Mycorrhizas for the Sustainable Managment of Agroecosystems. Field Studies on molecular and functional biodiversity of indigenous and introduced arbuscular mycorrhizal fungi.

We investigated the functional significance of extraradical mycorrhizal networks produced by geographically different isolates of the arbuscular mycorrhizal fungal (AMF) species Glomus mosseae and Glomus intraradices. A two-dimensional experimental system was utilized to visualize and quantify intact extraradical mycelium (ERM) spreading from Medicago sativa roots. Growth, P and N nutrition were assessed in M. sativa plants grown in microcosms. The AMF isolates were characterised by differences in extent and interconnectedness of ERM. Phenotypic fungal variables, such as total hyphal length, hyphal density, hyphal length per mm of total or colonized root length were positively correlated with M. sativa growth response variables, such as total shoot biomass and plant P content. The utilization of an experimental system, where size, growth rate, viability and interconnectedness of ERM extending from mycorrhizal roots are easily quantified under realistic conditions, allows the simultaneous evaluation of different isolates and provides data with a predictive value for selection of efficient AMF. Diversity and structure of arbuscular mychorrizal (AM) fungal communities were investigated within the root systems of eight plant species (Medicago sativa, Trifolium alexandrinum, Cicer arietinum, Sorghum halepense, Lolium perenne, Rubdeckia hirta, Matricaria camomilla, Plantago major) growing in natural soil microcosms, by using the terminal restriction fragment length polymorphism (T-RFLP) on the small-subunit rRNA (SSUrRNA). Since important differences were shown by T-RFLP patterns of the different plant species, the cloning and sequencing were accurately designed on a subset of representative samples (database T-RFLP). The AM fungal sequences were phylogenetically analyzed and used to highlight monophyletic sequence types. The AM fungal community – host plant interactions were investigated in monocultures and the growth responses were compared with non-mychorrizal controls. The AMF community composition was strongly influenced by the plant species, and plant species belonging to the same family showed closer AMF patterns. Detailed analysis of the eight plant species revealed that the AMF community diversity varied significantly, and that all the eight plant taxa hosted multiple fungal lineage phylotypes. Furthermore, some AMF phylotypes were systematically found within the roots of all analyzed plant species, while others were exclusively in symbiosis with certain host plants. A degree of fungal isolate - plant interaction was observed. This works suggests that not only the plant species but also the family may play a role in determining the AMF community diversity and structure. In addition, the T-RFLP relative abundance was comparable with the cloning results, although on a small subset of samples. The inter- and intraspecific genetic diversity was studied by using the small subunit nuclear ribosomal rRNA gene (SSU) and the internal transcribed spacer (ITS) on multisporal samples of four geografically different isolates belonging to Glomus mosseae and Glomus Intraradices species, introduced as fungal inoculum in the experimental site. AMF diversity and structure within the roots of eight plant species, grew in monocoltures, were assessed by using T-RFLP, which, with an appropriate sequences database, and T-RFLP simulations (database T-RFLP) (Dickie and Fitzjohn, 2007), allowed the detection of root samples abundantly colonized by the indigenous G. intraradices and G. mosseae. Afterwards, by using the intraradical DNA samples, inter- and intraspecific diversity of the indigenous G. intraradices and G. mosseae was evaluate on SSU and ITS regions. The sequences that in the SSU BLAST research were referred as G. intraradices e G. mosseae were exclusively used for the subsequent ITS analysis. The low variability shown by the SSU rDNA sequences did not allow the discrimination of indigenous and introduced AMF species, even tough two groups within G. intraradices were found. Only G. intraradices IMA5 isolates could be disciminated by using the rDNA SSU. ITS sequences of the introduced AMF was variable between and within isolates, except for G. intraradices IMA6. G. intraradices IMA6 ITS sequencse were not different from the other G. intraradices isolates, and showed a low intra-isolate variability. Thus, ITS is a suitable region to discriminate G. intraradices IMA5, and the G. mosseae isolates, from the natural AMF communities in the experimental site. A field study was carried out in order to assess the inter- and intraspecific functional diversity of geographically different isolates of the AMF species Glomus intraradices and Glomus mosseae, of indigenous (NN) and introduced (MIX) isolate mixtures in symbiosis with the plant species Cicer arietinum, Trifolium alexandrinum and Medicago sativa. A number of fungal and ecophysiological functional traits were taken into account. The inoculum infectivity was measured, in Lactuca sativa microcosm, as degree of root colonization, number of infective units, total number of entry points and spore density. The natural experimental soil infectivity was assessed, by using the same fungal functional traits and the degree of colonization of weeds. In the field, the degree of affinity between AM fungi and host plants was assessed in terms of degree of colonization, of the different AM fungal isolates in C. arietinum, T. alexandrinum and M. sativa. Ecophysiological parameters, such as biomass production and P and N uptake, in T. alexandrinum e M. sativa, were studied. Furthermore, on C. arietinum, the grain production, the number of chickpeas per plant and the collar diameter were assessed. The different AM fungal isolates used as inocula, showed a significantly higher infectivity compared to the natural experimental soil, and inter- and intraspecific differences were observed. Spore density did not change among different isolates, except in the case of NN. The infection-unit-with-entry-points parameter was chosen as the most appropriate character in order to evaluate the inoculum potential. The AM colonization degree of weeds was influenced by host plant species. After one month’s growth, mycorrhizal inoculation caused a significantly effect on root colonization in all plant species, although only in the case of M. sativa the ecophysiological parameters were affected. In the case of C. arietinum, mycorrhizal inoculation induced a significant effect on grain production and N content. Some interspecific functional differences were observed in G. mosseae IMA1 vs G. intraradices IMA5. In T. alexandrinum and M. sativa, AMF inoculum affected root colonization, even after two years’ growth in the case of M. sativa. Inter- and intraspecific differences in growth parameters, as well as in P and N uptake, were found in T. alexandrinum and M. sativa. G. mosseae AZ225C isolate, both in T. alexandrinum and M. sativa, and the introduced mix in symbiosis with M. sativa, showed highest functional performaces. A differential degree of host affinity was found in the field, enabling the selection of the most efficient plant – fungus combinations in terms of P and N uptake and growth parameters