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Weeds are responsible for major crop losses worldwide but can provide beneficial agroecosystem services. This study aimed to elucidate how arbuscular mycorrhizal fungi (AMF) in weeds respond to host identity and conservation agricultural practices. The study was carried out at two locations in Southern Africa during off-season and in-season maize cultivation. Off-season AMF root colonisation, diversity indices and community composition significantly differed among weed species at both locations. Glomus sp. VTX00280 explains most of the AMF community differences. In-season, implementation of conventional tillage with mulching alone (CT + M) or together with crop rotation (CT + M + R) resulted in a 20% increase in AMF colonisation of the constantly occurring weed species, Bidens pilosa (BIDPI) and Richardia scabra (RCHSC), compared with conventional tillage plus rotations (CT + R). The diversity of AMF was highest under no-tillage plus mulching (NT + M). Off-season and in-season AMF structures of both BIDPI and RCHSC were not related, but 39% of the taxa were shared. Structural equation modelling showed a significant effect of the cropping system on weed AMF diversity parameters and weed and maize root colonisation, but no significant influence of weed root AMF traits and maize colonisation was detected on maize yield. This may be explained by the improvement in weed competitive ability, which may have offset the AMF-mediated benefits on yield. Our findings highlight that implementing M and CR to CT and NT positively affected weed AMF colonisation and diversity. The similarity between the off-season and in-season AMF composition of weeds supports the fact that weeds functionally host AMF during the non-crop period.

Under conservation agriculture (CA), soil aggregates physically protect soil organic C, creating microhabitats with heterogeneities in nutrient availability. These may become rich in microbial taxa with structured interconnections, and thus maintain the equilibrium between C sources and sinks. A long-term experiment on tillage and N fertilization located in the Mediterranean was used to investigate the microbiota within small macroaggregates (sM), and occluded microaggregates (mM). At surface layer N fertilization was the main driver of diversity of prokaryotes and fungi in soil aggregates, whereas at subsurface layer tillage intensity was the primary driver. Moreover, although along the soil profile a conserved core microbial community was found across managements in soil aggregates, some taxa were unique to certain managements. At surface layer, N fertilization significantly modified the prokaryotic community structure in sM and mM under conventional tillage, whereas in the subsurface layer, tillage modified the community structure of prokaryotes in both soil aggregates, and of fungi in mM. The fungal community structure in sM was strongly modified by the interaction between tillage and N fertilization at both soil layers and in mM only at surface layer. Overall sM had a higher diversity of prokaryotes and a lower diversity of fungi than mM. Small macroaggregates and mM had distinctive microbial community structures. Prokaryotic taxa, such as Actinobacteria, Chloroflexi and Thermomicrobia, and fungi, such as Agaricomycetes, Dydimellaceae, and Mortierellaceae, characterized sM, whereas others prokaryotes (Betaproteobacteria, Sphingobacteriia, Blastocatellia) and fungi (Sordariales, Lasiosphaeriaceae and Glomeraceae) characterized mM. Within- and cross-domain network were more complex in mM than sM at surface layer, and the opposite occurred at subsurface. Some prokaryotic and fungal taxa (Chloroflexi and Sordariomycetes), found abundant in hubs within soil aggregate networks, were consistently positively related to C cycling and soil structuring. We can therefore conclude that soil aggregation should be included in a more complete ‘multifunctional’ perspective of soil ecology, and that a full understanding of soil processes requires analyses emphasizing feedbacks between soil structure and soil microbiota, rather than a unidirectional approach simply addressing single members in bulk soil. As CA systems and soil structure were strongly connected to soil microbiome and function, the application of CA practices should be supported for the restoration of disturbed soils, the prevention of soil erosion and the enhancement of SOC storage. Overall, the higher diversity and differentiated soil microbial structures observed in minimum and fertilized tillage systems may offer biological buffering capacity and maintain agriculturally relevant soil functions. This study allows to improve the knowledge on taxa resistant and sensitive to modifications induced by tillage and N fertilization, according to soil aggregation size. We also demonstrate that linking taxonomy to function is a priority for explaining the ecological interactions that promote SOC accumulation in soil aggregates.

Agricultural production in regions such as sub-Saharan Africa (SSA) is very low as compared to other regions such as the Mediterranean area (MED). Differences are mainly due to agricultural input use which is generally lower in SSA. Indeed, in both regions, where unsustainable agricultural practices are largely applied although varying in intensification, soil organic carbon degradation and soil biodiversity decline are widespread issues. However, whether changes of soil microbial diversity have consequences on agroecosystem services, like crop productivity, in such agro-ecological zones is still scarcely investigated at field level. This long-term field study aimed to understand how different agricultural practices, such as conservation agriculture (CA)-based systems, affect soil microbiome (i.e., arbuscular mycorrhizal fungi (AMF) and bacteria) and their implications on crop productivity. We selected two contrasting soils and agro-ecological zones, in Italy (Centre of Agro-Environmental Research “Enrico Avanzi” – CiRAA – in Pisa) and in Zambia (Msekera Research Station – MRS – in Chipata), to investigate the responses of AMF and bacterial community to CA practices. The experiment at CiRAA was started in 1993 on a silt loam soil and was set up as a split plot design to test tillage regime as the main plot factor and nitrogen (N) fertilization rate as the sub-plot factor. Tillage regimes were conventional tillage (CT) and minimum tillage (MT), while the N fertilization rates were no fertilization (N0) and fertilized with 200 kg N ha-1 split into three applications (N200). For all treatments, wheat (Triticum aestivum L.) was rotated with soybean (Glycine max L.) in one-year rotations. The experiment at MRS was started in 2012 on a sandy clay loam soil and tested three treatments: CT and maize (Zea mays L.) as sole crop, no-tillage plus mulch (NT+M), and no-tillage plus mulch and rotation (NT+M+R) with maize in rotation with soybean. The climate is cold humid Mediterranean (Csa) at CiRAA and warm temperate with dry winters and hot summers (Cwa) at MRS. DNA was extracted from soil: for AMF PCRs were carried out amplifying part of the SSU, ITS1, 5.8S, ITS2 and part of the LSU of the 18S rRNA region, while for bacteria PCRs were carried out amplifying the V3 and V4 regions of the 16S rRNA region. AMF were characterized by a cloning and Sanger sequencing approach (ca. 1700 bp), whereas bacteria by an Illumina sequencing approach (ca. 630 bp). In both zones, AMF and bacterial composition was similar among CA systems, whereas the long-term implementation of the CA systems resulted in more diverse microbial communities across the agro-ecological zones. CA systems led to positive interactions between AMF and bacterial communities and more complex soil microbial networks. This ultimately led to an improved crop yield. At MRS, soybean as a rotational crop enriched bacterial diversity and within the AMF communities, members of the family Gigasporaceae were more dominant. We finally identified the microbial taxa highly related to crop productivity, providing cause-effect relationships for the involvement of microbes in crop productivity.

Hydroxyapatite nanoparticles (nHAs) deriving from by-products have gained increasing interest as novel phosphorus (P)-based fertilisers, since they can provide a slow P release, minimising P losses and adverse environmental side-effects, and reducing the dependency of agriculture on mineral fertiliser inputs. Phosphate solubilising bacteria (PSB) have proven to release P available for crop uptake from different inorganic sources (e.g. tricalcium phosphate, TCP, hydroxyapatite, HA). In the present study, nHAs were prepared from salmon (S-nHAs) and tuna (T-nHAs) bones by a calcination process, followed by a high energy ball milling. The obtained fine powders were characterised by scanning electron microscopy (SEM) for size and shape and by X-ray diffraction (XRD) for crystal phase composition. The phosphate solubilisation activity of seven selected PSB strains belonging to Pseudomonas and Paraburkholderia genera was in vitro investigated under acidic (pH = 5.5) and alkaline (pH = 7.5) conditions by a quantitative assessment of the solubilised PO43- from TCP, S-nHAs and T-nHAs over time. Moreover, time trend of pH and organic acids in the liquid media were investigated. Characterization of S-nHAs by XRD and SEM revealed a biphasic composition of the material consisting of TCP and HA – about 50 wt% of each phase - and a heterogeneous rounded-shape (Ø < 50 nm) material. By contrast, XRD pattern of T-nHAs showed a single-phase composition mainly made of pure HA (> 95 wt%) and SEM micrographs exhibiting an elongated shape uniform in size (200 x 30 nm). At day seven, Pseudomonas graminis PG0319 solubilised the highest proportion of the total PO43- in the TCP substrate under acidic pH (83%), followed by Pseudomonas rhodesiae PR0393 and P. graminis PG1211 (79% and 72%, respectively). In S-nHAs under alkaline pH, Paraburkholderia terricola PT0405, PR0393, PG0319 and PG1211 solubilised from 53% to 57% of the total PO43-, whereas in T-nHAs under acidic pH the maximum solubilisation efficiency was 27% by PT0405 at day seven. The difference in the solubilisation of S-nHAs and T-nHAs is due to the lower solubility of HA in comparison with TCP. Values of pH in in the liquid media decreased over the time along with an increasing PO43- solubilisation activity, suggesting an extracellular secretion of organic acids by PSB. Accordingly, differential patterns of organic acids were detected among strains with TCP as well as S-nHAs and T-nHAs. Notably, gluconic, propionic, fumaric and acetic acids played key roles during P solubilisation with all the tested strains, substrates, and pH conditions. Our results indicate that the use of microbial inocula together with P-based nanofertilisers is a promising option for a sustainable agricultural transition.

Supplementary Figures 1-19. Supplementary Figure 1. Hypoxia pathways are enriched in ALCL. Supplementary Figure 2. Gene expression data on ALCL. Supplementary Figure 3. EML4-ALK NSCLC show enrichment in hypoxia pathway associated genes. Supplementary Figure 4. ALK regulates HIF-1α and HIF-2α expression in ALK-rearranged ALCL cell lines. Supplementary Figure 5. Oncogenic ALK regulates HIF-1α and HIF-2α expression levels in ALK-rearranged ALCL cell lines. Supplementary Figure 6. Oncogenic ALK-mediated control of HIF-1α and HIF-2α expression is not dependent on proteasome-dependent degradation. Supplementary Figure 7. ALK regulates HIF-1α and HIF-2α through STAT3 and C/EBPβ. Supplementary Figure 8. STAT3 and C/EBPβ mediated regulation of HIFs in ALCL. Supplementary Figure 9. Ectopic expression of NPM-ALK in ALK negative ALCL. Supplementary Figure 10. Validation of shRNA against HIF-1α and HIF-2α. Supplementary Figure 11. HIF-2α is essential for the growth of ALK-rearranged ALCL in vivo. Supplementary Figure 12. HIF-1α and HIF-2α are not required for the growth of ALK-negative T cell lymphoma in vivo. Supplementary Figure 13. ALK regulates VEGFA mRNA expression and production in vitro. Supplementary Figure 14. Anti-angiogenic treatment in ALK negative ALCL. Supplementary Figure 15. Oncogenic ALK regulates HIF-1α and HIF-2α expression levels in EML4-ALK NSCLC. Supplementary Figure 16. HIF-α regulation in NSCLC is specifically dependent on ALK activity. Supplementary Figure 17. Oncogenic ALK activity up-regulates VEGF expression in NSCLC. Supplementary Figure 18. HIF-α knock-down does not affect the growth of K-RAS mutated NSCLC. Supplementary Figure 19. HIF-1α and HIF-2α are essential for metastasis formation in EML4-ALK NSCLC.

Supplementary Figure Legends. File with Supplementary Figure Legends

Bioremediation of pharmaceuticals (PhCs) has gained large research efforts, but there is still a need to improve the performance of bioremediation systems by selecting effective organisms. In this study, we characterized the capability to remove clarithromycin (CLA) and diclofenac (DCF) by the bacterium Streptomyces rochei, and the fungi Phanerochaete chrysosporium and Trametes versicolor. Growth and content of the PhCs and a DCF metabolite (MET) by the energy crop Arundo donax L. were also evaluated under hydroponic conditions. The removal rate (RR) by S. rocheiincreased from 24 to 40% at 10 and 100 µg CLA L-1, respectively, averaged over incubation times. At 144 h, the RR by P. chrysosporium was 84%, while by T. versicolor was 70 and 45% at 10 and 100 CLA µg L-1. The RR by S. rochei did not exceed 30% at 1 mg DCF L-1 and reached 60% at 10 mg DCF L-1, whereas approached 95% and 63% by P. chrysosporium and T. versicolor, respectively, at both doses. Root biomass and length of A. donax were strongly affected at 100 µg CLA L-1. CLA concentration in roots and shoots increased with the increase of the dose and translocation factor (TF) was about 1. DCF severely affected both shoot fresh weight and root length at the highest dose and concentration in roots and shoots increased with the increase of the dose. DCF concentrations were 16-19 times higher in roots than in shoots and TF was about 0.1. MET was detected only in roots and its proportion over the parent compound decreased with the increase of the DCF dose. This study highlights the potential contribution of A. donax and the tested microbial inoculants for improving the effectiveness of bioremediation systems for CLA and DCF removal.

Purpose Plant microbial biostimulants, such as arbuscular mycorrhizal fungi (AMF), enhance nutrient concentration in fruits, including tomato. However, field studies on tomato AMF inoculation are scarce. AMF species belonging to Gigasporaceae and Glomeraceae families known to vary in life-history strategies may determine differential effects on plant nutrient benefits and residue decomposition. Despite this, the effect of different life-history strategies on nutrient acquisition of tomato fruits has not been investigated yet. Methods We studied the effect of inoculation of two tomato varieties with four AMF species belonging to Glomeraceae and Gigasporaceae. Fungal colonization, yield, fruit nutrient concentration, litter decomposition, and bacterial and fungal abundances in soil were assessed in the field under organic agriculture. Results Overall Gigasporaceae promoted the concentration of nutrients in tomato fruits compared to Glomeraceae. A variability in AM fungal colonization and fruit nutrient concentration was detected within Glomeraceae. Scutellospora pellucida increased the yield (+ 27%) of var. Rio Grande with respect to Gigaspora gigantea. In var. Rio Grande, inoculation with Funneliformis mosseae did not change litter decomposition as compared to non-inoculated controls, whereas it was lower than in Sclerocystis sinuosa and Gigasporaceae species, which showed the highest decomposition rates. AMF inoculation promoted soil total bacterial and fungal abundance and fungal:bacterial (F:B) ratio compared to controls, and members of Gigasporaceae had the highest F:B ratio. Conclusion These findings pointed at the inclusion of AM fungal life-history strategy within the selection criteria for the development of biofertilizers able to enhance the nutritional value of vegetables under organic farming systems.

Purpose: In the scenario of an increased atmospheric CO2 concentration, nutrients are reduced in all vegetables. In this context, arbuscular mycorrhizal fungi (AMF) could enhance nutrient concentration in edible portions of plants, including tomato. However, field studies on tomato AMF inoculation are scarse. AMF species belonging to Gigasporaceae and Glomeraceae families known to vary in life-history strategies may determine differential effects on plant nutrient benefits and residue decomposition. Despite this, the effect of different life-history strategies on nutrient acquisition of tomato fruits has not yet been investigated. Methods: We studied the effect of inoculation of two tomato varieties with AMF belonging to Glomeraceae and Gigasporaceae. Fungal establishment, yield, fruit nutrient concentration, litter decomposition, and bacterial and fungal abundances in soil were assessed in a two-year field trial under organic agriculture. Results: Overall Gigasporaceae promoted the concentration of nutrients in tomato fruits compared to Glomeraceae. A variability in AM fungal colonization and fruit nutrient concentration occurred within Glomeraceae. Scutellospora pellucida increased the yield (+27%) of var. Rio Grande respect to Gigaspora gigantea. In var. Rio Grande, Funneliformis mosseae determined a litter degradation similar to controls and lower than Sclerocystis sinuosa and Gigasporaceae species, which showed the highest decomposition rates. AMF inoculation promoted soil total bacterial and fungal abundance and fungal:bacterial (F:B) ratio compared to controls, and members of Gigasporaceae had the highest F:B ratio. Conclusion: These findings strongly support that AM fungal life-history strategy should be considered in biofertilizer development to enhance the nutritional value of vegetables under organic farming systems.

Summary The role that common mycorrhizal networks (CMNs) play in plant‐to‐plant transfer of zinc (Zn) has not yet been investigated, despite the proved functions of arbuscular mycorrhizal fungi (AMF) in crop Zn acquisition. Here, two autotrophic Medicago truncatula plants were linked by a CMN formed by Rhizophagus irregularis. Plants were grown in vitro in physically separated compartments (Donor‐C and Receiver‐C) and their connection ensured only by CMN. A symbiosis‐defective mutant of M. truncatula was used as control in Receiver‐C. Plants in both compartments were grown on Zn‐free medium, and only the leaves of the donor plants were Zn fertilized. A direct transfer of Zn was demonstrated from donor leaves to receiver shoots mediated by CMN. Direct transfer of Zn was supported by changes in the expression of fungal genes, RiZRT1 and RiZnT1, and plant gene MtZIP2 in roots and MtNAS1 in roots and shoots of the receiver plants. Moreover, Zn transfer was supported by the change in expression of MtZIP14 gene in AM fungal colonized roots. This work is the first evidence of a direct Zn transfer from a donor to a receiver plant via CMN, and of a triggering of transcriptional regulation of fungal‐plant genes involved in Zn transport‐related processes.

The effect of arbuscular mycorrhizal fungi (AMF) on yield and quality was investigated on a set of seven bread wheat genotypes with varying years of release, including five old genotypes and two modern varieties. A two-year field trial was conducted in central Italy under rainfed conditions. The effect of AM fungal seed coating was proved by assessing the AM fungal root colonization and studied on agronomic and quality traits, and in particular on gluten-forming proteins and grain mineral composition. AMF seed coating led to a general yield improvement in old genotypes (+24%). Concerning the effects on grain quality, while modern genotypes showed an increase in protein content (+16%), in the old ones an improvement of gluten quality was observed, with an increased proportion of HMW-GS from +17% to +92%. The gluten index results were mostly influenced by HMW-GS allelic configuration and amount, showing a significant correlation with gliadin-to-glutenin ratio and HMW-GS to LMW-GS. Concerning mineral uptake, AM fungal treatment determined a general increase in P content, which was more marked in the modern group (+44%). Furthermore, AMF significantly increased mean Fe concentration in Verna (+53%) and Bologna (+45%). Finally, phytate content did not increase with AMF, without affecting mineral bioavailability.

In this study, we examined whether the convergence in interlocutors’ vowel acoustics leads to decreasing discriminability between interlocutors’ voices. Ten pairs of Grison and Zürich German speakers produced lexical items before and after dialogue interactions with evidence of vowel convergence in post-dialogue productions. In Experiment 1, native and non-native Swiss German listeners discriminated pairs of speakers whose speech was obtained pre- and post-dialogue. Results showed that listeners’ sensitivity (A’) was higher for native than non-native listeners, but comparable for pre- and post-dialogue recordings. The observed negative correlation between voice discrimination and the acoustic distance in formant space was mainly driven by a single speaker pair. In Experiment 2, the speaker recognition performance of an i-vector-based software was compared in pre- and post-dialogue speech. Results revealed no difference in the system performance between the two conditions. The findings suggest that vowel convergence does not compromise voice discriminability under the given experimental conditions.

Arbuscular mycorrhizal fungi are promoted as biofertilizers due to potential benefits in crop productivity, and macro- and microelement uptake. However, crop response to arbuscular mycorrhizal fungi (AMF) inoculation is context-dependent, and AMF diversity and field establishment and persistence of inoculants can greatly contribute to variation in outcomes. This study was designed to test the hypotheses that multiple and local AMF inoculants could enhance alfalfa yield and fatty acids (FA) compared to exotic isolates either single or in the mixture. We aimed also to verify the persistence of inoculated AMF, and which component of the AMF communities was the major driver of plant traits. Therefore, a field experiment of AMF inoculation of alfalfa (

Foreign-accented speech typically deviates segmentally and suprasegmentally from native-accented speech. Two experiments were conducted to investigate the role of amplitude envelope (ENV), segment duration (DUR), and speech rate (SR) on Italian listeners' ability to identify native-accented Italian in utterances produced by Zurich German speakers. In experiment 1, listeners judged in a two-alternative forced-choice perception task which of the two stimuli in a trial they perceived as more native-like. Stimuli in each trial only varied in ENV and DUR, which were retrieved either from a native Italian speaker [first language (L1) donor] or from a German speaker of Italian [second language (L2) donor]. Results revealed that listeners make use of both DUR and ENV to identify the more native-like stimuli, but the effect of ENV was more subtle. In experiment 2, SR differences (resulting from native and non-native segment duration differences in experiment 1) were normalized for. It was found that this drastically reduced the effect of segment durations in terms of perceived nativeness; however, the ENV effect still remained. This was not the case in a control group of listeners without competence in Italian. Though effects were subtle, the study shows that ENV cues contribute to the percept of nativeness in L2 speech.

Human voices are individual and humans have elaborate skills in recognizing speakers by their voice, phenomena that are deeply rooted in the evolution of human behavior. To date, the mechanisms of speaker recognition are not well understood because of the high variability of the acoustic cues to a speaker’s identity. We wondered what role the speaker plays in making his/her voice more or less well recognizable. While it is evident from the literature that humans can control vocal properties to enhance their intelligibility, it is unclear whether speakers can and/or do control vocal characteristics to be better recognizable and whether such control mechanisms play a role in the communication process. In this paper, we reviewed results from the literature supporting the view that speaker idiosyncratic information is dynamic and that humans have the ability to control how well they can be recognized. We suggest possible experimental setups by which the control over identity in voice can be tested and present pilot acoustic characteristics of speech that was produced to be either targeted at being (a) intelligible (clear speech) and (b) suitable for person recognition (identity marked speech). Results revealed that there is reason to believe that speakers apply different mechanisms when making their individuality identifiable as opposed to making their speech better understood. We discuss predictions that a control of recognizability and intelligibility has within major theories of speech perception.

Zinc (Zn) is an essential micronutrient for plants and animals, and Zn deficiency is a widespread problem for agricultural production. Although many studies have been performed on biofortification of staple crops with Zn, few studies have focused on forages. Here, the molecular mechanisms of Zn transport in alfalfa (Medicago sativa L.) were investigated following foliar Zn applications. Zinc uptake and redistribution between shoot and root were determined following application of six Zn doses to leaves. Twelve putative genes encoding proteins involved in Zn transport (MsZIP1-7, MsZIF1, MsMTP1, MsYSL1, MsHMA4, and MsNAS1) were identified and changes in their expression following Zn application were quantified using newly designed RT-qPCR assays. These assays are the first designed specifically for alfalfa and resulted in being more efficient than the ones already available for Medicago truncatula (i.e., MtZIP1-7 and MtMTP1). Shoot and root Zn concentration was increased following foliar Zn applications ≥ 0.1 mg plant−1. Increased expression of MsZIP2, MsHMA4, and MsNAS1 in shoots, and of MsZIP2 and MsHMA4 in roots was observed with the largest Zn dose (10 mg Zn plant−1). By contrast, MsZIP3 was downregulated in shoots at Zn doses ≥ 0.1 mg plant−1. Three functional gene modules, involved in Zn uptake by cells, vacuolar Zn sequestration, and Zn redistribution within the plant, were identified. These results will inform genetic engineering strategies aimed at increasing the efficiency of crop Zn biofortification.

Conservation agriculture has been promoted to sustainably intensify food production in smallholder farming systems in southern Africa. However, farmers have rarely fully implemented all its components, resulting in different combinations of no-tillage, crop rotation, and permanent soil cover being practiced, thus resulting in variable yield responses depending on climatic and soil conditions. Therefore, it is crucial to assess the effect of conservation agriculture components on yield stability. We hypothesized that the use of all three conservation agriculture components would perform the best, resulting in more stable production in all environments. We evaluated at, eight trial locations across southern Africa, how partial and full implementation of these components affected crop yield and yield stability compared with conventional tillage alone or combined with mulching and/or crop rotation. Grain yield and shoot biomass of maize and cowpea were recorded along with precipitation for 2 to 5 years. Across different environments, the addition of crop rotation and mulch to no-tillage increased maize grain by 6%, and the same practices added to conventional tillage led to 13% yield increase. Conversely, adding only mulch or crop rotation to no-tillage or conventional tillage led to lower or equal maize yield. Stability analyses based on Shukla’s index showed for the first time that the most stable systems are those in which mulch is added without crop rotation. Moreover, the highest yielding systems were the least stable. Finally, additive main effects and multiplicative interaction analysis allowed clarifying that mulch added to no-tillage gives stable yields on sandy soil with high rainfall. Similarly, mulch added to conventional tillage gives stable yield on sandy soil, but under low rainfall. This is the first study that highlighted the crucial role of mulch to enhance the stability and resilience of cropping systems in southern Africa, supporting their adaptability to climate change.

The application of next generation sequencing (NGS) technique has a great impact on complex disease studies. Indeed, genetic heterogeneity, phenotypic variability, and disease rarity are all factors that make the traditional diagnostic approach to genetic disorders, whereby a specific gene is selected for sequencing based on the clinical phenotype, very challenging and obsolete.Exome sequencing, which sequences the protein-coding region of the genome, has been rapidly applied to variant discovery in research settings. Recent coverage and accuracy improvements have accelerated the development of clinical exome sequencing (CES) platforms targeting disease-related genes and enabling variant identification in patients with suspected genetic diseases. Nowadays, CES is rapidly becoming the diagnostic test of choice in patients with suspected Mendelian diseases, especially for those with heterogeneous etiology and clinical presentation. Reporting large CES series can improve guidelines on best practices for test utilization, and a better variant interpretation through clinically oriented data sharing.Herein, we suggest a feasible CES procedure for the genetic testing of Cerebral Cavernous Malformation (CCM) disease, including proband identification, library preparation, data analysis, and variant interpretation.

Zinc (Zn) is an essential micronutrient for both plants and animals, and Zn deficiency is one of the most widespread problems for agricultural production. Although many studies have been performed on the biofortification of staple crops with Zn, few studies have focused on forage crops. In this study the molecular mechanisms of Zn transport-related in Medicago sativa L. were investigated following foliar Zn applications aimed at increasing the accumulation of Zn in edible tissues. Zinc uptake and redistribution between shoot and root were determined following the application of six Zn doses to leaves (0, 0.01, 0.1, 0.5, 1, 10 mg Zn plant-1). Twelve putative genes encoding proteins involved in Zn transport (MsZIP1-7, MsZIF1, MsMTP1, MsYSL1, MsHMA4 and MsNAS1) were identified and the changes in their expression following foliar Zn application were quantified using newly designed RT-qPCR assays. Shoot and root Zn concentration was increased following foliar Zn applications ≥ 0.1 mg plant-1. Increased expression of MsZIP2, MsHMA4 and MsNAS1 in shoots, and of MsZIP2 and MsHMA4 in roots, was observed with the largest Zn dose. By contrast, MsZIP3 was downregulated in shoots at Zn doses ≥ 0.1 mg plant-1. Three functional modules were identified in the M. sativa response to foliar Zn application: genes involved in Zn uptake by cells, genes involved in vacuolar Zn sequestration and genes involved in Zn redistribution within the plant. These results will inform genetic engineering strategies aimed at increasing the efficiency of crop Zn biofortification.One-sentence summaryUpregulation of ZIP2, NASI and HMA4 and downregulation of ZIP3 are associated with Zn sequestration and shoot-to-root translocation in Medicago sativa following foliar Zn biofortification

Arbuscular mycorrhizal fungi (AMF) promote crop growth and yield by increasing N and P uptake and disease resistance, but the role of field AMF inoculation on the uptake of micronutrients, such as Fe and Zn, and accumulation in plant edible portions is still not clarified. Therefore, we studied the effect of field inoculation with Rhizophagus irregularis in an organic system on 11 old genotypes and a modern variety of bread wheat. Inoculation increased root colonization, root biomass and shoot Zn concentration at early stage and grain Fe concentration at harvest, while it did not modify yield. Genotypes widely varied for shoot Zn concentration at early stage, and for plant height, grain yield, Zn and protein concentration at harvest. Inoculation differentially modified root AMF community of the genotypes Autonomia B, Frassineto and Bologna. A higher abundance of Rhizophagus sp., putatively corresponding to the inoculated isolate, was only proved in Frassineto. The increase of plant growth and grain Zn content in Frassineto is likely linked to the higher R. irregularis abundance. The AMF role in increasing micronutrient uptake in grain was proved. This supports the introduction of inoculation in cereal farming, if the variable response of wheat genotypes to inoculation is considered.

In the Mediterranean, long-term impact of typical land uses on soil fertility have not been quantified yet on replicated mixed crop-livestock farms and considering the variability of soil texture. Here, we report the effects, after 15 years of practice, of two legume-winter cereal rotations, olive orchards and vineyards on microbiological and chemical indicators of soil fertility and the communities of arbuscular mycorrhizal fungi (AMF). We compare the changes among these four agricultural land-use types to woodland reference sites. Root colonization by AMF of English ryegrass (Lolium perenne L.), a grass that occurred under all land use types, was only half as heavy in biannual berseem clover (Trifolium alexandrinum L.)-winter cereal rotations than in 4-year alfalfa (Medicago sativa L.)-winter cereal rotations. In olive (Olea europaea L.) orchards and vineyards (Vitis vinifera L.), where weeds are controlled by frequent surface tillage, the AMF root colonization of ryegrass was again much lower than in the legume-cereal rotations and at the woodland reference sites. All the microbial parameters and soil organic carbon correlated most strongly with differences in occurrence and relative abundance (β-diversity) of AMF genera in soil. The soil pH and mineral nutrients in soil strongly correlated with differences in AMF root colonization and AMF genus richness (α-diversity) in soil. Diversity of AMF was much less affected by soil texture than land use, while the opposite was true for microbial and chemical soil fertility indicators. Land uses that guaranteed a continuous ground cover of herbaceous plants and that involved only infrequent tillage, such as multiyear alfalfa-winter cereal rotation, allowed members of the AMF genus Scutellospora to persist and remain abundant. On the contrary, under land uses accompanied by frequent tillage and hence discontinuous presence of herbaceous plants, such as tilled olive orchard and vineyard, members of the genus Funneliformis dominated. These results suggest that multiyear alfalfa-winter cereal rotation with active plant growth throughout the year is the least detrimental agricultural land use in soil carbon and AMF abundance and diversity, relative to the woodland reference.

Conservation agriculture is globally recommended for increasing soil C and reducing greenhouse gases emissions by modifying soil physical, chemical and biological properties and processes. We assessed the impact of long-term conservation tillage and N fertilization in wheat-soybean rotation on soil aggregate distribution, nutrients stocks and functions of soil microorganisms related to C, N, P and S cycles at different aggregation scales in the Mediterranean area. A long-term field experiment was set up in a silt loam soil comparing two tillage intensities to bread wheat (Triticum aestivum L.) - soybean (Glycine max L. Merr.) rotation: conventional tillage (CT) and minimum tillage (MT). Two N fertilization levels were applied on bread wheat: 0 and 200 kg N ha−1 (N0 and N200, respectively). Under CT, almost 100% of the crop residues were incorporated in the 0–25 cm soil layer, whereas under MT approximately 50% were incorporated at 0–15-cm depth. Tillage was the most discriminant factor explaining 72% and 60% of total variance of soil parameters at 0–15 cm and 15–30 cm soil depth, respectively, whereas N fertilization explained 22% and 29% of total variance, respectively. All enzyme activities were higher under MT, whereas the majority of soil chemical parameters were higher under N200. Under MT a higher proportion of free microaggregates (+51%) was recorded compared to CT suggesting that in a silt loam soil MT had a greater potential to form macroaggregates. The proportion of small macroaggregates was not changed by tillage, but when this fraction was fractionated a higher proportion of occluded microaggregates was found under MT at 15–30 cm (+21%). In the occluded microaggregates, interaction of tillage x N fertilization explained 52% of total variance of soil parameters at 0–15 cm. The most discriminant parameters were the biochemical ones and SOC. All those parameters were higher in MT-N200. Specifically, SOC in occluded microaggregates was increased by N fertilization under MT (+16% at 0–15 cm; +84% at 15–30 cm), whereas it was decreased under CT (-46% at 0–15 cm; -15% at 15–30 cm). In addition, the synthetic indexes for enzymatic activities and for those involved in C-cycle were reduced by N fertilization under MT up to 37% and increased up to 87% under CT. In the Mediterranean area, maintaining or even increasing SOC conservation may require both reduced tillage systems and N fertilization, shifting microbial community towards toward taxa more effective in contrasting soil degradation.