Phd in ENVIRONMENTAL AND EVOLUTIONARY BIOLOGY

Thesis title: Bioresources for a sustainable agriculture: potentialities of Minimedusa polyspora and Chaetomium globosum as plant growth promoting fungi

Feeding a constantly growing global population, while facing global change, without further impairing the environment is probably the greatest challenge our society is facing nowadays. Modern agriculture mostly depends on the use of agrochemicals, including chemical fertilizers, pesticides and herbicides, due to their determinant role in enhancing efficiently and economically crop production, to meet the constantly increasing food demand. However, modern agriculture pressures determine major detrimental impacts on the environment at different spatial and temporal scales, on all the natural matrices: air, soil, and water. Consequently, mitigating agriculture’s impacts on the environment represents an urgent need and a key strategy towards sustainability. Furthermore, this challenge is also concomitant with two other major challenges: increasing food production up to 60% by 2050 due to the world population growth, and adapting to a rapidly evolving climate change. In fact, due to climate change effects, plants are already more frequently subjected to severe abiotic (e.g. drought, flooding, extreme temperature) and biotic (e.g. pathogens and pest outbreaks) stresses, while future scenarios foresee these phenomena to become even more severe. In this context, plant growth promotion represents an interesting sustainable solution that may play a key role in increasing crop resilience and productivity in adverse environmental conditions, minimizing agrochemicals applications and tackling climate change effects. Indeed, in healthy ecosystems soil microorganisms, through the wide array of ecosystem services they provide, express a multifunctionality that support soil productivity and plant growth. In particular, microbial strains with high soil colonization potential and multiple plant growth promoting traits — such as the ability to stimulate the plant, increase nutrient availability, exert biocontrol activity against detrimental microorganisms, and biodegrade organic pollutant and detoxifying inorganic pollutants — present a higher efficacy due to their multipurpose applicability. In this context, fungi as multifunctional microorganisms, perfectly adapted to soil microhabitats, thanks to their functional traits, metabolic plasticity and highly potent and relatively non-specific enzymes, represent valuable and effective potential bioresources. This thesis aimed to characterize Minimedusa polyspora (Hotson) Weresub & P. M. LeClair and Chaetomium globosum Kunze, two strains of soil saprotrophic fungi, for multiple activities — including biostimulation, biocontrol and bioremediation — that may contribute to plant growth promotion, to assess their potential as multifunctional bioresources for biotechnological application aimed at promoting a more sustainable agriculture. Therefore, following a general introduction and literature review on the topic, three chapters, each one addressing these species characterization for a specific activity that may contribute to plant growth promotion, are reported. The first study presented in this thesis focused on assessing the efficacy of M. polyspora and C. globosum culture filtrates as biostimulant for the cultivation Cichorium intybus (L.), a plant of agricultural and medicinal interest. In a pot experiment set up in walk-in chambers, chicory plants, one month after the transfer of the seedlings in pots, were stimulated by soil drenching with 8 ml/pot (30 ml/kg of soil) of the culture filtrates obtained by a 14-days incubation of the fungal strains in Malt Extract Broth (MEB), or the same amount of uninoculated MEB in the control group. Fourteen days after the stimulation, plant biomasses were recovered to estimate several growth parameters and analyze the metabolomic variations occurred in roots and leaves through 1H-NMR 600 MHz. We observed for the first time that M. polyspora culture filtrate promotes an increase of biomass, both in shoots and roots, and of the leaf area, while no increase was observed in plants treated with C. globosum culture filtrate. Based on 1H-NMR metabolomics data, differential metabolites and their related metabolic pathways were highlighted. A common response in C. intybus roots involving the synthesis of 3-OH-butyrate through the decrease of the synthesis of fatty acids and sterols, as a mechanism balancing the NADPH/NADP+ ratio, was observed in both the treatments with C. globosum and M. polyspora culture filtrates. The phenylpropanoid pathway was differently triggered by the fungal culture filtrates. C. globosum culture filtrate increased phenylalanine and chicoric acid in the roots. Chicoric acid, whose biosynthetic pathway in chicory plant is putative and still not well known, is a very promising natural compound playing an important role in plant defense. Instead, M. polyspora culture filtrate interestingly stimulated an increase of 4-OH benzoate, being benzoic acids precursors for a wide variety of essential compounds playing crucial roles in plant fitness and defense response activation. Therefore, both C. globosum and M. polyspora culture filtrates affected C. intybus metabolome and, based on the findings of this study, could be considered as promising fungal bioresources for further studies aimed the development of new biostimulants. Moving on, in the second study presented in this thesis, M. polyspora and C. globosum biocontrol potential against the phytopathogenic fungi Alternaria alternata (Fr.) Keissl., Berkeleyomyces basicola (Berk. & Broome) W.J. Nel, Z.W. de Beer, T.A. Duong & M.J. Wingf. and Botrytis cinerea Pers. was investigated. Plant diseases, resulting in an annual estimated loss of 10–15% of world's major crops, represent a major threat to global crops production and social and political stability of nations. 70–80% of these diseases are caused by pathogenic fungi, numbers that are expected to increase in future years due to the effect of climate change on plant-pathogens interactions. In the effort to transition to a more sustainable and resilient agriculture, the application of biological control agents and their secondary metabolites represent a promising option to support the achievement of food security, without further compromise ecosystems’ health. Therefore, it is important deepening the potential of known fungal biocontrol agents against the existing fungal pathogens, shedding further light on their action mechanisms and discovering new efficient fungal strains suitable for biotechnological applications. In vitro screenings, despite presenting several limitations, constitute valuable methods for the identification of potential biocontrol agents. Therefore, through an array of in vitro plate assays M. polyspora and C. globosum were assessed for their ability to inhibit A. alternata, B. basicola and B. cinerea growth, aiming also to gain insight on possible antimicriobial mechanism/s involved in their biological control action. More specifically, a dual culture assay, a dual culture for volatile antimicrobial compounds (performed in two different conditions), and a culture filtrate antifungal activity assay were designed to try to discriminate the impact of direct and indirect biological control mechanisms. This study results show that both M. polyspora and C. globosum were able to inhibit, to a different extent, all the pathogens’ growth in the dual culture assay, suggesting a mechanism of biocontrol involving competition for nutrients and space. M. polyspora, based on the culture filtrate antifungal activity assay, was found to exert its inhibition on all the pathogens thanks also to an antibiosis mechanism through the release of diffusible compounds. Moreover, M. polyspora culture filtrate resulted to be particularly effective especially against B. basicola whose growth was completely inhibited; furthermore, its high inhibition effect against this species was also observed in the dual culture for volatile antimicrobial compounds assay, suggesting that M. polyspora antagonism against B. basicola occurs through multiple or mixed mechanisms. Therefore, based on this preliminary study’s results M. polyspora and C. globosum are promising biocontrol agents of three fungal phytopathogens of economical and agronomical relevance, and consequently species of interest for further studies in this area aimed at validating their potential as antagonists in in vivo conditions. Finally, the last study focused on evaluating M. polyspora and C. globosum bioremediation potentialities towards glyphosate. Addressing, in particular, their ability to tolerate and utilize glyphosate as a nutritional source and eventually degrade it. Indeed, glyphosate is the most commonly used herbicide worldwide. Its improper use during recent decades has resulted in glyphosate contamination of soils and waters. Fungal bioremediation is an environmentally friendly, cost effective, and feasible solution to glyphosate contamination in soils. In this study, M. polyspora and C. globosum together with other 16 saprotrophic fungal strains were screened in vitro for their ability to tolerate and eventually utilize Roundup at two different concentrations (1 mM and 10 mM) in different cultural conditions as a nutritional source. M. polyspora and C. globosum were found to be tolerant to RoundUp, a glyphosate-based herbicide, only at the concentration of 1 mM, while a concentration of 10 mM completely inhibited their growth. Moreover, Purpureocillium lilacinum was further screened to evaluate the ability to break down and utilize glyphosate as a P source in a liquid medium. The dose-response effect for Roundup, and the difference in toxicity between pure glyphosate and Roundup were also studied. This study’s results highlight the ability of several strains to tolerate 1 mM and 10 mM Roundup and to utilize it as a nutritional source. P. lilacinum was reported for the first time for its ability to degrade glyphosate to a considerable extent (80%) and to utilize it as a P source, without showing dose-dependent negative effects on growth. Pure glyphosate was found to be more toxic than Roundup for P. lilacinum. Our results showed that pure glyphosate toxicity can be only partially addressed by the pH decrease determined in the culture medium. In conclusion, despite the strains studied in this thesis were not able to degrade glyphosate, experimental results emphasized the in vitro noteworthy potential in glyphosate degradation of P. lilacinum, another fungal strain of biotechnological interest. In conclusion, based on this thesis’ results M. polyspora and C. globosum showed promising potentialities as plant growth promoting fungi and should be further studied as bioresources for eventual biotechnological applications towards a sustainable agriculture. This thesis, in addition to the studies addressing its aim, includes also an additional section composed of three published papers dealing with topics regarding fungal species conservation applying IUCN red-listing criteria and the biotechnological potentialities of strains preserved in the culture collection of the Fungal Biodiversity Laboratory of the Department of Environmental Biology, Sapienza University of Rome.