Fungal Communities of Eucalyptus grandis Leaves Are Influenced by the Insect Pest Leptocybe invasa.

Fungal communities in above-ground tree tissues are hyperdiverse and are influenced by biotic interactions with other organisms living in or on these tissues. These biotic interactions are, however, still poorly understood. In this study, we aimed to understand how insect-associated gall formation on Eucalyptus foliage correlates with the diversity of foliar fungal communities in surrounding healthy leaf tissue, as well as the co-occurrence patterns among the members of the fungal community. We used ITS metabarcoding to characterise the foliar fungal communities of 179 individual E. grandis trees. These trees were assigned to infestation levels of the wasp Leptocybe invasa (Eulophidae: Hymenoptera), which causes gall formation on shoot tips and leaves of its host. Fungal community networks were calculated using a Pearson correlation coefficient. The composition and diversity of fungal communities were influenced by the severity of L. invasa infestations. We identified potential Eucalyptus pathogens with high sequence abundance at all disease severity levels, but network analysis indicated that the co-occurrence of potential pathogens between no to mild and medium to heavy infestation differed significantly. A better understanding of microbial interactions, especially the role of pathogens, can be useful for controlling disease- and beneficial host-associated microbial communities.

Fungal diversity notes 1512-1610: taxonomic and phylogenetic contributions on genera and species of fungal taxa.

This article is the 14th in the Fungal Diversity Notes series, wherein we report 98 taxa distributed in two phyla, seven classes, 26 orders and 50 families which are described and illustrated. Taxa in this study were collected from Australia, Brazil, Burkina Faso, Chile, China, Cyprus, Egypt, France, French Guiana, India, Indonesia, Italy, Laos, Mexico, Russia, Sri Lanka, Thailand, and Vietnam. There are 59 new taxa, 39 new hosts and new geographical distributions with one new combination. The 59 new species comprise Angustimassarina kunmingense, Asterina lopi, Asterina brigadeirensis, Bartalinia bidenticola, Bartalinia caryotae, Buellia pruinocalcarea, Coltricia insularis, Colletotrichum flexuosum, Colletotrichum thasutense, Coniochaeta caraganae, Coniothyrium yuccicola, Dematipyriforma aquatic, Dematipyriforma globispora, Dematipyriforma nilotica, Distoseptispora bambusicola, Fulvifomes jawadhuvensis, Fulvifomes malaiyanurensis, Fulvifomes thiruvannamalaiensis, Fusarium purpurea, Gerronema atrovirens, Gerronema flavum, Gerronema keralense, Gerronema kuruvense, Grammothele taiwanensis, Hongkongmyces changchunensis, Hypoxylon inaequale, Kirschsteiniothelia acutisporum, Kirschsteiniothelia crustaceum, Kirschsteiniothelia extensum, Kirschsteiniothelia septemseptatum, Kirschsteiniothelia spatiosum, Lecanora immersocalcarea, Lepiota subthailandica, Lindgomyces guizhouensis, Marthe asmius pallidoaurantiacus, Marasmius tangerinus, Neovaginatispora mangiferae, Pararamichloridium aquisubtropicum, Pestalotiopsis piraubensis, Phacidium chinaum, Phaeoisaria goiasensis, Phaeoseptum thailandicum, Pleurothecium aquisubtropicum, Pseudocercospora vernoniae, Pyrenophora verruculosa, Rhachomyces cruralis, Rhachomyces hyperommae, Rhachomyces magrinii, Rhachomyces platyprosophi, Rhizomarasmius cunninghamietorum, Skeletocutis cangshanensis, Skeletocutis subchrysella, Sporisorium anadelphiae-leptocomae, Tetraploa dashaoensis, Tomentella exiguelata, Tomentella fuscoaraneosa, Tricholomopsis lechatii, Vaginatispora flavispora and Wetmoreana blastidiocalcarea. The new combination is Torula sundara. The 39 new records on hosts and geographical distribution comprise Apiospora guiyangensis, Aplosporella artocarpi, Ascochyta medicaginicola, Astrocystis bambusicola, Athelia rolfsii, Bambusicola bambusae, Bipolaris luttrellii, Botryosphaeria dothidea, Chlorophyllum squamulosum, Colletotrichum aeschynomenes, Colletotrichum pandanicola, Coprinopsis cinerea, Corylicola italica, Curvularia alcornii, Curvularia senegalensis, Diaporthe foeniculina, Diaporthe longicolla, Diaporthe phaseolorum, Diatrypella quercina, Fusarium brachygibbosum, Helicoma aquaticum, Lepiota metulispora, Lepiota pongduadensis, Lepiota subvenenata, Melanconiella meridionalis, Monotosporella erecta, Nodulosphaeria digitalis, Palmiascoma gregariascomum, Periconia byssoides, Periconia cortaderiae, Pleopunctum ellipsoideum, Psilocybe keralensis, Scedosporium apiospermum, Scedosporium dehoogii, Scedosporium marina, Spegazzinia deightonii, Torula fici, Wiesneriomyces laurinus and Xylaria venosula. All these taxa are supported by morphological and multigene phylogenetic analyses. This article allows the researchers to publish fungal collections which are important for future studies. An updated, accurate and timely report of fungus-host and fungus-geography is important. We also provide an updated list of fungal taxa published in the previous fungal diversity notes. In this list, erroneous taxa and synonyms are marked and corrected accordingly.

The evolving species concepts used for yeasts: from phenotypes and genomes to speciation networks.

Here we review how evolving species concepts have been applied to understand yeast diversity. Initially, a phenotypic species concept was utilized taking into consideration morphological aspects of colonies and cells, and growth profiles. Later the biological species concept was added, which applied data from mating experiments. Biophysical measurements of DNA similarity between isolates were an early measure that became more broadly applied with the advent of sequencing technology, leading to a sequence-based species concept using comparisons of parts of the ribosomal DNA. At present phylogenetic species concepts that employ sequence data of rDNA and other genes are universally applied in fungal taxonomy, including yeasts, because various studies revealed a relatively good correlation between the biological species concept and sequence divergence. The application of genome information is becoming increasingly common, and we strongly recommend the use of complete, rather than draft genomes to improve our understanding of species and their genome and genetic dynamics. Complete genomes allow in-depth comparisons on the evolvability of genomes and, consequently, of the species to which they belong. Hybridization seems a relatively common phenomenon and has been observed in all major fungal lineages that contain yeasts. Note that hybrids may greatly differ in their post-hybridization development. Future in-depth studies, initially using some model species or complexes may shift the traditional species concept as isolated clusters of genetically compatible isolates to a cohesive speciation network in which such clusters are interconnected by genetic processes, such as hybridization.

Active Fungal Communities in Asymptomatic Eucalyptus grandis Stems Differ between a Susceptible and Resistant Clone.

Fungi represent a common and diverse part of the microbial communities that associate with plants. They also commonly colonise various plant parts asymptomatically. The molecular mechanisms of these interactions are, however, poorly understood. In this study we use transcriptomic data from Eucalyptus grandis, to demonstrate that RNA-seq data are a neglected source of information to study fungal-host interactions, by exploring the fungal transcripts they inevitably contain. We identified fungal transcripts from E. grandis data based on their sequence dissimilarity to the E. grandis genome and predicted biological functions. Taxonomic classifications identified, amongst other fungi, many well-known pathogenic fungal taxa in the asymptomatic tissue of E. grandis. The comparison of a clone of E. grandis resistant to Chrysoporthe austroafricana with a susceptible clone revealed a significant difference in the number of fungal transcripts, while the number of fungal taxa was not substantially affected. Classifications of transcripts based on their respective biological functions showed that the fungal communities of the two E. grandis clones associate with fundamental biological processes, with some notable differences. To shield the greater host defence machinery in the resistant E. grandis clone, fungi produce more secondary metabolites, whereas the environment for fungi associated with the susceptible E. grandis clone is more conducive for building fungal cellular structures and biomass growth. Secreted proteins included carbohydrate active enzymes that potentially are involved in fungal-plant and fungal-microbe interactions. While plant transcriptome datasets cannot replace the need for designed experiments to probe plant-microbe interactions at a molecular level, they clearly hold potential to add to the understanding of the diversity of plant-microbe interactions.

Flooding Duration Affects the Structure of Terrestrial and Aquatic Microbial Eukaryotic Communities.

The increasing number and duration of inundations is reported to be a consequence of climate change and may severely compromise non-adapted macroorganisms. The effect of flooding events on terrestrial and aquatic microbial communities is, however, less well understood. They may respond to the changed abiotic properties of their native habitat, and the native community may change due to the introduction of alien species. We designed an experiment to investigate the effect of five different flooding durations on the terrestrial and aquatic communities of eukaryotic microorganism, using the AquaFlow mesocosms. With amplicon sequencing of the small subunit (SSU) and internal transcribed spacer (ITS) rRNA gene regions, we analyzed community compositions directly before and after flooding. Subsequently, they were monitored for another 28 days, to determine the sustainability of community changes. Our results revealed a temporary increase in similarity between terrestrial and aquatic communities according to OTU composition (operational taxonomic unit, serves as a proxy for species). Increased similarity was mainly caused by the transmission of OTUs from water to soil. A minority of these were able to persist in soil until the end of the experiment. By contrast, the vast majority of soil OTUs was not transmitted to water. Flooding duration affected the community structure (abundance) more than composition (occurrence). Terrestrial communities responded immediately to flooding and the flooding duration influenced the community changes. Independent from flooding duration, all terrestrial communities recovered largely after flooding, indicating a remarkable resilience to the applied disturbances. Aquatic communities responded immediately to the applied inundations too. At the end of the experiment, they grouped according to the applied flooding duration and the amount of ammonium and chloride that leached from the soil. This indicates a sustained long-term response of the aquatic communities to flooding events.

Botryosphaeria dothidea: a latent pathogen of global importance to woody plant health.

Botryosphaeria dothidea is the type species of Botryosphaeria (Botryosphaeriaceae, Botryosphaeriales). Fungi residing in this order are amongst the most widespread and important canker and dieback pathogens of trees worldwide, with B. dothidea one of the most common species on a large number of hosts. Its taxonomic circumscription has undergone substantial change in the past decade, making it difficult to interpret the large volume of literature linked to the name B. dothidea. This pathogen profile synthesizes the current understanding of B. dothidea pertaining to its distribution, host associations and role as a pathogen in managed and natural woody environments. The prolonged latent infection or endophytic phase is of particular importance, as it implies that the fungus can easily pass undetected by quarantine systems in traded living plants, fruits and other plant parts. Infections typically become obvious only under conditions of host stress, when disease symptoms develop. This study also considers the knowledge emerging from the recently sequenced B. dothidea genome, elucidating previously unknown aspects of the species, including mating and host infection strategies. Despite more than 150 years of research on B. dothidea, there is clearly much to be learned regarding this global tree pathogen. This is increasingly important given the stresses imposed on various woody hosts as a result of climate change. TAXONOMY: Botryosphaeria dothidea (Moug. ex Fr) Ces. & De Not, 1863. Kingdom Fungi, Phylum Ascomycota, Class Dothideomycetes, Order Botryosphaeriales, Family Botryosphaeriaceae, Genus Botryosphaeria, Species dothidea. HOST RANGE: Confirmed on more than 24 host genera, including woody plants, such as Acacia (= Vachellia), Eucalyptus, Vitis and Pistachio. DISEASE SYMPTOMS: Associated with twig, branch and stem cankers, tip and branch dieback, fruit rot, blue stain and plant death. USEFUL WEBSITES: The Botryosphaeria site for detailed morphological descriptions (http://www.crem.fct.unl.pt/botryosphaeria_site/); Systematic Mycology and Microbiology Laboratory Fungal Database for all literature and associated hosts (https://nt.ars-grin.gov/fungaldatabases/); TreeBASE link for the combined ITS and TEF-1α tree (http://purl.org/phylo/treebase/phylows/study/TB2:S18906); DOE Joint Genome Institute, JGI Mycocosm for the Botryosphaeria dothidea genome (http://genome.jgi.doe.gov/Botdo1_1/Botdo1_1.home.html).

Maternal effects on tree phenotypes: considering the microbiome.

The biotic and abiotic environmental experience of plants can influence the offspring without any changes in DNA sequence. These effects can modulate the development of the progeny and their interaction with microorganisms. This interaction includes fungal endophytic communities which have significant effects on trees and their associated ecosystems. In this opinion article, we highlight potential maternal mechanisms through which endophytes could influence the progeny. We argue that a better understanding of these interactions might help to predict the response of trees to stress conditions and enhance the efficiency of tree breeding programs.

Identification of a new order of root-colonising fungi in the Entorrhizomycota: Talbotiomycetales ord. nov. on eudicotyledons.

The recently described fungal phylum Entorrhizomycota was established solely for the genus Entorrhiza, species of which cause root-galls in Cyperaceae and Juncaceae. Talbotiomyces calosporus (incertae sedis) shares morphological characteristics and an ecological niche with species of Entorrhiza. We investigated the higher classification of T. calosporus to determine whether it belongs in Entorrhizomycota. Ribosomal DNA sequences showed Talbotiomyces to be a close relative of Entorrhiza and both taxa form a highly supported monophyletic group. Based on molecular phylogenetic analyses and in congruence with existing morphological and ecological data, Entorrhiza and Talbotiomyces represent a deep dichotomy within the Entorrhizomycota. While species of Entorrhiza are characterised by dolipores and occur on monocotyledons, members of Talbotiomyces are characterised by simple pores and are associated with eudicotyledons. This expands the host range of the recently described Entorrhizomycota from Poales to other angiosperms. Higher taxa, namely Talbotiomycetales ord. nov. and Talbotiomycetaceae fam. nov., are proposed here to accommodate Talbotiomyces.

Ion Torrent PGM as tool for fungal community analysis: a case study of endophytes in Eucalyptus grandis reveals high taxonomic diversity.

The Kingdom Fungi adds substantially to the diversity of life, but due to their cryptic morphology and lifestyle, tremendous diversity, paucity of formally described specimens, and the difficulty in isolating environmental strains into culture, fungal communities are difficult to characterize. This is especially true for endophytic communities of fungi living in healthy plant tissue. The developments in next generation sequencing technologies are, however, starting to reveal the true extent of fungal diversity. One of the promising new technologies, namely semiconductor sequencing, has thus far not been used in fungal diversity assessments. In this study we sequenced the internal transcribed spacer 1 (ITS1) nuclear encoded ribosomal RNA of the endophytic community of the economically important tree, Eucalyptus grandis, from South Africa using the Ion Torrent Personal Genome Machine (PGM). We determined the impact of various analysis parameters on the interpretation of the results, namely different sequence quality parameter settings, different sequence similarity cutoffs for clustering and filtering of databases for removal of sequences with incomplete taxonomy. Sequence similarity cutoff values only had a marginal effect on the identified family numbers, whereas different sequence quality filters had a large effect (89 vs. 48 families between least and most stringent filters). Database filtering had a small, but statistically significant, effect on the assignment of sequences to reference sequences. The community was dominated by Ascomycota, and particularly by families in the Dothidiomycetes that harbor well-known plant pathogens. The study demonstrates that semiconductor sequencing is an ideal strategy for environmental sequencing of fungal communities. It also highlights some potential pitfalls in subsequent data analyses when using a technology with relatively short read lengths.

Microbotryum silenes-saxifragae sp. nov. sporulating in the anthers of Silene saxifraga in southern European mountains.

Currently, the monophyletic lineage of anther smuts on Caryophyllaceae includes 22 species classified in the genus Microbotryum. They are model organisms studied in many disciplines of fungal biology. A molecular phylogenetic approach was used to resolve species boundaries within the caryophyllaceous anther smuts, as species delimitation based solely on phenotypic characters was problematic. Several cryptic species were found amongst the anther smuts on Caryophyllaceae, although some morphologically distinct species were discernible, and most species were characterized by high host-specificity. In this study, anther smut specimens infecting Silene saxifraga were analysed using rDNA sequences (ITS and LSU) and morphology to resolve their specific status and to discuss their phylogenetic position within the lineage of caryophyllaceous anther smuts. The molecular phylogeny revealed that all specimens form a monophyletic lineage that is supported by the morphological trait of reticulate spores with tuberculate interspaces (observed in certain spores). This lineage cannot be attributed to any of the previously described species, and the anther smut on Silene saxifraga is described and illustrated here as a new species, Microbotryum silenes-saxifragae. This species clusters in a clade that includes Microbotryum species, which infect both closely and distantly related host plants growing in diverse ecological habitats. It appears possible that host shifts combined with changes to ecological host niches drove the evolution of Microbotryum species within this clade.

Microbotryum heliospermae, a new anther smut fungus parasitic on Heliosperma pusillum in the mountains of the European Alpine System.

The members of the smut genus Microbotryum are pathogens of a wide range of host plant species from nine dicotyledonous families. Within the genus, the species sporulating in anthers of Caryophyllaceae form a monophyletic group that in recent years attracted much interest in various biological studies. The phylogenetic framework developed for species delimitation within Microbotryum revealed that high level host-specificity is a major feature of most caryophyllaceous anther smuts. However, the great number of anther smut specimens on diverse host plant species reported worldwide has still not been included in phylogenetic analyses due to the inaccessibility of recently collected specimens, and thus many species remain still undiscovered. In this study, anther smut specimens on Heliosperma pusillum originating from all main mountain ranges of the European Alpine System were examined using partial rDNA sequence and/or morphological analyses. The investigation revealed that all specimens are morphologically uniform and phylogenetically represent a monophyletic lineage, sister to Microbotryum lagerheimii complex on Atocion rupestre/Silene lacera/Silene vulgaris/Viscaria vulgaris. This lineage cannot be attributed to any of the previously described species, and therefore the smut in anthers of H. pusillum is described and illustrated here as a new species, Microbotryum heliospermae. The species is known from subalpine zone of the Alps, the Carpathians, the Dinaric Alps, and the Pyrenees, inhabiting host plants growing in open spring communities or semihumid mountain meadows.

Species accumulation curves and incidence-based species richness estimators to appraise the diversity of cultivable yeasts from beech forest soils.

BACKGROUND: Yeast-like fungi inhabit soils throughout all climatic zones in a great abundance. While recent estimations predicted a plethora of prokaryotic taxa in one gram of soil, similar data are lacking for fungi, especially yeasts. METHODOLOGY/PRINCIPAL FINDINGS: We assessed the diversity of soil yeasts in different forests of central Germany using cultivation-based techniques with subsequent identification based on rDNA sequence data. Based on experiments using various pre-cultivation sample treatment and different cultivation media we obtained the highest number of yeasts by analysing mixed soil samples with a single nutrient-rich medium. Additionally, several species richness estimators were applied to incidence-based data of 165 samples. All of them predicted a similar range of yeast diversity, namely 14 to 16 species. Randomized species richness curves reached saturation in all applied estimators, thus indicating that the majority of species is detected after approximately 30 to 50 samples analysed. CONCLUSIONS/SIGNIFICANCE: In this study we demonstrate that robust species identification as well as mathematical approaches are essential to reliably estimate the sampling effort needed to describe soil yeast communities. This approach has great potential for optimisation of cultivation techniques and allows high throughput analysis in the future.

Anther smuts of Caryophyllaceae: molecular analyses reveal further new species.

Recent collections of Microbotryum (Pucciniomycotina, Basidiomycota) specimens inhabiting anthers of different caryophyllaceous host plants were analysed using LM and electron microscopy, as well as molecular phylogenetic analyses using rDNA (ITS and LSU) sequences. The phylogenetic relationships of caryophyllaceous anther parasites are discussed. Three new species, Microbotryum adenopetalae, M. minuartiae, and M. silenes-acaulis, are described based on morphological, ecological, and molecular characteristics. New host plants are reported for Microbotryum dianthorum (Dianthus jacquemontii and Petrorhagia saxifraga) and M. lychnidis-dioicae (Cucubalus baccifer and Silene zawadskii). For Microbotryum violaceum, a neotype is selected.

Implications of molecular characters for the phylogeny of the Microbotryaceae (Basidiomycota: Urediniomycetes).

BACKGROUND: Anther smuts of the basidiomycetous genus Microbotryum on Caryophyllaceae are important model organisms for many biological disciplines. Members of Microbotryum are most commonly found parasitizing the anthers of host plants in the family Caryophyllaceae, however they can also be found on the anthers of members of the Dipsacaceae, Lamiaceae, Lentibulariaceae, and Portulacaceae. Additionally, some members of Microbotryum can be found infecting other organs of mainly Polygonaceae hosts. Based on ITS nrDNA sequences of members of almost all genera in Microbotryaceae, this study aims to resolve the phylogeny of the anther smuts and their relationship to the other members of the family of plant parasites. A multiple analysis strategy was used to correct for the effects of different equally possible ITS sequence alignments on the phylogenetic outcome, which appears to have been neglected in previous studies. RESULTS: The genera of Microbotryaceae were not clearly resolved, but alignment-independent moderate bootstrap support was achieved for a clade containing the majority of the Microbotryum species. The anther parasites appeared in two different well-supported lineages whose interrelationship remained unresolved. Whereas bootstrap support values for some clades were highly vulnerable to alignment conditions, other clades were more robustly supported. The differences in support between the different alignments were much larger than between the phylogenetic optimality criteria applied (maximum parsimony and maximum likelihood). CONCLUSION: The study confirmed, based on a larger dataset than previous work, that the anther smuts on Caryophyllaceae are monophyletic and that there exists a native North American group that diverged from the European clade before the radiation of the European species. Also a second group of anther smuts was revealed, containing parasites on Dipsacaceae, Lamiaceae, and Lentibulariaceae. At least the majority of the parasites of Asteraceae appeared as a monophylum, but delimitations of some species in this group should be reconsidered. Parasitism on Polygonaceae is likely to be the ancestral state for the Microbotryaceae on Eudicot hosts.