Ectomycorrhizal fungi integrate nitrogen mobilisation and mineral weathering in boreal forest soil.

Tree growth in boreal forests is driven by ectomycorrhizal fungal mobilisation of organic nitrogen and mineral nutrients in soils with discrete organic and mineral horizons. However, there are no studies of how ectomycorrhizal mineral weathering and organic nitrogen mobilisation processes are integrated across the soil profile. We studied effects of organic matter (OM) availability on ectomycorrhizal functioning by altering the proportions of natural organic and mineral soil in reconstructed podzol profiles containing Pinus sylvestris plants, using 13 CO2 pulse labelling, patterns of naturally occurring stable isotopes (26 Mg and 15 N) and high-throughput DNA sequencing of fungal amplicons. Reduction in OM resulted in nitrogen limitation of plant growth and decreased allocation of photosynthetically derived carbon and mycelial growth in mineral horizons. Fractionation patterns of 26 Mg indicated that magnesium mobilisation and uptake occurred primarily in the deeper mineral horizon and was driven by carbon allocation to ectomycorrhizal mycelium. In this horizon, relative abundance of ectomycorrhizal fungi, carbon allocation and base cation mobilisation all increased with increased OM availability. Allocation of carbon through ectomycorrhizal fungi integrates organic nitrogen mobilisation and mineral weathering across soil horizons, improving the efficiency of plant nutrient acquisition. Our findings have fundamental implications for sustainable forest management and belowground carbon sequestration.

Widespread bacterial responses and their mechanism of bacterial metallogenic detoxification under high concentrations of heavy metals.

Microbial mineralization is increasingly used in bioremediation of heavy metal pollution, but better mechanistic understanding of the processes involved and how they are regulated are required to improve the practical application of microorganisms in bioremediation. We used a combination of morphological (TEM) and analytical (XRD, XPS, FTIR) methods, together with novel proteomic analyses, to investigate the detoxification mechanisms, used by a range of bacteria, including the strains Bacillus velezensis LB002, Escherichia coli DH5α, B. subtilis 168, Pseudomonas putida KT2440, and B. licheniformis MT-1, exposed to elevated concentrations of Cd2+ and combinations of Cd2+, Pb2+, Cu2+, and Zn2+, in the presence and absence of added CaCl2. Common features of detoxification included biomineralization, including the production of biological vaterite, up-regulation of proteins involved in flagellar movement and chemotaxis, biofilm synthesis, transmembrane transport of small molecules and organic matter decomposition. The putative roles of differentially expressed proteins in detoxification are discussed in relation to chemical and morphological data and together provide important tools to improve screening, selection, and practical application of bacterial isolates in bioremediation of polluted environments.

A tipping point in carbon storage when forest expands into tundra is related to mycorrhizal recycling of nitrogen.

Tundra ecosystems are global belowground sinks for atmospheric CO2 . Ongoing warming-induced encroachment by shrubs and trees risks turning this sink into a CO2 source, resulting in a positive feedback on climate warming. To advance mechanistic understanding of how shifts in mycorrhizal types affect long-term carbon (C) and nitrogen (N) stocks, we studied small-scale soil depth profiles of fungal communities and C-N dynamics across a subarctic-alpine forest-heath vegetation gradient. Belowground organic stocks decreased abruptly at the transition from heath to forest, linked to the presence of certain tree-associated ectomycorrhizal fungi that contribute to decomposition when mining N from organic matter. In contrast, ericoid mycorrhizal plants and fungi were associated with organic matter accumulation and slow decomposition. If climatic controls on arctic-alpine forest lines are relaxed, increased decomposition will likely outbalance increased plant productivity, decreasing the overall C sink capacity of displaced tundra.

Root associated fungi respond more strongly than rhizosphere soil fungi to N fertilization in a boreal forest.

Nitrogen (N) fertilization is a routine practice in boreal forests but its effects on fungal functional guilds in Pinus sylvestris forests are still incompletely understood. Sampling is often restricted to the upper organic horizons and based on DNA extracted from mixtures of soil and roots without explicitly analysing different spatial niches. Fungal community structure in soil and roots of an 85-y-old Pinus sylvestris forest was investigated using high throughput sequencing. Fertilized plots had been treated with a single dose of N fertilizer, 15 months prior to sampling. Species richness of fungi colonizing roots was reduced in all horizons by N fertilization. In contrast, species richness of soil fungi in the organic horizon was increased by N fertilization, but unaffected in the mineral horizons. Community composition of fungi colonizing roots differed from that of soil fungi, and both communities were significantly influenced by soil horizon and N. The ectomycorrhizal community composition in both roots and soil was significantly affected by N fertilization but no significant effect was found on saprotrophic fungi. The results highlight the importance of analysing the rhizosphere soil and root compartments separately since the fungal communities in these two niches appear to respond differently to environmental perturbations involving the addition of nitrogen.

Contrasting effects of ectomycorrhizal fungi on early and late stage decomposition in a boreal forest.

Symbiotic ectomycorrhizal fungi have received increasing attention as regulators of below-ground organic matter storage. They are proposed to promote organic matter accumulation by suppressing saprotrophs, but have also been suggested to play an active role in decomposition themselves. Here we show that exclusion of tree roots and associated ectomycorrhizal fungi in a boreal forest increased decomposition of surface litter by 11% by alleviating nitrogen limitation of saprotrophs-a "Gadgil effect". At the same time, root exclusion decreased Mn-peroxidase activity in the deeper mor layer by 91%. Our results show that ectomycorrhizal fungi may hamper short-term litter decomposition, but also support a crucial role of ectomycorrhizal fungi in driving long-term organic matter oxidation. These observations stress the importance of ectomycorrhizal fungi in regulation of below-ground organic matter accumulation. By different mechanisms they may either hamper or stimulate decomposition, depending upon stage of decomposition and location in the soil profile.

The second International Symposium on Fungal Stress: ISFUS.

The topic of 'fungal stress' is central to many important disciplines, including medical mycology, chronobiology, plant and insect pathology, industrial microbiology, material sciences, and astrobiology. The International Symposium on Fungal Stress (ISFUS) brought together researchers, who study fungal stress in a variety of fields. The second ISFUS was held in May 8-11 2017 in Goiania, Goiás, Brazil and hosted by the Instituto de Patologia Tropical e Saúde Pública at the Universidade Federal de Goiás. It was supported by grants from CAPES and FAPEG. Twenty-seven speakers from 15 countries presented their research related to fungal stress biology. The Symposium was divided into seven topics: 1. Fungal biology in extreme environments; 2. Stress mechanisms and responses in fungi: molecular biology, biochemistry, biophysics, and cellular biology; 3. Fungal photobiology in the context of stress; 4. Role of stress in fungal pathogenesis; 5. Fungal stress and bioremediation; 6. Fungal stress in agriculture and forestry; and 7. Fungal stress in industrial applications. This article provides an overview of the science presented and discussed at ISFUS-2017.

Fungal strategies for dealing with environment- and agriculture-induced stresses.

The Fungal Kingdom is responsible for many ecosystem services as well as many industrial and agricultural products. Nevertheless, how these fungal species function and carry out these services is dependent on their capacity to grow under different stress conditions caused by a variety of abiotic factors such as ionizing radiation, UV radiation, extremes of temperature, acidity and alkalinity, and environments of low nutritional status, low water activity, or polluted with, e.g. toxic metals or xenobiotics. This article reviews some natural or synthetic environments where fungi thrive under stress and have important impacts in agriculture and forestry.

Bacterial microbiomes of individual ectomycorrhizal Pinus sylvestris roots are shaped by soil horizon and differentially sensitive to nitrogen addition.

Plant roots select non-random communities of fungi and bacteria from the surrounding soil that have effects on their health and growth, but we know little about the factors influencing their composition. We profiled bacterial microbiomes associated with individual ectomycorrhizal Pinus sylvestris roots colonized by different fungi and analyzed differences in microbiome structure related to soils from distinct podzol horizons and effects of short-term additions of N, a growth-limiting nutrient commonly applied as a fertilizer, but known to influence patterns of carbon allocation to roots. Ectomycorrhizal roots growing in soil from different horizons harboured distinct bacterial communities. The fungi colonizing individual roots had a strong effect on the associated bacterial communities. Even closely related species within the same ectomycorrhizal genus had distinct bacterial microbiomes in unfertilized soil, but fertilization removed this specificity. Effects of N were rapid and context dependent, being influenced by both soil type and the particular ectomycorrhizal fungi involved. Fungal community composition changed in soil from all horizons, but bacteria only responded strongly to N in soil from the B horizon where community structure was different and bacterial diversity was significantly reduced, possibly reflecting changed carbon allocation patterns.

Growing evidence for facultative biotrophy in saprotrophic fungi: data from microcosm tests with 201 species of wood-decay basidiomycetes.

Ectomycorrhizal (ECM) symbioses have evolved a minimum of 78 times independently from saprotrophic lineages, indicating the potential for functional overlap between ECM and saprotrophic fungi. ECM fungi have the capacity to decompose organic matter, and although there is increasing evidence that some saprotrophic fungi exhibit the capacity to enter into facultative biotrophic relationships with plant roots without causing disease symptoms, this subject is still not well studied. In order to determine the extent of biotrophic capacity in saprotrophic wood-decay fungi and which systems may be useful models, we investigated the colonization of conifer seedling roots in vitro using an array of 201 basidiomycete wood-decay fungi. Microtome sectioning, differential staining and fluorescence microscopy were used to visualize patterns of root colonization in microcosm systems containing Picea abies or Pinus sylvestris seedlings and each saprotrophic fungus. Thirty-four (16.9%) of the tested fungal species colonized the roots of at least one tree species. Two fungal species showed formation of a mantle and one showed Hartig net-like structures. These features suggest the possibility of an active functional symbiosis between fungus and plant. The data indicate that the capacity for facultative biotrophic relationships in free-living saprotrophic basidiomycetes may be greater than previously supposed.

Changes in turnover rather than production regulate biomass of ectomycorrhizal fungal mycelium across a Pinus sylvestris chronosequence.

In boreal forest soils, ectomycorrhizal fungi are fundamentally important for carbon (C) dynamics and nutrient cycling. Although their extraradical mycelium (ERM) is pivotal for processes such as soil organic matter build-up and nitrogen cycling, very little is known about its dynamics and regulation. In this study, we quantified ERM production and turnover, and examined how these two processes together regulated standing ERM biomass in seven sites forming a chronosequence of 12- to 100-yr-old managed Pinus sylvestris forests. This was done by determining ERM biomass, using ergosterol as a proxy, in sequentially harvested in-growth mesh bags and by applying mathematical models. Although ERM production declined with increasing forest age from 1.2 to 0.5 kg ha-1  d-1 , the standing biomass increased from 50 to 112 kg ha-1 . This was explained by a drastic decline in mycelial turnover from seven times to one time per year with increasing forest age, corresponding to mean residence times from 25 d up to 1 yr. Our results demonstrate that ERM turnover is the main factor regulating biomass across differently aged forest stands. Explicit inclusion of ERM parameters in forest ecosystem C models may significantly improve their capacity to predict responses of mycorrhiza-mediated processes to management and environmental changes.

Fractionation and assimilation of Mg isotopes by fungi is species dependent.

Symbiotic ectomycorrhizal fungi mobilize nutrients from both organic and inorganic substrates and supply them to their host plants. Their role in mobilizing base cations and phosphorus from mineral substrates through weathering has received increasing attention in recent years but the processes involved remain to be elucidated. We grew selected ectomycorrhizal and nonmycorrhizal fungi in axenic systems containing mineral and organic substrates and examined their capacity to fractionate and assimilate stable isotopes of magnesium. The mycorrhizal fungi were significantly depleted in heavy isotopes with the lowest Δ26 Mg values (the difference between δ26 Mg in fungal tissue and δ26 Mg in the substrate) compared with nonmycorrhizal fungi, when grown on mineral substrates containing granite particles. The ectomycorrhizal fungi accumulated significantly higher concentrations of Mg, K and P than the nonmycorrhizal fungi. There was a highly significant statistical relationship between δ26 Mg tissue signature and mycelial concentration of Mg, with a clear separation between most ectomycorrhizal fungi and the nonmycorrhizal fungi. These results are consistent with the idea that ectomycorrhizal fungi have evolved efficient mechanisms to mobilize, transport and store Mg within their mycelia.

Analysis of single root tip microbiomes suggests that distinctive bacterial communities are selected by Pinus sylvestris roots colonized by different ectomycorrhizal fungi.

Symbiotic ectomycorrhizal tree roots represent an important niche for interaction with bacteria since the fungi colonizing them have a large surface area and receive a direct supply of photosynthetically derived carbon. We examined individual root tips of Pinus sylvestris at defined time points between 5 days and 24 weeks, identified the dominant fungi colonizing each root tip using Sanger sequencing and the bacterial communities colonizing individual root tips by 454 pyrosequencing. Bacterial colonization was extremely dynamic with statistically significant variation in time and increasing species richness until week 16 (3477 operational taxonomic units). Bacterial community structure of roots colonized by Russula sp. 6 GJ-2013b, Piloderma spp., Meliniomyces variabilis and Paxillus involutus differed significantly at weeks 8 and 16 but diversity declined and significant differences were no longer apparent at week 24. The most common genera were Burkholderia, Sphingopyxsis, Dyella, Pseudomonas, Acinetobacter, Actinospica, Aquaspirillum, Acidobacter Gp1, Sphingomonas, Terriglobus, Enhydrobacter, Herbaspirillum and Bradyrhizobium. Many genera had high initial abundance at week 8, declining with time but Dyella and Terriglobus increased in abundance at later time points. In roots colonized by Piloderma spp. several other bacterial genera, such as Actinospica, Bradyrhizobium, Acidobacter Gp1 and Rhizomicrobium appeared to increase in abundance at later sampling points.

Transcriptomic changes in the plant pathogenic fungus Rhizoctonia solani AG-3 in response to the antagonistic bacteria Serratia proteamaculans and Serratia plymuthica.

BACKGROUND: Improved understanding of bacterial-fungal interactions in the rhizosphere should assist in the successful application of bacteria as biological control agents against fungal pathogens of plants, providing alternatives to chemicals in sustainable agriculture. Rhizoctonia solani is an important soil-associated fungal pathogen and its chemical treatment is not feasible or economic. The genomes of the plant-associated bacteria Serratia proteamaculans S4 and Serratia plymuthica AS13 have been sequenced, revealing genetic traits that may explain their diverse plant growth promoting activities and antagonistic interactions with R. solani. To understand the functional response of this pathogen to different bacteria and to elucidate whether the molecular mechanisms that the fungus exploits involve general stress or more specific responses, we performed a global transcriptome profiling of R. solani Rhs1AP anastomosis group 3 (AG-3) during interaction with the S4 and AS13 species of Serratia using RNA-seq. RESULTS: Approximately 104,504 million clean 75-100 bp paired-end reads were obtained from three libraries, each in triplicate (AG3-Control, AG3-S4 and AG3-AS13). Transcriptome analysis revealed that approximately 10% of the fungal transcriptome was differentially expressed during challenge with Serratia. The numbers of S4- and AS13-specific differentially expressed genes (DEG) were 866 and 292 respectively, while there were 1035 common DEGs in the two treatment groups. Four hundred and sixty and 242 genes respectively had values of log2 fold-change > 3 and for further analyses this cut-off value was used. Functional classification of DEGs based on Gene Ontology enrichment analysis and on KEGG pathway annotations revealed a general shift in fungal gene expression in which genes related to xenobiotic degradation, toxin and antioxidant production, energy, carbohydrate and lipid metabolism and hyphal rearrangements were subjected to transcriptional regulation. CONCLUSIONS: This RNA-seq profiling generated a novel dataset describing the functional response of the phytopathogen R. solani AG3 to the plant-associated Serratia bacteria S4 and AS13. Most genes were regulated in the same way in the presence of both bacterial isolates, but there were also some strain-specific responses. The findings in this study will be beneficial for further research on biological control and in depth exploration of bacterial-fungal interactions in the rhizosphere.

Fungal stress biology: a preface to the Fungal Stress Responses special edition.

There is currently an urgent need to increase global food security, reverse the trends of increasing cancer rates, protect environmental health, and mitigate climate change. Toward these ends, it is imperative to improve soil health and crop productivity, reduce food spoilage, reduce pesticide usage by increasing the use of biological control, optimize bioremediation of polluted sites, and generate energy from sustainable sources such as biofuels. This review focuses on fungi that can help provide solutions to such problems. We discuss key aspects of fungal stress biology in the context of the papers published in this Special Issue of Current Genetics. This area of biology has relevance to pure and applied research on fungal (and indeed other) systems, including biological control of insect pests, roles of saprotrophic fungi in agriculture and forestry, mycotoxin contamination of the food-supply chain, optimization of microbial fermentations including those used for bioethanol production, plant pathology, the limits of life on Earth, and astrobiology.

The International Symposium on Fungal Stress: ISFUS.

Fungi play central roles in many biological processes, influencing soil fertility, decomposition, cycling of minerals, and organic matter, plant health, and nutrition. They produce a wide spectrum of molecules, which are exploited in a range of industrial processes to manufacture foods, food preservatives, flavoring agents, and other useful biological products. Fungi can also be used as biological control agents of microbial pathogens, nematodes or insect pests, and affect plant growth, stress tolerance, and nutrient acquisition. Successful exploitation of fungi requires better understanding of the mechanisms that fungi use to cope with stress as well as the way in which they mediate stress tolerance in other organisms. It is against this backdrop that a scientific meeting on fungal stress was held in São José dos Campos, Brazil, in October 2014. The meeting, hosted by Drauzio E. N. Rangel and Alene E. Alder-Rangel, and supported by the São Paulo Research Foundation (FAPESP), brought together more than 30 young, mid-career, and highly accomplished scientists from ten different countries. Here we summarize the highlights of the meeting.

Transcriptional responses of the bacterial antagonist Serratia plymuthica to the fungal phytopathogen Rhizoctonia solani.

Rhizobacteria with biocontrol ability exploit a range of mechanisms to compete successfully with other microorganisms and to ensure their growth and survival in the rhizosphere, ultimately promoting plant growth. The rhizobacterium Serratia plymuthica AS13 is able to promote oilseed rape growth and improve seedling survival in the presence of the fungal pathogen, Rhizoctonia solani AG 2-1; however, our understanding of the mechanisms underlying the antagonism of Serratia is limited. To elucidate possible mechanisms, genome-wide gene expression profiling of S. plymuthica AS13 was carried out in the presence or absence of R. solani. We used RNA sequencing methodology to obtain a comprehensive overview of Serratia gene expression in response to R. solani. The differential gene expression profiles of S. plymuthica AS13 revealed significantly increased expression of genes related to the biosynthesis of the antibiotic pyrrolnitrin (prnABCD), protease production and transporters. The results presented here provide evidence that antibiosis is a major functional mechanism underlying the antagonistic behaviour of S. plymuthica AS13.

Carbon sequestration is related to mycorrhizal fungal community shifts during long-term succession in boreal forests.

Boreal forest soils store a major proportion of the global terrestrial carbon (C) and below-ground inputs contribute as much as above-ground plant litter to the total C stored in the soil. A better understanding of the dynamics and drivers of root-associated fungal communities is essential to predict long-term soil C storage and climate feedbacks in northern ecosystems. We used 454-pyrosequencing to identify fungal communities across fine-scaled soil profiles in a 5000 yr fire-driven boreal forest chronosequence, with the aim of pinpointing shifts in fungal community composition that may underlie variation in below-ground C sequestration. In early successional-stage forests, higher abundance of cord-forming ectomycorrhizal fungi (such as Cortinarius and Suillus species) was linked to rapid turnover of mycelial biomass and necromass, efficient nitrogen (N) mobilization and low C sequestration. In late successional-stage forests, cord formers declined, while ericoid mycorrhizal ascomycetes continued to dominate, potentially facilitating long-term humus build-up through production of melanized hyphae that resist decomposition. Our results suggest that cord-forming ectomycorrhizal fungi and ericoid mycorrhizal fungi play opposing roles in below-ground C storage. We postulate that, by affecting turnover and decomposition of fungal tissues, mycorrhizal fungal identity and growth form are critical determinants of C and N sequestration in boreal forests.

Influence of soil type, cultivar and Verticillium dahliae on the structure of the root and rhizosphere soil fungal microbiome of strawberry.

Sustainable management of crop productivity and health necessitates improved understanding of the ways in which rhizosphere microbial populations interact with each other, with plant roots and their abiotic environment. In this study we examined the effects of different soils and cultivars, and the presence of a soil-borne fungal pathogen, Verticillium dahliae, on the fungal microbiome of the rhizosphere soil and roots of strawberry plants, using high-throughput pyrosequencing. Fungal communities of the roots of two cultivars, Honeoye and Florence, were statistically distinct from those in the rhizosphere soil of the same plants, with little overlap. Roots of plants growing in two contrasting field soils had high relative abundance of Leptodontidium sp. C2 BESC 319 g whereas rhizosphere soil was characterised by high relative abundance of Trichosporon dulcitum or Cryptococcus terreus, depending upon the soil type. Differences between different cultivars were not as clear. Inoculation with the pathogen V. dahliae had a significant influence on community structure, generally decreasing the number of rhizosphere soil- and root-inhabiting fungi. Leptodontidium sp. C2 BESC 319 g was the dominant fungus responding positively to inoculation with V. dahliae. The results suggest that 1) plant roots select microorganisms from the wider rhizosphere pool, 2) that both rhizosphere soil and root inhabiting fungal communities are influenced by V. dahliae and 3) that soil type has a stronger influence on both of these communities than cultivar.

Nitrogen and carbon reallocation in fungal mycelia during decomposition of boreal forest litter.

Boreal forests are characterized by spatially heterogeneous soils with low N availability. The decomposition of coniferous litter in these systems is primarily performed by basidiomycete fungi, which often form large mycelia with a well-developed capacity to reallocate resources spatially- an advantageous trait in heterogeneous environments. In axenic microcosm systems we tested whether fungi increase their biomass production by reallocating N between Pinus sylvestris (Scots pine) needles at different stages of decomposition. We estimated fungal biomass production by analysing the accumulation of the fungal cell wall compound chitin. Monospecific systems were compared with systems with interspecific interactions. We found that the fungi reallocated assimilated N and mycelial growth away from well-degraded litter towards fresh litter components. This redistribution was accompanied by reduced decomposition of older litter. Interconnection of substrates increased over-all fungal C use efficiency (i.e. the allocation of assimilated C to biomass rather than respiration), presumably by enabling fungal translocation of growth-limiting N to litter with higher C quality. Fungal connection between different substrates also restricted N-mineralization and production of dissolved organic N, suggesting that litter saprotrophs in boreal forest ecosystems primarily act to redistribute rather than release N. This spatial integration of different resource qualities was hindered by interspecific interactions, in which litters of contrasting quality were colonised by two different basidiomycete species. The experiments provide a detailed picture of how resource reallocation in two decomposer fungi leads to a more efficient utilisation of spatially separated resources under N-limitation. From an ecosystem point of view, such economic fungal behaviour could potentially contribute to organic matter accumulation in the litter layers of boreal forests.

Non-contiguous finished genome sequence of plant-growth promoting Serratia proteamaculans S4.

Serratia proteamaculans S4 (previously Serratia sp. S4), isolated from the rhizosphere of wild Equisetum sp., has the ability to stimulate plant growth and to suppress the growth of several soil-borne fungal pathogens of economically important crops. Here we present the non-contiguous, finished genome sequence of S. proteamaculans S4, which consists of a 5,324,944 bp circular chromosome and a 129,797 bp circular plasmid. The chromosome contains 5,008 predicted genes while the plasmid comprises 134 predicted genes. In total, 4,993 genes are assigned as protein-coding genes. The genome consists of 22 rRNA genes, 82 tRNA genes and 58 pseudogenes. This genome is a part of the project "Genomics of four rapeseed plant growth-promoting bacteria with antagonistic effect on plant pathogens" awarded through the 2010 DOE-JGI's Community Sequencing Program.