Fungal Bioresources for Mycoremediation of Glyphosate- and AMPA-Contaminated Agricultural Soils

Francesca Emili ¹, Veronica Spinelli ¹, Andrea Ceci ¹, Valeria Crosara ¹, Massimo Reverberi ¹, Domenico Davolos ², Anna Maria Persiani ¹

Department of Environmental Biology, Sapienza University of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy ¹, Department of Technological Innovations and Safety of Plants, Products and Anthropic Settlements (DIT), INAIL, Research Area, Via R. Ferruzzi 38/40, 00143 Rome, Italy ²

Glyphosate (GP) is a widely used herbicide in agriculture, well known for its broad-spectrum effectiveness. However, its extensive use has caused significant contamination of soils and water resources. Despite concerns about its environmental and health risks, glyphosate remains authorized for use in the European Union. This situation calls for sustainable solutions to reduce GP concentration in agricultural soils. Recent research has shown that certain fungi, isolated from contaminated matrices, can degrade GP and its primary metabolite aminomethylphosphonic acid (AMPA), which exhibits a greater toxicity and persistence than GP. This study aims to explore the fungal communities in GP-contaminated agricultural soil and isolate strains with potential in mycoremediation. Here, six soil samples were collected from an agricultural field with a history of glyphosate use. DNA metabarcoding analisys of ITS region using Oxford Nanopore technology was carried out to analyze the fungal communities in soil samples. Biodiversity was assessed using the Shannon-Wiener and Pielou’s Evenness indices. Selective enrichment procedure with a mixture of GP and AMPA as only phosphorus source was carried out to isolate fungal strains suitable for GP and AMPA degradation. Moreover, 22 strains of Clonostachys, which was the most adbundant isolated genus, were tested in liquid media with 10, 20, 30 mM GP and AMPA concentrations as the sole phosphorus source. Growth was monitored for 10 days, through spectrophotometric readings at 620 nm every 48 hours to select the most efficient degrading strains. DNA metabarcoding analisys revealed a high fungal diversity, with Linnemannia elongata, Mortierella globalpina, and Funneliformis mosseae abundantly present in all soil samples and few exclusive species were highlighted. The Shannon-Wiener index showed high biodiversity values, but the Pielou’s Evenness index suggested that a few species were more abundant than others. Enrichment isolation revealed a high presence of entomopathogenic fungi including Clonostachys spp. and Paecilomyces spp., which were instead scarcely detected or absent in DNA metabarcoding data. These results suggested potential hormetic responses, such as increased growth and spore germination, in fungi exposed to low doses of GP and AMPA. The 22 Clonostachys strains showed variable growth responses to GP and AMPA exposure. At 10 mM GP, growth was observed but with reduced biomass development compared to AMPA exposure, while at 20 and 30 mM GP treatments spore germination was inhibited. For some Clonostachys strains, a slow growth was observed at 10 mM AMPA, while at 20 and 30 mM AMPA for 48 hours a stimulated growth was evident compared to the control, although this was less defined at the experiment endpoint. This work highlights the importance of integrating molecular and culturomics approaches to gain knowledge on fungi exposed to GP and on their abilities to degrade GP and AMPA. Further activities will focus on molecular perspective to study mechanism involved. In conclusion the insights gained in our study will benefit future efforts for mycoremediation of contaminated agricultural soils. The research was funded by the projects “INAIL-DIT, PAR 2022-2024, Ricerca Scientifica P8 O7“ and "Glyphosate mycoremediation of agricultural soils: a broad spectrum of nature-based solutions" -protocol no.RM123188F7AAFA9E”.

Main author career stage: PhD student

Contribution type: Poster

First choice session: 6. Plants, Fungi and Society

Second choice session: 2. Ecology