ENVIRONMENTALLY SUSTAINABLE TOOLS FOR GRAPEVINE PROTECTION AGAINST FUNGAL AND OOMYCETES DISEASES Activity of peptaibol analogs and a grape chitinase against the grape pathogens B. cinerea and P. viticola

Grapevine productions is worldwide affected by fungal pathogens both in the field and during post-harvest storage. In addition to yield losses, even the quality of grape and wine is affected. The management of these pathogens mainly relies on chemicals and agronomical practices. The majority of the plant protection products (PPPs) distributed in the vineyard are directed to control Botrytis cinerea and Plasmopara viticola, causal agents of gray mold and downy mildew disease, respectively. However, pathogens can develop resistance to synthetic fungicides. Moreover, the high use of PPPs poses serious risks to the environment and the health of operators and consumers. In this regard, European Union (UE) policies foresee a 50% reduction in the use of chemical pesticides by 2030 and the implementation of integrated approaches for crop protection. The research studies presented in this thesis share the final purpose to find out new alternative strategies and molecules to counteract grapevine pathogens. To this aim, a deep knowledge of the pathogenic mechanism is fundamental. Therefore, in the first part of the thesis the interaction between B. cinerea and grapevine was investigated by characterizing the ability of this fungus to detoxify plant defense proteins. During ripening, grape berries accumulate a class IV chitinase, a pathogenesis related (PR) protein with antifungal activity. However, the protease activity of B. cinerea is able to cleave this chitinase impairing its antifungal activity. This research could represent a first step into the identification of new fungal virulence factors to be counteracted. The second part of the thesis focused on the development of new biopesticides active against P. viticola and B. cinerea. The effectiveness of several water-soluble analogs produced by targeted amino acid substitutions of an antimicrobial peptide (peptaibol) naturally produced by Trichoderma longibrachiatum, was evaluated against the two pathogens. The assays allowed to identify a peptide highly effective against both pathogens that may be developed as biopesticide.Grapevine productions is worldwide affected by fungal pathogens both in the field and during post-harvest storage. In addition to yield losses, even the quality of grape and wine is affected. The management of these pathogens mainly relies on chemicals and agronomical practices. The majority of the plant protection products (PPPs) distributed in the vineyard are directed to control Botrytis cinerea and Plasmopara viticola, causal agents of gray mold and downy mildew disease, respectively. However, pathogens can develop resistance to synthetic fungicides. Moreover, the high use of PPPs poses serious risks to the environment and the health of operators and consumers. In this regard, European Union (UE) policies foresee a 50% reduction in the use of chemical pesticides by 2030 and the implementation of integrated approaches for crop protection. The research studies presented in this thesis share the final purpose to find out new alternative strategies and molecules to counteract grapevine pathogens. To this aim, a deep knowledge of the pathogenic mechanism is fundamental. Therefore, in the first part of the thesis the interaction between B. cinerea and grapevine was investigated by characterizing the ability of this fungus to detoxify plant defense proteins. During ripening, grape berries accumulate a class IV chitinase, a pathogenesis related (PR) protein with antifungal activity. However, the protease activity of B. cinerea is able to cleave this chitinase impairing its antifungal activity. This research could represent a first step into the identification of new fungal virulence factors to be counteracted. The second part of the thesis focused on the development of new biopesticides active against P. viticola and B. cinerea. The effectiveness of several water-soluble analogs produced by targeted amino acid substitutions of an antimicrobial peptide (peptaibol) naturally produced by Trichoderma longibrachiatum, was evaluated against the two pathogens. The assays allowed to identify a peptide highly effective against both pathogens that may be developed as biopesticide

Peptaibol analogs as new effective fungicides against Botrytis cinerea

In the last decades the search for new effective and sustainable fungicides has gained much importance in the European political agenda. The well-known biocontrol agent Trichoderma spp. produces short hydrophobic non-ribosomal peptides, named peptaibols, with antibiotic properties given by their ability to permeabilize lipid bilayers such as the cell membrane. Specifically, the peptaibol trichogin produced by T. longibrachiatum, was used as a model to synthesize several water-soluble analogs (De Zotti et al., 2020 International Journal of Molecular Sciences, 21, 7521). These analogs present one or more substitutions of the glycine residues, which improve their water-solubility while maintaining their thermal and chemical stability. The aim of this study was to assess the fungicidal activity of trichogin analogs against Botrytis cinerea. With an in vitro screening, four peptides were identified as effective in inhibiting conidia germination at 15 µM concentration and the most effective peptide displayed a Minimal Inhibitory Concentration of 1-5 µM. A microscopy analysis confirmed conidia cell death at 15 µM. This peptide was used in further experiments to assess its efficacy in controlling B. cinerea infection on different plant tissues. On bean leaves, this peptide determined a significant reduction of disease symptoms (higher than 95%) at 50 µM, being effective also at 15 µM (75% symptom reduction). Treatment of grapevine leaves and berries at 50 µM showed a significant reduction of disease symptoms of about 70% and 45%, respectively. Several analogs of this peptide, differing in sequence length or C-terminus to decrease synthesis costs, have also been produced and tested. Both changes did not affect peptide efficacy either in vitro and against infection on bean leaves. These results show the potential given by small structural modifications of natural secondary metabolites. Ongoing trials are now focusing on the combination of peptides and other natural antimicrobial compounds, with the aim to identify any synergistic effect against B. cinerea. Synergy would allow to reduce the fungicide dosage with many benefits, including the reduction of treatment cost, a better toxicity profile and a minimized probability of resistance development

Botrytis cinerea cleaves a grapevine chitinase reducing its enzymatic activity and its detrimental effects on fungal growth

Chitin represents the main fibrillary component of the cell wall in filamentous fungi. During plant infection, chitin apposition to the fungal cell wall is counteracted by host chitinases. Following Botrytis cinerea infection, a chitinase IV is highly expressed in grapevine leaves and is also abundantly contained in grapevine berries. The fungus decreases the molecular size of this protein (Favaron et al., 2009, Journal of Plant Pathology, 91, 579-588) by its proteolytic activity (Marcato et al., 2017, Physiological and Molecular Plant Pathology, 99, 7-15). The cleavage of the chitinase occurs early during the in vitro fungal growth. The N-terminal sequencing of the cleaved chitinase shows that the fragment removed is the chitin-binding domain (CBD). Without the CBD, the chitinase decreases its activity by about 50%. To investigate the possible effects of the native and cleaved chitinase on B. cinerea, the two purified proteins were administered at 100 \ub5g/ml to the fungal spores providing the proper conditions to avoid the chitinase cleavage. The native chitinase significantly decreased the conidia germination rate and the length of the germination tube while the cleaved chitinase did not. Protease inhibition assays provided evidence that metalloprotease activity is involved in the chitinase cleavage. To ascertain whether the native and cleaved chitinase can differently affect the expression of genes involved in B. cinerea cell wall modeling, we analyzed the expression of five fungal chitin synthase (Chs) and four chitin deacetylase (Cda) genes. By quantitative PCR we observed that only one Chs gene decreased its expression in presence of the native chitinase and three Cda genes were slightly down-regulated by both native and cleaved chitinase. In conclusion, the removal of the CBD by B. cinerea proteases appears as a mechanism preserving fungal growth from plant chitinase activity as highlighted also with other fungi (Jashni et al. 2015, Frontiers of plant science, 6, 1-7). Further experiments will better clarify the type of B. cinerea protease activity capable to disarm the plant chitinases

Efficacy of <i>Trichoderma longibrachiatum</i> Trichogin GA IV Peptaibol analogs against the Black Rot Pathogen <i>Xanthomonas campestris</i> pv. <i>campestris</i> and other Phytopathogenic Bacteria

Black rot caused by the Gram-negative bacterial pathogen Xanthomonas campestris pv. campestris (Xcc) is considered one of the most destructive diseases affecting crucifers. Xcc is a seedborne pathogen able to infect the host at any growth stage. The management of the pathogen mainly relies on the use of copper-based products with possible negative effects on human health and the environment. Searching for protection alternatives is crucial for achieving a sustainable management of Xcc. Trichoderma spp. has been largely used as a biocontrol agent against several phytopathogens. Among Trichoderma species, Trichoderma longibrachiatum produces the peptaibol trichogin GA IV, a secondary metabolite with antimicrobial activity against Gram-positive bacteria, as well as filamentous and yeast-like fungi. In this work, we tested, at micromolar concentrations, 25 synthetic analogs of the peptaibol trichogin GA IV for their bacteriostatic and bactericidal activity toward the bacterium Xcc. One of the most effective peptides (4r) was also tested against the Gram-negative bacteria Xanthomonas arboricola, Pseudomonas corrugata, Pseudomonas savastanoi pv. savastanoi, Agrobacterium tumefaciens, Ralstonia solanacearum, and Erwinia carotovora subsp. carotovora, as well as the Gram-positive bacterium Bacillus subtilis. The peptide 4r reduced black rot symptoms on cauliflower plants when administered both before and 24 h after inoculation with Xcc. The cytotoxic activity of the peptide 4r was also evaluated towards suspensions of tobacco cells by Evans Blue assay

Peptide analogs of a Trichoderma peptaibol effectively control downy mildew in the vineyard

Plasmopara viticola, the agent of grapevine downy mildew, causes enormous economic damage and its control is primarily based on synthetic fungicides. The European Union (EU) policies promote reducing reliance on synthetic plant protection products. Biocontrol agents (BCA) such as Trichoderma constitute a resource for the development of biopesticides. Trichoderma species produce secondary metabolites such as peptaibols, whose poor water solubility hampers their practical use as agrochemicals. To identify new bio-inspired molecules effective against P. viticola, some water-soluble peptide analogs of the peptaibol trichogin were synthesized. In grapevine leaf disk assays, various peptides at 50 µM completely prevented P. viticola infection after zoosporangia inoculation. Microscopic observations carried out with one of the most effective peptides showed that it causes membrane lysis and cytoplasm granulation of both zoosporangia and zoospores. Among the effective peptides, 4r was selected for a two-year field trial experiment. In the vineyard, the peptide administered at 100 µM (equivalent to 129.3 g/ha) overall reduced significantly disease incidence and severity on both leaves and bunches, allowing protection levels similar to those obtained with a cupric fungicide. In the second-year trial, reduced dosages were also tested, and results indicated that even by reducing the peptide concentration by 50 or 75%, a significant decrease in the disease level was obtained at the end of the trial. The peptide did not show any phytotoxic effect. Previously, peptide 4r had been demonstrated to be active against other fungal pathogens, including the grapevine fungus Botrytis cinerea. Thus, this peptide may be a candidate for broad-spectrum fungicide whose biological properties deserve further investigation

Targeted Amino Acid Substitutions in a Trichoderma Peptaibol Confer Activity against Fungal Plant Pathogens and Protect Host Tissues from Botrytis cinerea Infection

Fungal species belonging to the Trichoderma genus are commonly used as biocontrol agents against several crop pathogens. Among their secondary metabolites, peptaibols are helical, antimicrobial peptides, which are structurally stable even under extreme pH and temperature conditions. The promise of peptaibols as agrochemicals is, however, hampered by poor water solubility, which inhibits efficient delivery for practical use in crop protection. Using a versatile synthetic strategy, based on green chemistry procedures, we produced water-soluble analogs of the short-length peptaibol trichogin. Although natural trichogin was inactive against the tested fungal plant pathogens (Botrytis cinerea, Bipolaris sorokiniana, Fusarium graminearum, and Penicillium expansum), three analogs completely inhibited fungal growth at low micromolar concentrations. The most effective peptides significantly reduced disease symptoms by B. cinerea on common bean and grapevine leaves and ripe grape berries without visible phytotoxic effects. An in-depth conformational analysis featuring a 3D-structure&ndash;activity relationship study indicated that the relative spatial position of cationic residues is crucial for increasing peptide fungicidal activity

Two-Dimensional Electronic Spectroscopy Reveals Dynamics and Mechanisms of Solvent-Driven Inertial Relaxation in Polar BODIPY Dyes

In this work, we demonstrate the use of two-dimensional electronic spectroscopy (2DES) to study the mechanism and time scale of the femtosecond Stokes shift dynamics in molecules characterized by intramolecular charge transfer, such as distyryl-functionalized boron dipyrromethene (BODIPY) molecules. The obtained results demonstrate that 2DES allows clear and direct visualization of the phenomenon. The analysis of the 2D data in terms of 2D frequency-frequency decay associated maps provides indeed not only the time scale of the relaxation process but also the starting and the final point of the energy flow and the associated reorganization energy, identified by looking at the coordinates of a negative signature below the diagonal. The sensitivity of the 2DES technique to vibrational coherence dynamics also allowed the identification of a possible relaxation mechanism involving specific interaction between a vibrational mode of the dye and the solvent

Looking for novel control measures against the rice fungal pathogen Pyricularia oryzae

The filamentous fungus Pyricularia oryzae is the main causal agent of the rice blast disease, which accounts for 10-30% yield losses per year globally. The objective of this project, which is part of the Scientific and Technological Cooperation Agreement between the Italian Ministry of Foreign Affairs and International Cooperation and the Department of International Cooperation of the Ministry of Science and Technology of Vietnam, is to identify new pathogen targets and new molecules to control rice blast disease. P. oryzae uses a large number of degrading enzymes active on cell wall polysaccharides and lignin to penetrate and invade the rice plant tissues. Since these enzymes could be potential targets for plant inhibitors, candidate P. oryzae genes encoding enzymes particularly expressed during the infection process have been deleted from the fungal genome and the characterization of the obtained mutants is in progress to identify enzymes essential for fungal virulence on rice. In addition, new ecofriendly antimicrobial peptides, analogs of the natural Trichoderma longibrachiatum peptaibol, have been synthesized and tested in vitro against several P. oryzae strains from different geographic origin. The screening has allowed to identify some peptides very effective in inhibiting spore germination and fungal growth that could be used in vivo to confirm their efficacy in protecting rice from the blast disease