Roberto Borriello

Disclosing arbuscular mycorrhizal fungal biodiversity in soil through a land-use gradient using a pyrosequencing approach.

The biodiversity of arbuscular mycorrhizal fungi (AMF) communities present in five Sardinian soils (Italy) subjected to different land-use (tilled vineyard, covered vineyard, pasture, managed meadow and cork-oak formation) was analysed using a pyrosequencing-based approach for the first time. Two regions of the 18S ribosomal RNA gene were considered as molecular target. The pyrosequencing produced a total of 10924 sequences: 6799 from the first and 4125 from the second target region. Among these sequences, 3189 and 1003 were selected to generate operational taxonomic units (OTUs) and to evaluate the AMF community richness and similarity: 117 (37 of which were singletons) and 28 (nine of which were singletons) unique AMF OTUs were detected respectively. Within the Glomeromycota OTUs, those belonging to the Glomerales order were dominant in all the soils. Diversisporales OTUs were always detected, even though less frequently, while Archaeosporales and Paraglomerales OTUs were exclusive of the pasture soil. Eleven OTUs were shared by all the soils, but each of the five AMF communities showed particular features, suggesting a meaningful dissimilarity among the Glomeromycota populations. The environments with low inputs (pasture and covered vineyard) showed a higher AMF biodiversity than those subjected to human input (managed meadow and tilled vineyard). A reduction in AMF was found in the cork-oak formation because other mycorrhizal fungal species, more likely associated to trees and shrubs, were detected. These findings reinforce the view that AMF biodiversity is influenced by both human input and ecological traits, illustrating a gradient of AMF communities which mirror the land-use gradient. The high number of sequences obtained by the pyrosequencing strategy has provided detailed information on the soil AMF assemblages, thus offering a source of light to shine on this crucial soil microbial group.

Effects of different management practices on arbuscular mycorrhizal fungal diversity in maize fields by a molecular approach

As obligate mutualistic symbionts, arbuscular mycorrhizal fungi (AMF) colonize the roots of many agricultural crops, and it is often claimed that agricultural practices are detrimental to AMF. As a result, agroecosystems impoverished in AMF may not get the fully expected range of benefits from these fungi. Using molecular markers on DNA extracted directly from soil and roots, we studied the effects of different management practices (tillage and N fertilization) on the AMF communities colonizing an experimental maize field in Central Italy. Our molecular analysis based on three different nuclear rRNA regions (18S, 28S and ITS) allowed us to assess AMF biodiversity. Glomeraceae members were the main colonizer, and they co-occurred with Gigasporaceae and Paraglomus regardless of the management practices applied. Diversisporaceae and Entrophosporaceae members were instead detected in the N-fertilized soils and in the untreated soil, respectively. The results obtained indicated that the general AMF assemblages structure and composition in the maize field plots appear to be primarily influenced by N fertilization and, to a lesser extent, by tillage. This study also validates the usefulness of multiple molecular markers to consolidate and refine the assessment of the environmental AMF diversity.

Application of laser microdissection to identify the mycorrhizal fungi that establish arbuscules inside root cells

Obligate symbiotic fungi that form arbuscular mycorrhizae (AMF; belonging to the Glomeromycota phylum) are some of the most important soil microorganisms. AMFs facilitate mineral nutrient uptake from the soil, in exchange for plant-assimilated carbon, and promote water-stress tolerance and resistance to certain diseases. AMFs colonize the root by producing inter- and intra-cellular hyphae. When the fungus penetrates the inner cortical cells, it produces a complex ramified structure called arbuscule, which is considered the preferential site for nutrient exchange. Direct DNA extraction from the whole root and sequencing of ribosomal gene regions are commonly carried out to investigate intraradical AMF communities. Nevertheless, this protocol cannot discriminate between the AMFs that actively produce arbuscules and those that do not. To solve this issue, the authors have characterized the AMF community of arbusculated cells (AC) through a laser microdissection (LMD) approach, combined with sequencing-based taxa identification. The results were then compared with the AMF community that was found from whole root DNA extraction. The AMF communities originating from the LMD samples and the whole root samples differed remarkably. Five taxa were involved in the production of arbuscules, while two taxa were retrieved inside the root but not in the AC. Unexpectedly, one taxon was found in the AC, but its detection was not possible when extracting from the whole root. Thus, the LMD technique can be considered a powerful tool to obtain more precise knowledge on the symbiotically active intraradical AMF community.

Arbuscular Mycorrhizal Fungi and their Value for Ecosystem Management

Arbuscular Mycorrhizal Fungi (AMF) are a group of obligate biotrophs, to the extent that they must develop a close symbiotic association with the roots of a living host plant in order to grow and complete their life cycle [1]. The term “mycorrhiza” literally derives from the Greek mykes and rhiza, meaning fungus and root, respectively. AMF can symbiotically interact with almost all the plants that live on the Earth. They are found in the roots of about 80-90% of plant species (mainly grasses, agricultural crops and herbs) and exchange benefits with their partners, as is typical of all mutual symbiotic relationships [2]. They represent an interface between plants and soil, growing their mycelia both inside and outside the plant roots. AMF provide the plant with water, soil mineral nutrients (mainly phosphorus and nitrogen) and pathogen protection. In exchange, photosynthetic compounds are transferred to the fungus [3].

Sequencing and comparison of the mitochondrial COI gene from isolates of Arbuscular Mycorrhizal Fungi belonging to Gigasporaceae and Glomeraceae families.

Arbuscular Mycorrhizal Fungi (AMF) are well known for their ecological importance and their positive influence on plants. The genetics and phylogeny of this group of fungi have long been debated. Nuclear markers are the main tools used for phylogenetic analyses, but they have sometimes proved difficult to use because of their extreme variability. Therefore, the attention of researchers has been moving towards other genomic markers, in particular those from the mitochondrial DNA. In this study, 46 sequences of different AMF isolates belonging to two main clades Gigasporaceae and Glomeraceae have been obtained from the mitochondrial gene coding for the Cytochrome c Oxidase I (COI), representing the largest dataset to date of AMF COI sequences. A very low level of divergence was recorded in the COI sequences from the Gigasporaceae, which could reflect either a slow rate of evolution or a more recent evolutionary divergence of this group. On the other hand, the COI sequence divergence between Gigasporaceae and Glomeraceae was high, with synonymous divergence reaching saturated levels. This work also showed the difficulty in developing valuable mitochondrial markers able to effectively distinguish all Glomeromycota species, especially those belonging to Gigasporaceae, yet it represents a first step towards the development of a full mtDNA-based dataset which can be used for further phylogenetic investigations of this fungal phylum.

Application of nonspecific commercial AMF inocula results in poor mycorrhization in Camellia japonica L.

Camellia japonica L. (Theaceae, Theales) is an acidophilic evergreen flowering shrub and is traded worldwide. Symbiotic associations between the roots of this plant species and arbuscular mycorrhizal fungi (AMF), commonly recognized as natural biofertilizers and biocontrol agents, have been poorly studied so far. The aim of our study was to verify whether the application of nonspecific commercial AMF-based inocula could succeed and improve the growth of C. japonica. An experiment was conducted concerning the application of commercial inocula constituted by a specific AMF isolate (Funneliformis mosseae) or a consortium of different fungi and bacteria as alternatives to fertilization in pot cultivated C. japonica ‘Dr. Burnside’. Several growth parameters, plant nutrition and mycorrhization levels were monitored at the end of plant cultivation. Generally, increases in some macroelements (Ca, Mg, K) and microelements (Cu, Mn, Fe, Zn) were detected in the root system of inoculated camellias, while only Cu and Mn accumulation was increased in the leaves. Plants inoculated with the consortium inoculum exhibited increased chlorophyll content and foliage diameter while plants inoculated with F. mosseae exhibited increased number of flowers. Based on the polymorphism of an 18S rDNA region, we assessed the inoculated AMF that colonized C. japonica roots. The experiments showed that the applied AMF poorly colonized the root system of C. japonica. We suggest that commercial AMF formulations should be more targeted and host-specific in order to successfully colonize the host root and, potentially, fully express their benefits.

Rationalization of Camellia japonica L. pot cultivation: a multidisciplinary approach

Camellia japonica L. is an evergreen flowering perennial with more than 3,000 named cultivars of great ornamental value. These are nowadays traded worldwide as containerized small sized plants. Cultivation of ornamental plants is strongly oriented to sustainable production on the one hand and on a consumer-oriented high quality product on the other hand. In this context, the present work concerned a series of critical points of the cultivation cycle of potted C. japonica: the reduction of fertilization; the choice of a peat alternative substrate; the regulation of plant growth for size and flowering control; the control of the dormancy release of flower buds. Experiment 1 concerned the application of commercial biofertilizer inocula which included a specific arbuscular mycorrhizal fungus (AMF) isolate or a consortium of microorganisms (AMF, saprophytic fungi and helper bacteria) as alternatives to inorganic fertilization of pot cultivated C. japonica. To deepen AMF role, Experiment 2 evaluated potential specific isolates associated with camellia roots (active population) or surrounding soil (potential and soil exploring population) of centennial specimens found in natural or semi-natural ecosystems. Experiment 3 assessed the efficacy of flurprimidol and three peat alternatives (nutshells, rice husk, coconut fiber) on camellia growth control. Finally, Experiment 4 used a multidisciplinary approach to describe and characterize the effects of cold treatments on the dormancy release of flower buds. Overall, the results highlighted that C. japonica is susceptible to AMF inoculation and that a series of benefits can be achieved by inoculating pots with specific symbiotic fungi. Moreover, two newly tested materials (nutshells and rice husk) were suitable as partial peat alternatives (30% v/v), and the efficacy of very low concentrations of flurprimidol to control growth and enhance flowering was underlined. Lastly, the use of cold treatment allowed the achievement of an earlier, uniform and enhanced flowering.