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

Modelli di stress ossidativo per la medicina personalizzata delle patologie croniche epato-gastrointestinali umane: il modello della malattia diverticolare

La patofisiologia della malattia diverticolare non è ancora del tutto chiara, tuttavia recenti studi condotti su modelli ex-vivo di colon umano hanno evidenziato la presenza di stress ossidativo che interessa l’intero spessore della parete colica nonostante la sua origine resti ancora ignota. Lo scopo principale del progetto è lo studio approfondito sullo stress ossidativo nei diversi quadri clinici della malattia diverticolare che vanno dalla diverticolosi asintomatica alla malattia diverticolare complicata per chiarire il possibile ruolo patogenetico nei vari stadi di malattia e quindi identificare nuove possibile strategie terapeutiche. Nel dettaglio lo studio è stato condotto parallelamente sia in modelli ex-vivo di malattia che in-vivo per ricercare i biomarcatori diretti di danno ossidativo, sia biomarcatori che dimostrino la perdita di antiossidanti legati al danno. In ultimo, per rafforzare il valore traslazionale dello studio è stato predisposto un modello in-vitro con il fine di valutare il potere benefico dell’antiossidante N-acetil cisteina nella reversione del danno ossidativo. Complessivamente le analisi dei tessuti del modello ex-vivo condotte hanno evidenziato sia in campioni di diverticolosi che di malattia diverticolare complicata un incremento di fibrosi parallelamente alla perdita dell’architettura del muscolo, accompagnati dal rilievo dei marker di switch fenotipico a livello cellulare. Nei tessuti di entrambi i fenotipi della patologia è stato riscontrato l’incremento di marker ossidativi quali proteine ossidate e presenza di nitrotirosina, parallelamente alla perdita di molecole legate al mondo antiossidante come Tioredossina-1 e fattore di trascrizione nucleare eritroide-2. Queste alterazioni si accompagnano all’incremento del fattore di ipossia HIF-1a riscontrato sia nella diverticolosi che nella malattia diverticolare complicata. Le alterazioni ossidative riscontrate si confermano inoltre anche a livello sistemico nei sieri del modello in-vivo. In dettaglio, rispetto al controllo, i campioni di diverticolosi e malattia diverticolare complicata hanno evidenziato l’incremento di H2O2 parallelamente alla perdita del potere antiossidante e l’incremento di LPS circolante e zonulina. Considerando il ruolo preponderante del mitocondrio nella genesi della malattia diverticolare particolare rilevo è stato dato allo studio di questo organello. Analisi ex-vivo condotte tramite microscopia elettronica hanno evidenziato un incremento del numero di mitocondri nei campioni patologici e l’aumento di fenomeni quali frammentazione e fusione mitocondriale. Analisi della catena di trasporto degli elettroni hanno evidenziato l’alterazione del complesso proteico II e IV della catena facendo presupporre un danno di tipo funzionale confermato a livello cellulare dall’incremento della massa mitocondriale, perdita del potenziale di membrane e perdita di antiossidanti. In modo interessante l’analisi del modello in-vitro, che prevedeva l’incubazione delle cellule con N-acetil cisteina, ha mostrato dei dati positivi in merito al ripristino della funzionalità delle cellule e del contenuto di antiossidante sia nella diverticolosi che nella malattia diverticolare complicata in seguito all’incubazione con questo antiossidante. In conclusione, le informazioni ottenute da questo studio ci hanno permesso di confermare la presenza di cambiamenti morfologici nel muscolo dovuti a cambiamenti dello stato ossidativo e perdita di antiossidanti attraverso un modello ex-vivo. Si noti che tutti questi cambiamenti influenzano gradualmente sia la diverticolosi che la malattia diverticolare complessa, suggerendo per la prima volta che non si tratta di due patologie diverse, ma di due condizioni con lo stesso quadro clinico. Di fondamentale importanza traslazionale, i cambiamenti di cui sopra sono parzialmente reversibili mediante il trattamento con antiossidanti, aprendo potenzialmente nuove strade per l’uso terapeutico di queste molecole nel trattamento della MD

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

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

Draft genome sequence of the apple pathogen Colletotrichum chrysophilum strain M932

Colletotrichum chrysophilum (Ascomycota, Sordariomycetes, Glomerellaceae) is a species belonging to the C. gloeosporioides complex. Described in 2017 as responsible for anthracnose on Musa acuminata (banana plants; Vieira et al. 2017), C. chrysophilum has been associated with Persea americana (avocado) and Prunus persica (peach) (Talhinhas and Baroncelli 2021). Moreover, together with Colletotrichum fructicola and C. noveboracense, it is considered one of the major causal agents of Glomerella leaf spot (GLS) and Apple bitter rot (ABR) diseases on Malus domestica (apple) (Astolfi et al. 2022; Khodadadi et al. 2020). Originally, C. chrysophilum was presumed to be limited to the American and Asian continents (Astolfi et al. 2022; Talhinhas and Baroncelli 2021), however, reports of GLS and ABR caused by this pathogen in European apple orchards, such as in Italy and Spain, start emerging in 2022 (Cabrefiga et al. 2022; Deltedesco and Oettl 2022)

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

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

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

The actinomycete Kitasatospora sp. SeTe27, subjected to adaptive laboratory evolution (ALE) in the presence of selenite, varies its cellular morphology, redox stability, and tolerance to the toxic oxyanion

The effects of oxyanions selenite (SeO32−) in soils are of high concern in ecotoxicology and microbiology as they can react with mineral particles and microorganisms. This study investigated the evolution of the actinomycete Kitasatospora sp. SeTe27 in response to selenite. To this aim, we used the Adaptive Laboratory Evolution (ALE) technique, an experimental approach that mimics natural evolution and enhances microbial fitness for specific growth conditions. The original strain (wild type; WT) isolated from uncontaminated soil gave us a unique model system as it has never encountered the oxidative damage generated by the prooxidant nature of selenite. The WT strain exhibited a good basal level of selenite tolerance, although its growth and oxyanion removal capacity were limited compared to other environmental isolates. Based on these premises, the WT and the ALE strains, the latter isolated at the end of the laboratory evolution procedure, were compared. While both bacterial strains had similar fatty acid profiles, only WT cells exhibited hyphae aggregation and extensively produced membrane-like vesicles when grown in the presence of selenite (challenged conditions). Conversely, ALE selenite-grown cells showed morphological adaptation responses similar to the WT strain under unchallenged conditions, demonstrating the ALE strain improved resilience against selenite toxicity. Whole-genome sequencing revealed specific missense mutations in genes associated with anion transport and primary and secondary metabolisms in the ALE variant. These results were interpreted to show that some energy-demanding processes are attenuated in the ALE strain, prioritizing selenite bioprocessing to guarantee cell survival in the presence of selenite. The present study indicates some crucial points for adapting Kitasatospora sp. SeTe27 to selenite oxidative stress to best deal with selenium pollution. Moreover, the importance of exploring non-conventional bacterial genera, like Kitasatospora, for biotechnological applications is emphasized