Non-pathogenic endophytic bacteria can affect host plants growth and health by inducing changes in plant physiological responses and metabolic activities, improving their response to biotic and abiotic stresses. At the same time, supporting the interaction between plants and plant growth-promoting bacteria (PGPB) can improve the qualitative and nutraceutical properties of agri-food products. The aim of the present research work was to identify microbial endophytes with a possible application as biocontrol and biofortification agents. In the first part of the work, the volatile organic compounds (VOCs) produced by Streptomyces sp. SA51 were tested as biocontrol agent of Fusarium oxysporum f.sp. lactucae (FOL) race 1, both in vitro and against the development of fusarium wilt symptoms in lettuce plants. In vitro, an 8% inhibition of fungal growth was observed. In lettuce plants the health conditions were estimated through the McKinney index (MKI), which showed a decrease of 25% when plants infected with FOL grew in the presence of bacterial VOCs. Even plants not infected with FOL benefited from the presence of bacterial VOCs, in fact the MKI decreased by 48 %. To identify the VOCs produced by Streptomyces sp. SA51, an extraction of volatile compounds with HS-SPME technique and a GC–MS analysis were performed. The analysis revealed the presence of several compounds with already known antifungal properties, such as Germacrene D and Phenylethyl Alcohol. In the second part, the interactions between three species of baby leaf vegetables destined for the fresh cut market (red mustard, lettuce and bull’s blood) and three endophytic bacteria (Streptomyces sp. SA51, Pseudomonas sp. DLS65 and Pantoea sp. S1) were investigated. The purpose was to evaluate whether these interactions could be exploited to increase the polyphenol content of the plants. For each plant species, the seeds were treated separately with the three bacterial cell suspensions. The total polyphenolic content of the plants was extracted and estimated with the Folin-Ciocâlteu assay. Significant differences were observed in lettuce plants treated with Streptomyces sp. SA51 and in lettuce and red mustard treated with Pantoea sp. S1. Then, LC-ESI-IT-MS/MS analysis was performed to assess the qualitative variations of the polyphenolic content. The last part of the research was conducted at the Austrian Institute of Technology (AIT). The vitamin B12 is an essential nutrient for humans that can only be produced by certain bacteria and archaea. The aim of the study was to identify endophytic bacteria able of synthetising vitamin B12 de novo and to verify whether they could be exploited to enrich edible plants with this vitamin. An in-silico analysis of 69 bacterial genomes was carried out to verify the presence of the metabolic pathway of vitamin B12. Based on the presence and the completeness of the pathway in their genomes, 30 strains were selected to test their actual ability to produce vitamin B12 de novo through an HPLC-DAD analysis on pure culture extracts. Ten strains showed to be able to produce detectable amounts of vitamin B12. The best candidates were further tested to evaluate their efficacy in producing vitamin B12 in lettuce plants. Vitamin B12 was extracted from the edible parts of plants and purified with immunoaffinity columns. Detection and quantification of vitamin B12 were performed by HPLC-DAD analysis. One bacterial strain was proved to be able of producing vitamin B12 in lettuce plants. In conclusion, it is possible to assess that some endophytic bacteria have a great potential to be exploited both as biological control against fungal pathogens and to implement the content of molecules of nutraceutical interest in plants.

I batteri endofiti non patogeni possono influenzare la crescita e la salute delle piante inducendo cambiamenti nelle loro risposte fisiologiche e attività metaboliche, migliorandone la risposta agli stress biotici e abiotici. Inoltre, l'interazione tra piante e batteri promotori della crescita (PGPB) può migliorare le proprietà qualitative e nutraceutiche dei prodotti agroalimentari. Dunque, lo scopo del presente lavoro di ricerca è identificare endofiti potenzialmente applicabili come agenti di biocontrollo e biofortificazione. Nella prima parte del lavoro, i composti organici volatili (VOC) prodotti da Streptomyces sp. SA51 sono stati testati come agenti di biocontrollo del Fusarium oxysporum f.sp. lactucae (FOL) razza 1, sia in vitro che contro lo sviluppo dei sintomi da fusariosi in piante di lattuga. In vitro è stata osservata un'inibizione della crescita fungina dell'8%. Nelle piante di lattuga le condizioni di salute sono state valutate attraverso l'indice McKinney (MKI), che ha mostrato una diminuzione del 25% quando le piante infettate da FOL sono cresciute in presenza di VOC batterici. Anche le piante non infettate da FOL hanno beneficiato della presenza dei VOC, infatti in questo caso l'MKI è diminuito del 48%. Per identificare i VOC prodotti da Streptomyces sp. SA51, sono state eseguite un'estrazione dei composti volatili con tecnica HS-SPME e un'analisi con GC-MS. L’analisi ha rivelato la presenza di diversi composti, come Germacrene D e Phenylethyl Alcohol, già noti per le loro proprietà antimicotiche. Nella seconda parte, è stata osservata l’interazione tra tre specie di baby leaf destinate al mercato della IV gamma (red mustard, lattuga e bull’s blood) e tre batteri endofiti (Streptomyces sp. SA51, Pseudomonas sp. DLS65 e Pantoea sp. S1). Lo scopo era valutare se gli endofiti potessero essere sfruttati per aumentare il contenuto di polifenoli delle piante. Per ciascuna specie vegetale, i semi sono stati trattati separatamente con le tre sospensioni di cellule batteriche. Il contenuto polifenolico totale delle piante è stato estratto e stimato con il saggio Folin-Ciocâlteu. Sono state osservate differenze significative nelle piante di lattuga trattate con Streptomyces sp. SA51 e in lattuga e red mustard trattate con Pantoea sp. S1. Quindi, è stata eseguita l'analisi LC-ESI-IT-MS/MS per valutare le variazioni qualitative del contenuto polifenolico. Nell’ultima parte della ricerca lo scopo era quello di identificare batteri endofiti in grado di sintetizzare de novo la vitamina B12, un nutriente essenziale per l'uomo che può essere prodotto solo da alcuni batteri e archaea, e verificare se questi endofiti possono essere sfruttati per arricchire le piante edibili con vitamina B12. È stata effettuata un'analisi in-silico su 69 genomi batterici per verificare la presenza della via metabolica della vitamina B12. Sulla base di questa analisi, sono stati selezionati 30 ceppi per testare la loro effettiva capacità di produrre vitamina B12 de novo, attraverso un'analisi HPLC-DAD su estratti di coltura pura. Dieci ceppi hanno dimostrato di essere in grado di produrre quantità rilevabili di vitamina B12. I migliori produttori sono stati ulteriormente testati per valutare la loro efficacia nella produzione di vitamina B12 in piante di lattuga. La vitamina è stata estratta dalle parti commestibili delle piante e purificata con colonne di immunoaffinità. Il rilevamento e la quantificazione sono stati effettuati mediante analisi HPLC-DAD, e un ceppo batterico si è rivelato in grado di produrre vitamina B12 in piante di lattuga. In conclusione, è possibile affermare che alcuni batteri endofiti hanno un grande potenziale sia nel controllo biologico sia nell’implementare il contenuto di molecole di interesse nutraceutico nelle piante.

APPLICAZIONE DI ENDOFITI MICROBICI NEL BIOCONTROLLO DELLE MALATTIE FUNGINE E PER LA PRODUZIONE DI ALIMENTI FUNZIONALI / Sara Pipponzi , 2023 Apr 21. 35. ciclo, Anno Accademico 2021/2022.

APPLICAZIONE DI ENDOFITI MICROBICI NEL BIOCONTROLLO DELLE MALATTIE FUNGINE E PER LA PRODUZIONE DI ALIMENTI FUNZIONALI

PIPPONZI, SARA
2023

Abstract

Non-pathogenic endophytic bacteria can affect host plants growth and health by inducing changes in plant physiological responses and metabolic activities, improving their response to biotic and abiotic stresses. At the same time, supporting the interaction between plants and plant growth-promoting bacteria (PGPB) can improve the qualitative and nutraceutical properties of agri-food products. The aim of the present research work was to identify microbial endophytes with a possible application as biocontrol and biofortification agents. In the first part of the work, the volatile organic compounds (VOCs) produced by Streptomyces sp. SA51 were tested as biocontrol agent of Fusarium oxysporum f.sp. lactucae (FOL) race 1, both in vitro and against the development of fusarium wilt symptoms in lettuce plants. In vitro, an 8% inhibition of fungal growth was observed. In lettuce plants the health conditions were estimated through the McKinney index (MKI), which showed a decrease of 25% when plants infected with FOL grew in the presence of bacterial VOCs. Even plants not infected with FOL benefited from the presence of bacterial VOCs, in fact the MKI decreased by 48 %. To identify the VOCs produced by Streptomyces sp. SA51, an extraction of volatile compounds with HS-SPME technique and a GC–MS analysis were performed. The analysis revealed the presence of several compounds with already known antifungal properties, such as Germacrene D and Phenylethyl Alcohol. In the second part, the interactions between three species of baby leaf vegetables destined for the fresh cut market (red mustard, lettuce and bull’s blood) and three endophytic bacteria (Streptomyces sp. SA51, Pseudomonas sp. DLS65 and Pantoea sp. S1) were investigated. The purpose was to evaluate whether these interactions could be exploited to increase the polyphenol content of the plants. For each plant species, the seeds were treated separately with the three bacterial cell suspensions. The total polyphenolic content of the plants was extracted and estimated with the Folin-Ciocâlteu assay. Significant differences were observed in lettuce plants treated with Streptomyces sp. SA51 and in lettuce and red mustard treated with Pantoea sp. S1. Then, LC-ESI-IT-MS/MS analysis was performed to assess the qualitative variations of the polyphenolic content. The last part of the research was conducted at the Austrian Institute of Technology (AIT). The vitamin B12 is an essential nutrient for humans that can only be produced by certain bacteria and archaea. The aim of the study was to identify endophytic bacteria able of synthetising vitamin B12 de novo and to verify whether they could be exploited to enrich edible plants with this vitamin. An in-silico analysis of 69 bacterial genomes was carried out to verify the presence of the metabolic pathway of vitamin B12. Based on the presence and the completeness of the pathway in their genomes, 30 strains were selected to test their actual ability to produce vitamin B12 de novo through an HPLC-DAD analysis on pure culture extracts. Ten strains showed to be able to produce detectable amounts of vitamin B12. The best candidates were further tested to evaluate their efficacy in producing vitamin B12 in lettuce plants. Vitamin B12 was extracted from the edible parts of plants and purified with immunoaffinity columns. Detection and quantification of vitamin B12 were performed by HPLC-DAD analysis. One bacterial strain was proved to be able of producing vitamin B12 in lettuce plants. In conclusion, it is possible to assess that some endophytic bacteria have a great potential to be exploited both as biological control against fungal pathogens and to implement the content of molecules of nutraceutical interest in plants.
APPLICATION OF MICROBIAL ENDOPHYTES IN THE BIOCONTROL OF FUNGAL DISEASES AND FOR THE PRODUCTION OF FUNCTIONAL FOOD
21-apr-2023
MANDRIOLI, Mauro
STEFANI, Emilio
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11380/1303015
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