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.

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).

Fungi in hair roots of Vaccinium spp. (Ericaceae) growing on decomposing wood: colonization patterns, identity, and in vitro symbiotic potential.

Most of our knowledge on the ericoid mycorrhizal (ErM) symbiosis comes from temperate heathlands characterized by acidic peaty soils and many experiments with a few ascomycetous fungi. However, ericaceous plants thrive in many other ecosystems and in temperate coniferous forests, their seedlings often prosper on decomposing wood. While wood is typically exploited by basidiomycetous ectomycorrhizal (EcM) and saprobic fungi, the role of ErM fungi (ErMF) is much less clear. We explored the cultivable mycobiota of surface sterilized hair roots of Vaccinium spp. growing on decomposing wood in two coniferous forests in Mid-Norway (Scandinavia) and Northern Bohemia (Central Europe). Obtained isolates were identified using molecular tools and their symbiotic potential was tested in vitro. While the detected community lacked the archetypal ErMF Hyaloscypha hepaticicola and the incidence of dark septate endophytes and EcM fungi was negligible, it comprised other frequent asexual ascomycetous ErMF, namely H. variabilis and Oidiodendron maius, together with several isolates displaying affinities to sexual saprobic H. daedaleae and H. fuckelii. Ascomycete-suppressing media revealed representatives of the saprobic basidiomycetous genera Coprinellus, Gymnopilus, Mycena (Agaricales), and Hypochnicium (Polyporales). In the resyntheses, the tested basidiomycetes occasionally penetrated the rhizodermal cells of their hosts but never formed ericoid mycorrhizae and in many cases overgrew and killed the inoculated seedlings. In contrast, a representative of the H. daedaleae/H. fuckelii-related isolates repeatedly formed what morphologically appears as the ErM symbiosis and supported host's growth. In conclusion, while basidiomycetous saprobic fungi have a potential to colonize healthy-looking ericaceous hair roots, the mode(-s) of their functioning remain obscure. For the first time, a lineage in Hyaloscypha s. str. (corresponding to the former Hymenoscyphus ericae aggregate) where sexual saprobes are intermingled with root symbionts has been revealed, shedding new light on the ecology and evolution of these prominent ascomycetous ErMF.

Molecular identification and antibacterial properties of an ericoid associated mycorrhizal fungus.

BACKGROUND: The quest for novel sources of antibacterial compounds have necessitated the inclusion of ericoid mycorrhizal fungi (ERM) commonly found within the root of ericaceous plants. Agar-well diffusion method was used to detect antibacterial activity and was followed by the microbroth diffusion method [minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)]. RESULTS: The results of the phytochemical screening indicated that only alkaloids, flavonoids, phenols, saponins, cardiac glycosides and terpenoids were present, while steroids and tannins were absent. The MIC of the extracts ranged between 2 and 16 mg/mL, and the lowest MIC was obtained with Staphylococcus aureus. Also, the result of the MBC study indicated that the fungal extract was most active at concentrations of 2 and 4 mg/mL against Bacillus subtilis and S. aureus, respectively. CONCLUSIONS: This bioassay showed, for the first time, antibacterial activity of L. incrustata against some bacterial species. Subsequently, ERM fungi should be given attention when searching for antimicrobial agents because they could provide a solution to solve problems associated with conventional disease treatments (i.e. pathogenic microorganisms resistance).

A novel proof of concept for capturing the diversity of endophytic fungi preserved in herbarium specimens.

Herbarium specimens represent important records of morphological and genetic diversity of plants that inform questions relevant to global change, including species distributions, phenology and functional traits. It is increasingly appreciated that plant microbiomes can influence these aspects of plant biology, but little is known regarding the historic distribution of microbes associated with plants collected in the pre-molecular age. If microbiomes can be observed reliably in herbarium specimens, researchers will gain a new lens with which to examine microbial ecology, evolution, species interactions. Here, we describe a method for accessing historical plant microbiomes from preserved herbarium specimens, providing a proof of concept using two plant taxa from the imperiled boreal biome (Andromeda polifolia and Ledum palustre subsp. groenlandicum, Ericaceae). We focus on fungal endophytes, which occur within symptomless plant tissues such as leaves. Through a three-part approach (i.e. culturing, cloning and next-generation amplicon sequencing via the Illumina MiSeq platform, with extensive controls), we examined endophyte communities in dried, pressed leaves that had been processed as regular herbarium specimens and stored at room temperature in a herbarium for four years. We retrieved only one endophyte in culture, but cloning and especially the MiSeq analysis revealed a rich community of foliar endophytes. The phylogenetic distribution and diversity of endophyte assemblages, especially among the Ascomycota, resemble endophyte communities from fresh plants collected in the boreal biome. We could distinguish communities of endophytes in each plant species and differentiate likely endophytes from fungi that could be surface contaminants. Taxa found by cloning were observed in the larger MiSeq dataset, but species richness was greater when subsets of the same tissues were evaluated with the MiSeq approach. Our findings provide a proof of concept for capturing endophyte DNA from herbarium specimens, supporting the importance of herbarium records as roadmaps for understanding the dynamics of plant-associated microbial biodiversity in the Anthropocene.This article is part of the theme issue 'Biological collections for understanding biodiversity in the Anthropocene'.

Sphagnum Species Modulate their Phenolic Profiles and Mycorrhizal Colonization of Surrounding Andromeda polifolia along Peatland Microhabitats.

Sphagnum mosses mediate long-term carbon accumulation in peatlands. Given their functional role as keystone species, it is important to consider their responses to ecological gradients and environmental changes through the production of phenolics. We compared the extent to which Sphagnum phenolic production was dependent on species, microhabitats and season, and how surrounding dwarf shrubs responded to Sphagnum phenolics. We evaluated the phenolic profiles of aqueous extracts of Sphagnum fallax and Sphagnum magellanicum over a 6-month period in two microhabitats (wet lawns versus dry hummocks) in a French peatland. Phenolic profiles of water-soluble extracts were measured by UHPLC-QTOF-MS. Andromeda polifolia mycorrhizal colonization was quantified by assessing the intensity of global root cortex colonization. Phenolic profiles of both Sphagnum mosses were species-, season- and microhabitat- dependant. Sphagnum-derived acids were the phenolics mostly recovered; relative quantities were 2.5-fold higher in S. fallax than in S. magellanicum. Microtopography and vascular plant cover strongly influenced phenolic profiles, especially for minor metabolites present in low abundance. Higher mycorrhizal colonization of A. polifolia was found in lawns as compared to hummocks. Mycorrhizal abundance, in contrast to environmental parameters, was correlated with production of minor phenolics in S. fallax. Our results highlight the close interaction between mycorrhizae such as those colonizing A. polifolia and the release of Sphagnum phenolic metabolites and suggest that Sphagnum-derived acids and minor phenolics play different roles in this interaction. This work provides new insight into the ecological role of Sphagnum phenolics by proposing a strong association with mycorrhizal colonization of shrubs.

New 1,3-benzodioxin-4-ones from Synnemapestaloides ericacearum sp. nov., a biosynthetic link to remarkable compounds within the Xylariales.

Surveys of foliar endophytes from the Acadian forest region over the past three decades have identified numerous phylogenetically diverse fungi producing natural products toxic to forest pests and diseases. The life histories of some conifer endophytes can be restricted to plant foliage or may include saprotrophic phases on other plants tissues or even alternate hosts. Considering the potentially broad host preferences of conifer endophytes we explored fungi isolated from understory species and their metabolites as part of an ongoing investigation of fungal biodiversity from the Acadian forest. We report a hitherto unidentified Xylariomycetidae species isolated from symptomatic Labrador tea (Rhododendron groenlandicum) leaves and mountain laurel (Kalmia latifolia) collected in coastal southern New Brunswick, Canada. Morphological and phylogenetic evidence demonstrated the unknown species was a novel Synnemapestaloides (Sporocadaceae) species, described here as Syn. ericacearum. A preliminary screening assay indicated that the culture filtrate extract of the new species was potently antifungal towards the biotrophic pathogen Microbotryum violaceum, warranting an investigation of its natural products. Two natural products possessing a rare 1,3-benzodioxin-4-one scaffold, synnemadoxins A-B (1-2), and their postulated precursor, synnemadiacid A (3), were characterized as new structures and assessed for antimicrobial activity. All isolated compounds elicited in vitro inhibitory antifungal activity towards M. violaceum at 2.3 µg mL-1 and moderate antibiotic activity. Further, the characterization of synnemadoxins A-B provided a perspective on the biosynthesis of some related 1,3-benzodioxin-4-ones produced by other fungi within the Xylariales.

Gamarada debralockiae gen. nov. sp. nov.-the genome of the most widespread Australian ericoid mycorrhizal fungus.

This study describes a novel ericoid mycorrhizal fungus (ErMF), Gamarada debralockiae Midgley and Tran-Dinh gen. nov. sp. nov. Additionally, catabolism was explored from a genomic perspective. The nuclear and mitochondrial genomes of G. debralockiae were sequenced. Morphological characteristics were assessed on various media. Catabolic genes of G. debralockiae were explored using SignalP and dbCAN. Phylogenetic comparisons were undertaken using Phylogeny.fr. The 58.5-Mbp draft genome of G. debralockiae contained 17,075 putative genes. The complete mitochondrial genome was 28,168 bp in length. In culture, G. debralockiae produces slow-growing non-sporulating colonies. Gamarada debralockiae has many putative secreted catabolic enzymes. Phylogeny indicated G. debralockiae was distinct from known ascomycetous ErMF: Pezoloma ericae, Meliniomyces spp., Oidiodendron spp., and Cairneyella variabilis. It is closely related to many undescribed plant root-associated fungi and its nearest described relative is Hyphodiscus brevicollaris. Gamarada debralockiae has been recovered from virtually all Australian ericoid mycorrhizal studies and biogeographic data suggests the taxon is widespread in Australia. Gamarada debralockiae has similar catabolic potential to C. variabilis and co-occurs with C. variabilis at Australian sites. Plants that host multiple ErMF may benefit from subtle differences in catabolism that improve access to nitrogen and phosphorus from within recalcitrant organic matter.

Ericoid plant species and Pinus sylvestris shape fungal communities in their roots and surrounding soil.

Root-colonizing fungi can form mycorrhizal or endophytic associations with plant roots, the type of association depending on the host. We investigated the differences and similarities of the fungal communities of three boreal ericoid plants and one coniferous tree, and identified the community structure of fungi utilizing photosynthates from the plants studied. The fungal communities of roots and soils of Vaccinium myrtillus, Vaccinium vitis-idaea, Calluna vulgaris and Pinus sylvestris were studied in an 18-month-long experiment where the plants were grown individually in natural substrate. Photosynthates utilizing fungi were detected with DNA stable-isotope probing using 13 CO2 (13 C-DNA-SIP). The results indicated that the plants studied provide different ecological niches preferred by different fungal species. Those fungi which dominated the community in washed roots had also the highest 13 C-uptake. In addition, a common root endophyte without confirmed mycorrhizal status also obtained 13 C from all the plants, indicating close plant-association of this fungal species. We detect several fungal species inhabiting the roots of both ericoid mycorrhizal and ectomycorrhizal plants. Our results highlight that the ecological role of co-occurrence of fungi with different life styles (e.g. mycorrhizal or endophytic) in plant root systems should be further investigated.

Mixotrophy in Pyroleae (Ericaceae) from Estonian boreal forests does not vary with light or tissue age.

Background and Aims: In temperate forests, some green plants, namely pyroloids (Pyroleae, Ericaceae) and some orchids, independently evolved a mode of nutrition mixing photosynthates and carbon gained from their mycorrhizal fungi (mixotrophy). Fungal carbon is more enriched in 13C than photosynthates, allowing estimation of the proportion of carbon acquired heterotrophically from fungi in plant biomass. Based on 13C enrichment, mixotrophic orchids have previously been shown to increase shoot autotrophy level over the growth season and with environmental light availability. But little is known about the plasticity of use of photosynthetic versus fungal carbon in pyroloids. Methods: Plasticity of mixotrophy with leaf age or light level (estimated from canopy openness) was investigated in pyroloids from three Estonian boreal forests. Bulk leaf 13C enrichment of five pyroloid species was compared with that of control autotrophic plants along temporal series (over one growth season) and environmental light gradients (n=405 samples). Key Results: Mixotrophic 13C enrichment was detected at studied sites for Pyrola chlorantha and Orthilia secunda (except at one site for the latter), but not for Chimaphila umbellata, Pyrola rotundifolia and Moneses uniflora. Enrichment with 13C did not vary over the growth season or between leaves from current and previous years. Finally, although one co-occurring mixotrophic orchid showed 13C depletion with increasing light availability, as expected for mixotrophs, all pyroloids responded identically to autotrophic control plants along light gradients. Conclusions: A phylogenetic trend previously observed is further supported: mixotrophy is rarely supported by 13C enrichment in the Chimaphila + Moneses clade, whereas it is frequent in the Pyrola + Orthilia clade. Moreover, pyroloid mixotrophy does not respond plastically to ageing or to light level. This contrasts with the usual view of a convergent evolution with orchids, and casts doubt on the way pyroloids use the carbon gained from their mycorrhizal fungi, especially to replace photosynthetic carbon.

Communities of Putative Ericoid Mycorrhizal Fungi Isolated from Alpine Dwarf Shrubs in Japan: Effects of Host Identity and Microhabitat.

Dwarf shrubs of the family Ericaceae are common in arctic and alpine regions. Many of these plants are associated with ericoid mycorrhizal (ERM) fungi, which allow them to take nutrients and water from the soil under harsh environmental conditions and, thus, affect host plant survival. Despite the importance of ERM fungi to alpine plant communities, limited information is available on the effects of microhabitat and host identity on ERM fungal communities. We investigated the communities of putative ERM fungi isolated from five dwarf shrub species (Arcterica nana, Diapensia lapponica, Empetrum nigrum, Loiseleuria procumbens, and Vaccinium vitis-idaea) that co-occur in an alpine region of Japan, with reference to distinct microhabitats provided by large stone pine (Pinus pumila) shrubs (i.e. bare ground, the edge of stone pine shrubs, and the inside of stone pine shrubs). We obtained 703 fungal isolates from 222 individual plants. These isolates were classified into 55 operational taxonomic units (OTUs) based on the sequencing of internal transcribed spacer regions in ribosomal DNA. These putative ERM fungal communities were dominated by Helotiales fungi for all host species. Cistella and Trimmatostroma species, which have rarely been detected in ERM roots in previous studies, were abundant. ERM fungal communities were significantly different among microhabitats (R2=0.28), while the host effect explained less variance in the fungal communities after excluding the microhabitat effect (R2=0.17). Our results suggest that the host effect on ERM fungal communities is minor and the distributions of hosts and fungal communities may be assessed based on microhabitat conditions.

Increased phylogenetic resolution within the ecologically important Rhizopogon subgenus Amylopogon using 10 anonymous nuclear loci.

Rhizopogon species are ecologically significant ectomycorrhizal fungi in conifer ecosystems. The importance of this system merits the development and utilization of a more robust set of molecular markers specifically designed to evaluate their evolutionary ecology. Anonymous nuclear loci (ANL) were developed for R. subgenus Amylopogon. Members of this subgenus occur throughout the United States and are exclusive fungal symbionts associated with Pterospora andromedea, a threatened mycoheterotrophic plant endemic to disjunct eastern and western regions of North America. Candidate ANL were developed from 454 shotgun pyrosequencing and assessed for positive amplification across targeted species, sequencing success, and recovery of phylogenetically informative sites. Ten ANL were successfully developed and were subsequently used to sequence representative taxa, herbaria holotype and paratype specimens in R. subgenus Amylopogon. Phylogenetic reconstructions were performed on individual and concatenated data sets by Bayesian inference and maximum likelihood methods. Phylogenetic analyses of these 10 ANL were compared with a phylogeny traditionally constructed using the universal fungal barcode nuc rDNA ITS1-5.8S-ITS2 region (ITS). The resulting ANL phylogeny was consistent with most of the species designations delineated by ITS. However, the ANL phylogeny provided much greater phylogenetic resolution, yielding new evidence for cryptic species within previously defined species of R. subgenus Amylopogon. Additionally, the rooted ANL phylogeny provided an alternate topology to the ITS phylogeny, which inferred a novel set of evolutionary relationships not identified in prior phylogenetic studies.

First evidence of Pezoloma ericae in Australia: using the Biomes of Australia Soil Environments (BASE) to explore the Australian phylogeography of known ericoid mycorrhizal and root-associated fungi.

The prominent ericoid mycorrhizal fungus, Pezoloma ericae, has not been found in Australia to date. In the present study, internal transcribed spacer (ITS) data from the Biomes of Australia Soil Environments (BASE) was searched for evidence of P. ericae and other known ericoid mycorrhizal and root-associated taxa. ITS sequences with high identity to P. ericae, Meliniomyces bicolor, Meliniomyces variabilis, Cairneyella sp. 2, Cadophora finlandica and Woollsia mycorrhizal fungus VI were identified and their distribution in Australia visualised. This is the first evidence that P. ericae, M. bicolor and M. variabilis very likely occur on the Australian continent and provides a set of locations from which to seek isolates for further characterisation. The presence of P. ericae in South America, South Africa, and now Australia suggests a broad and ancient Gondwanan distribution for this well-studied species.

Secondary metabolites from the endophytic fungus Talaromyces pinophilus.

Endophytic fungi have a great influence on plant health and growth, and are an important source of bioactive natural compounds. Organic extracts obtained from the culture filtrate of an endophytic strain of Talaromyces pinophilus isolated from strawberry tree (Arbutus unedo) were studied. Metabolomic analysis revealed the presence of three bioactive metabolites, the siderophore ferrirubin, the platelet-aggregation inhibitor herquline B and the antibiotic 3-O-methylfunicone. The latter was the major metabolite produced by this strain and displayed toxic effects against the pea aphid Acyrthosiphon pisum (Homoptera Aphidiidae). This toxicity represents an additional indication that the widespread endophytic occurrence of T. pinophilus may be related to a possible role in defensive mutualism. Moreover, the toxic activity on aphids could promote further study on 3-O-methylfunicone, or its derivatives, as an alternative to synthetic chemicals in agriculture.

Does warming by open-top chambers induce change in the root-associated fungal community of the arctic dwarf shrub Cassiope tetragona (Ericaceae)?

Climate change may alter mycorrhizal communities, which impact ecosystem characteristics such as carbon sequestration processes. These impacts occur at a greater magnitude in Arctic ecosystems, where the climate is warming faster than in lower latitudes. Cassiope tetragona (L.) D. Don is an Arctic plant species in the Ericaceae family with a circumpolar range. C. tetragona has been reported to form ericoid mycorrhizal (ErM) as well as ectomycorrhizal (ECM) symbioses. In this study, the fungal taxa present within roots of C. tetragona plants collected from Svalbard were investigated using DNA metabarcoding. In light of ongoing climate change in the Arctic, the effects of artificial warming by open-top chambers (OTCs) on the fungal root community of C. tetragona were evaluated. We detected only a weak effect of warming by OTCs on the root-associated fungal communities that was masked by the spatial variation between sampling sites. The root fungal community of C. tetragona was dominated by fungal groups in the Basidiomycota traditionally classified as either saprotrophic or ECM symbionts, including the orders Sebacinales and Agaricales and the genera Clavaria, Cortinarius, and Mycena. Only a minor proportion of the operational taxonomic units (OTUs) could be annotated as ErM-forming fungi. This indicates that C. tetragona may be forming mycorrhizal symbioses with typically ECM-forming fungi, although no characteristic ECM root tips were observed. Previous studies have indicated that some saprophytic fungi may also be involved in biotrophic associations, but whether the saprotrophic fungi in the roots of C. tetragona are involved in biotrophic associations remains unclear. The need for more experimental and microscopy-based studies to reveal the nature of the fungal associations in C. tetragona roots is emphasized.

Diversity of fungal assemblages in roots of Ericaceae in two Mediterranean contrasting ecosystems.

The plants belonging to the Ericaceae family are morphologically diverse and widely distributed groups of plants. They are typically found in soil with naturally poor nutrient status. The objective of the current study was to identify cultivable mycobionts from roots of nine species of Ericaceae (Calluna vulgaris, Erica arborea, Erica australis, Erica umbellate, Erica scoparia, Erica multiflora, Arbutus unedo, Vaccinium myrtillus, and Vaccinium corymbosum). The sequencing approach was used to amplify the Internal Transcribed Spacer (ITS) region. Results from the phylogenetic analysis of ITS sequences stored in the Genbank confirmed that most of strains (78) were ascomycetes, 16 of these were closely related to Phialocephala spp, 12 were closely related to Helotiales spp and 6 belonged to various unidentified ericoid mycorrhizal fungal endophytes. Although the isolation frequencies differ sharply according to regions and ericaceous species, Helotiales was the most frequently encountered order from the diverse assemblage of associated fungi (46.15%), especially associated with C. vulgaris (19.23%) and V. myrtillus (6.41%), mostly present in the Loge (L) and Mellousa region (M). Moreover, multiple correspondence analysis (MCA) showed three distinct groups connecting fungal order to ericaceous species in different regions.

Specificity of fungal associations of Pyroleae and Monotropa hypopitys during germination and seedling development.

Mycoheterotrophic plants obtain organic carbon from associated mycorrhizal fungi, fully or partially. Angiosperms with this form of nutrition possess exceptionally small 'dust seeds' which after germination develop 'seedlings' that remain subterranean for several years, fully dependent on fungi for supply of carbon. Mycoheterotrophs which as adults have photosynthesis thus develop from full to partial mycoheterotrophy, or autotrophy, during ontogeny. Mycoheterotrophic plants may represent a gradient of variation in a parasitism-mutualism continuum, both among and within species. Previous studies on plant-fungal associations in mycoheterotrophs have focused on either germination or the adult life stages of the plant. Much less is known about the fungal associations during development of the subterranean seedlings. We investigated germination and seedling development and the diversity of fungi associated with germinating seeds and subterranean seedlings (juveniles) in five Monotropoideae (Ericaceae) species, the full mycoheterotroph Monotropa hypopitys and the putatively partial mycoheterotrophs Pyrola chlorantha, P. rotundifolia, Moneses uniflora and Chimaphila umbellata. Seedlings retrieved from seed sowing experiments in the field were used to examine diversity of fungal associates, using pyrosequencing analysis of ITS2 region for fungal identification. The investigated species varied with regard to germination, seedling development and diversity of associated fungi during juvenile ontogeny. Results suggest that fungal host specificity increases during juvenile ontogeny, most pronounced in the fully mycoheterotrophic species, but a narrowing of fungal associates was found also in two partially mycoheterotrophic species. We suggest that variation in specificity of associated fungi during seedling ontogeny in mycoheterotrophs represents ongoing evolution along a parasitism-mutualism continuum.

Effect of soil moisture on root-associated fungal communities of Erica dominans in Drakensberg mountains in South Africa.

Ericoid mycorrhiza represents a key adaptation of the Ericaceae plants to facilitate their establishment in harsh conditions. The Ericaceae are a large family of flowering plants, with global distribution. However, our current knowledge about the ericoid mycorrhizal fungal diversity and ecology largely relates to the Northern Hemisphere. Our study focused on the assembly of root-associated fungal (RAF) communities of Erica dominans in two types of microhabitats of contrasting moisture along an elevation gradient in Drakensberg mountains in South Africa. RAF communities were determined by 454-sequencing of the internal transcribed spacer (ITS) region of ribosomal DNA. The majority of RAF showed affinity to the orders Helotiales, Pezizales, and Pleosporales. Microhabitat type as well as elevation had significant but weak effect on RAF community composition. We identified two putative ericoid mycorrhizal fungi, the ecological niches of which were differentiated between the studied microhabitats. Our study also provides one of the first comprehensive data about RAF communities of Ericaceae on African continent and shows the occurrence of the most studied ericoid mycorrhizal fungus Pezoloma ericae (belonging to P. ericae aggregate) in roots of Ericaceae host plant in Africa.

First record of Rhizoscyphus ericae in Southern Hemisphere's Ericaceae.

Ericoid mycorrhiza is arguably the least investigated mycorrhizal type, particularly when related to the number of potential hosts and the ecosystems they inhabit. Little is known about the global distribution of ericoid mycorrhizal (ErM) fungi, and this holds true even for the prominent ErM mycobiont Rhizoscyphus ericae. Earlier studies suggested R. ericae might be low in abundance or absent in the roots of Southern Hemisphere's Ericaceae, and our previous investigations in two Argentine Patagonian forests supported this view. Here, we revisited the formerly investigated area, albeit at a higher altitude, and screened fungi inhabiting hair roots of Gaultheria caespitosa and Gaultheria pumila at a treeless alpine site using the same methods as previously. We obtained 234 isolates, most of them belonging to Ascomycota. In contrast to previous findings, however, among 37 detected operational taxonomic units (OTUs), OTU 1 (=R. ericae s. str.) comprised the highest number of isolates (87, ∼37 %). Most of the OTUs and isolates belonged to the Helotiales, and 82.5 % of isolates belonged to OTUs shared between both Gaultheria species. At the alpine site, ericoid mycorrhizal fungi dominated, followed by dark septate endophytes and aquatic hyphomycetes probably acting as root endophytes. Our results suggest that the distribution of R. ericae is influenced, among others, by factors related to altitude such as soil type and presence/absence and type of the neighboring vegetation. Our study is the first report on R. ericae colonizing Ericaceae roots in the Southern Hemisphere and extends the known range of this prominent ErM species to NW Patagonia.

Identification of antibacterial peptides from endophytic microbiome.

Endophytes, microorganisms living inside plant tissues, are promising producers of lead compounds for the pharmaceutical industry. However, the majority of endophytes are unculturable and therefore inaccessible for functional studies. To evaluate genetic resources of endophytes, we analyzed the biodiversity of fungal microbiome of black crowberry (Empetrum nigrum L.) by next-generation sequencing and found that it consists mainly of unknown taxa. We then separated the host and the endophyte genomes and constructed a fosmid expression library from the endophytic DNA. This library was screened for antibacterial activity against Staphylococcus aureus. A unique antibacterial clone was selected for further analysis, and a gene En-AP1 was identified with no similarity to known sequences. The expressed, folded protein En-AP1 was not active against S. aureus, while tryptic digests exhibited antimicrobial activity. Seven out of twelve synthesized peptides, predicted antibacterial in silico, exhibited in vitro activity towards both S. aureus and Escherichia coli. We propose that the En-AP1 protein is degraded in the library host E. coli and antimicrobial fragments are released from the cell, explaining the in vitro antibacterial activity of the clone. This is the first report of a novel gene expressed in vitro derived from an endophytic microbiome, demonstrating the potential of finding novel genes and compounds from unculturable endophytes.