Plant-bacteria interactions: identification, characterization and localization of beneficial bacterial endophytes isolated from Vitis vinifera cv. Glera

This thesis reports the results obtained during the three years PhD course focused on the study of culturable bacterial endophytes of Vitis vinifera Glera and their beneficial activities. The study, part of a large project named “EndoFlorVit project” (FEARS-UE and Regione Del Veneto), aims at investigate the biodiversity and the plant growth promoting activities of culturable endophytes isolated from Glera grapevine in vineyards of Conegliano-Valdobbiadene DOCG production area. This thesis reports the results of the isolation of culturable bacterial endophytes from surface-sterilized Glera grapevine tissues. 381 culturable strains were successfully isolated from roots, shoots and leaves of Vitis vinifera Glera, sampled from six different vineyards in the Conegliano-Valdobbiadene DOCG area (Veneto, Italy). The community was investigated by Amplified Ribosomal DNA Restriction Analysis (ARDRA) and nucleotide sequencing to identify the most representative genera of the Glera microbiome. Approximately 30% of the isolates belonged to the genus Bacillus, which was the most represented; other genera such as Staphylococcus, Microbacterium, Paenibacillus, Curtobacterium, Stenotrophomonas, Variovorax, Micrococcus and Agrococcus were identified. The composition of the communities isolated from different vineyards was not the same; moreover we reported that endophyte biodiversity inside plants was influenced by the season. After molecular characterization, we focused our attention to investigate the plant growth promoting abilities of the culturable strains. Using biochemical tests we assayed some of the most important and effective properties in order to investigate the physiology of these bacteria and identify some strains that could have a strong beneficial effect on plant nutrition and growth. In this work, using Carboxymethyl Cellulose degradation test, we demonstrated that 85 strains secreted cellulolytic enzymes; this trait could confer to these bacteria an advantage in plant penetration and tissues colonization. By qualitative biochemical assays, we demonstrated that many strains were able to solubilize phosphate (127 strains), produce ammonia (142 strains) and secrete siderophores (155 strains). Using the colorimetric Salkowsky assay, we determined that 17 strains produced the phytohormone Indol 3-acetic acid (IAA) ; using Arabidopsis thaliana DR5:GUS, where the β-glucuronidase reporter gene is expressed under control of a IAA-induced promoter, we demonstrated that bacterial IAA was recognised by the Arabidopsis plants and caused morphological alteration on the root architecture. It is known that IAA is not the only bacterial molecule that influence the plant growth and the root morphology. To investigate the effects of the Glera endophytes on the plant morphology we used the model system Arabidopsis thaliana co-cultured in vitro with every single strain. Morphological parameters (root length, surface and diameter) were measured by a software and statistically analysed by cluster analysis. Plants were thus clustered according with the effect of the strain on the root parameters, demonstrating the effects of the strains on roots morphology. In particular, some strains caused an enhanced root length displaying a plant growth promotion effect. By this large-scale characterization we selected two of the most promising strains, one for the putative plant growth effect (Pantoea agglomerans GL83) and one as putative biocontrol agent (Bacillus licheniformis GL174) that were transformed with a DNA cassette containing a gfp reporter gene. Using Laser Scanning Confocal Microscopy, we demonstrate the colonization of the stem endosphere of Glera cuttings 20 and 30 days after the inoculum of the fluorescent strains. In this thesis, the evidences of the colonization are reported demonstrating that Pantoea agglomerans GL83 and Bacillus licheniformis GL174 are true Glera endophytes able to colonize cuttings when re-inoculated. After we demonstrated that B. licheniformis GL174 is a true endophyte of Glera, we investigated the biocontrol abilities of the strain. Results of antagonism tests against plant pathogenic fungi are shown, demonstrating that the strain is able to reduce and inhibit the mycelia growth of the grapevine pathogens Phaeoacremonium aleophilum, Phaeomoniella spp., Botryosphaeria spp., Botrytis cinerea and for the more generic plant pathogens Sclerotinia sclerotiorum and Phytophthora infestans. After that, we demonstrated by PCR and DNA sequencing that GL174 has the operons coding for lipopeptide synthetase enzymes and that the strain produced cyclic lipopeptide belonging to surfactin and lichenysin families. These molecules with antimicrobial effects were identified and characterized by mass spectrometric analysis and the results are reported and discussed in this thesis. The genome of the strain was thus sequenced to better investigate the strain and the sequences were preliminary analysed identifying the presence of many genes coding for lytic enzymes. The production of lipopeptides, the inhibition of fungal growth and the ability to colonize inner tissues of Glera indicated GL174 as a good candidate for biocontrol. Another aspect of endophyte-plant symbiosis that is not well explained is the ecology of these bacterial strains and the interactions between different bacterial species in the rhizosphere and inside plants are poorly described. Bacteria-bacteria interactions are likely to be an important factor that defines the composition of the endophyte community. The study of these interactions is essential to understand plant-bacteria relationship; moreover, the study of how the native community of rhizobacteria and endophytes may change after the inoculation of other bacteria is important for a safe and aware use of commercial biocontrol or bio-fertilizer products containing endophytes. A preliminary ecological study is presented in this thesis: some ecological aspects of endophyte of grapevine, endophytes of other plant species and some bacteria commercialized as beneficial strains, called “biofector strains” were analysed using tomato, a model plant for agriculture and horticulture. In this work, we demonstrated that these strains were able to colonize tomato plants and the population densities of the diverse tissues sampled are reported in the Chapter number 5. This work, that is still ongoing, aims to evaluate the impact of these endophytic strains investigating if the inoculum of the bacteria on tomato plants leads to a different endophytic community in comparison to uninoculated plants. This study is essential to unravel the effects of the bio effector strains on natural endophytic populations of plants: from peeled stems of all the inoculated plants the total DNA was extracted; this material will be used as template for the 16S rDNA amplification of all the endophytes present in the plants. Many sets of primers are being tested to select the best combination for this approach. The amplicons will be sequenced and analysed to determine if the community of endophytes has been changed by the inoculum of the external endophytic bio-effector strain. In conclusion, the results presented in this thesis are an overview of the composition of the endophytic community of Glera plants cultivated in the Conegliano-Valdobbiadene DOCG area. The isolation of the culturable strains has provided a large collection of bacteria that, during the PhD course, was characterized investigating plant growth promoting activities and bacteria effects on plant morphology considering different mechanisms underlying plant-microbe interactions. The evidence obtained in this work describes a clear and novel background to understand Glera endophytes biology and ecology and, when confirmed in planta by field trials, will permit the selection of some efficient strains to use as safe endophytic bio-fertilizers and biocontrol agents, for a sustainable production of Glera grapes

Beneficial Bacteria Isolated from Grapevine Inner Tissues Shape Arabidopsis thaliana Roots

We investigated the potential plant growth-promoting traits of 377 culturable endophytic bacteria, isolated from Vitis vinifera cv. Glera, as good biofertilizer candidates in vineyard management. Endophyte ability in promoting plant growth was assessed in vitro by testing ammonia production, phosphate solubilization, indole-3-acetic acid (IAA) and IAA-like molecule biosynthesis, siderophore and lytic enzyme secretion. Many of the isolates were able to mobilize phosphate (33%), release ammonium (39%), secrete siderophores (38%) and a limited part of them synthetized IAA and IAA-like molecules (5%). Effects of each of the 377 grapevine beneficial bacteria on Arabidopsis thaliana root development were also analyzed to discern plant growth-promoting abilities (PGP) of the different strains, that often exhibit more than one PGP trait. A supervised model-based clustering analysis highlighted six different classes of PGP effects on root architecture. A. thaliana DR5::GUS plantlets, inoculated with IAA-producing endophytes, resulted in altered root growth and enhanced auxin response. Overall, the results indicate that the Glera PGP endospheric culturable microbiome could contribute, by structural root changes, to obtain water and nutrients increasing plant adaptation and survival. From the complete cultivable collection, twelve promising endophytes mainly belonging to the Bacillus but also to Micrococcus and Pantoea genera, were selected for further investigations in the grapevine host plants towards future application in sustainable management of vineyards

Next Generation Sequencing of Ancient Fungal Specimens: The Case of the Saccardo Mycological Herbarium

Despite their essential role in the environment, the number of known fungal species is still low compared to the recent estimates of the fungal biodiversity, principally because of their often cryptic or ambiguous morphological traits. Recent studies have reported that the number of fungal DNA sequences deposited in public DNA databases and representing correctly a species appears dramatically low (approx. 120,000) when compared with the estimated total number of species (approx. from 2.2 to 3.8 million). Thus, the linkage of curated DNA sequence data to expertly identified voucher specimens is of fundamental importance to fill the present gap between the different sizes of described and sequenced fungal diversity. To this purpose, the mycological herbarium collections are considered an important source for fungal DNA-barcoding, and collection-based sequencing is a relevant priority for the coming decades. Unfortunately, ancient herbarium samples have both time and conservation related DNA damages, besides exogenous DNA contamination, that make nucleic acid extraction and amplification challenging. Here, we present the results of DNA extraction, ITS2 amplification and Illumina MiSeq sequencing of 36 specimens from the Saccardo Mycological Herbarium that were collected in the late XIX century and assigned to the genus Peziza. High-throughput sequencing was chosen as an alternative to the conventional Sanger- and cloning-based methods to overcome the high fragmentation of the ancient DNA and the massive occurrence of non-target DNA from fungal contaminants. Our approach has permitted to assign ITS2 sequences to 23 out of the 36 specimens studied in this work, thus providing a univocal DNA sequence for those one century old samples. Furthermore, the ITS2 sequence analysis has permitted a taxonomic study of the samples that has resulted in a revaluation of 5 samples at the species level and 18 samples at genus or higher level. Our results highlight the possibility to apply the technique presented in this work also to the old and more precious type specimens in order to relate a DNA sequence to the species distinctive sample, coupling the traditional morphological description of the species with a DNA sequence

Plant-bacteria interactions: identification, characterization and localization of beneficial bacterial endophytes isolated from Vitis vinifera cv. Glera

This thesis reports the results obtained during the three years PhD course focused on the study of culturable bacterial endophytes of Vitis vinifera Glera and their beneficial activities. The study, part of a large project named “EndoFlorVit project” (FEARS-UE and Regione Del Veneto), aims at investigate the biodiversity and the plant growth promoting activities of culturable endophytes isolated from Glera grapevine in vineyards of Conegliano-Valdobbiadene DOCG production area. This thesis reports the results of the isolation of culturable bacterial endophytes from surface-sterilized Glera grapevine tissues. 381 culturable strains were successfully isolated from roots, shoots and leaves of Vitis vinifera Glera, sampled from six different vineyards in the Conegliano-Valdobbiadene DOCG area (Veneto, Italy). The community was investigated by Amplified Ribosomal DNA Restriction Analysis (ARDRA) and nucleotide sequencing to identify the most representative genera of the Glera microbiome. Approximately 30% of the isolates belonged to the genus Bacillus, which was the most represented; other genera such as Staphylococcus, Microbacterium, Paenibacillus, Curtobacterium, Stenotrophomonas, Variovorax, Micrococcus and Agrococcus were identified. The composition of the communities isolated from different vineyards was not the same; moreover we reported that endophyte biodiversity inside plants was influenced by the season. After molecular characterization, we focused our attention to investigate the plant growth promoting abilities of the culturable strains. Using biochemical tests we assayed some of the most important and effective properties in order to investigate the physiology of these bacteria and identify some strains that could have a strong beneficial effect on plant nutrition and growth. In this work, using Carboxymethyl Cellulose degradation test, we demonstrated that 85 strains secreted cellulolytic enzymes; this trait could confer to these bacteria an advantage in plant penetration and tissues colonization. By qualitative biochemical assays, we demonstrated that many strains were able to solubilize phosphate (127 strains), produce ammonia (142 strains) and secrete siderophores (155 strains). Using the colorimetric Salkowsky assay, we determined that 17 strains produced the phytohormone Indol 3-acetic acid (IAA) ; using Arabidopsis thaliana DR5:GUS, where the β-glucuronidase reporter gene is expressed under control of a IAA-induced promoter, we demonstrated that bacterial IAA was recognised by the Arabidopsis plants and caused morphological alteration on the root architecture. It is known that IAA is not the only bacterial molecule that influence the plant growth and the root morphology. To investigate the effects of the Glera endophytes on the plant morphology we used the model system Arabidopsis thaliana co-cultured in vitro with every single strain. Morphological parameters (root length, surface and diameter) were measured by a software and statistically analysed by cluster analysis. Plants were thus clustered according with the effect of the strain on the root parameters, demonstrating the effects of the strains on roots morphology. In particular, some strains caused an enhanced root length displaying a plant growth promotion effect. By this large-scale characterization we selected two of the most promising strains, one for the putative plant growth effect (Pantoea agglomerans GL83) and one as putative biocontrol agent (Bacillus licheniformis GL174) that were transformed with a DNA cassette containing a gfp reporter gene. Using Laser Scanning Confocal Microscopy, we demonstrate the colonization of the stem endosphere of Glera cuttings 20 and 30 days after the inoculum of the fluorescent strains. In this thesis, the evidences of the colonization are reported demonstrating that Pantoea agglomerans GL83 and Bacillus licheniformis GL174 are true Glera endophytes able to colonize cuttings when re-inoculated. After we demonstrated that B. licheniformis GL174 is a true endophyte of Glera, we investigated the biocontrol abilities of the strain. Results of antagonism tests against plant pathogenic fungi are shown, demonstrating that the strain is able to reduce and inhibit the mycelia growth of the grapevine pathogens Phaeoacremonium aleophilum, Phaeomoniella spp., Botryosphaeria spp., Botrytis cinerea and for the more generic plant pathogens Sclerotinia sclerotiorum and Phytophthora infestans. After that, we demonstrated by PCR and DNA sequencing that GL174 has the operons coding for lipopeptide synthetase enzymes and that the strain produced cyclic lipopeptide belonging to surfactin and lichenysin families. These molecules with antimicrobial effects were identified and characterized by mass spectrometric analysis and the results are reported and discussed in this thesis. The genome of the strain was thus sequenced to better investigate the strain and the sequences were preliminary analysed identifying the presence of many genes coding for lytic enzymes. The production of lipopeptides, the inhibition of fungal growth and the ability to colonize inner tissues of Glera indicated GL174 as a good candidate for biocontrol. Another aspect of endophyte-plant symbiosis that is not well explained is the ecology of these bacterial strains and the interactions between different bacterial species in the rhizosphere and inside plants are poorly described. Bacteria-bacteria interactions are likely to be an important factor that defines the composition of the endophyte community. The study of these interactions is essential to understand plant-bacteria relationship; moreover, the study of how the native community of rhizobacteria and endophytes may change after the inoculation of other bacteria is important for a safe and aware use of commercial biocontrol or bio-fertilizer products containing endophytes. A preliminary ecological study is presented in this thesis: some ecological aspects of endophyte of grapevine, endophytes of other plant species and some bacteria commercialized as beneficial strains, called “biofector strains” were analysed using tomato, a model plant for agriculture and horticulture. In this work, we demonstrated that these strains were able to colonize tomato plants and the population densities of the diverse tissues sampled are reported in the Chapter number 5. This work, that is still ongoing, aims to evaluate the impact of these endophytic strains investigating if the inoculum of the bacteria on tomato plants leads to a different endophytic community in comparison to uninoculated plants. This study is essential to unravel the effects of the bio effector strains on natural endophytic populations of plants: from peeled stems of all the inoculated plants the total DNA was extracted; this material will be used as template for the 16S rDNA amplification of all the endophytes present in the plants. Many sets of primers are being tested to select the best combination for this approach. The amplicons will be sequenced and analysed to determine if the community of endophytes has been changed by the inoculum of the external endophytic bio-effector strain. In conclusion, the results presented in this thesis are an overview of the composition of the endophytic community of Glera plants cultivated in the Conegliano-Valdobbiadene DOCG area. The isolation of the culturable strains has provided a large collection of bacteria that, during the PhD course, was characterized investigating plant growth promoting activities and bacteria effects on plant morphology considering different mechanisms underlying plant-microbe interactions. The evidence obtained in this work describes a clear and novel background to understand Glera endophytes biology and ecology and, when confirmed in planta by field trials, will permit the selection of some efficient strains to use as safe endophytic bio-fertilizers and biocontrol agents, for a sustainable production of Glera grapes.Questo lavoro di tesi presenta e discute i risultati sperimentali ottenuti durante il corso di dottorato in Biologia Evoluzionistica presso la Scuola di Dottorato di Bioscienze e Biotecnologie dell’Università degli Studi di Padova. Questa ricerca, parte di un progetto più ampio denominato “EndoFlorVit” (FEARS-UE e Regione del Veneto), ha come scopo la caratterizzazione molecolare e lo studio delle proprierà di promozione della crescita vegetale e di bio-controllo di batteri endofiti isolati da piante di Vitis vinifera di cultivar Glera, coltivate nell’area di produzione del Prosecco di Conegliano-Valdobbiadene DOCG. Le comunità di microrganismi isolate sono state studiate utilizzando la tecnica ARDRA (Amplified Ribosomal DNA Restriction Analysis) e il sequenziamento di porzioni del 16S rDNA identificando che circa il 30% dei ceppi isolati appartengono al genere Bacillus, il quale risulta essere il più rappresentato nelle piante campionate. Altri generi a cui appartengono numerosi ceppi isolati sono Staphylococcus, Microbacterium, Paenibacillus, Curtobacterium, Stenotrophomonas, Variovorax, Micrococcus e Agrococcus. La composizione delle comunità endofite isolate da differenti piante non è uniforme: esse variano nei differenti vigneti e sono inoltre influenzate dalla stagionalità. Oltre alla descrizione dei ceppi isolati, in questo lavoro di tesi sono presentati e discussi i risultati dello studio delle proprietà di promozione della crescita vegetale dei ceppi isolati. Utilizzando saggi biochimici sono state investigate alcune delle principali attività benefiche che hanno un effetto di miglioramento della nutrizione vegetale. Mediante il test di degradazione della carbossimetil-cellulosa sono stati identificati 85 ceppi capaci di secernere enzimi degradanti la cellulosa: questa capacità può conferire ai ceppi che la esprimono un vantaggio nella colonizzazione dei tessuti vegetali facilitando loro il processo di penetrazione nei tessuti della pianta. Attraverso saggi biochimici qualitativi è stato possibile dimostrare che numerosi ceppi sono in grado di solubilizzare il fosfato insolubile (127 ceppi), produrre ammoniaca (142 ceppi) e secernere siderofori (155 ceppi). Inoltre, utilizzando il saggio di Salkowski, è stato dimostrato che 17 ceppi batterici producono l’ormone vegetale Acido 3-indolacetico (IAA). Per investigare l’effetto dei ceppi produttori di IAA sulla fisiologia e morfologia della pianta è stato utilizzata la pianta modello Arabidopsis thaliana DR5:GUS, una linea mutante esprimenti l’enzima β-glucuronidasi sotto controllo di un promotore indotto da IAA. Utilizzando questo sistema sperimentale è stato dimostrato che l’IAA prodotto dai batteri viene riconosciuto dalle piante di Arabidopsis e causa alterazioni alla morfologia e architettura radicale. È noto tuttavia che l’IAA non è l’unica molecola batterica che influenza la crescita vegetale e la morfologia vegetale. In tal senso, è stato valutato l’effetto di ciascun ceppo isolato sulla pianta Arabidopsis thaliana Col-0 wild tipe. Tre parametri morfologici della radice (lunghezza superficie e diametro) sono considerati e analizzati statisticamente mediante cluster analysis. Le piante quindi sono state raggruppate secondo gli effetti che i batteri hanno provocato sull’apparato radicale assegnando in questo modo a ciascun ceppo l’effetto corrispondente. In questo modo è stato dimostrato che alcuni ceppi hanno causato allungamento della radice, un effetto ascrivibile come promozione della crescita. Da questa caratterizzazione ed analisi su larga scala dei ceppi isolati sono stati selezionati due ceppi particolarmente promettenti per la promozione della crescita vegetale e per il biocontrollo: Pantoea agglomerans GL83 e Bacillus licheniformis GL174. Questi due ceppi sono stati trasformati geneticamente con un costrutto contenente il gene che esprime la proteina fluorescente GFP. Utilizzando tecniche di microscopia confocale è stato dimostrato che entrambi i ceppi fluorescenti sono in grado di ricolonizzare talee di vite Glera quando inoculate e che persistono all’interno dei tessuti del fusto dopo 20 e 30 giorni dopo l’inoculo. In questa tesi quindi sono presentate le evidenze sperimentali che questi due ceppi, Pantoea agglomerans GL83 e Bacillus licheniformis GL174, sono veri endofiti di vite Glera e risultano quindi interessanti per le loro proprietà benefiche. Dopo aver confermato che GL174 è endofita della vite Glera, il ceppo è stato investigato per evidenziarne alcune capacità utili per il biocontrollo dei patogeni. In questo lavoro di tesi sono presentati i risultati di saggi di antagonismo in vitro nei quali il ceppo in esame ha effetto di inibizione della crescita del micelio di alcuni funghi patogeni della vite (Phaeoacremonium aleophilum, Paeomoniella spp., Botryosphaeria spp., Botrytis cinerea) e di due patogeni più generalisti (Sclerotinia sclerotiorum e Phytophtora infestans). Lo studio del ceppo GL174 si è successivamente focalizzato sulla capacità del batterio di produrre i lipopeptidi ciclici, una classe di molecole con forte attività antimicrobica e surfattante. Per prima cosa, attraverso PCR e sequenziamento del DNA, è stata identificata la presenza nel genoma batterico degli operoni codificanti per alcune lipopepide sintetasi, gli enzimi deputati alla sintesi di queste molecole. La produzione di lipopeptidi è stata successivamente dimostrata utilizzando tecniche di spettrometria di massa; le quali hanno permesso di identificare le molecole prodotte e di ricostruirne la struttura chimica. La produzione di queste molecole e la capacità inibitoria di funghi patogeni rendono il ceppo GL174 un buon candidato come agente di biocontrollo nella coltivazione della vite e di altre specie economicamente rilevanti. L’ ecologia dei batteri endofiti è un tema che ancora non è stato del tutto investigato all’interno dello studio dell’interazione tra piante ed endofiti. Inoltre, le interazioni che avvengono a livello di rizosfera ed endosfera non sono ancora ben descritte ma evidenze sperimentali suggeriscono che esse siano un fattore importante nella definizione della composizione delle comunità endofite. Lo studio di come un inoculo batterico esogeno può modificare la composizione della comunità nativa di endofiti è essenziale per un uso consapevole di formulati commerciali a base di batteri endofiti. In questa tesi viene quindi presentato un lavoro preliminare che analizza l’ecologia di ceppi isolati da Glera, ceppi isolati da altre specie vegetali, e batteri commercializzati come biostimolanti. La colonizzazione di piante di pomodoro, pianta modello per lo studio delle specie orticole, da parte di questi ceppi microbici è stata dimostrata e quantificata per radici, fusto e foglie di piante coltivate per 3 e 5 settimane. Questo lavoro ha come scopo inoltre l’analisi delle comunità di endofiti di queste piante inoculate per confrontarne la composizione con piante non inoculate. Lo studio, che è ancora in corso, ha comportato l’estrazione del DNA totale della endosfera di porzioni di fusto; da questo DNA saranno amplificati i 16S rDNA dei batteri endofiti presenti e sequenziati con tecniche di NGS. In conclusione, i risultati presentati in questa tesi descrivono la composizione delle comunità di endofiti coltivabili isolate da piante di vite Glera della zona del Prosecco Conegliano-Valdobbiadene DOCG. L’isolamento di tali batteri ha fornito una larga collezione di ceppi batterici, le cui proprietà benefiche che promuovono la crescita vegetale e gli effetti dei batteri sulla morfologia radicale di Arabidopsis thaliana sono stati analizzati e presentati criticamente coinvolgendo più aspetti importanti nell’interazione pianta-endofiti. I risultati ottenuti in questo lavoro descrivono le proprietà di alcuni ceppi isolati da Glera che, quando confermato da prove sperimentali in campo, potranno essere utilizzati in sicurezza come agenti endofiti di biofertilizzazione e/o biocontrollo nella produzione dell’uva Glera e di altre specie vegetali economicamente importanti

Is the bacterial endophyte community, living in Glera (Vitis vinifera) plants, active in biocontrol?

This project aims at characterizing the molecular and functional properties of endophytic bacteria that colonize Vitis vinifera L. cv. Glera (Prosecco) in order to select plant growth-promoting bacteria as biocontrol agents to stimulate plant growth and improve soil and plant health. This work is focused on two antimicrobial abilities of endophytes: cyclic lipopeptides (LPs) and siderophores production. LPs are small cyclic peptides, belonging to fengycin, surfactin and mycosubtilin families, with known antimicrobial activities. The analysis of genes coding for LP synthetase, the enzyme which synthesize LPs, has demonstrated that 20% of the analyzed strains carry the gene encoding for at least one of the LP synthetases investigated. In addition, CAS-agar microbiological assay indicated that some of the isolated endophytic strains were able to produce siderophores. Future work will be required to verify the antimicrobial/antifungal activity in vivo and in vitro of selected strains and to determine their ability to re-colonize and move within plant tissues, in order to develop biocontrol growth-enhancing inoculants for Glera grapevine cultivations