429 research outputs found
Plant-bacteria interactions: identification, characterization and localization of beneficial bacterial endophytes isolated from Vitis vinifera cv. Glera
No full textThis 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
Caratterizzazione molecolare di funghi della Collezione Saccardo conservata nell'erbario dell'Orto Botanico di Padova
No full textInsights into the grapevine inner tissue life: characterization of the bacterial endophyte community
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Analisi della comunità di endofiti batterici isolata da Vitis vinifera per la selezione di potenziali agenti di biocontrollo.
No full textValutazione di proprietà biochimiche di endofiti batterici e del loro ruolo nella promozione della crescita in Arabidopsis thaliana
No full textStudy of the effects of compost and its microbiota on the resistance of Solanum lycopersicum to water stress
No full textreservedMolti microorganismi che vivono nel terreno interagiscono con le piante fornendo
loro diversi effetti benefici. La biodiversità del suolo è dunque molto importante
per la crescita delle piante e una sua riduzione può provocare un calo delle relazioni
benefiche tra piante e microbiota. Il compost, ricco di nutrienti, ma anche di
microorganismi, può allora rappresentare un’eccellente soluzione per sopperire a
tale problema e di conseguenza aiutare gli organismi vegetali a resistere ad
eventuali stress ambientali di tipo abiotico. In particolare con l’esperimento che
abbiamo condotto abbiamo voluto determinare se il compost aiuti le piante di
pomodoro a resistere maggiormente allo stress idrico. I dati ottenuti ci hanno
permesso di affermare che le piante hanno giovato di in un terreno contenente il
compost quando sottoposte ad uno scarso apporto di acqua. Il lavoro ci dimostra
pertanto, non solo che l’utilizzo del compost aiuti le piante nella crescita, ma anche
che probabilmente è il microbiota in lui presente che vive nella rizosfera a conferire
una maggiore resistenza allo stress indotto
Is the bacterial endophyte community, living in Glera (Vitis vinifera) plants, active in biocontrol?
No full textThis 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
Molecular and genetic traits of Bacillus licheniformis GL174, a culturable endophyte of Vitis vinifera cv. Glera.
No full textSustainable aquaculture: new technology for treatment, disposal and possible reuse of suspended solids present in the effluent of intensive land based fish farms.
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