Exploration of the Production of Three Thiodiketopiperazines by an Endophytic Fungal Strain of Cophinforma mamane.

Endophytic fungi possess a versatile metabolism which is related to their ability to live in diverse ecological niches. While culturing under laboratory conditions, their metabolism is mainly influenced by the culture media, time of incubation and other physicochemical factors. In this study, we focused on the production of 3 thiodiketopiperazines (TDKPs) botryosulfuranols A-C produced by an endophytic strain of Cophinforma mamane isolated from the leaves of Bixa orellana L collected in the Peruvian Amazon. We studied the time-course production of botryosulfuranols A-C during 28 days and evaluated the variations in the production of secondary metabolites, including the TDKPs, produced by C. mamane in response to different culture media, light versus dark conditions and different incubation times. We observed a short time-frame production of botryosulfuranol C while its production was significantly affected by the light conditions and nutrients of the culture media. Botryosulfuranols A and B showed a similar production pattern and a similar response to culturing conditions. Molecular networking allowed us to detect three compounds related to TDKPs that will be the focus of future experiments.

Untargeted metabolomics to evaluate antifungal mechanism: a study of Cophinforma mamane and Candida albicans interaction.

Microbial interactions between filamentous fungi and yeast are still not fully understood. To evaluate a potential antifungal activity of a filamentous fungus while highlighting metabolomic changes, co-cultures between an endophytic strain of Cophinforma mamane (CM) and Candida albicans (CA) were performed. The liquid cultures were incubated under static conditions and metabolite alterations during the course were investigated by ultra-performance liquid chromatography-tandem mass spectrophotometry (UPLC-MS/MS). Results were analyzed using MS-DIAL, MS-FINDER, METLIN, Xcalibur, SciFinder, and MetaboAnalyst metabolomics platforms. The metabolites associated with catabolic processes, including the metabolism of branched-chain amino acids, carnitine, and phospholipids were upregulated both in the mono and co-cultures, indicating fungal adaptability to environmental stress. Several metabolites, including C20 sphinganine 1-phosphate, myo-inositol, farnesol, gamma-undecalactone, folinic acid, palmitoleic acid, and MG (12:/0:0/0:0) were not produced by CA during co-culture with CM, demonstrating the antifungal mechanism of CM. Our results highlight the crucial roles of metabolomics studies to provide essential information regarding the antifungal mechanism of C. mamane against C. albicans, especially when the lost/undetected metabolites are involved in fungal survival and pathogenicity.

Metabotyping of Andean pseudocereals and characterization of emerging mycotoxins.

Pseudocereals are best known for three crops derived from the Andes: quinoa (Chenopodium quinoa), canihua (C. pallidicaule), and kiwicha (Amaranthus caudatus). Their grains are recognized for their nutritional benefits; however, there is a higher level of polyphenism. Meanwhile, the chemical food safety of pseudocereals remains poorly documented. Here, we applied untargeted and targeted metabolomics approaches by LC-MS to achieve both: i) a comprehensive chemical mapping of pseudocereal samples collected in the Andes; and ii) a quantification of their contents in emerging mycotoxins. An inventory of the fungal community was also realized to better know the fungi present in these grains. Metabotyping permitted to add new insights into the chemotaxonomy of pseudocereals, confirming the previously established phylotranscriptomic clades. Sixteen samples from Peru (out of 27) and one from France (out of one) were contaminated with Beauvericin, an emerging mycotoxin. Several mycotoxigenic fungi were detected, including Aspergillus sp., Penicillium sp., and Alternaria sp.

Microbial community associated with the crustose lichen Rhizocarpon geographicum L. (DC.) living on oceanic seashore: A large source of diversity revealed by using multiple isolation methods.

Recently, the study of the interactions within a microcosm between hosts and their associated microbial communities drew an unprecedented interest arising from the holobiont concept. Lichens, a symbiotic association between a fungus and an alga, are redefined as complex ecosystems considering the tremendous array of associated microorganisms that satisfy this concept. The present study focuses on the diversity of the microbiota associated with the seashore located lichen Rhizocarpon geographicum, recovered by different culture-dependent methods. Samples harvested from two sites allowed the isolation and the molecular identification of 68 fungal isolates distributed in 43 phylogenetic groups, 15 bacterial isolates distributed in five taxonomic groups and three microalgae belonging to two species. Moreover, for 12 fungal isolates belonging to 10 different taxa, the genus was not described in GenBank. These fungal species have never been sequenced or described and therefore non-studied. All these findings highlight the novel and high diversity of the microflora associated with R. geographicum. While many species disappear every day, this work suggests that coastal and wild environments still contain an unrevealed variety to offer and that lichens constitute a great reservoir of new microbial taxa which can be recovered by multiplying the culture-dependent techniques.

Dynamics of Chemical Diversity during Co-Cultures: An Integrative Time-Scale Metabolomics Study of Fungal Endophytes Cophinforma mamane and Fusarium solani.

A rapid and efficient metabolomic study of Cophinforma mamane and Fusarium solani co-cultivation in time-series based analysis was developed to study metabolome variations during their fungal interactions. The fungal metabolomes were studied through the integration of four metabolomic tools: MS-DIAL, a chromatographic deconvolution of liquid-chromatography-mass spectrometry (LC/MS); MS-FINDER, a structure-elucidation program with a wide range metabolome database; GNPS, an effective method to organize MS/MS fragmentation spectra, and MetaboAnalyst, a comprehensive web application for metabolomic data analysis and interpretation. Co-cultures of C. mamane and F. solani induced different patterns of metabolite production over 10 days of incubation and induced production of five de novo compounds not occurring in monocultures. These results emphasize that co-culture in time-frame analysis is an interesting method to unravel hidden metabolome in the investigation of fungal chemodiversity.

Lipo-chitooligosaccharides as regulatory signals of fungal growth and development.

Lipo-chitooligosaccharides (LCOs) are signaling molecules produced by rhizobial bacteria that trigger the nodulation process in legumes, and by some fungi that also establish symbiotic relationships with plants, notably the arbuscular and ecto mycorrhizal fungi. Here, we show that many other fungi also produce LCOs. We tested 59 species representing most fungal phyla, and found that 53 species produce LCOs that can be detected by functional assays and/or by mass spectroscopy. LCO treatment affects spore germination, branching of hyphae, pseudohyphal growth, and transcription in non-symbiotic fungi from the Ascomycete and Basidiomycete phyla. Our findings suggest that LCO production is common among fungi, and LCOs may function as signals regulating fungal growth and development.

Do Host Plant and Associated Ant Species Affect Microbial Communities in Myrmecophytes?

Ant-associated microorganisms can play crucial and often overlooked roles, and given the diversity of interactions that ants have developed, the study of the associated microbiomes is of interest. We focused here on specialist plant-ant species of the genus Allomerus that grow a fungus to build galleries on their host-plant stems. Allomerus-inhabited domatia, thus, might be a rich arena for microbes associated with the ants, the plant, and the fungus. We investigated the microbial communities present in domatia colonised by four arboreal ants: Allomerus decemarticulatus, A. octoarticulatus, A. octoarticulatus var. demerarae, and the non-fungus growing plant-ant Azteca sp. cf. depilis, inhabiting Hirtella physophora or Cordia nodosa in French Guiana. We hypothesized that the microbial community will differ among these species. We isolated microorganisms from five colonies of each species, sequenced the 16S rRNA or Internal TranscribedSpacer (ITS) regions, and described both the alpha and beta diversities. We identified 69 microbial taxa, which belong to five bacterial and two fungal phyla. The most diverse phyla were Proteobacteria and Actinobacteria. The microbial community of Azteca cf. depilis and Allomerus spp. differed in composition and richness. Geographical distance affected microbial communities and richness but plant species did not. Actinobacteria were only associated with Allomerus spp.

The Ectomycorrhizal Fungus Laccaria bicolor Produces Lipochitooligosaccharides and Uses the Common Symbiosis Pathway to Colonize Populus Roots.

Mycorrhizal fungi form mutualistic associations with the roots of most land plants and provide them with mineral nutrients from the soil in exchange for fixed carbon derived from photosynthesis. The common symbiosis pathway (CSP) is a conserved molecular signaling pathway in all plants capable of associating with arbuscular mycorrhizal fungi. It is required not only for arbuscular mycorrhizal symbiosis but also for rhizobia-legume and actinorhizal symbioses. Given its role in such diverse symbiotic associations, we hypothesized that the CSP also plays a role in ectomycorrhizal associations. We showed that the ectomycorrhizal fungus Laccaria bicolor produces an array of lipochitooligosaccharides (LCOs) that can trigger both root hair branching in legumes and, most importantly, calcium spiking in the host plant Populus in a CASTOR/POLLUX-dependent manner. Nonsulfated LCOs enhanced lateral root development in Populus in a calcium/calmodulin-dependent protein kinase (CCaMK)-dependent manner, and sulfated LCOs enhanced the colonization of Populus by L. bicolor Compared with the wild-type Populus, the colonization of CASTOR/POLLUX and CCaMK RNA interference lines by L. bicolor was reduced. Our work demonstrates that similar to other root symbioses, L. bicolor uses the CSP for the full establishment of its mutualistic association with Populus.

How Histone Deacetylase Inhibitors Alter the Secondary Metabolites of Botryosphaeria mamane, an Endophytic Fungus Isolated from Bixa orellana.

Fungi are talented organisms able to produce several natural products with a wide range of structural and pharmacological activities. The conventional fungal cultivation used in laboratories is too poor to mimic the natural habitats of fungi, and this can partially explain why most of the genes responsible for the production of metabolites are transcriptionally silenced. The use of Histone Deacetylase inhibitors (HDACis) to perturb fungal secondary biosynthetic machinery has proven to be an effective approach for discovering new fungal natural products. The present study relates the effects of suberoylanilide hydroxamic acid (SAHA) and sodium valproate (VS) on the metabolome of Botryosphaeria mamane, an endophytic fungus isolated from Bixa orellana L. UHPLC/HR-MS analysis, integrated with four metabolomics tools: MS-DIAL, MS-FINDER, MetaboAnalyst and GNPS molecular networking, was established. This study highlighted that SAHA and VS changed metabolites in B. mamane, causing upregulation and downregulation of metabolites production. In addition, twelve compounds were detected in the extracts as metabolites structurally correlated to SAHA, indicating its important reactivity in the medium or its metabolism by the fungus. An addition of SAHA induced the production of eight metabolites while VS induced only two metabolites undetected in the control strain. This result illustrates the importance of adding HDACis to a fungal culture in order to induce metabolite production.

Thiodiketopiperazines with two spirocyclic centers extracted from Botryosphaeria mamane, an endophytic fungus isolated from Bixa orellana L.

Three thiodiketopiperazines, botryosulfuranols A-C (1-3) were isolated from the endophytic fungus Botryosphaeria mamane. The three compounds present sulfur atoms on α- and ß-positions of phenylalanine derived residues and unprecedented two spirocyclic centers at C-4 and C-2'. Their planar structures were determined by spectroscopic analysis and absolute configurations were achieved by X-ray diffraction analysis and ECD and NMR chemical shifts calculations. Botryosulfuranol A (1) was the most cytotoxic compound against four cancer cell lines (HT-29, HepG2, Caco-2, HeLa) and two healthy cell lines (IEC6, Vero) highlighting the importance of an electrophilic center for cell growth inhibition.

Fungal communities associated with Evernia prunastri, Ramalina fastigiata and Pleurosticta acetabulum: Three epiphytic lichens potentially active against Candida biofilms.

Fungal communities associated to three epiphytic lichens active against Candida, were investigated using culture-based methods We hypothetized that associated fungi would contribute to lichens activities. The ability of specific fungi to grow inside or outside lichens was investigated. To detect biogenesis pathways involved in the production of secondary metabolites, genes coding for nonribosomal peptide synthetase (NRPS) and polyketide synthase I (PKS I) were screened by PCR from fungal DNA extracts. Both endo and epilichenic communities were isolated from two fructicose (Evernia prunastri and Ramalina fastigiata) and one foliose (Pleurosticta acetabulum) lichens. A total of 86 endolichenic and 114 epilichenic isolates were obtained, corresponding to 18 and 24 phylogenetic groups respectively suggesting a wide diversity of fungi. The communities and the species richness were distinct between the three lichens which hosted potentially new fungal species. Additionally, the endo- and epilichenic communities differed in their composition: Sordariomycetes were particularly abundant among endolichenic fungi and Dothideomycetes among epilichenic fungi. Only a few fungi colonized both habitats, such as S. fimicola, Cladosporium sp1 and Botrytis cinerea. Interestingly, Nemania serpens (with several genotypes) was the most abundant endolichenic fungus (53% of isolates) and was shared by the three lichens. Finally, 12 out of 36 phylogenetic groups revealed the presence of genes coding for nonribosomal peptide synthetase (NRPs) and polyketide synthase I (PKS I). This study shows that common lichens are reservoirs of diverse fungal communities, which could potentially contribute to global activity of the lichen and, therefore, deserve to be isolated for further chemical studies.

Alder and the Golden Fleece: high diversity of Frankia and ectomycorrhizal fungi revealed from Alnus glutinosa subsp. barbata roots close to a Tertiary and glacial refugium.

BACKGROUND: Recent climatic history has strongly impacted plant populations, but little is known about its effect on microbes. Alders, which host few and specific symbionts, have high genetic diversity in glacial refugia. Here, we tested the prediction that communities of root symbionts survived in refugia with their host populations. We expected to detect endemic symbionts and a higher species richness in refugia as compared to recolonized areas. METHODS: We sampled ectomycorrhizal (EM) root tips and the nitrogen-fixing actinomycete Frankia communities in eight sites colonized by Alnus glutinosa subsp. barbata close to the Caucasus in Georgia. Three sites were located in the Colchis, one major Eurasian climatic refugia for Arcto-Tertiary flora and alders, and five sites were located in the recolonized zone. Endemic symbionts and plant ITS variants were detected by comparing sequences to published data from Europe and another Tertiary refugium, the Hyrcanian forest. Species richness and community structure were compared between sites from refugia and recolonized areas for each symbionts. RESULTS: For both symbionts, most MOTUs present in Georgia had been found previously elsewhere in Europe. Three endemic Frankia strains were detected in the Colchis vs two in the recolonized zone, and the five endemic EM fungi were detected only in the recolonized zone. Frankia species richness was higher in the Colchis while the contrary was observed for EM fungi. Moreover, the genetic diversity of one alder specialist Alnicola xanthophylla was particularly high in the recolonized zone. The EM communities occurring in the Colchis and the Hyrcanian forests shared closely related endemic species. DISCUSSION: The Colchis did not have the highest alpha diversity and more endemic species, suggesting that our hypothesis based on alder biogeography may not apply to alder's symbionts. Our study in the Caucasus brings new clues to understand symbioses biogeography and their survival in Tertiary and ice-age refugia, and reveals that isolated host populations could be of interest for symbiont diversity conservation.

Paxillus rubicundulus (Boletales, Paxillaceae) and two new alder-specific ectomycorrhizal species, Paxillus olivellus and Paxillus adelphus, from Europe and North Africa.

Paxillus rubicundulus P.D. Orton has been shown to be a complex of at least three ectomycorrhizal taxa strictly associated with alders (Alnus) in Europe, P. rubicundulus s. str., and two undescribed clades. To assess the taxonomic status of these three clades and their phylogenetic relationships, phylogenetic analyses of two independent gene regions (ITS and gpd), combined with macro- and micromorphological comparisons of genetically identified specimens, were carried out. A total of 85 sequences were successfully obtained from basidiomata and alder mycorrhizae collected in France and Algeria and combined with GenBank and UNITE sequences. The phylogenetic results and estimates of genetic diversity confirmed that the three clades are distinct species, often found in sympatry. As a result, P. rubicundulus s. str. was redefined based on the revision of type material, and Paxillus adelphus and Paxillus olivellus are introduced as new Linnaean names. The often used name Paxillus filamentosus is rejected since it could not be applied to any of the new species. The three species are distinguished micromorphologically by spore size and shape. They are widely distributed in Europe, North Africa and western Asia; P. rubicundulus is rare, and all species have a limited host range.

The Paxillus involutus (Boletales, Paxillaceae) complex in Europe: genetic diversity and morphological description of the new species Paxillus cuprinus, typification of P. involutus s.s., and synthesis of species boundaries.

Paxillus involutus is a model species for ecological or physiological studies of ectomycorrhizal agaricomycetes. Three to six groups or species linked to it have been ecologically and morphologically distinguished. Phylogenetic studies have revealed the existence of four species in Europe: Paxillus ammoniavirescens, Paxillus obscurisporus, P. involutus, and a fourth as yet not described species. We studied 47 collections from 24 French and Italian locations, supplemented with GenBank data, in order to genetically and taxonomically delineate these species. Phylogenetic analyses of three nuclear DNA regions (rDNA internal transcribed spacer (ITS), tef1-α, and gpd) confirmed the four European species. Morphology, culture, and ecology features allowed us to delineate species boundaries and to describe the fourth species we named Paxillus cuprinus since it turns coppery with age. As there is no existing original herbarium specimen for P. involutus, one of our collections was chosen as the epitype. The low genetic diversity found in P. cuprinus correlates with stable morphological traits (basidiome colour, ovoid-amygdaliform spores with an apical constriction) and with ecological preferences (association with Betulaceae in open and temperate areas). In contrast, P. ammoniavirescens is characterized by a high genetic diversity and a high variation of its morphological and ecological features.

What determines Alnus-associated ectomycorrhizal community diversity and specificity? A comparison of host and habitat effects at a regional scale.

· Global-scale analyses of ectomycorrhizal (ECM) fungi communities emphasize host plant families as the main drivers of diversity. This study aims to test, on Alnus-ECM communities, which fungi are said to be 'host-specific', to what extent host species, habitat and distance explain their alpha and beta diversity variations, and their specificity. · In France, ECM communities associated with two subgenera and five species of Alnus, were sampled on 165 trees from 39 lowland to subalpine sites. In all, 1178 internal transcribed spacer (ITS) sequences of ECM fungi clustered in 86 molecular operational taxonomic units (MOTUs). · The species richness was low but still variable, and the evenness of communities was lower on organic soils and in Corsica. Similarity between communities was influenced both by host, soil parameters, altitude and longitude, but not by climate and distance. A large majority of 'specific' fungi were shared between host species within a subgenus, and showed habitat preferences within the subgenus distribution range. · Our study confirms that Alnus ECM communities are low in diversity, highly conserved at a regional scale, and partly shared between congeneric host species. A large part of alpha and beta diversity variations remained unexplained, and other processes may shape these communities.

An efficient protocol for isolating melanised chaetothyrialean anamorphic fungi associated with plant-ants.

Because of their ecological characteristics, slow growth rates and the presence of contaminants, Chaetothyriales fungi associated with structures built by tropical plant-ants can be difficult to isolate with standard procedures. Here, we describe an easy-to-use protocol for obtaining pure cultures by using cotton as a first substrate. We have further found by means of fluorescent stains that nuclei concentrate either in young hyphae or in the tips of the hyphae.

Specific, non-nutritional association between an ascomycete fungus and Allomerus plant-ants.

Ant-fungus associations are well known from attine ants, whose nutrition is based on a symbiosis with basidiomycete fungi. Otherwise, only a few non-nutritional ant-fungus associations have been recorded to date. Here we focus on one of these associations involving Allomerus plant-ants that build galleried structures on their myrmecophytic hosts in order to ambush prey. We show that this association is not opportunistic because the ants select from a monophyletic group of closely related fungal haplotypes of an ascomycete species from the order Chaetothyriales that consistently grows on and has been isolated from the galleries. Both the ants' behaviour and an analysis of the genetic population structure of the ants and the fungus argue for host specificity in this interaction. The ants' behaviour reveals a major investment in manipulating, growing and cleaning the fungus. A molecular analysis of the fungus demonstrates the widespread occurrence of one haplotype and many other haplotypes with a lower occurrence, as well as significant variation in the presence of these fungal haplotypes between areas and ant species. Altogether, these results suggest that such an interaction might represent an as-yet undescribed type of specific association between ants and fungus in which the ants cultivate fungal mycelia to strengthen their hunting galleries.

Phylogenetic species delimitation in ectomycorrhizal fungi and implications for barcoding: the case of the Tricholoma scalpturatum complex (Basidiomycota).

Population studies have revealed that the fungal ectomycorrhizal morphospecies Tricholoma scalpturatum consists of at least two genetically distinct groups that occur sympatrically in several geographical areas. This discovery prompted us to examine species boundaries and relationships between members formerly assigned to T. scalpturatum and allied taxa using phylogenetic analyses. Sequence data were obtained from three nuclear DNA regions [internal transcribed spacer (ITS), gpd and tef], from 101 carpophores collected over a large geographical range in Western Europe, and some reference sequences from public databases. The ITS was also tested for its applicability as DNA barcode for species delimitation. Four highly supported phylogenetic clades were detected. The two previously detected genetic groups of T. scalpturatum were assigned to the phylospecies Tricholoma argyraceum and T. scalpturatum. The two remaining clades were referred to as Tricholoma cingulatum and Tricholoma inocybeoides. Unexpectedly, T. cingulatum showed an accelerated rate of evolution that we attributed to narrow host specialization. This study also reveals recombinant ITS sequences in T. inocybeoides, suggesting a hybrid origin. The ITS was a useful tool for the determination of species boundaries: the mean value of intraspecific genetic distances in the entire ITS region (including 5.8S rDNA) was <0.2%, whereas interspecific divergence estimates ranged from 1.78% to 4.22%. Apart from giving insights into the evolution of the T. scalpturatum complex, this study contributes to the establishment of a library of taxonomically verified voucher specimens, an a posteriori correlation between phenotype and genotype, and DNA barcoding of ectomycorrhizal fungi.

High sexual reproduction and limited contemporary dispersal in the ectomycorrhizal fungus Tricholoma scalpturatum: new insights from population genetics and spatial autocorrelation analysis.

Dispersal and establishment are fundamental processes influencing the response of species to environmental changes, and the long-term persistence of populations. A previous study on the symbiotic ectomycorrhizal fungus Tricholoma scalpturatum revealed strong genetic differentiations between populations in Western Europe, suggesting restricted dispersal for this wind-dispersed cosmopolitan fungus. Two distinct genetic groups (genetic groups 1 and 2), co-occurring in some locations, were also identified and could correspond to cryptic species. In the present work, we examine the reproductive strategy and dispersal biology of the two T. scalpturatum's genetic groups. Variable molecular markers (intersimple sequence repeats and intergenic spacer 2-restriction fragment length polymorphisms) and spatial autocorrelation analyses were used to examine fine-scale patterns (< 140 m) of genetic structure, in an effort to determine the physical scale at which genetic structure exists. A total of 473 fruit bodies were mapped and collected over 3 years from two plots located in the south of France, including 219 and 254 samples from group 1 and group 2, respectively. High genetic diversity and the presence of numerous small genets were observed in both groups. Autocorrelation analyses revealed significant positive spatial genetic structures of genets at close distances (up to few metres for both groups). Mantel tests confirmed this isolation-by-distance pattern. These results clearly demonstrate high sexual reproduction and spatial structuring of genets at very small geographical scales in this wind-dispersed ectomycorrhizal fungal species, a pattern consistent with restricted contemporary dispersal of spores.

Molecular evolution of mitochondrial ribosomal DNA in the fungal genus Tricholoma: barcoding implications.

The molecular evolution of the V6 and V9 domains of the mitochondrial SSU-rDNA was investigated to evaluate the use of these sequences for DNA barcodes in the Basidiomycota division. The PCR products from 27 isolates belonging to 11 Tricholoma species were sequenced. Both domains in the isolates belonging to the same species had identical sequences. All the species possess distinctive V9 sequences due to point mutations and insertion/deletion events. Secondary structures revealed that the insertion-deletion events occurred in regions not directly involved in the maintenance of the standard SSU-rRNA structure. The inserted sequences possess conserved motifs that enable their alignment among phylogenetically distant species. Hence, the V9 domain by displaying identical sequences within species, an adequate divergence level, easy amplification, and alignment represents an alternative molecular marker for the Basidiomycota division and opens the way for this sequence to be used as specific molecular markers of the fungal kingdom.