Andrea Berruti

Arbuscular Mycorrhizal Fungi as Natural Biofertilizers: Let's Benefit from Past Successes

Arbuscular Mycorrhizal Fungi (AMF) constitute a group of root obligate biotrophs that exchange mutual benefits with about 80% of plants. They are considered natural biofertilizers, since they provide the host with water, nutrients, and pathogen protection, in exchange for photosynthetic products. Thus, AMF are primary biotic soil components which, when missing or impoverished, can lead to a less efficient ecosystem functioning. The process of re-establishing the natural level of AMF richness can represent a valid alternative to conventional fertilization practices, with a view to sustainable agriculture. The main strategy that can be adopted to achieve this goal is the direct re-introduction of AMF propagules (inoculum) into a target soil. Originally, AMF were described to generally lack host- and niche-specificity, and therefore suggested as agriculturally suitable for a wide range of plants and environmental conditions. Unfortunately, the assumptions that have been made and the results that have been obtained so far are often worlds apart. The problem is that success is unpredictable since different plant species vary their response to the same AMF species mix. Many factors can affect the success of inoculation and AMF persistence in soil, including species compatibility with the target environment, the degree of spatial competition with other soil organisms in the target niche and the timing of inoculation. Thus, it is preferable to take these factors into account when “tuning” an inoculum to a target environment in order to avoid failure of the inoculation process. Genomics and transcriptomics have led to a giant step forward in the research field of AMF, with consequent major advances in the current knowledge on the processes involved in their interaction with the host-plant and other soil organisms. The history of AMF applications in controlled and open-field conditions is now long. A review of biofertilization experiments, based on the use of AMF, has here been proposed, focusing on a few important factors that could increase the odds or jeopardize the success of the inoculation process.

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].

ITS fungal barcoding primers vs 18S AMF-specific primers reveal similar AMF-based diversity patterns in roots and soils of three mountain vineyards

ITS primers commonly used to describe soil fungi are flawed for AMF although it is unknown the extent to which they distort the interpretation of community patterns. Here, we focus on how the use of a specific ITS2 fungal barcoding primer pair biased for AMF changes the interpretation of AMF community patterns from three mountain vineyards compared to a novel AMF-specific approach on the 18S. We found that although discrepancies were present in the taxonomic composition of the two resulting datasets, the estimation of diversity patterns among AMF communities was similar and resulted in both primer systems being able to correctly assess the community-structuring effect of location, compartment (root vs. soil) and environment. Both methodologies made it possible to detect the same alpha-diversity trend among the locations under study but not between root and soil transects. We show that the ITS2 primer system for fungal barcoding provides a good estimate of both AMF community structure and relation to environmental variables. However, this primer system does not fit in with cross-compartment surveys (roots vs. soil) as it can underestimate AMF diversity in soil samples. When specifically focusing on AMF, the 18S primer system resulted in wide coverage and marginal non-target amplification.

AMF components from a microbial inoculum fail to colonize roots and lack soil persistence in an arable maize field

Arbuscular mycorrhizal fungi (AMF) are root obligate biotrophs that provide the host with nutrients and pathogen protection, in exchange of photosynthetic products. A decline in AMF diversity can reduce the overall benefit for host plants. A sustainable strategy to re-establish AMF diversity is to supply the target soil with AMF inoculants. After inoculation, it is essential to verify whether the inoculants successfully colonize the host plant and persist, and if the resident AMF community is affected. The AMF components of a microbial inoculum (including other saprotrophs) that was applied to maize were identified and traced in field by 454-pyrosequencing of the partial rRNA 18S gene. In addition, mycorrhizal colonization and plant biomass were monitored in inoculated and non-inoculated maize. The inoculated AMF taxa failed to colonize roots and lacked soil persistence. Nevertheless, the inoculation process reduced species dominance and increased diversity in the pre-existing AMF community. No differences were seen between mycorrhizal colonization in treated and control maize. We suggest that the slightly significant increase in treated plant biomass was potentially due to (i) marginally colonizing inoculated AMF that remained unseen and other saprotroph inoculants applied and/or (ii) the effect of inoculation on the pre-existing AMF community in treated maize roots.

Cold Treatment Breaks Dormancy but Jeopardizes Flower Quality in Camellia japonica L.

Camellia japonica L. is an evergreen shrub whose cultivars are of great ornamental value. In autumn, after flower bud differentiation, dormancy is initiated. As in many other spring flowering woody ornamentals, winter low temperatures promote dormancy release of both flower and vegetative buds. However, warm spells during late autumn and winter can lead to unfulfilled chilling requirements leading to erratic and delayed flowering. We hypothesized that storing plants at no light and low temperature could favor dormancy breaking and lead to early and synchronized flowering in response to forcing conditions in C. japonica ‘Nuccio’s Pearl’. Plants with fully developed floral primordia were stored at dark, 7°C, and RH > 90% for up to 8 weeks. To monitor endodormancy release during the storage, we evaluated the content of abscisic acid (ABA) in flower buds and the expression profiles of five putative genes related to dormancy and cold acclimation metabolism in leaves and flower buds. In addition, the expression of four anthocyanin biosynthesis pathway genes was profiled in flower buds to assess the effect of the treatment on flower pigment biosynthesis. At 0, 4, 6, and 8 weeks of cold treatment, 10 plants were transferred to the greenhouse and forced to flower. Forced plant flower qualities and growth were observed. The ABA content and the expression profiles of two dormancy-related genes (CjARP and CjDEH) suggested that dormancy breaking occurred after 6–8 weeks of cold treatment. Overall, plants treated for 6–8 weeks showed earlier vegetative sprouting, enhanced, and homogeneous flowering with reduced forcing time. Prolonged cold treatments also reduced flower size and longevity, anthocyanin content, and pigment biosynthesis-related gene transcripts. In conclusion, the cold treatment had a promotive effect on dormancy breaking but caused severe drawbacks on flower quality.

Differential biodiversity responses between kingdoms (plants, fungi, bacteria and metazoa) along an Alpine succession gradient

Biological diversities of multiple kingdoms potentially respond in similar ways to environmental changes. However, studies either compare details of microbial diversity across general vegetation or land use classes or relate details of plant community diversity with the extent of microbially governed soil processes, via physiological profiling. Here, we test the hypothesis of shared responses of plant and rhizosphere bacterial, fungal and metazoan biodiversities (especially across‐habitat β‐diversity patterns) along a disturbance gradient encompassing grazed to abandoned Alpine pasture, on acid soil in the European Central Alps. Rhizosphere biological diversity was inferred from eDNA fractions specific to bacteria, fungi and metazoans from contrasting plant habitats indicative of different disturbance levels. We found that soil β‐diversity patterns were weakly correlated with plant diversity measures and similarly ordinated along an evident edaphic (pH, C:N, assimilable P) and disturbance gradient but, contrary to our hypothesis, did not demonstrate the same diversity patterns. While plant communities were well separated along the disturbance gradient, correlating with fungal diversity, the majority of bacterial taxa were shared between disturbance levels (75% of bacteria were ubiquitous, cf. 29% plant species). Metazoa exhibited an intermediate response, with communities at the lowest levels of disturbance partially overlapping. Thus, plant and soil biological diversities were only loosely dependent and did not exhibit strictly linked environmental responses. This probably reflects the different spatial scales of organisms (and their habitats) and capacity to invest resources in persistent multicellular tissues, suggesting that vegetation responses to environmental change are unreliable indicators of below‐ground biodiversity responses.

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.

An in vitro bioassay for the evaluation of cold treatment on flower bud dormancy in Camellia

Camellia japonica L. is an evergreen shrub whose cultivars are of great ornamental value. Initiation and early differentiation of flower buds in C. japonica starts from late spring on while flower bud development and visible bud enlargement sequel until autumn. In many temperate woody ornamentals dormancy is installed after flower bud differentiation. The exposure of floral buds to cold temperatures (between 2-7°C) is supposed to stimulate the initiation of normal growth and anthesis during the next spring. As an attempt to quicken the fulfillment of the cold need, as well as to homogenize flowering, five low temperature regimes, consisting of darkness and a constant temperature of 7°C applied for 0, 2, 4, 6 and 8 weeks, were tested on budded plants of cultivar ‘Nuccio’s Pearl’. As indicator of dormancy release, an inexpensive and easygoing test was conducted by forcing excised flower buds with an in vitro bioassay. To infer about the reliability of this test, a possible hormonal basis was assessed by measuring the concentration of abscisic acid in floral buds. Four groups of camellias of ten specimens each, treated respectively for 0, 4, 6 and 8 weeks, were then forced in the greenhouse with supplementary lighting and semi-controlled temperature for qualitative and quantitative evaluations of flowering. Our results indicated that the bioassay is a suitable indicator of the moment of dormancy release as it was able to highlight a reduced amount of dormancy when buds stayed longer at cold. Abscisic acid content in floral buds showed to be reduced and homogenized after 6 and 8 weeks of cold, thereby promoting a more uniform flowering. Hence, the bioassay appears as a costeffective tool that could be of interest for breeders, to characterize the chilling requirements of flowers of parental plants or their offspring.