thailand and Lao PDR, and phylogenetic relationships with other trematodes

The present study compared the genetic variation among 14 different geographical isolates of Opisthorchis viverrini sensu lato from Thailand and Lao PDR using sequence data for 2 mitochondrial DNA genes, the subunit 1 of NADH dehydrogenase gene (nad1) and cytochrome c oxidase gene (cox1). Four different nad1 haplotypes were detected among isolates, all of which were identical at the amino acid sequence level. Nucleotide sequence variation among 14 isolates ranged from 0 to 0.3% for nad1. Two different cox1 haplotypes were detected among isolates. These two haplotypes differed at 2 nucleotide positions, one of which resulted in a change in the amino acid sequence. Nucleotide sequence variation among isolates for cox1 ranged from 0 to 0.5%. Comparison of cox1 sequences of O. viverrini to those of other trematodes revealed nucleotide differences of 13-31%. A phylogenetic analysis of the cox1 sequence data revealed strong statistical support for a clade containing O. viverrini and 2 other species of opisthorchid trematodes; O. felineus and Clonorchis sinsensis.     

Population structure of the African savannah elephant inferred from mitochondrial control region sequences and nuclear microsatellite loci

Two hundred and thirty-six mitochondrial DNA nucleotide sequences were used in combination with polymorphism at four nuclear microsatellite loci to assess the amount and distribution of genetic variation within and between African savannah elephants. They were sampled from 11 localities in eastern, western and southern Africa. In the total sample, 43 haplotypes were identified and an overall nucleotide diversity of 2.0% was observed. High levels of polymorphism were also observed at the microsatellite loci both at the level of number of alleles and gene diversity. Nine to 14 alleles per locus across populations and 44 alleles in the total sample were found. The gene diversity ranged from 0.51 to 0.72 in the localities studied. An analysis of molecular variance showed significant genetic differentiation between populations within regions and also between regions. The extent of subdivision between populations at the mtDNA control region was approximately twice as high as shown by the microsatellite loci (mtDNA F(ST) = 0.59; microsatellite R(ST) = 0.31). We discuss our results in the light of Pleistocene refugia and attribute the observed pattern to population divergence in allopatry accompanied by a recent population admixture following a recent population expansion.     

Genetic characterization of Sabin types 1 and 3 poliovaccine virus following serial passage in the human intestinal tract.

Poliovirus isolates types 1 and 3 were obtained from five and seven successive passages respectively, in infants who had been fed monovalent OPV in two separate clinical trials conducted in 1960. The purpose of these trials was to answer the question how much the vaccine virus would revert to its original neurovirulent phenotype following multiplication in the intestinal tract. Human passages were performed either by contact exposure or by feeding the excreted virus while the infants were maintained in isolation. Several virus isolates were obtained at each passage level. Infants participating in both studies showed no symptoms of disease. Antigenic studies (McBride, van Wezel) and protein analysis (PAGE) of the isolates, reported earlier from this laboratory, had shown that the isolates remained vaccine-like, although isolates from the later passages revealed some differences. Monkey neurovirulence test results showed that for both types 1 and 3 viruses the loss of attenuation of the vaccine strain upon passage was gradual, although the loss was faster for type 3. Examination of the oligonucleotide maps demonstrated that the oligonucleotide configuration of the isolates remained the same as for the vaccine strain but there was an increase of individual spot differences with increasing passage. The nucleotide sequence analysis of selected regions of the virus genomes revealed that there was no change from a G to A in nucleotide 480 of type 1 isolates; however, nucleotide 476 changed from a U to an A in type 1 passages 3, 4 and 5. Conversely, for type 3 the change of nucleotide 472 from a U to a C changed at the early first passage (4 days following administration of OPV), and remained a C in the six following passages; type 3 nucleotide 2034 did not change in the first passage from a U to a C, but it became a C in all further passages tested. The nucleotide changes mentioned for both virus types remained stable in successive passages. However, there was another nucleotide change for type 3 from a U to a C at position 1973 only for passages 5 and 6 which reverted to a U for passages 7L and 7LL. Study of selected human passage virus strains could further contribute to the identification of the critical nucleotides that are responsible for the attenuation of these two polio types of vaccine viruses.     

Development of molecular markers and preliminary investigation of the population structure and mating system in one lineage of black morel (Morchella elata) in the Pacific Northwestern USA

Phylogenetic analysis of LSU/ITS sequence data revealed two distinct lineages among 44 morphologically similar fruiting bodies of natural black morels (Morchella elata group) sampled at three non-burn locations in the St Joe and Kanisku National Forests in northern Idaho. Most of the sampled isolates (n = 34) represented a dominant LSU/ITS haplotype present at all three sites and identical to the Mel-12 phylogenetic lineage (GU551425) identified in a previous study. Variation at 1-3 nucleotide sites was detected among a small number of isolates (n = 6) within this well supported clade (94%). Four isolates sampled from a single location were in a well supported clade (97%) distinct from the dominant haplotypes and may represent a previously un-sampled, cryptic phylogenetic species. Species-specific SNP and SCAR markers were developed for Mel-12 lineage isolates by cloning and sequencing AFLP amplicons, and segregation of AFLP markers were studied from single ascospore isolates from individual fruiting bodies. Based on the segregation of AFLP markers within single fruiting bodies, split decomposition analyses of two SCAR markers, and population genetic analyses of SNP, SCAR, and AFLP markers, it appears that members of the Morchella sp. Mel-12 phylogenetic lineage are heterothallic and outcross in nature similar to yellow morels. This is the first set of locus-specific molecular markers that has been developed for any Morchella species, to our knowledge. These markers will prove to be valuable tools to study mating system, gene flow and genetic structure of black morels at various spatial scales with field-collected fruiting bodies and eliminate the need to culture samples in vitro.     

The diversity of Rhizobia, Sinorhizobia and novel non-Rhizobial Paenibacillus nodulating wild herbaceous legumes

The objective of the present study was to isolate and characterize nodulating bacteria associated with wild legumes. For this purpose, we recovered twenty isolates from root nodules of five wild legume species: Melilotus alles, Melilotus officinalis, Trifolium pratense, Trifolium repens and Medicago sp. Most of the isolates were morphologically analogous with only few exceptions in colony shape, appearance and incubation time. All isolates were Gram negative except T.P2-4. Random amplification of polymorphic DNA showed genetic variation among isolates. The 16S rRNA sequence analysis revealed these isolates as Rhizobium, Sinorhizobium and Paenibacillus. Each of these was also screened for nod D and nod F genes with marked variation at these loci; however, the nucleotide sequence analysis confirmed the presence of nod genes. The assignment of strains to their hosts revealed a unique symbiotic association of Paenibacillus sp. nodulating T .pratense which is being reported here for the first time.     

Complex mutational patterns and size homoplasy at maize microsatellite loci

Microsatellite markers have become one of the most popular tools for germplasm characterization, population genetics and evolutionary studies. To investigate the mutational mechanisms of maize microsatellites, nucleotide sequence information was obtained for ten loci. In addition, Single-Strand Conformation Polymorphism (SSCP) analysis was conducted to assess the occurrence of size homoplasy. Sequence analysis of 54 alleles revealed a complex pattern of mutation at 8/10 loci, with only 2 loci showing allele variation strictly consistent with stepwise mutations. The overall allelic diversity resulted from changes in the number of repeat units, base substitutions, and indels within repetitive and non-repetitive segments. Thirty-one electromorphs sampled from six maize landraces were considered for SSCP analysis. The number of conformers per electromorph ranged from 1 to 7, with 74.2% of the electromorphs showing more than one conformer. Size homoplasy was apparent within landraces and populations. Variation in the amount of size homoplasy was observed within and between loci, although no differences were detected among populations. The results of the present study provide useful information on the interpretation of genetic data derived from microsatellite markers. Further efforts are still needed to determine the impact of these findings on the estimation of population parameters and on the inference of phylogenetic relationships in maize investigations. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Isolation and characterization of microsatellite markers from the entomopathogenic fungus Metarhizium anisopliae

Fourteen polymorphic microsatellite markers were isolated from the entomopathogenic fungus, Metarhizium anisopliae, based on enriched genomic libraries. In order to assess allelic variability, the microsatellite loci were analysed in a collection of 34 isolates sampled from across Switzerland. The number of detected alleles in 14 loci ranged from two to eight and expected heterozygosity from 0.265 to 0.808. Because of the high expected heterozygosity, the 14 microsatellite loci are very useful for ecological studies and analysis of population diversity, and to identifying, monitoring, and tracking M. anisopliae strains applied as biological control agents.     

中国落叶松-杨栅锈菌遗传多样性研究

采用SSR分子标记技术对采自全国19个地区、不同年份的落叶松-杨栅锈菌Melampsora larici-populina（简称MLP）36个单孢子堆菌系的遗传多样性和种群遗传结构进行了研究。用5对SSR多态性引物共检测到62个观察等位基因（Na）,有效等位基因数（Ne）为5.42–9.82,平均有效等位基因数为7.05。供试MLP样本在5个SSR位点上的多态性信息量（PIC）变化范围为0.64–0.89,平均值0.79。平均观察杂合度（Ho）、平均期望杂合度（He）分别为0.36和0.86,不同位点的S