Comparison of two DNA sequence-based typing schemes for the Fusarium solani Species Complex and proposal of a new consensus method

Multilocus sequence typing (MLST) is a widely used approach for differentiating microbial isolates presenting many advantages such as easy access through online databases and straightforward interpretation. For the Fusarium solani species complex (FSSC), three gene regions have been widely used to investigate phylogenetic relationships at the interspecific level (ITS-nuLSU, EF1a, RPB2) and a nomenclature system has been proposed for the different known haplotypes. More recently, a MLST scheme was proposed for this species complex based on the polymorphisms of five housekeeping genes (ACC, ICL, GDP, MDP, SOD). Here, we compare the phylogenetic resolution and sequence discriminatory powers of these two sets of loci on 50 epidemiologically unrelated FSSC strains. Although the widely used gene set offers better phylogenetic resolution, the newly developed gene set is slightly better at discriminating isolates using a MLST method. A consensus scheme of eight loci is proposed for typing FSSC strains combining the advantages of the two previous gene sets and offering the best typing efficiency.     

Comparison of a semi-automated rep-PCR system and multilocus sequence typing for differentiation of Salmonella enterica isolates

The accurate sub-typing of Salmonella enterica isolates is essential for epidemiological investigations and surveillance of Salmonella infections. Salmonella isolates are currently identified using the Kauffman-White serotyping scheme. Multilocus sequence typing (MLST) schemes have been developed for the major bacterial pathogens including Salmonella and have assisted in understanding the molecular epidemiology and population biology of these organisms. Recently, the DiversiLab rep-PCR system has been developed using micro-fluidic chips to provide standardized, semi-automated fingerprinting for pathogens including S. enterica. In the current study, 71 isolates of S. enterica, representing 21 serovars, were analyzed using MLST and the DiversiLab rep-PCR system. MLST was able to identify 31 sequence types (STs), while the DiversiLab system revealed 38 DiversiLab types (DTs). The rep-PCR distinguished isolates of different serovars and showed greater discriminatory power (0.95) than MLST typing (0.89). Rep-PCR exhibited 92% concordance with MLST and 90% with serotyping, while the concordance level of MLST typing with serotyping was 96%, representing a strong association. Comparison of rep-PCR profiles with those held in an online library database led to the accurate prediction of serovar in 63% of cases and resulted in inaccurate predictions for 10% of profiles. MLST and the rep-PCR system may provide useful additional informative techniques for the molecular identification of S. enterica. We conclude that the DiversiLab rep-PCR system may provide a rapid (less than 4 h) and standardized method for sub-typing isolates of S. enterica.     

Multilocus Sequence Typing of Pathogenic Candida albicans Isolates Collected from a Teaching Hospital in Shanghai,China: A Molecular Epidemiology Study

Molecular typing of Candida albicans is important for studying the population structure and epidemiology of this opportunistic yeast, such as population dynamics, nosocomial infections, multiple infections and microevolution. The genetic diversity of C. albicans has been rarely studied in China. In the present study, multilocus sequence typing (MLST) was used to characterize the genetic diversity and population structure of 62 C. albicans isolates collected from 40 patients from Huashan Hospital in Shanghai, China. A total of 50 diploid sequence types (DSTs) were identified in the 62 C. albicans isolates, with 41 newly identified DSTs. Based on cluster analysis, the 62 isolates were classified into nine existing clades and two new clades (namely clades New 1 and New 2). The majority of the isolates were clustered into three clades, clade 6 (37.5%), clade 1 (15.0%) and clade 17 (15.0%). Isolates of clade New 2 were specifically identified in East Asia. We identified three cases of potential nosocomial transmission based on association analysis between patients’ clinical data and the genotypes of corresponding isolates. Finally, by analyzing the genotypes of serial isolates we further demonstrated that the microevolution of C. albicans was due to loss of heterozygosity. Our study represents the first molecular typing of C. albicans in eastern China, and we confirmed that MLST is a useful tool for studying the epidemiology and evolution of C. albicans.     

Construction of a deletion strain and expression vector for the Escherichia coli NADH:ubiquinone oxidoreductase (Complex I)

Complex I of Escherichia coli is encoded by 13 consecutive genes, called the nuo operon. A chromosomal deletion of all nuo genes has been achieved by homologous recombination. A vector that encodes all of the nuo genes has been constructed, and it expresses a functional enzyme.     

Development of species-specific primers for rapid identification by PCR of the ecologically important pathogen Fusarium keratoplasticum from isolated and environmental samples

The genus Fusarium contains many fungal species known to be pathogenic to animals and plants alike. One species complex within this genus, the Fusarium solani species complex (FSSC), is of particular concern due to its high numbers of pathogenic members. FSSC members are known to contribute significantly to plant, human and other animal fungal disease. One member of the FSSC, Fusarium keratoplasticum, is of particular ecological concern and has been implicated in low hatching success of endangered sea turtle eggs, as well as contribute to human and other animal Fusarium pathogenesis. Species-specific primers for molecular identification of F. keratoplasticum currently do not exist to our knowledge, making rapid identification, tracking and quantitation of this pathogenic fungus difficult. The objective of this study was to develop primers specific to F. keratoplasticum that could be applied to DNA from isolated cultures as well as total (mixed) DNA from environmental samples. RPB2 sequence from 109 Fusarium isolates was aligned and analysed to determine nucleotide polymorphisms specific to F. keratoplasticum useful for primer design. A set of primers were generated and found to be effective for identification of F. keratoplasticum from total DNA extracted from sand surrounding sea turtle nesting sites.     

Limitations of variable number of tandem repeat typing identified through whole genome sequencing of Mycobacterium avium subsp. paratuberculosis on a national and herd level

Background

Mycobacterium avium subsp. paratuberculosis (MAP), the causative bacterium of Johne’s disease in dairy cattle, is widespread in the Canadian dairy industry and has significant economic and animal welfare implications. An understanding of the population dynamics of MAP can be used to identify introduction events, improve control efforts and target transmission pathways, although this requires an adequate understanding of MAP diversity and distribution between herds and across the country. Whole genome sequencing (WGS) offers a detailed assessment of the SNP-level diversity and genetic relationship of isolates, whereas several molecular typing techniques used to investigate the molecular epidemiology of MAP, such as variable number of tandem repeat (VNTR) typing, target relatively unstable repetitive elements in the genome that may be too unpredictable to draw accurate conclusions. The objective of this study was to evaluate the diversity of bovine MAP isolates in Canadian dairy herds using WGS and then determine if VNTR typing can distinguish truly related and unrelated isolates.

Results

Phylogenetic analysis based on 3,039 SNPs identified through WGS of 124 MAP isolates identified eight genetically distinct subtypes in dairy herds from seven Canadian provinces, with the dominant type including over 80% of MAP isolates. VNTR typing of 527 MAP isolates identified 12 types, including “bison type” isolates, from seven different herds. At a national level, MAP isolates differed from each other by 1–2 to 239–240 SNPs, regardless of whether they belonged to the same or different VNTR types. A herd-level analysis of MAP isolates demonstrated that VNTR typing may both over-estimate and under-estimate the relatedness of MAP isolates found within a single herd.

Conclusions

The presence of multiple MAP subtypes in Canada suggests multiple introductions into the country including what has now become one dominant type, an important finding for Johne’s disease control. VNTR typing often failed to identify closely and distantly related isolates, limiting the applicability of using this typing scheme to study the molecular epidemiology of MAP at a national and herd-level.     

Suppression of Alfalfa Growth by Concommitant Populations of Pratylenchus penetrans and two Fusarium species

Growth of alfalfa (Medicago sativa cv. Vernal) seedlings was compared after inoculation with combinations of either Pratylenchus penetrans and Fusarium soloni or P. penetrans and F. oxysporum f. sp. medicaginis. A synergistic disease interaction occurred in alfalfa when F. oxysporum and P. penetrans were added simultaneously to the soil. Alfalfa growth was suppressed at all inoculum levels of P. penetrans and F. oxysporum, but not with F. solani. Seedlings inoculated with the nematode alone gave lower yields than when inoculated with either Fusarium species alone. Fusarium oxysporum, but not F. solani, was pathogenic to alfalfa under similar experimental conditions. Fusarium oxysporum did not alter the populations of P. penetrans in alfalfa roots, whereas the presence of F. solani was associated with a diminished number of P. penetrans in the roots.     

Comparison of DNA Microarray,Loop-Mediated Isothermal Amplification (LAMP) and Real-Time PCR with DNA Sequencing for Identification of <Emphasis Type="Italic">Fusarium</Emphasis> spp. Obtained from Patients with Hematologic Malignancies

The performance of three molecular biology techniques, i.e., DNA microarray, loop-mediated isothermal amplification (LAMP), and real-time PCR were compared with DNA sequencing for properly identification of 20 isolates of Fusarium spp. obtained from blood stream as etiologic agent of invasive infections in patients with hematologic malignancies. DNA microarray, LAMP and real-time PCR identified 16 (80%) out of 20 samples as Fusarium solani species complex (FSSC) and four (20%) as Fusarium spp. The agreement among the techniques was 100%. LAMP exhibited 100% specificity, while DNA microarray, LAMP and real-time PCR showed 100% sensitivity. The three techniques had 100% agreement with DNA sequencing. Sixteen isolates were identified as FSSC by sequencing, being five Fusarium keratoplasticum, nine Fusarium petroliphilum and two Fusarium solani. On the other hand, sequencing identified four isolates as Fusarium non-solani species complex (FNSSC), being three isolates as Fusarium napiforme and one isolate as Fusarium oxysporum. Finally, LAMP proved to be faster and more accessible than DNA microarray and real-time PCR, since it does not require a thermocycler. Therefore, LAMP signalizes as emerging and promising methodology to be used in routine identification of Fusarium spp. among cases of invasive fungal infections.     

Identification of 1 a-hydroxy phaseollone,a phaseollin metabolite produced by Fusarium solani

A structure for the phaseollin metabolite of Fusarium solani f. sp phaseoli has been proposed and assigned the name 1 a-hydroxyphaseollone.     

Interaction of Rotylenchulus reniformis with Seedling Disease Pathogens of Cotton

The impact of 10 Fusarium species in concomitant association with Rotylenchulus reniformis on cotton seedling disease was examined under greenhouse conditions. In experiment 1, fungal treatments consisted of Fusarium chlamydosporum, F. equiseti, F. lateritium, F. moniliforme, F. oxysporum, F. oxysporum f.sp. vasinfectum, F. proliferatum, F. semitectum, F. solani, and F. sporotrichioides; Rhizoctonia solani; and Thielaviopsis basicola. The experimental design was a 2 × 14 factorial consisting of the presence or absence of R. reniformis and the 12 fungal treatments plus two controls in autoclaved field soil. In experiment 2, the same fungal and nematode treatments were examined in autoclaved or non-autoclaved soil. This experimental design was a 2 × 2 × 14 factorial consisting of field or autoclaved soil, presence or absence of R. reniformis, and the 12 fungal treatments plus two controls. In both tests, Fusarium oxysporum f. sp. vasinfectum, F. solani, R. solani, and T. basicola consistently displayed extensive root and hypocotyl necrosis that was more severe (P ≤ 0.05) in the presence of R. reniformis. Soil treatment (autoclaved vs. non-autoclaved) influenced the impact of the Fusarium species on cotton seedling disease, with disease being more severe in the autoclaved soil. Rotylenchulus reniformis reproduction on cotton seedlings was greater in field soil compared to autoclaved soil (P ≤ 0.05). This study suggests the importance of Fusarium species and R. reniformis in cotton seedling disease.     

Typing of four genetic loci discriminates among closely related species of New World Leishmania

All New World Leishmania species can cause cutaneous lesions, while only Leishmania (Viannia) braziliensis has been associated with mucosal metastases. Multilocus enzyme electrophoresis (MLEE) is the optimal standard for species identification but is slow and costly. New methods for species identification are needed to ensure proper identification and therapy. The coding regions of four metabolic enzyme markers in the MLEE typing method: mannose phosphate isomerase (MPI), malate dehydrogenase (MDH), glucose-6-phosphate isomerase (GPI), and 6-phosphogluconate dehydrogenase (6PGD), were analysed from seven species of New World Leishmania isolated from patients with either cutaneous or mucosal lesions to identify specific genetic polymorphisms responsible for the phenotypic variations observed in the MLEE typing scheme. We identified species-specific polymorphisms and determined that a combination of sequencing of the mpi and 6pgd genes was sufficient to differentiate among seven closely related species of New World Leishmania and among isolates of L. braziliensis shown previously to have atypical MLEE patterns. When DNA isolated from 10 cutaneous lesion biopsies were evaluated, the sequence typing method was 100% concordant with the published MLEE/monoclonal antibody identification methods. The identification of species-specific polymorphisms can be used to design a DNA-based test with greater discriminatory power that requires shorter identification times. When the causative agent of the disease is L. braziliensis, this method ensures correct species identification, even when the agent is a genetic variant. Proper identification could facilitate adequate treatment, preventing the onset of the disfiguring mucosal form of the disease.     

Multilocus phylogeny and MALDI-TOF analysis of the plant pathogenic species Alternaria dauci and relatives

The genus Alternaria includes numerous phytopathogenic species, many of which are economically relevant. Traditionally, identification has been based on morphology, but is often hampered by the tendency of some strains to become sterile in culture and by the existence of species-complexes of morphologically similar taxa. This study aimed to assess if strains of four closely-related plant pathogens, i.e., accurately Alternaria dauci (ten strains), Alternaria porri (six), Alternaria solani (ten), and Alternaria tomatophila (ten) could be identified using multilocus phylogenetic analysis and Matrix-Assisted Laser Desorption Ionisation Time of Flight (MALDI-TOF) profiling of proteins. Phylogenetic analyses were performed on three loci, i.e., the internal transcribed spacer (ITS) region of rRNA, and the glyceraldehyde-3-phosphate dehydrogenase (gpd) and Alternaria major antigen (Alt a 1) genes. Phylogenetic trees based on ITS sequences did not differentiate strains of A. solani, A. tomatophila, and A. porri, but these three species formed a clade separate from strains of A. dauci. The resolution improved in trees based on gpd and Alt a 1, which distinguished strains of the four species as separate clades. However, none provided significant bootstrap support for all four species, which could only be achieved when results for the three loci were combined. MALDI-TOF-based dendrograms showed three major clusters. The first comprised all A. dauci strains, the second included five strains of A. porri and one of A. solani, and the third included all strains of A. tomatophila, as well as all but one strain of A. solani, and one strain of A. porri. Thus, this study shows the usefulness of MALDI-TOF mass spectrometry as a promising tool for identification of these four species of Alternaria which are closely-related plant pathogens.     

The dorsal chaetotaxy of Trogolaphysa (Collembola,Paronellidae), with descriptions of two new species?from caves in Belize

Species diagnosis in Trogolaphysa has been based, until now, almost exclusively on number of eyes and shape of claws and mucro. Chaetotaxy, a character system important to diagnose species in other genera of scaled Entomobryoidea, has been described only for a few Trogolaphysa species. Here the complete dorsal chaetotaxy of six species of Trogolaphysa is described using the AMS and Szeptycki’s systems for head and body, respectively. A morphology-based parsimony analysis was performed to evaluate whether chaetotaxic characters overcome the influence of putatively cave adaptive convergent characters to resolve species level relationships, and to evaluate the evolution of the dorsal macrochaetae of the head. Phylogenetic analysis using only putative cave-adaptive characters support clades of unrelated taxa, but the addition of chaetotaxy overcomes the influence of convergent characters. A phylogeny based on all characters supports a trend towards reduced head macrochaetae number. Head macrochaetae are lost beginning with A3 and followed, in order, by S5, S3 and M3. In addition, a checklist of New World Trogolaphysa is provided and two new species, Trogolaphysa giordanoae sp. n. and Trogolaphysa jacobyi sp. n., are described on the basis of material collected in six caves in southern Belize.     

Presidential address: rediscovering parasites using molecular tools--towards revising the taxonomy of Echinococcus, Giardia and Cryptosporidium

Molecular biology has provided parasitologists with a fantastic variety of techniques that have had a major impact on research into parasites and parasitism. Molecular tools have revealed the extent and nature of genetic diversity in parasites and this information has made a significant contribution to studies on the population genetics and evolutionary biology of parasites. Similarly, epidemiology has benefited enormously from the application of molecular tools in terms of studying parasite life cycles and transmission, and in the development of specific and sensitive methods for diagnosis and surveillance. However, the theme I wish to develop in this paper is concerned with the contribution molecular tools have made to parasite taxonomy and systematics, and in particular, the fact that in many cases molecular tools are validating the proposals made many years ago by taxonomists and biologists which were discounted or not fully accepted at the time. To do this I have chosen four examples (Echinococcus, Entamoeba, Giardia, Cryptosporidium) where recent research involving molecular characterisation has confirmed observations made many years ago and has resulted in a need to revise the taxonomy of different groups of parasites.     

Comparative Molecular docking analysis of DNA Gyrase subunit A in Pseudomonas aeruginosaPAO1

Pseudomonas aeruginosa is an opportunistic bacterium known for causing chronic infections in cystic fibrosis and chronic obstructive pulmonary disease (COPD) patients. Recently, several drug targets in Pseudomonas aeruginosa PAO1 have been reported using network biology approaches on the basis of essentiality and topology and further ranked on network measures viz. degree and centrality. Till date no drug/ligand molecule has been reported against this targets.In our work we have identified the ligand /drug molecules, through Orthologous gene mapping against Bacillus subtilis subsp. subtilis str. 168 and performed modelling and docking analysis. From the predicted drug targets in PA PAO1, we selected those drug targets which show statistically significant orthology with a model organism and whose orthologs are present in all the selected drug targets of PA PAO1.Modeling of their structure has been done using I-Tasser web server. Orthologous gene mapping has been performed using Cluster of Orthologs (COGs) and based on orthology; drugs available for Bacillus sp. have been docked with PA PAO1 protein drug targets using MoleGro virtual docker version 4.0.2.Orthologous gene for PA3168 gyrA is BS gyrAfound in Bacillus subtilis subsp. subtilis str. 168. The drugs cited for Bacillus sp. have been docked with PA genes and energy analyses have been made. Based on Orthologous gene mapping andin-silico studies, Nalidixic acid is reported as an effective drug against PA3168 gyrA for the treatment of CF and COPD.     

Reactive Oxygen Species Play a Role in the Infection of the Necrotrophic Fungi,Rhizoctonia solani in Wheat

Rhizoctonia solani is a nectrotrophic fungal pathogen that causes billions of dollars of damage to agriculture worldwide and infects a broad host range including wheat, rice, potato and legumes. In this study we identify wheat genes that are differentially expressed in response to the R. solani isolate, AG8, using microarray technology. A significant number of wheat genes identified in this screen were involved in reactive oxygen species (ROS) production and redox regulation. Levels of ROS species were increased in wheat root tissue following R. solani infection as determined by Nitro Blue Tetrazolium (NBT), 3,3''-diaminobenzidine (DAB) and titanium sulphate measurements. Pathogen/ROS related genes from R. solani were also tested for expression patterns upon wheat infection. TmpL, a R. solani gene homologous to a gene associated with ROS regulation in Alternaria brassicicola, and OAH, a R. solani gene homologous to oxaloacetate acetylhydrolase which has been shown to produce oxalic acid in Sclerotinia sclerotiorum, were highly induced in R. solani when infecting wheat. We speculate that the interplay between the wheat and R. solani ROS generating proteins may be important for determining the outcome of the wheat/R. solani interaction.     

Description of Quinisulcius solani n.sp. (Nematoda: Tylenchorhynchidae) with a Key to the Species and Data on Scutylenchus koreanus from Pakistan

A new species. Quinisulcius solani, is described and illustrated from specimens on Solanum tuberosum from Murree Hills, Pakistan. Q. solani n.sp. differs from its closest relative, Q. acutus (Allen, 1955) Siddiqi, 1971, by its spiral to open ''C'' shaped body and stylet length of 19 μn, vs. 17 μm in Q. acutus. In Q. acutus the stylet knobs project anteriorly but slope posteriorly in Q. solani n.sp. Tail annules number 17 in Q. acutus but 38 in Q. solani n.sp.; also the phasmids in the former species are at mid-tail and in the latter are in anterior half of tail, at about 34%. Q. solani n.sp. is also closely related to Q. capitatus (Allen, 1955) Siddiqi, 1971 but differs in some characters. Head annules number eight and stylet length is 17 μm in Q. capitatus, but head annules are six and stylet length is 19 μm in Q. solani n.sp. In Q. capitatus T/ABW is 3 vs. 2.2 in Q. solani n.sp. Also, phasmids are located at mid-tail on Q. capitatus but at ahout one-third of the tail on Q. solani n.sp. A key to the 10 species of Quinisulcius is also presented. Scutylenchus koreanus (Choi &Geraert, 1971) Siddiqi, 1979 is recorded for the first time in Pakistan and morphometric data and illustrations given.     

In silico analyses of penicillin binding proteins in Burkholderia pseudomallei uncovers SNPs with utility for phylogeography,species differentiation,and sequence typing

Burkholderia pseudomallei causes melioidosis. Sequence typing this pathogen can reveal geographical origin and uncover epidemiological associations. Here, we describe B. pseudomallei genes encoding putative penicillin binding proteins (PBPs) and investigate their utility for determining phylogeography and differentiating closely related species. We performed in silico analysis to characterize 10 PBP homologs in B. pseudomallei 1026b. As PBP active site mutations can confer β-lactam resistance in Gram-negative bacteria, PBP sequences in two resistant B. pseudomallei strains were examined for similar alterations. Sequence alignments revealed single amino acid polymorphisms (SAAPs) unique to the multidrug resistant strain Bp1651 in the transpeptidase domains of two PBPs, but not directly within the active sites. Using BLASTn analyses of complete assembled genomes in the NCBI database, we determined genes encoding PBPs were conserved among B. pseudomallei (n = 101) and Burkholderia mallei (n = 26) strains. Within these genes, single nucleotide polymorphisms (SNPs) useful for predicting geographic origin of B. pseudomallei were uncovered. SNPs unique to B. mallei were also identified. Based on 11 SNPs identified in two genes encoding predicted PBP-3s, a dual-locus sequence typing (DLST) scheme was developed. The robustness of this typing scheme was assessed using 1,523 RefSeq genomes from B. pseudomallei (n = 1,442) and B. mallei (n = 81) strains, resulting in 32 sequence types (STs). Compared to multi-locus sequence typing (MLST), the DLST scheme demonstrated less resolution to support the continental separation of Australian B. pseudomallei strains. However, several STs were unique to strains originating from a specific country or region. The phylogeography of Western Hemisphere B. pseudomallei strains was more highly resolved by DLST compared to internal transcribed spacer (ITS) typing, and all B. mallei strains formed a single ST. Conserved genes encoding PBPs in B. pseudomallei are useful for strain typing, can enhance predictions of geographic origin, and differentiate strains of closely related Burkholderia species.     

Chitosan as a Component of Pea-Fusarium solani Interactions

Chitosan, a polymer of β-1,4-linked glucosamine residues with a strong affinity for DNA, was implicated in the pea pod-Fusarium solani interaction as an elicitor of phytoalexin production, an inhibitor of fungal growth and a chemical which can protect pea tissue from infection by F. solani f. sp. pisi. Purified Fusarium fungal cell walls can elicit phytoalexin production in pea pod tissue. Enzymes from acetone powders of pea tissue release eliciting components from the F. solani f. sp. phaseoli cell walls. Hydrochloric acid-hydrolyzed F. solani cell walls are about 20% glucosamine. The actual chitosan content of F. solani cell walls is about 1%. However, chitosan assays and histochemical observations indicate that chitosan content of F. solani spores and adjacent pea cells increases following inoculation. Dormant F. solani spores also accumulate chitosan. Concentrations of nitrous acid-cleaved chitosan as low as 0.9 microgram per milliliter and 3 micrograms per milliliter elicit phytoalexin induction and inhibit germination of F. solani macroconidia, respectively. When chitosan is applied to pea pod tissue with or prior to F. solani f. sp. pisi, the tissue is protected from infection.