• AMF functional diversity
• biosphere reserve
• local AMF diversity
• native AMF community composition

The present thesis aimed at characterizing native communities and single isolates of arbuscular mycorrhizal fungi (AMF) occurring in an experimental field representing a “hot spot” of AMF diversity located inside the UNESCO Biosphere Reserve called “Selva Pisana”, near San Piero a Grado (Pisa), a natural reserve on the east coast of Italy, in the northwest of Tuscany. The functional and molecular study of native communities provided new insights concerning the impact of AMF local species richness and community composition variations on host plant growth and nutrition. Moreover, the isolation of the largest possible number of AMF living in the protected experimental site, followed by functional and molecular characterization, represented a key step towards their potential use as natural sources of beneficial biofertilizers and bioenhancers for sustainable agriculture. A conceptual framework, summarizing the objective aforementioned was pursued through the following integrated set of tasks: a) characterization of the chemical-physical properties and of the mycorrhizal inoculum potential (MIP) of the experimental site soil collected in different sampling points; b) characterization of molecular and functional diversity of AMF communities occurring in the same soil samples; c) isolation and reproduction in trap-cultures of AMF occurring in the soil samples; d) morphological and molecular characterization of the reproduced AMF; e) functional characterization of the reproduced AMF for infectivity, by assessing spore dormancy and germination, extent of asymbiotic mycelium, formation of appressoria and intraradical colonization; and f) functional characterization of infective AMF isolates for efficiency, by assessing mycorrhizal responses of different plant species.
In Chapter 1, analyses of soil samples revealed that, in spite of an overall low variability of the experimental soils, physical and chemical properties differed among samples. Biological characterization of the soil through MIP analysis, determined as percentages of root colonization of test plants, showed that in most samples values were higher than 40%.
In Chapter 2, the native AMF communities of 12 soil samples were functionally characterized using different host plants. Growth responses and N and P shoot content significantly differed among native AMF treatments within the three tested plant species (Allium cepa, Capsicum annuum and Lactuca sativa). Among AMF native treatments, 2 (named 2 and 22) showed significantly different host responses, despite the similarity of soil properties.
In Chapter 3, the molecular characterization of the two root native AMF communities (2 and 22) is reported. Results indicated large variations when considering both the two native AMF treatments and the identity of host plants. Sequences obtained matched those of AMF belonging to eight genera of Glomeromycotina. Analysis of sequences showed the occurrence of phylotypes with high similarity to Claroideoglomus etunicatum, Dominikia iranica, Funneliformis geosporus Funneliformis mosseae, Paraglomus laccatum, Rhizoglomus irregulare and Racocetra fulgida.
In Chapter 4, the isolation and identification in trap-cultures of native AMF from the different experimental soils are described. A total of 36 trap cultures were established with different host plants, Zea mays, Petroselinum crispum and spontaneous plants from the seed bank of the original site. Different morphotypes were reproduced in cultures and characterized by morphological and molecular analyses. Isolates were assigned to F. mosseae, C. etunicatum, R. fulgida, D. aurea, F. geosporum and Septoglomus constrictum and maintained in pure cultures to obtain spores for functional characterization.
In Chapter 5, infectivity, defined as the ability of an isolate to quickly establish an extensive mycorrhizal colonization in the roots of test plants, was evaluated on selected AMF isolated from the experimental field. Different experiments were performed to assess spore germination and hyphal growth, using either the “sandwich” method or multiwell plates. In F. mosseae isolates, sporocarp germination rates ranged between 52% and 89%, whereas spores of R. fulgida showed 69% germination percentage. The ability of AMF isolates to form appressoria giving rise to entry points colonizing roots was variable, and values ranged from 1.6 to 6.1 entry points cm root-1. Accordingly, mycorrhizal colonization ranged from 2% to 31%.
Finally, in Chapter 6, efficiency, as the ability of AMF to improve host nutritional status, was evaluated on selected AMF isolates, by four different experimental systems: in sandwich systems, with the host plant Cichorium intybus, both in the greenhouse and in growth chamber; in the greenhouse on soil-based substrate, with the host plants C. intybus, Medicago sativa and Lactuca sativa; in the greenhouse on soil-based substrate, with the host plants Fragaria vesca and M. sativa. The selected isolates showed differential behaviour in symbiosis with diverse host plants, suggesting host preference phenomena. In particular, an isolate of F. mosseae was able to enhance biomass in both L. sativa and M. sativa and nutrient uptake in M. sativa, whereas other isolates of the same species induced larger growth in F. vesca and C. intybus. The isolation, characterization, and evaluation of single native species from AMF communities of the “hot spot” field carried out in this work allowed the detection of symbionts establishing different functional relationships with different host plants. Such data may be useful for a correct management of AMF biotechnology, to obtain positive effects when specific AMF isolates are inoculated on the diverse crop species and varieties. Future research could provide knowledge with predictive value on AMF traits involved in symbiotic efficiency, necessary to improve the successful use of such inoculants as bioenhancers in sustainable agriculture.