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 ß-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 ß-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.

Seasonal variation in winter wheat field soil arbuscular mycorrhizal fungus communities after non-mycorrhizal crop cultivation.

Intensive farming practices that implement deep and frequent tillage, high input inorganic fertilization, cultivation with non-host species, and pesticide use are widely reported to be detrimental for arbuscular mycorrhizal fungi (AMF), which are one of the most important plant biofertilizers. The effect of the reduction of agricultural input on AMF community dynamics following conversion from conventional non-mycorrhizal to lower input mycorrhizal crop cultivation has not yet been fully elucidated. We investigated the effect of the reduction of agricultural input, rotation, and season on AMF communities in winter wheat field soil after conversion from long-term (more than 20 years) non-mycorrhizal (sugar beet) crop cultivation. We described AMF communities from bulk soil samples by specifically targeting the 18S ribosomal gene using a combination of AMF specific primers and 454 pyrosequencing. No effect was found after 3 years' reduction of agricultural input, and only marginal effects were due to rotation with specific crops preceding winter wheat. Instead, season and year of sampling had the most appreciable influence on the AMF community. We suggest that, after conversion from long-term non-mycorrhizal to mycorrhizal crop cultivation, AMF diversity is low if compared to similar agroecosystems. Seasonal and successional dynamics play an important role as determinants of community structure.

Edaphic factors trigger diverse AM fungal communities associated to exotic camellias in closely located Lake Maggiore (Italy) sites.

Camellia japonica L. is an acidophilic ornamental shrub of high economic value that has its center of origin in Japan and has been introduced in several European environmental niches. This exotic species forms arbuscular mycorrhizas (AM), known for their ability to positively affect plant growth. However, AM fungal communities associated to C. japonica in the field have never been characterized. For the first time, the AM fungal community naturally selected by C. japonica was screened in three sites located on the shores of Lake Maggiore (Italy), where specimens of this plant were introduced in the nineteenth century. Mycorrhizal levels were assessed, and the AM fungal communities associated to roots and soil were molecularly characterized based on the small subunit (SSU) rDNA region. The frequency of mycorrhizal roots was high in all sampled root systems (>90 %). Overall, 39 Operational Taxonomic Units (OTUs; 22 Glomerales, 9 Paraglomerales, 7 Archaeosporales, and 1 Diversisporales) were found in the root and soil samples. OTU richness did not significantly differ between the root and the soil niche (5.7 ± 0.6 and 8.0 ± 1.1 average OTUs per sample, respectively) and the three sites analyzed (7.5 ± 0.7, 5.2 ± 1.0, and 7.8 ± 1.5 average OTUs per sample in the three sites, respectively). The AM fungal community composition significantly differed between root-colonizing and soil-dwelling communities and among the three sites under study. Data show a major involvement of edaphic factors, such as available N sources, P, Mg, and K content in soil and soil compaction, in the structuring of the AM fungal communities.

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.

Paleomicrobiology: Tracking the past microbial life from single species to entire microbial communities.

By deciphering information encoded in degraded ancient DNA extracted from up to million-years-old samples, molecular paleomicrobiology enables to objectively retrace the temporal evolution of microbial species and communities. Assembly of full-length genomes of ancient pathogen lineages allows not only to follow historical epidemics in space and time but also to identify the acquisition of genetic features that represent landmarks in the evolution of the host-microbe interaction. Analysis of microbial community DNA extracted from essentially human paleo-artefacts (paleofeces, dental calculi) evaluates the relative contribution of diet, lifestyle and geography on the taxonomic and functional diversity of these guilds in which have been identified species that may have gone extinct in today's human microbiome. As for non-host-associated environmental samples, such as stratified sediment cores, analysis of their DNA illustrates how and at which pace microbial communities are affected by local or widespread environmental disturbance. Description of pre-disturbance microbial diversity patterns can aid in evaluating the relevance and effectiveness of remediation policies. We finally discuss how recent achievements in paleomicrobiology could contribute to microbial biotechnology in the fields of medical microbiology and food science to trace the domestication of microorganisms used in food processing or to illustrate the historic evolution of food processing microbial consortia.

Identification of DNA Viruses in Ancient DNA from Herbarium Samples.

Herbaria encompass millions of plant specimens, mostly collected in the nineteenth and twentieth centuries that can represent a key resource for investigating the history and evolution of phytopathogens. In the last years, the application of high-throughput sequencing technologies for the analysis of ancient nucleic acids has revolutionized the study of ancient pathogens including viruses, allowing the reconstruction of historical genomic viral sequences, improving phylogenetic based molecular dating, and providing essential insight into plant virus ecology. In this chapter, we describe a protocol to reconstruct ancient plant and soil viral sequences starting from highly fragmented ancient DNA extracted from herbarium plants and their associated rhizospheric soil. Following Illumina high-throughput sequencing, sequence data are de novo assembled, and DNA viral sequences are selected, according to their similarity with known viruses.

Herbaria preserve plant microbiota responses to environmental changes.

Interaction between plants and their microbiota is a central theme to understand adaptation of plants to their environment. Considering herbaria as repositories of holobionts that preserved traces of ancient plant-associated microbial communities, we propose to explore these historical collections to evaluate the impact of long-lasting global changes on plant-microbiota interactions.

Different farming and water regimes in Italian rice fields affect arbuscular mycorrhizal fungal soil communities.

Arbuscular mycorrhizal fungi (AMF) comprise one of the main components of soil microbiota in most agroecosystems. These obligate mutualistic symbionts colonize the roots of most plants, including crop plants. Many papers have indicated that different crop management practices could affect AMF communities and their root colonization. However, there is little knowledge available on the influence of conventional and low-input agriculture on root colonization and AMF molecular diversity in rice fields. Two different agroecosystems (continuous conventional high-input rice monocropping and organic farming with a five-year crop rotation) and two different water management regimes have been considered in this study. Both morphological and molecular analyses were performed. The soil mycorrhizal potential, estimated using clover trap cultures, was high and similar in the two agroecosystems. The diversity of the AMF community in the soil, calculated by means of PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) and 18S rDNA sequencing on clover trap cultures roots, was higher for the organic cultivation. The rice roots cultivated in the conventional agrosystem or under permanent flooding showed no AMF colonization, while the rice plants grown under the organic agriculture system showed typical mycorrhization patterns. Considered together, our data suggest that a high-input cropping system and conventional flooding depress AMF colonization in rice roots and that organic managements could help maintain a higher diversity of AMF communities in soil.

Metabarcoding of Soil Fungal Communities Associated with Alpine Field-Grown Saffron (Crocus sativus L.) Inoculated with AM Fungi.

Soil fungi strongly influence ecosystem structure and functioning, playing a key role in many ecological services as decomposers, plant mutualists and pathogens. Arbuscular mycorrhizal fungi (AMF) establish mutualistic symbiotic associations with plant roots and act as biofertilizers by enhancing plant nutrients and water uptake. Information about the AMF association with Crocus sativus L. (saffron) and their impact on crop performances and spice quality has been increasing in recent years. Instead, there is still little data on the biodiversity of soil microbial communities associated with this crop in the Alpine environments. The aims of this study were to investigate the fungal communities of two Alpine experimental sites cultivated with saffron, and to rank the relative impact of two AMF inocula, applied to soil as single species (R = Rhizophagus intraradices, C. Walker & A. Schüßler) or a mixture of two species (M = R. intraradices and Funneliformis mosseae, C. Walker & A. Schüßler), on the resident fungal communities which might be influenced in their diversity and composition. We used Illumina MiSeq metabarcoding on nuclear ribosomal ITS2 region to characterize the fungal communities associated to Crocus sativus cultivation in two fields, located in the municipalities of Saint Christophe (SC) and Morgex (MG), (Aosta Valley, Italy), treated or not with AMF inocula and sampled for two consecutive years (Y1; Y2). Data analyses consistently indicated that Basidiomycota were particularly abundant in both sites and sampling years (Y1 and Y2). Significant differences in the distribution of fungal taxa assemblages at phylum and class levels between the two sites were also found. The main compositional differences consisted in significant abundance changes of OTUs belonging to Dothideomycetes and Leotiomycetes (Ascomycota), Agaricomycetes and Tremellomycetes (Basidiomycota), Mortierellomycetes and Mucoromycetes. Further differences concerned OTUs, of other classes, significantly represented only in the first or second year of sampling. Concerning Glomeromycota, the most represented genus was Claroideoglomus always detected in both sites and years. Other AMF genera such as Funneliformis, Septoglomus and Microdominikia, were retrieved only in MG site. Results highlighted that neither sites nor inoculation significantly impacted Alpine saffron-field fungal communities; instead, the year of sampling had the most appreciable influence on the resident communities.

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.

High-Throughput DNA Sequence-Based Analysis of AMF Communities.

Arbuscular mycorrhizal fungi (AMF) are obligate symbionts of most land plants. They have great ecological and economic impacts as they support plant nutrition and water supply, soil structure, and plant resistance to pathogens. Investigating AMF presence and distribution at small and large scales is critical. Therefore, research requires standard protocols to be easily implemented. In this chapter, we describe a workflow for AMF identification by high-throughput sequencing through Illumina MiSeq platform of two DNA target regions: small subunit (SSU) and internal transcribed spacer (ITS). The protocol can apply to both soil and root AMF communities.

Impact of land use history on the arbuscular mycorrhizal fungal diversity in arid soils of Argentinean farming fields.

Arbuscular mycorrhizal fungi (AMF) are a key soil functional group, with an important potential to increase crop productivity and sustainable agriculture including food security. However, there is clear evidence that land uses, crop rotations and soil features affect the AMF diversity and their community functioning in many agroecosystems. So far, the information related to AMF biodiversity in ecosystems like the Argentinean Puna, an arid high plateau where plants experience high abiotic stresses, is still scarce. In this work, we investigated morphological and molecular AMF diversity in soils of native corn, bean and native potato Andean crops, under a familiar land use, in Chaupi Rodeo (Jujuy, Argentina), without agrochemical supplements but with different histories of crop rotation. Our results showed that AMF morphological diversity was not only high and variable among the three different crop soils but also complemented by Illumina MiSeq data. The multivariate analyses highlighted that total fungal diversity is significantly affected by the preceding crop plants and the rotation histories, more than from the present crop species, while AMF communities are significantly affected by preceding crop only in combination with the effect of nitrogen and calcium soil concentration. This knowledge will give useful information on appropriate familiar farming.

Native Arbuscular Mycorrhizal Fungi Characterization from Saline Lands in Arid Oases, Northwest China.

Arbuscular mycorrhizal fungi (AMF) colonize land plants in almost every ecosystem, even in extreme conditions, such as saline soils. In the present work, we report the mycorrhizal capacity of rhizosphere soils collected in the dry desert region of the Minqin Oasis, located in the northwest of China (Gansu province), which is characterized by several halophytes. Lycium spp. and Peganum nigellastrum were used as trap plants in a greenhouse experiment to identify autochthonous AMF associated with the halophytes' rhizospheres. Morphological observations showed the typical AMF structures inside roots. Twenty-six molecularly distinct AMF taxa were recovered from soil and root DNA. The taxonomical diversity mirrors the several AMF adapted to extreme environmental conditions, such as the saline soil of central China. Knowledge of the AMF associated with halophytes may contribute to select specific fungal isolates to set up agriculture strategies for protecting non-halophyte crop plants in saline soils.

The ftsZ gene of the endocellular bacterium 'Candidatus Glomeribacter gigasporarum' is preferentially expressed during the symbiotic phases of its host mycorrhizal fungus.

The arbuscular mycorrhizal fungus (AM) Gigaspora margarita consistently hosts bacteria, named 'Candidatus Glomeribacter gigasporarum,' inside its cytoplasm. Endobacteria have a positive impact on fungal fitness during the presymbiotic phase, prior to plant roots colonization. We tested the hypothesis that the endobacterium and its cell divisions depend on fungal metabolism, mirroring also the events of the fungal life cycle which are influenced by plant signals. We first cloned a fragment of ftsZ, a marker gene for bacterial division, and then analyzed its expression along the different stages of fungus development. The bacterial gene transcripts showed the highest values when the fungus was associated to the plant, and peaked in the extraradical mycelium. Strigolactones, which are known to stimulate the AM fungal growth, caused a significant transcript increase in the germinated spores in the absence of the plant. The quantitative real-time reverse-transcription polymerase chain reaction data were strengthened by the quantification of the dividing bacteria, which were increasing in number in germinating spores after the strigolactone treatment. The bioactive molecule alone did not cause any change in the number of bacteria after their isolation from the fungus, thus showing that the strigolactone alone cannot confer free-living capacities to the bacterium.

Simultaneous detection and quantification of the unculturable microbe Candidatus Glomeribacter gigasporarum inside its fungal host Gigaspora margarita.

A combined approach based on quantitative and nested polymerase chain reaction (qPCR and nPCR, respectively) has been set up to detect and quantify the unculturable endobacterium Candidatus Glomeribacter gigasporarum inside the spores of its fungal host Gigaspora margarita. Four genes were targeted, two of bacterial origin (23S rRNA gene and rpoB) and two from the fungus (18S rRNA gene and EF1-alpha). The sensitivity of the qPCR protocol has proved to be comparable to that of nPCR, both for the fungal and the bacterial detection. It has been demonstrated that the last detected dilution in qPCR corresponded, in each case, to 10 copies of the target sequences, suggesting that the method is equally sensitive for the detection of both fungal and bacterial targets. As the two targeted bacterial genes are predicted to be in single copy, it can be concluded that the detection limit is of 10 bacterial genomes for each mixture. The protocol was then successfully applied to amplify fungal and bacterial DNA from auxiliary cells and extraradical and intraradical mycelium. For the first time qPCR has been applied to a complex biological system to detect and quantify fungal and bacterial components using single-copy genes, and to monitor the bacterial presence throughout the fungal life cycle.

Interactions between engineered tomato plants expressing antifungal enzymes and nontarget fungi in the rhizosphere and phyllosphere.

The introduction of genetically modified (GM) plants in agroecosystems raises concern about possible effects on nontarget species. The impact of a tomato line transformed for constitutive expression of tobacco beta-1,3-glucanase and chitinase on indigenous nonpathogenic fungi was investigated. In greenhouse experiments, no significant differences were found in the colonization by arbuscular mycorrhizal fungi. Diversity indices computed from over 20 500 colonies of culturable rhizosphere and phyllosphere saprotrophic microfungi, assigned to 165 species (plus > 80 sterile morphotypes), showed no significant differences between GM and wild-type plants. Differences were found by discriminant analysis in both the rhizosphere and the phyllosphere, but such effects were minor compared with those linked to different plant growth stages.

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.

Arbuscular mycorrhizal fungal diversity in the Tuber melanosporum brûlé.

The development of the fruiting body (truffle) of the ectomycorrhizal fungus Tuber melanosporum is associated with the production of an area (commonly referred to with the French word brûlé) around its symbiotic plant that has scanty vegetation. As truffles produce metabolites that can mediate fungal-plant interactions, the authors wondered whether the brûlé could affect the arbuscular mycorrhizal fungi (AMF) that colonize the patchy herbaceous plants inside the brûlé. A morphological evaluation of the roots of plants collected in 2009 from a T. melanosporum/Quercus pubescens brûlé in France has shown that the herbaceous plants are colonized by AMF to a great extent. An analysis of the 18S rRNA sequences obtained from roots and soil inside the brûlé has shown that the AMF community structure seemed to be affected in the soil inside the brûlé, where less richness was observed compared to outside the brûlé.

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.

Unravelling soil fungal communities from different Mediterranean land-use backgrounds.

BACKGROUND: Fungi strongly influence ecosystem structure and functioning, playing a key role in many ecological services as decomposers, plant mutualists and pathogens. The Mediterranean area is a biodiversity hotspot that is increasingly threatened by intense land use. Therefore, to achieve a balance between conservation and human development, a better understanding of the impact of land use on the underlying fungal communities is needed. METHODOLOGY/PRINCIPAL FINDINGS: We used parallel pyrosequencing of the nuclear ribosomal its regions to characterize the fungal communities in five soils subjected to different anthropogenic impact in a typical mediterranean landscape: a natural cork-oak forest, a pasture, a managed meadow, and two vineyards. Marked differences in the distribution of taxon assemblages among the different sites and communities were found. Data analyses consistently indicated a sharp distinction of the fungal community of the cork oak forest soil from those described in the other soils. Each soil showed features of the fungal assemblages retrieved which can be easily related to the above-ground settings: ectomycorrhizal phylotypes were numerous in natural sites covered by trees, but were nearly completely missing from the anthropogenic and grass-covered sites; similarly, coprophilous fungi were common in grazed sites. CONCLUSIONS/SIGNIFICANCE: Data suggest that investigation on the below-ground fungal community may provide useful elements on the above-ground features such as vegetation coverage and agronomic procedures, allowing to assess the cost of anthropogenic land use to hidden diversity in soil. Datasets provided in this study may contribute to future searches for fungal bio-indicators as biodiversity markers of a specific site or a land-use degree.