Barrage formation is independent from heterokaryon incompatibility in Verticillium dahliae

Barrage formation has been traditionally used for the assessment of mycelial compatibility in many fungi and has often been assumed to represent a non-self recognition phenotype that is directly associated with vegetative incompatibility in these organisms. In this work, the optimal growth conditions for large-scale studies of barrage formation in the asexual fungus Verticillium dahliae were determined, and they were used for the analysis of a diverse collection comprising 69 isolates of V. dahliae and related species. Barrage formation was very frequent on a defined complete agar medium within V. dahliae and between species of the genus. However, it was not correlated with the classification of V. dahliae isolates into Vegetative Compatibility Groups (VCGs) (based on the standard method using complementary nit mutants), as it was recorded at high frequencies both within and between V. dahliae VCGs. The high overall frequency of barrage formation demonstrated the presence of a higher level of mycelial incompatibility in V. dahliae than heterokaryon incompatibility assessed by forcing complementary nit mutants to form heterokaryons under selective conditions. The possible association of barrage formation with morphological characteristics of the fungal colonies was investigated, and a negative correlation of frequency and intensity of barrages with the isolates’ capacity for pigment production was detected. Real-time quantitative PCR VCG discriminatio

High-Throughput Assessment and Genetic Investigation of Vegetative Compatibility in Verticillium dahliae

Classification of isolates into vegetative compatibility groups (VCGs) using nitrate-non-utilizing (nit) mutants has been widely used for the characterization of Verticillium dahliae populations. However, certain methodological limitations prevent its application on a large scale. Furthermore, systematic investigations into the genetics underlying complementation tests between nit mutants of fungal isolates (i.e. heterokaryon formation) are lacking for Verticillium species. In this work, a diverse collection of 27 V. dahliae isolates – including representatives of all VCGs, both mating types, and heterokaryon self-incompatible isolates – was employed for the development and optimization of (i) a protocol for the rapid generation of nit mutants of V. dahliae isolates using UV-irradiation and (ii) a reproducible high-throughput procedure for complementation tests between nit mutants in liquid cultures using 96-well microplates. The genetic analysis of selected heterokaryons demonstrated that the frequently encountered ‘weak’ cross-reactions between VCGs and their subgroups can be actually heterokaryotic, implying the absence of strict genetic barriers between VCGs. In conclusion, we provide in this work an optimized method for the high-throughput VCG assignment of V. dahliae populations and a genetic analysis of heterokaryons that may have serious implications for the interpretation of VCG classification data. These advancements in the available methodology and the genetic background of vegetative compatibility grouping may contribute to a better understanding of the population biology of V. dahliae and possibly other mitosporic fun

‘‘Cryptic’’ group-I introns in the nuclear SSU-rRNA gene of Verticillium dahliae

Group-I introns are widespread—though irregularly distributed—in eukaryotic organisms, and they have been extensively used for discrimination and phylogenetic analyses. Within the Verticillium genus, which comprises important phytopathogenic fungi, a group-I intron was previously identified in the SSU-rRNA (18S) gene of only V. longisporum. In this work, we aimed at elucidating the SSU-located intron distribution in V. dahliae and other Verticillium species, and the assessment of heterogeneity regarding intron content among rDNA repeats of fungal strains. Using conserved PCR primers for the amplification of the SSU gene, a structurally similar novel intron (sub-group IC1) was detected in only a few V. dahliae isolates. However, when intron-specific primers were used for the screening of a diverse collection of Verticillium isolates that originally failed to produce intron-containing SSU amplicons, most were found to contain one or both intron types, at variable rDNA repeat numbers. This marked heterogeneity was confirmed with qRT-PCR by testing rDNA copy numbers (varying from 39 to 70 copies per haploid genome) and intron copy ratios in selected isolates. Our results demonstrate that (a) IC1 group-I introns are not specific to V. longisporum within the Verticillium genus, (b) V. dahliae isolates of vegetative compatibility groups (VCGs) 4A and 6, which bear the novel intron at most of their rDNA repeats, are closely related, and (c) there is considerable intra-genomic heterogeneity for the presence or absence of introns among the ribosomal repeats. These findings underline that distributions of introns in the highly heterogeneous repetitive rDNA complex should always be verified with sensitive methods to avoid misleading conclusions for the phylogeny of fungi and other organisms

Structural and phylogenetic analysis of the rDNA intergenic spacer region of Verticillium dahliae

The nuclear ribosomal intergenic spacer (IGS) region was structurally analyzed and exploited for molecular discrimination and phylogenetic analysis of vegetative compatibility groups (VCGs) of Verticillium dahliae. A structural study of 201 available IGS sequences of the fungus was performed, and four classes of ubiquitous repetitive elements, organized in higher-order repetitive structures or composite blocks, were detected in a variable IGS subregion. This subregion was amplified from an international collection of 59 V. dahliae isolates covering all VCGs, together with nine representative V. albo-atrum and V. longisporum isolates, and sequenced. Structural and phylogenetic analyses of the sequences of this polymorphic IGS subregion were consistently informative and allowed the identification of two main lineages in V. dahliae, that is, clade I including VCGs 1A, 1B, 2A, 4B, and 3 and clade II containing VCGs 2B, 4A, and 6. Analysis of IGS sequences proved a highly suitable molecular tool for (a) rapid interspecific differentiation, (b) intraspecific discrimination among VCGs of V. dahliae, facilitating high-throughput VCG confirmation and prediction/profiling, and (c) phylogenetic analysis within and among V. dahliae VCGs

Transcriptomic Adjustments of Staphylococcus aureus COL (MRSA) Forming Biofilms Under Acidic and Alkaline Conditions

© Copyright © 2019 Efthimiou, Tsiamis, Typas and Pappas. Methicillin-resistant Staphylococcus aureus (MRSA) strains are important human pathogens and a significant health hazard for hospitals and the food industry. They are resistant to β-lactam antibiotics including methicillin and extremely difficult to treat. In this study, we show that the Staphylococcus aureus COL (MRSA) strain, with a known complete genome, can easily survive and grow under acidic and alkaline conditions (pH5 and pH9, respectively), both planktonically and as a biofilm. A microarray-based analysis of both planktonic and biofilm cells was performed under acidic and alkaline conditions showing that several genes are up- or down-regulated under different environmental conditions and growth modes. These genes were coding for transcription regulators, ion transporters, cell wall biosynthetic enzymes, autolytic enzymes, adhesion proteins and antibiotic resistance factors, most of which are associated with biofilm formation. These results will facilitate a better understanding of the physiological adjustments occurring in biofilm-associated S. aureus COL cells growing in acidic or alkaline environments, which will enable the development of new efficient treatment or disinfection strategies

Effect of rocket (Eruca sativa) extract on MRSA growth and proteome: Metabolic adjustments in plant-based media

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) in food has provoked a great concern about the presence of MRSA in associated foodstuff. Although MRSA is often detected in various retailed meat products, it seems that food handlers are more strongly associated with this type of food contamination. Thus, it can be easily postulated that any food could be contaminated with this pathogen in an industrial environment or in household and cause food poisoning. To this direction, the effect of rocket (Eruca sativa) extract on MRSA growth and proteome was examined in the present study. This goal was achieved with the comparative study of the MRSA strain COL proteome, cultivated in rocket extract versus the standard Luria-Bertani growth medium. The obtained results showed that MRSA was able to grow in rocket extract. In addition, proteome analysis using 2-DE method showed that MRSA strain COL is taking advantage of the sugar-, lipid-, and vitamin-rich substrate in the liquid rocket extract, although its growth was delayed in rocket extract compared to Luria-Bertani medium. This work could initiate further research about bacterial metabolism in plant-based media and defense mechanisms against plant-derived antibacterials

The role of the β-1,6-endoglucanase gene vegB in physiology and virulence of Verticillium dahliae

The β-1,6-endoglucanase gene (vegB) of Verticillium dahliae was isolated using a genome walking technique. Nucleotide and deduced amino acid sequences of the gene showed high identity with the PAN1 sequence deposited at the Verticillium genome database (Broad Institute), but significant differences in intron numbers and sites of insertion. Detailed in silico analysis, accompanied by sequencing of both genomic and cDNA, as well as RT-PCR experiments, provided the correct size of the gene and the exact number, length and positions of introns. The putative protein of this gene was compared with corresponding β-1,6-endoglucanases from other fungi, and sequences were used to construct a phylogenetic tree. A clear differentiation between enzymes derived from plant pathogenic and mycoparasitic fungi was observed, fully supported by bootstrap data. An internal fragment (1.2kb) of vegB was used to disrupt the wild-type gene of a V. dahliae tomato race 2 strain, and the mutant strain, vegB-, was tested for pathogenicity on tomato plants. Results showed a small but constant reduction in disease symptoms only on eggplants for the vegB- strain in comparison with the wild type. Growth on minimal medium supplemented with different carbon sources showed reduced ability of the mutant to breakdown cellulose, whereas growth on glucose, pectin and sucrose was similar to the wild type

Revisiting the phylogeny and taxonomy of the genus Sidera (Hymenochaetales, Basidiomycota) with particular emphasis on S. vulgaris

The genus Sidera (Hymenochaetales, Basidiomycota) comprises white-rot, mono- or dimitic fungi with poroid or hydnoid hymenophore. It has a worldwide distribution albeit with fewer species present in the Southern Hemisphere. Although recent studies revealed the existence of several new Sidera species, there are still taxonomic inconsistencies and obscure phylogenetic relationships amongst certain taxa of the genus. In this work, a large number of Sidera collections were used to obtain an updated phylogeny, based on ITS and 28S rDNA sequences by including new material from Mediterranean Europe. The monophyly of the genus was strongly supported and all species with poroid hymenophore formed a highly-supported lineage with two major subclades. In total, 23 putative species were recognised. Amongst those, five are considered to possibly represent entities new to science, but further work is required since they are represented by single specimens or environmental sequences. Examined collections originally named S. lenis from southern Europe were grouped within S. vulgaris. Similarly, several collections under various names were hereby identified as S. vulgaris, including those of the recently described species S. tibetica. Furthermore, a critical discussion (based on morphoanatomical findings) is made on the key features that could be used to distinguish S. lenis from S. vulgaris

Revisiting the phylogeny and taxonomy of the genus Sidera (Hymenochaetales, Basidiomycota) with particular emphasis on S. vulgaris

The genus Sidera (Hymenochaetales, Basidiomycota) comprises white-rot, mono- or dimitic fungi with poroid or hydnoid hymenophore. It has a worldwide distribution albeit with fewer species present in the Southern Hemisphere. Although recent studies revealed the existence of several new Sidera species, there are still taxonomic inconsistencies and obscure phylogenetic relationships amongst certain taxa of the genus. In this work, a large number of Sidera collections were used to obtain an updated phylogeny, based on ITS and 28S rDNA sequences by including new material from Mediterranean Europe. The monophyly of the genus was strongly supported and all species with poroid hymenophore formed a highly-supported lineage with two major subclades. In total, 23 putative species were recognised. Amongst those, five are considered to possibly represent entities new to science, but further work is required since they are represented by single specimens or environmental sequences. Examined collections originally named S. lenis from southern Europe were grouped within S. vulgaris. Similarly, several collections under various names were hereby identified as S. vulgaris, including those of the recently described species S. tibetica. Furthermore, a critical discussion (based on morphoanatomical findings) is made on the key features that could be used to distinguish S. lenis from S. vulgaris