19 research outputs found
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
No full textGrapevine 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
Role of the chitin-binding domain (CBD) of a grapevine class IV chitinase against Botrytis cinerea
No full textDuring plant infection, fungal chitin deposition at the hyphal tip is counteracted by host chitinases. We studied the interaction between Botrytis cinerea and a class IV chitinase, that is constitutively expressed in mature grapevine berries. Early during fungal growth, the fungus proteolytically removes the chitin-binding domain (CBD). This removal results in a 50% reduction in the enzymatic activity of the chitinase and a complete loss of its botryticidal activity. Despite this, the pattern of chitin degradation by the native and the cleaved chitinase remains similar. We also detected the expression of this class IV chitinase in grapevine leaves during B. cinerea infection. Analysis of chitinase activity in leaf samples at various infection stages revealed that while this chitinase is not the most active among all chitinases, it is the most abundant at the protein level, with its expression correlating with infection progression. MALDI-TOF analysis of specific bands after SDS-PAGE confirmed the presence of both the native and CBD-deprived forms of the chitinase in the infected leaves, indicating that CBD removal observed in vitro also occurs during infection. Given the relatively low activity of this chitinase, as shown by zymogram, we further investigated the role of the CBD against B. cinerea. We transiently expressed the CBD in Nicotiana tabacum and heterologously expressed it in Pichia pastoris to assess its function. These findings highlight the significant role of the CBD in the chitinase's antifungal activity and its potential application in enhancing plant resistance to fungal infections
Peptaibol analogs as new effective fungicides against Botrytis cinerea
No full textIn 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
No full textChitin 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 μg/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
Drought, Waterlogging and Co‐Infection Influence the Severity of Coniella granati and Phytophthora palmivora in Pomegranate and the Biocontrol Efficacy of Bacillus amyloliquefaciens
No full textCultivation of pomegranate (Punica granatum) is threatened by the emerging pathogens Coniella granati and Phytophthora palmivora. Whether these microorganisms increase their damage due to simultaneous infection and abiotic stress in pomegranate is unknown. Amylo-X LC is an effective biofungicide based on Bacillus amyloliquefaciens but its efficacy to protect pomegranate from C. granati and P. palmivora if plants are under abiotic stress has not been tested. Therefore, the effects of drought, waterlogging and co-infection on the aggressiveness of these two pathogens and the efficacy of Amylo-X LC were evaluated. Pomegranate trees were subjected to regular watering, drought and waterlogging conditions, treated with Amylo-X LC, and individually or simultaneously inoculated in the stem by C. granati and P. palmivora. Drought experienced by trees limited P. palmivora, while waterlogging favoured C. granati. In combined infections, the presence of P. palmivora amplified the damage caused by C. granati in trees subjected to regular watering and drought. Amylo-X LC proved effective in preventing P. palmivora damage and did not allow the synergistic effect of P. palmivora on C. granati to occur in trees under regular watering and drought. Drought further reduced P. palmivora lesions in plants pretreated with Amylo-X LC compared to those under regular watering. The impact of abiotic stress in pomegranate during C. granati and P. palmivora infections and the efficacy of B. amyloliquefaciens as a potential agent against pomegranate dieback were discussed. We conclude that further efforts to evaluate the efficacy of biological control agents on crops exposed to different environmental scenarios are needed
Botrytis cinerea cleaves a grapevine chitinase decreasing its enzymatic activity and its detrimental effects on fungal growth
No full textPeptide analogs of a Trichoderma peptaibol effectively control downy mildew in the vineyard
No full textPlasmopara 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
Two-Dimensional Electronic Spectroscopy Reveals Dynamics and Mechanisms of Solvent-Driven Inertial Relaxation in Polar BODIPY Dyes
No full textIn
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.
No full textThe 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
Looking for novel control measures against the rice fungal pathogen Pyricularia oryzae
No full textThe 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
