Detection and quantification of a mycorrhization helper bacterium and a mycorrhizal fungus in plant-soil microcosms at different levels of complexity

BACKGROUND: Host plant roots, mycorrhizal mycelium and microbes are important and potentially interacting factors shaping the performance of mycorrhization helper bacteria (MHB). We investigated the impact of a soil microbial community on the interaction between the extraradical mycelium of the ectomycorrhizal fungus Piloderma croceum and the MHB Streptomyces sp. AcH 505 in both the presence and the absence of pedunculate oak microcuttings. RESULTS: Specific primers were designed to target the internal transcribed spacer of the rDNA and an intergenic region between two protein encoding genes of P. croceum and the intergenic region between the gyrA and gyrB genes of AcH 505. These primers were used to perform real-time PCR with DNA extracted from soil samples. With a sensitivity of 10 genome copies and a linear range of 6 orders of magnitude, these real-time PCR assays enabled the quantification of purified DNA from P. croceum and AcH 505, respectively. In soil microcosms, the fungal PCR signal was not affected by AcH 505 in the absence of the host plant. However, the fungal signal became weaker in the presence of the plant. This decrease was only observed in microbial filtrate amended microcosms. In contrast, the PCR signal of AcH 505 increased in the presence of P. croceum. The increase was not significant in sterile microcosms that contained plant roots. CONCLUSIONS: Real-time quantitative PCR assays provide a method for directly detecting and quantifying MHB and mycorrhizal fungi in plant microcosms. Our study indicates that the presence of microorganisms and plant roots can both affect the nature of MHB-fungus interactions, and that mycorrhizal fungi may enhance MHB growth

Molecular analysis of co-infection of Scots pine seedlings with actinobacteria Streptomyces and Heterobasidion annosum

Heterobasidion annosum is a pathogenic fungus that causes extensive damage to many trees in temperate forests including Scots pine (Pinus Sylvestris). Various microbes have been studied for potential use as bio-control agents to inhibit or reduce the H. annosum infection of trees. This study examined the potential use of bacterial isolates, belonging to streptomyces genus, as a bio-control agent for Scots pine seedlings against H. annosum. Streptomyces species were isolated from surface of mycorrhizal fungi in the forest and they are known to have a mutualistic relationship with mycorrhizal fungi. The goal of this study was to understand the relationship between H. annosum and Streptomyces sp. A11. This was tested in in two settings, in presence of pine seedlings, growing in soil and in dual cultures. In the first setting, seedlings from different treatments were inoculated with Streptomyces sp. A11, H. annosum, co-inoculated with both species or grown in sterile conditions as control. After incubation period, growth and root development of seedlings were analysed. H. annosum grown in dual culture against Streptomyces sp. A11 was utilised for gene expression using quantitative real time PCR method. The results indicated that pine seedlings inoculated with both Streptomyces sp. A11 and H. annosum, had more severe infection compared to the seedlings infected with H. annosum alone. This implies that Streptomyces sp. A11 can interfere with pine’s defence response during interaction with H. annosum. Moreover, Streptomyces sp. A11 suppressed the growth of H. annosum in dual culture. The Suppression of H. annosum was potentially because of antifungal secondary metabolites that were produced by Streptomyces sp. A11. These secondary metabolites caused disruption in glucose metabolism and cell wall integrity of H. annosum. Future experiments should include mycorrhizal fungal species along with species tested in this study. Streptomyces sp. are known to respond differently in presence of specific species. The results for this study should be considered for studies of mycorrhizal associated streptomyces species and can be built upon for broader future investigation

Bioactive properties of streptomyces may affect the dominance of Tricholoma matsutake in shiro

Tricholoma matsutake is known to be the dominant fungal species in matsutake fruitbody neighboring (shiro) soil. To understand the mechanisms behind matsutake dominance, we studied the bacterial communities in matsutake dominant shiro soil and non-shiro soil, isolated the strains of Streptomyces from matsutake mycorrhizal root tips both from shiro soil and from the Pinus densiflora seedlings cultivated in shiro soil. Further, we investigated three Streptomyces spp. for their ability to inhibit fungal growth and Pinus densiflora seedling root elongation as well as two strains for their antifungal and antioxidative properties. Our results showed that Actinobacteria was the most abundant phylum in shiro soil. However, the differences in the Actinobacterial community composition (phylum or order level) between shiro and non-shiro soils were not significant, as indicated by PERMANOVA analyses. A genus belonging to Actinobacteria, Streptomyces, was present on the matsutake mycorrhizas, although in minority. The two antifungal assays revealed that the broths of three Streptomyces spp. had either inhibitory, neutral or promoting effects on the growth of different forest soil fungi as well as on the root elongation of the seedlings. The extracts of two strains, including one isolated from the P. densiflora seedlings, inhibited the growth of either pathogenic or ectomycorrhizal fungi. The effect depended on the medium used to cultivate the strains, but not the solvent used for the extraction. Two Streptomyces spp. showed antioxidant activity in one out of three assays used, in a ferric reducing antioxidant power assay. The observed properties seem to have several functions in matsutake shiro soil and they may contribute to the protection of the shiro area for T. matsutake dominance.Peer reviewe

Multitrophic interactions in oak

Several studies, including the present dissertation, have illustrated the ecological importance of trophic interactions between species. Centred on a plants point of view, this includes defense responses, nutrient supply supported by symbionts and decomposers, as well as interacting effects linking different kinds of interaction. Due to the complexity of natural habitats, a suitable way for studying trophic interactions in a multi-factorial way was of urgent need. The second chapter is devoted to the construction of a reliable reference data base for large scale gene expression analysis. The OakContigDF159.1 provides a reliable reference to study gene expression patterns, e.g. triggered by different trophic or multitrophic interactions. In order to provide a reliable reference which covers the largest possible number of gene transcripts, we treated micropropagated cuttings of the pedunculate oak (Quercus robur) with seven different interacting species plus control treatment. These microcuttings had to cope with an ectomycorrhizal fungus (Piloderma croceum), a leaf herbivore (Lymantria dispar), a Collembolan species (Protaphorura armata), a leaf pathogen (Microsphaera alphitoides), a root feeding nematode (Pratylenchus penetrans), a root pathogen (Phytophthora quercina) and a mycorrhiza helper bacterium (Streptomyces AcH 505) in experiments under controlled laboratory conditions. After an initial normalisation of RNA templates, sequencing using the two architectures, 454 pyrosequencing and Illumina sequencing took place. Further, we used the Mimicking Intelligent Read Assembly (MIRA) and the Trinity assembler, to construct the OakContigDF159.1 hybrid assembly, containing 65,712 contigs with a mean length of 1003 bp. Illumina constructed cDNA libraries were used to examine gene expression induced by mycorrhiza formation. After aligning the cDNA libraries of fine roots against the reference, reads were quantified by RSEM, differences in gene expression was measured in R, revealing 3018 differentially expressed contigs, of which 1399 were up-regulated and 1619 down-regulated in oak EMs with P .croceum. Our data support and confirm the view that gene families are precisely regulated to adjust the plants metabolism to mycorrhizal symbiosis, rather than by expression of symbiosis-specific genes. Furthermore, our findings of e.g. down-regulated chitinases confirmes that defense-related genes are attenuated in mature mycorrizal symbiosis. taking account to the mycorrhizal symbiosis, the expression of a SWEET1 glucose transporter could indicate a direct export of hexose into the plant apoplast to support the fungus and may suggest the existence of a complementary sugar exchange mechanism in mycorrhizal symbiosis in oak. As mycorrhizal fungi and herbivores interactively influence the growth, resource utilization, and defense of plants, the third chapter shall provide a deeper insight in actual gene expression changes and metabolic adjustments of oak facing herbivory in a system including mycorrhizal fungi. Again, microcuttings of the Quercus robur clone DF159 served as a experimental platform for a full factorial experiment. In order to examine the separate and combined effects of a mycorrhizal fungus and a leaf chewing caterpillar on oak, I analysed changes in gene expression and nutrient allocation patterns on a total of 71 13C and 15N labelled microcuttings, of which 35 were in root flush and 36 in shoot flush. First, I extracted total RNA templates of pooled samples which were further used to produce 100-bp paired-end libraries and sequenced using an Illumina HiSeq 2000. After alignment against the reference transcriptome OakContigDF159.1, significance of differences in gene expression was measured via pairwise comparisons between contigs of control vs. herbivory, control vs. inoculation with P. croceum, control vs. inoculation/herbivory, separately for plants in root flush and for plants in shoot flush. Furthermore, I analysed changes in nutrient incorporation using calculated differences of 13C and 15N excess over unlabelled control plants. Significance of effects was tested using a three-way ANOVA. I found an increased expression of genes related to compensatory growth and direct defense in oaks in root flush, an effect that was attenuated in presence of P. croceum. Further was the expression of genes related to the production of VOC increased in oak inoculated with P. croceum and exposed to L. dispar. I found diverse effects of the growth stage, inoculation and herbivory on the incorporation of nutrients. Additionally I found, on the one hand an increased incorporation of 13C and 15N in sink leaves of oaks exposed to herbivory, on the other hand the incorporation of 13C decreased in source leaves of oaks in shoot flush and exposed to herbivory. In stems, the incorporation of both, 13C and 15N was decreased in oak exposed to herbivory, an effect that was reversed in inoculated oak exposed to herbivory. Taken together, these findings indicate that oaks may switches from direct herbivore defense mechanism, including compensatory growth, during root flush to the indirect defense mechanism of producing chemicals attractive to natural enemies of herbivores when the plant benefits from pre-mycorrhizal effects. The experiment described in the fourth chapter was designed to investigate above-ground and below-ground interaction of herbivory, collembola and mycorrhization. I used a four-factorial system comprising seedlings of Quercus robur, Lymantria dispar, Protaphorura armata and Piloderma croceum. Under controlled laboratory conditions I applied a 13C and 15N labelling prior to the feeding experiment with the leaf chewing caterpillars. I found strong pre-mycorrhizal effects, reflecting an increased root and shoot biomass but also an increased incorporation of currently assimilated carbon into the root system. The results showed further, that oaks indeed respond sensitively to the studied biological interactions, including changes in morphology, C and N concentrations and nutrient and carbon allocation to different plant fractions. I didn’t found evidence for hyphal consumtion of P. aramta and no negative effects of collembola presence on performance of oak inoculated with a mycorrhizal fungus. Furthermore, I found none of the expected interactive effects of above-ground herbivory and below-ground collembolan activity. However, this study suggests a strong compensatory growth reaction of oak seedlings after exposure to leaf chewing caterpillars with a soothing effect of P. croceum on this reaction.Mit der vorliegenden Dissertation konnte ich die Bedeutung von trophischen Interaktion, als Wechselwirkung zwischen Arten darstellen. Betrachtet man dies von der Seite der Pflanzen aus, zeigen sich sowohl Effekte wie Verteidigungsreaktionen, Änderungen in der Nährstoffverteilung innerhalb der Pflanze als auch interagierende Effekte. Die Komplexität natürlicher Habitate hat es bisher sehr schwierig gemacht solche Interaktionen zu untersuchen. Die Möglichkeit ein komplexes System ins Labor zu bringen und Untersuchungen unter kontrollierten Bedingungen mit mehreren Faktoren durchführen zu können, stellt einen großen Vorteil dar und soll im Folgenden näher beleuchtet werden. Das erste Kapitel dieser Dissertation ist einer allgemeinen Einleitung in das Thema gewidmet. Im zweiten Kapitel stelle ich das experimentelle System näher vor. Des Weiteren beschreibe ich die Konstruktion der verlässlichen Referenzdatenbank OakContigDF159.1, welche das Studium von Genexpression auf Gesamttranskriptom-Ebene ermöglicht. Damit das Referenztranskriptom eine möglichst hohe Anzahl an Transkripten aus dem Genom der Stieleiche (Quercus robur) beinhaltet, behandelten wir microcuttings der Stieleiche mit insgesamt sieben Arten, welche jeweils eine andere Form der trophischen Interaktion mit der Eiche eingehen. Zusätzlich einer Kontrolle wurden die microcuttings Piloderma croceum, Lymantria dispar, Protaphorura armata, Microsphaera alphitoides, Pratylenchus penetrans, Phytophthora quercina and Streptomyces AcH 505 ausgesetzt. Normalisierung und anschließende Sequenzierung mittels Illumina und 454 Pyro-Sequenzierung resultierten, nach Nachbereitung mit MIRA und Trinity assembler, in der OakContigDF159.1 Datenbank. Im dritten Kapitel untersuche ich mit Hilfe der Referenzdatenbank OakContigDF159.1 differenzielle Genexpression, sowie Änderungen in der Nährstoffverteilung. Hierzu habe ich ein multifaktorielles Experiment mit Piloderma croceum und Lymantria dispar durchgeführt. Die Versuchspflanzen wurden im Vorfeld mit den stabilen Isotopen 13C und 15N markiert, um nach der Ernte den Isotopen-Überschuss mit einer unmarkierten Kontrolle zu vergleichen und mittels einer 3-faktoriellen ANOVA zu analysieren. Zusätzlich wurde die Gesamt-RNA extrahiert und mittels Illumina sequenziert. Die enhaltenen cDNA-Bibliotheken habe ich an die Referenzdatenbank angepasst und erhielt so 35 Transcriptome welche ich weiterhin zur paarweisen Berechnung von differentieller Genexpression verwendete. Pflanzen die mit dem Mykorrhizapilz Piloderma croceum inokuliert waren, zeigten starke prämykorrhizale Effekte, welche sich z.B. in einer Erhöhung der Biomasse zeigten. Herbivorie verursachte von Genen die mit kompensatorischem Wachstum in Verbindung stehen, sowie eine verstärkte Expression von Primär-Verteidigungs Genen. Dieser Effekt wurde durch inokulierung mit P. croceum abgeschwächt. Die Expressionsdaten ließen zudem bei inokulierten und befressenen Eichen auf eine erhöhte produktion von volativen organischen Substanzen schließen, die der Attraktion von Fraßfeinden der Herbivoren dienen. Zusätzlich zeigte sich bei Pflanzen mit Herbivorenfraß eine erhöhte Aufnahme von 13C and 15N in junge Blätter, wohingegen sich die Aufnahme von 13C in reife Blätter durch Herbivory verringert zeigte. Auf diesen Ergebnissen folgerte ich, dass eine Interaktion mit einem Ekto-Mykorrhiza Pilz, selbst in einem frühen Stadium der Symbiose, deutliche Effekte auf das Abwehrverhalten einer Pflanze haben kann. Hier wurde die heftige Abwehrreaktion, inkl. kompensatorischen Wachstums, durch den Pilz abgeschwächt und sogar verlagert auf Mechanismen der sekundären Abwehr. Die Gegenwart des Pilzes scheint eine “beruhigende“ Wirkung auf die Pflanze auszuüben. Das vierte Kapitel ist der genaueren Untersuchung der ober- und unterirdischen Wechselwirkungen der verschieden trophischen Partner gewidmet. Hierzu führte ich ein multifaktorielles Experiment mit den trophischen Partnern Piloderma croceum, Lymantria dispar und Protaphorura armata durch. Auch hier fand ich starke prä-mykorrhizale Effekte, sich die in einer Erhöhung der Biomasse, analog zu einer erhöhten Einlagerung von 13C and 15N in P. croceum inokulierten Eichen zeigten. Weiterhin zeigte sich eine Erhöhung der Biomasse bei Eichen die L. dispar ausgesetzt waren. Obwohl ich keine Anzeichen für eine Interaktion der ober- und unterirdischen Interaktoren der Eiche fand, hatten alle Faktoren einzeln betrachtet deutlichen Einfluss auf die Nährstoffverteilung. Ich konnte keine Hinweise von Hyphenfraß durch Collembolen finden, ebenso konnte ich keine negativen Effekte der Collembolenpopulation auf inokulierte Eichen feststellen. Im Gegenteil zeigte sich eine erhöhte Nährstoffaufnahme, was ich als Hinweis auf gesteigerte Nährstoffmineralisierung durch die Collembolen deutete

Metagenome sequence of Elaphomyces granulatus from sporocarp tissue reveals Ascomycota ectomycorrhizal fingerprints of genome expansion and a Proteobacteria‐rich microbiome

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/113145/1/emi12840.pd

Mechanisms that promote bacterial fitness in fungal-affected soil microhabitats

Soil represents a very heterogeneous environment for its microbiota. Among the soil inhabitants, bacteria and fungi are important organisms as they are involved in key biogeochemical cycling processes. A main energy source driving the system is formed by plants through the provision of plant-fixed (reduced) carbon to the soil, whereas soil nitrogen and phosphorus may move from the soil back to the plant. The carbonaceous compounds released form the key energy and nutrient sources for the soil microbiota. In the grossly carbon-limited soil, the emergence of plant roots and the formation of their associated mycorrhizae thus create nutritional hot spots for soil-dwelling bacteria. As there is natural (fitness) selection on bacteria in the soil, those bacteria that are best able to benefit from the hot spots have probably been selected. The purpose of this review is to examine the interactions of bacteria with soil fungi in these hot spots and to highlight the key mechanisms involved in the selection of fungal-responsive bacteria. Salient bacterial mechanisms that are involved in these interactions have emerged from this examination. Thus, the efficient acquisition for specific released nutrients, the presence of type-III secretion systems and the capacity of flagellar movement and to form a biofilm are pinpointed as key aspects of bacterial life in the mycosphere. The possible involvement of functions present on plasmid-borne genes is also interrogated

Study of the growth, artemisinin production and leaf volatilome of some micropropagated Artemisia annua clones inoculated or not with beneficial soil microorganisms

Artemisia annua is a medicinal plant appreciated for the production of many volatile compounds and for the artemisinin, a molecule recommended by the World Health Organization for the malaria treatment. However, artemisinin in planta concentration is very low and one strategy to improve plant biomass and artemisinin production may be the use of beneficial soil microorganisms, such as Arbuscular Mycorrhizal Fungi (AMF) and Plant Growth-Promoting Bacteria (PGPB). The effectiveness of these microbes varies according to the genetic compatibility between plant and microorganisms. The aim of this study was to evaluate the effect of clonal variability and beneficial soil microorganisms on 5 different genotypes of A. annua plant, propagated in vitro through the micropropagation technique. The different clone plants were inoculated or not with several beneficial soil microorganisms, and cultivated in controlled conditions. In a first phase, different experiments were performed to test clone in vivo stability, and to evaluate the mycorrhizal colonization in the root and plant growth responses after 60 days of cultivation. Afterwards, in the last experiment, the two most stable clones were inoculated and two samplings at different times (30 and 60 days) were carried out, and artemisinin concentration was also evaluated, using a HPLC method. The mycorrhizal colonization values varied in all the clones, according to the different used AMF and PGPB. Furthermore, the use of different combinations of microorganisms led to positive or negative effects on plant growth, in the different clones, also after 30 days of cultivation. The artemisinin concentration did not vary in the presence of microorganisms, but it showed different values between the two considered clones. Finally, a characterization of leaf volatilome was performed through GC-MS analysis in one clone, and the leaf volatile profile varied according to the plant age and to the different combinations of microbes

Metagenome sequence of Elaphomyces granulatus from sporocarp tissue reveals Ascomycota ectomycorrhizal fingerprints of genome expansion and a Proteobacteria-rich microbiome

Many obligate symbiotic fungi are difficult to maintain in culture, and there is a growing need for alternative approaches to obtaining tissue and subsequent genomic assemblies from such species. In this study, the genome of Elaphomyces granulatus was sequenced from sporocarp tissue. The genome assembly remains on many contigs, but gene space is estimated to be mostly complete. Phylogenetic analyses revealed that the Elaphomyces lineage is most closely related to Talaromyces and Trichocomaceae s.s. The genome of E. granulatus is reduced in carbohydrate-active enzymes, despite a large expansion in genome size, both of which are consistent with what is seen in Tuber melanosporum, the other sequenced ectomycorrhizal ascomycete. A large number of transposable elements are predicted in the E. granulatus genome, especially Gypsy-like long terminal repeats, and there has also been an expansion in helicases. The metagenome is a complex community dominated by bacteria in Bradyrhizobiaceae, and there is evidence to suggest that the community may be reduced in functional capacity as estimated by KEGG pathways. Through the sequencing of sporocarp tissue, this study has provided insights into Elaphomyces phylogenetics, genomics, metagenomics and the evolution of the ectomycorrhizal association.This is the publisher’s final pdf. The article is copyrighted by the Society for Applied Microbiology and John Wiley & Sons, Ltd. It is published by John Wiley & Sons, Ltd. and can be found at: http://onlinelibrary.wiley.com/journal/10.1111/%28ISSN%291462-292