Molecular characterization of the complete genome of a novel ormycovirus infecting the ectomycorrhizal fungus Hortiboletus rubellus.

Advancements in high-throughput sequencing and the development of new bioinformatics tools for large-scale data analysis play a crucial role in uncovering virus diversity and enhancing our understanding of virus evolution. The discovery of the ormycovirus clades, a group of RNA viruses that are phylogenetically distinct from all known Riboviria members and are found in fungi, highlights the value of these tools for the discovery of novel viruses. The aim of this study was to examine viral populations in fungal hosts to gain insights into the diversity, evolution, and classification of these viruses. Here, we report the molecular characterization of a newly discovered ormycovirus, which we have named "Hortiboletus rubellus ormycovirus 1" (HrOMV1), that was found in the ectomycorrhizal fungus Hortiboletus rubellus. The bipartite genome of HrOMV1, whose nucleotide sequence was determined by HTS and RLM-RACE, consists of two RNA segments (RNA1 and RNA2) that exhibit similarity to those of previously studied ormycoviruses in their organization and the proteins they encode. The presence of upstream, in-frame AUG triplets in the 5' termini of both RNA segments suggests that HrOMV1, like certain other ormycoviruses, employs a non-canonical translation initiation strategy. Phylogenetic analysis showed that HrOMV1 is positioned within the gammaormycovirus clade. Its putative RNA-dependent RNA polymerase (RdRp) exhibits sequence similarity to those of other gammaormycovirus members, the most similarity to that of Termitomyces ormycovirus 1, with 33.05% sequence identity. This protein was found to contain conserved motifs that are crucial for RNA replication, including the distinctive GDQ catalytic triad observed in gammaormycovirus RdRps. The results of this study underscore the significance of investigating the ecological role of mycoviruses in mycorrhizal fungi. This is the first report of an ormycovirus infecting a member of the ectomycorrhizal genus Hortiboletus.

Suillus: an emerging model for the study of ectomycorrhizal ecology and evolution.

Research on mycorrhizal symbiosis has been slowed by a lack of established study systems. To address this challenge, we have been developing Suillus, a widespread ecologically and economically relevant fungal genus primarily associated with the plant family Pinaceae, into a model system for studying ectomycorrhizal (ECM) associations. Over the last decade, we have compiled extensive genomic resources, culture libraries, a phenotype database, and protocols for manipulating Suillus fungi with and without their tree partners. Our efforts have already resulted in a large number of publicly available genomes, transcriptomes, and respective annotations, as well as advances in our understanding of mycorrhizal partner specificity and host communication, fungal and plant nutrition, environmental adaptation, soil nutrient cycling, interspecific competition, and biological invasions. Here, we highlight the most significant recent findings enabled by Suillus, present a suite of protocols for working with the genus, and discuss how Suillus is emerging as an important model to elucidate the ecology and evolution of ECM interactions.

The influence of tree genus, phylogeny, and richness on the specificity, rarity, and diversity of ectomycorrhizal fungi.

Partner specificity is a well-documented phenomenon in biotic interactions, yet the factors that determine specificity in plant-fungal associations remain largely unknown. By utilizing composite soil samples, we identified the predictors that drive partner specificity in both plants and fungi, with a particular focus on ectomycorrhizal associations. Fungal guilds exhibited significant differences in overall partner preference and avoidance, richness, and specificity to specific tree genera. The highest level of specificity was observed in root endophytic and ectomycorrhizal associations, while the lowest was found in arbuscular mycorrhizal associations. The majority of ectomycorrhizal fungal species showed a preference for one of their partner trees, primarily at the plant genus level. Specialist ectomycorrhizal fungi were dominant in belowground communities in terms of species richness and relative abundance. Moreover, all tree genera (and occasionally species) demonstrated a preference for certain fungal groups. Partner specificity was not related to the rarity of fungi or plants or environmental conditions, except for soil pH. Depending on the partner tree genus, specific fungi became more prevalent and relatively more abundant with increasing stand age, tree dominance, and soil pH conditions optimal for the partner tree genus. The richness of partner tree species and increased evenness of ectomycorrhizal fungi in multi-host communities enhanced the species richness of ectomycorrhizal fungi. However, it was primarily the partner-generalist fungi that contributed to the high diversity of ectomycorrhizal fungi in mixed forests.

Decoding the chemical language of Suillus fungi: genome mining and untargeted metabolomics uncover terpene chemical diversity.

Ectomycorrhizal fungi establish mutually beneficial relationships with trees, trading nutrients for carbon. Suillus are ectomycorrhizal fungi that are critical to the health of boreal and temperate forest ecosystems. Comparative genomics has identified a high number of non-ribosomal peptide synthetase and terpene biosynthetic gene clusters (BGC) potentially involved in fungal competition and communication. However, the functionality of these BGCs is not known. This study employed co-culture techniques to activate BGC expression and then used metabolomics to investigate the diversity of metabolic products produced by three Suillus species (Suillus hirtellus EM16, Suillus decipiens EM49, and Suillus cothurnatus VC1858), core members of the pine microbiome. After 28 days of growth on solid media, liquid chromatography-tandem mass spectrometry identified a diverse range of extracellular metabolites (exometabolites) along the interaction zone between Suillus co-cultures. Prenol lipids were among the most abundant chemical classes. Out of the 62 unique terpene BGCs predicted by genome mining, 41 putative prenol lipids (includes 37 putative terpenes) were identified across the three Suillus species using metabolomics. Notably, some terpenes were significantly more abundant in co-culture conditions. For example, we identified a metabolite matching to isomers isopimaric acid, sandaracopimaric acid, and abietic acid, which can be found in pine resin and play important roles in host defense mechanisms and Suillus spore germination. This research highlights the importance of combining genomics and metabolomics to advance our understanding of the chemical diversity underpinning fungal signaling and communication.IMPORTANCEUsing a combination of genomics and metabolomics, this study's findings offer new insights into the chemical diversity of Suillus fungi, which serve a critical role in forest ecosystems.

Ectomycorrhizal fungal communities in natural and urban ecosystems: Quercus humboldtii as a study case in the tropical Andes.

Worldwide urban landscapes are expanding because of the growing human population. Urban ecosystems serve as habitats to highly diverse communities. However, studies focusing on the diversity and structure of ectomycorrhizal communities are uncommon in this habitat. In Colombia, Quercus humboldtii Bonpl. is an ectomycorrhizal tree thriving in tropical montane forests hosting a high diversity of ectomycorrhizal fungi. Q. humboldtii is planted as an urban tree in Bogotá (Colombia). We studied how root-associated fungal communities of this tree change between natural and urban areas. Using Illumina sequencing, we amplified the ITS1 region and analyzed the resulting data using both OTUs and Amplicon Sequence Variants (ASVs) bioinformatics pipelines. The results obtained using both pipelines showed no substantial differences between OTUs and ASVs for the community patterns of root-associated fungi, and only differences in species richness were observed. We found no significant differences in the species richness between urban and rural sites based on Fisher's alpha or species-accumulation curves. However, we found significant differences in the community composition of fungi present in the roots of rural and urban trees with rural communities being dominated by Russula and Lactarius and urban communities by Scleroderma, Hydnangium, and Trechispora, suggesting a high impact of urban disturbances on ectomycorrhizal fungal communities. Our results highlight the importance of urban trees as reservoirs of fungal diversity and the potential impact of urban conditions on favoring fungal species adapted to more disturbed ecosystems.

Year-round dynamics of arbuscular mycorrhizal fungi communities in the roots and surrounding soils of Cryptomeria japonica.

Arbuscular mycorrhizal fungi (AMF) live simultaneously inside and outside of host plant roots for a functional mycorrhizal symbiosis. Still, the year-round dynamics and relationships between soil properties and AMF communities of trees in forest ecosystems remain unclear. We collected paired root and soil samples of the same Cryptomeria japonica trees at two forest sites (five trees at each site) every 2 months over a year. Total DNA was extracted from roots and soil separately and soil physicochemical properties were measured. With Illumina's next-generation amplicon sequencing targeting the small subunit of fungal ribosomal DNA, we clarified seasonal dynamics of soil properties and AMF communities. Soil pH and total phosphorus showed significant seasonality while total carbon, nitrogen, and C/N did not. Only pH was a good predictor of the composition and dynamics of the AMF community. The total AMF community (roots + soil) showed significant seasonality because of variation from May to September. Root and soil AMF communities were steady year-round, however, with similar species richness but contained significantly different AMF assemblages in any sampling month. Despite the weak seasonality in the communities, the top two dominant OTUs showed significant but different shifts between roots and soils across seasons with strong antagonistic relationships. In conclusion, few dominant AMF taxa are dynamically shifting between the roots and soils of C. japonica to respond to seasonal and phenological variations in their microhabitats. AMF inhabiting forest ecosystems may have high environmental plasticity to sustain a functional symbiosis regardless of seasonal variations that occur in the soil.

Mycorrhizas in South American Ericaceae.

Mycorrhizal symbioses (mycorrhizas) of Ericaceae, including ericoid mycorrhiza (ErM), have been mainly studied in the Northern Hemisphere, although the highest diversity of ericaceous plants is located in the Southern Hemisphere, where several regions remain largely unexplored. One of them is South America, which harbors a remarkably high diversity of Ericaceae (691 species and 33 genera) in a wide range of environmental conditions, and a specific mycorrhizal type called cavendishioid. In this review, we compile all available information on mycorrhizas of Ericaceae in South America. We report data on the mycorrhizal type and fungal diversity in 17 and 11 ericaceous genera, respectively. We show that South American Ericaceae exhibit a high diversity of habitats and life forms and that some species from typical ErM subfamilies may also host arbuscular mycorrhiza. Also, a possible geographical pattern in South American ErM fungal communities is suggested, with Sebacinales being the dominant mycorrhizal partners of the Andean clade species from tropical mountains, while archetypal ErM fungi are common partners in southern South America species. The gathered information challenges some common assumptions about ErM and suggests that focusing on understudied regions would improve our understanding of the evolution of mycorrhizal associations in this intriguing family.

Organic farming promotes the abundance of fungi keystone taxa in bacteria-fungi interkingdom networks.

Soil bacteria-fungi interactions are essential in the biogeochemical cycles of several nutrients, making these microbes major players in agroecosystems. While the impact of the farming system on microbial community composition has been extensively reported in the literature, whether sustainable farming approaches can promote associations between bacteria and fungi is still unclear. To study this, we employed 16S, ITS, and 18S DNA sequencing to uncover how microbial interactions were affected by conventional and organic farming systems on maize crops. The Bray-Curtis index revealed that bacterial, fungal, and arbuscular mycorrhizal fungi communities were significantly different between the two farming systems. Several taxa known to thrive in healthy soils, such as Nitrosophaerales, Orbiliales, and Glomus were more abundant in the organic farming system. Constrained ordination revealed that the organic farming system microbial community was significantly correlated with the ß-glucosidase activity, whereas the conventional farming system microbial community significantly correlated with soil pH. Both conventional and organic co-occurrence interkingdom networks exhibited a parallel node count, however, the former had a higher number of edges, thus being denser than the latter. Despite the similar amount of fungal nodes in the co-occurrence networks, the organic farming system co-occurrence network exhibited more than 3-fold the proportion of fungal taxa as keystone nodes than the conventional co-occurrence network. The genera Bionectria, Cercophora, Geastrum, Penicillium, Preussia, Metarhizium, Myceliophthora, and Rhizophlyctis were among the fungal keystone nodes of the organic farming system network. Altogether, our results uncover that beyond differences in microbial community composition between the two farming systems, fungal keystone nodes are far more relevant in the organic farming system, thus suggesting that bacteria-fungi interactions are more frequent in organic farming systems, promoting a more functional microbial community.

The mycorrhizal symbiosis: research frontiers in genomics, ecology, and agricultural application.

Mycorrhizal symbioses between plants and fungi are vital for the soil structure, nutrient cycling, plant diversity, and ecosystem sustainability. More than 250 000 plant species are associated with mycorrhizal fungi. Recent advances in genomics and related approaches have revolutionized our understanding of the biology and ecology of mycorrhizal associations. The genomes of 250+ mycorrhizal fungi have been released and hundreds of genes that play pivotal roles in regulating symbiosis development and metabolism have been characterized. rDNA metabarcoding and metatranscriptomics provide novel insights into the ecological cues driving mycorrhizal communities and functions expressed by these associations, linking genes to ecological traits such as nutrient acquisition and soil organic matter decomposition. Here, we review genomic studies that have revealed genes involved in nutrient uptake and symbiosis development, and discuss adaptations that are fundamental to the evolution of mycorrhizal lifestyles. We also evaluated the ecosystem services provided by mycorrhizal networks and discuss how mycorrhizal symbioses hold promise for sustainable agriculture and forestry by enhancing nutrient acquisition and stress tolerance. Overall, unraveling the intricate dynamics of mycorrhizal symbioses is paramount for promoting ecological sustainability and addressing current pressing environmental concerns. This review ends with major frontiers for further research.

Extreme environments simplify reassembly of communities of arbuscular mycorrhizal fungi.

The ecological impacts of long-term (press) disturbance on mechanisms regulating the relative abundance (i.e., commonness or rarity) and temporal dynamics of species within a community remain largely unknown. This is particularly true for the functionally important arbuscular mycorrhizal (AM) fungi; obligate plant-root endosymbionts that colonize more than two-thirds of terrestrial plant species. Here, we use high-resolution amplicon sequencing to examine how AM fungal communities in a specific extreme ecosystem-mofettes or natural CO2 springs caused by geological CO2 exhalations-are affected by long-term stress. We found that in mofettes, specific and temporally stable communities form as a subset of the local metacommunity. These communities are less diverse and dominated by adapted, "stress tolerant" taxa. Those taxa are rare in control locations and more benign environments worldwide, but show a stable temporal pattern in the extreme sites, consistently dominating the communities in grassland mofettes. This pattern of lower diversity and high dominance of specific taxa has been confirmed as relatively stable over several sampling years and is independently observed across multiple geographic locations (mofettes in different countries). This study implies that the response of soil microbial community composition to long-term stress is relatively predictable, which can also reflect the community response to other anthropogenic stressors (e.g., heavy metal pollution or land use change). Moreover, as AM fungi are functionally differentiated, with different taxa providing different benefits to host plants, changes in community structure in response to long-term environmental change have the potential to impact terrestrial plant communities and their productivity.IMPORTANCEArbuscular mycorrhizal (AM) fungi form symbiotic relationships with more than two-thirds of plant species. In return for using plant carbon as their sole energy source, AM fungi improve plant mineral supply, water balance, and protection against pathogens. This work demonstrates the importance of long-term experiments to understand the effects of long-term environmental change and long-term disturbance on terrestrial ecosystems. We demonstrated a consistent response of the AM fungal community to a long-term stress, with lower diversity and a less variable AM fungal community over time under stress conditions compared to the surrounding controls. We have also identified, for the first time, a suite of AM fungal taxa that are consistently observed across broad geographic scales in stressed and anthropogenically heavily influenced ecosystems. This is critical because global environmental change in terrestrial ecosystems requires an integrative approach that considers both above- and below-ground changes and examines patterns over a longer geographic and temporal scale, rather than just single sampling events.

Populus MYC2 orchestrates root transcriptional reprogramming of defence pathway to impair Laccaria bicolor ectomycorrhizal development.

The jasmonic acid (JA) signalling pathway plays an important role in the establishment of the ectomycorrhizal symbiosis. The Laccaria bicolor effector MiSSP7 stabilizes JA corepressor JAZ6, thereby inhibiting the activity of Populus MYC2 transcription factors. Although the role of MYC2 in orchestrating plant defences against pathogens is well established, its exact contribution to ECM symbiosis remains unclear. This information is crucial for understanding the balance between plant immunity and symbiotic relationships. Transgenic poplars overexpressing or silencing for the two paralogues of MYC2 transcription factor (MYC2s) were produced, and their ability to establish ectomycorrhiza was assessed. Transcriptomics and DNA affinity purification sequencing were performed. MYC2s overexpression led to a decrease in fungal colonization, whereas its silencing increased it. The enrichment of terpene synthase genes in the MYC2-regulated gene set suggests a complex interplay between the host monoterpenes and fungal growth. Several root monoterpenes have been identified as inhibitors of fungal growth and ECM symbiosis. Our results highlight the significance of poplar MYC2s and terpenes in mutualistic symbiosis by controlling root fungal colonization. We identified poplar genes which direct or indirect control by MYC2 is required for ECM establishment. These findings deepen our understanding of the molecular mechanisms underlying ECM symbiosis.

Soil microbial communities along elevational gradients in the Madrean Sky Islands.

The Madrean Sky Islands are mountain ranges isolated by a 'desert sea'. This area is a biodiversity hotspot currently threatened by climate change. Here, we studied soil microbial communities along elevational gradients in eight Madrean Sky Islands in southeastern Arizona (USA). Our results showed that while elevational microbial richness gradients were weak and not consistent across different mountains, soil properties strongly influenced microbial community composition (overall composition and the abundance of key functional groups) along elevational gradients. In particular, warming is associated with a higher abundance of soil-borne fungal plant pathogens that concomitantly might facilitate upward elevational shifts of plant species released from negative plant-soil feedbacks. Furthermore, projected warming and drought in the area aggravated by anthropogenic nitrogen deposition on mountain tops (and thus, decreasing nitrogen limitation) can enhance a shift from ectomycorrhizal to arbuscular mycorrhizal fungi. Overall, these results indicate that climate change effects on plant-soil interactions might have profound ecosystem consequences.

Heterogeneity in Arbuscular Mycorrhizal Fungi and Plant Communities of the Brazilian Cerrado, Transitional Areas toward the Caatinga, and the Atlantic Forest.

The Cerrado is the most diverse tropical savanna worldwide and the second-largest biome in South America. The objective of this study was to understand the heterogeneity and dynamics of arbuscular mycorrhizal fungi (AMF) in different types of natural Cerrado vegetation and areas that are transitioning to dryer savannas or tropical rainforests and to elucidate the driving factors responsible for the differences between these ecosystems. Twenty-one natural sites were investigated, including typical Cerrado forest, typical Caatinga, Atlantic Rainforest, transitions between Cerrado and Caatinga, Cerrado areas near Caatinga or rainforest, and Carrasco sites. Spores were extracted from the soils, counted, and morphologically analyzed. In total, 82 AMF species were detected. AMF species richness varied between 36 and 51, with the highest richness found in the area transitioning between Cerrado and Caatinga, followed by areas of Cerrado close to Caatinga and typical Cerrado forest. The types of Cerrado vegetation and the areas transitioning to the Caatinga shared the highest numbers of AMF species (32-38). Vegetation, along with chemical and physical soil parameters, affected the AMF communities, which may also result from seasonal rainfall patterns. The Cerrado has a great AMF diversity and is, consequently, a natural refuge for AMF. The plant and microbial communities as well as the diversity of habitats require urgent protection within the Cerrado, as it represents a key AMF hotspot.

Metatranscriptomics sheds light on the links between the functional traits of fungal guilds and ecological processes in forest soil ecosystems.

Soil fungi belonging to different functional guilds, such as saprotrophs, pathogens, and mycorrhizal symbionts, play key roles in forest ecosystems. To date, no study has compared the actual gene expression of these guilds in different forest soils. We used metatranscriptomics to study the competition for organic resources by these fungal groups in boreal, temperate, and Mediterranean forest soils. Using a dedicated mRNA annotation pipeline combined with the JGI MycoCosm database, we compared the transcripts of these three fungal guilds, targeting enzymes involved in C- and N mobilization from plant and microbial cell walls. Genes encoding enzymes involved in the degradation of plant cell walls were expressed at a higher level in saprotrophic fungi than in ectomycorrhizal and pathogenic fungi. However, ectomycorrhizal and saprotrophic fungi showed similarly high expression levels of genes encoding enzymes involved in fungal cell wall degradation. Transcripts for N-related transporters were more highly expressed in ectomycorrhizal fungi than in other groups. We showed that ectomycorrhizal and saprotrophic fungi compete for N in soil organic matter, suggesting that their interactions could decelerate C cycling. Metatranscriptomics provides a unique tool to test controversial ecological hypotheses and to better understand the underlying ecological processes involved in soil functioning and carbon stabilization.

The evolution of ectomycorrhizal symbiosis in the Late Cretaceous is a key driver of explosive diversification in Agaricomycetes.

Ectomycorrhizal (EcM) symbiosis, a ubiquitous plant-fungus interaction in forests, evolved in parallel in fungi. Why the evolution of EcM fungi did not necessarily increase ecological opportunities for explosive diversification remains unclear. This study aimed to reveal the driving mechanism of the evolutionary diversification in the fungal class Agaricomycetes, specifically by testing whether the evolution of EcM symbiosis in the Late Cretaceous increased ecological opportunities. The historical character transitions of trophic state and fruitbody form were estimated based on phylogenies inferred from fragments of 89 single-copy genes. Moreover, five analyses were used to estimate the net diversification rates (speciation rate minus extinction rate). The results indicate that the unidirectional evolution of EcM symbiosis occurred 27 times, ranging in date from the Early Triassic to the Early Paleogene. The increased diversification rates appeared to occur intensively at the stem of EcM fungal clades diverging in the Late Cretaceous, coinciding with the rapid diversification of EcM angiosperms. By contrast, the evolution of fruitbody form was not strongly linked with the increased diversification rates. These findings suggest that the evolution of EcM symbiosis in the Late Cretaceous, supposedly with coevolving EcM angiosperms, was the key drive of the explosive diversification in Agaricomycetes.

Metagenomic Analyses of Viruses in the Orchid Mycorrhizal Interaction Using Improved Assemble Tools.

In nature, mycorrhizal association with soil-borne fungi is indispensable for orchid species. Compatible mycorrhizal fungi form endo-mycorrhizal structures in orchid cells, and the fungal structures are digested in orchid cells to be supplied to orchids as nutrition. Because orchid seeds lack the reserves for germination, they keep receiving nutrition through mycorrhizal formation from seed germination until nonphotosynthetic young seedlings develop leaves and become photoautotrophic. Seeds of all orchids germinate with the help of their own fungal partners, and therefore, specific partnership has been acquired in a long evolutionary history between orchids and fungi. Assuming that horizontal transmission of viruses may occur in such a close relationship, we are focusing on viruses that infect orchids and their mycorrhizal fungi. We prepared aseptically germinated orchid plants (i.e., fungi-free plants) together with pure-cultured fungal isolates and conducted transcriptome analyses (RNA-seq) by next-generation sequencing (NGS) approach. To reconstruct virus-related sequences that would have been present in the RNA sample of interest, de novo assembly process is required using short read sequences obtained from RNA-seq. In the previous version of our protocol (see Viral Metagenomics, first edition 2018), virus searches were conducted using contig sets constructed by a single assembler, but this time we devised a method to construct more reliable contigs using multiple assemblers and again reinvestigated that viruses could be detected. Because the virus detection efficiency and number of detected virus species clearly differed depending on the assembly pipeline and the number of the input data, multiple methods should be used to identify viral infection, if possible.

Arbuscular mycorrhizal fungi alter rhizosphere fungal community characteristics of Acorus calamus to improve Cr resistance.

To investigate changes in fungal community characteristics under different Cr(VI) concentration stresses and the advantages of adding arbuscular mycorrhizal fungi (AMF), we used high throughput sequencing to characterize the fungal communities. Cr(VI) stress reduced rhizosphere soil SOM (soil organic matter) content and AMF addition improved this stress phenomenon. There were significant differences in fungal community changes under different Cr(VI) concentrations. The fungal community characteristics changed through inhibition of fungal metabolic ability, as fungal abundance increased after AMF addition, and the fungal diversity increased under high Cr(VI) concentration. The dominant phyla were members of the Ascomycota, Basidiomycota, Mortierellomycota, and Rozellomycota. Dominant groups relevant to Cr resistance were Ascomycota and Basidiomycota fungi. Moreover, Fungal community characteristics were analyzed using high-throughput sequencing of the cytochrome c metabolic pathway, NADH dehydrogenase, and NADH: ubiquinone reductase and all these functions were enhanced after AMF addition. Therefore, Cr(VI) stress significantly affects fungal community structure, while AMF addition could increase its SOM content, and metabolic capacity, and improve fungal community tolerance to Cr stress. This study contributed to the understanding response of rhizosphere fungal community in AMF-assisted wetland phytoremediation under Cr stress.

Visualization of root extracellular traps in an ectomycorrhizal woody plant (Pinus densiflora) and their interactions with root-associated bacteria.

MAIN CONCLUSION: Extracellular traps in the primary root of Pinus densiflora contribute to root-associated bacterial colonization. Trapped rhizobacteria induce the production of reactive oxygen species in root-associated, cap-derived cells. Ectomycorrhizal (ECM) woody plants, such as members of Pinaceae and Fagaceae, can acquire resistance to biotic and abiotic stresses through the formation of mycorrhiza with ECM fungi. However, germinated tree seedlings do not have mycorrhizae and it takes several weeks for ectomycorrhizae to form on their root tips. Therefore, to confer protection during the early growth stage, bare primary roots require defense mechanisms other than mycorrhization. Here, we attempted to visualize root extracellular traps (RETs), an innate root defense mechanism, in the primary root of Pinus densiflora and investigate the interactions with root-associated bacteria isolated from ECM and fine non-mycorrhizal roots. Histological and histochemical imaging and colony-forming unit assays demonstrated that RETs in P. densiflora, mainly consisting of root-associated, cap-derived cells (AC-DCs) and large amounts of root mucilage, promote bacterial colonization in the rhizosphere, despite also having bactericidal activity via extracellular DNA. Four rhizobacterial strains retarded the mycelial growth of a pathogenic strain belonging to the Fusarium oxysporum species complex in dual culture assay. They also induced the production of reactive oxygen species (ROS) from host tree AC-DCs without being excluded from the rhizosphere of P. densiflora. Applying three Paraburkholderia strains, especially PM O-EM8 and PF T-NM22, showed significant differences in the ROS levels from the control group. These results reveal the indirect contributions of rhizobacteria to host root defense and suggest that root-associated bacteria could be a component of RETs as a first line of defense against root pathogens in the early growth stage of ECM woody plants.

Species composition of root-associated mycobiome of ruderal invasive Anthemis cotula L. varies with elevation in Kashmir Himalaya / La composición de especies del micobioma asociado a las raíces de la invasora ruderal Anthemis cotula L. varía con la elevación en Cachemira Himalaya

Investigating the microbial communities associated with invasive plant species can provide insights into how these species establish and thrive in new environments. Here, we explored the fungal species associated with the roots of the invasive species Anthemis cotula L. at 12 sites with varying elevations in the Kashmir Himalaya. Illumina MiSeq platform was used to identify the species composition, diversity, and guild structure of these root-associated fungi. The study found a total of 706 fungal operational taxonomic units (OTUs) belonging to 8 phyla, 20 classes, 53 orders, 109 families, and 160 genera associated with roots of A. cotula, with the most common genus being Funneliformis. Arbuscular mycorrhizal fungi (AMF) constituted the largest guild at higher elevations. The study also revealed that out of the 12 OTUs comprising the core mycobiome, 4 OTUs constituted the stable component while the remaining 8 OTUs comprised the dynamic component. While α-diversity did not vary across sites, significant variation was noted in β-diversity. The study confirmed the facilitative role of the microbiome through a greenhouse trial in which a significant effect of soil microbiome on height, shoot biomass, root biomass, number of flower heads, and internal CO2 concentration of the host plant was observed. The study indicates that diverse fungal mutualists get associated with this invasive alien species even in nutrient-rich ruderal habitats and may be contributing to its spread into higher elevations. This study highlights the importance of understanding the role of root-associated fungi in invasion dynamics and the potential use of mycobiome management strategies to control invasive species.(AU)

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).