Fungal response to abruptly or gradually delivered antifungal agent amphotericin B is growth stage dependent

Anthropogenic disturbances pose a multitude of novel challenges to ecosystems. While many experiments have tested effects using abrupt treatment applications, most environmental changes in fact are gradual. Since ecosystem responses might be highly dependent on the temporal nature of stressors, it is crucial to differentiate the effects of abrupt vs gradual treatment application. Antifungal agents, which are widely used in disease control both for humans and in agriculture, are becoming a new class of environmental contaminants. In this study, we examined the effect of a sub-lethal application of one antifungal agent, amphotericin B. We applied different rates of delivery, e.g. gradual and abrupt, and monitored biomass and sporulation of the model fungus Neurospora crassa in a batch culture. Our results demonstrate that: (i) the effect size difference between abrupt and gradual treatments is fungal growth stage dependent and (ii) the gradual treatment clearly had a higher sporulation level compared with all types of abrupt treatments. Our findings highlight the importance of considering the rate of change in environmental change research and point to a new research direction for future global change studies. Furthermore, our results also have important implications for avoiding treatment-induced spore production in agriculture and medical practise

Rapid evolution of trait correlation networks during bacterial adaptation to the rhizosphere

There is a growing awareness that traits do not evolve individually but rather are organized as modular networks of covarying traits. Although the importance of multi-trait correlation has been linked to the ability to evolve in response to new environmental conditions, the evolvability of the network itself has to date rarely been assessed experimentally. By following the evolutionary dynamics of a model bacterium adapting to plant roots, we demonstrate that the whole structure of the trait correlation network is highly dynamic. We experimentally evolved Pseudomonas protegens, a common rhizosphere dweller, on the roots of Arabidopsis thaliana. We collected bacteria at regular intervals and determined a range of traits linked to growth, stress resistance, and biotic interactions. We observed a rapid disintegration of the original trait correlation network. Ancestral populations showed a modular network, with the traits linked to resource use and stress resistance forming two largely independent modules. This network rapidly was restructured during adaptation, with a loss of the stress resistance module and the appearance of new modules out of previously disconnected traits. These results show that evolutionary dynamics can involve a deep restructuring of phenotypic trait organization, pointing to the emergence of novel life history strategies not represented in the ancestral phenotype

Combined application of up to ten pesticides decreases key soil processes

Natural systems are under increasing pressure by a range of anthropogenic global change factors. Pesticides represent a nearly ubiquitously occurring global change factor and have the potential to affect soil functions. Currently the use of synthetic pesticides is at an all-time high with over 400 active ingredients being utilized in the EU alone, with dozens of these pesticides occurring concurrently in soil. However, we presently do not understand the impacts of the potential interaction of multiple pesticides when applied simultaneously. Using soil collected from a local grassland, we utilize soil microcosms to examine the role of both rate of change and number of a selection of ten currently used pesticides on soil processes, including litter decomposition, water stable aggregates, aggregate size, soil pH, and EC. Additionally, we used null models to enrich our analyses to examine potential patterns caused by interactions between pesticide treatments. We find that both gradual and abrupt pesticide application have negative consequences for soil processes. Notably, pesticide number plays a significant role in affecting soil health. Null models also reveal potential synergistic behavior between pesticides which can further their consequences on soil processes. Our research highlights the complex impacts of pesticides, and the need for environmental policy to address the threats posed by pesticides

Plant-microbe eco-evolutionary dynamics in a changing world

Both plants and their associated microbiomes can respond strongly to anthropogenic environmental changes. These responses can be both ecological (e.g. a global change affecting plant demography or microbial community composition) and evolutionary (e.g. a global change altering natural selection on plant or microbial populations). As a result, global changes can catalyse eco-evolutionary feedbacks. Here, we take a plant-focused perspective to discuss how microbes mediate plant ecological responses to global change and how these ecological effects can influence plant evolutionary response to global change. We argue that the strong and functionally important relationships between plants and their associated microbes are particularly likely to result in eco-evolutionary feedbacks when perturbed by global changes and discuss how improved understanding of plant-microbe eco-evolutionary dynamics could inform conservation or even agriculture.</p

Functionality of root-associated bacteria along a salt marsh primary succession

Plant-associated bacteria are known for their high functional trait diversity, from which many are likely to play a role in primary and secondary succession, facilitating plant establishment in suboptimal soils conditions. Here we used an undisturbed salt marsh chronosequence that represents over 100 years of soil development to assess how the functional traits of plant associated bacteria respond to soil type, plant species and plant compartment. We isolated and characterized 808 bacterial colonies from the rhizosphere soil and root endosphere of two salt marsh plants, Limonium vulgare and Artemisia maritima, along the chronosequence. From these, a set of 59 strains (with unique BOX-PCR patterns, 16S rRNA sequence and unique to one of the treatments) were further screened for their plant growth promoting traits (siderophore production, IAA production, exoprotease production and biofilm formation), traits associated with bacterial fitness (antibiotic and abiotic stress resistance – pH, osmotic and oxidative stress, and salinity) and metabolic potential. An overall view of functional diversity (multivariate analysis) indicated that the distributional pattern of bacterial functional traits was driven by soil type. Samples from the late succession (Stage 105 year) showed the most restricted distribution, harboring strains with relatively low functionalities, whereas the isolates from intermediate stage (35 year) showed a broad functional profiles. However, strains with high trait performance were largely from stage 65 year. Grouping the traits according to category revealed that the functionality of plant endophytes did not vary along the succession, thus being driven by plant rather than soil type. In opposition, the functionality of rhizosphere isolates responded strongly to variations in soil type as observed for antibiotic resistance (P = 0.014). Specifically, certain Pseudomonas sp. and Serratia sp. strains revealed high resistance against abiotic stress and antibiotics and produce more siderophores, confirming the high plant-growth promoting activity of these two genera. Overall, this study contributes to a better understanding of the functional diversity and adaptation of the microbiome at typical salt marsh plant species across soil types. Specifically, soil type was influential only in the rhizosphere but not on the endosphere, indicating a strong plant-driven effect on the functionality of endophytes

Myristate and the ecology of AM fungi : significance, opportunities, applications and challenges

A recent study by Sugiura and coworkers reported the nonsymbiotic growth and spore production of an arbuscular mycorrhizal (AM) fungus, Rhizophagus irregularis, when the fungus received an external supply of certain fatty acids, myristates (C:14). This discovery follows the insight that AM fungi receive fatty acids from their hosts when in symbiosis. If this result holds up and can be repeated under nonsterile conditions and with a broader range of fungi, it has numerous consequences for our understanding of AM fungal ecology, from the level of the fungus, at the plant community level, and to functional consequences in ecosystems. In addition, myristate may open up several avenues from a more applied perspective, including improved fungal culture and supplementation of AM fungi or inoculum in the field. We here map these potential opportunities, and additionally offer thoughts on potential risks of this potentially new technology. Lastly, we discuss the specific research challenges that need to be overcome to come to an understanding of the potential role of myristate in AM ecology

Experimental evolution of mutualistic plant-microbe interactions in the rhizosphere

We are living on a hungry planet and securing food supply for the steadily increasing human population is a major challenge for mankind. The productivity of agricultural and horticultural crops is constrained by lack of nutrients, abiotic stressors as well as pests and diseases. Together, all these limitations substantially prevent the accomplishment of the full genetic potential of plants for growth and fitness. The use of pesticides and chemical fertilizers to alleviate restrictions on plant performance causes serious environmental problems. Plant growth and health depend to a large extent on soil microbes that are associated with plant roots. These microbes can supply the plant with nutrients, alleviate effects of abiotic stresses, and protect against pathogens and pests. Thus such beneficial microorganisms may be essential allies to improve crop yields in a sustainable way. However, a major problem for large scale and long term applications in agriculture is that the efficacy of such beneficial microbes is unpredictable, often resulting from insufficient population densities on plant roots. In this study I provide evidence that we can use an evolutionary framework to engineer beneficial microbes to perform better in novel host rhizospheres. I attempted to obtain better colonizers by introducing them on plant roots and allowing the population to grow and evolve over many generations during a period of 8 months. In this simplified experimental evolution setup, we used five independently evolving bacterial populations. We then tracked population densities on plant roots at each cycle, and passed bacteria to a new plant for 8 cycles in total. At the end of each growth cycle bacterial colonies were picked and characterized for a wide range of traits. The results in chapter 2 reveal that plants can domesticate root-associated bacteria. Initially the bacteria had a detrimental effect on plant performance, but within a few generations, mutualists that promote plant growth accumulated in the bacterial population. In chapter 3 changes in the evolving bacterial populations were tracked by sequencing the genomes of bacterial colonies that were randomly picked at the end of different plant growth cycles. Mutations that accumulated in parallel in the independently evolving populations targeted global regulators and bacterial cell surface structures. This suggests that there are different strategies of bacterial adaptation to the plant root environment. Networks of co-varying bacterial traits were the focus of the last experimental chapter. Rather than traits evolving individually, trait co-variation has been linked to the ability to rapidly evolve to adapt to new conditions. In chapter 4 it is shown that whereas the network of traits linked to growth, stress resistance and biotic interactions was modular in the ancestral bacterial population, it rapidly restructured during adaptation to the rhizosphere. The most important knowledge we obtained from this work is that plants have the ability to breed their associated microbiome. This may explain the prevalence of beneficial plant-microbe interactions in nature. This study sets the stage for evolutionary microbiome management by steering the evolution of mutualism out of the existing species pool instead of changing species composition

Experimental evolution of mutualistic plant-microbe interactions in the rhizosphere

We are living on a hungry planet and securing food supply for the steadily increasing human population is a major challenge for mankind. The productivity of agricultural and horticultural crops is constrained by lack of nutrients, abiotic stressors as well as pests and diseases. Together, all these limitations substantially prevent the accomplishment of the full genetic potential of plants for growth and fitness. The use of pesticides and chemical fertilizers to alleviate restrictions on plant performance causes serious environmental problems. Plant growth and health depend to a large extent on soil microbes that are associated with plant roots. These microbes can supply the plant with nutrients, alleviate effects of abiotic stresses, and protect against pathogens and pests. Thus such beneficial microorganisms may be essential allies to improve crop yields in a sustainable way. However, a major problem for large scale and long term applications in agriculture is that the efficacy of such beneficial microbes is unpredictable, often resulting from insufficient population densities on plant roots. In this study I provide evidence that we can use an evolutionary framework to engineer beneficial microbes to perform better in novel host rhizospheres. I attempted to obtain better colonizers by introducing them on plant roots and allowing the population to grow and evolve over many generations during a period of 8 months. In this simplified experimental evolution setup, we used five independently evolving bacterial populations. We then tracked population densities on plant roots at each cycle, and passed bacteria to a new plant for 8 cycles in total. At the end of each growth cycle bacterial colonies were picked and characterized for a wide range of traits. The results in chapter 2 reveal that plants can domesticate root-associated bacteria. Initially the bacteria had a detrimental effect on plant performance, but within a few generations, mutualists that promote plant growth accumulated in the bacterial population. In chapter 3 changes in the evolving bacterial populations were tracked by sequencing the genomes of bacterial colonies that were randomly picked at the end of different plant growth cycles. Mutations that accumulated in parallel in the independently evolving populations targeted global regulators and bacterial cell surface structures. This suggests that there are different strategies of bacterial adaptation to the plant root environment. Networks of co-varying bacterial traits were the focus of the last experimental chapter. Rather than traits evolving individually, trait co-variation has been linked to the ability to rapidly evolve to adapt to new conditions. In chapter 4 it is shown that whereas the network of traits linked to growth, stress resistance and biotic interactions was modular in the ancestral bacterial population, it rapidly restructured during adaptation to the rhizosphere. The most important knowledge we obtained from this work is that plants have the ability to breed their associated microbiome. This may explain the prevalence of beneficial plant-microbe interactions in nature. This study sets the stage for evolutionary microbiome management by steering the evolution of mutualism out of the existing species pool instead of changing species composition

Functionality of Root-Associated Bacteria along a Salt Marsh Primary Succession

Plant-associated bacteria are known for their high functional trait diversity, from which many are likely to play a role in primary and secondary succession, facilitating plant establishment in suboptimal soils conditions. Here we used an undisturbed salt marsh chronosequence that represents over 100 years of soil development to assess how the functional traits of plant associated bacteria respond to soil type, plant species and plant compartment. We isolated and characterized 808 bacterial colonies from the rhizosphere soil and root endosphere of two salt marsh plants, Limonium vulgare and Artemisia maritima, along the chronosequence. From these, a set of 59 strains (with unique BOX-PCR patterns, 16S rRNA sequence and unique to one of the treatments) were further screened for their plant growth promoting traits (siderophore production, IAA production, exoprotease production and biofilm formation), traits associated with bacterial fitness (antibiotic and abiotic stress resistance - pH, osmotic and oxidative stress, and salinity) and metabolic potential. An overall view of functional diversity (multivariate analysis) indicated that the distributional pattern of bacterial functional traits was driven by soil type. Samples from the late succession (Stage 105 year) showed the most restricted distribution, harboring strains with relatively low functionalities, whereas the isolates from intermediate stage (35 year) showed a broad functional profiles. However, strains with high trait performance were largely from stage 65 year. Grouping the traits according to category revealed that the functionality of plant endophytes did not vary along the succession, thus being driven by plant rather than soil type. In opposition, the functionality of rhizosphere isolates responded strongly to variations in soil type as observed for antibiotic resistance (P = 0.014). Specifically, certain Pseudomonas sp. and Serratia sp. strains revealed high resistance against abiotic stress and antibiotics and produce more siderophores, confirming the high plant-growth promoting activity of these two genera. Overall, this study contributes to a better understanding of the functional diversity and adaptation of the microbiome at typical salt marsh plant species across soil types. Specifically, soil type was influential only in the rhizosphere but not on the endosphere, indicating a strong plant-driven effect on the functionality of endophytes

Rapid evolution of trait correlation networks during bacterial adaptation to the rhizosphere

There is a growing awareness that traits do not evolve individually but rather are organized as modular networks of covarying traits. Although the importance of multi-trait correlation has been linked to the ability to evolve in response to new environmental conditions, the evolvability of the network itself has to date rarely been assessed experimentally. By following the evolutionary dynamics of a model bacterium adapting to plant roots, we demonstrate that the whole structure of the trait correlation network is highly dynamic. We experimentally evolved Pseudomonas protegens, a common rhizosphere dweller, on the roots of Arabidopsis thaliana. We collected bacteria at regular intervals and determined a range of traits linked to growth, stress resistance, and biotic interactions. We observed a rapid disintegration of the original trait correlation network. Ancestral populations showed a modular network, with the traits linked to resource use and stress resistance forming two largely independent modules. This network rapidly was restructured during adaptation, with a loss of the stress resistance module and the appearance of new modules out of previously disconnected traits. These results show that evolutionary dynamics can involve a deep restructuring of phenotypic trait organization, pointing to the emergence of novel life history strategies not represented in the ancestral phenotype