Reduction of antimicrobial resistance: Advancements in nature-based wastewater treatment.

Water scarcity, affecting one-fifth of the global population, is exacerbated by industrial, agricultural, and population growth pressures on water resources. Wastewater, containing Contaminants of Emerging Concern (CECs) such as antibiotics, presents environmental and health hazards. This study explores a Nature-Based Solution (NBS) using Constructed Wetlands (CWs) for wastewater reclamation and CECs removal. Two CW configurations (Vertical-VCW and Hybrid-HCW) were tested for their efficacy. Results show significant reduction in for all the chemico-physical and biological parameters meeting Italian water reuse standards. Furthermore, Antibiotic Resistant Bacteria (ARB) and Antibiotic Resistant Genes (ARGs) were effectively reduced, emphasizing the potential of the CWs in mitigating Antimicrobial Resistance (AMR). Lettuce seedlings irrigated with the treated wastewater exhibited no ARB/ARGs transfer, indicating the safety of the reclaimed wastewater for agricultural use. Overall, CWs emerge as sustainable Nature Based Solutions (NBS) for wastewater treatment, contributing to global water conservation efforts amid escalating water scarcity challenges.

Counteracting action of Bacillus stratosphericus and Staphylococcus succinus strains against deleterious salt effects on Zea mays L.

The salinization of soil is the process of progressive accumulation of salts such as sulfates, sodium, or chlorides into the soil. The increased level of salt has significant effects on glycophyte plants, such as rice, maize, and wheat, which are staple foods for the world's population. Consequently, it is important to develop biotechnologies that improve crops and clean up the soil. Among other remediation methods, there is an environmentally friendly approach to ameliorate the cultivation of glycophyte plants in saline soil, namely, the use of microorganisms tolerant to salt with growth-promoting features. Plant growth-promoting rhizobacteria (PGPR) can improve plant growth by colonizing their roots and playing a vital role in helping plants to establish and grow in nutrient-deficient conditions. Our research aimed to test in vivo halotolerant PGPR, isolated and characterized in vitro in a previous study conducted in our laboratory, inoculating them on maize seedlings to improve their growth in the presence of sodium chloride. The bacterial inoculation was performed using the seed-coating method, and the produced effects were evaluated by morphometric analysis, quantization of ion contents (sodium, potassium), produced biomass, both for epigeal (shoot) and hypogeal (root) organs, and by measuring salt-induced oxidative damage. The results showed an increase in biomass and sodium tolerance and even a reduction of oxidative stress in seedlings pretreated with a PGPR bacterial consortium (Staphylococcus succinus + Bacillus stratosphericus) over the control. Moreover, we observed that salt reduces growth and alters root system traits of maize seedlings, while bacterial treatment improves plant growth and partially restores the root architecture system in saline stress conditions. Therefore, the PGPR seed-coating or seedling treatment could be an effective strategy to enhance sustainable agriculture in saline soils due to the protection of the plants from their inhibitory effect.

Exploring the crop epigenome: a comparison of DNA methylation profiling techniques.

Epigenetic modifications play a vital role in the preservation of genome integrity and in the regulation of gene expression. DNA methylation, one of the key mechanisms of epigenetic control, impacts growth, development, stress response and adaptability of all organisms, including plants. The detection of DNA methylation marks is crucial for understanding the mechanisms underlying these processes and for developing strategies to improve productivity and stress resistance of crop plants. There are different methods for detecting plant DNA methylation, such as bisulfite sequencing, methylation-sensitive amplified polymorphism, genome-wide DNA methylation analysis, methylated DNA immunoprecipitation sequencing, reduced representation bisulfite sequencing, MS and immuno-based techniques. These profiling approaches vary in many aspects, including DNA input, resolution, genomic region coverage, and bioinformatics analysis. Selecting an appropriate methylation screening approach requires an understanding of all these techniques. This review provides an overview of DNA methylation profiling methods in crop plants, along with comparisons of the efficacy of these techniques between model and crop plants. The strengths and limitations of each methodological approach are outlined, and the importance of considering both technical and biological factors are highlighted. Additionally, methods for modulating DNA methylation in model and crop species are presented. Overall, this review will assist scientists in making informed decisions when selecting an appropriate DNA methylation profiling method.

Landfill leachate treatment by a combination of a multiple plant hybrid constructed wetland system with a solar photoFenton process in a raceway pond reactor.

The sustainable and green treatment of landfill leachate (LL), produced by municipal solid waste, represents one of the most relevant challenges in the integrated waste management systems. Accordingly, in this work a green solution was investigated by coupling an innovative hybrid constructed wetland (HCW) to a solar photo-Fenton (SPF) process. A multiple layers HCW pilot plant including different medium substrates (sand, solid compost and carriers) and plant species (Phragmites australis, Arundo donax and A. plinii) was designed. The HCW was functionalised with compost tea solution to simultaneously provide high nutrient content for plants and increase the microorganism biodiversity. Process efficiency was investigated using different real LLs (young and mature) in terms of chemical oxygen demand (COD), nitrogen compounds, chlorides and metals. Removals in the range 75-95% were observed for all the parameters after ten days of leachate recirculation in the pilot plant. Subsequently, the SPF process was carried out in a raceway pond reactor (RPR) as polishing step, significantly improving COD removal (further 49%). HCW combined with SPF in RPR would allow to meet the corresponding limits according to the final use/fate of the effluent by modulating the main parameters of the process.

Transgenerational effects of chromium stress at the phenotypic and molecular level in Arabidopsis thaliana.

In this study, we describe the results obtained in a study of the transgenerational phenotypic effects of chromium (Cr) stress on the model plant species Arabidopsis thaliana. The F1 generation derived from parents grown under chronic and medium chronic stress showed significantly higher levels of the maximal effective concentration (EC50) compared with F1 plants generated from unstressed parents. Moreover, F1 plants from Cr-stressed parents showed a higher germination rate when grown in the presence of Cr. F1 plants derived from parents cultivated under chronic Cr stress displayed reduced hydrogen peroxide levels under Cr stress compared to controls. At lower Cr stress levels, F1 plants were observed to activate promptly more genes involved in Cr stress responses than F0 plants, implying a memory effect linked to transgenerational priming. At higher Cr levels, and at later stages, F1 plants modulated significantly fewer genes than F0 plants, implying a memory effect leading to Cr stress adaptation. Several bHLH transcription factors were induced by Cr stress in F1 but not in F0 plants, including bHLH100, ORG2 and ORG3. F1 plants optimized gene expression towards pathways linked to iron starvation response. A model of the transcriptional regulation of transgenerational memory to Cr stress is presented here, and could be applied for other heavy metal stresses.

Normalized Workflow to Optimize Hybrid De Novo Transcriptome Assembly for Non-Model Species: A Case Study in Lilium ledebourii (Baker) Boiss.

A high-quality transcriptome is required to advance numerous bioinformatics workflows. Nevertheless, the effectuality of tools for de novo assembly and real precision assembled transcriptomes looks somewhat unexplored, particularly for non-model organisms with complicated (very long, heterozygous, polyploid) genomes. To disclose the performance of various transcriptome assembly programs, this study built 11 single assemblies and analyzed their performance on some significant reference-free and reference-based criteria. As well as to reconfirm the outputs of benchmarks, 55 BLAST were performed and compared using 11 constructed transcriptomes. Concisely, normalized benchmarking demonstrated that Velvet-Oases suffer from the worst results, while the EvidentialGene strategy can provide the most comprehensive and accurate transcriptome of Lilium ledebourii (Baker) Boiss. The BLAST results also confirmed the superiority of EvidentialGene, so it could capture even up to 59% more (than Velvet-Oases) unique gene hits. To promote assembly optimization, with the help of normalized benchmarking, PCA and AHC, it is emphasized that each metric can only provide part of the transcriptome status, and one should never settle for just a few evaluation criteria. This study supplies a framework for benchmarking and optimizing the efficiency of assembly approaches to analyze RNA-Seq data and reveals that selecting an inefficient assembly strategy might result in less identification of unique gene hits.

An Epigenetic Alphabet of Crop Adaptation to Climate Change.

Crop adaptation to climate change is in a part attributed to epigenetic mechanisms which are related to response to abiotic and biotic stresses. Although recent studies increased our knowledge on the nature of these mechanisms, epigenetics remains under-investigated and still poorly understood in many, especially non-model, plants, Epigenetic modifications are traditionally divided into two main groups, DNA methylation and histone modifications that lead to chromatin remodeling and the regulation of genome functioning. In this review, we outline the most recent and interesting findings on crop epigenetic responses to the environmental cues that are most relevant to climate change. In addition, we discuss a speculative point of view, in which we try to decipher the "epigenetic alphabet" that underlies crop adaptation mechanisms to climate change. The understanding of these mechanisms will pave the way to new strategies to design and implement the next generation of cultivars with a broad range of tolerance/resistance to stresses as well as balanced agronomic traits, with a limited loss of (epi)genetic variability.

Botanical, Phytochemical, Anti-Microbial and Pharmaceutical Characteristics of Hawthorn (Crataegusmonogyna Jacq.), Rosaceae.

Hawthorn (Crataegus monogyna Jacq.) is a wild edible fruit tree of the genus Crataegus, one of the most interesting genera of the Rosaceae family. This review is the first to consider, all together, the pharmaceutical, phytochemical, functional and therapeutic properties of C. monogyna based on numerous valuable secondary metabolites, including flavonoids, vitamin C, glycoside, anthocyanin, saponin, tannin and antioxidants. Previous reviews dealt with the properties of all species of the entire genera. We highlight the multi-therapeutic role that C. monogyna extracts could have in the treatment of different chronic and degenerative diseases, mainly focusing on flavonoids. In the first part of this comprehensive review, we describe the main botanical characteristics and summarize the studies which have been performed on the morphological and genetic characterization of the C. monogyna germplasm. In the second part, the key metabolites and their nutritional and pharmaceutical properties are described. This work could be an essential resource for promoting future therapeutic formulations based on this natural and potent bioactive plant extract.

Air quality biomonitoring through Olea europaea L.: The study case of "Land of pyres".

The "Land of pyres", namely "La Terra dei Fuochi", is an area of Campania region (South-Italy), highly inhabited and comprises between the Provinces of Naples and Caserta, sadly known worldwide for the criminal activities related to the illegal waste disposal and burning. These fires, concomitantly with traffic emissions, might be the source of potential toxic element (PTE) dangerous for the human health and causing pathologies. In the framework of Correlation Health-Environment project, funded by the Campania region, eight municipalities (of area "Land of pyres") and three remote sites have been bio-monitored using the olive (Olea europaea L.) plants as biomonitors. Leaves of olive plants were collected in each assayed municipality and the concentration of 11 metal(loid)s was evaluated by means of ICP-OES. Our findings revealed that the air of these municipalities was limitedly contaminated by PTE; in fact, only Sb, Al and Mn were detected in the olive leaves collected in some of the assayed municipalities and showed a high enrichment factors (EC) manly due, probably, to the vehicular traffic emissions. Furthermore, the concentrations of the other assayed PTEs were lower than those of Sb, Al and Mn. For these reasons we suppose that their emissions in the troposphere have been and are limited, and they mainly have a crustal origin. Even if our data are very comforting for those urban area, regarded by many as one of the most contaminated one in Italy, a great environment care, in any case, is always needed.

Genetic Diversity and Differentiation of Eleven Medicago Species from Campania Region Revealed by Nuclear and Chloroplast Microsatellites Markers.

The species belonging to the genus Medicago are considered a very important genetic resource at global level both for planet's food security and for sustainable rangelands management. The checklist of the Italian flora (2021) includes a total number of 40 Medicago species for Italy, and 27 for Campania region, with a number of doubtful records or related to species no more found in the wild. In this study, 10 Medicago species native to Campania region, and one archaeophyte (M. sativa), identified by means of morphological diagnostic characters, were analyzed in a blind test to assay the efficacy of nine microsatellite markers (five cp-SSRs and four n-SSRs). A total number of 33 individuals from 6 locations were sampled and genotyped. All markers were polymorphic, 40 alleles were obtained with n-SSRs ranging from 8-12 alleles per locus with an average of 10 alleles per marker, PIC values ranged from 0.672 to 0.847, and the most polymorphic SSR was MTIC 564. The cp-SSRs markers were highly polymorphic too; PIC values ranged from 0.644 to 0.891 with an average of 0.776, the most polymorphic cp-SSR was CCMP10. 56 alleles were obtained with cp-SSRs ranging from 7 to 17 alleles per locus with an average of 11. AMOVA analysis with n-SSR markers highlighted a great level of genetic differentiation among the 11 species, with a statistically significant fixation index (FST). UPGMA clustering and Bayesian-based population structure analysis assigned these 11 species to two main clusters, but the distribution of species within clusters was not the same for the two analyses. In conclusion, our results demonstrated that the combination of the used SSRs well distinguished the 11 Medicago species. Moreover, our results demonstrated that the use of a limited number of SSRs might be considered for further genetic studies on other Medicago species.

Epigenetic Analysis through MSAP-NGS Coupled Technology: The Case Study of White Poplar Monoclonal Populations/Stands.

Over the last several decades, several lines of evidence have shown that epigenetic modifications modulate phenotype and mediate an organism's response to environmental stimuli. Plant DNA is normally highly methylated, although notable differences exist between species. Many biomolecular techniques based on PCR have been developed to analyse DNA methylation status, however a qualitative leap was made with the advent of next-generation sequencing (NGS). In the case of large, repetitive, or not-yet-sequenced genomes characterised by a high level of DNA methylation, the NGS analysis of bisulphite pre-treated DNA is expensive and time consuming, and moreover, in some cases data analysis is a major challenge. Methylation-sensitive amplification polymorphism (MSAP) analysis is a highly effective method to study DNA methylation. The method is based on the comparison of double DNA digestion profiles (EcoRI-HpaII and EcoRI-MspI) to reveal methylation pattern variations. These are often attributable to pedoclimatic and stress conditions which affect all organisms during their lifetime. In our study, five white poplar (Populus alba L.) specimens were collected from different monoclonal stands in the Maltese archipelago, and their DNA was processed by means of an innovative approach where MSAP analysis was followed by NGS. This allowed us to identify genes that were differentially methylated among the different specimens and link them to specific biochemical pathways. Many differentially methylated genes were found to encode transfer RNAs (tRNAs) related to photosynthesis or light reaction pathways. Our results clearly demonstrate that this combinatorial method is suitable for epigenetic studies of unsequenced genomes like P. alba (at the time of study), and to identify epigenetic variations related to stress, probably caused by different and changing pedoclimatic conditions, to which the poplar stands have been exposed.

Effects of Zinc Pollution and Compost Amendment on the Root Microbiome of a Metal Tolerant Poplar Clone.

Until recently, many phytoremediation studies were focused solely on a plants ability to reclaim heavy metal (HM) polluted soil through a range of different processes, such as phytoextraction and phytostabilization. However, the interaction between plants and their own rhizosphere microbiome represents a new research frontier for phytoremediation. Our hypothesis is that rhizomicrobiome might play a key role in plant wellness and in the response to external stimuli; therefore, this study aimed to shed light the rhizomicrobiome dynamics after an organic amendment (e.g., compost) and/or HM pollution (e.g., Zn), and its relation with plant reclamation ability. To reach this goal we set up a greenhouse experiment cultivating in pot an elite black poplar clone (N12) selected in the past for its excellent ability to reclaim heavy metals. N12 saplings were grown on a soil amended with compost and/or spiked with high Zn doses. At the end of the experiment, we observed that the compost amendment strongly increased the foliar size but did not affect significantly the Zn accumulation in plant. Furthermore, the rhizomicrobiome communities (bacteria and fungi), investigated through NGS, highlighted how α diversity increased in all treatments compared to the untreated N12 saplings. Soil compost amendment, as well as Zn pollution, strongly modified the bacterial rhizomicrobiome structure. Conversely, the variation of the fungal rhizomicrobiome was only marginally affected by soil Zn addition, and only partially impaired by compost. Nevertheless, substantial alterations of the fungal community were due to both compost and Zn. Together, our experimental results revealed that organic amendment increased the bacterial resistance to external stimuli whilst, in the case of fungi, the amendment made the fungi microbiome more susceptible. Finally, the greater microbiome biodiversity does not imply, in this case, a better plant wellness or phytoremediation ability, although the microbiome plays a role in the external stimuli response supporting plant life.

Arsenic phytovolatilization and epigenetic modifications in Arundo donax L. assisted by a PGPR consortium.

Arsenic-(As) pollution is an increasing threat across the globe and it is reaching harmful values in several areas of the world. In this perspective, we assayed bio-phyto-remediation technology using Arundo donax L., assisted by Plant Growth Promoting Bacteria (PGPB) consortium (BC) constituted of two strains of Stenotrophomonas maltophilia sp. and one of Agrobacterium sp.; furthermore, we assayed the epigenetic response to As pollution. The three bacterial strains initially evaluated for their As tolerance, revealed different resistance to both forms of As[As(III) and As(V)] however at concentration greater than those foreseen in the phytoremediation experiment (2.0, 10.0, 20.0 mgL-1 of NaAsO2). At the end of the trial plant biomass and As concentration were measured. Plants did not show any visible signs of toxicity, rather the leaf and stem biomass slightly increased in the presence of As and/or PGPBs; moreover, although the Bioaccumulation Factor was double in the presence of BC, the absolute values of As accumulation in the Arundo plants were very low, both in the presence or absence of BC and only detectable in the presence of the highest As dose (20 mgL-1 As). In this case, regardless the presence of PGPB, ≈25% of As remained in the sand and ≈0.15% was accumulated in the plant, whilst the remaining 75% was volatilized by transpiration. Finally, the methylation sensitive amplified polymorphisms (MSAP) of leaves were analyzed in order to investigate their epigenetic response to As and/or BC. Our results suggest that epigenetic modifications are involved in stress response and As detoxification.

The combined effect of Cr(III) and NaCl determines changes in metal uptake, nutrient content, and gene expression in quinoa (Chenopodium quinoa Willd.).

Many areas of the world are affected simultaneously by salinity and heavy metal pollution. Halophytes are considered as useful candidates in remediation of such soils due to their ability to withstand both osmotic stress and ion toxicity deriving from high salt concentrations. Quinoa (Chenopodium quinoa Willd) is a halophyte with a high resistance to abiotic stresses (drought, salinity, frost), but its capacity to cope with heavy metals has not yet been fully investigated. In this pot experiment, we investigated phytoextraction capacity, effects on nutrient levels (P and Fe), and changes in gene expression in response to application of Cr(III) in quinoa plants grown on saline or non-saline soil. Plants were exposed for three weeks to 500 mg kg-1 soil of Cr(NO3)3·9H2O either in the presence or absence of 150 mM NaCl. Results show that plants were able tolerate this soil concentration of Cr(III); the metal was mainly accumulated in roots where it reached the highest concentration (ca. 2.6 mg g-1 DW) in the presence of NaCl. On saline soil, foliar Na concentration was significantly reduced by Cr(III). Phosphorus translocation to leaves was reduced in the presence of Cr(III), while Fe accumulation was enhanced by treatment with NaCl alone. A real-time RT-qPCR analysis was conducted on genes encoding for sulfate, iron, and phosphate transporters, a phytochelatin, a metallothionein, glutathione synthetase, a dehydrin, Hsp70, and enzymes responsible for the biosynthesis of proline (P5CS), glycine betaine (BADH), tocopherols (TAT), and phenolic compounds (PAL). Cr(III), and especially Cr(III)+NaCl, affected transcript levels of most of the investigated genes, indicating that tolerance to Cr is associated with changes in phosphorus and sulfur allocation, and activation of stress-protective molecules. Moderately saline conditions, in most cases, enhanced this response, suggesting that the halophytism of quinoa could contribute to prime the plants to respond to chromium stress.

Heavy metal tolerance of orchid populations growing on abandoned mine tailings: A case study in Sardinia Island (Italy).

Understanding how environmental pollutants influence plant occurrence, growth, and development is key for effective management plans and potential bioremediation. Rare plants, such as orchids, may occur in modified habitats and on soils containing heavy metals, yet their ecological and physiological responses to heavy metals is poorly understood. We investigated the influence of heavy metal pollution on orchid growth rates and interactions with soil fungal mutualists by comparing a large population of the orchid Epipactis helleborine (L.) Crantz subsp. tremolsii (Pau) E. Klein that grows on mine tailings in south-west Sardinia (Italy) with a population that grows on non-contaminated soils in central Sardinia. Soils of the contaminated site had high levels of heavy metals and low organic matter and nutritive elements content. We performed a morphological analysis on twenty individuals that have been subjected to measurement of bioaccumulation and translocation of heavy metals. Fungi associated with the roots of plants from the contaminated and uncontaminated site were grown and identified by DNA barcoding approach. Plants from the contaminated site were smaller than the ones growing in the uncontaminated site and were found to be able to tolerate heavy metals from the soil and to accumulate and translocate them into their organs. Fungi belonging to the genus Ilyonectria (Ascomycota) were found both in contaminated and uncontaminated sites, while an unidentified fungus was isolated from roots in the contaminated site only. These results are discussed in terms of orchids' tolerance to heavy metals and its physiological and ecological mechanisms. The role of contaminated habitats in harbouring orchids and peculiar taxa is also discussed.

High Biodiversity Arises from the Analyses of Morphometric, Biochemical and Genetic Data in Ancient Olive Trees of South of Italy.

Morphometric, biochemical and genetic analyses were conducted on Olea europaea L. of Campania, an area of Southern Italy highly suited to the cultivation of olive trees and the production of extra virgin olive oil (EVOO). We aimed to characterize the distribution of morphological, biochemical and genetic diversity in this area and to develop a practical tool to aid traceability of oils. Phenotypes were characterized using morphometric data of drupes and leaves; biochemical and genetic diversity were assessed on the basis of the fatty acid composition of the EVOOs and with microsatellite markers, respectively. We provide an open-source tool as a novel R package titled 'OliveR', useful in performing multivariate data analysis using a point and click interactive approach. These analyses highlight a clear correlation among the morphological, biochemical and genetic profiles of samples with four collection sites, and confirm that Southern Italy represents a wide reservoir of phenotypic and genetic variability.

Ecotype-Level Genetic Biodiversity of Five Italian Traditional Crops.

Italy displays a high level of agrobiodiversity due to its diversified pedoclimatic zones. The Administrative Region of Campania includes several and divergent biomes, occurring close to each other. In fact, the distance between a sea level environment and that of high mountains can be less than 20 km. These environmental conditions allow the cultivation of many different crops and vegetables, represented by diverse ecotypes and varieties that are well adapted to the distribution range where they have been selected and grown. Efforts to maintain and further increase biodiversity in farming systems require a better understanding of the existing diversity created by traditional farming practices. The aim of our study was to identify and molecularly characterize several ecotypes belonging to five horticultural species commonly cultivated in Campania. In particular, we analysed five ecotypes of maize, two of garlic, four of onion, one of escarole, and two of courgette by means of simple sequence repeat (SSR) markers in order to evaluate their level of genetic biodiversity. The results reveal, for the first time, the high genetic biodiversity of horticultural ecotypes of the Campania Region. This feature is very important to improve the quality and productivity of agroecosystems.

The use of MSAP reveals epigenetic diversity of the invasive clonal populations of Arundo donax L.

Among the most widespread plant species with clonal reproduction Arundo donax L. represents one of most studied one characterized by very low genetic biodiversity. Although it is a perennial rhizomatous tall grass native to eastern and southern Asia, it spreads only asexually in the invaded range all over the world thriving very well in a large array of pedo-climatic conditions. This ability to morphologically or physiologically adapt to a broad array of conditions could be attributed to epigenetic mechanisms. To shade light on this relevant issue, 96 stems of A. donax from spontaneous populations distributed across the Italian invaded range (island of Sardinia, Northern and Southern Italy) were analysed. Leaf DNAs were extracted and processed through AFLPs and MSAPs for defining either genetic and epigenetic profiles. Both analyses clearly showed that the A. donax populations of Sardinia island are genetically distinct from those of Italian mainland; AFLPs showed an extremely low genetic biodiversity due to vegetative reproduction, whilst, epi-biodiversity, estimated through MSAP marker, increased within the analyzed populations. These results suggest that the capability of A. donax to invade and thrive in diverse environmental conditions can be, at least, partially attributed to a higher epigenetic variability. Therefore, the different DNA methylation status may have significant and important biological meaning, in particular, in the case of invasive clonal plants such as A. donax, also for the biodiversity definition, and MSAP marker can be considered an useful and cost effective marker to reveal it.