Diverse Collection Of Isolates Research Articles

Exploring the competitive potential of Ralstonia pseudosolanacearum and Ralstonia solanacearum: Insights from a comparative adaptability study

AbstractBacterial wilt, caused by Ralstonia pseudosolanacearum (Rpsol) and R. solanacearum (Rsol), poses a significant challenge to solanaceous plant cultivation worldwide, particularly in tropical and subtropical regions. Even though Brazil is recognized as one of the centres of origin and diversity of Rsol, in certain regions of this large country there is an emerging prevalence of Rpsol in production fields. Therefore, this study aimed to comprehensively investigate the adaptive traits of Rpsol and Rsol using a polyphasic approach. A diverse collection of isolates from both species was assessed for their physiological, biochemical, ecological and pathogenic traits. Rsol isolates demonstrated greater adaptability to a broader range of temperature, salinity and pH. They also exhibited enhanced abilities in biofilm formation and bacteriocin production. Conversely, Rpsol isolates exhibited a broader utilization of carbon sources and displayed a wider spectrum of resistance to inhibitory substances. Moreover, they demonstrated higher infectivity towards different solanaceous hosts, showing a faster invasion and colonization process in the roots and stems of tomato plants compared to Rsol isolates. Based on our findings, we concluded that Rsol exhibited greater physiological and ecological adaptability, while Rpsol showed greater pathogenic and biochemical adaptability. These results suggest that the coexistence of both species is maintained through a balance of distinct traits within each species.

Genome Sequencing and Transcriptome Analysis of the Hop Downy Mildew Pathogen Pseudoperonospora humuli Reveal Species-Specific Genes for Molecular Detection.

Pseudoperonospora humuli is an obligate oomycete pathogen of hop (Humulus lupulus) that causes downy mildew, an important disease in most production regions in the Northern Hemisphere. The pathogen can cause a systemic infection in hop, overwinter in the root system, and infect propagation material. Substantial yield loss may occur owing to P. humuli infection of strobiles (seed cones), shoots, and cone-bearing branches. Fungicide application and cultural practices are the primary methods to manage hop downy mildew. However, effective, sustainable, and cost-effective management of downy mildew can be improved by developing early detection systems to inform on disease risk and timely fungicide application. However, no species-specific diagnostic assays or genomic resources are available for P. humuli. The genome of the P. humuli OR502AA isolate was partially sequenced using Illumina technology and assembled with ABySS. The assembly had a minimum scaffold length of 500 bp and an N50 (median scaffold length of the assembled genome) of 19.2 kbp. A total number of 18,656 genes were identified using MAKER standard gene predictions. Additionally, transcriptome assemblies were generated using RNA-seq and Trinity for seven additional P. humuli isolates. Bioinformatics analyses of next generation sequencing reads of P. humuli and P. cubensis (a closely related sister species) identified 242 candidate species-specific P. humuli genes that could be used as diagnostic molecular markers. These candidate genes were validated using polymerase chain reaction against a diverse collection of isolates from P. humuli, P. cubensis, and other oomycetes. Overall, four diagnostic markers were found to be uniquely present in P. humuli. These candidate markers identified through comparative genomics can be used for pathogen diagnostics in propagation material, such as rhizomes and vegetative cuttings, or adapted for biosurveillance of airborne sporangia, an important source of inoculum in hop downy mildew epidemics.

Whole genome analysis reveals the diversity and evolutionary relationships between necrotic enteritis-causing strains of Clostridium perfringens

BackgroundClostridium perfringens causes a range of diseases in animals and humans including necrotic enteritis in chickens and food poisoning and gas gangrene in humans. Necrotic enteritis is of concern in commercial chicken production due to the cost of the implementation of infection control measures and to productivity losses. This study has focused on the genomic analysis of a range of chicken-derived C. perfringens isolates, from around the world and from different years. The genomes were sequenced and compared with 20 genomes available from public databases, which were from a diverse collection of isolates from chickens, other animals, and humans. We used a distance based phylogeny that was constructed based on gene content rather than sequence identity. Similarity between strains was defined as the number of genes that they have in common divided by their total number of genes. In this type of phylogenetic analysis, evolutionary distance can be interpreted in terms of evolutionary events such as acquisition and loss of genes, whereas the underlying properties (the gene content) can be interpreted in terms of function. We also compared these methods to the sequence-based phylogeny of the core genome.ResultsDistinct pathogenic clades of necrotic enteritis-causing C. perfringens were identified. They were characterised by variable regions encoded on the chromosome, with predicted roles in capsule production, adhesion, inhibition of related strains, phage integration, and metabolism. Some strains have almost identical genomes, even though they were isolated from different geographic regions at various times, while other highly distant genomes appear to result in similar outcomes with regard to virulence and pathogenesis.ConclusionsThe high level of diversity in chicken isolates suggests there is no reliable factor that defines a chicken strain of C. perfringens, however, disease-causing strains can be defined by the presence of netB-encoding plasmids. This study reveals that horizontal gene transfer appears to play a significant role in genetic variation of the C. perfringens chromosome as well as the plasmid content within strains.

Genomic analysis of the multi-host pathogen Erysipelothrix rhusiopathiae reveals extensive recombination as well as the existence of three generalist clades with wide geographic distribution.

BackgroundKnowledge about how bacterial populations are structured is an important prerequisite for studying their ecology and evolutionary history and facilitates inquiry into host specificity, pathogenicity, geographic dispersal and molecular epidemiology. Erysipelothrix rhusiopathiae is an opportunistic pathogen that is currently reemerging in both the swine and poultry industries globally. This bacterium sporadically causes mortalities in captive marine mammals, and has recently been implicated in large-scale wildlife die-offs. However, despite its economic relevance and broad geographic and host distribution, including zoonotic potential, the global diversity, recombination rates, and population structure of this bacterium remain poorly characterized. In this study, we conducted a broad-scale genomic comparison of E. rhusiopathiae based on a diverse collection of isolates in order to address these knowledge gaps.ResultsEighty-three E. rhusiopathiae isolates from a range of host species and geographic origins, isolated between 1958 and 2014, were sequenced and assembled using both reference-based mapping and de novo assembly. We found that a high proportion of the core genome (58 %) had undergone recombination. Therefore, we used three independent methods robust to the presence of recombination to define the population structure of this species: a phylogenetic tree based on a set of conserved protein sequences, in silico chromosome painting, and network analysis. All three methods were broadly concordant and supported the existence of three distinct clades within the species E. rhusiopathiae. Although we found some evidence of host and geographical clustering, each clade included isolates from diverse host species and from multiple continents.ConclusionsUsing whole genome sequence data, we confirm recent suggestions that E. rhusiopathiae is a weakly clonal species that has been shaped extensively by homologous recombination. Despite frequent recombination, we can reliably identify three distinct clades that do not clearly segregate by host species or geographic origin. Our results provide an essential baseline for future molecular epidemiological, ecological and evolutionary studies of E. rhusiopathiae and facilitate comparisons to other recombinogenic, multi-host bacteria.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2643-0) contains supplementary material, which is available to authorized users.

The difficult road to new vaccines for pertussis and serogroup B meningococcal disease

AbstractDevelopment of vaccines should be a well‐trodden path in which possible diversions and roadblocks should be clearly understood. However, challenges and unexpected twists and turns are not uncommon in modern vaccine development. In this perspective the considerable uncertainties for development of third generation pertussis vaccines will be discussed along with related issues in the development of new vaccines for the prevention of serogroup B meningococcal (MenB) disease. Acellular pertussis vaccines developed in the 1980s showed good efficacy in large clinical trials and whooping cough was at historically low levels in the last decade of the 20th century in most countries where the vaccines are used. However, the unexpected resurgence in cases in several countries has questioned the ability of current acellular pertussis vaccines to provide long‐term protection against the disease. For MenB vaccines the initial challenge has been to identify antigens that will induce protection against the diverse collection of isolates that cause disease. Following this there have been significant challenges to determine potential breadth of strain coverage. Furthermore, there has been a drop in the incidence of MenB disease in countries considering implementation of the new MenB vaccines making cost effectiveness a more difficult case to argue. It is likely that lessons learnt in the development and use of both pertussis and MenB vaccines will inform the future of vaccines for these two diseases. © 2015 Crown copyright. Journal of Chemical Technology & Biotechnology © 2015 Society of Chemical Industry

Development of a FTIR-ATR based model for typing clinically relevant Acinetobacter baumannii clones belonging to ST98, ST103, ST208 and ST218

In this work we developed and validated a FTIR-ATR (Fourier transform infrared spectroscopy with attenuated total reflectance) based model for typing Acinetobacter baumannii clinical isolates belonging to ST98, ST208 and ST218 included into the worldwide spread clonal complex (CC) 92 and ST103. FTIR-ATR spectra of seventy-seven previously characterized isolates (Multi Locus Sequence Type-MLST, Pulsed-Field Gel Electrophoresis-PFGE and carbapenem-hydrolyzing class D β-lactamase-CHDL) were acquired and modeled by partial least squares discriminant analysis (PLSDA). The model was tested and successfully validated with a diverse collection of isolates (n=148) recovered from different countries and periods of time belonging to modeled and non-modeled STs.

Identification of a new variable number tandem repeat locus in Mycobacterium ulcerans for potential strain discrimination among African isolates

Intra-species discrimination in the highly clonal pathogen Mycobacterium ulcerans was studied in a diverse collection of isolates by PCR amplification of a short sequence repeat locus containing heterogeneous arrays of tri-nucleotide repeats with an ACC consensus pattern. Post-amplification analysis indicated excellent typeability and identified five M. ulcerans alleles, including a unique Angolan type differentiated for the first time among a genetically conserved cluster of African isolates. These results are consistent with previously investigated independent markers, and provide an additional locus for variable number tandem repeat-based typing of M. ulcerans.

Intensive and extensive sampling techniques used to measure genetic diversity of Ustilago tritici, using virulence and DNA polymorphism

Assessment of genetic variability within a natural population of a pathogen is dependent on obtaining a genetically diverse collection of isolates for analysis. Collections of isolates of Ustilago tritici are traditionally made by sampling one smutted head of wheat per field from a large number of fields over a large area. However, there is little evidence that extensively sampling one smutted head from each of many fields over a large area yields a more genetically diverse collection of isolates than intensively sampling many smutted heads from each of a few fields. The objective of this study was to compare genetic diversity derived from intensive sampling of U. tritici (80 isolates from 4 fields) with that derived from extensive sampling (81 isolates from 81 fields). Genetic diversity was measured using virulence data and amplified fragment length polymorphism (AFLP). The virulence of the isolates was assessed on five differential hosts, and the AFLP data were obtained using 10 selective primers that yielded 23 polymorphic bands. Genetic diversity was similar within the extensive- and the intensive-sampling collections, when measured using virulence data, but it was more variable within the intensive-sampling collection than within the extensive-sampling collection, when measured using AFLP. In general, the results of AFLP analysis indicated a higher degree of genetic variability than did the virulence data. The results also indicated that host genotypes can strongly influence the genetic variability within populations of an obligate pathogen like U. tritici when this variability is measured on virulence data, but that host genotype has a weaker influence on diversity measured by AFLP.

Using multilocus sequence data to define the pneumococcus.

We investigated the genetic relationships between serotypeable pneumococci and nonserotypeable presumptive pneumococci using multilocus sequence typing (MLST) and partial sequencing of the pneumolysin gene (ply). Among 121 nonserotypeable presumptive pneumococci from Finland, we identified isolates of three classes: those with sequence types (STs) identical to those of serotypeable pneumococci, suggesting authentic pneumococci in which capsular expression had been downregulated or lost; isolates that clustered among serotypeable pneumococci on a tree based on the concatenated sequences of the MLST loci but which had STs that differed from those of serotypeable pneumococci in the MLST database; and a more diverse collection of isolates that did not cluster with serotypeable pneumococci. The latter isolates typically had sequences at all seven MLST loci that were 5 to 10% divergent from those of authentic pneumococci and also had distinct and divergent ply alleles. These isolates are proposed to be distinct from pneumococci but cannot be resolved from them by optochin susceptibility, bile solubility, or the presence of the ply gene. Complete resolution of pneumococci from the related but distinct population is problematic, as recombination between them was evident, and a few isolates of each population possessed alleles at one or occasionally more MLST loci from the other population. However, a tree based on the concatenated sequences of the MLST loci in most cases unambiguously distinguished whether a nonserotypeable isolate was or was not a pneumococcus, and the sequence of the ply gene fragment was found to be useful to resolve difficult cases.

Ribosomal DNA Sequence Divergence and Group I Introns within the Leucostoma Species L. cinctum, L. persoonii, and L. parapersoonii sp. nov., Ascomycetes That Cause Cytospora Canker of Fruit Trees

Leucostoma species that are the causal agents of Cytospora canker of stone and pome fruit trees were studied in detail. DNA sequence of the internal transcribed spacer regions and the 5.8S of the nuclear ribosomal DNA operon (ITS rDNA) supplied sufficient characters to assess the phylogenetic relationships among species of Leucostoma, Valsa, Valsella, and related anamorphs in Cytospora. Parsimony analysis of the aligned sequence divided Cytospora isolates from fruit trees into clades that generally agreed with the morphological species concepts, and with some of the phenetic groupings (PG 1-6) identified previously by isozyme analysis and cultural characteristics. Phylogenetic analysis inferred that isolates of L. persoonii formed two well-resolved clades distinct from isolates of L. cinctum. Phylogenetic analysis of the ITS rDNA, isozyme analysis, and cultural characteristics supported the inference that L. persoonii groups PG 2 and PG 3 were populations of a new species apparently more genetically different from L. persoonii PG 1 than from isolates representative of L. massariana, L. niveum, L. translucens, and Valsella melastoma. The new species, L. parapersoonii, was described. A diverse collection of isolates of L. cinctum, L. persoonii, and L. parapersoonii were examined for genetic variation using restriction fragment length polymorphism (RFLP) analysis of the ITS rDNA and the five prime end of the large subunit of the rDNA (LSU rDNA). HinfI and HpaII endonucleases were each useful in dividing the Leucostoma isolates into RFLP profiles corresponding to the isozyme phenetic groups, PG 1-6. RFLP analysis was more effective than isozyme analysis in uncovering variation among isolates of L. persoonii PG 1, but less effective within L. cinctum populations. Isolates representative of seven of the L. persoonii formae speciales proposed by G. Défago in 1935 were found to be genetically diverse isolates of PG 1. Two large insertions, 415 and 309 nucleotides long, in the small subunit (SSU) of the nuclear rDNA of L. cinctum were identified as Group 1 introns; intron 1 at position 943 and intron 2 at position 1199. The two introns were found to be consistently present in isolates of L. cinctum PG 4 and PG 5 and absent from L. cinctum PG 6 isolates, despite the similarity of the ITS sequence and teleomorph morphology. Intron 1 was of subgroup 1C1 whereas intron 2 was of an unknown subgroup. RFLP patterns and presence/absence of introns were useful characters for expediting the identification of cultures of Leucostoma isolated from stone and pome fruit cankers. RFLP patterns from 13 endonucleases provided an effective method for selecting an array of diverse PG 1 isolates useful in screening plant germplasm for disease-resistance.

Ribosomal DNA sequence divergence and group I introns within the Leucostoma species L. cinctum, L. persoonii, and L. parapersoonii sp. nov., ascomycetes that cause Cytospora canker of fruit trees

Leucostoma species that are the causal agents of Cytospora canker of stone and pome fruit trees were studied in detail. DNA sequence of the internal transcribed spacer regions and the 5.8S of the nuclear ribosomal DNA operon (ITS rDNA) supplied sufficient characters to assess the phylogenetic relationships among species of Leucostoma, Valsa, Valsella, and related anamorphs in Cytospora. Parsimony analysis of the aligned sequence divided Cytospora isolates from fruit trees into clades that generally agreed with the morphological species concepts, and with some of the phenetic groupings (PG 1–6) identified previously by isozyme analysis and cultural characteristics. Phylogenetic analysis inferred that isolates of L. persoonii formed two well-resolved clades distinct from isolates of L. cinctum. Phylogenetic analysis of the ITS rDNA, isozyme analysis, and cultural characteristics supported the inference that L. persoonii groups PG 2 and PG 3 were populations of a new species apparently more genetically different from L. persoonii PG 1 than from isolates representative of L. massariana, L. niveum, L. translucens, and Valsella melastoma. The new species, L. parapersoonii, was described. A diverse collection of isolates of L. cinctum, L. persoonii, and L. parapersoonii were examined for genetic variation using restriction fragment length polymorphism (RFLP) analysis of the ITS rDNA and the five prime end of the large subunit of the rDNA (LSU rDNA). HinfI and HpaII endonucleases were each useful in dividing the Leucostoma isolates into RFLP profiles corresponding to the isozyme phenetic groups, PG 1–6. RFLP analysis was more effective than isozyme analysis in uncovering variation among isolates of L. persoonii PG 1, but less effective within L. cinctum populations. Isolates representative of seven of the L. persoonii formae speciales proposed by G. Défago in 1935 were found to be genetically diverse isolates of PG 1. Two large insertions, 415 and 309 nucleotides long, in the small subunit (SSU) of the nuclear rDNA of L. cinctum were identified as Group 1 introns; intron 1 at position 943 and intron 2 at position 1199. The two introns were found to be consistently present in isolates of L. cinctum PG 4 and PG 5 and absent from L. cinctum PG 6 isolates, despite the similarity of the ITS sequence and teleomorph morphology. Intron 1 was of subgroup 1C1 whereas intron 2 was of an unknown subgroup. RFLP patterns and presence/absence of introns were useful characters for expediting the identification of cultures of Leucostoma isolated from stone and pome fruit cankers. RFLP patterns from 13 endonucleases provided an effective method for selecting an array of diverse PG 1 isolates useful in screening plant germplasm for disease-resistance.

Specific detection of Phytophthora nicotianae using the polymerase chain reaction and primers based on the DNA sequence of its elicitin gene ParA1

Six primers based on the sequence of the flanking and coding regions of the elicitin gene ParA1 of Phytophthora nicotianae were tested for specific detection of the fungus by the polymerase chain reaction (PCR). One combination, IL7/IL8, with IL7 in a flanking region and IL8 in a coding region of the gene, gave an intense 378 bp signal with a diverse collection of isolates of P. nicotianae, that included some from black shank disease of tobacco and others from a variety of hosts. The sequence of the amplification product obtained with an isolate that produces elicitin and one that does not, was homologous with the known sequence of the ParA1 gene. The same primer combination gave no signal with sixteen other Phytophthora species tested except for two isolates P. palmivora with which it gave a weak 800 bp signal. It gave no signal with DNA from healthy tobacco and tomato plants but P. nicotianae was detected in inoculated tobacco and tomato plants. Small numbers of zoospores (>100) trapped onto a nitrocellulose membrane after filtration from suspension were also detected after two successive rounds of PCR.