Metabarcoding of fungal assemblages in Vaccinium myrtillus endosphere suggests colonization of above-ground organs by some ericoid mycorrhizal and DSE fungi.

Plants harbor in their external surfaces and internal tissues a highly diverse and finely structured microbial assembly, the microbiota. Each plant compartment usually represents a unique ecological niche hosting a distinct microbial community and niche differentiation, which may mirror distinct functions of a specialized microbiota, has been mainly investigated for bacteria. Far less is known for the fungal components of the plant-associated microbiota. Here, we applied a metabarcoding approach to describe the fungal assemblages in different organs of Vaccinium myrtillus plants (Ericaceae) collected in a subalpine meadow in North-West Italy, and identified specific taxa enriched in internal tissues of roots, stems, leaves and flowers. We also traced the distribution of some important fungi commonly associated with plants of the family Ericaceae, namely the ericoid mycorrhizal (ErM) fungi and the dark septate endophytes (DSE), both playing important roles in plant growth and health. Operational taxonomic units attributed to established ErM fungal species in the genus Hyaloscypha and to DSE species in the Phialocephala-Acephala applanata complex (PAC) were found in all the plant organs. Mycorrhizal fungi are thought to be strictly associated with the plant roots, and this first observation of ErM fungi in the above-ground organs of the host plant may be explained by the evolutionary closeness of ErM fungi in the genus Hyaloscypha with non mycorrhizal fungal endophytes. This is also witnessed by the closer similarities of the ErM fungal genomes with the genomes of plant endophytes than with those of other mycorrhizal fungi, such as arbuscular or ectomycorrhizal fungi.

Effect of anthocyanins on lipid oxidation and microbial spoilage in value-added emulsions with bilberry seed oil, anthocyanins and cold set whey protein hydrogels.

The objective of this work was to explore the storage properties of a structured oil-in-water emulsion containing both water- and fat-soluble bioactive compounds from bilberries (Vaccinium myrtillus L.). Bilberry seed oil (BSO) was dispersed in a continuous aqueous phase of anthocyanins (AC) and whey protein isolate. The microstructure was evaluated using light microscopy and the effect of anthocyanins on lipid oxidation and microbial growth was investigated. The results showed that it was possible to generate a stable emulsion structure that resisted phase separation during 25 weeks of storage. Gas chromatography-mass spectrometry measurements of the fatty acids in the BSO during storage showed that AC had a protective effect against lipid oxidation. The AC did not have an antimicrobial effect against the investigated strains Zygosaccharomyces bailii (ATCC 42476) and Aspergillus niger (ATCC 6275 (M68)).

Influence of Different Fermentation Strategies on the Phenolic Profile of Bilberry Wine (Vaccinium myrtillus L.).

Polyphenol rich and especially anthocyanin rich berries like bilberries (Vaccinium myrtillus L.) and derived products such as wine have enjoyed increasing popularity. During winemaking and aging, the phenolic profile undergoes distinct changes, a phenomenon that has been well investigated in grape wine but not in bilberry wine. The present study determined the influence of different fermentation strategies including various pre- and postfermentative heating and cooling concepts on the phenolic profile of bilberry wine. Besides significant differences in total anthocyanin and tannin concentrations, the different fermentation strategies resulted in distinguishable anthocyanin profiles. A very fast aging manifested by a rapid decrease in monomeric anthocyanins of up to 98% during a 12 week storage and a coincident formation of polymeric pigments and pyranoanthocyanins was observed. Several well-known processes associated with production and aging of wine were much more pronounced in bilberry wine compared to grape wine.

OmGOGAT-disruption in the ericoid mycorrhizal fungus Oidiodendron maius induces reorganization of the N pathway and reduces tolerance to heavy-metals.

Mycorrhizal fungi are key mediators of soil-to-plant movement of mineral nutrients, including essential and non-essential metals. In soil conditions that facilitate mobilization of metal ions, potentially toxic metals can interfere with nitrogen metabolism in both plants and microorganisms. Less is known about possible relationships between nitrogen metabolism and responses to heavy metals. Aim of this study was to investigate this aspect in the ericoid mycorrhizal fungus Oidiodendron maius strain Zn, a metal tolerant ascomycete. Growth of O. maius Zn on zinc and cadmium containing media was significantly affected by the nitrogen source. Screening of a library of O. maius Zn random genetic transformants for sensitivity to heavy metals (zinc and cadmium) and oxidative stress (menadione) yielded a mutant strain that carried a partial deletion of the glutamate synthase (NADH-GOGAT EC 1.4.1.14) gene and its adjacent gene, the APC15 subunit of the anaphase promoting complex. Comparison of WT and OmGOGAT-OmAPC15 mutant strains indicated an impaired N-metabolism and altered stress tolerance, and assays on the OmAPC15-recomplemented strains ascribed the observed phenotypes to the deletion in the OmGOGAT gene. OmGOGAT disruption modified the nitrogen pathway, with a strong reduction of the associated glutamine synthetase (GS, EC 6.3.1.2) activity and an up-regulation of the alternative NADP-glutamate dehydrogenase (NADP-GDH, EC 1.4.1.4) pathway for glutamate biosynthesis. Unless they were supplemented with glutamine, O. maius Zn transformants lacking OmGOGAT were very sensitive to zinc. These results highlight the importance of nitrogen metabolism not only for nitrogen assimilation and transformation, but also for stress tolerance. For mycorrhizal fungi, such as O. maius, this may bear consequences not only to the fungus, but also to the host plant.

SOD1-targeted gene disruption in the ericoid mycorrhizal fungus Oidiodendron maius reduces conidiation and the capacity for mycorrhization.

The genome sequences of mycorrhizal fungi will provide new opportunities for studying the biology and the evolution underlying this symbiotic lifestyle. The generation of null mutants at the wild-type loci is one of the best methods for gene-function assignment in the post-genomic era. To our knowledge, the generation of superoxide dismutase 1 (SOD1)-null mutants in the ericoid mycorrhizal fungus Oidiodendron maius is the first example of a gene-targeted disruption via homologous recombination in a mycorrhizal fungus. The disruption of OmSOD1 by Agrobacterium-mediated transformation resulted in the presence of oxidative stress markers, even in the absence of external superimposed stresses, and an increased sensitivity to reactive oxygen species (ROS)-generating substances, especially to menadione. A reduction in conidiation and in the percentage of mycorrhization of Vaccinium myrtillus roots was also observed. The latter findings establish the pivotal role of SOD1 as an important factor in the relationship between O. maius and its symbiotic partner. The lack of this ROS-scavenger may cause an imbalance in the redox homeostasis during host colonization and an alteration in the delicate dialogue between the fungus and its host plant.

Diversity of fungi in hair roots of Ericaceae varies along a vegetation gradient.

Ericaceous dwarf shrubs including Calluna vulgaris and Vaccinium spp. occur both in open heathland communities and in forest ecosystems as understory vegetation. Ericaceous shrubs were once thought to form ericoid mycorrhizal associations with a relatively narrow range of ascomycetous fungi closely related to, and including, Rhizoscyphus ericae. However, perceptions have recently changed since the realization that a broader range of ascomycete fungi, and in some cases basidiomycete fungi, can also form associations with the roots of ericaceous plants. We used a combination of molecular approaches, including denaturing gradient gel electrophoresis, terminal restriction fragment length polymorphism, cloning and sequencing, to investigate the diversity of fungi associated with C. vulgaris roots collected across a heathland/native Scots pine forest vegetation gradient. We also determined differences in fungal community composition between roots of co-occurring C. vulgaris and Vaccinium myrtillus in the forest understory. Collectively, the data show that a large diversity of potentially ericoid mycorrhizal fungal taxa associate with roots of C. vulgaris and V. myrtillus, and that ascomycetes were about 2.5 times more frequent than basidiomycetes. The assemblages of fungi associated with C. vulgaris and V. myrtillus were different. In addition, the community of fungi associated with C. vulgaris hair roots was different for samples collected from the forest, open heathland and a transition zone between the two. This separation was partly, but not entirely, due to the occurrence of typical ectomycorrhizal basidiomycetes associated with the hair roots of C. vulgaris in the forest understory. These data demonstrate that forest understory ericaceous shrubs associate with a diverse range of ascomycete and basidiomycete taxa, including typical ectomycorrhizal basidiomycetes.

Use of multiplex terminal restriction fragment length polymorphism for rapid and simultaneous analysis of different components of the soil microbial community.

A multiplex terminal restriction fragment length polymorphism (M-TRFLP) fingerprinting method was developed and validated for simultaneous analysis of the diversity and community structure of two or more microbial taxa (up to four taxa). The reproducibility and robustness of the method were examined using soil samples collected from different habitats. DNA was PCR amplified separately from soil samples using individual taxon-specific primers for bacteria, archaea, and fungi. The same samples were also subjected to a multiplex PCR with the primers for all three taxa. The terminal restriction fragment length polymorphism profiles generated for the two sets of PCR products were almost identical not only in terms of the presence of peaks but also in terms of the relative peak intensity. The M-TRFLP method was then used to investigate rhizosphere bacterial, fungal, and rhizobial/agrobacterial communities associated with the dwarf shrub Calluna vulgaris growing in either open moorland, a mature pine forest, or a transition zone between these two habitats containing naturally regenerating pine trees. Rhizosphere microbial communities associated with Vaccinium myrtillus collected from the native pine forest were also investigated. In this study, individual PCR products from the three taxa were also pooled before restriction digestion and fragment size analysis. The terminal restriction fragment length polymorphism profiles obtained with PCR products amplified individually and with multiplexed and pooled PCR products were found to be consistent with each other in terms of the number, position, and relative intensity of peaks. The results presented here confirm that M-TRFLP analysis is a highly reproducible and robust molecular tool for simultaneous investigation of multiple taxa, which allows more complete and higher resolution of microbial communities to be obtained more rapidly and economically.

Population genetic analysis of Phialocephala fortinii s.l. and Acephala applanata in two undisturbed forests in Switzerland and evidence for new cryptic species.

The genetic structure of the root endophytes Phialocephala fortinii s.l. and Acephala applanata was analyzed in two undisturbed forests. A total of 606 strains isolated from surface-sterilized, fine roots of Picea abies and Vaccinium myrtillus were examined. Two new cryptic species of P. fortinii were recognized and host specialization of A. applanata was confirmed. This species was almost exclusively isolated from roots of P. abies. The index of association did not deviate significantly from zero within any population, suggesting that recombination occurs or had occurred. Significant gene but no genotype flow was detected among study sites for P. fortinii s.l. In contrast, several isolates of A. applanata with both identical multi-locus haplotype and identical ISSR fingerprint were found in both study sites indicating genotype flow or a recent common history.

Polyamine profiles of healthy and parasite-infected Vaccinium myrtillus plants under nitrogen enrichment.

Addition of nitrogen (N) to the field layer of boreal forests has been shown to increase the occurrence of the parasitic fungus Valdensia heterodoxa on Vaccinium myrtillus plants. We investigated whether N addition to soil alters the levels of polyamines in V. myrtillus shoots, and discuss here whether such changes could promote the spread of the parasitic fungus on V myrtillus. Using HPLC, we analyzed the concentrations of free and conjugated polyamines in healthy and naturally V. heterodoxa-infected V. myrtillus plants, which had received a moderate or high dose of N fertilizer, or no additional N. Fertilization with N increased the concentrations of free diamines (putrescine and diaminopropane), but had no significant effect on conjugated amines. Thus, N-induced changes in the constitutive levels of soluble conjugated amines do not seem to explain the increased parasite susceptibility of V. myrtillus under N enrichment. Generally, the concentrations of free diamines and insoluble conjugated putrescine were higher in diseased than in healthy shoots, suggesting parasite-induced accumulation of diamines. Free spermine seemed to accumulate in unfertilized, diseased plants, but in fertilized plants this induction was dampened, suggesting that N-induced alterations in spermine metabolism may promote the spread of parasites on V. myrtillus under N-enrichment.

Do multitrophic interactions override N fertilization effects on Operophtera larvae?

We examined how performance of Operophtera brumata (Lepidoptera) larvae was affected by nitrogen (N) fertilization of boreal forest understorey vegetation. We monitored larval densities on Vaccinium myrtillus plants for a period of 7 years in a field experiment. Preliminary results indicated that the N effect on larval densities was weak. To examine if this was due to indirect interactions with a plant pathogen, Valdensia heterodoxa, that share the same host plant, or due to top-down effects of predation, we performed both a laboratory feeding experiment (individual level) and a bird exclusion experiment (population level) in the field. At the individual level, altered food plant quality (changes in plant concentration of carbon, N, phenolics, or condensed tannins) due to repeated infection by the pathogen had no effect on larval performance, but both survival to the adult stage and adult weight were positively affected by N fertilization. Exclusion of insectivorous birds increased the frequency of larval damage on V. myrtillus shoots, indicating higher larval densities. This effect was stronger in fertilized than in unfertilized plots, indicating higher bird predation in fertilized plots. Predation may thus explain the lack of fertilization effect on larval densities in the field experiment. Our results suggest that top-down effects are more important for larval densities than bottom-up effects, and that bird predation may play an important role in population regulation of O. brumata in boreal forests.

Gene expression of the ericoid mycorrhizal fungus Oidiodendron maius in the presence of high zinc concentrations.

A heavy metal tolerant strain of the ericoid mycorrhizal species Oidiodendron maius, isolated from roots of Vaccinium myrtillus growing in soil heavily contaminated with zinc, was previously shown to tolerate high concentrations of zinc and cadmium ions in the growth medium. We have investigated the genetic basis of this fungal strain tolerance to high zinc concentrations by using an untargeted approach. From a cDNA library constructed by using mRNA from Zn-treated O. maius mycelia, 444 clones were randomly selected and 318 were sequenced. Sequence analysis identified 219 unique clones: 117 showed homology to previously identified genes, 26 matched unknown protein coding regions found in other organisms, and 76 were novel. Variation in the gene expression level after a 20-day treatment with high concentrations of Zn was monitored on 130 unigenes by reverse northern blot hybridisation. Sixteen unigenes were shown to be either up- (9) or down- (7) regulated. The putative function of these genes and their involvement in stress tolerance is discussed.