Tesi etd-09302025-111431
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Tipo di tesi
Corso di Dottorato (D.M.226/2021)
Autore
PAPPALETTERE, LIVIA
URN
etd-09302025-111431
Titolo
Studying new polymicrobial inoculants to improve herbaceous and arboreal crops’ yield and quality
Settore scientifico disciplinare
AGR/03
Corso di studi
Ph.D. in Agrobioscienze - Ph.D. in Agrobioscienze
Relatori
relatore BARTOLINI, SUSANNA
Parole chiave
- agamic propagation
- hydrophonic
- Plant growth–promoting bacteria
- Root exudate profiling
- Seed priming and rooting
- Sustainable agriculture
Data inizio appello
12/02/2026;
Disponibilità
parziale
Riassunto analitico
Abstract
Plant growth–promoting bacteria (PGPB) are increasingly regarded as a sustainable alternative to chemical fertilizers, rooting agents, and pesticides, providing multiple benefits to both annual and perennial crops. Their activity is often mediated by the production of phytohormones such as auxins, by biocontrol compounds, and by their ability to colonize plant tissues and interact with root exudates. This thesis explored the potential of different auxin-producing bacterial strains—Azospirillum baldaniorum Sp245, A. brasilense Sp7 and Cd, Methylobacterium symbioticum SB0023, and Bacillus spp. (B. subtilis, B. amyloliquefaciens, B. licheniformis)—to improve the performance of tomato and olive plants across multiple experimental systems. The work integrated physiological assays, histological studies, metabolomic profiling, strain-specific molecular tracking, and genomic characterization, with the aim of building a comprehensive framework for the use of PGPB in sustainable agriculture.
In tomato (Solanum lycopersicum cvs. Canestrino di Lucca and Pisanello), seed priming with seven microbial strains resulted in improved germination rates, and seedling development. Treated plants exhibited higher fresh and dry biomass, increased root number and length, and enhanced overall growth under greenhouse conditions. Hydroponic trials confirmed that bacterial inoculation at early developmental stages promoted both root and shoot growth. The most efficient IAA producers were Azospirillum strains, which strongly stimulated root proliferation, while Bacillus amyloliquefaciens and B. licheniformis preferentially enhanced stem growth. A positive correlation was consistently observed between IAA production and root/shoot traits, confirming the central role of bacterial auxins in mediating growth promotion. These results provide evidence that microbial priming and inoculation could serve as effective, chemical-free tools to boost tomato plants establishment in organic and low-input systems.
In olive (Olea europaea cv. Leccino), experiments focused on two critical stages: vegetative propagation via cuttings and growth in nursery conditions. Semi-hardwood cuttings collected in different seasons were treated with A. baldaniorum Sp245 and compared with indole-3-butyric acid (IBA), the synthetic auxin widely used in nursery production. Histological assays revealed that Azospirillum treatment triggered cellular changes comparable to those induced by IBA, particularly in spring cuttings and when grown in organic substrates such as Elepot®. The bacterial inoculum also improved the rooting process, supporting its potential as an alternative rooting agent for organic nurseries where synthetic compounds are prohibited. In parallel, young nursery plants treated periodically with bacterial suspensions showed improved hypogeal and epigeal growth compared to controls, further confirming the biostimulant role of A. baldaniorum Sp245 beyond the propagation stage.
A central objective of the thesis was the development of molecular tools to monitor PGPB colonization in planta. To this end, a novel tetraplex qPCR assay was designed and optimized for the simultaneous detection of three Azospirillum strains and Methylobacterium symbioticum. Primer–probe sets were developed through a multi-step bioinformatic pipeline and validated against plant DNA backgrounds to ensure specificity and sensitivity. The assay demonstrated robust linearity and sensitivity down to 30–40 genome copies per reaction, making it the first multiplex platform enabling strain-resolved quantification of these taxa in olive tissues. When applied to greenhouse trials, the assay revealed that A. brasilense Sp7 established at the highest densities in roots (~10³–10⁴ genome equivalents g⁻¹ fresh tissue), while A. baldaniorum Sp245 and A. brasilense Cd were present at lower levels. Soil-applied M. symbioticum showed limited persistence, whereas foliar application successfully colonized leaves, with little systemic movement to other tissues. These findings demonstrate that colonization outcomes are shaped by both strain identity and inoculation method.
To complement molecular analyses, the chemical footprint of PGPB inoculation was investigated through root exudate profiling. Using thermal desorption GC–MS with in situ HMDS derivatization, the first chemically resolved characterization of olive root exudates under microbial treatments was achieved. Four main metabolite classes—organic acids (glycolic, lactic, hydroxybutyric), carbohydrates (glucose, levoglucosan, aldonic acid lactones), glycerol, and seven inositol isomers—were consistently detected. Microbial treatments significantly modulated these profiles, with the Azospirillum–Methylobacterium consortium (MIX A) inducing the strongest increases in acids, sugars, and inositols, suggesting that microbial inoculants not only promote plant growth but also remodel root exudation patterns that may further influence rhizosphere interactions. The thesis also included the genomic characterization of Bacillus subtilis BS101, a strain with demonstrated antifungal activity against Fusarium oxysporum. Hybrid genome assembly produced a high-quality draft (4.13 Mb, 97.3% complete) revealing biosynthetic gene clusters for lipopeptides such as surfactin, iturin, and bacillomycin, as well as genes associated with biofilm formation and plant growth promotion. Antimicrobial resistance determinants were also identified. The availability of this genome expands the molecular basis for understanding the dual role of B. subtilis as a biocontrol and biostimulant agent, supporting its application in sustainable agriculture.
Overall, this thesis provides an integrated view of PGPB action across multiple plant systems and experimental scales. Combining physiological, histological, metabolomic, molecular, and genomic approaches, this work advances the understanding of plant–microbe interactions and supports the employment of PGPB-based strategies as viable, sustainable tools for improving plant growth and development, in two representative herbaceous and woody crops namely tomato and olive.
Plant growth–promoting bacteria (PGPB) are increasingly regarded as a sustainable alternative to chemical fertilizers, rooting agents, and pesticides, providing multiple benefits to both annual and perennial crops. Their activity is often mediated by the production of phytohormones such as auxins, by biocontrol compounds, and by their ability to colonize plant tissues and interact with root exudates. This thesis explored the potential of different auxin-producing bacterial strains—Azospirillum baldaniorum Sp245, A. brasilense Sp7 and Cd, Methylobacterium symbioticum SB0023, and Bacillus spp. (B. subtilis, B. amyloliquefaciens, B. licheniformis)—to improve the performance of tomato and olive plants across multiple experimental systems. The work integrated physiological assays, histological studies, metabolomic profiling, strain-specific molecular tracking, and genomic characterization, with the aim of building a comprehensive framework for the use of PGPB in sustainable agriculture.
In tomato (Solanum lycopersicum cvs. Canestrino di Lucca and Pisanello), seed priming with seven microbial strains resulted in improved germination rates, and seedling development. Treated plants exhibited higher fresh and dry biomass, increased root number and length, and enhanced overall growth under greenhouse conditions. Hydroponic trials confirmed that bacterial inoculation at early developmental stages promoted both root and shoot growth. The most efficient IAA producers were Azospirillum strains, which strongly stimulated root proliferation, while Bacillus amyloliquefaciens and B. licheniformis preferentially enhanced stem growth. A positive correlation was consistently observed between IAA production and root/shoot traits, confirming the central role of bacterial auxins in mediating growth promotion. These results provide evidence that microbial priming and inoculation could serve as effective, chemical-free tools to boost tomato plants establishment in organic and low-input systems.
In olive (Olea europaea cv. Leccino), experiments focused on two critical stages: vegetative propagation via cuttings and growth in nursery conditions. Semi-hardwood cuttings collected in different seasons were treated with A. baldaniorum Sp245 and compared with indole-3-butyric acid (IBA), the synthetic auxin widely used in nursery production. Histological assays revealed that Azospirillum treatment triggered cellular changes comparable to those induced by IBA, particularly in spring cuttings and when grown in organic substrates such as Elepot®. The bacterial inoculum also improved the rooting process, supporting its potential as an alternative rooting agent for organic nurseries where synthetic compounds are prohibited. In parallel, young nursery plants treated periodically with bacterial suspensions showed improved hypogeal and epigeal growth compared to controls, further confirming the biostimulant role of A. baldaniorum Sp245 beyond the propagation stage.
A central objective of the thesis was the development of molecular tools to monitor PGPB colonization in planta. To this end, a novel tetraplex qPCR assay was designed and optimized for the simultaneous detection of three Azospirillum strains and Methylobacterium symbioticum. Primer–probe sets were developed through a multi-step bioinformatic pipeline and validated against plant DNA backgrounds to ensure specificity and sensitivity. The assay demonstrated robust linearity and sensitivity down to 30–40 genome copies per reaction, making it the first multiplex platform enabling strain-resolved quantification of these taxa in olive tissues. When applied to greenhouse trials, the assay revealed that A. brasilense Sp7 established at the highest densities in roots (~10³–10⁴ genome equivalents g⁻¹ fresh tissue), while A. baldaniorum Sp245 and A. brasilense Cd were present at lower levels. Soil-applied M. symbioticum showed limited persistence, whereas foliar application successfully colonized leaves, with little systemic movement to other tissues. These findings demonstrate that colonization outcomes are shaped by both strain identity and inoculation method.
To complement molecular analyses, the chemical footprint of PGPB inoculation was investigated through root exudate profiling. Using thermal desorption GC–MS with in situ HMDS derivatization, the first chemically resolved characterization of olive root exudates under microbial treatments was achieved. Four main metabolite classes—organic acids (glycolic, lactic, hydroxybutyric), carbohydrates (glucose, levoglucosan, aldonic acid lactones), glycerol, and seven inositol isomers—were consistently detected. Microbial treatments significantly modulated these profiles, with the Azospirillum–Methylobacterium consortium (MIX A) inducing the strongest increases in acids, sugars, and inositols, suggesting that microbial inoculants not only promote plant growth but also remodel root exudation patterns that may further influence rhizosphere interactions. The thesis also included the genomic characterization of Bacillus subtilis BS101, a strain with demonstrated antifungal activity against Fusarium oxysporum. Hybrid genome assembly produced a high-quality draft (4.13 Mb, 97.3% complete) revealing biosynthetic gene clusters for lipopeptides such as surfactin, iturin, and bacillomycin, as well as genes associated with biofilm formation and plant growth promotion. Antimicrobial resistance determinants were also identified. The availability of this genome expands the molecular basis for understanding the dual role of B. subtilis as a biocontrol and biostimulant agent, supporting its application in sustainable agriculture.
Overall, this thesis provides an integrated view of PGPB action across multiple plant systems and experimental scales. Combining physiological, histological, metabolomic, molecular, and genomic approaches, this work advances the understanding of plant–microbe interactions and supports the employment of PGPB-based strategies as viable, sustainable tools for improving plant growth and development, in two representative herbaceous and woody crops namely tomato and olive.
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