UPDATE ON THE BIOPESTICIDE REGULATION AND DEVELOPMENT OF PROTEIN-BASED PRODUCTS FOR THE BIOCONTROL OF CROP DISEASES

In the last decades, concerns on the negative drawbacks of chemical pesticides on human health and environment have raised interest in safer alternatives; biopesticides, such as biogenic elicitors, represent an encouraging solution. The regulatory approach in the European Union does not distinguish biopesticides as a specific category of plant protection, and for this reason they are subjected to the same regulations as synthetic chemicals, requiring several authorization steps for the final approval and marketing. Among others, protein-based products and peptide fragments can stimulate plant growth, and represent a wide category of elicitors able to reduce the symptoms of common crop diseases, by acting as stimulators of plant defence and influencing systemic resistance processes, which can be regarded as effective alternative to synthetic chemical pesticides. The final objective of the current doctoral project was to provide new insights on the use of bioactive protein-based products against crop diseases, to further develop new sustainable strategies for organic agricultural practices. More in details, as well as providing an updated overview of the regulatory procedures for the authorization of biopesticides, the specific goals of the present research were i) to characterize the mode of action of protein-based products against crop diseases and ii) to optimize a method for the low-cost production of bioactive protein-based products. For this purpose, we analysed the mechanisms of action of a protein derivative called nutrient broth (NB) against grapevine downy mildew (caused by Plasmopara viticola), focusing on its roles as resistance inducer and nutritional source for phyllosphere microbial populations (Chapter 2). We showed that NB reduced downy mildew symptoms and induced the expression of defence-related genes in greenhouse- and in vitro-grown plants, indicating the activation of grapevine defence processes. Furthermore, NB increased the number of culturable phyllosphere bacteria, and altered the composition of bacterial and fungal populations on grapevine leaves. Thus, modifications in the structure of leaf populations caused by NB application could partially contribute to downy mildew control by competition for space/nutrients with the pathogen or other biocontrol strategies. Particularly, changes in the abundance of phyllosphere microorganisms may provide a cont ribution to the resistance induction, partially affecting the hormone-mediated signalling pathways involved. Later, we optimized an experimental procedure to develop low-cost protein hydrolysates starting from plant agro-industrial by-products, since animal-derived protein derivatives may create concerns about food safety (Chapter 3). Particularly, we compared the effect of enzymatic and acid hydrolysis on different plant protein sources (soybean, rapeseed and guar protein meals), in terms of efficacy against the powdery mildew of Cucurbitaceae (caused on courgette plants by Podosphaera xanthii), investigating the potential contribution of amino acids and peptide fragment s generated during the hydrolysis to the activation of plant resistance. Our results showed that the original protein source affected the biocontrol properties of protein hydrolysates, and two hydrolysis processes improved the functional properties of guar protein meal against powdery mildew. A positive correlation was found between the efficacy and degree of hydrolysis of guar acid hydrolysates, suggesting that the hydrolysis method may enhance the functional properties of the original protein source. In addition, significant correlations were revealed between the efficacy of guar hydrolysates and concentrations of specific peptide fragments and amino acids, which may be involved in the regulation of the plant defence response. Specifically, guar enzymatic hydrolysates did not present a direct toxic effect against the germination of pathogenic conidia, suggesting a mode of action mainly based on the stimulation of plant resistance mechanisms, as observed for NB. The possibility of controlling crop diseases with the preventive foliar application of protein-based products represents an innovative approach, especially in a view of reducing harmful chemical pesticides in integrated pest management programs. However, further studies are required to fully clarify their modes of action and the impact on phyllosphere microorganisms under field conditions

Ruptured middle cerebral artery aneurysms. retrospective study and multivariate analysis of 105 patients treated by surgical clipping

Objective of the study: We analyze in this study only patients with surgically treated ruptured aneurysms in order to identify statistical significance of each predictive factor in terms of outcome of patients with ruptured MCAAs. Materials and methods: In this retrospective study, we analyzed 105 cases of ruptured MCAAs, admitted from January 2001 to December 2015 at Neurosurgical Department of Umberto I University Hospital of Rome, Italy. Predictive factors evaluated are: Patient’s features (age, sex, co-morbidities), aneurysmal location (proximal, bifurcation or distal) and size of aneurysmal dome (small, large or giant); surgical timing (ultra-early, early, delayed), and Intracerebral Hemorrhage (ICH) volume. For each parameter we calculated mean and standard deviation, covariance and relation coefficient (through the linear regression model). Results: The clinical evaluation of patients assessed through the World Federation of Neurological Surgeons (WFSN) grading scale, that is 5 for 37 patients (35.3%), 4 for 28 patients. In 40% of cases the maximum sac diameter was between 7 mm and 12 mm, while in 67% of the cases the aneurysms concerned the bifurcation of the middle cerebral artery. ICH was associated in 57 cases (54.3%). As far as outcome is concerned, at 3 months, 32 patients (30.47%) had a favourable outcome, while 73 (69.52%) patients had not favourable outcome. To one year, 46 patients (43.8%) had favourable outcomes, while 59 patients (56.19%) had not favourable outcome. The mean outcomes as mean mRS are significantly less favourable in patients with ICH. Conclusion: In MCAAs patients, the presence of ICH strongly affects the outcome with a marked increase in mortality and morbidity. Surgical timing significantly influences the outcomes and ultra-early surgery should always be taken into account

Unraveling the Metabolic Potential of Asgardarchaeota in a Sediment from the Mediterranean Hydrocarbon-Contaminated Water Basin Mar Piccolo (Taranto, Italy)

Increasing number of metagenome sequencing studies have proposed a central metabolic role of still understudied Archaeal members in natural and artificial ecosystems. However, their role in hydrocarbon cycling, particularly in the anaerobic biodegradation of aliphatic and aromatic hydrocarbons, is still mostly unknown in both marine and terrestrial environments. In this work, we focused our study on the metagenomic characterization of the archaeal community inhabiting the Mar Piccolo (Taranto, Italy, central Mediterranean) sediments heavily contaminated by petroleum hydrocarbons and polychlorinated biphenyls (PCB). Among metagenomic bins reconstructed from Mar Piccolo microbial community, we have identified members of the Asgardarchaeota superphylum that has been recently proposed to play a central role in hydrocarbon cycling in natural ecosystems under anoxic conditions. In particular, we found members affiliated with Thorarchaeota, Heimdallarchaeota, and Lokiarchaeota phyla and analyzed their genomic potential involved in central metabolism and hydrocarbon biodegradation. Metabolic prediction based on metagenomic analysis identified the malonyl-CoA and benzoyl-CoA routes as the pathways involved in aliphatic and aromatic biodegradation in these Asgardarchaeota members. This is the first study to give insight into the archaeal community functionality and connection to hydrocarbon degradation in marine sediment historically contaminated by hydrocarbons

Editorial: Microbial Life Under Stress: Biochemical, Genomic, Transcriptomic, Proteomic, Bioinformatics, Evolutionary Aspects, and Biotechnological Applications of Poly-Extremophilic Bacteria, Volume II

Habitats defined as “extremes” exist across the entire planet. They can be widely different in their physico-chemical features as they include a diverse array of harsh parameters thought to preclude the existence of living organisms, such as temperature, pH, salinity, radiation, pressure, low water activity, low nutrients, and even the presence of toxic agents such as metals and/or metalloids. Organisms capable of surviving or thriving in those habitats are named “extremophiles” and the vast majority of them are prokaryotes, which is not surprising as they show a remarkable reservoir of genomes allowing them to grow in a great variety of hostile niches. Interestingly, several harsh conditions may occur simultaneously and the microorganisms able to withstand them are called “poly-extremophiles”. Although many bacterial species from all kinds of extreme environments have been isolated and described in the last decades, very little is known about the molecular strategies and physiology that allow them to grow in such critical conditions. The aim of this Research Topic on Microbial Life Under Stress (Volume II) is to address this issue by applying multidisciplinary approaches for integrating data from biochemical, genomic, transcriptomic, proteomic, bioinformatics, and evolutionary studies of bacteria from extreme and poly-extreme environments. This Research Topic consists of 14 original articles by numerous authors actively engaged in the study of microbiology, biochemistry, and omic-research of extremophiles. The present Editorial can be divided into four sections which include groups of articles on different genera and species of acidophiles, studies on microorganisms from arid/desiccated environments but also from habitats at low and high temperatures, and finally, a set of papers on extremophiles capable of coping with extreme levels of radiation, pressure, and toxic metals

Leaf treatments with a protein-based resistance inducer partially modify phyllosphere microbial communities of grapevine

Protein derivatives and carbohydrates can stimulate plant growth, increase stress tolerance, and activate plant defense mechanisms. However, these molecules can also act as a nutritional substrate for microbial communities living on the plant phyllosphere and possibly affect their biocontrol activity against pathogens. We investigated the mechanisms of action of a protein derivative (nutrient broth, NB) against grapevine downy mildew, specifically focusing on the effects of foliar treatments on plant defense stimulation and on the composition and biocontrol features of the phyllosphere microbial populations. NB reduced downy mildew symptoms and induced the expression of defense-related genes in greenhouse- and in vitro-grown plants, indicating the activation of grapevine resistance mechanisms. Furthermore, NB increased the number of culturable phyllosphere bacteria and altered the composition of bacterial and fungal populations on leaves of greenhouse-grown plants. Although, NB-induced changes on microbial populations were affected by the structure of indigenous communities originally residing on grapevine leaves, degrees of disease reduction and defense gene modulation were consistent among the experiments. Thus, modifications in the structure of phyllosphere populations caused by NB application could partially contribute to downy mildew control by competition for space or other biocontrol strategies. Particularly, changes in the abundance of phyllosphere microorganisms may provide a contribution to resistance induction, partially affecting the hormone-mediated signaling pathways involved. Modifying phyllosphere populations by increasing natural biocontrol agents with the application of selected nutritional factors can open new opportunities in terms of sustainable plant protection strategie

Microbial inactivation of raw chicken meat by supercritical carbon dioxide treatment alone and in combination with fresh culinary herbs

The objective of the present study was to assess the potential synergistic effect between supercritical carbon dioxide (SC-CO2) and fresh culinary herbs (Coriandrum sativum and Rosmarinus officinalis) on the microbial inactivation of raw chicken meat. The microbiological inactivation was performed on Escherichia coli and natural flora (total mesophilic bacteria, yeasts, and molds). High pressure treatments were carried out at 40\ub0C, 80 or 140 bar from 15 to 45 min. Microbial inactivation had a strong dependence on treatment time, achieving 1.4 log CFU/g reduction of E. coli after 15 min, and up to 5 log after 45 min, while a pressure increase from 80 up to 140 bar was not significant on the microbial inactivation. Mesophilic microorganisms were strongly reduced (>2.6 log CFU/g) after 45 min, and yeasts and molds were below the detection limits of the technique (<100 CFU/g) in most cases. The combination of fresh herbs together with SC-CO2 treatment did not significantly increase the inactivation of either E. coli or natural flora, which was similar to the SC-CO2 alone. The synergistic effect was obtained on the inactivation of E. coli using a proper concentration of coriander essential oil (EO) (0.5% v/w), while rosemary EO did not show a significant effect. Color analysis after the treatment showed an increment of lightness (L*), and a decrease of redness (a*) on the surface of the sample, making the product visually similar to cooked meat. Texture analysis demonstrated the modification of the texture parameters as a function of the process pressure making the meat more similar to the cooked one

Antimicrobial activity of different nanocellulose films embedded with thyme, cinnamon, and oregano essential oils for active packaging application on raspberries

The study focuses on the antimicrobial activity of nanocellulose films (NFC) embedded with thyme, cinnamon, and oregano essential oils for active packaging application. The activity against model pathogenic bacteria was first tested to obtain each oil's minimum inhibitory concentration (MIC). The tests showed that the surface area of the films and the quantity of essential oil strongly influenced the antimicrobial effect. Then, the different active packaging systems were tested directly on packed raspberries to study their actual commercial packaging conditions. Through 12 days of storage at 1 & DEG;C, it was observed that thyme and oregano essential oils were more effective in maintaining the firmness and reducing the weight loss of the fruits compared to cinnamon essential oil or the control; no significant effect was observed on the other quality parameters that were considered. The essential oils independently proved effective in preventing fungal growth. However, the combined impact of thyme+oregano (NAP_TO) demonstrated the best performance.COST ACTION 19124 CIRCULA-BILITYinfo:eu-repo/semantics/publishedVersio

Customized biofilm device for antibiofilm and antibacterial screening of newly developed nanostructured silver and zinc coatings

Background Bacterial colonisation on implantable device surfaces is estimated to cause more than half of healthcare-associated infections. The application of inorganic coatings onto implantable devices limits/prevents microbial contaminations. However, reliable and high-throughput deposition technologies and experimental trials of metal coatings for biomedical applications are missing. Here, we propose the combination of the Ionized Jet Deposition (IJD) technology for metal-coating application, with the Calgary Biofilm Device (CBD) for high-throughput antibacterial and antibiofilm screening, to develop and screen novel metal-based coatings. Results The films are composed of nanosized spherical aggregates of metallic silver or zinc oxide with a homogeneous and highly rough surface topography. The antibacterial and antibiofilm activity of the coatings is related with the Gram staining, being Ag and Zn coatings more effective against gram-negative and gram-positive bacteria, respectively. The antibacterial/antibiofilm effect is proportional to the amount of metal deposited that influences the amount of metal ions released. The roughness also impacts the activity, mostly for Zn coatings. Antibiofilm properties are stronger on biofilms developing on the coating than on biofilms formed on uncoated substrates. This suggests a higher antibiofilm effect arising from the direct contact bacteria-coating than that associated with the metal ions release. Proof-of-concept of application to titanium alloys, representative of orthopaedic prostheses, confirmed the antibiofilm results, validating the approach. In addition, MTT tests show that the coatings are non-cytotoxic and ICP demonstrates that they have suitable release duration (&gt; 7 days), suggesting the applicability of these new generation metal-based coatings for the functionalization of biomedical devices.Conclusions The combination of the Calgary Biofilm Device with the Ionized Jet Deposition technology proved to be an innovative and powerful tool that allows to monitor both the metal ions release and the surface topography of the films, which makes it suitable for the study of the antibacterial and antibiofilm activity of nanostructured materials. The results obtained with the CBD were validated with coatings on titanium alloys and extended by also considering the anti-adhesion properties and biocompatibility. In view of upcoming application in orthopaedics, these evaluations would be useful for the development of materials with pleiotropic antimicrobial mechanisms

Photoidentification as a complementary tool to evaluate whale shark movements between different areas: the case of Nosy Be in Madagascar and the Gulf of Tadjoura in Djibouti

The whale shark Rhincodon typus, the world’s largest fish, is highly mobile, and is capable of migrating for thousands of kilometers each year. According to the IUCN Red List, areas where 500 or more individuals have been documented through either counts or model estimates include the Arabian Gulf and Gulf of Oman, Ningaloo Reef in Western Australia, Darwin Island in the Galapagos, Quintana Roo in Mexico, Inhambane province in Mozambique, Philippines, and Mahe in the Seychelles (Pierce &amp; Norman, 2016). However, information on the global population structure and movement patterns remains limited. Recently, the whale shark population in Madagascar has been estimated at more than 400 individuals (Diamant et al., 2021), while in Djibouti at about 190 individuals (Boldrocchi et al., 2020). The aim of the study is to compare in the long term the two populations with a non-invasive system of identification of individual specimens, the I3S Classic program, to evaluate the possible presence of movements of individuals between these two areas