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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.

Tropical ecosystems have an important role in global change scenarios, in part because they serve as a large terrestrial carbon pool. Carbon protection is mediated by soil aggregation processes, whereby biotic and abiotic factors influence the formation and stability of aggregates. Nutrient additions may affect soil structure indirectly by simultaneous shifts in biotic factors, mainly roots, and fungal hyphae, but also via impacts on abiotic soil properties. Here, we tested the hypothesis that soil aggregation will be affected by nutrient additions primarily via changes in arbuscular mycorrhizal fungal (AMF) hyphae and root length in a pristine tropical forest system. Therefore, the percentage of water-stable macroaggregates (> 250 μm) (WSA) and the soil mean weight diameter (MWD) was analyzed, as well as nutrient contents, pH, root length, and AMF abundance. Phosphorus additions significantly increased the amount of WSA, which was consistent across two different sampling times. Despite a positive effect of phosphorus additions on extra-radical AMF biomass, no relationship between WSA and extra-radical AMF nor roots was revealed by regression analyses, contrary to the proposed hypothesis. These findings emphasize the importance of analyzing soil structure in understudied tropical systems, since it might be affected by increasing nutrient deposition expected in the future.

Tropical rainforests – characterized by an exceptional biodiversity, important for the regulation of climatic cycles and storing an estimate of 30% of terrestrial carbon – are severely endangered by human impacts. Increased nutrient depositions expected in the future by anthropogenic activities might affect these ecosystems primarily limited by nitrogen (N) and phosphorus (P). In Southern Ecuador consequences of these effects on a tropical montane forest are evaluated in the framework of a nutrient manipulation experiment. As part of this project we evaluated effects of moderate N and P additions on an important organism group involved in plant nutrient uptake – the arbuscular mycorrhizal fungi (AMF). Though AMF are known to represent the dominant mycorrhizal form in tropical forests, only few studies on AMF ecology were conducted in this diverse system. The main function of AMF represents increased nutrient uptake for the plant mainly of P, but also of N and other nutrients. Thus, since plants invest their carbon towards structures that are most limited (functional equilibrium model), theory predicts that increased nutrient availability will decrease AMF abundance. Based on this model and previous work conducted in temperate areas we tested our main hypotheses that (1) AMF abundance will be reduced following nutrient amendments and (2) AMF diversity will decrease and the AMF community shift towards lineages favored by fertilized conditions. In the case of AMF abundance we analyzed a time series from the beginning of the experiment up to five years of constant nutrient applications. Furthermore, effects were tested along an altitudinal gradient (1000 – 3000m above seal level) which is characterized by decreasing nutrient availability and a shift from N/P- towards N-limitation with elevation. For molecular analyses, after two years of nutrient applications the AMF community was analyzed in-depth by 454-pyrosequencing at 2000m. As part of this experiment, we aimed to characterize AMF in more detail in this tropical montane forest. Here, we focused on AMF distribution in the organic soil layer, since the dominance of AMF in such soils is contrary to common theories on AMF occurrence. Generally, we found some interesting patterns associated to this study site in AMF morphology and diversity, which we confirmed to be the dominant mycorrhizal form. Along the altitudinal gradient, as predicted by the functional equilibrium model, root and AMF abundance increased in parallel with decreasing nutrient availability. A more detailed molecular analysis at 2000m revealed a diverse community (74 operational taxonomic units) with a large proportion of Diversisporales. At this site, morphological analyses revealed a close association of AMF hyphae with barely decomposed plant material in the organic layer, though a great amount of hyphae was also detected on root surfaces. Within the nutrient manipulation experiment and contrary to our first hypothesis, results show an overall positive effect of P additions on both, extra- and intraradical AMF biomass. However, N additions decreased AMF abundance. These effects were most pronounced at the intermediate site (2000m). This pattern is unusual and might be explained either by a predominant role of AMF in N uptake in this system or direct P-limitation of the fungus itself. Regarding AMF diversity, we observed a significant richness decrease in all treatments, with the strongest effect by concurrent application of N and P. Interestingly, Diversisporales and Glomerales were differentially affected by N and P, respectively, potentially pointing out group-specific traits. The findings presented here reveal insights in interesting patterns in AMF and their interrelations in this tropical montane forest, a biome where detailed knowledge on AMF is scarce. Direct conclusions on AMF functionality cannot be based solely on these observational results, but they allow the development of specific experimental designs for future studies. Nevertheless, the observed results indicate impacts of future nutrient depositions expected for the study area on the abundance and diversity of AMF, an important group influencing whole ecosystem processes., Tropische Regenwälder – gekennzeichnet von außergewöhnlicher Biodiversität, wichtig für die Regulierung von Klimazyklen, Speicher von geschätzten 30 % des terrestrischen Kohlenstoffs – sind gefährdet durch menschliches Eingreifen. Zukünftige erhöhte Nährstoffdepositionen, verursacht durch anthropogene Aktivitäten könnten diese Ökosysteme, welche hauptsächlich durch Stickstoff (N) und auch Phosphor (P) limitiert werden nachhaltig verändern. Im Süden Ecuadors werden im Rahmen eines NährstoffManipulationsexperimentes die Auswirkungen dieser Nährstoffdepositionen auf einen tropischen Bergregenwald getestet. Als Teil dieses Projektes haben wir den Einfluss von moderaten N und P Zugaben auf eine wichtige Organismengruppe untersucht, welche stark in die pflanzliche Nährstoffaufnahme involviert ist – die arbuskulären Mykorrhizapilze (AMF). Obwohl es bekannt ist, dass AMF die dominante Mykorrhizaform in tropischen Regenwäldern darstellen, gibt es nur wenige Studien zur Ökologie dieser Pilze in diesem diversen Ökosystem. Die Hauptfunktion von AMF für die Pflanze ist die verbesserte Nährstoffaufnahme, hauptsächlich von P, jedoch auch von N und anderen Nährstoffen. In Anbetracht der Grundlage, dass Pflanzen ihren Kohlenstoff an Strukturen verteilen welche am stärksten limitiert sind („Functional equilibrium model“), besagt die Theorie, dass erhöhte Nährstoffverfügbarkeit die AMF-Abundanz verringern wird. Basierend auf diesem Modell und vorherigen Ergebnissen aus temperaten Gebieten haben wir vorrangig die folgenden Hypothesen getestet: (1) AMF Abundanz wird in Folge von Nährstoffzugaben reduziert; (2) AMF-Diversität wird geringer und die Zusammensetzung der AMF Lebensgemeinschaft verschiebt sich zugunsten von Arten/Gruppen, welche durch die veränderten Bedingungen begünstigt werden. Basierend auf einer Zeitreihe von 5 Jahren ab Beginn des Experimentes wurden Untersuchungen zur AMF-Abundanz durchgeführt. Außerdem wurden Proben entlang eines Höhengradienten genommen (1000 – 3000 m ü. d. M.), welcher durch abnehmende Nährstoffverfügbarkeit parallel zu einer Verschiebung der Pflanzenlimitierung von P- bzw. N/P- hin zu N-Limitierung charakterisiert ist. Für die molekulare Analyse der AMF Lebensgemeinschaft auf 2000 m wurde nach zwei Jahren kontinuierlicher Nährstoffzugaben die detaillierte Methode der 454-Pyrosequenzierung angewandt. Weiterhin war es Teil dieses Projektes AMF im Allgemeinen in diesem besonderen System des tropischen Bergregenwaldes besser zu verstehen. Dafür lag der Fokus auf der AMF-Anpassung an die organische Auflage im Boden, da die Dominanz von AMF in solchen Böden früheren Theorien zum AMF-Vorkommen widerspricht. Zusammenfassung Insgesamt konnten einige interessante Muster bezüglich der AMF-Morphologie und -Diversität im Zusammenhang mit dem Untersuchungsgebiet gefunden werden. Außerdem wurden AMF als vorherrschende Mykorrhizaform im Gebiet bestätigt. Im Einklang mit dem „functional equilibrium model“, nimmt die Wurzellänge sowie die AMF-Abundanz parallel zur abnehmenden Nährstoffverfügbarkeit entlang des Höhengradienten zu. Eine detaillierte molekulare Analyse auf 2000 m offenbarte eine diverse Lebensgemeinschaft (74 „operational taxonomic units“) mit einem großen Anteil an Diversisporales. Auf der gleichen Höhenstufe zeigte die morphologische Untersuchung des extraradikalen AMF-Mycels eine enge Assoziation der Hyphen mit im Abbauprozess befindlichem Pflanzenmaterial in der organischen Auflage, obwohl ein sehr großer Anteil an Hyphen auch auf der Wurzeloberfläche lokalisiert ist. Im Gegensatz zu unserer ersten Hypothese, zeigten die Ergebnisse des NährstoffManipulationsexperiments einen insgesamt positiven Effekt von P-Zugaben sowohl auf extra- als auch auf intraradikale AMF- Abundanz. Im Gegensatz dazu verringerten N-Zugaben die AMF-Abundanz eher. Die gefundenen Effekte waren auf der mittleren Höhenstufe (2000 m) am stärksten ausgeprägt. Dieses Muster ist ungewöhnlich und könnte entweder durch eine vorherrschende Rolle von AMF in der N-Aufnahme in diesem System, oder durch direkte P-Limitierung der Pilze selbst begründet werden. In Bezug auf die AMF- Diversität wurde in Folge aller Nährstoffbehandlungen ein signifikantes Absinken der Artenzahl beobachtet, mit dem stärksten Effekt bei gleichzeitiger Applikation von N und P. Interessanterweise wurden Diversisporales und Glomerales jeweils von N und P unterschiedlich beeinflusst, was auf bestimmte gruppenspezifische Eigenschaften hindeutet. Zusammengenommen geben diese Ergebnisse Einblick in interessante Eigenschaften von AMF und ihren Wechselbeziehungen in diesem tropischen Bergregenwald, einem Biom für welches es an Detailwissen zu AMF noch mangelt. Direkte Schlussfolgerungen zur AMFFunktionalität können alleinig basierend auf diesen Ergebnissen nicht getroffen werden, jedoch erlauben sie die Entwicklung von spezifischen experimentellen Designs für zukünftige Studien. Nichtsdestotrotz deuten die beobachteten Effekte daraufhin, dass zukünftige Nährstoffdepositionen, welche im Untersuchungsgebiet erwartet werden, Einfluss auf die Abundanz und Diversität von AMF haben werden, einer wichtigen Organismengruppe mit Einfluss auf ganze Ökosystemprozesse.

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.

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.

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.

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 functionality