Periodic Reporting for period 1 - MYTUNETOOL (A mycorrhizal fungi tunning tool for adaptation to harsh environment)
Reporting period: 2023-10-01 to 2026-04-30
Summary of the context and overall objectives of the project
Soil contamination by heavy metals (HMs) represents a major environmental challenge worldwide, threatening ecosystem functioning, agricultural productivity, and human health. In Europe, the restoration of degraded and contaminated soils has become a strategic priority, particularly within the framework of the European Green Deal and the EU Soil Strategy for 2030, which aim to improve soil health, reduce land degradation, and promote sustainable land management. Conventional approaches for remediation of HM-contaminated soils, including soil replacement and chemical treatments, are often costly, disruptive, and difficult to apply at large spatial scales. Therefore, sustainable biological solutions that enhance natural soil restoration processes are urgently needed.
Arbuscular mycorrhizal fungi (AMF) are key soil microorganisms that form symbiotic associations with most terrestrial plants and contribute to plant nutrition, stress tolerance, and ecosystem resilience. AMF-based inoculation has significant potential for improving plant establishment and phytoremediation in degraded environments. However, currently available AMF inocula are generally derived from natural populations or long-term cultivation systems and often lack sufficient tolerance to extreme environmental stresses, limiting their effectiveness in highly contaminated soils. A major scientific bottleneck is the absence of controlled approaches to generate stress-adapted AMF strains and the limited understanding of the molecular mechanisms underlying acquired stress tolerance in these fungi.
The MYTUNETOOL project addresses this challenge by developing a novel strategy to artificially adapt AMF to harsh environments through controlled, multi-generational exposure to specific heavy metal stresses. The project aims to establish an innovative in vitro adaptation platform that enables the generation of HM-tolerant AMF strains, investigate the genetic, epigenetic, and transcriptomic mechanisms responsible for stress adaptation, and translate these adapted strains into effective biological products for soil restoration.
The project is structured along a clear pathway to impact. During the outgoing phase at the University of Lausanne (UNIL), a controlled AMF adaptation system is developed and used to generate HM-adapted fungal lines, followed by molecular analyses to identify mechanisms associated with acquired tolerance. During the return phase at the Institute of Botany of the Czech Academy of Sciences (IBOT), the ecological relevance of adapted AMF will be evaluated through plant growth experiments under HM-contaminated soil conditions. Finally, during the non-academic placement at Symvitro Ltd., the most promising adapted strains will be transferred towards scalable production, supporting their future application as commercial AMF inoculants.
By integrating fungal biotechnology, molecular biology, soil ecology, and industrial translation, MYTUNETOOL aims to establish a new paradigm for producing stress-resilient beneficial microorganisms. The expected outcomes include (i) a transferable methodology for generating AMF adapted to specific environmental stresses, (ii) fundamental insights into the mechanisms of fungal stress adaptation, and (iii) the development of improved microbial inocula for sustainable soil remediation and climate-resilient agriculture. Beyond heavy metal contamination, the developed adaptation framework has the potential to be extended to other major environmental challenges, including drought, salinity, and organic pollutants, thereby contributing to sustainable ecosystem restoration and the EU objectives for healthy soils and circular bio-based solutions.
Arbuscular mycorrhizal fungi (AMF) are key soil microorganisms that form symbiotic associations with most terrestrial plants and contribute to plant nutrition, stress tolerance, and ecosystem resilience. AMF-based inoculation has significant potential for improving plant establishment and phytoremediation in degraded environments. However, currently available AMF inocula are generally derived from natural populations or long-term cultivation systems and often lack sufficient tolerance to extreme environmental stresses, limiting their effectiveness in highly contaminated soils. A major scientific bottleneck is the absence of controlled approaches to generate stress-adapted AMF strains and the limited understanding of the molecular mechanisms underlying acquired stress tolerance in these fungi.
The MYTUNETOOL project addresses this challenge by developing a novel strategy to artificially adapt AMF to harsh environments through controlled, multi-generational exposure to specific heavy metal stresses. The project aims to establish an innovative in vitro adaptation platform that enables the generation of HM-tolerant AMF strains, investigate the genetic, epigenetic, and transcriptomic mechanisms responsible for stress adaptation, and translate these adapted strains into effective biological products for soil restoration.
The project is structured along a clear pathway to impact. During the outgoing phase at the University of Lausanne (UNIL), a controlled AMF adaptation system is developed and used to generate HM-adapted fungal lines, followed by molecular analyses to identify mechanisms associated with acquired tolerance. During the return phase at the Institute of Botany of the Czech Academy of Sciences (IBOT), the ecological relevance of adapted AMF will be evaluated through plant growth experiments under HM-contaminated soil conditions. Finally, during the non-academic placement at Symvitro Ltd., the most promising adapted strains will be transferred towards scalable production, supporting their future application as commercial AMF inoculants.
By integrating fungal biotechnology, molecular biology, soil ecology, and industrial translation, MYTUNETOOL aims to establish a new paradigm for producing stress-resilient beneficial microorganisms. The expected outcomes include (i) a transferable methodology for generating AMF adapted to specific environmental stresses, (ii) fundamental insights into the mechanisms of fungal stress adaptation, and (iii) the development of improved microbial inocula for sustainable soil remediation and climate-resilient agriculture. Beyond heavy metal contamination, the developed adaptation framework has the potential to be extended to other major environmental challenges, including drought, salinity, and organic pollutants, thereby contributing to sustainable ecosystem restoration and the EU objectives for healthy soils and circular bio-based solutions.
Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far
During the reporting period (outgoing phase, M1–M18), the project activities were primarily conducted at the University of Lausanne (UNIL), focusing on the establishment of an in vitro AMF adaptation platform, generation of heavy metal-adapted fungal lines, and preparation for downstream molecular and ecological validation.
A major achievement was the successful design, fabrication, and validation of a compartmented split-plate cultivation system for controlled AMF adaptation. This innovative system physically separates the root compartment from the hyphal compartment, allowing fungal hyphae to be directly exposed to defined heavy metal stress while maintaining plant host growth under non-contaminated conditions. The system provides a controlled and reproducible platform to drive fungal adaptation while avoiding the limitations associated with traditional soil-based selection approaches. The functionality of the system was successfully demonstrated for four target heavy metals: copper (Cu), zinc (Zn), cadmium (Cd), and lead (Pb).
Using this adaptation platform, a multi-generational exposure experiment was conducted with Rhizophagus irregularis. Preliminary experiments were first performed to determine the tolerance thresholds of different AMF isolates to each metal and to establish suitable starting concentrations and exposure gradients. Subsequently, five generations of fungal cultures were exposed independently to increasing concentrations of Cu, Zn, Cd, and Pb. This step-wise selection process resulted in the establishment of independent AMF adaptation lines for each heavy metal treatment. The final-generation adapted fungal materials were successfully harvested.
To investigate the mechanisms underlying acquired heavy metal tolerance, molecular analyses were initiated. DNA extraction from the adapted fungal lines was successfully completed, and samples were prepared for subsequent whole-genome sequencing, bisulphite sequencing, and RNA sequencing. These analyses will enable the identification of genetic, epigenetic, and transcriptomic changes associated with AMF adaptation and will help distinguish stable evolutionary adaptation from reversible physiological acclimation.
In parallel, experiments were initiated to examine the stability and reversibility of heavy metal tolerance. The adapted AMF lines are currently being cultivated under reduced or absent heavy metal conditions to evaluate whether tolerance is maintained after removal of the selective pressure. Fungal performance, including hyphal growth and sporulation capacity, is being monitored and will be complemented by molecular analyses to determine the mechanisms responsible for tolerance maintenance or loss.
Preparatory activities for the subsequent ecological validation phase were also initiated. In collaboration with the return host institution, the Institute of Botany of the Czech Academy of Sciences (IBOT), experimental planning for greenhouse trials using heavy metal-contaminated soils was completed, including soil preparation and selection of suitable host plants. These activities will allow rapid implementation of plant-based validation experiments during the return phase.
Overall, the project has successfully achieved the major technical milestones planned for the outgoing phase. The development of the AMF adaptation system and the generation of independently adapted AMF lines for four heavy metals represent important advances towards producing stress-resilient fungal inocula. The generated biological materials and molecular resources provide the foundation for understanding AMF stress adaptation mechanisms and for future development of sustainable microbial solutions for contaminated soil restoration.
A major achievement was the successful design, fabrication, and validation of a compartmented split-plate cultivation system for controlled AMF adaptation. This innovative system physically separates the root compartment from the hyphal compartment, allowing fungal hyphae to be directly exposed to defined heavy metal stress while maintaining plant host growth under non-contaminated conditions. The system provides a controlled and reproducible platform to drive fungal adaptation while avoiding the limitations associated with traditional soil-based selection approaches. The functionality of the system was successfully demonstrated for four target heavy metals: copper (Cu), zinc (Zn), cadmium (Cd), and lead (Pb).
Using this adaptation platform, a multi-generational exposure experiment was conducted with Rhizophagus irregularis. Preliminary experiments were first performed to determine the tolerance thresholds of different AMF isolates to each metal and to establish suitable starting concentrations and exposure gradients. Subsequently, five generations of fungal cultures were exposed independently to increasing concentrations of Cu, Zn, Cd, and Pb. This step-wise selection process resulted in the establishment of independent AMF adaptation lines for each heavy metal treatment. The final-generation adapted fungal materials were successfully harvested.
To investigate the mechanisms underlying acquired heavy metal tolerance, molecular analyses were initiated. DNA extraction from the adapted fungal lines was successfully completed, and samples were prepared for subsequent whole-genome sequencing, bisulphite sequencing, and RNA sequencing. These analyses will enable the identification of genetic, epigenetic, and transcriptomic changes associated with AMF adaptation and will help distinguish stable evolutionary adaptation from reversible physiological acclimation.
In parallel, experiments were initiated to examine the stability and reversibility of heavy metal tolerance. The adapted AMF lines are currently being cultivated under reduced or absent heavy metal conditions to evaluate whether tolerance is maintained after removal of the selective pressure. Fungal performance, including hyphal growth and sporulation capacity, is being monitored and will be complemented by molecular analyses to determine the mechanisms responsible for tolerance maintenance or loss.
Preparatory activities for the subsequent ecological validation phase were also initiated. In collaboration with the return host institution, the Institute of Botany of the Czech Academy of Sciences (IBOT), experimental planning for greenhouse trials using heavy metal-contaminated soils was completed, including soil preparation and selection of suitable host plants. These activities will allow rapid implementation of plant-based validation experiments during the return phase.
Overall, the project has successfully achieved the major technical milestones planned for the outgoing phase. The development of the AMF adaptation system and the generation of independently adapted AMF lines for four heavy metals represent important advances towards producing stress-resilient fungal inocula. The generated biological materials and molecular resources provide the foundation for understanding AMF stress adaptation mechanisms and for future development of sustainable microbial solutions for contaminated soil restoration.
Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)
Current approaches to obtain stress-tolerant arbuscular mycorrhizal fungi (AMF) mainly rely on isolation and screening of naturally occurring tolerant strains from contaminated environments. However, these approaches are often time-consuming, difficult to control experimentally, and provide limited insights into how tolerance is acquired and maintained. In addition, naturally isolated AMF strains may lose their stress tolerance during subsequent cultivation under laboratory conditions.
MYTUNETOOL introduces a new strategy by applying a controlled in vitro multi-generational adaptation approach to actively enhance AMF tolerance to environmental stress. During the project, a compartmented split-plate cultivation system was optimized and validated as an experimental platform that enables direct exposure of fungal hyphae to heavy metal (HM) stress while maintaining plant growth in a non-contaminated compartment. Although compartmented cultivation systems have previously been used for AMF research, their application as a controlled evolutionary platform for driving targeted AMF adaptation to specific environmental contaminants represents a new methodological direction.
Using this approach, the project successfully generated independent Rhizophagus irregularis adaptation lines exposed to four individual heavy metals: copper (Cu), zinc (Zn), cadmium (Cd), and lead (Pb). In particular, Cd- and Pb-adapted lines demonstrated enhanced tolerance compared with non-adapted controls, providing unique biological materials to investigate the mechanisms underlying acquired stress tolerance in AMF. These adapted fungal lines represent a significant advancement beyond traditional environmental screening approaches, as they allow controlled investigation of whether stress tolerance is associated with stable genetic changes, epigenetic modifications, or reversible physiological acclimation.
The results have the potential to generate important scientific and practical impacts. From a scientific perspective, the adapted AMF lines combined with ongoing whole-genome sequencing, bisulphite sequencing, and transcriptome analyses will provide new insights into the molecular mechanisms of environmental adaptation in AMF, an area that remains poorly understood. From an applied perspective, HM-tolerant AMF strains could contribute to the development of more effective biological tools for phytoremediation, ecological restoration, and the improvement of plant performance in degraded or contaminated soils.
Further actions are required to ensure the uptake and successful application of these results. These include validation of the adapted AMF strains under greenhouse and field conditions during the returning phase (in collaboration with IBOT), assessment of their effectiveness in improving plant growth and HM remediation, and evaluation of their scalability for future applications during the planned industrial secondment with Symvitro Ltd. Additional assessment of regulatory requirements, intellectual property opportunities, and pathways towards potential commercialization will also be needed to translate the scientific outcomes into practical solutions.
MYTUNETOOL introduces a new strategy by applying a controlled in vitro multi-generational adaptation approach to actively enhance AMF tolerance to environmental stress. During the project, a compartmented split-plate cultivation system was optimized and validated as an experimental platform that enables direct exposure of fungal hyphae to heavy metal (HM) stress while maintaining plant growth in a non-contaminated compartment. Although compartmented cultivation systems have previously been used for AMF research, their application as a controlled evolutionary platform for driving targeted AMF adaptation to specific environmental contaminants represents a new methodological direction.
Using this approach, the project successfully generated independent Rhizophagus irregularis adaptation lines exposed to four individual heavy metals: copper (Cu), zinc (Zn), cadmium (Cd), and lead (Pb). In particular, Cd- and Pb-adapted lines demonstrated enhanced tolerance compared with non-adapted controls, providing unique biological materials to investigate the mechanisms underlying acquired stress tolerance in AMF. These adapted fungal lines represent a significant advancement beyond traditional environmental screening approaches, as they allow controlled investigation of whether stress tolerance is associated with stable genetic changes, epigenetic modifications, or reversible physiological acclimation.
The results have the potential to generate important scientific and practical impacts. From a scientific perspective, the adapted AMF lines combined with ongoing whole-genome sequencing, bisulphite sequencing, and transcriptome analyses will provide new insights into the molecular mechanisms of environmental adaptation in AMF, an area that remains poorly understood. From an applied perspective, HM-tolerant AMF strains could contribute to the development of more effective biological tools for phytoremediation, ecological restoration, and the improvement of plant performance in degraded or contaminated soils.
Further actions are required to ensure the uptake and successful application of these results. These include validation of the adapted AMF strains under greenhouse and field conditions during the returning phase (in collaboration with IBOT), assessment of their effectiveness in improving plant growth and HM remediation, and evaluation of their scalability for future applications during the planned industrial secondment with Symvitro Ltd. Additional assessment of regulatory requirements, intellectual property opportunities, and pathways towards potential commercialization will also be needed to translate the scientific outcomes into practical solutions.