Sub-lethal fungicide concentrations both reduce and stimulate the growth rate of non-target soil fungi from a natural grassland

Conventional agriculture has relied extensively on the use of fungicides to prevent or control crop diseases. However, some fungicides, particularly broad-spectrum fungicides, not only eliminate target pathogens but also non-target and beneficial soil microbes. This scenario is not only limited to agricultural soil, but this may also potentially occur when neighboring environments are contaminated by fungicides through spray drift. Although concentrations may be sub-lethal, the chemicals may accumulate in the soil when used continuously resulting in more toxic effects. In this study, the effect on the colony extension rate of 31 filamentous soil saprobic fungi, initially isolated from a protected grassland ecosystem, were analyzed under fungicide treatment. These isolates were considered naive (no deliberate exposure), hence presumed to have not developed resistance. Two currently used fungicides with different modes of action were added to Potato Dextrose Agar at varying concentrations. Results showed a wide range of tolerance and sensitivity to isopyrazam and prothioconazole. Fungi belonging to the phylum Basidiomycota were most negatively affected by both fungicides. Phylum Mucoromycota were the most tolerant to prothioconazole while isolates belonging to phylum Ascomycota differed in their responses to both fungicides. Negative effects on the growth rate were more pronounced at higher concentrations except for a few isolates that were inhibited at 1 mg·L−1. A slightly positive effect was also observed in three of the isolates under fungicide treatment. Lastly, the negative impact of fungicides was not associated with the growth strategy of the fungi, whether fast growing or slow growing, rather it is isolate-specific and phylogenetically conserved. The results of this study demonstrate that co-occurring fungi differ in their sensitivity to fungicides even without prior exposure. This difference in sensitivity among co-occurring fungi may result in shifts in community composition of the soil fungal community to the detriment of the more sensitive isolates

Soil fungal mycelia have unexpectedly flexible stoichiometric C:N and C:P ratios

Soil ecological stoichiometry provides powerful theories to integrate the complex interplay of element cycling and microbial communities into biogeochemical models. One essential assumption is that microbes maintain stable C:N:P (carbon:nitrogen:phosphorus) ratios independent of resource supply, although such homeostatic regulations have rarely been assessed in individual microorganisms. Here, we report an unexpected high flexibility in C:N and C:P values of saprobic fungi along nutrient supply gradients, overall ranging between 7-126 and 20-1488, respectively, questioning microbial homeostasis. Fungal N:P varied comparatively less due to simultaneous reductions in mycelial N and P contents. As a mechanism, internal recycling processes during mycelial growth and an overall reduced N and P uptake appear more relevant than element storage. The relationships among fungal stoichiometry and growth disappeared in more complex media. These findings affect our interpretation of stoichiometric imbalances among microbes and soils and are highly relevant for developing microbial soil organic carbon and nitrogen models

Response of protists to nitrogen addition, arbuscular mycorrhizal fungi manipulation, and mesofauna reduction in a tropical montane rainforest in southern Ecuador

The tropical Andes are a species-rich and nitrogen-limited system, susceptible to increased nitrogen (N) inputs from the atmosphere. However, our understanding of the impacts of increased N input on belowground systems, in particular on protists and their role in nutrient cycling, remains limited. We explored how increased N affects protists in tropical montane rainforests in Ecuador using high-throughput sequencing (HTS) of environmental DNA from two litter layers. In addition, we manipulated the amount of arbuscular mycorrhizal fungi (AMF) and mesofauna, both playing a significant role in N cycling and interacting in complex ways with protist communities. We found that N strongly affected protist community composition in both layers, while mesofauna reduction had a stronger effect on the lower layer. Changes in concentration of the AMF marker lipid had little effect on protists. In both layers, the addition of N increased phagotrophs and animal parasites and decreased plant parasites, while mixotrophs decreased in the upper layer but increased in the lower layer. In the upper layer with higher AMF concentration, mixotrophs decreased, while in the lower layer, photoautotrophs increased and plant parasites decreased. With reduced mesofauna, phagotrophs increased and animal parasites decreased in both layers, while plant parasites increased only in the upper layer. The findings indicate that to understand the intricate response of protist communities to environmental changes, it is critical to thoroughly analyze these communities across litter and soil layers, and to include HTS

Galactosamine and mannosamine are integral parts of bacterial and fungal extracellular polymeric substances

Extracellular polymeric substances (EPS) are produced by microorganisms and interact to form a complex matrix called biofilm. In soils, EPS are important contributors to the microbial necromass and, thus, to soil organic carbon (SOC). Amino sugars (AS) are used as indicators for microbial necromass in soil, although the origin of galactosamine and mannosamine is largely unknown. However, indications exist that they are part of EPS. In this study, two bacteria and two fungi were grown in starch medium either with or without a quartz matrix to induce EPS production. Each culture was separated in two fractions: one that directly underwent AS extraction (containing AS from both biomass and EPS), and another that first had EPS extracted, followed then by AS determination (exclusively containing AS from EPS). We did not observe a general effect of the quartz matrix neither of microbial type on AS production. The quantified amounts of galactosamine and mannosamine in the EPS fraction represented on average 100% of the total amounts of these two AS quantified in cell cultures, revealing they are integral parts of the biofilm. In contrast, muramic acid and glucosamine were also quantified in the EPS, but with much lower contribution rates to total AS production, of 18% and 33%, respectively, indicating they are not necessarily part of EPS. Our results allow a meaningful ecological interpretation of mannosamine and galactosamine data in the future as indicators of microbial EPS, and also attract interest of future studies to investigate the role of EPS to SOC and its dynamics

Moderate phosphorus additions consistently affect community composition of arbuscular mycorrhizal fungi in tropical montane forests in southern Ecuador

Summary: Anthropogenic atmospheric deposition can increase nutrient supply in the most remoteecosystems, potentially affecting soil biodiversity. Arbuscular mycorrhizal fungal (AMF) com-munities rapidly respond to simulated soil eutrophication in tropical forests. Yet the limitedspatio-temporal extent of such manipulations, together with the often unrealistically high fer-tilization rates employed, impedes generalization of such responses. We sequenced mixed root AMF communities within a seven year-long fully factorial nitro-gen (N) and phosphorus (P) addition experiment, replicated at three tropical montane forestsin southern Ecuador with differing environmental characteristics. We hypothesized: strongshifts in community composition and species richness after long-term fertilization, site- andclade-specific responses to N vs P additions depending on local soil fertility and clade life his-tory traits respectively. Fertilization consistently shifted AMF community composition across sites, but only reducedrichness of Glomeraceae. Compositional changes were mainly driven by increases in P supplywhile richness reductions were observed only after combined N and P additions. We conclude that moderate increases of N and P exert a mild but consistent effect on tropi-cal AMF communities. To predict the consequences of these shifts, current results need to besupplemented with experiments that characterize local species-specific AMF functionalit

Revisiting soil fungal biomarkers and conversion factors: Interspecific variability in phospholipid fatty acids, ergosterol and rDNA copy numbers

- Refined conversion factors for soil fungal biomarkers are proposed. - High interspecific variability is present in all fungal biomarkers. - A modeling approach supports the validity of biomarker estimates in diverse soils. - ITS1 copies vary strongly, but are fungal-specific with least phylogenetic bias. - A combination of fungal biomarkers will reveal soil fungal physiology and activity. The abundances of fungi and bacteria in soil are used as simple predictors for carbon dynamics, and represent widely available microbial traits. Soil biomarkers serve as quantitative estimates of these microbial groups, though not quantifying microbial biomass per se. The accurate conversion to microbial carbon pools, and an understanding of its comparability among soils is therefore needed. We refined conversion factors for classical fungal biomarkers, and evaluated the application of quantitative PCR (qPCR, rDNA copies) as a biomarker for soil fungi. Based on biomarker contents in pure fungal cultures of 30 isolates tested here, combined with comparable published datasets, we propose average conversion factors of 95.3 g fungal C g−1 ergosterol, 32.0 mg fungal C µmol−1 PLFA 18:2ω6,9 and 0.264 pg fungal C ITS1 DNA copy−1. As expected, interspecific variability was most pronounced in rDNA copies, though qPCR results showed the least phylogenetic bias. A modeling approach based on exemplary agricultural soils further supported the hypothesis that high diversity in soil buffers against biomarker variability, whereas also phylogenetic biases impact the accuracy of comparisons in biomarker estimates. Our analyses suggest that qPCR results cover the fungal community in soil best, though with a variability only partly offset in highly diverse soils. PLFA 18:2ω6,9 and ergosterol represent accurate biomarkers to quantify Ascomycota and Basidiomycota. To conclude, the ecological interpretation and coverage of biomarker data prior to their application in global models is important, where the combination of different biomarkers may be most insightful

Revisiting soil fungal biomarkers and conversion factors: Interspecific variability in phospholipid fatty acids, ergosterol and rDNA copy numbers

- Refined conversion factors for soil fungal biomarkers are proposed. - High interspecific variability is present in all fungal biomarkers. - A modeling approach supports the validity of biomarker estimates in diverse soils. - ITS1 copies vary strongly, but are fungal-specific with least phylogenetic bias. - A combination of fungal biomarkers will reveal soil fungal physiology and activity. The abundances of fungi and bacteria in soil are used as simple predictors for carbon dynamics, and represent widely available microbial traits. Soil biomarkers serve as quantitative estimates of these microbial groups, though not quantifying microbial biomass per se. The accurate conversion to microbial carbon pools, and an understanding of its comparability among soils is therefore needed. We refined conversion factors for classical fungal biomarkers, and evaluated the application of quantitative PCR (qPCR, rDNA copies) as a biomarker for soil fungi. Based on biomarker contents in pure fungal cultures of 30 isolates tested here, combined with comparable published datasets, we propose average conversion factors of 95.3 g fungal C g−1 ergosterol, 32.0 mg fungal C µmol−1 PLFA 18:2ω6,9 and 0.264 pg fungal C ITS1 DNA copy−1. As expected, interspecific variability was most pronounced in rDNA copies, though qPCR results showed the least phylogenetic bias. A modeling approach based on exemplary agricultural soils further supported the hypothesis that high diversity in soil buffers against biomarker variability, whereas also phylogenetic biases impact the accuracy of comparisons in biomarker estimates. Our analyses suggest that qPCR results cover the fungal community in soil best, though with a variability only partly offset in highly diverse soils. PLFA 18:2ω6,9 and ergosterol represent accurate biomarkers to quantify Ascomycota and Basidiomycota. To conclude, the ecological interpretation and coverage of biomarker data prior to their application in global models is important, where the combination of different biomarkers may be most insightful

Fungal traits help to understand the decomposition of simple and complex plant litter

Litter decomposition is a key ecosystem process, relevant for the release and storage of nutrients and carbon in soil. Soil fungi are one of the dominant drivers of organic matter decomposition, but fungal taxa differ substantially in their functional ability to decompose plant litter. Knowledge is mostly based on observational data and subsequent molecular analyses and in vitro studies have been limited to forest ecosystems. In order to better understand functional traits of saprotrophic soil fungi in grassland ecosystems, we isolated 31 fungi from a natural grassland and performed several in vitro studies testing for i) leaf and wood litter decomposition, ii) the ability to use carbon sources of differing complexity, iii) the enzyme repertoire. Decomposition strongly varied among phyla and isolates, with Ascomycota decomposing the most and Mucoromycota decomposing the least. The phylogeny of the fungi and their ability to use complex carbon were the most important predictors for decomposition. Our findings show that it is crucial to understand the role of individual members and functional groups within the microbial community. This is an important way forward to understand the role of microbial community composition for the prediction of litter decomposition and subsequent potential carbon storage in grassland soils

Tropical Andean Forests Are Highly Susceptible to Nutrient Inputs - Rapid Effects of Experimental N and P Addition to an Ecuadorian Montane Forest

Tropical regions are facing increasing atmospheric inputs of nutrients, which will have unknown consequences for the structure and functioning of these systems. Here, we show that Neotropical montane rainforests respond rapidly to moderate additions of N (50 kg ha−1 yr−1) and P (10 kg ha−1 yr−1). Monitoring of nutrient fluxes demonstrated that the majority of added nutrients remained in the system, in either soil or vegetation. N and P additions led to not only an increase in foliar N and P concentrations, but also altered soil microbial biomass, standing fine root biomass, stem growth, and litterfall. The different effects suggest that trees are primarily limited by P, whereas some processes—notably aboveground productivity—are limited by both N and P. Highly variable and partly contrasting responses of different tree species suggest marked changes in species composition and diversity of these forests by nutrient inputs in the long term. The unexpectedly fast response of the ecosystem to moderate nutrient additions suggests high vulnerability of tropical montane forests to the expected increase in nutrient inputs

Trait‐based approaches reveal fungal adaptations to nutrient‐limiting conditions

The dependency of microbial activity on nutrient availability in soil is only partly understood, but highly relevant for nutrient cycling dynamics. In order to achieve more insight on microbial adaptations to nutrient limiting conditions, precise physiological knowledge is needed. Therefore, we developed an experimental system assessing traits of 16 saprobic fungal isolates in nitrogen (N) limited conditions. We tested the hypotheses that (1) fungal traits are negatively affected by N deficiency to a similar extent and (2) fungal isolates respond in a phylogenetically conserved fashion. Indeed, mycelial density, spore production and fungal activity (respiration and enzymatic activity) responded similarly to limiting conditions by an overall linear decrease. By contrast, mycelial extension and hyphal elongation peaked at lowest N supply (C:N 200), causing maximal biomass production at intermediate N contents. Optimal N supply rates differed among isolates, but only the extent of growth reduction was phylogenetically conserved. In conclusion, growth responses appeared as a switch from explorative growth in low nutrient conditions to exploitative growth in nutrient‐rich patches, as also supported by responses to phosphorus and carbon limitations. This detailed trait‐based pattern will not only improve fungal growth models, but also may facilitate interpretations of microbial responses observed in field studies