A functional assessment of root endophytic fungal diversity and their context dependent effect on plants

The fungal interaction with plants is a 400 million years old phenomenon, which presumably assisted in the plants’ establishment on land. In a natural ecosystem, all plant-ranging from large trees to sea-grasses-are colonized by fungal endophytes, which can be detected inter- and intracellularly within the tissues of apparently healthy plants, without causing obvious negative effects on their host. These ubiquitous and diverse microorganisms are likely playing important roles in plant fitness and development. However, the knowledge on the ecological functions of fungal root endophytes is scarce. Among possible functions of endophytes, they are implicated in mutualisms with plants, which may increase plant resistance to biotic stressors like herbivores and pathogens, and/or to abiotic factors like soil salinity and drought. Also, endophytes are fascinating microorganisms in regard to their high potential to produce a great spectrum of secondary metabolites with expected ecological functions. However, evidences suggest that the interactions between host plants and endophytes are not static and endophytes express different symbiotic lifestyles ranging from mutualism to parasitism, which makes difficult to predict the ecological roles of these cryptic microorganisms. To reveal the ecological function of fungal root endophytes, this doctoral thesis aims at assessing fungal root endophytes interactions with different plants and their effects on plant fitness, based on their phylogeny, traits, and competition potential in settings encompassing different abiotic contexts. To understand the cryptic implication of nonmycorrhizal endophytes in ecosystem processes, we isolated a diverse spectrum of fungal endophytes from roots of several plant species growing in different natural contexts and tested their effects on different model plants under axenic laboratory conditions. Additionally,we aimed at investigating the effect of abiotic and biotic variables on the outcome of interactions between fungal root endophytes and plants. In summary, the morphological and physiological traits of 128 fungal endophyte strains within ten fungal orders were studied and artificial experimental systems were used to reproduce their interactions with three plant species under laboratory conditions. Under defined axenic conditions, most endophytes behaved as weak parasites, but their performance varied across plant species and fungal taxa. The variation in the interactions was partly explained by convergent fungal traits that separate groups of endophytes with potentially different niche preferences. According to my findings, I predict that the functional complementarity of strains is essential in structuring natural root endophytic communities. Additionally, the responses of plant-endophyte interactions to different abiotic factors, namely nutrient availability, light intensity, and substrate’s pH, indicate that the outcome of plant-fungus relationships may be robust to changes in the abiotic environment. The assessment of the responses of plant endophyte interactions to biotic context, as combinations of selected dominant root fungal endophytes with different degrees of trait similarity and shared evolutionary history, indicates that frequently coexisting root-colonizing fungi may avoid competition in inter-specific interactions by occupying specific niches, and that their interactions likely define the structure of root-associated fungal communities and influence the microbiome impacts on plant fitness. In conclusion, my findings suggest that dominant fungal lineages display different ecological preferences and complementary sets of functional traits, with different niche preferences within root tissues to avoid competition. Also, their diverse effects on plant fitness is likely host-isolate dependent and robust to changes in the abiotic environment when these encompass the tolerance range of either symbiont.Die Wechselbeziehung zwischen Pilzen und Pflanzen ist ein 400 Millionen Jahre altes Phänomen, das vermutlich zur Etablierung von Pflanzen an Land beigetragen hat. Im natürlichen Ökosystem werden alle Pflanzen – von großen Bäumen bis zu Seegräsern – von Pilzen als Endophyten besiedelt. Diese können inter- und intrazellulär in Gewebe scheinbar gesunder Pflanzen nachgewiesen werden, ohne sichtbare negative Auswirkungen auf die Wirtspflanzen auszulösen. Dabei haben die allgegenwärtigen und vielfältigen Mikroorganismen sehr wahrscheinlich einen Einfluss auf die Pflanzengesundheit und entwicklung. Allerdings ist das Wissen über die ökologischen Funktionen von Wurzelpilz-Endophyten gering. Als mögliche Funktion wurde unter anderem eine mutualistische Lebensweise in Betracht gezogen, durch die die Wirtspflanze Resistenzen gegenüber biotischen Stressoren, wie Herbivoren und Pathogenen, sowie abiotischen Stressoren wie Salzen und Trockenheit erlangt. Des Weiteren macht die Fähigkeit der Endophyten, ein breites Spektrum sekundärer Metabolite zu produzieren, die eventuell ökologische Funktionen ausüben, sie zu faszinierenden Mikroorganismen. Bisherige Untersuchungsergebnisse weisen jedoch darauf hin, dass die Beziehung zwischen Wirtspflanze und Endophyt nicht statischer Natur ist. Vielmehr reichen die symbiotischen Lebensweisen von Parasitismus bis hin zu Mutualismus. Dies erschwert es, die ökologische Rolle der Mikroorganismen einzuschätzen. Um die ökologische Funktion von Wurzelpilz-Endophyten näher zu untersuchen, war es das Ziel dieser Doktorarbeit, die Wechselwirkungen zwischen Wurzelpilz-Endophyten und verschiedenen Pflanzen zu untersuchen. Dabei wurde spezielles Augenmerk auf den Einfluss der Endophyten auf die Pflanzengesundheit gelegt, basierend auf Phylogenie, Eigenschaften, und Durchsetzungsfähigkeit in Umgebungen mit unterschiedlichen abiotischen Bedingungen. Um den Einfluss nicht-mykorrhizierender Endophyten auf Vorgänge im Ökosystem zu entschlüsseln, haben wir eine Vielfalt von Pilz-Endophyten aus den Wurzeln diverser Pflanzen isoliert, die unter verschiedenen Bedingungen gewachsen sind. Getestet wurden dann deren Effekte auf verschiedene Modelpflanzen unter axenischen Laborbedingungen. Des Weiteren wurden die Effekte mehrerer biotischer und abiotischer Parameter auf die Symbiose zwischen Wurzelpilz-Endophyt und den Pflanzen untersucht. Die morphologischen und physiologischen Eigenschaften von 128 Wurzelpilz-Endophytenstämmen 10 verschiedener Ordnungen wurden untersucht. Dafür wurden unter kontrollierten Laborbedingungen ihre Wechselwirkungen mit drei Pflanzenarten nachgestellt. Unter definierten axenischen Bedingungen verhielten sich die meisten Endophyten schwach parasitär, wobei sich in Abhängigkeit von Pflanzenart und Pilztaxon Variationen im Verhalten zeigten. Ein Teil der Unterschiede in den Wechselwirkungen kann mit konvergenten Eigenschaften der Pilzarten erklärt werden, die Klassen von Endophyten mit potentiell unterschiedlichen Präferenzen für Nischen abgrenzen. Von meinen Ergebnissen ausgehend, lautet meine Hypothese, dass die funktionelle Komplementarität verschiedener Stämme für die Strukturierung natürlicher Gemeinschaften von Wurzelendophyten essentiell ist. Des Weiteren implizieren die Reaktionen der Interaktion von Pflanzen mit Endophyten auf verschiedene abiotische Faktoren wie Nährstoffangebot, Lichtintensität, und Substrat-pH eine hohe Anpassungsfähigkeit der Gemeinschaften aus Pflanzen und Pilzen an Änderungen ihrer abiotischen Umwelt. Die Untersuchungen der Reaktion der Pflanzen-Endophyt-Beziehung auf biotische Faktoren, wie die Kombination ausgewählter, dominanter Endophyten mit verschiedenen Graden der Ähnlichkeit und geteilter Evolution implizieren, dass wurzelbesiedelnde Endophyten, die häufig in natürlich vorkommenden Wurzeln koexistieren, innerartliche Konkurrenz vermeiden, indem sie indem sie spezifische Nischen besiedeln. Sehr wahrscheinlich definieren ihre Wechselwirkungen die Struktur wurzelassoziierter Pilzgemeinschaften und beeinflussen die Einwirkungen des Mikrobioms auf die Pflanzengesundheit. Zusammenfassend ist zu sagen, dass unsere Ergebnisse darauf hinweisen, dass dominante Pilzarten voneinander abweichende ökologische Präferenzen und komplementäre funktionelle Eigenschaften zeigen. Somit entwickeln sie Vorlieben für unterschiedliche Nischen innerhalb des Wurzelgewebes um Konkurrenz zu vermeiden. Ihre unterschiedlichen Einflüsse auf die Pflanzengesundheit sind wahrscheinlich Host-Isolate abhängig und robust gegenüber Veränderungen der abiotischen Umwelt, wenn diese innerhalb der Toleranzgrenzen der jeweiligen Symbionten liegen

The effects of fungal root endophytes on plant growth are stable along gradients of abiotic habitat conditions

Plant symbioses with fungal root endophytes span a continuum from mutualistic to parasitic outcomes, and are highly variable depending on the genotype of each symbiont. The abiotic context in which interactions occur also seems to influence the outcome of plant-endophyte symbioses, but we lack understanding of its relative importance. We aimed to assess if changes in abiotic variables determine the effects of fungal root endophytes on plant growth. We used in vitro co-cultivation assays to test the impact of a selection of endophytic strains from diverse lineages on the growth of Arabidopsis thaliana, Microthlaspi erraticum and Hordeum vulgare along gradients of nutrient availability, light intensity or substrate pH. Most fungi showed a negative but weak effect on plant growth, whereas only a few had persistent detrimental effects across plants and conditions. Changes in abiotic factors affected plant growth but had little influence on their response to fungal inoculation. Of the factors tested, variation in nutrient availability resulted in the most variable plant-endophyte interactions, although changes were feeble and strain-specific. Our findings suggest that the effects of root endophytes on plant growth are robust to changes in the abiotic environment when these encompass the tolerance range of either symbiont

Data from: Genotypic diversity in root-endophytic fungi reflects efficient dispersal and environmental adaptation

Studying community structure and dynamics of plant-associated fungi is the basis for unravelling their interactions with hosts and ecosystem functions. A recent sampling revealed that only a few fungal groups, as defined by internal transcribed spacer region (ITS) sequence similarity, dominate culturable root endophytic communities of nonmycorrhizal Microthlaspi spp. plants across Europe. Strains of these fungi display a broad phenotypic and functional diversity, which suggests a genetic variability masked by ITS clustering into operational taxonomic units (OTUs). The aims of this study were to identify how genetic similarity patterns of these fungi change across environments and to evaluate their ability to disperse and adapt to ecological conditions. A first ITS-based haplotype analysis of ten widespread OTUs mostly showed a low to moderate genotypic differentiation, with the exception of a group identified as Cadophora sp. that was highly diverse. A multilocus phylogeny based on additional genetic loci (partial translation elongation factor 1α, beta-tubulin and actin) and amplified fragment length polymorphism profiling of 185 strains representative of the five dominant OTUs revealed a weak association of genetic differences with geography and environmental conditions, including bioclimatic and soil factors. Our findings suggest that dominant culturable root endophytic fungi have efficient dispersal capabilities, and that their distribution is little affected by environmental filtering. Other processes, such as inter- and intraspecific biotic interactions, may be more important for the local assembly of their communities

Details of all fungal isolates in this study, including GenBank sequence accessions, classification into ITS-based haplotypes, and data on their location of origin.

Details of all fungal isolates in this study, including GenBank sequence accessions, classification into ITS-based haplotypes, and data on their location of origin

DNA alignments and phylogenetic trees

DNA alignments and phylogenetic tree

Supplementary Material for: The effects of fungal root endophytes on plant growth are stable along gradients of abiotic habitat conditions

Supplementary files for paper 'The effects of fungal root endophytes on plant growth are stable along gradients of abiotic habitat conditions.'<div><br></div><div>Includes data files and script with R commands to reproduce the statistical analyses.</div><div><br></div

Matrices with binary AFLP data generated in this study

Matrices with binary AFLP data generated in this stud

Metabolomics-based chemotaxonomy of root endophytic fungi for natural products discovery

Fungi are prolific producers of natural products routinely screened for biotechnological applications, and those living endophytically within plants attract particular attention because of their purported chemical diversity. However, the harnessing of their biosynthetic potential is hampered by a large and often cryptic phylogenetic and ecological diversity, coupled with a lack of large-scale natural products' dereplication studies. To guide efforts to discover new chemistries among root-endophytic fungi, we analyzed the natural products produced by 822 strains using an untargeted UPLC-ESI-MS/MS-based approach and linked the patterns of chemical features to fungal lineages. We detected 17 809 compounds of which 7951 were classified in 1992 molecular families, whereas the remaining were considered unique chemistries. Our approach allowed to annotate 1191 compounds with different degrees of accuracy, many of which had known fungal origins. Approximately 61% of the compounds were specific of a fungal order, and differences were observed across lineages in the diversity and characteristics of their chemistries. Chemical profiles also showed variable chemosystematic values across lineages, ranging from relative homogeneity to high heterogeneity among related fungi. Our results provide an extensive resource to dereplicate fungal natural products and may assist future discovery programs by providing a guide for the selection of target fungi

Abutilon theophrasti’s Defense Against the AllelochemicalBenzoxazolin-2(3H)-One: Support by Actinomucor elegans

Abutilon theophrasti Medik., previously found tobe rather insensitive to benzoxazinoid containing rye mulchand the allelochemical benzoxazolin-2(3H)-one (BOA), canbe associated with the zygomycete Actinomucor elegans,whereby the fungus colonizes the root relatively superficiallyand mainly in the maturation zone. The fungus mitigatesnecrosis of the cotyledons when seedlings are incubated with2 mMBOA, in contrast to those that lack the fungus. In liquidcultures of the fungus, tryptophan was identified. The accumulationof tryptophan is increased in presence of BOA. Thisamino acid seems to be important in protecting Abutilonagainst BOA and its derivatives since it suppressed the accumulationof BOA derived, highly toxic 2-aminophen-oxazin-3-one (APO) in the medium and on the root surface duringBOA incubations of Abutilon seedlings. Although A. elegansis insensitive to BOA and APO, the fungus is not able toprotect the plant against harmful effects of APO, when seedlingsare treated with the compound. Abutilon can detoxifyBOA via BOA-6-OH glucosylation probably by a cell wallassociated glucosyltransferase, but only low amounts of theproduct accumulate. Low tryptophan concentrations can contributeto a degradation of the toxic intermediate BOA-6-OHby Fenton reactions, whereby the amino acid is oxidized. Oneof the oxidation products was identified as 4(1H)-quinolinone,which is the core substructure of the quorum sensing molecule2-heptyl-3-hydroxy-4-quinolone. The mutualistic associationof Abutilon theophrasti with Actinomucor elegans is consideredas opportunistic and facultative. Such plant-fungus associationsdepend rather likely on environmental conditions,such as the mode of fertilization

Supplementary material for: Metabolomics-based chemotaxonomy of root endophytic fungi for natural products discovery

Supplementary files for Maciá-Vicente et al. (2018) Metabolomics-based chemotaxonomy of root endophytic fungi for natural products discovery. Environmental Microbiology, doi: 10.1111/1462-2920.1407