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Digital archive of theses discussed at the University of Pisa


Thesis etd-12052016-120517

Thesis type
Tesi di dottorato di ricerca
Thesis title
The production of nutraceutical compounds in food plants as affected by mycorrhizal symbionts and associated microbiota
Academic discipline
Course of study
tutor Dott.ssa Agnolucci, Monica
  • Arbuscular mycorrhizal fungi
  • Mycorrhizosphere
  • Phytochemicals
  • Plant Growth Promoting Bacteria (PGPB)
  • Spore-associated bacteria
Graduation session start date
The general objective of this PhD thesis was the study of the molecular and functional diversity of mycorrhizal symbionts and associated microbiota for their potential use as a natural source of beneficial biofertilizers and bioenhancers. Firstly, the diversity of bacterial communities associated with the spores of six arbuscular mycorrhizal (AM) fungi (AMF) isolates, belonging to different genera and species was investigated by PCR denaturing gradient gel electrophoresis (DGGE). The results showed that the six AMF isolates displayed diverse bacterial community profiles unrelated with their taxonomic position, suggesting that each AMF isolate recruits on its spores a different microbiota. After sequence analysis of DGGE relevant bands, bacterial species affiliated with Actinomycetales, Bacillales, Pseudomonadales, Burkholderiales, Rhizobiales and with Mollicutes-related endobacteria (Mre) were identified. By using culture-dependent methods the microbiota strictly associated to Rhizophagus intraradices spores was isolated and functionally characterized. The strains with best potential plant growth promoting (PGP) activities were molecularly identified by 16S rDNA sequence analysis. In particular, 374 bacterial strains belonging to different functional groups—actinobacteria, spore-forming, chitinolytic and N2-fixing bacteria— were isolated: among them 122 strains were screened for their potential PGP activities. The most common PGP trait was represented by P solubilisation from phytate (69.7%), followed by siderophore production (65.6%), mineral P solubilization (49.2%) and IAA production (42.6%). About 76% of actinobacteria and 65% of chitinolytic bacteria displayed multiple PGP activities. The 19 strains showing the best potential PGP activities were further identified as Sinorhizobium meliloti, Streptomyces spp., Arthrobacter phenanthrenivorans, Nocardiodes albus, Bacillus sp. pumilus group, Fictibacillus barbaricus and Lysinibacillus fusiformis. Ten of such PGP strains were tested alone or in combination with AMF in order to evaluate their effect on maize growth and P uptake. The results showed that AMF colonization produced large plant growth responses, while bacterial inoculants increased the length density of AMF hyphae in soil and also root dry weight and root P content. Afterwards, a greenhouse experiment was carried out to investigate the ability of the AM symbiont R. intraradices and its associated PGP bacteria S. meliloti TSA41 and Streptomyces sp. W43N to improve the antioxidant activity, the production of health-promoting phytochemicals and plant growth regulators in two different cultivars of sweet basil (Ocimum basilicum) under commercial growth conditions: Tigullio, with green leaves and Dark Opal with purple leaves. Dark Opal was more responsive than Tigullio to the dual inoculation treatment, showing higher antioxidant activity, anthocyanin and rosmarinic acid leaf levels. Finally, the mechanisms by which AMF and PGP bacteria differentially modulate the biochemical pathways leading to the synthesis of the relevant phytochemicals, were investigated for the first time. The genes encoding key enzymes of the biochemical pathways leading to the production of rosmarinic acid (RA), a bioactive compound showing multiple beneficial properties for human health, were differentially expressed in Ocimum basilicum cv. Tigullio (sweet basil) inoculated with the AM symbiont R. intraradices and/or its associated PGP bacteria S. meliloti TSA41 and Streptomyces sp. W43N. In particular, results showed that the selected PGP bacteria were able to trigger the overexpression of tyrosine amino-transferase (TAT), hydroxyphenylpyruvate reductase (HPPR) and p-coumaroyl shikimate 3′-hydroxylase isoform 1 (CS3′H iso1) genes, 5.7- fold, 2-fold and 2.4-fold, respectively, in O. basilicum leaves. By contrast, inoculation with R. intraradices triggered TAT upregulation and HPPR and CS3′H iso1 downregulation.