From 13C-lignin to 13C-mycelium: Agaricus bisporus uses polymeric lignin as a carbon source.

Plant biomass conversion by saprotrophic fungi plays a pivotal role in terrestrial carbon (C) cycling. The general consensus is that fungi metabolize carbohydrates, while lignin is only degraded and mineralized to CO2. Recent research, however, demonstrated fungal conversion of 13C-monoaromatic compounds into proteinogenic amino acids. To unambiguously prove that polymeric lignin is not merely degraded, but also metabolized, carefully isolated 13C-labeled lignin served as substrate for Agaricus bisporus, the world's most consumed mushroom. The fungus formed a dense mycelial network, secreted lignin-active enzymes, depolymerized, and removed lignin. With a lignin carbon use efficiency of 0.14 (g/g) and fungal biomass enrichment in 13C, we demonstrate that A. bisporus assimilated and further metabolized lignin when offered as C-source. Amino acids were high in 13C-enrichment, while fungal-derived carbohydrates, fatty acids, and ergosterol showed traces of 13C. These results hint at lignin conversion via aromatic ring-cleaved intermediates to central metabolites, underlining lignin's metabolic value for fungi.

Accumulation of Health-Promoting Compounds in Upland Black Rice by Interacting Mycorrhizal and Endophytic Fungi.

There is an increasing interest in finding eco-friendly and safe approaches to increase agricultural productivity and deliver healthy foods. Arbuscular mycorrhizal fungi (AMF) and endophytic fungi (EPF) are important components of sustainable agriculture in view of their ability to increase productivity and various plant secondary metabolites with health-promoting effects. In a pot experiment, our main research question was to evaluate the additive and synergistic effects of an AMF and four root-endophytic fungi on plant performance and on the accumulation of health-promoting secondary compounds. Plant growth varied between the treatments with both single inoculants and co-inoculation of an AMF and four EPF strains. We found that inoculation with a single EPF positively affected the growth and biomass production of most of the plant-endophyte consortia examined. The introduction of AMF into this experiment (dual inoculation) had a beneficial effect on plant growth and yield. AMF, Rhizophagus variabilis KS-02 co-inoculated with EPF, Trichoderma zelobreve PBMP16 increased the highest biomass, exceeding the growth rate of non-inoculated plants. Co-inoculated R. variabilis KS-02 and T. zelobreve PBMP16 had significantly greater beneficial effects on almost all aspects of plant growth, photosynthesis-related parameters, and yield. It also promoted root growth quality and plant nutrient uptake. The phenolic compounds, anthocyanin, and antioxidant capacity in rice seeds harvested from plants co-inoculated with AMF and EPF were dramatically increased compared with those from non-inoculated plants. In conclusion, our results indicated that EPF and AMF contributed to symbiosis in Maled Phai cultivar and were coordinately involved in promoting plant growth performance under a pot trial.

Responses of rhizosphere fungi to the root economics space in grassland monocultures of different age.

Recent studies on root traits have shown that there are two axes explaining trait variation belowground: the collaboration axis with mycorrhizal partners and the conservation ('fast - slow') axis. However, it is yet unknown whether these trait axes affect the assembly of soilborne fungi. We expect saprotrophic fungi to link to the conservation axis of root traits, whereas pathogenic and arbuscular mycorrhizal fungi link to the collaboration axis, but in opposite directions, as arbuscular mycorrhizal fungi might provide pathogen protection. To test these hypotheses, we sequenced rhizosphere fungal communities and measured root traits in monocultures of 25 grassland plant species, differing in age. Within the fungal guilds, we evaluated fungal species richness, relative abundance and community composition. Contrary to our hypotheses, fungal diversity and relative abundance were not strongly related to the root trait axes. However, saprotrophic fungal community composition was affected by the conservation gradient and pathogenic community composition by the collaboration gradient. The rhizosphere AMF community composition did not change along the collaboration gradient, even though the root trait axis was in line with the root mycorrhizal colonization rate. Overall, our results indicate that in the long term, the root trait axes are linked with fungal community composition.

The secretome of Agaricus bisporus: Temporal dynamics of plant polysaccharides and lignin degradation.

Despite substantial lignocellulose conversion during mycelial growth, previous transcriptome and proteome studies have not yet revealed how secretomes from the edible mushroom Agaricus bisporus develop and whether they modify lignin models in vitro. To clarify these aspects, A. bisporus secretomes collected throughout a 15-day industrial substrate production and from axenic lab-cultures were subjected to proteomics, and tested on polysaccharides and lignin models. Secretomes (day 6-15) comprised A. bisporus endo-acting and substituent-removing glycoside hydrolases, whereas ß-xylosidase and glucosidase activities gradually decreased. Laccases appeared from day 6 onwards. From day 10 onwards, many oxidoreductases were found, with numerous multicopper oxidases (MCO), aryl alcohol oxidases (AAO), glyoxal oxidases (GLOX), a manganese peroxidase (MnP), and unspecific peroxygenases (UPO). Secretomes modified dimeric lignin models, thereby catalyzing syringylglycerol-ß-guaiacyl ether (SBG) cleavage, guaiacylglycerol-ß-guaiacyl ether (GBG) polymerization, and non-phenolic veratrylglycerol-ß-guaiacyl ether (VBG) oxidation. We explored A. bisporus secretomes and insights obtained can help to better understand biomass valorization.

Oxidation-driven lignin removal by Agaricus bisporus from wheat straw-based compost at industrial scale.

Fungi are main lignin degraders and the edible white button mushroom, Agaricus bisporus, inhabits lignocellulose-rich environments. Previous research hinted at delignification when A. bisporus colonized pre-composted wheat straw-based substrate in an industrial setting, assumed to aid subsequent release of monosaccharides from (hemi-)cellulose to form fruiting bodies. Yet, structural changes and specific quantification of lignin throughout A. bisporus mycelial growth remain largely unresolved. To elucidate A. bisporus routes of delignification, at six timepoints throughout mycelial growth (15 days), substrate was collected, fractionated, and analyzed by quantitative pyrolysis-GC-MS, 2D-HSQC NMR, and SEC. Lignin decrease was highest between day 6 and day 10 and reached in total 42 % (w/w). The substantial delignification was accompanied by extensive structural changes of residual lignin, including increased syringyl to guaiacyl (S/G) ratios, accumulated oxidized moieties, and depleted intact interunit linkages. Hydroxypropiovanillone and hydroxypropiosyringone (HPV/S) subunits accumulated, which are indicative for ß-|O-4' ether cleavage and imply a laccase-driven ligninolysis. We provide compelling evidence that A. bisporus is capable of extensive lignin removal, have obtained insights into mechanisms at play and susceptibilities of various substructures, thus we were contributing to understanding fungal lignin conversion.

Mycorrhiza-mediated recruitment of complete denitrifying Pseudomonas reduces N2O emissions from soil.

BACKGROUND: Arbuscular mycorrhizal fungi (AMF) are key soil organisms and their extensive hyphae create a unique hyphosphere associated with microbes actively involved in N cycling. However, the underlying mechanisms how AMF and hyphae-associated microbes may cooperate to influence N2O emissions from "hot spot" residue patches remain unclear. Here we explored the key microbes in the hyphosphere involved in N2O production and consumption using amplicon and shotgun metagenomic sequencing. Chemotaxis, growth and N2O emissions of isolated N2O-reducing bacteria in response to hyphal exudates were tested using in vitro cultures and inoculation experiments. RESULTS: AMF hyphae reduced denitrification-derived N2O emission (max. 63%) in C- and N-rich residue patches. AMF consistently enhanced the abundance and expression of clade I nosZ gene, and inconsistently increased that of nirS and nirK genes. The reduction of N2O emissions in the hyphosphere was linked to N2O-reducing Pseudomonas specifically enriched by AMF, concurring with the increase in the relative abundance of the key genes involved in bacterial citrate cycle. Phenotypic characterization of the isolated complete denitrifying P. fluorescens strain JL1 (possessing clade I nosZ) indicated that the decline of net N2O emission was a result of upregulated nosZ expression in P. fluorescens following hyphal exudation (e.g. carboxylates). These findings were further validated by re-inoculating sterilized residue patches with P. fluorescens and by an 11-year-long field experiment showing significant positive correlation between hyphal length density with the abundance of clade I nosZ gene. CONCLUSIONS: The cooperation between AMF and the N2O-reducing Pseudomonas residing on hyphae significantly reduce N2O emissions in the microsites. Carboxylates exuded by hyphae act as attractants in recruiting P. fluorescens and also as stimulants triggering nosZ gene expression. Our discovery indicates that reinforcing synergies between AMF and hyphosphere microbiome may provide unexplored opportunities to stimulate N2O consumption in nutrient-enriched microsites, and consequently reduce N2O emissions from soils. This knowledge opens novel avenues to exploit cross-kingdom microbial interactions for sustainable agriculture and for climate change mitigation. Video Abstract.

A trade-off between space exploration and mobilization of organic phosphorus through associated microbiomes enables niche differentiation of arbuscular mycorrhizal fungi on the same root.

Ecology seeks to explain species coexistence, but experimental tests of mechanisms for coexistence are difficult to conduct. We synthesized an arbuscular mycorrhizal (AM) fungal community with three fungal species that differed in their capacity of foraging for orthophosphate (P) due to differences in soil exploration. We tested whether AM fungal species-specific hyphosphere bacterial assemblages recruited by hyphal exudates enabled differentiation among the fungi in the capacity of mobilizing soil organic P (Po). We found that the less efficient space explorer, Gigaspora margarita, obtained less 13C from the plant, whereas it had higher efficiencies in Po mobilization and alkaline phosphatase (AlPase) production per unit C than the two efficient space explorers, Rhizophagusintraradices and Funneliformis mosseae. Each AM fungus was associated with a distinct alp gene harboring bacterial assemblage, and the alp gene abundance and Po preference of the microbiome associated with the less efficient space explorer were higher than those of the two other species. We conclude that the traits of AM fungal associated bacterial consortia cause niche differentiation. The trade-off between foraging ability and the ability to recruit effective Po mobilizing microbiomes is a mechanism that allows co-existence of AM fungal species in a single plant root and surrounding soil habitat.

Promoting growth and production of sunchoke (Helianthus tuberosus) by co-inoculation with phosphate solubilizing bacteria and arbuscular mycorrhizal fungi under drought.

Due to different functions of phosphate solubilizing bacteria (PSB) and arbuscular mycorrhizal fungi (AMF), their potential synergistic effects on enhancing plant growth and yield are worth investigating, especially under adverse conditions. This work focused on the isolation of PSB and characterization for their plant growth promoting properties under drought. The most efficient P solubilizing bacterium was isolated and identified as Burkholderia vietnamiensis strain KKUT8-1. Then, a factorial experiment on the performance of sunchoke (Helianthus tuberosus) was set up with four factors, viz., PSB (presence or absence of KKUT8-1), AMF (presence or absence of Rhizophagus aggregatus), rock phosphate (RP; added or not) and moisture (well-watered (WW) or drought (DS) conditions). Sunchoke performance was enhanced by the presence of AMF, whereas addition of PSB had a positive effect on SPAD values and inulin concentration. Drought reduced plant performance, while addition of RP reduced photosynthetic rate. There was little evidence for synergistic effects between PSB and AMF, except for SPAD values and inulin concentration. Plants that were co-inoculated with AMF and PSB had highest SPAD value, shoot diameter, leaf area, leaf number, chlorophyll concentration, plant biomass, tuber production, root growth and total soluble sugar concentration. Co-inoculated plants also had increased plant water status, reduced electrolyte leakage, and reduced malondialdehyde and proline concentration. Strain KKUT8-1 is the first strain of B. vietnamiensis capable of promoting growth and yield of sunchoke. Enhanced production of sunchoke by a combination of AMF and PSB was much better than the application of RP. Our finding offers an opportunity to develop combinations of biological inoculants for increasing the growth and production of sunchoke under drought in the future.

Enhancement of growth and Cannabinoids content of hemp (Cannabis sativa) using arbuscular mycorrhizal fungi.

This study aimed to investigate the efficiency of arbuscular mycorrhizal fungi (AMF) to promote growth and cannabinoid yield of Cannabis sativa KKU05. A completely randomized design (CRD) was conducted with six replications for 60 days. Two different species of AMF, Rhizophagus prolifer PC2-2 and R. aggregatus BM-3 g3 were selected as inocula and compared with two non-mycorrhizal controls, one without synthetic fertilizer and one with synthetic NPK fertilizer. The unfertilized non-mycorrhizal plants had the lowest performance, whereas plants inoculated with R. aggregatus BM-3 g3 performed best, both in terms of plant biomass and concentrations of CBD and THC. There were no significant differences in plant biomass and cannabinoid concentrations between non-mycorrhizal plants that received synthetic fertilizer and mycorrhizal plants with inoculum of R. prolifer PC2-2. Our data demonstrate the great potential for cannabis cultivation without risking deterioration of soil structure, such as soil hardening and increased acidity, which might be induced by long-term use of synthetic fertilizer.

Nitrogen availability mediates the priming effect of soil organic matter by preferentially altering the straw carbon-assimilating microbial community.

Straw incorporation into soil increases carbon (C) sequestration but can induce priming effects (PE), the enhanced breakdown of soil organic matter. The direction and magnitude of PE and the consequences for the C balance induced by straw addition depend on nitrogen (N) availability and soil management history. Using 13C-labeled maize straw, we conducted a 56-day incubation to determine the dynamics of PE and the underlying microbial mechanisms after straw and/or mineral N addition to three soils with contrasting cultivation and fertilization histories, i) unfertilized soil (Unfertilized), ii) 8 years farmyard manure amended soil (Manured), and iii) abandoned cropland soil (Abandoned). 13C-PLFAs (phospholipid fatty acids) were analyzed to identify microbial groups utilizing straw and to explore their contribution to the PE. Straw addition increased microbial biomass (MBC), activities of enzymes related to the C and N cycles, and changed microbial community composition. SOC decomposition was enhanced by microbes activated by straw addition, leading to a positive cumulative PE ranging from 494 to 789 µg C g-1 soil. The magnitude of positive PE and straw decomposition in the manured soil was higher than that in the unfertilized and abandoned soils due to larger MBC and higher enzyme activities, resulting in a lower net SOC gain. Compared with straw only, the combination of straw addition with N fertilizer did not influence MBC, but increased positive PE (average increase of 18.1%) and straw decomposition (17.1%), further limiting SOC gain. 13C-labeled fungi: bacteria ratios and Gram-positive (G+): negative (G-) bacteria ratios increased with the increasing PE after N fertilization, but soil-derived (un-labeled) PLFAs remained stable. Random forest analysis further showed that straw C-assimilating microbial attributes are important predictors in driving the greater PE after N addition. Our study highlights the importance of straw C-assimilating fungi and G+ bacteria in mediating N-induced PE in arable soils.

Breeding Beyond Monoculture: Putting the "Intercrop" Into Crops.

Intercropping is both a well-established and yet novel agricultural practice, depending on one's perspective. Such perspectives are principally governed by geographic location and whether monocultural practices predominate. Given the negative environmental effects of monoculture agriculture (loss of biodiversity, reliance on non-renewable inputs, soil degradation, etc.), there has been a renewed interest in cropping systems that can reduce the impact of modern agriculture while maintaining (or even increasing) yields. Intercropping is one of the most promising practices in this regard, yet faces a multitude of challenges if it is to compete with and ultimately replace the prevailing monocultural norm. These challenges include the necessity for more complex agricultural designs in space and time, bespoke machinery, and adapted crop cultivars. Plant breeding for monocultures has focused on maximizing yield in single-species stands, leading to highly productive yet specialized genotypes. However, indications suggest that these genotypes are not the best adapted to intercropping systems. Re-designing breeding programs to accommodate inter-specific interactions and compatibilities, with potentially multiple different intercropping partners, is certainly challenging, but recent technological advances offer novel solutions. We identify a number of such technology-driven directions, either ideotype-driven (i.e., "trait-based" breeding) or quantitative genetics-driven (i.e., "product-based" breeding). For ideotype breeding, plant growth modeling can help predict plant traits that affect both inter- and intraspecific interactions and their influence on crop performance. Quantitative breeding approaches, on the other hand, estimate breeding values of component crops without necessarily understanding the underlying mechanisms. We argue that a combined approach, for example, integrating plant growth modeling with genomic-assisted selection and indirect genetic effects, may offer the best chance to bridge the gap between current monoculture breeding programs and the more integrated and diverse breeding programs of the future.

Ancestral predisposition toward a domesticated lifestyle in the termite-cultivated fungus Termitomyces.

The ancestor of termites relied on gut symbionts for degradation of plant material, an association that persists in all termite families.1,2 However, the single-lineage Macrotermitinae has additionally acquired a fungal symbiont that complements digestion of food outside the termite gut.3 Phylogenetic analysis has shown that fungi grown by these termites form a clade-the genus Termitomyces-but the events leading toward domestication remain unclear.4 To address this, we reconstructed the lifestyle of the common ancestor of Termitomyces using a combination of ecological data with a phylogenomic analysis of 21 related non-domesticated species and 25 species of Termitomyces. We show that the closely related genera Blastosporella and Arthromyces also contain insect-associated species. Furthermore, the genus Arthromyces produces asexual spores on the mycelium, which may facilitate insect dispersal when growing on aggregated subterranean fecal pellets of a plant-feeding insect. The sister-group relationship between Arthromyces and Termitomyces implies that insect association and asexual sporulation, present in both genera, preceded the domestication of Termitomyces and did not follow domestication as has been proposed previously. Specialization of the common ancestor of these two genera on an insect-fecal substrate is further supported by similar carbohydrate-degrading profiles between Arthromyces and Termitomyces. We describe a set of traits that may have predisposed the ancestor of Termitomyces toward domestication, with each trait found scattered in related taxa outside of the termite-domesticated clade. This pattern indicates that the origin of the termite-fungus symbiosis may not have required large-scale changes of the fungal partner.

Ectomycorrhizas and tipping points in forest ecosystems.

The resilience of forests is compromised by human-induced environmental influences pushing them towards tipping points and resulting in major shifts in ecosystem state that might be difficult to reverse, are difficult to predict and manage, and can have vast ecological, economic and social consequences. The literature on tipping points has grown rapidly, but almost exclusively based on aquatic and aboveground systems. So far little effort has been made to make links to soil systems, where change is not as drastically apparent, timescales may differ and recovery may be slower. Predicting belowground ecosystem state transitions and recovery, and their impacts on aboveground systems, remains a major scientific, practical and policy challenge. Recently observed major changes in aboveground tree condition across European forests are probably causally linked to ectomycorrhizal (EM) fungal changes belowground. Based on recent breakthroughs in data collection and analysis, we apply tipping point theory to forests, including their belowground component, focusing on EM fungi; link environmental thresholds for EM fungi with nutrient imbalances in forest trees; explore the role of phenotypic plasticity in EM fungal adaptation to, and recovery from, environmental change; and propose major positive feedback mechanisms to understand, address and predict forest ecosystem tipping points.

Mycorrhizal associations change root functionality: a 3D modelling study on competitive interactions between plants for light and nutrients.

Recent studies show that the variation in root functional traits can be explained by a two-dimensional trait framework, containing a 'collaboration' axis in addition to the classical fast-slow 'conservation' axis. This collaboration axis spans from thin and highly branched roots that employ a 'do-it-yourself' strategy to thick and sparsely branched roots that 'outsource' nutrient uptake to symbiotic arbuscular mycorrhizal fungi (AMF). Here, we explore the functionality of this collaboration axis by quantifying how interactions with AMF change the impact of root traits on plant performance. To this end, we developed a novel functional-structural plant (FSP) modelling approach that simulates plants competing for light and nutrients in the presence or absence of AMF. Our simulation results support the notion that in the absence of AMF, plants rely on thin, highly branched roots for their nutrient uptake. The presence of AMF, however, promotes thick, unbranched roots as an alternative strategy for uptake of immobile phosphorus, but not for mobile nitrogen. This provides further support for a root trait framework that accommodates for the interactive effect of roots and AMF. Our modelling study offers unique opportunities to incorporate soil microbial interactions into root functionality as it integrates consequences of belowground trait expression.

Colonization by arbuscular mycorrhizal fungi improves salinity tolerance of eucalyptus (Eucalyptus camaldulensis) seedlings.

Soil salinity affects soil quality and reduces plant performance. Arbuscular mycorrhizal fungi (AMF) can enhance the tolerance of plants under salinity stress. Cultivation of eucalyptus (Eucalyptus camaldulensis), which exhibits high water use efficiency, is possible in saline areas to produce raw materials for the pulp industry. We determined the effects of arbuscular mycorrhizal fungi (AMF) on the growth and survival of eucalyptus seedlings under saline conditions. Three different clones of eucalyptus seedlings were pre-inoculated with three salt-tolerant AMF species, namely Glomus sp.2, Gigaspora albida and G. decipiens, and without pre-inoculation. The seedlings were grown in a greenhouse for 45 days. They were then transferred to individual pots, filled with field soil and subsequently treated with NaCl solution until electro-conductivity (EC) reached 10, 15 and 20 dS m-1. They were watered for 90 days under nursery conditions. The results show that increased salinity levels reduced plant performance, fractional AMF root colonization, spore number, and eucalypt K/Na ratio. AMF significantly increased chlorophyll and decreased leaf proline concentrations by more than 50% and 20% respectively and increased the K/Na ratio three- to six-fold compared with non-inoculated plants. Pre-inoculation with AMF before outplanting also improved plant performance by more than 30% under salinity stress compared to non-inoculated plants. We conclude that AMF can alleviate the negative impacts of salinity on plant physiological and biochemical parameters.

High microbial diversity stabilizes the responses of soil organic carbon decomposition to warming in the subsoil on the Tibetan Plateau.

Soil microbes are directly involved in soil organic carbon (SOC) decomposition, yet the importance of microbial biodiversity in regulating the temperature sensitivity of SOC decomposition remains elusive, particularly in alpine regions where climate change is predicted to strongly affect SOC dynamics and ecosystem stability. Here we collected topsoil and subsoil samples along an elevational gradient on the southeastern Tibetan Plateau to explore the temperature sensitivity (Q10 ) of SOC decomposition in relation to changes in microbial communities. Specifically, we tested whether the decomposition of SOC would be more sensitive to warming when microbial diversity is low. The estimated Q10 value ranged from 1.28 to 1.68, and 1.80 to 2.10 in the topsoil and subsoil, respectively. The highest Q10 value was observed at the lowest altitude of forests in the topsoil, and at the highest altitude of alpine meadow in the subsoil. Variations in Q10 were closely related to changes in microbial properties. In the topsoil the ratio of gram-positive to gram-negative bacteria (G+:G-) was the predominant factor associated with the altitudinal variations in Q10 . In the subsoil, SOC decomposition showed more resilience to warming when the diversity of soil bacteria (both whole community and major groups) and fungi was higher. Our results partly support the positive biodiversity-ecosystem stability hypothesis. Structural equation modeling further indicates that variations in Q10 in the subsoil were directly related to changes in microbial diversity and community composition, which were affected by soil pH. Collectively our results provide compelling evidence that microbial biodiversity plays an important role in stabilizing SOC decomposition in the subsoil of alpine montane ecosystems. Conservation of belowground biodiversity is therefore of great importance in maintaining the stability of ecosystem processes under climate change in high-elevation regions of the Tibetan Plateau.

Syndromes of production in intercropping impact yield gains.

Intercropping, the simultaneous production of multiple crops on the same field, provides opportunities for the sustainable intensification of agriculture if it can provide a greater yield per unit land and fertilizer than sole crops. The worldwide absolute yield gain of intercropping as compared with sole crops has not been analysed. We therefore performed a global meta-analysis to quantify the effect of intercropping on the yield gain, exploring the effects of crop species combinations, temporal and spatial arrangements, and fertilizer input. We found that the absolute yield gains, compared with monocultures, were the greatest for mixtures of maize with short-grain cereals or legumes that had substantial temporal niche differentiation from maize, when grown with high nutrient inputs, and using multirow strips of each species. This approach, commonly practised in China, provided yield gains that were (in an absolute sense) about four times as large as those in another, low-input intercropping strategy, commonly practised outside China. The alternative intercropping strategy consisted of growing mixtures of short-stature crop species, often as full mixtures, with the same growing period and with low to moderate nutrient inputs. Both the low- and high-yield intercropping strategies saved 16-29% of the land and 19-36% of the fertilizer compared with monocultures grown under the same management as the intercrop. The two syndromes of production in intercropping uncovered by this meta-analysis show that intercropping offers opportunities for the sustainable intensification of both high- and low-input agriculture.