Multilocus Gene Research Articles

Multilocus and SSU rRNA gene phylogenetic analyses of available cyanobacterial genomes, and their relation to the current taxonomic system

Despite the global significance of cyanobacteria, their taxonomy is still heavily lagging behind the progress in genome sequencing. Besides nomenclatural issues and difficulties in species identification, this problem is largely a consequence of missing reference strains and sequences for many cyanobacterial taxa. In this study, a robust multilocus phylogenetic tree based on 23 conserved proteins from the current set of available genomes is presented and compared to a respective 16S rRNA gene tree. To facilitate further progress in achieving a stable system of monophyletic taxa, the phylogenetic status of eight cyanobacterial orders recently proposed in the botanical system was evaluated. While several orders addressed by previous studies (Gloeobacterales, Spirulinales, Chroococcidiopsidales, Nostocales) retain their monophyly, the remaining big groups (Synechococcales, Pleurocapsales, Chroococcales, Oscillatoriales) are polyphyletic and require thorough revision. Comparison of the 16S rRNA tree to the multilocus tree supports the careful use of this gene in distinguishing cyanobacterial genera, whereas its application at other taxonomic levels is limited.

Vibrio aphrogenes sp. nov., in the Rumoiensis clade isolated from a seaweed.

A novel strain Vibrio aphrogenes sp. nov. strain CA-1004T isolated from the surface of seaweed collected on the coast of Mie Prefecture in 1994 [1] was characterized using polyphasic taxonomy including multilocus sequence analysis (MLSA) and a genome based comparison. Both phylogenetic analyses on the basis of 16S rRNA gene sequences and MLSA based on eight protein-coding genes (gapA, gyrB, ftsZ, mreB, pyrH, recA, rpoA, and topA) showed the strain could be placed in the Rumoiensis clade in the genus Vibrio. Sequence similarities of the 16S rRNA gene and the multilocus genes against the Rumoiensis clade members, V. rumoiensis, V. algivorus, V. casei, and V. litoralis, were low enough to propose V. aphrogenes sp. nov. strain CA-1004T as a separate species. The experimental DNA-DNA hybridization data also revealed that the strain CA-1004T was separate from four known Rumoiensis clade species. The G+C content of the V. aphrogenes strain was determined as 42.1% based on the genome sequence. Major traits of the strain were non-motile, halophilic, fermentative, alginolytic, and gas production. A total of 27 traits (motility, growth temperature range, amylase, alginase and lipase productions, and assimilation of 19 carbon compounds) distinguished the strain from the other species in the Rumoiensis clade. The name V. aphrogenes sp. nov. is proposed for this species in the Rumoiensis clade, with CA-1004T as the type strain (JCM 31643T = DSM 103759T).

First Report of Neofabraea kienholzii Causing Bull’s Eye Rot on Pear (Pyrus communis) in the Netherlands

Pear (Pyrus communis L.) is an important fruit crop in the Netherlands, with a total production of 349,000 tons in 2014, and ‘Conference’ is the main cultivar. In the Netherlands, pears are kept in controlled atmosphere cold storage up to 11 months after harvest. Symptoms of bull’s eye rot were observed in 2015 on ‘Conference’ pears in storage in the Netherlands. Bull’s eye lesions on apple and pear fruits are generally caused by four Neofabraea species: N. alba Jacks, N. malicorticis Guthrie, N. perennans Kienholz, and N. kienholzii Seifert, Spotts & Levesque (Gariepy et al. 2005). N. alba is the major pathogen causing bull’s eye rot on pear fruits in the Netherlands. Independent of the species, the symptoms appear as flat or slightly sunken lesions, which are brown, often lighter brown in the center (Spotts et al. 2009). To isolate the causal agent, fruit were rinsed with sterile water, lesions were sprayed with 70% ethanol until droplet runoff, the skin was removed aseptically with a scalpel, and tissue under the lesion was isolated and placed onto potato dextrose agar (PDA). PDA plates were incubated at 20°C in the dark, and single spores were transferred to fresh PDA plates. The isolates produced colonies with white-yellowish to brownish mycelium. Microconidia were produced on feathery fascicles of aerial mycelium, with a white, powdery, or sugary appearance on the surface of the agar colony. Microconidia were 2.5 to 6.5 × 1.5 to 2.5 µm, ellipsoidal, slightly asymmetrical to a curved form. The identity of a representative isolate (PPO 45010) was confirmed by means of multilocus gene sequencing. To this end, genomic DNA was extracted using the LGC Mag Plant Kit (Berlin) in combination with the Kingfisher method (Waltham, MA). Segments of the internal transcribed spacer region (ITS), 28S ribosomal RNA (28S rRNA) and beta-tubulin (TUB2) loci were amplified, sequenced with primers ITS1/ITS4, LR0R/LR5, and Btub2Fd/Btub4Rd (Chen et al. 2016), and deposited in GenBank under accession nos. KX424942 (ITS), KX424941 (28S rRNA), and KX424940 (TUB2). MegaBLAST analysis revealed that the ITS, 28S rRNA, and TUB2 sequences matched with 99 to 100% identity to N. kienholzii isolates in GenBank (KR859082 and KR859083 [ITS], KR858873 and KR858874 [28S rRNA], KR859288 and KR859289 [TUB2]). Alcohol surface sterilized fruits were inoculated in pathogenicity tests in two ways: (i) with an agar disk (10 mm diameter) with actively growing mycelium of N. kienholzii prepared from a 14-day-old culture grown on PDA; and (ii) with 20 μl of a spore suspension (105 conidia ml-1) prepared from a 21-day-old PDA culture after wounding with a needle. Both experiments were performed on 10 ‘Conference’ pears. Inoculated fruits were sealed in plastic bags and were incubated in darkness at 20°C. Typical symptoms appeared between 7 and 14 days. Mock-inoculated controls with water and PDA-only controls remained symptomless. Fungi isolated from the lesions had morphological characteristics that resembled the original isolates from infected pears. The identity of these isolates was confirmed as N. kienholzii by sequencing, thus completing Koch’s postulates. Bull’s eye rot of apple and pear is an important postharvest disease, occurring in major fruit growing areas of North America, Chile, Australia, and Europe (Henriquez et al. 2004; Spotts et al. 2009). N. kienholzii was reported twice on apple in Europe (Michalecka et al. 2016). To the best of our knowledge, this is the first report of N. kienholzii causing bull’s eye rot of pear in Europe.

Identification of acetic acid bacteria through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and report of Gluconobacter nephelii Kommanee et al. 2011 and Gluconobacter uchimurae Tanasupawat et al. 2012 as later heterotypic synonyms of Gluconobacter japonicus Malimas et al. 2009 and Gluconobacter oxydans (Henneberg 1897) De Ley 1961 (Approved Lists 1980) emend. Gosselé et al. 1983, respectively

The classification and identification of acetic acid bacteria (AAB) underwent much change during the last two decades. However, several closely related AAB species are difficult to differentiate and rapid and correct species level identification remains challenging. In the present study, 281 well-characterized AAB strains representing AAB species of the family Acetobacteraceae were used to construct a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) identification database. The database was evaluated using 270 poorly characterized AAB strains. 16S rRNA and/or multilocus gene sequence analysis and whole-genome sequencing for the calculation of average nucleotide identity values were performed on a selection of strains to verify the accuracy of the obtained results. Virtually all poorly characterized AAB strains were accurately identified to the species level. Most of the remaining strains were considered to represent novel AAB species or belonged to species represented by too few strains in the identification database. The data also revealed that Gluconobacter nephelii and Gluconobacter uchimurae are later heterotypic synonyms of Gluconobacter japonicus and Gluconobacter oxydans, respectively.

In Vitro Variant Surface Antigen Expression in Plasmodium falciparum Parasites from a Semi-Immune Individual Is Not Correlated with Var Gene Transcription.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is considered to be the main variant surface antigen (VSA) of Plasmodium falciparum and is mainly localized on electron-dense knobs in the membrane of the infected erythrocyte. Switches in PfEMP1 expression provide the basis for antigenic variation and are thought to be critical for parasite persistence during chronic infections. Recently, strain transcending anti-PfEMP1 immunity has been shown to develop early in life, challenging the role of PfEMP1 in antigenic variation during chronic infections. In this work we investigate how P. falciparum achieves persistence during a chronic asymptomatic infection. The infected individual (MOA) was parasitemic for 42 days and multilocus var gene genotyping showed persistence of the same parasite population throughout the infection. Parasites from the beginning of the infection were adapted to tissue culture and cloned by limiting dilution. Flow cytometry using convalescent serum detected a variable surface recognition signal on isogenic clonal parasites. Quantitative real-time PCR with a field isolate specific var gene primer set showed that the surface recognition signal was not correlated with transcription of individual var genes. Strain transcending anti-PfEMP1 immunity of the convalescent serum was demonstrated with CD36 selected and PfEMP1 knock-down NF54 clones. In contrast, knock-down of PfEMP1 did not have an effect on the antibody recognition signal in MOA clones. Trypsinisation of the membrane surface proteins abolished the surface recognition signal and immune electron microscopy revealed that antibodies from the convalescent serum bound to membrane areas without knobs and with knobs. Together the data indicate that PfEMP1 is not the main variable surface antigen during a chronic infection and suggest a role for trypsin sensitive non-PfEMP1 VSAs for parasite persistence in chronic infections.

A permutation approach for inferring species networks from gene trees in polyploid complexes by minimising deep coalescences

SummaryContext and purpose. Due to ubiquity of polyploid species formation in angiosperms, reconstructing the evolutionary history of polyploid complexes is at the forefront of methodological field of activity in molecular phylogenetics and systematic botany. The advent of multilocus species tree reconstruction approaches motivated by next‐generation sequencing methods pave the way to species network inference methods, with which phylogenetics could accommodate to the necessity for reconstructing reticulate relationships among diploids and (allo‐)polyploids in an objective manner, simultaneously taking into account gene tree incongruences caused by incomplete lineage sorting and hybridisation.Methods used. The present contribution describes a relatively simple and fast workflow pipeline for the reconstruction of species networks in polyploid complexes based on multilocus gene trees. It uses a permutation strategy and a parsimony‐based principle in species tree reconstruction (Minimising Deep Coalescences,MDC) for the assignment of homoeologs to parental genomes in allopolyploids, which is a crucial step in species network inference for polyploid complexes. Simulations with varying effective population sizes, different temporal scenarios for diploid or polyploid formations and different types of allopolyploid speciation (sister‐species diploids, non‐sister diploids or polyploidisation under participation of ancestral diploids) are used to assess the reliability of the reconstruction.Main results. Simulations show that the proposed method leads to trustworthy reconstructions if effective populations sizes are not too large and divergences among ancestral taxa are not too shallow. The applicability of our method to a real data set is demonstrated by inferring the reticulate relationships among diploid, tetraploid and hexaploid representatives of the genusLeucanthemopsis(Compositae, Anthemideae) based on gene tree topologies from four nuclear single‐copy genes and two plastidic intergenic spacer regions.Conclusion. The proposedMDC‐based method for species network reconstruction in polyploids is fast and manages to trustworthily reconstruct parentage in polyploidisation events. Concerns with and possible improvements of the described method are discussed.

Antifungal Streptomyces spp. Associated with the Infructescences of Protea spp. in South Africa.

Common saprophytic fungi are seldom present in Protea infructescences, which is strange given the abundance of mainly dead plant tissue in this moist protected environment. We hypothesized that the absence of common saprophytic fungi in Protea infructescences could be due to a special symbiosis where the presence of microbes producing antifungal compounds protect the infructescence. Using a culture based survey, employing selective media and in vitro antifungal assays, we isolated antibiotic producing actinomycetes from infructescences of Protea repens and P. neriifolia from two geographically separated areas. Isolates were grouped into three different morphological groups and appeared to be common in the Protea spp. examined in this study. The three groups were supported in 16S rRNA and multi-locus gene trees and were identified as potentially novel Streptomyces spp. All of the groups had antifungal activity in vitro. Streptomyces sp. Group 1 had inhibitory activity against all tested fungi and the active compound produced by this species was identified as fungichromin. Streptomyces spp. Groups 2 and 3 had lower inhibition against all tested fungi, while Group 3 showed limited inhibition against Candida albicans and Sporothrix isolates. The active compound for Group 2 was also identified as fungichromin even though its production level was much lower than Group 1. The antifungal activity of Group 3 was linked to actiphenol. The observed antifungal activity of the isolated actinomycetes could contribute to protection of the plant material against common saprophytic fungi, as fungichromin was also detected in extracts of the infructescence. The results of this study suggest that the antifungal Streptomyces spp. could play an important role in defining the microbial population associated with Protea infructescences.

Potential Relationship between Phenotypic and Molecular Characteristics in Revealing Livestock-Associated Staphylococcus aureus in Chinese Humans without Occupational Livestock Contact.

While some studies have defined Staphylococcus aureus based on its clonal complex and resistance pattern, few have explored the relations between the genetic lineages and antibiotic resistance patterns and immune evasion cluster (IEC) genes. Our aim was to investigate the potential relationship between phenotypic and molecular characteristics so as to reveal livestock-associated S. aureus in humans. The study participants were interviewed, and they provided two nasal swabs for S. aureus analysis. All S. aureus and methicillin-resistant S. aureus (MRSA) were tested for antibiotic susceptibility, multilocus sequence type and IEC genes. Of the 1162 participants, 9.3% carried S. aureus, including MRSA (1.4%) and multidrug-resistant S. aureus (MDRSA, 2.8%). The predominant multidrug-resistant pattern among MDRSA isolates was non-susceptibility to erythromycin, clindamycin and tetracycline. The most common S. aureus genotypes were ST7, ST6, ST188, and ST59, and the predominant MRSA genotype was ST7. Notably, the livestock-associated S. aureus isolates (IEC-negative CC9, IEC-negative tetracycline-resistant CC398, and IEC-negative tetracycline-resistant CC5) were found in people with no occupational livestock contact. These findings reveal a potential relationship between S. aureus CCs and IEC genes and antibiotic resistance patterns in defining livestock-associated S. aureus in humans and support growing concern about the potential livestock-to-human transmission of livestock-associated S. aureus by non-occupational livestock contact.

First Report of Fusarium avenaceum Causing Postharvest Decay on ‘Conference’ Pears in the Netherlands

Pear (Pyrus communis) is an important fruit crop in the Netherlands, with a total production of 349,000 tons in 2014. ‘Conference’ is the main pear cultivar that occupies 75% of the total pear production area in the Netherlands. In the Netherlands, pears are kept in controlled atmosphere cold storage for 9 to 11 months after harvest. Lesions were observed on pears of the cultivar Conference in a survey carried out from 2012 to 2014 in packing houses in the Netherlands. In general, low incidences of 1 to 5% were recorded. Lesions showed brown and watery circular necrosis, were slightly sunken, often with visible whitish, yellowish, or pink mycelia covering the lesions. Fruits were rinsed with sterile water, and lesions were sprayed with 70% ethanol until droplet runoff. The skin was removed aseptically with a scalpel, and tissue under the lesion was isolated and placed onto potato dextrose agar (PDA). The PDA plates were incubated at 20°C in the dark, and single spore isolates were propagated on fresh PDA plates. These isolates produced fast-growing colonies with extensive aerial mycelium which was initially white, then turning yellow to pink and a dark pink to red reverse. Macroconidia were slightly falcate, thin-walled, usually 5 septate, with a tapering apical cell, and measured 40 to 80 × 3.5 to 5 μm. Both cultural and morphological characteristics of the pathogen were similar to those described for Fusarium spp. The identity of two representative isolates (Fu5-44261 and Fu6-44280) from different pear lots was confirmed by means of multilocus gene sequencing. Genomic DNA was extracted using the LGC Mag Plant Kit (Berlin) in combination with the Kingfisher method (Waltham, MA). Sequences of ITS region, translation elongation factor 1-alpha (TEF1), and histone H3 (HIS3) loci were amplified and sequenced. The sequences have been deposited in GenBank under accession numbers KT350588 and 89 (ITS), KT350605 and 06 (TEF1), and KT935569 and 70 (HIS3). MegaBLAST analysis revealed that our ITS sequences matched with 99.8 to 100% identity to Fusarium spp. belonging to the Fusarium tricinctum species complex. The TEF1 sequences of both isolates were 99 to 100% identical with F. avenaceum culture collection sequences in GenBank (JQ429374 and GQ915502). The HIS3 sequences of both isolates were 97 to 99% identical with the sequences of accessions JQ435857 and GQ915469, confirming the identity of these isolates as F. avenaceum. Koch’s postulates were performed on 15 ‘Conference’ pears per isolate. Surface sterilized fruit were inoculated with 20 μl of a suspension of 105 macroconidia ml–1 prepared from a 15-day-old PDA culture, after wounding with a needle. Inoculated fruits were sealed in a plastic bag and incubated in darkness at 20°C. Symptoms appeared after 4 to 6 days on 100% of the fruits while mock-inoculated controls with water remained symptomless. Fungal colonies isolated from the lesions and cultured on PDA morphologically resembled the original isolate from the infected pears. Symptoms observed on artificially inoculated ‘Conference’ pear fruit were identical to the decay observed on ‘Conference’ pears that were obtained from cold storage. Only few reports describe symptoms associated with F. avenaceum on apple in Europe and the United States (Kou et al. 2014; Sever et al. 2012; Sorensen et al. 2009), while F. avenaceum infections of pears have not been reported. Thus, this is the first report of storage decay caused by F. avenaceum on pear. F. avenaceum infections may constitute a safety issue due to the potential production of mycotoxins such as moniliformin (Sorensen et al. 2009).

Beta-hemolytic Streptococcus dysgalactiae strains isolated from horses are a genetically distinct population within the Streptococcus dysgalactiae taxon.

The pathogenic role of beta-hemolytic Streptococcus dysgalactiae in the equine host is increasingly recognized. A collection of 108 Lancefield group C (n = 96) or L (n = 12) horse isolates recovered in the United States and in three European countries presented multilocus sequence typing (MLST) alleles, sequence types and emm types (only 56% of the isolates could be emm typed) that were, with few exceptions, distinct from those previously found in human Streptococcus dysgalactiae subsp. equisimilis. Characterization of a subset of horse isolates by multilocus sequence analysis (MLSA) and 16S rRNA gene sequence showed that most equine isolates could also be differentiated from S. dysgalactiae strains from other animal species, supporting the existence of a horse specific genomovar. Draft genome information confirms the distinctiveness of the horse genomovar and indicates the presence of potentially horse-specific virulence factors. While this genomovar represents most of the isolates recovered from horses, a smaller MLST and MLSA defined sub-population seems to be able to cause infections in horses, other animals and humans, indicating that transmission between hosts of strains belonging to this group may occur.

First Report of Neonectria candida Causing Postharvest Decay on ‘Conference’ Pears in the Netherlands

Pear (Pyrus communis) is an important fruit crop in the Netherlands, with a total production of 349,000 tons in 2014, and ‘Conference’ is the main pear cultivar that occupies 75% of the total pear production area. In the Netherlands, pears are kept in controlled atmosphere cold storage up to 11 months after harvest. Occasionally, storage rots are observed when storage crates are contaminated with orchard soil. In a storage trial (2012 to 2013), boxes with ‘Conference’ pears were amended with soil particles from the same orchard from which the pears were harvested (four orchards), and stored for 11 months. Boxes without amended soil were included as controls. In contrast to the control boxes, up to 15% of the pears stored in boxes with soil particles showed typical rot symptoms (lesions) of an unknown causal agent. The lesions showed brown and watery circular necrosis, were slightly sunken, and displayed whitish to yellowish mycelia covering the lesions. To isolate the causal agent, fruit were rinsed with sterile water, lesions were sprayed with 70% ethanol until droplet runoff, the skin was removed aseptically with a scalpel, and tissue under the lesion was isolated and placed onto potato dextrose agar (PDA). The PDA plates were incubated at 20°C in the dark, and single spore isolates were transferred to fresh PDA plates. These isolates produced fast-growing colonies with white-yellowish mycelium. Conidia were hyaline, cylindrical, 1 to 3 septate, and 15.8 to 26.4 × 5.3 to 7.9 µm. The fungus was morphologically identical to Neonectria candida (syn. N. ramulariae; anamorph Cylindrocarpon obtusiusculum) (Lombard et al. 2015). The identity of a representative isolate (VTN10Bs3) was confirmed by means of multilocus gene sequencing. To this end, genomic DNA was extracted using the LGC Mag Plant Kit (Berlin, Germany) in combination with the Kingfisher method (Waltham, USA). Sequences of the ITS region, translation elongation factor 1-alpha (TEF1), and actin (ACT2) loci were amplified, sequenced, and deposited in GenBank under accessions KU588183 (ITS), KU588186 (TEF1), and KU588184 (ACT2). MegaBLAST analysis revealed that our ITS, TEF1, and ACT2 sequences matched with >99 to 100% identity to N. candida isolates in GenBank (KM249079 and JF735314 [ITS], JF735791 and HM054091 [TEF1], and KM231146 [ACT2]). Subsequently, Koch’s postulates were performed on 15 ‘Conference’ pears. Surface sterilized fruits were inoculated with 20 µl of a suspension of 105 conidiospores ml–1 water, prepared from a 15-day-old PDA culture, after wounding with a needle. Inoculated fruits were sealed in a plastic bag and incubated in darkness at 20°C. Symptoms appeared after 7 days on 100% of the fruits while mock-inoculated controls with water remained symptomless. Fungal colonies isolated from the lesions and cultured on PDA morphologically resembled the original isolate from the infected pears. Moreover, symptoms observed on artificially inoculated ‘Conference’ pear fruit were identical to the decay observed on ‘Conference’ pears that were obtained from the cold storage experiment. The identity of the reisolations was confirmed as N. candida by sequencing. N. candida (syn. N. ramulariae) is known as a globally distributed soilborne fungus (Domsch et al. 2007), but only few studies have identified the fungus as plant pathogen (Hirooka 2012). This is the first report of N. candida causing storage rot of pears. Importantly, we note that the occurrence of storage rots may be enhanced by contamination of storage crates or fruit with orchard soil.

First Report of Fusarium avenaceum Causing Wet Core Rot of ‘Elstar’ Apples in the Netherlands

Apple (Malus domestica) is an important fruit crop in the Netherlands. ‘Elstar’, the main cultivar, occupies 40% of the apple production area. Symptoms of apple wet core rot were observed on Elstar in January to March 2013 after 4 to 6 months’ storage in different packing houses at controlled atmosphere. The disease was present in a large number of lots harvested at orchards from different locations across the Netherlands, and incidences up to 25% were recorded. Apples exhibited light-brown wet rot, initially developing in the core and subsequently spreading into the surrounding cortex, often with a white to rose-reddish mycelium. Apples from five lots with infections (four apples per lot; 20 in total) were rinsed with sterile water, sprayed with 70% ethanol until droplet runoff and halved aseptically with a scalpel. The tissue next to the core rot was isolated and placed onto potato dextrose agar (PDA). The PDA plates were incubated at 20°C in the dark, and single-spore isolates were propagated on fresh PDA plates. All cultures formed abundant white aerial mycelium with yellow to rose pigment and a dark pink to red reverse. Macroconidia were slightly falcate, thin-walled, usually 5 septate, with a tapering apical cell, and 40 to 80×3.5 to 5 μm. Pathogen characteristics were similar to those described for F. avenaceum, causal agent of wet core rot of apple (Sanzani et al. 2013;Sorenson et al. 2009). The identity of two representative isolates (Fu1-44145 and Fu2-44145) from different apple lots was confirmed by means of multilocus gene sequencing. Genomic DNA was extracted and sequences of ITS region, TEF1-α, and histone H3 loci were amplified and sequenced. The sequences have been deposited in GenBank (Accession Nos. KT350586 and KT350587 [ITS], KT350603 and KT350604 [TEF1], KT935567 and KT935568 [HIS3]). MegaBLAST analysis revealed 99.8 to 100% identity to Fusarium spp. belonging to the F. avenaceum-F. tricinctumspecies complex. The TEF1 and HIS3 sequences of both isolates were 100% identical with F.avenaceum sequences JQ429374, GQ915502, JQ435857, and GQ915469, confirming their identity as F. avenaceum. Koch’s postulates were satisfied in two experiments, with 15 Elstar apples per isolate. In the first experiment, surface-sterilized fruits were inoculated with 20 μl of a suspension of 105conidiospores/ml prepared from a 15-day-old PDA culture after wounding with a needle. Inoculated fruits were sealed in a plastic bag and incubated in darkness at 20°C. In the second experiment, apples were surface sterilized, cut in half longitudinally, and one half was inoculated with 20 μl of the conidiospore suspension into the core of the apple. Control apple halves were inoculated with sterilized water. After inoculation, the halves were covered with plastic foil and incubated in darkness at 20°C. In both experiments, symptoms appeared after 4 to 6 days on 100% of the fruits; controls remained symptomless. Fungal colonies isolated from the lesions and cultured on PDA plates morphologically resembled the original isolates. Fusarium avenaceum is a wound pathogen that has been isolated from apple fruit in Croatia and in the United States (Kou et al. 2014; Sever et al. 2012). Only few reports describe wet core symptoms associated with F. avenaceum on apple (Sanzani et al. 2013; Sorenson et al. 2009). This is the first report of wet core rot caused by F. avenaceum on apple fruit in the Netherlands. As wet core of apple is undetectable until the fruit is cut or consumed, it affects consumer confidence. Due to potential mycotoxin production during infection, F. avenaceum infections potentially pose a safety issue (Sorenson et al. 2009).

Colwellia and sulfur-oxidizing bacteria: An unusual dual symbiosis in a Terua mussel (Mytilidae: Bathymodiolinae) from whale falls in the Antilles arc

Seven individuals of a single morphotype of mussels (Bivalvia: Mytilidae) were found attached to a naturally sunken whale intervertebral disk collected in Guadeloupe (Caribbean) at 800m depth. These specimens resemble small Idas mussels which are found worldwide at cold seeps and hydrothermal vents, and typically harbor ectosymbiotic bacteria on their gills upon which they depend for nutrition. Based on multi-locus gene sequencing, these specimens appear to belong to a new species closely related to two species now included within the genus Terua. Unexpectedly, its closest relatives are found in the Pacific, questioning how this species has reached the Antilles arc. Based on marker gene sequence analysis, electron and fluorescence microscopy, Terua n. sp. harbors two distinct and abundant extracellular bacterial symbionts located between microvilli at the apical surface of host gill epithelial cells. One is a sulfur-oxidizing bacterium similar to the symbionts previously identified in several deep-sea mussels, while the other is related to Colwellia species, a group of cold-adapted heterotrophic bacteria able to degrade organic compounds. This study provides the first evidence for the existence of a dual symbiosis in mussels from whale fall ecosystems in the Caribbean. The evolutionary history of Terua n. sp. and potential role of its Colwellia symbionts are discussed.

Imbalanced presence of Borrelia burgdorferi s.l. multilocus sequence types in clinical manifestations of Lyme borreliosis

In this study we used typing based on the eight multilocus sequence typing scheme housekeeping genes (MLST) and 5S-23S rDNA intergenic spacer (IGS) to explore the population structure of Borrelia burgdorferi sensu lato isolates from patients with Lyme borreliosis (LB) and to test the association between the B. burgdorferi s.l. sequence types (ST) and the clinical manifestations they cause in humans. Isolates of B. burgdorferi from 183 LB cases across Europe, with distinct clinical manifestations, and 257 Ixodes ricinus lysates from The Netherlands, were analyzed for this study alone. For completeness, we incorporated in our analysis also 335 European B. burgdorferi s.l. MLST profiles retrieved from literature. Borrelia afzelii and Borrelia bavariensis were associated with human cases of LB while Borrelia garinii, Borrelia lusitaniae and Borrelia valaisiana were associated with questing I. ricinus ticks. B. afzelii was associated with acrodermatitis chronica atrophicans, while B. garinii and B. bavariensis were associated with neuroborreliosis. The samples in our study belonged to 251 different STs, of which 94 are newly described, adding to the overall picture of the genetic diversity of Borrelia genospecies. The fraction of STs that were isolated from human samples was significantly higher for the genospecies that are known to be maintained in enzootic cycles by mammals (B. afzelii, B. bavariensis, and Borrelia spielmanii) than for genospecies that are maintained by birds (B. garinii and B. valaisiana) or lizards (B. lusitaniae). We found six multilocus sequence types that were significantly associated to clinical manifestations in humans and five IGS haplotypes that were associated with the human LB cases. While IGS could perform just as well as the housekeeping genes in the MLST scheme for predicting the infectivity of B. burgdorferi s.l., the advantage of MLST is that it can also capture the differential invasiveness of the various STs.

Engineering super mycovirus donor strains of chestnut blight fungus by systematic disruption of multilocus vic genes

Transmission of mycoviruses that attenuate virulence (hypovirulence) of pathogenic fungi is restricted by allorecognition systems operating in their fungal hosts. We report the use of systematic molecular gene disruption and classical genetics for engineering fungal hosts with superior virus transmission capabilities. Four of five diallelic virus-restricting allorecognition [vegetative incompatibility (vic)] loci were disrupted in the chestnut blight fungus Cryphonectria parasitica using an adapted Cre-loxP recombination system that allowed excision and recycling of selectable marker genes (SMGs). SMG-free, quadruple vic mutant strains representing both allelic backgrounds of the remaining vic locus were then produced through mating. In combination, these super donor strains were able to transmit hypoviruses to strains that were heteroallelic at one or all of the virus-restricting vic loci. These results demonstrate the feasibility of modulating allorecognition to engineer pathogenic fungi for more efficient transmission of virulence-attenuating mycoviruses and enhanced biological control potential.

First Report of Colletotrichum godetiae Causing Bitter Rot on ‘Golden Delicious’ Apples in the Netherlands

Apple (Malus domestica) is an important fruit crop in the Netherlands, with a total production of 418,000 tons in 2011. Symptoms of apple bitter rot were observed on ‘Golden Delicious’ apples in the Netherlands in July 2013 after 9 months of storage in a packing house at controlled atmosphere. Lesions were round, 1 to 5 cm in diameter, gray and dry with acervuli, producing orange spore masses in concentric rings. Fruit were rinsed with sterile water, and lesions were sprayed with 70% ethanol until droplet runoff. The skin was removed aseptically with a scalpel, and tissue under the lesion was isolated and placed onto Potato Dextrose Agar (PDA). The PDA plates were incubated at 20°C in the dark, and single-spore isolates were propagated on PDA. The isolates were identified as Colletotrichum sp. based on culture morphology, having light gray to pale orange mycelium and, when viewed from the reverse side, ranged from pink to reddish orange. The cultures carried yellowish spore masses and dark melanized structures similar to acervuli that oozed orange conidia. Conidia were cylindrical to fusiform, pointed at one or both ends, and measured 8.0 to 17.0 μm × 3.5 to 5.0 μm. Both cultural and morphological characteristics of the pathogen were similar to those described for C. acutatum, causal agent of bitter rot of apple. A representative isolate (PPO-44377) was used for multilocus gene sequencing (Damm et al. 2012). Genomic DNA was extracted using the LGC Mag Plant Kit (Berlin, Germany) in combination with the KingFisher method (Waltham, USA) and six loci were amplified and sequenced. Primer pairs ACT-512F + ACT-783R, CHS-354R + CHS-79F, GDF1 + GDR1, CYLH3F + CYLH3R, BT2Fd + BT4R, and ITS1 and ITS4 (White et al. 1990) were used for amplification of parts of the actin (ACT), chitin synthase (CHS-1) gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone H3 (HIS3), beta-tubulin (TUB2) and ITS region of the rDNA gene, respectively. One sequence for each locus has been deposited in GenBank under Accession Nos. KR180290 (ACT), KR180292 (CHS-1), KR180293 (GAPDH), KR180294 (HIS3), KR180295 (TUB2), and KR180296 (ITS). MegaBLAST analysis revealed that the ITS sequences matched with 98.9 to 100% identity to Colletotrichum spp. belonging to C. acutatum species complex (including C. godetiae). The phylogenetic trees constructed using sequences of ACT, CHS-1, GAPDH, HIS3, and TUB2 of our strain (PPO-44377), and available sequences from GenBank confirmed the identity of this strain as C. godetiae. Koch’s postulates were performed on 15 ‘Golden Delicious’ apples. Surface-sterilized fruit were inoculated with 20 μl of a spore suspension (105 conidia/ml) prepared from a 15-day-old PDA culture after wounding with a needle. Inoculated fruits were sealed in a plastic bag and were incubated in darkness at 20°C. Symptoms appeared after 4 to 6 days on 80% of the fruits while mock-inoculated controls with water remained symptomless. Fungal colonies isolated from the lesions and cultured on PDA had morphological characteristics that resembled the original isolate from the infected apples. There are few reports of symptoms associated with C. godetiae on apple in Europe (Baroncelli et al. 2014; Ivic et al. 2013; Munda 2014). This is the first report of bitter rot caused by C. godetiae on apple fruit in the Netherlands. Currently, bitter rot is not an important disease in apples in the Netherlands. However, it is ranges worldwide and is considered one of the most important diseases, causing considerable crop losses, and may become an emerging problem in the Netherlands in the near future.

The complete mitochondrial genome sequence of Japanese murrelet (Aves: Alcidae) and its phylogenetic position in Charadriiformes

The Japanese murrelet (Synthliboramphus wumizusume) is a threatened bird endemic to Japan, Korea, and Russia. We generated the complete mitochondrial genome sequence to provide molecular genetic information for phylogeny and conservation of the species. The S. wumizusume mitochondrial genome is 16 714 bp in length and comprises 13 protein-coding genes, 22 transfer RNAs, two ribosomal RNAs, a non-coding control region, and a repeat region. Gene composition and order in the genome is consistent with that of other mitochondrial genomes of the order Charadriiformes currently available in the GenBank. Phylogenetic analysis using all available Charadriiform mitochondrial genomes revealed that interfamilial relationships of the birds based on mitochondrial genes were in agreement with those based on multilocus nuclear genes. The complete mitochondrial genome of S. wumizusume we sequenced might be a useful genetic resource for phylogenetic relationships, evolutionary biology, and conservation of the species.

Exoproteome analysis reveals higher abundance of proteins linked to alkaline stress in persistent Listeria monocytogenes strains

The foodborne pathogen Listeria monocytogenes, responsible for listeriosis a rare but severe infection disease, can survive in the food processing environment for month or even years. So-called persistent L. monocytogenes strains greatly increase the risk of (re)contamination of food products, and are therefore a great challenge for food safety. However, our understanding of the mechanism underlying persistence is still fragmented.In this study we compared the exoproteome of three persistent strains with the reference strain EGDe under mild stress conditions using 2D differential gel electrophoresis. Principal component analysis including all differentially abundant protein spots showed that the exoproteome of strain EGDe (sequence type (ST) 35) is distinct from that of the persistent strain R479a (ST8) and the two closely related ST121 strains 4423 and 6179. Phylogenetic analyses based on multilocus ST genes showed similar grouping of the strains.Comparing the exoproteome of strain EGDe and the three persistent strains resulted in identification of 22 differentially expressed protein spots corresponding to 16 proteins. Six proteins were significantly increased in the persistent L. monocytogenes exoproteomes, among them proteins involved in alkaline stress response (e.g. the membrane anchored lipoprotein Lmo2637 and the NADPH dehydrogenase NamA). In parallel the persistent strains showed increased survival under alkaline stress, which is often provided during cleaning and disinfection in the food processing environments. In addition, gene expression of the proteins linked to stress response (Lmo2637, NamA, Fhs and QoxA) was higher in the persistent strain not only at 37°C but also at 10°C. Invasion efficiency of EGDe was higher in intestinal epithelial Caco2 and macrophage-like THP1 cells compared to the persistent strains. Concurrently we found higher expression of proteins involved in virulence in EGDe e.g. the actin-assembly-inducing protein ActA and the surface virulence associated protein SvpA. Furthermore proteins involved in cell wall modification, such as the lipoteichonic acid primase LtaP and the N-acetylmuramoyl-l-alanine amidase (Lmo2591) are more abundant in EGDe than in the persistent strains and could indirectly contribute to virulence.In conclusion this study provides information about a set of proteins that could potentially support survival of L. monocytogenes in abiotic niches in food processing environments. Based on these data, a more detailed analysis of the role of the identified proteins under stresses mimicking conditions in food producing environment is essential for further elucidate the mechanism of the phenomenon of persistence of L. monocytogenes.

High genetic similarity of ciprofloxacin-resistant Campylobacter jejuni in central Europe.

Campylobacteriosis is the leading zoonosis in the European Union with the majority of cases attributed to Campylobacter jejuni. Although the disease is usually self-limiting, some severe cases need to be treated with antibiotics, primarily macrolides and quinolones. However, the resistance to the latter is reaching alarming levels in most of the EU countries. To shed light on the expansion of antibiotic resistance in central Europe, we have investigated genetic similarity across 178 ciprofloxacin-resistant C. jejuni mostly isolated in Slovenia, Austria and Germany. We performed comparative genetic similarity analyses using allelic types of seven multilocus sequence typing housekeeping genes, and single nucleotide polymorphisms of a quinolone resistance determining region located within the DNA gyrase subunit A gene. This analysis revealed high genetic similarity of isolates from clonal complex ST-21 that carry gyrA allelic type 1 in all three of these central-European countries, suggesting these ciprofloxacin resistant isolates arose from a recent common ancestor and are spread clonally.

“You Can’t Always Get What You Want…” The Mycobacterium abscessus Issue. Name versus Function

“You Can’t Always Get What You Want…” The <i>Mycobacterium abscessus</i> Issue. Name versus Function