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Digital archive of theses discussed at the University of Pisa


Thesis etd-12052016-122425

Thesis type
Tesi di dottorato di ricerca
Thesis title
Biofortification of food plants by mycorrhizal symbionts: the role of mycelial networks
Academic discipline
Course of study
tutor Prof.ssa Giovannetti, Manuela
  • anastomosing ability
  • arbuscular mycorrhizal fungi
  • extraradical mycelium
  • functional diversity
  • gene expression
Graduation session start date
Arbuscular mycorrhizas are widespread mutualistic symbioses between plant roots and fungi of the phylum Glomeromycota. Arbuscular mycorrhizal fungi (AMF) colonize root cells forming typical tree-like structures, the arbuscules, representing the main site where nutrient exchanges between host plants and fungal symbionts occur. The association is essential for plant ecosystem functioning because the great majority of plant species depends on AMF for soil mineral nutrient uptake and translocation. The improved mineral nutrient supply may directly contribute to biofortification and to increase the accumulation of secondary metabolites in plant-derived foods. AMF ability to enhance the biosynthesis of compounds with nutraceutical value in leaves, roots, and fruits of plants used for human nutrition suggests that the inoculation of selected AM fungal species and isolates may represent a suitable biotechnological tool to produce safe and healthy food. The most important AM fungal structure for plant nutrition is represented by the extraradical mycelium spreading from mycorrhizal roots into the surrounding soil, which is able to uptake mineral nutrients and to transfer them to root cells.
The aim of this project is to gain knowledge on the role played by mycorrhizal symbionts in mineral nutrition and biofortification of food plants, by assessing the relationships among structural and functional features of extraradical mycelium (ERM) produced in vivo. ERM structural traits, such as extent, density, interconnectedness and viability, may represent significant parameters of fungal efficiency, since they measure and characterize the nutrient-absorbing network, where specific receptors and transporters of mineral elements are expressed.
By using a microcosm system, this work investigated whether fungal genotype can affect patterns of interconnections and structural traits of ERM, by comparing three AM fungal species growing in symbiosis with five host plants. A Funneliformis coronatus isolate showed low ability to form interconnected ERM, with percentages of perfect hyphal fusions of 1.2-7.7, whereas those of Rhizoglomus irregulare and Funneliformis mosseae ranged from 25.8-48 to 35.6-53.6, respectively. F. coronatus consistently formed low interconnected networks, both in symbiotic and asymbiotic stages, and showed the occurrence of hyphal self-incompatibility (up to 21% of hyphal contacts). Differences in interconnectedness were correlated with different density and extent values characterizing ERM produced by the three isolates and suggesting the need of further studies on such structural traits, which may affect fungal interactions with the environment and fungal-plant nutritional relationships.
The ability of the previously characterized ERM produced by the three AMF species F. mosseae, F. coronatus and R. irregulare, to transfer the macronutrients P and N from soil to host plants was assessed in vivo, in symbiosis with Cichorium intybus var. foliosum. Moreover, the relationship between nutrient transfer and fungal ability to form appressoria, key colonization structures connecting extra- and intraradical mycelium, was studied in the same experimental system. Results revealed a differential ability of the three AMF to transfer P and N to the host plant: most of the mycorrhizal efficiency parameters (plant biomass, P and N uptake) recorded for F. coronatus showed low values, compared with those obtained for F. mosseae and R. irregulare. A positive correlation was found between P and N content of mycorrhizal plants and the number of entry points, which was significantly lower in roots colonized by F. coronatus, compared with the other isolates.
With the aim of performing gene expression analyses on the characterized ERM obtained in vivo, the bi-dimensional experimental system (sandwich system) was modified by wrapping Cichorium intybus var. foliosum colonized roots in a nylon net to facilitate ERM collection. ERM produced by F. mosseae, R. irregulare and F. coronatus was used for RNA extraction and the experimental system was validated by analysing cDNA for ammonium transporter (AMT)s gene expression by RT-qPCR, using specific primers designed on available (R. irregulare) and new (F. mosseae and F. coronatus) AMT sequences. The analyses showed that AMT genes were differentially expressed in the three AMF and that relative transcription levels in F. coronatus ERM were significantly lower compared with the other two AMF. These results suggest a relationship among nutrient uptake and transfer and ERM structural traits, indicating that network levels of density, extent and interconnectedness may play an important role in determining AMF functional diversity.
The structural and functional traits of ERM formed by F. mosseae and R. irregulare in symbiosis with Cichorium intybus var. foliosum have been here assessed in ERM connected to living host roots. Nevertheless, it is not known whether ERM viability and ability to establish new mycorrhizal symbioses may be maintained after host shoot death or harvest. Further time-course experiments showed that ERM was able to maintain viability and colonization ability for at least five months after host shoot detachment, suggesting that ERM lifespan is not limited by the presence of viable host plants. Such findings could have important implication for AMF dispersal and survival and for the maintenance of mycorrhizal inoculum potential of soils.