Environmental heterogeneity structures root-associated fungal communities in Daphne arbuscula (Thymelaeaceae), a shrub adapted to extreme rocky habitats.

Rocky habitats, globally distributed ecosystems, harbour diverse biota, including numerous endemic and endangered species. Vascular plants thriving in these environments face challenging abiotic conditions, requiring diverse morphological and physiological adaptations. Their engagement with the surrounding microbiomes is, however, equally vital for their adaptation, fitness, and long-term survival. Nevertheless, there remains a lack of understanding surrounding this complex interplay within this fascinating biotic ecosystem. Using microscopic observations and metabarcoding analyses, we examined the fungal abundance and diversity in the root system of the rock-dwelling West Carpathian endemic shrub, Daphne arbuscula (Thymelaeaceae). We explored the diversification of root-associated fungal communities in relation to microclimatic variations across the studied sites. We revealed extensive colonization of the Daphne roots by diverse taxonomic fungal groups attributed to different ecological guilds, predominantly plant pathogens, dark septate endophytes (DSE), and arbuscular mycorrhizal fungi (AMF). Notably, differences in taxonomic composition and ecological guilds emerged between colder and warmer microenvironments. Apart from omnipresent AMF, warmer sites exhibited a prevalence of plant pathogens, while colder sites were characterized by a dominance of DSE. This mycobiome diversification, most likely triggered by the environment, suggests that D. arbuscula populations in warmer areas may be more vulnerable to fungal diseases, particularly in the context of global climate change.

GlobalAMFungi: a global database of arbuscular mycorrhizal fungal occurrences from high-throughput sequencing metabarcoding studies.

Arbuscular mycorrhizal (AM) fungi are crucial mutualistic symbionts of the majority of plant species, with essential roles in plant nutrient uptake and stress mitigation. The importance of AM fungi in ecosystems contrasts with our limited understanding of the patterns of AM fungal biogeography and the environmental factors that drive those patterns. This article presents a release of a newly developed global AM fungal dataset (GlobalAMFungi database, https://globalamfungi.com) that aims to reduce this knowledge gap. It contains almost 50 million observations of Glomeromycotinian AM fungal amplicon DNA sequences across almost 8500 samples with geographical locations and additional metadata obtained from 100 original studies. The GlobalAMFungi database is built on sequencing data originating from AM fungal taxon barcoding regions in: i) the small subunit rRNA (SSU) gene; ii) the internal transcribed spacer 2 (ITS2) region; and iii) the large subunit rRNA (LSU) gene. The GlobalAMFungi database is an open source and open access initiative that compiles the most comprehensive atlas of AM fungal distribution. It is designed as a permanent effort that will be continuously updated by its creators and through the collaboration of the scientific community. This study also documented applicability of the dataset to better understand ecology of AM fungal taxa.

Exploring structural and molecular diversity of Ericaceae hair root mycobionts: a comparison between Northern Bohemia and Argentine Patagonia.

Core Ericaceae produce delicate hair roots with inflated rhizodermal cells that host plethora of fungal symbionts. These poorly known mycobionts include various endophytes, parasites, saprobes, and the ericoid mycorrhizal (ErM) fungi (ErMF) that form the ErM symbiosis crucial for the fitness of their hosts. Using microscopy and high-throughput sequencing, we investigated their structural and molecular diversity in 14 different host × site combinations in Northern Bohemia (Central Europe) and Argentine Patagonia (South America). While we found typical ericoid mycorrhiza in all combinations, we did not detect ectomycorrhiza and arbuscular mycorrhiza. Superficial mantles of various thickness formed by non-clamped hyphae were observed in all combinations except Calluna vulgaris from N. Bohemia. Some samples contained frequent intercellular hyphae while others possessed previously unreported intracellular haustoria-like structures linked with intracellular hyphal coils. The 711 detected fungal OTU were dominated by Ascomycota (563) and Basidiomycota (119), followed by four other phyla. Ascomycetes comprised Helotiales (255), Pleosporales (53), Chaetothyriales (42), and other 19 orders, while basidiomycetes Sebacinales (42), Agaricales (28), Auriculariales (7), and other 14 orders. While many dominant OTU from both hemispheres lacked close relatives in reference databases, many were very similar to identical to unnamed sequences from around the world. On the other hand, several significant ericaceous mycobionts were absent in our dataset, incl. Cairneyella, Gamarada, Kurtia, Lachnum, and Leohumicola. Most of the detected OTU could not be reliably linked to a particular trophic mode, and only two could be reliably assigned to the archetypal ErMF Hyaloscypha hepaticicola. Probable ErMF comprised Hyaloscypha variabilis and Oidiodendron maius, both detected only in N. Bohemia. Possible ErMF comprised sebacinoid fungi and several unnamed members of Hyaloscypha s. str. While H. hepaticicola was dominant only in C. vulgaris, this model ErM host lacked O. maius and sebacinoid mycobionts. Hyaloscypha hepaticicola was absent in two and very rare in six combinations from Patagonia. Nine OTU represented dark septate endophytes from the Phialocephala fortinii s. lat.-Acephala applanata species complex, including the most abundant OTU (the only detected in all combinations). Statistical analyses revealed marked differences between N. Bohemia and Patagonia, but also within Patagonia, due to the unique community detected in a Valdivian temperate rainforest. Our results show that the ericaceous hair roots may host diverse mycobionts with mostly unknown functions and indicate that many novel ErMF lineages await discovery. Transhemispheric differences (thousands of km) in their communities may be evenly matched by local differences (scales of km, m, and less).

PacBio sequencing of Glomeromycota rDNA: a novel amplicon covering all widely used ribosomal barcoding regions and its applicability in taxonomy and ecology of arbuscular mycorrhizal fungi.

There is no consensus barcoding region for determination of arbuscular mycorrhizal fungal (AMF) taxa. To overcome this obstacle, we have developed an approach to sequence an AMF marker within the ribosome-encoding operon (rDNA) that covers all three widely applied variable molecular markers. Using a nested PCR approach specific to AMF, we amplified a part (c. 2.5 kb) of the rDNA spanning the majority of the small subunit rRNA (SSU) gene, the complete internal transcribed spacer (ITS) region and a part of the large subunit (LSU) rRNA gene. The PCR products were sequenced on the PacBio platform utilizing Single Molecule Real Time (SMRT) sequencing. Employing this method for selected environmental DNA samples, we were able to describe complex AMF communities consisting of various glomeromycotan lineages. We demonstrate the applicability of this new 2.5 kb approach to provide robust phylogenetic assignment of AMF lineages without known sequences from pure cultures and to consolidate information about AMF taxon distributions coming from three widely used barcoding regions into one integrative dataset.

Symbiosis of isoetid plant species with arbuscular mycorrhizal fungi under aquatic versus terrestrial conditions.

Arbuscular mycorrhizal fungi (AMF) colonize the roots of numerous aquatic and wetland plants, but the establishment and functioning of mycorrhizal symbiosis in submerged habitats have received only little attention. Three pot experiments were conducted to study the interaction of isoetid plants with native AMF. In the first experiment, arbuscular mycorrhizal (AM) symbiosis did not establish in roots of Isoëtes echinospora and I. lacustris, while Littorella uniflora roots were highly colonized. Shoot and root biomass of L. uniflora were, however, not affected by AMF inoculation, and only one of nine AMF isolates significantly increased shoot P concentration. In the second experiment, we compared colonization by three Glomus tetrastratosum isolates of different cultivation history and origin (aquatic versus terrestrial) and their effects on L. uniflora growth and phosphorus nutrition under submerged versus terrestrial conditions. The submerged cultivation considerably slowed, but did not inhibit mycorrhizal root colonization, regardless of isolate identity. Inoculation with any AMF isolate improved plant growth and P uptake under terrestrial, but not submerged conditions. In the final experiment, we compared the communities of AMF established in two cultivation regimes of trap cultures with lake sediments, either submerged on L. uniflora or terrestrial on Zea mays. After 2-year cultivation, we did not detect a significant effect of cultivation regime on AMF community composition. In summary, although submerged conditions do not preclude the development of functional AM symbiosis, the contribution of these symbiotic fungi to the fitness of their hosts seems to be considerably less than under terrestrial conditions.

Sympatric diploid and tetraploid cytotypes of Centaurea stoebe s.l. do not differ in arbuscular mycorrhizal communities and mycorrhizal growth response.

PREMISE OF THE STUDY: Genome duplication is associated with multiple changes at different levels, including interactions with pollinators and herbivores. Yet little is known whether polyploidy may also shape belowground interactions. METHODS: To elucidate potential ploidy-specific interactions with arbuscular mycorrhizal fungi (AMF), we compared mycorrhizal colonization and assembly of AMF communities in roots of diploid and tetraploid Centaurea stoebe s.l. (Asteraceae) co-occurring in a Central European population. In a follow-up greenhouse experiment, we tested inter-cytotype differences in mycorrhizal growth response by combining ploidy, substrate, and inoculation with native AMF in a full-factorial design. KEY RESULTS: All sampled plants were highly colonized by AMF, with the Glomeraceae predominating. AMF-community composition revealed by 454-pyrosequencing reflected the spatial distribution of the hosts, but not their ploidy level or soil characteristics. In the greenhouse experiment, the tetraploids produced more shoot biomass than the diploids did when grown in a more fertile substrate, while no inter-cytotype differences were found in a less fertile substrate. AMF inoculation significantly reduced plant growth and improved P uptake, but its effects did not differ between the cytotypes. CONCLUSIONS: The results do not support our hypotheses that the cytotype structure in a mixed-ploidy population of C. stoebe is mirrored in AMF-community composition and that ploidy-specific fungal communities contribute to cytotype co-existence. Causes and implications of the observed negative growth response to AMF are discussed.

Geosmithia associated with bark beetles and woodborers in the western USA: taxonomic diversity and vector specificity.

Fungi in the genus Geosmithia (Ascomycota: Hypocreales) are frequent associates of bark beetles and woodborers that colonize hardwood and coniferous trees. One species, Geosmithia morbida, is an economically damaging invasive species. The authors surveyed the Geosmithia species of California and Colorado, USA, to (i) provide baseline data on taxonomy of Geosmithia and beetle vector specificity across the western USA; (ii) investigate the subcortical beetle fauna for alternative vectors of the invasive G. morbida; and (iii) interpret the community composition of this region within the emerging global biogeography of Geosmithia. Geosmithia was detected in 87% of 126 beetle samples obtained from 39 plant species. Twenty-nine species of Geosmithia were distinguished, of which 13 may be new species. Bark beetles from hardwoods, Cupressus, and Sequoia appear to be regular vectors, with Geosmithia present in all beetle gallery systems examined. Other subcortical insects appear to vector Geosmithia at lower frequencies. Overall, most Geosmithia have a distinct level of vector specificity (mostly high, sometimes low) enabling their separation to generalists and specialists. Plant pathogenic Geosmithia morbida was not found in association with any other beetle besides Pityophthorus juglandis. However, four additional Geosmithia species were found in P. juglandis galleries. When integrated with recent data from other continents, a global pattern of Geosmithia distribution across continents, latitudes, and vectors is emerging: of the 29 Geosmithia species found in the western USA, 12 have not been reported outside of the USA. The most frequently encountered species with the widest global distribution also had the broadest range of beetle vectors. Several Geosmithia spp. with very narrow vector ranges in Europe exhibited the similar degree of specialization in the USA. Such strong canalization in association could reflect an ancient origin of each individual association, or a recent origin and a subsequent diversification in North America.

Plant community stability is associated with a decoupling of prokaryote and fungal soil networks.

Soil microbial networks play a crucial role in plant community stability. However, we lack knowledge on the network topologies associated with stability and the pathways shaping these networks. In a 13-year mesocosm experiment, we determined links between plant community stability and soil microbial networks. We found that plant communities on soil abandoned from agricultural practices 60 years prior to the experiment promoted destabilising properties and were associated with coupled prokaryote and fungal soil networks. This coupling was mediated by strong interactions of plants and microbiota with soil resource cycling. Conversely, plant communities on natural grassland soil exhibited a high stability, which was associated with decoupled prokaryote and fungal soil networks. This decoupling was mediated by a large variety of past plant community pathways shaping especially fungal networks. We conclude that plant community stability is associated with a decoupling of prokaryote and fungal soil networks and mediated by plant-soil interactions.

Extensive sampling and high-throughput sequencing reveal Posidoniomycesatricolor gen. et sp. nov. (Aigialaceae, Pleosporales) as the dominant root mycobiont of the dominant Mediterranean seagrass Posidoniaoceanica.

Seagrasses provide invaluable ecosystem services yet very little is known about their root mycobiont diversity and distribution. Here we focused on the dominant Mediterranean seagrass Posidoniaoceanica and assessed its root mycobiome at 32 localities covering most of the ecoregions in the NW Mediterranean Sea using light and scanning electron microscopy and tag-encoded 454-pyrosequencing. Microscopy revealed that the recently discovered dark septate endophytic association specific for P.oceanica is present at all localities and pyrosequencing confirmed that the P.oceanica root mycobiome is dominated by a single undescribed pleosporalean fungus, hitherto unknown from other hosts and ecosystems. Its numerous slow-growing isolates were obtained from surface-sterilised root segments at one locality and after prolonged cultivation, several of them produced viable sterile mycelium. To infer their phylogenetic relationships we sequenced and analysed the large (LSU) and small (SSU) subunit nrDNA, the ITS nrDNA and the DNA-directed RNA polymerase II (RPB2). The fungus represents an independent marine biotrophic lineage in the Aigialaceae (Pleosporales) and is introduced here as Posidoniomycesatricolor gen. et sp. nov. Its closest relatives are typically plant-associated saprobes from marine, terrestrial and freshwater habitats in Southeast Asia and Central America. This study expands our knowledge and diversity of the Aigialaceae, adds a new symbiotic lifestyle to this family and provides a formal name for the dominant root mycobiont of the dominant Mediterranean seagrass.