Cistus salviifolius Toxicity in Cattle.

Toxicity related to consumption of Cistus sp. pl. has been described in ruminants in some countries. This report describes the clinical and pathological findings of Cistus salviifolius toxicosis in 3 beef cattle herds located in 2 different areas of Sicily, Italy. Outbreaks were observed after grazing in poor winter pasture where C. salviifolius was abundant. Mean morbidity and mortality were 29% and 21%, respectively. Most of the affected animals (6 to 36 months old) showed anorexia, weight loss, and pollakiuria culminating in recumbency and death. Occasionally, abortion and neurological signs were observed. In animals with acute signs, there was a moderate decrease of sodium and chloride concentrations in serum. Animals with chronic signs showed an increase of serum urea, creatine phosphokinase (CPK), aspartate transaminase (AST), lactate dehydrogenase (LDH), and phosphorus and a decrease in total serum protein, calcium, chloride, and magnesium concentrations. Moderate anemia and slight neutropenia, lymphocytosis, and eosinophilia were detected in all groups. At necropsy, the main lesion was severe distention of the urinary bladder with turbid hemorrhagic urine and crystalluria. Histologically, chronic cystitis, interstitial nephritis, eosinophilic enteritis, and nonsuppurative necrotizing hepatitis were observed. To our knowledge, this is the first report of C. salviifolius toxicosis in cattle in Italy.

Complete plastome sequence of Narcissus pseudonarcissus L., one of the most iconic European plants.

Narcissus pseudonarcissus L. is one of the most iconic plants of the European flora. It is a species of great horticultural interest, but also an endangered and protected plant in the wild as a consequence of loss of natural habitats. Complete plastid genome was assembled from next-generation sequencing data obtaining a circular genome of 160,008 bp long assembly. It comprises a pair of inverted repeat regions, a large single-copy region (108,400 bp), and a small single-copy region (16,434 bp). It encodes 131 genes, including 87 protein coding genes, 37 tRNA genes and seven rRNA genes. Phylogeny showed the strict relationship between N. pseudonarcissus and Narcissus poeticus L. The complete plastome will provide a useful genetic resource for future conservation programmes, phylogenetic studies and horticultural applications.

The cultivation of rye in marginal Alpine environments: a comparison of the agronomic, technological, health and sanitary traits of local landraces and commercial cultivars.

Rye is a secondary crop that is characterized by a higher tolerance to climatically less favorable conditions than other cereal species. For this reason, rye was historically used as a fundamental raw material for bread production and as a supply of straw in northern parts of Europe as well as in mountain environments, such as Alpine valleys, where locally adapted landraces have continued to be cultivated over the years. In this study, rye landraces collected in different valleys in the Northwest Italian Alps have been selected as the most genetically isolated within their geographical contexts and cultivated in two different marginal Alpine environments. The traits concerning their agronomy, mycotoxin contamination, bioactive content, as well as their technological and baking quality were assessed to characterize and compare rye landraces with commercial wheat and rye cultivars. Rye cultivars showed the same grain yield level as wheat in both environments. Only the genotype selected from the Maira Valley was characterized by tall and thin culms and a proneness to lodging, thereby resulting in a lower yield capacity. Among the rye cultivars, the hybrid one presented the highest yield potential, but also the highest susceptibility to the occurrence of ergot sclerotia. However, the rye cultivars, especially the landraces, were characterized by higher concentrations of minerals, soluble fibers, and soluble phenolic acids, and thus both their flours and breads had superior antioxidant properties. A 40% substitution of refined wheat flour with whole-grain rye flour led to a higher dough water absorption and a lower stability, thereby resulting in lower loaf volumes and darker products. Agronomically and qualitatively speaking, the rye landraces diverged significantly from the conventional rye cultivars, thus reflecting their genetic distinctiveness. The landrace from the Maira Valley shared a high content in phenolic acids and good antioxidant properties with the one from the Susa Valley and, when combined with wheat flour, turned out to be the most suitable for bread making. Overall, the results have highlighted the suitability of reintroducing historic rye supply chains, based on the cultivation of local landraces in marginal environments and the production of value-added bakery goods.

Alpine constructed wetlands: A metagenomic analysis reveals microbial complementary structure.

Constructed wetlands (CWs) are used to water treatment worldwide, however their application at high-altitude is poorly studied. In order to survive mountain winters, CWs rely on native flora and associated microbial communities. However, the choice of plant-microbes pairs more suitable for water treatment is challenging in alpine environments. Using a metagenomic approach, we investigated the composition of prokaryotes and fungal communities, through extensive sampling inside a constructed wetland in the SW-Alps. Best performing plant species were searched among those hosting the most diverse and resilient microbial communities and to this goal, we analysed them in the natural environment also. Our results showed that microbial communities were less diverse in the CW than at natural conditions, and they differed from plant to plant, revealing a clear variation in taxonomic composition between forbs and gramineous plants. Carex rostrata, Deschampsia caespitosa and Rumex alpinus hosted bacteria very active in N-cycles. Moreover, fungal and prokaryotic communities associated to R. alpinus (Polygonaceae) turned to be the richest and stable among the studied species. In our opinion, this species should be prioritized in CWs at high elevations, also in consideration of its low maintenance requirements.

A ddRADseq Survey of the Genetic Diversity of Rye (Secale cereale L.) Landraces from the Western Alps Reveals the Progressive Reduction of the Local Gene Pool.

Rye (Secale cereale L.) has been at the basis of agriculture for centuries in most mountainous and northern areas of Eurasia, because it is more resistant than other cereals to low temperatures and poor soils. Rye deserves to be re-evaluated as a source of "environmentally resilient" genes in the future as well, and particularly in a perspective to grow cereals able to withstand global warming. According to recent studies, modern rye varieties have a relatively narrow genetic pool, a condition that is worsening in the most recent breeding processes. The preservation of local landraces as unique sources of genetic diversity has therefore become important, in order to preserve the genetic heritage of rye. In this study, genetic diversity of rye landraces collected in a sector of the Italian Alps particularly suited to traditional agriculture was investigated using the ddRADseq technique. A few landraces still managed with family farming turned out to be genetically distant from the commercial varieties currently in use, highlighting that the phenomenon of homogenization of the local genetic pool can be still circumvented. Ex situ conservation of genetically divergent landraces is a valid tool to avoid the dissipation of an as yet unexplored genetic potential.

Integrating Multiple Lines of Evidence to Explore Intraspecific Variability in a Rare Endemic Alpine Plant and Implications for Its Conservation.

We studied the ecology, distribution, and phylogeography of Tephroseris balbisiana, a rare plant whose range is centered to the South-Western Alps. Our aim was to assess the extent of intraspecific variability within the nominal species and the conservation status of isolated populations. We studied genetic diversity across the whole species range. We analyzed leaf traits, which are distinctive morphological characters within the Tephroseris genus. A clear pattern of genetic variation was found among populations of T. balbisiana, which clustered according to their geographic position. On the contrary, there was a strong overlap in the morphological space of individuals across the species' range, with few peripheral populations diverging in their leaf morphology. Studying habitat suitability by means of species distribution models, we observed that T. balbisiana range is primarily explained by solar radiation and precipitation seasonality. Environmental requirements could explain the genetic and morphological uniformity of T. balbisiana in its core distribution area and justify genetic, morphological, and ecological divergences found among the isolated populations of the Apennines. Our findings emphasize the need to account for the whole diversity of a species, comprising peripheral populations, in order to better estimate its status and to prioritize areas for its conservation.

Comparative Germination Ecology of Two Endemic Rhaponticum Species (Asteraceae) in Different Climatic Zones of the Ligurian and Maritime Alps (Piedmont, Italy).

Comparative studies of seed germination of closely related taxa can help increase our understanding of the ecological limitations of cold-adapted plants and forecast how they might respond to global warming. No studies exist on the relationship between thermoclimatic belts that classify mountain life zones according to bioclimatic criteria and the germination strategy of alpine plants. The aim of this study was to assess this relationship using two closely related species growing in different thermotypes and to test whether their germination responses were related to the climate at natural sites. Fresh Rhaponticum bicknellii and R. scariosum seeds were cold stratified for 0, 30, 60 and 90 days and tested for germination at 10, 15 and 20 °C. At the same time, seed burial experiments were run in the field and in the plant nursery. A GLM analysis showed that the length of cold stratification affected significantly only the germination of R. bicknellii seeds, while increasing temperatures prompted germination in both species. We found that R. bicknellii adopts a drought-avoiding germination strategy, while R. scariosum germination is favoured by warm temperatures. Our findings support the general view that alpine plants do not share common germination requirements and that any conclusions should be interpreted from a biogeographical and bioclimatic perspective. Therefore, seed germination and seedling establishment of endemic alpine species can also be predicted by looking at the bioclimate of the species' range.

The microbial community of Vetiver root and its involvement into essential oil biogenesis.

Vetiver is the only grass cultivated worldwide for the root essential oil, which is a mixture of sesquiterpene alcohols and hydrocarbons, used extensively in perfumery and cosmetics. Light and transmission electron microscopy demonstrated the presence of bacteria in the cortical parenchymatous essential oil-producing cells and in the lysigen lacunae in close association with the essential oil. This finding and the evidence that axenic Vetiver produces in vitro only trace amounts of oil with a strikingly different composition compared with the oils from in vivo Vetiver plants stimulated the hypothesis of an involvement of these bacteria in the oil metabolism. We used culture-based and culture-independent approaches to analyse the microbial community of the Vetiver root. Results demonstrate a broad phylogenetic spectrum of bacteria, including alpha-, beta- and gamma-Proteobacteria, high-G+C-content Gram-positive bacteria, and microbes belonging to the Fibrobacteres/Acidobacteria group. We isolated root-associated bacteria and showed that most of them are able to grow by using oil sesquiterpenes as a carbon source and to metabolize them releasing into the medium a large number of compounds typically found in commercial Vetiver oils. Several bacteria were also able to induce gene expression of a Vetiver sesquiterpene synthase. These results support the intriguing hypothesis that bacteria may have a role in essential oil biosynthesis opening the possibility to use them to manoeuvre the Vetiver oil molecular structure.

The AlpArray Seismic Network: A Large-Scale European Experiment to Image the Alpine Orogen.

The AlpArray programme is a multinational, European consortium to advance our understanding of orogenesis and its relationship to mantle dynamics, plate reorganizations, surface processes and seismic hazard in the Alps-Apennines-Carpathians-Dinarides orogenic system. The AlpArray Seismic Network has been deployed with contributions from 36 institutions from 11 countries to map physical properties of the lithosphere and asthenosphere in 3D and thus to obtain new, high-resolution geophysical images of structures from the surface down to the base of the mantle transition zone. With over 600 broadband stations operated for 2 years, this seismic experiment is one of the largest simultaneously operated seismological networks in the academic domain, employing hexagonal coverage with station spacing at less than 52 km. This dense and regularly spaced experiment is made possible by the coordinated coeval deployment of temporary stations from numerous national pools, including ocean-bottom seismometers, which were funded by different national agencies. They combine with permanent networks, which also required the cooperation of many different operators. Together these stations ultimately fill coverage gaps. Following a short overview of previous large-scale seismological experiments in the Alpine region, we here present the goals, construction, deployment, characteristics and data management of the AlpArray Seismic Network, which will provide data that is expected to be unprecedented in quality to image the complex Alpine mountains at depth.

The abundance and diversity of arbuscular mycorrhizal fungi are linked to the soil chemistry of screes and to slope in the Alpic paleo-endemic Berardia subacaulis.

Berardia subacaulis Vill. is a monospecific genus that is endemic to the South-western Alps, where it grows on alpine screes, which are extreme habitats characterized by soil disturbance and limiting growth conditions. Root colonization by arbuscular mycorrhizal fungi (AMF) is presumably of great importance in these environments, because of its positive effect on plant nutrition and stress tolerance, as well as on structuring the soil. However, there is currently a lack of information on this topic. In this paper, we tested which soil characteristics and biotic factors could contribute to determining the abundance and community composition of AMF in the roots of B. subacaulis, which had previously been found to be mycorrhizal. For such a reason, the influence of soil properties and environmental factors on AMF abundance and community composition in the roots of B. subacaulis, sampled on three different scree slopes, were analysed through microscopic and molecular analysis. The results have shown that the AMF community of Berardia roots was dominated by Glomeraceae, and included a core of AMF taxa, common to all three scree slopes. The vegetation coverage and dark septate endophytes were not related to the AMF colonization percentage and plant community did not influence the root AMF composition. The abundance of AMF in the roots was related to some chemical (available extractable calcium and potassium) and physical (cation exchange capacity, electrical conductivity and field capacity) properties of the soil, thus suggesting an effect of AMF on improving the soil quality. The non-metric multidimensional scaling (NMDS) ordination of the AMF community composition showed that the diversity of AMF in the various sites was influenced not only by the soil quality, but also by the slope. Therefore, the slope-induced physical disturbance of alpine screes may contribute to the selection of disturbance-tolerant AMF taxa, which in turn may lead to different plant-fungus assemblages.

In Vitro Morphogenesis of Arabidopsis to Search for Novel Endophytic Fungi Modulating Plant Growth.

Fungal endophytes have shown to affect plant growth and to confer stress tolerance to the host; however, effects of endophytes isolated from water plants have been poorly investigated. In this study, fungi isolated from stems (stem-E) and roots (root-E) of Mentha aquatica L. (water mint) were identified, and their morphogenetic properties analysed on in vitro cultured Arabidopsis (L.) Heynh., 14 and 21 days after inoculation (DAI). Nineteen fungi were analysed and, based on ITS analysis, 17 isolates showed to be genetically distinct. The overall effect of water mint endophytes on Arabidopsis fresh (FW) and dry weight (DW) was neutral and positive, respectively, and the increased DW, mainly occurring 14 DAI, was possibly related to plant defence mechanism. Only three fungi increased both FW and DW of Arabidopsis at 14 and 21 DAI, thus behaving as plant growth promoting (PGP) fungi. E-treatment caused a reduction of root depth and primary root length in most cases and inhibition-to-promotion of root area and lateral root length, from 14 DAI. Only Phoma macrostoma, among the water mint PGP fungi, increased both root area and depth, 21 DAI. Root depth and area 14 DAI were shown to influence DWs, indicating that the extension of the root system, and thus nutrient uptake, was an important determinant of plant dry biomass. Reduction of Arabidopsis root depth occurred to a great extent when plants where treated with stem-E while root area decreased or increased under the effects of stem-E and root-E, respectively, pointing to an influence of the endophyte origin on root extension. M. aquatica and many other perennial hydrophytes have growing worldwide application in water pollution remediation. The present study provided a model for directed screening of endophytes able to modulate plant growth in the perspective of future field applications of these fungi.

In vitro and in vivo peppermint (Mentha piperita) growth promotion by nonmycorrhizal fungal colonization.

• Here peppermint growth and terpene production of in vitro generated plants (Mentha piperita) in response to inoculation with a leaf fungal endophyte were characterized. • Peppermint plants were studied by means of morphometric, biochemical and image analysis, employing both in vitro and in pot cultures. Leaf essential oils were analysed by gascromatography-mass spectrometry. • The endophyte induced profound effects on the growth of peppermint, which responded with taller plants bearing more expanded leaves. The observed increase of leaf dry matter over leaf area suggested a real improvement of peppermint metabolic and photosynthetic apparatus. Root architecture was of the herring-bone type, showing greater dry biomass percentage over the total. A sustained lowering of (+)-menthofuran and an increase of (+)-menthol percentage concentrations were found in plants from both in vitro and pot cultures. • The study represents the first report on specialized endophytic fungi in peppermint green tissues and highlights some of the principal morphological and biochemical aspects of this mutualism. Effects exerted on plant growth and essential oil production in peppermint suggest further biotechnological applications.

An ascomycetous endophyte isolated from Mentha piperita L.: biological features and molecular studies.

A hyaline sterile fungus forming epiphyllous mycelial nets was isolated from meristem cultures of Mentha piperita. Histological studies indicated that the culture isolate is able to colonize stems and leaves with no damage to the host plant. In vitro-grown peppermint plants displayed enhanced vegetative growth when infected by the fungus, with mycelium extending from green tissues to growing rootlets. The production of very thin hyphae growing away from host meristems and the asymptomatic nature of the symbiosis were commonly observed in cultures, where the isolate never sporulated. No attribution to a precise morphospecies was therefore possible and the fungal culture was named sterile mycelium PGP-HSF. Through comparison of the 18 S rDNA sequence of the epibiont to those available in literature and in GenBank we were able to determine that the mutualist of peppermint is a member of the Pyrenomycetes, belonging to the subclass Sordariomycetidae.

Genetic variability and population divergence in the rare Fritillaria tubiformis subsp. moggridgei Rix (Liliaceae) as revealed by RAPD analysis.

Fritillaria tubiformis subsp. moggridgei Rix. is a rare alpine geophyte with shiny yellow flowers. This plant is sporadically distributed across the southwestern Alps where it is biogeographically close to F. tubiformis var. burnatii Planch. The latter has dark purple flowers and ranges in the majority of the Western and Central Alps. In order to develop appropriate strategies of conservation, a RAPD based analysis was conducted to study the genetic status of these taxa and the distribution of genetic variability of the subspecies by sampling seven populations distributed across the subspecies' range. Four populations of var. burnatii were chosen within this range and included in the genetic analysis. Some 264 individuals were analysed and 201 polymorphic loci were scored. Genetic diversity scored in the subspecies was in line with expectations for endemic species (He  = 0.194). F. tubiformis var. burnatii showed lower intraspecific diversity (He  = 0.173), notwithstanding a wider range than the subspecies. Most of the total phenotypic variation (about 83%) was allocated within populations, and significant lower proportions between taxa (6.45%) and between populations of the same taxon (10.64%). Moreover, PCoA analysis and Bayesian clustering separated populations into two genetically differentiated groups corresponding with the subspecific taxa. However, three populations ascribed to the subsp. moggridgei repeatedly showed genetic admixture with var. burnatii populations. Our findings suggest that: i) although the different flower colour, the two taxa are genetically very similar and share a consistent part of their gene pool, ii) the majority of genetic variability is allocated within populations rather than among them, iii) a representative amount of genetic diversity can be preserved by sampling from a restricted number of populations. The efficacy of RAPD markers in analysing genetic variation, and the contribution of the results to the preservation of biodiversity of the species, are discussed.

Volatile terpenoids of endophyte-free and infected peppermint (Mentha piperita L.): chemical partitioning of a symbiosis.

The study reports the effects on volatiles of an endophytic fungus inhabiting asymptomatically the leaves of peppermint. By means of headspace solid-phase microextraction (HS-SPME) and gaschromatography-mass spectrometry (GC-MS) terpenoids were sampled in time course from the head space of peppermint leaves and roots. After removal of the mycelium from peppermint tissues, fungal volatiles were analyzed and compared with those of pure fungal cultures. In the presence of the endophyte, the relative amount of all main compounds increased in leaves. Starting from the first 14 d of culture, (-)-menthone and (+)-neomenthol were consistently higher than in control plants. On the contrary, (+)-menthofuran increased only by 28 d of culture. Root volatiles were also dramatically altered by the presence of the fungus, with (+)-pulegone accounting for at least 44% of the total volatile emission. (+)-Pulegone was also the main compound of PGP-HSF mycelium isolated from peppermint roots. The sesquiterpenoid cuparene was found as a novel compound of peppermint leaf headspace and was a main volatile of ex planta and pure culture mycelia. The chemical spectrum of terpenoids and their distribution among peppermint roots, leaves, and mycelia are likely to account for a fine regulation of the mutualism in planta and for the acquisition by the fungus of novel metabolic competences.