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Evolutionary change in 5S rRNA secondary structure and a phylogenic tree of 352 5S rRNA species.

The secondary structure models of 5S rRNA have been constructed from the primary structure of 352 5S rRNA species available at present. All the 5S rRNAs examined can take essentially the same secondary structure, however they reveal characteristic differences between eukaryotes, metabacteria (= archaebacteria) and eubacteria. These three types of models can be further subgrouped by minor but characteristic differences. A phylogenic tree of organisms has been constructed using these 5S rRNA sequences by the weighted pairing method (WPG method). The tree reveals that there exist several major groups of eubacteria which seem to have diverged into different directions in the early stages of bacterial evolution. After emergence of eubacteria, metabacteria and eukaryotes separated from each other from their common ancestor. In the eukaryotic evolution, red algae (Rhodophyta) emerged first, and thereafter, thraustocytrids-Proctista, Ascomycota, green plants (green algae and land plants), Basidiomycota, Chromophyta (brown algae, diatoms and golden-yellow algae), slime- and water molds, various protozoans, and animals emerged in this order.

Animals↗

Biodegradation of phenol and phenol-related compounds by psychrophilic and cold-tolerant alpine yeasts.

We characterized 32 cold-adapted, psychrophilic and cold-tolerant, yeast strains isolated from alpine habitats with regard to their taxonomy, growth temperature profile, and ability to degrade phenol and 18 phenol-related mono-aromatic compounds at 10 degrees C. Twenty of the strains were identified by sequencing of the ribosomal ITS region as seven species of the basidiomycota: Cryptococcus terreus (three strains), Cryptococcus terricola (one strain), Rhodosporidium lusitaniae (two strains), Rhodotorula creatinivora (10 strains), Rhodotorula ingeniosa (one strain), Mastigobasidium intermedium (one strain), and Sporobolomyces roseus (two strains). Twelve strains sharing closely related ITS sequences could not be identified to the species level; according to their ITS sequence they are included in the Microbotryomycetidae. These 12 strains were psychrophilic (no growth at temperatures above 20 degrees C); one-third of these strains did not grow above 15 degrees C. None of the 32 strains utilized any of the highly volatile mono-aromatic compounds (benzene, toluene, ethylbenzene, nitrobenzene, o-xylene, m-xylene, and p-xylene) as the sole carbon source. Non/low volatile aromatic compounds were degraded in the following order: phenol>hydroquinone>resorcinol>benzoate>catechol>salicylate>>p-cresol>m-cresol. o-Cresol, guaiacol, p-nitrophenol, or p-nitrotoluene were not utilized for growth. R. creatinivora strains degraded up to seven compounds, whereas C. terricola and S. roseus strains degraded only two compounds. The toxicity of the compounds was determined via growth inhibition in the presence of toxicants and nutrients at 10 degrees C. R. creatinivora strains were characterized by higher IC50 values than other species, S. roseus was the most sensitive species. The most toxic compounds were the xylene isomers, ethylbenzene, p-nitrophenol, and m-cresol. There was a relation between the chemical structure of the compounds and their toxicity, whereas a relation between the toxicity of the compounds and the ability of the yeasts strains to utilize these compounds for growth was only detected in some cases.

Adaptation, Physiological↗

Glutamic protease distribution is limited to filamentous fungi.

Glutamic proteases are a distinct, and recently re-classified, group of peptidases that are thought to be found only in fungi. We have identified and analysed the distribution of over 20 putative glutamic proteases from all fungal species whose genomes have been sequenced so far. Although absent from the Saccharomycetales class, glutamic proteases appear to be present in all other ascomycetes species examined. A large number of coding regions for glutamic proteases were also found clustered together in the Phanerochaete chrysosporium genome, despite apparently being absent from three other species of Basidiomycota.

Fungi↗

Molecular detection of Fusarium oxysporum f. sp. niveum and Mycosphaerella melonis in infected plant tissues and soil.

We developed two species-specific PCR assays for rapid and accurate detection of the pathogenic fungi Fusarium oxysporum f. sp. niveum and Mycosphaerella melonis in diseased plant tissues and soil. Based on differences in internal transcribed spacer (ITS) sequences of Fusarium spp. and Mycosphaerella spp., two pairs of species-specific primers, Fn-1/Fn-2 and Mn-1/Mn-2, were synthesized. After screening 24 isolates of F. oxysporum f. sp. niveum, 22 isolates of M. melonis, and 72 isolates from the Ascomycota, Basidiomycota, Deuteromycota, and Oomycota, the Fn-1/Fn-2 primers amplified only a single PCR band of approximately 320 bp from F. oxysporum f. sp.niveum, and the Mn-1/Mn-2 primers yielded a PCR product of approximately 420 bp from M. melonis. The detection sensitivity with primers Fn-1/Fn-2 and Mn-1/Mn-2 was 1fg of genomic DNA. Using ITS1/ITS4 as the first-round primers, combined with either Fn-1/Fn-2 and or Mn-1/Mn-2, two nested PCR procedures were developed, and the detection sensitivity increased 1000-fold to 1ag. The detection sensitivity for the soil pathogens was 100-microconidia/g soil. A duplex PCR method, combining primers Fn-1/Fn-2 and Mn-1/Mn-2, was used to detect F. oxysporum f. sp. niveum and M. melonis in plant tissues infected by the pathogens. Real-time fluorescent quantitative PCR assays were developed to detect and monitor the pathogens directly in soil samples. The PCR-based methods developed here could simplify both plant disease diagnosis and pathogen monitoring as well as guide plant disease management.

Ascomycota↗

Application of temperature gradient gel electrophoresis to the study of yeast diversity in the estuary of the Tagus river, Portugal.

Temperature gradient gel electrophoresis (TGGE) was employed for the assessment of yeast diversity in the estuary of the Tagus river (Portugal). The molecular detection of yeasts was carried out directly from water samples and, in parallel, a cultivation approach by means of an enrichment step was employed. A nested PCR was employed to obtain a fungal amplicon containing the D2 domain of the 26S rRNA gene. For identification the TGGE bands were extracted, re-amplified, and sequenced. Fourteen fungal taxa were detected and all except one were yeasts. Most yeast sequences corresponded to members of the Ascomycota and only three belonged to the Basidiomycota. Five yeasts (four ascomycetes and one basidiomycete) could not be identified to the species level due to the uniqueness of their sequences. The number of species detected after enrichment was higher than the number of taxa found using the direct detection method. This suggests that some yeast populations are present in densities that are below the detection threshold of the method. With respect to the analysis of the yeast community structure, our results indicate that the dominant populations belong to Debaryomyces hansenii, Rhodotorula mucilaginosa, Cryptococcus longus, and to an uncultured basidiomycetous yeast phylogenetically close to Cr. longus. The combined analysis of direct detection and cultivation approaches indicates a similar community structure at the two sampled sites since nine species were present at both localities.

Base Sequence↗

Restoration contexts shape the bacterial and fungal soil communities in desertification hotspots in the Brazilian semiarid region.

Desertification in the Brazilian semiarid has compromised ecosystem functionality, impacting soil microbial biodiversity. Thus, restoration strategies have been implemented, aiming to mitigate the negative impacts. However, little is known about their effects on soil microbial communities. In this study, we hypothesized that the two restoration contexts would promote distinct trajectories of soil microbial community recovery. We evaluated 36 soil samples collected from two desertification hotspots in the Brazilian semiarid, representing active (Gilbués) and passive (Irauçuba) restoration contexts. Soil DNA was extracted and subjected to 16S and ITS amplicon sequencing to characterize bacterial and fungal communities, respectively. Community differences were assessed using alpha-diversity metrics, redundancy analysis (RDA), and PERMANOVA. The results showed that within Gilbués (active restoration), bacterial and fungal community composition differed among soils under desertification and restoration. In Irauçuba (passive restoration), only native soils differed from both soils under desertification and restoration. Proteobacteria, Actinobacteriota, and Firmicutes (bacteria), and Ascomycota and Basidiomycota (fungi), were the dominant phyla in both hotspots. Bacterial and fungal communities showed distinct taxonomic patterns among native, degraded, and restored soils within each restoration context. Niche occupancy patterns also differed between restoration contexts. In conclusion, the two hotspots followed contrasting microbial recovery trajectories, demonstrating that restoration responses are context-dependent and vary according to the microbial groups, rather than supporting the universal superiority of one restoration strategy over the other.

Soil Microbiology↗

Dynamics of soil fungal communities restored with biochar from a quarry site.

Quarrying activities have intensified due to population expansion, leading to landscape degradation and ecological destruction. Quarry restoration is usually mandatory in Hong Kong, China. Although biochar is used for sustainable soil amendment, its effectiveness in restoring quarry soil with poor properties has rarely been investigated. A 24-month field study was conducted to evaluate the ecological feasibility of restoring a quarry site by using native species (that is, Castanopsis fissa and Cyclobalanopsis edithiae) and biochar amendment. The results revealed that after 24 months, the application of biochar increased the organic carbon, phosphorus and potassium of the vegetated soil by at least 120 %, 31 % and 12 %, respectively, due to higher cation exchange capacity and better plant growth. The relative abundance of Ascomycota and Basidiomycota increased by 24 % and 47 % with biochar application when C. fissa was planted, which was likely associated with the improved nutrient cycling and soil fertility. Even though adding biochar to bare soil was found to increase the complexity of fungal co-occurrence networks, biochar application only increased fungal diversity in vegetated quarry soil but usually reduced its fungal richness. Moreover, fungal co-occurrence networks in vegetated soil became less complex, suggesting that biochar potentially helped plants to assemble specific, beneficial fungal communities. This effect is most pronounced in the soil planted with C. edithiae, where the structure of fungal communities after 24 months was significantly different from that at other restoration times. This study identifies key fungal phyla enhanced by biochar in quarry soil and provides an effective strategy for facilitating the restoration and management of degraded lands, especially quarry sites.

Charcoal↗

Raman spectroscopic study of Lactarius spores (Russulales, Fungi).

Fungi are important organisms in ecosystems, in industrial and pharmaceutical production and are valuable food sources as well. Classical identification is often time-consuming and specialistic. In this study, Raman spectroscopy is applied to the analysis of fungal spores of Lactarius, an economically and ecologically important genus of Basidiomycota. Raman spectra of spores of Lactarius controversus Pers.: Fr., Lactarius lacunarum (Romagn.) ex Hora, Lactarius quieticolor Romagn. and Lactarius quietus (Fr.: Fr.) Fr. are reported for the first time. The spectra of these species show large similarity. These spectra are studied and compared with the Raman spectra of reference substances known to occur in macrofungi, including saccharides, lipids and some minor compounds that may serve as specific biomarkers (adenine, ergosterol and glycine). Most Raman bands could be attributed to specific components. In agreement with the biological role of fungal spores, high amounts of lipids were observed, the main fatty acid being oleate. In addition to different types of lipids and phospholipids, the polysaccharides chitin and amylopectin could be detected as well. The presence of trehalose is not equivocally shown, due to overlapping bands. Raman band positions are reported for the observed bands of the different species and reference products.

Agaricales↗

Conflicting phylogenetic position of Schizosaccharomyces pombe.

The phylogenetic position of the fission yeast Schizosaccharomyces pombe in the fungal Tree of Life is still controversial. Three alternative phylogenetic positions have been proposed in the literature, namely (1) a position basal to the Hemiascomycetes and Euascomycetes, (2) a position as a sister group to the Euascomycetes with the Hemiascomycetes as a basal branch, or (3) a sister group to the Hemiascomycetes with Euascomycetes as a basal branch. Here we compared 91 clusters of orthologous proteins containing a single orthologue that are shared by 19 eukaryote genomes. The major part of these 91 orthologues supports a phylogenetic position of S. pombe as a basal lineage among the Ascomycota, thus supporting the second proposition. Interestingly, part of the orthologous proteins supported a fourth, not yet described alternative, in which S. pombe is basal to both Basidiomycota and Ascomycota. Both topologies of phylogenetic trees are well supported. We believe that both reflect correctly the phylogenetic history of the species concerned. This apparent paradox may point to a heterogeneous nuclear genome of the fungi. Importantly, this needs to be taken in consideration for a correct understanding of the fungal Tree of Life.

Genetic Speciation↗

Improving phylogenetic inference of mushrooms with RPB1 and RPB2 nucleotide sequences (Inocybe; Agaricales).

Approximately 3000 bp across 84 taxa have been analyzed for variable regions of RPB1, RPB2, and nLSU-rDNA to infer phylogenetic relationships in the large ectomycorrhizal mushroom genus Inocybe (Agaricales; Basidiomycota). This study represents the first effort to combine variable regions of RPB1 and RPB2 with nLSU-rDNA for low-level phylogenetic studies in mushroom-forming fungi. Combination of the three loci increases non-parametric bootstrap support, Bayesian posterior probabilities, and resolution for numerous clades compared to separate gene analyses. These data suggest the evolution of at least five major lineages in Inocybe-the Inocybe clade, the Mallocybe clade, the Auritella clade, the Inosperma clade, and the Pseudosperma clade. Additionally, many clades nested within each major lineage are strongly supported. These results also suggest the family Crepiodataceae sensu stricto is sister to Inocybe. Recognition of Inocybe at the family level, the Inocybaceae, is recommended.

Agaricales↗

Phylogenetic comparison of protein-coding versus ribosomal RNA-coding sequence data: a case study of the Lecanoromycetes (Ascomycota).

The resolving power and statistical support provided by two protein-coding (RPB1 and RPB2) and three ribosomal RNA-coding (nucSSU, nucLSU, and mitSSU) genes individually and in various combinations were investigated based on maximum likelihood bootstrap analyses on lichen-forming fungi from the class Lecanoromycetes (Ascomycota). Our results indicate that the optimal loci (single and combined) to use for molecular systematics of lichen-forming Ascomycota are protein-coding genes (RPB1 and RPB2). RPB1 and RPB2 genes individually were phylogenetically more efficient than all two- and three-locus combinations of ribosomal loci. The 3rd codon position of each of these two loci provided the most characters in support of phylogenetic relationships within the Lecanoromycetes. Of the three ribosomal loci we used in this study, mitSSU contributed the most to phylogenetic analyses when combined with RPB1 and RPB2. Except for the mitSSU, ribosomal genes were the most difficult to recover because they often contain many introns, resulting in PCR bias toward numerous and intronless co-extracted contaminant fungi (mainly Dothideomycetes, Chaetothyriomycetes, and Sordariomycetes in the Ascomycota, and members of the Basidiomycota), which inhabit lichen thalli. Maximum likelihood analysis on the combined five-locus data set for 82 members of the Lecanoromycetes provided a well resolved and well supported tree compared to existing phylogenies. We confirmed the monophyly of three recognized subclasses in the Lecanoromycetes, the Acarosporomycetidae, Ostropomycetidae, and Lecanoromycetideae; the latter delimited as monophyletic for the first time, with the exclusion of the family Umbilicariaceae and Hypocenomyce scalaris. The genus Candelariella (formerly in the Candelariaceae, currently a member of the Lecanoraceae) represents the first evolutionary split within the Lecanoromycetes, before the divergence of the Acarosporomycetidae. This study provides a foundation necessary to guide the selection of loci for future multilocus phylogenetic studies on lichen-forming and allied ascomycetes.

Ascomycota↗

Extraordinary proliferation of microorganisms in aposymbiotic pea aphids, Acyrthosiphon pisum.

Aposymbiotic pea aphids, which were deprived of their intracellular symbiotic bacterium, Buchnera, exhibit growth retardation and no fecundity. High performance liquid chromatographic (HPLC) analysis revealed that these aposymbiotic aphids, when reared on broad bean plants, accumulated a large amount of histamine. To assess the possibility of extraordinary proliferation of microorganisms other than Buchnera, we enumerated eubacteria and fungi in aphids using the real-time quantitative PCR method that targets genes encoding small-subunit rRNAs. The result showed that these microorganisms were extremely abundant in the aposymbiotic aphids reared on plants. Microbial communities in aposymbiotic aphids were further profiled by phylogenetic analysis of small-subunit rDNAs. Of 172 nonchimeric sequences of fungal 18S rDNAs, 138 (80.2%) belonged to the phylum Ascomycota. Among them, 21 clustered within a monophyletic group consisting of insect-pathogenic fungi and yeast-like symbionts of homopteran insects. Thirty-one (18.0%), two (1.2%), and one (0.6%) clones were clustered within the Basidiomycota, Zygomycota, and Oomycota, respectively. Of 167 nonchimeric sequences of eubacterial 16S rDNAs, 84 (50.3%) belonged to the gamma-subdivision of Proteobacteria to which most primary endosymbionts of insects and prolific histamine producers belong. Forty (24.0%), 25 (15.0%), 10 (6.0%), and five (3.0%) clones were clustered within alpha-Proteobacteria, Cytophaga-Flavobacterium-Bacteroides (CFB) group, Actinobacteria, and beta-Proteobacteria, respectively. Three had no phylogenetic association with known taxonomic divisions. None of the sequences studied in this study coincided exactly with those deposited in GenBank.

Animals↗

Mycorrhizal evolution.

Two recent studies have cast new light on the evolutionary history of MYCORRHIZAE. New fossil evidence has pushed the date for the appearance of ARBUSCULAR MYCORRHIZAE back to 460 million years ago, predating vascular plants. A phylogenetic tree for the Basidiomycota shows that these fungi have gained (and lost) the ectomycorrhizal condition repeatedly during their history. These studies help highlight the paleoecological importance of mycorrhizae and add to our understanding of the evolution of mutualisms.

Journal Article↗

High-level production of erythritol by strain 618A-01 isolated from pollen.

We have isolated a microorganism (strain 618A-01) from pollen which has the ability to produce erythritol when grown in the presence of glucose as the carbon source. When cultivated in a medium consisting of 20% glucose and 1% dried bouillon in a shake flask, 75 g/l erythritol was produced after 950 h, corresponding to a 37.5% yield against glucose consumption. No other polyols, including glycerol, were detected in the medium. Positive-ion fast atom bombardment mass spectrometry and 1H- and 13C-NMR analyses confirmed that the fermentation product was erythritol. Scanning electron microscopic analysis clearly demonstrated that the cells grown on YPD medium at 30 degrees C showed yeast-like morphology, while they appeared like hyphae at 37 degrees C. The complete 18S rRNA sequence of the isolate was determined, which showed high identity (99.5%) with the genus Ustilago of the phylum Basidiomycota. The data strongly suggest that strain 618A-01 belongs to the class Ustilaginomycetes. The culture conditions for the production of erythritol by the isolate were examined. The use of medium containing 1% tryptone, 0.5% yeast extract and 0.5% NaCl yielded the highest cell growth and erythritol productivity among the media tested. Continuous glucose feeding at 6-7% to the fermentor further increased the production of erythritol, and we obtained a maximal 100 g/l erythritol after 530 h, with a 39.3% yield.

Journal Article↗

Differential effect of Agaricus host species on the population development of Megaselia halterata (Diptera: Phoridae).

Twelve isolates from the genus Agaricus (Fungi, Basidiomycota) were investigated for their ability to support development of the phorid fly, Megaselia halterata (Wood), which is an important pest of the commercial white button mushroom Agaricus bisporus. Combined effects of oviposition of adult female M. halterata and larval development in mushroom compost inoculated with Agaricus mycelium were determined using bioassays. The numbers of M. halterata offspring that developed were affected by the Agaricus isolate used, and there was a significant separation between resistant and susceptible isolates. In a bioassay where the female phorids had a choice of all 12 isolates for oviposition, three isolates produced >200 adults per 100 g compost pot while the remaining nine isolates had <20 adults per pot. Where there was no choice of Agaricus isolate for oviposition, five isolates resulted in >100 adults per 100 g compost pot while the remainder resulted in <4 adults per pot. With the susceptible isolates, there was a positive correlation between increasing concentration of mycelium in the substrate and phorid development until the concentration exceeded 40% after which numbers of emerging phorids declined. Genetic identity of Agaricus isolates was determined using ITS sequencing and phylogenetic methods, which revealed two major cluster groups. Isolates supporting the development of large populations of M. halterata were located in one of these clusters (group I), and were either Agaricus bisporus or other species from the same Agaricus section Duploannulatae. Isolates that did not support the development of M. halterata populations were located in a different cluster (group II) and were more genetically distant from A. bisporus, e.g. Agaricus sections Arvenses, Minores and Xanthodermatei. Species of Agaricus with resistance to M. halterata could have significant potential for the breeding and cultivation of phorid-free mushrooms.

Agaricus↗

Molecular phylogeny of Ustilago, Sporisorium, and related taxa based on combined analyses of rDNA sequences.

Combined analyses of ITS and LSU rDNA sequences were utilized to resolve the phylogenetic relationships of 98 members of the smut genera Lundquistia, Melanopsichium, Moesziomyces, Macalpinomyces, Sporisorium, and Ustilago (Basidiomycota: Ustilaginales). Minimum Evolution and Bayesian inference of phylogeny resolve three major groups of almost identical composition: Sporisorium, Ustilago, and a basal assemblage of both Ustilago and Sporisorium species. Macalpinomyces deserves generic rank regarding its type species M. eriachnes; all other Macalpinomyces species of our study clearly turn out to be part of Ustilago or Sporisorium. Lundquistia evidently belongs to Sporisorium. Moesziomyces, probably paraphyletic, stands basal to all other genera. Interestingly, Melanopsichium belongs to the Ustilago clade, being the only member of the ingroup not parasitizing on Poaceae. The patchy distribution of commonly used morphological characters along our phylograms points to their variability and dependence on the host's morphological traits instead of being valuable for resolving parasite phylogeny. The new combination: Sporisorium fascicularis comb. nov. (syn. Lundquistia.fascicularis) is made.

DNA, Fungal↗

A revision of the descriptions of ectomycorrhizas published since 1961.

All available publications providing descriptions of ectomycorrhizas (ECM) were reviewed in order to build a database containing details on fungus forming the ECM, host tree, country where the material for description was collected, and habitat of the ECM. Other secondary data were also recorded. In all 1244 descriptions of ECM published since 1961 in 479 papers were reviewed. The number of different ECM morphotypes described was 814. Most ECM described were collected in Europe and North America. Gymnosperms were the most common tree associates, and boreal and temperate forests the most studied ecosystems. Fungal symbionts were mostly Basidiomycota, epigeous, and with mushroom-like morphology. The paper also addresses the gaps in ECM knowledge that mycorrhizologists should address in future studies.

Ecosystem↗

Potential bias of fungal 18S rDNA and internal transcribed spacer polymerase chain reaction primers for estimating fungal biodiversity in soil.

Four fungal 18S rDNA and internal transcribed spacer (ITS) polymerase chain reaction (PCR) primer pairs were tested for their specificity towards target fungal DNA in soil DNA extracts, and their ability to assess the diversity of fungal communities in a natural grassland soil was compared. Amplified PCR products were cloned, and approximately 50 clones from each library were sequenced. Phylogenetic analysis and database searches indicated that each of the sequenced cloned DNA fragments was of fungal origin for each primer pair, with the exception of the sequences generated using the 18S rDNA primers nu-SSU-0817 and nu-SSU-1196, where 35 of the 50 sequenced clones represented soil invertebrates. Although some of the primers have previously been suggested to be biased towards certain fungal taxonomic groups, the ratio of sequences representing each of the four main fungal phyla, Ascomycota, Basidiomycota, Chytridiomycota and Zygomycota, was similar for each of the primer pairs, suggesting that primer bias may be less significant than previously thought. Collector's curves were plotted to estimate the coverage obtained for each of the clone libraries after clustering the sequences into operational taxonomic units at a level of 99% sequence similarity. The curves indicated that good coverage of diversity was achieved, with the exception of the clone library constructed using primers nu-SSU-0817 and nu-SSU-1196, on account of the high number of non-fungal sequences obtained. The work demonstrates the usefulness of 18S rDNA and ITS PCR primers for assessing fungal diversity in environmental samples, and it also highlights some potential limitations of the approach with respect to PCR primer specificity and bias.

Bias↗