Enhancement of soil aggregation and physical properties through fungal amendments under varying moisture conditions.

Soil structure and aggregation are crucial for soil functionality, particularly under drought conditions. Saprobic soil fungi, known for their resilience in low moisture conditions, are recognized for their influence on soil aggregate dynamics. In this study, we explored the potential of fungal amendments to enhance soil aggregation and hydrological properties across different moisture regimes. We used a selection of 29 fungal isolates, recovered from soils treated under drought conditions and varying in colony density and growth rate, for single-strain inoculation into sterilized soil microcosms under either low or high moisture (≤-0.96 and -0.03 MPa, respectively). After 8 weeks, we assessed soil aggregate formation and stability, along with soil properties such as soil water content, water hydrophobicity, sorptivity, total fungal biomass and water potential. Our findings indicate that fungal inoculation altered soil hydrological properties and improved soil aggregation, with effects varying based on the fungal strains and soil moisture levels. We found a positive correlation between fungal biomass and enhanced soil aggregate formation and stabilization, achieved by connecting soil particles via hyphae and modifying soil aggregate sorptivity. The improvement in soil water potential was observed only when the initial moisture level was not critical for fungal activity. Overall, our results highlight the potential of using fungal inoculation to improve the structure of agricultural soil under drought conditions, thereby introducing new possibilities for soil management in the context of climate change.

Visualising long distance sugar transport in fungi using infrared fluorescence scanning imaging.

Mycelia of saprotrophic basidiomycetes can cover large areas in nature that are typified by their heterogeneous nutrient availability. This heterogeneity is overcome by long distance transport of nutrients within the hyphal network to sites where they are needed. It is therefore key to be able to study nutrient transport and its underlying mechanisms. An IRDye-conjugate was used for the first time for imaging transport in fungi. A method was set up for time-lapse, high spatial resolution infrared imaging of IRDye-labelled deoxyglucose (IRDye-DG) in Schizophyllum commune and Agaricus bisporus. Scanning imaging visualised the tracer in individual hyphae as well as deeper tissues in mushrooms (mm-cm depth). The advantage of using fluorescence scanning imaging of IRDye in contrast to radiolabelled tracers studies, is that a higher spatial resolution and higher sensitivity (244 fg/ml) can be obtained. Moreover, it has a large field of view (25 × 25 cm) compared to microscopy (µm-mm range), allowing relatively fast and detailed imaging of large dimension samples.

Cycling in degradation of organic polymers and uptake of nutrients by a litter-degrading fungus.

Wood and litter degrading fungi are the main decomposers of lignocellulose and thus play a key role in carbon cycling in nature. Here, we provide evidence for a novel lignocellulose degradation strategy employed by the litter degrading fungus Agaricus bisporus (known as the white button mushroom). Fusion of hyphae allows this fungus to synchronize the activity of its mycelium over large distances (50 cm). The synchronized activity has a 13-h interval that increases to 20 h before becoming irregular and it is associated with a 3.5-fold increase in respiration, while compost temperature increases up to 2°C. Transcriptomic analysis of this burst-like phenomenon supports a cyclic degradation of lignin, deconstruction of (hemi-) cellulose and microbial cell wall polymers, and uptake of degradation products during vegetative growth of A. bisporus. Cycling in expression of the ligninolytic system, of enzymes involved in saccharification, and of proteins involved in nutrient uptake is proposed to provide an efficient way for degradation of substrates such as litter.

Subpopulations of hyphae secrete proteins or resist heat stress in Aspergillus oryzae colonies.

Hyphae at the outer part of colonies of Aspergillus niger and Aspergillus oryzae are heterogeneous with respect to transcriptional and translational activity. This heterogeneity is maintained by Woronin body mediated closure of septal pores that block interhyphal mixing of cytoplasm. Indeed, heterogeneity between hyphae is abolished in ΔhexA strains that lack Woronin bodies. The subpopulation of hyphae with high transcriptional and translational activity secretes enzymes that degrade the substrate resulting in breakdown products that serve as nutrients. The role of hyphae with low transcriptional and translational activity was not yet known. Here, we show that this subpopulation is more resistant to environmental stress in A. oryzae, in particular to temperature stress, when compared to hyphae with high transcriptional and translational activity. Notably, all hyphae of the ΔhexA strain of A. oryzae were sensitive to heat stress explained by the reduced heterogeneity in this strain. Together, we show that different subpopulations of hypha secrete proteins and resist heat stress showing the complexity of a fungal mycelium.

Selective transport between heterogeneous hyphal compartments via the plasma membrane lining septal walls of Aspergillus niger.

Hyphae of ascomycetes are compartmentalized by septa. The central pore in these septa allows for cytoplasmic streaming. However, many of these pores are closed by Woronin bodies in Aspergillus, which prevents cytoplasmic mixing and thus maintains hyphal heterogeneity. Here, glucose uptake and transport was studied in Aspergillus niger. Glucose uptake was higher in the hyphal population with high transcriptional activity when compared to the population with low transcriptional activity. Glucose was transported from the colony center to the periphery, but not vice versa. This unidirectional flow was similar in the wild-type and the ΔhexA strain that does not form Woronin bodies. This indicated that septal plugging by Woronin bodies does not impact long distance glucose transport. Indeed, the glucose analogue 2-NBDG (2-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino)-2-deoxyglucose) translocated to neighboring hyphal compartments despite Woronin body mediated plugging of the septum that separated these compartments. Notably, 2-NBDG accumulated in septal cross walls, indicating that intercompartmental glucose transport is mediated by transporters that reside in the plasma membrane lining the septal cross-wall. The presence of such transporters would thus enable selective transport between heterogeneous compartments.

VeA of Aspergillus niger increases spore dispersing capacity by impacting conidiophore architecture.

Aspergillus species are highly abundant fungi worldwide. Their conidia are among the most dominant fungal spores in the air. Conidia are formed in chains on the vesicle of the asexual reproductive structure called the conidiophore. Here, it is shown that the velvet protein VeA of Aspergillus niger maximizes the diameter of the vesicle and the spore chain length. The length and width of the conidiophore stalk and vesicle were reduced nearly twofold in a ΔveA strain. The latter implies a fourfold reduced surface area to develop chains of spores. Over and above this, the conidial chain length was approximately fivefold reduced. The calculated 20-fold reduction in formation of conidia by ΔveA fits the 8- to 17-fold decrease in counted spore numbers. Notably, morphology of the ΔveA conidiophores of A. niger was very similar to that of wild-type Aspergillus sydowii. This suggests that VeA is key in conidiophore architecture diversity in the fungal kingdom. The finding that biomass formation of the A. niger ΔveA strain was reduced twofold shows that VeA not only impacts dispersion capacity but also colonization capacity of A. niger.

Hyphal heterogeneity in Aspergillus oryzae is the result of dynamic closure of septa by Woronin bodies.

Hyphae of higher fungi are compartmentalized by septa. These septa contain a central pore that allows for inter-compartmental and inter-hyphal cytoplasmic streaming. The cytoplasm within the mycelium is therefore considered to be a continuous system. In this study, however, we demonstrate by laser dissection that 40% of the apical septa of exploring hyphae of Aspergillus oryzae are closed. Closure of septa correlated with the presence of a peroxisome-derived organelle, known as Woronin body, near the septal pore. The location of Woronin bodies in the hyphae was dynamic and, as a result, plugging of the septal pore was reversible. Septal plugging was abolished in a ΔAohex1 strain that cannot form Woronin bodies. Notably, hyphal heterogeneity was also affected in the ΔAohex1 strain. Wild-type strains of A. oryzae showed heterogeneous distribution of GFP between neighbouring hyphae at the outer part of the colony when the reporter was expressed from the promoter of the glucoamylase gene glaA or the α-glucuronidase gene aguA. In contrast, GFP fluorescence showed a normal distribution in the case of the ΔAohex1 strain. Taken together, it is concluded that Woronin bodies maintain hyphal heterogeneity in a fungal mycelium by impeding cytoplasmic continuity.

Heterogeneity in Spore Aggregation and Germination Results in Different Sized, Cooperative Microcolonies in an Aspergillus niger Culture.

The fungus Aspergillus niger is among the most abundant fungi in the world and is widely used as a cell factory for protein and metabolite production. This fungus forms asexual spores called conidia that are used for dispersal. Notably, part of the spores and germlings aggregate in an aqueous environment. The aggregated conidia/germlings give rise to large microcolonies, while the nonaggregated spores/germlings result in small microcolonies. Here, it is shown that small microcolonies release a larger variety and quantity of secreted proteins compared to large microcolonies. Yet, the secretome of large microcolonies has complementary cellulase activity with that of the small microcolonies. Also, large microcolonies are more resistant to heat and oxidative stress compared to small microcolonies, which is partly explained by the presence of nongerminated spores in the core of the large microcolonies. Together, it is proposed that heterogeneity in germination and aggregation has evolved to form a population of different sized A. niger microcolonies, thereby increasing stress survival and producing a meta-secretome more optimally suited to degrade complex substrates. IMPORTANCE Aspergillus niger can form microcolonies of different size due to partial aggregation of spores and germlings. So far, this heterogeneity was considered a negative trait by the industry. We here, however, show that heterogeneity in size within a population of microcolonies is beneficial for food degradation and stress survival. This functional heterogeneity is not only of interest for the industry to make blends of enzymes (e.g., for biofuel or bioplastic production) but could also play a role in nature for effective nutrient cycling and survival of the fungus.

Single Cell Analysis and Sorting of Aspergillus fumigatus by Flow Cytometry.

Experimental results in fungal biology research are usually obtained as average measurements across whole populations of cells, whilst ignoring what is happening at the single cell level. Microscopy has allowed us to study single-cell behavior, but it has low throughput and cannot be used to select individual cells for downstream experiments. Here we present a method that allows for the analysis and selection of single fungal cells in high throughput by flow cytometry and fluorescence activated cell sorting (FACS), respectively. This protocol can be adapted for every fungal species that produces cells of up to 70 microns in diameter. After initial setting of the flow cytometry gates, which takes a single day, accurate single cell analysis and sorting can be performed. This method yields a throughput of thousands of cells per second. Selected cells can be subjected to downstream experiments to study single-cell behavior.

Growth induced translocation effectively directs an amino acid analogue to developing zones in Agaricus bisporus.

The vegetative mycelium of Agaricus bisporus supplies developing white button mushrooms with water and nutrients. However, it is not yet known which part of the mycelium contributes to the feeding of the mushrooms and how this depends on growth conditions. Here we used photon counting scintillation imaging to track translocation of the 14C-radiolabeled metabolically inert amino acid analogue α-aminoisobutyric acid (14C-AIB). Translocation to the periphery of the mycelium was observed in actively growing vegetative mycelium with a velocity of up to 6.6 mm h-1, which was 30-fold higher than the growth rate. Furthermore, 14C-AIB translocated to neighboring colonies after fusion by anastomosis depending on the relative growth rate in these colonies. When mushrooms started to develop, translocation of 14C-AIB was redirected to the fruiting bodies via mycelium and hyphal cords. More abundant mycelial cord formation and a 5-fold higher rate of translocation was observed for cultures growing directionally from inoculum located at one side of the substrate, when compared to non-directional growth (inoculum mixed throughout the substrate). The maximum translocation distance was also greater (≥50 and 22 cm, respectively). In conclusion, 14C-AIB translocation switches between vegetative growth and towards developing mushrooms, especially via cords and when source-sink relationships change.

Cell Wall Composition Heterogeneity between Single Cells in Aspergillus fumigatus Leads to Heterogeneous Behavior during Antifungal Treatment and Phagocytosis.

Aspergillus fumigatus can cause a variety of lung diseases in immunocompromised patients, including life-threatening invasive aspergillosis. There are only three main classes of antifungal drugs currently used to treat aspergillosis, and antifungal resistance is increasing. Experimental results in fungal biology research are usually obtained as average measurements across whole populations while ignoring what is happening at the single cell level. In this study, we show that conidia with the same genetic background in the same cell population at a similar developmental stage show heterogeneity in their cell wall labeling at the single cell level. We present a rigorous statistical method, newly applied to quantify the level of cell heterogeneity, which allows for direct comparison of the heterogeneity observed between treatments. We show the extent of cell wall labeling heterogeneity in dormant conidia and how the level of heterogeneity changes during germination. The degree of heterogeneity is influenced by deletions of cell wall synthesizing genes and environmental conditions, including medium composition, method of inoculation, age of conidia, and the presence of antifungals. This heterogeneity results in subpopulations of germinating conidia with heterogeneous fitness to the antifungal caspofungin, which targets cell wall synthesis and heterogeneous sensitivity of dormant conidia to phagocytosis by macrophages.IMPORTANCE The fungus Aspergillus fumigatus can cause invasive lung diseases in immunocompromised patients resulting in high mortality. Treatment using antifungal compounds is often unsuccessful. Average population measurements hide what is happening at the individual cell level. We set out to test what impact individual differences between the cell walls of fungal conidia have on their behavior. We show that a population of cells having the same genetic background gives rise to subpopulations of cells that exhibit distinct behavior (phenotypic heterogeneity). This cell heterogeneity is dependent on the strain type, gene deletions, cell age, and environmental conditions. By looking at the individual cell level, we discovered subpopulations of cells that show differential fitness during antifungal treatment and uptake by immune cells.

Switching from a unicellular to multicellular organization in an Aspergillus niger hypha.

UNLABELLED: Pores in fungal septa enable cytoplasmic streaming between hyphae and their compartments. Consequently, the mycelium can be considered unicellular. However, we show here that Woronin bodies close ~50% of the three most apical septa of growing hyphae of Aspergillus niger. The incidence of closure of the 9th and 10th septa was even ≥94%. Intercompartmental streaming of photoactivatable green fluorescent protein (PA-GFP) was not observed when the septa were closed, but open septa acted as a barrier, reducing the mobility rate of PA-GFP ~500 times. This mobility rate decreased with increasing septal age and under stress conditions, likely reflecting a regulatory mechanism affecting septal pore diameter. Modeling revealed that such regulation offers effective control of compound concentration between compartments. Modeling also showed that the incidence of septal closure in A. niger had an even stronger impact on cytoplasmic continuity. Cytoplasm of hyphal compartments was shown not to be in physical contact when separated by more than 4 septa. Together, data show that apical compartments of growing hyphae behave unicellularly, while older compartments have a multicellular organization. IMPORTANCE: The hyphae of higher fungi are compartmentalized by porous septa that enable cytosolic streaming. Therefore, it is believed that the mycelium shares cytoplasm. However, it is shown here that the septa of Aspergillus niger are always closed in the oldest part of the hyphae, and therefore, these compartments are physically isolated from each other. In contrast, only part of the septa is closed in the youngest part of the hyphae. Still, compartments in this hyphal part are physically isolated when separated by more than 4 septa. Even open septa act as a barrier for cytoplasmic mixing. The mobility rate through such septa reduces with increasing septal age and under stress conditions. Modeling shows that the septal pore width is set such that its regulation offers maximal control of compound concentration levels within the compartments. Together, we show for the first time that Aspergillus hyphae switch from a unicellular to multicellular organization.

Biotechnological applications and potential of fungal feruloyl esterases based on prevalence, classification and biochemical diversity.

Feruloyl esterases are part of the enzymatic spectrum employed by fungi and other microorganisms to degrade plant polysaccharides. They release ferulic acid and other aromatic acids from these polymeric structures and have received an increasing interest in industrial applications such as in the food, pulp and paper and bio-fuel industries. This review provides an overview of the current knowledge on fungal feruloyl esterases focussing in particular on the differences in substrate specificity, regulation of their production, prevalence of these enzymes in fungal genomes and industrial applications.