PRODUZIONE DI MICRORGANISMI BENEFICI DALLE CARATTERISTICHE MIGLIORATE

I funghi del genere Trichoderma sono considerati tra i principali microrganismi benefici impiegati in agricoltura grazie alla loro capacità di controllare numerosi agenti fitopatogeni, unita agli effetti benefici esercitati sulle piante, come la promozione della crescita (PGP) o l'induzione di meccanismi di resistenza (ISR). Per migliorare l'efficienza di questi funghi sono state adottate diverse tecniche di manipolazione genetica, come la trasformazione, la mutagenesi o la fusione di protoplasti (PF). Tra queste, la PF presenta numerosi vantaggi; in particolare permette di superare i vincoli dello scambio genetico che, nella maggior parte dei casi, conduce ad un'inevitabile incompatibilità vegetativa della progenie. Lo scopo di questo progetto di dottorato è stato quello di produrre mediante PF ibridi intragenici di Trichoderma con caratteristiche migliorate rispetto ai ceppi parentali, selezionati tra quelli che già presentavano capacità di biocontrollo, PGP o ISR. Sono state allestite due PF, la prima tra T. harzianum ceppo M10 e T. virens ceppo GV41 (fusione "MG"); la seconda tra T. harzianum ceppo T22 e T. asperellum ceppo KV906 (fusione "TK"). La progenie ottenuta è stata selezionata sfruttando un innovativo approccio high throgouhtput basato sull'osservazione in vivo dell'attività contro diversi agenti fitopatogeni (Rhizoctonia solani, Botrytis cinerea, Fusarium graminearum) e/o PGP. Dalla rizosfera di piante di Solanum lycopersicum inoculate con le miscele di fusione sono stati isolati in coltura monosporica 13 ibridi, successivamente caratterizzati sotto il profilo morfologico, genetico, metabolomico e funzionale. Dall'analisi delle sequenze ITS1-4 è emerso che 5 degli 8 ibridi derivanti dalla fusione MG sono geneticamente più simili alla specie harzianum, mentre 4 dei 5 ibridi ottenuti dalla fusione TK sono risultati più affini al parentale T. asperellum. A dimostrazione dell'effettivo rimescolamento genetico ottenuto con le PF, l'analisi PCR-RAPD ha evidenziato differenze nei profili di espressione degli ibridi, anche rispetto ai ceppi parentali. Gli ibridi isolati hanno dimostrato in vitro attività antagonista contro R. solani, B. cinerea e F. graminearum, nonché di promuovere significativamente la crescita delle piante trattate rispetto al controllo e ai ceppi parentali (i.e. in termini di allungamento di fusto e radice e peso fresco/secco della pianta). L'analisi metabolomica degli ibridi, effettuata mediante LC-MS Q-TOF, ha indicato la produzione in coltura liquida di numerosi metaboliti secondari tipici dei ceppi di Trichoderma utilizzati in questo lavoro, ma anche di altri composti, come il 6- pentil-alfa-pirone (6PP), non riscontrati nelle colture dei parentali. Infine, in esperimenti in co-coltura con F. graminearum su mais, l'ibrido A3 ha inibito sia la crescita del patogeno, sia la produzione della micotossina deossinivalenolo (DON), come osservato dall'analisi GC-MS. I risultati ottenuti hanno dimostrato che la PF unita allo screening fenotipo-guidato può rappresentare un'utile strategia per l'ottenimento di ceppi di Trichoderma dalle caratteristiche migliorate. Gli individui così ottenuti possono trovare immediata applicazione quali ingredienti attivi di bioformulati utili in agricoltura e, inoltre, rappresentano una valida alternativa agli OGM prodotti mediante tecniche convenzionali di manipolazione genetica

Polychlorinated Biphenyls (PCBs) in the Environment: Occupational and Exposure Events, Effects on Human Health and Fertility

In the last decade or so, polychlorinated biphenyls (PCBs) garnered renewed attention in the scientific community due to new evidence pointing at their continued presence in the environment and workplaces and the potential human risks related to their presence. PCBs move from the environment to humans through different routes; the dominant pathway is the ingestion of contaminated foods (fish, seafood and dairy products), followed by inhalation (both indoor and outdoor air), and, to a lesser extent, dust ingestion and dermal contact. Numerous studies reported the environmental and occupational exposure to these pollutants, deriving from building materials (flame-retardants, plasticizers, paints, caulking compounds, sealants, fluorescent light ballasts, etc.) and electrical equipment. The highest PCBs contaminations were detected in e-waste recycling sites, suggesting the need for the implementation of remediation strategies of such polluted areas to safeguard the health of workers and local populations. Furthermore, a significant correlation between PCB exposure and increased blood PCB concentrations was observed in people working in PCB-contaminated workplaces. Several epidemiological studies suggest that environmental and occupational exposure to high concentrations of PCBs is associated with different health outcomes, such as neuropsychological and neurobehavioral deficits, dementia, immune system dysfunctions, cardiovascular diseases and cancer. In addition, recent studies indicate that PCBs bioaccumulation can reduce fertility, with harmful effects on the reproductive system that can be passed to offspring. In the near future, further studies are needed to assess the real effects of PCBs exposure at low concentrations for prolonged exposure in workplaces and specific indoor environments

Gut Microbiome Dysbiosis in Antibiotic-Treated COVID-19 Patients is Associated with Microbial Translocation and Bacteremia

Although microbial populations in the gut microbiome are associated with COVID-19 severity, a causal impact on patient health has not been established. Here we provide evidence that gut microbiome dysbiosis is associated with translocation of bacteria into the blood during COVID-19, causing life-threatening secondary infections. We first demonstrate SARS-CoV-2 infection induces gut microbiome dysbiosis in mice, which correlated with alterations to Paneth cells and goblet cells, and markers of barrier permeability. Samples collected from 96 COVID-19 patients at two different clinical sites also revealed substantial gut microbiome dysbiosis, including blooms of opportunistic pathogenic bacterial genera known to include antimicrobial-resistant species. Analysis of blood culture results testing for secondary microbial bloodstream infections with paired microbiome data indicates that bacteria may translocate from the gut into the systemic circulation of COVID-19 patients. These results are consistent with a direct role for gut microbiome dysbiosis in enabling dangerous secondary infections during COVID-19

Gender affects early psychomotor milestones and long-term neurodevelopment of preterm infants

Temporal differences of neurodevelopmental milestones' achievement are commonly taken into account in preterm infant assessment during the first year of life, especially when minor or none neurological signs arise from clinical examination. The influence of gender on neurodevelopment in preterm infants was examined by a milestones-based neurological approach. Two-hundred twenty-seven moderate and late preterm and full-term infants (51.9% male), without evidence of severe white matter insults, were early assessed by classic neurological examination. Griffiths Mental Developmental Scale was used at long-term observation. Children were sorted into three gestational age groups (Group 1: 32–33 weeks, Group 2: 34–36 weeks, and Group 3: ≥37 weeks) and compared according to their neurodevelopmental pathways and gender. The achievement of head control was slightly earlier in females. The pointing, one of the main communicative hand gesture, appeared significantly earlier in females across all the groups. At the Griffiths Scale, the majority of gender-related differences emerged in personal–social and eye and hand coordination subscale. An independent role of male gender was evidenced in personal–social, language, and eye and hand coordination subscales. Gender and gestational age likely addressed different temporal profiles of neurodevelopment in early and late assessments in preterm and full-term infants. The role of gender and gestational age on these findings has been discussed. Highlights: A classic, milestones-based neurological evaluation disclosed different developmental profiles in males versus females preterm infants. Moderate preterm and full-term infants showed similar developmental profiles. Gender and gestational age may influence neurodevelopmen

<i>Trichoderma</i> Species Problematic to the Commercial Production of <i>Pleurotus</i> in Italy: Characterization, Identification, and Methods of Control

Nine isolates of Trichoderma were obtained from the diverse phases of compost preparation and the production of different commercial varieties of oyster mushrooms Pleurotus spp. with the apparent presence of green mould disease. The isolates were morphologically and genetically characterized. Molecular fingerprinting indicated that the isolates belonged to the species T. pleuroticola and T. harzianum. In order to identify control measures, changes in temperature, pH, and application of fungicides were tested on the present isolates, using known Trichoderma biocontrol strains as a reference. Fungicide effects on the growth of Pleurotus isolates were also assessed. The optimal growth temperatures were 25 °C for Trichoderma isolates and 28 °C for Pleurotus isolates, and Trichoderma always grew faster than Pleurotus. In particular, a reduction of about 30% was recorded for some of the Trichoderma isolates when comparing the colony growth at 25 and 28 °C. Trichoderma isolates developed well within a wide pH range, with the best growth occurring between pH 5 and 7, whereas Pleurotus preferred more alkaline conditions (pH 8 to 9). Prochloraz and metrafenone were found to inhibit Trichoderma growth with different dose responses that did not affect the growth of Pleurotus spp. In particular, metrafenone was the most effective active ingredient that inhibited the majority of the Trichoderma isolates (1–25% growth reduction)

Trichoderma Strains and Metabolites Selectively Increase the Production of Volatile Organic Compounds (VOCs) in Olive Trees

Plants emit volatile organic compounds (VOCs) that induce metabolomic, transcriptomic, and behavioral reactions in receiver organisms, including insect pollinators and herbivores. VOCs’ composition and concentration may influence plant-insect or plant-plant interactions and affect soil microbes that may interfere in plant-plant communication. Many Trichoderma fungi act as biocontrol agents of phytopathogens and plant growth promoters. Moreover, they can stimulate plant defense mechanisms against insect pests. This study evaluated VOCs’ emission by olive trees (Olea europaea L.) when selected Trichoderma fungi or metabolites were used as soil treatments. Trichoderma harzianum strains M10, T22, and TH1, T. asperellum strain KV906, T. virens strain GV41, and their secondary metabolites harzianic acid (HA), and 6-pentyl-α-pyrone (6PP) were applied to olive trees. Charcoal cartridges were employed to adsorb olive VOCs, and gas chromatography mass spectrometry (GC-MS) analysis allowed their identification and quantification. A total of 45 volatile compounds were detected, and among these, twenty-five represented environmental pollutants and nineteen compounds were related to olive plant emission. Trichoderma strains and metabolites differentially enhanced VOCs production, affecting three biosynthetic pathways: methylerythritol 1-phosphate (MEP), lipid-signaling, and shikimate pathways. Multivariate analysis models showed a characteristic fingerprint of each plant-fungus/metabolite relationship, reflecting a different emission of VOCs by the treated plants. Specifically, strain M10 and the metabolites 6PP and HA enhanced the monoterpene syntheses by controlling the MEP pathway. Strains GV41, KV906, and the metabolite HA stimulated the hydrocarbon aldehyde formation (nonanal) by regulating the lipid-signaling pathway. Finally, Trichoderma strains GV41, M10, T22, TH1, and the metabolites HA and 6PP improve aromatic syntheses at different steps of the shikimate pathway

The Application of Trichoderma Strains or Metabolites Alters the Olive Leaf Metabolome and the Expression of Defense-Related Genes

Biocontrol fungal strains of the genus Trichoderma can antagonize numerous plant pathogens and promote plant growth using different mechanisms of action, including the production of secondary metabolites (SMs). In this work we analyzed the effects of repeated applications of selected Trichoderma strains or SMs on young olive trees on the stimulation of plant growth and on the development of olive leaf spot disease caused by Fusicladium oleagineum. In addition, metabolomic analyses and gene expression profiles of olive leaves were carried out by LC-MS Q-TOF and real-time RT-PCR, respectively. A total of 104 phenolic compounds were detected from olive leave extracts and 20 were putatively identified. Targeted and untargeted approaches revealed significant differences in both the number and type of phenolic compounds accumulated in olive leaves after Trichoderma applications, as compared to water-treated plants. Different secoiridoids were less abundant in treated plants than in controls, while the accumulation of flavonoids (including luteolin and apigenin derivatives) increased following the application of specific Trichoderma strain. The induction of defense-related genes, and of genes involved in the synthesis of the secoiridoid oleuropein, was also analyzed and revealed a significant variation of gene expression according to the strain or metabolite applied

Biodegradable Mulch Films and Bioformulations Based on <i>Trichoderma</i> sp. and Seaweed Extract Differentially Affect the Metabolome of Industrial Tomato Plants

The use of biostimulants and biofilms in agriculture is constantly increasing, as they may support plant growth and productivity by improving nutrient absorption, increasing stress resilience and providing sustainable alternatives to chemical management practices. In this work, two commercial products based on Trichoderma afroharzianum strain T22 (Trianum P®) and a seaweed extract from Ascophyllum nodosum (Phylgreen®) were tested on industrial tomato plants (Solanum lycopersicum var. Heinz 5108F1) in a field experiment. The effects of single and combined applications of microbial and plant biostimulants on plants grown on two different biodegradable mulch films were evaluated in terms of changes in the metabolic profiles of leaves and berries. Untargeted metabolomics analysis by LC-MS Q-TOF revealed the presence of several significantly accumulated compounds, depending on the biostimulant treatment, the mulch biofilm and the tissue examined. Among the differential compounds identified, some metabolites, belonging to alkaloids, flavonoids and their derivatives, were more abundant in tomato berries and leaves upon application of Trichoderma-based product. Interestingly, the biostimulants, when applied alone, similarly affected the plant metabolome compared to control or combined treatments, while significant differences were observed according to the mulch biofilm applied

Breakthrough Cancer Pain: Preliminary Data of The Italian Oncologic Pain Multisetting Multicentric Survey (IOPS-MS)

Introduction: An ongoing national multicenter survey [Italian Oncologic Pain multiSetting Multicentric Survey (IOPS-MS)] is evaluating the characteristics of breakthrough cancer pain (BTP) in different clinical settings. Preliminary data from the first 1500 cancer patients with BTP enrolled in this study are presented here. Methods: Thirty-two clinical centers are involved in the survey. A diagnosis of BTP was performed by a standard algorithm. Epidemiological data, Karnofsky index, stage of disease, presence and sites of metastases, ongoing oncologic treatment, and characteristics of background pain and BTP and their treatments were recorded. Background pain and BTP intensity were measured. Patients were also questioned about BTP predictability, BTP onset (≤10 or >10 min), BTP duration, background and BTP medications and their doses, time to meaningful pain relief after BTP medication, and satisfaction with BTP medication. The occurrence of adverse reactions was also assessed, as well as mucosal toxicity. Results: Background pain was well controlled with opioid treatment (numerical rating scale 3.0 ± 1.1). Patients reported 2.5 ± 1.6 BTP episodes/day with a mean intensity of 7.5 ± 1.4 and duration of 43 ± 40 min; 977 patients (65.1%) reported non-predictable BTP, and 1076 patients (71.7%) reported a rapid onset of BTP (≤10 min). Higher patient satisfaction was reported by patients treated with fast onset opioids. Conclusions: These preliminary data underline that the standard algorithm used is a valid tool for a proper diagnosis of BTP in cancer patients. Moreover, rapid relief of pain is crucial for patients’ satisfaction. The final IOPS-MS data are necessary to understand relationships between BTP characteristics and other clinical variables in oncologic patients. Funding: Molteni Farmaceutici, Italy