Antioxidants in Fruit Fractions of Mediterranean Ancient Pear Cultivars.

BACKGROUND: The genetic diversity of Sardinian pear germplasm has received limited attention regarding its chemical composition. Understanding this composition can aid in the setting up of resilient, extensive groves that offer multiple products and ecosystem services. This research aimed at investigating the antioxidant properties and phenolic compounds of ancient pear cultivars grown extensively in Sardinia (Italy); Methods: the cultivars Buttiru, Camusina, Spadona, and Coscia (as a reference) were compared. Fruit samples were manually peeled and cut. Their flesh, peel, core, and peduncle were frozen separately, lyophilized, and milled before being analysed; Results: The content of total phenolics (TotP), total flavonoids (TotF), condensed tannins (CT), and antioxidant capacity in each fruit part varied significantly among the cultivars. The TotP content was high in the peduncle (42.2-58.8 g GAE kg-1 DM) and low in flesh (6.4-17.7 g GAE kg-1 DM); Conclusions: the highest values of antioxidant capacity, TotP, NTP, TotF, and CT were found in the flesh of the cultivar Buttiru and in the peel of the cultivar Camusina. Chlorogenic acid was the major individual phenolic compound in peel, flesh and core, whereas arbutin was mostly present in the peduncle. Results can contribute to revise target exploitations of underutilized ancient pear cultivars.

Subterranean Clover and Sulla as Valuable and Complementary Sources of Bioactive Compounds for Rainfed Mediterranean Farming Systems.

Mediterranean pasture and forage legumes are important components of sustainable production systems. Subterranean clover and sulla represent key species having proven high agronomic value and traits for production and multiple services. Our research investigated the potential of the abovementioned species as a source of phenolic compounds and antioxidants for contributing to support their full exploitation in the fodder, animal welfare, and nutraceutical sectors. Antioxidant capacity, as well as the content of total phenolic compounds and individual phenolic compounds, was determined in subterranean clover and sulla shoots at the vegetative, flower bud, flowering, and seed ripening phenological stages. The antioxidant capacity and the phenolic content were affected significantly by harvest time. In subterranean clover, 10 individual phenolic compounds were detected, and isoflavones were the most abundant (3.19-18.27 mg·g-1 DM). Eleven phenolic compounds were identified in sulla shoots, and chlorogenic acid (0.76-3.43 mg·g-1 DM) and diosmin (3.64-4.94 mg·g-1 DM) were the most represented compounds at the vegetative and flower bud stage. On the basis of our findings, a complementary utilization of both legume species is suggested; this might ensure the exploitation of all phenolic compounds in view of the potential benefits for animal production and health.

Antioxidant Contents in a Mediterranean Population of Plantago lanceolata L. Exploited for Quarry Reclamation Interventions.

Plantago lanceolata L. (plantain) is an interesting multipurpose perennial species whose aerial parts are used in herbal medicine due to its precious phytochemicals and are palatable to animals. Moreover, peculiar traits such as drought tolerance, an extended growth season and a deep root system, make plantain a promising pioneer plant for quarry reclamation based on the use of native species. This study evaluated the effects of different environmental conditions and seasons on the accumulation of the bioactive compounds of its aerial organs. An autochthonous plantain population was grown in three locations in Sardinia (Italy). Leaves, peduncles and inflorescences were collected between October 2020 and July 2021. Phenolic contents and antioxidant capacity were determined. The analysis of the individual phenolic compounds was performed using liquid chromatography. In leaves, the content of total phenolics, antioxidant capacity and total flavonoids were significantly influenced by location and season. Total phenolic content ranged from 65 to 240 g gallic acid equivalent kg-1, whereas total flavonoids were from 16 to about 89 g catechin equivalent kg-1. Neochlorogenic, chlorogenic, cryptochlorogenic acids, verbascoside, diosmin and luteolin were identified in the methanolic extracts of leaves, peduncles and inflorescences. Verbascoside was the main antioxidant isolated from plantain extracts. Results evidenced an increasing accumulation pattern of phenolics from vegetative stage to flowering, followed by a decrement towards the seed ripening as well as site-specific differences with amounts of phenolics even 25% higher for same plantain accession.

Bioactive Compounds from Leaves and Twigs of Guayule Grown in a Mediterranean Environment.

Guayule (Parthenium argentatum) is one of the most promising alternatives to produce natural rubber. As the guayule leaves represent a waste byproduct in the rubber extraction process, their exploitation might contribute to the valorization of the whole plant biomass. The specific aim of our study was to determine the antioxidant capacity and the content of phenolic compounds in leaves and twigs of different guayule lines cultivated in a Mediterranean environment. The antioxidant capacity and the contents of phenolic were affected significantly by guayule accession and harvest time. Overall means in twigs represented about 60% of the corresponding leaf values for antioxidant capacity as DPPH (1,1-diphenyl-2-picrylhydrazyl) and not tannic phenolic and about 55% as ABTS (2,2'-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt), total phenolic, tannic phenolic and total flavonoid contents. Five individual phenolic compounds were identified in methanolic extracts of leaves. Neochlorogenic acid (62.5-174.8 mg g-1 DW) and chlorogenic acid (28.6-41.1 mg g-1 DW) were the most abundant phenolic acids. In addition to the compounds quantified in leaves, cynarin was identified only in twigs and for the first time in guayule biomass. Our results indicate that guayule leaves and twigs represent a rich source of antioxidants for potential applications in fodder, nutraceutical and pharmacological sectors.

Phenolic compounds content and antioxidant capacity in cardoon achenes from different head orders.

Cardoon inflorescences (heads) contain achenes, which are used for the production of edible oil and biodiesel but also represent a phenolic source. This research aimed at determining the level of bioactive compounds and antioxidant capacity of cardoon achenes and investigating their qualitative and quantitative variations in achenes harvested from heads having different insertion order on main stem. The head order significantly affected the number of achenes per head and one thousand weight but did not affect the achene values of antioxidant capacity and concentrations of total phenolic (107.7-117.4 g gallic acid equivalent kg-1 dry weight), as well as the concentration of chlorogenic and 3,5-di-O-E-caffeoylquinic acids. Conversely, the absolute amounts of these metabolites differed significantly among head order when referred to a single achene or head. About 75% of their absolute values per cardoon plant were produced by achenes belonging to secondary and tertiary heads.

Antioxidant Sources from Leaves of Russian Dandelion.

Taraxacum kok-saghyz (TKS) is a dandelion species native to Kazakhstan, Uzbekistan and north-west China, considered as a promising alternative source of natural rubber from its roots. The aim of this study was to investigate the possible exploitation of TKS leaves, a rubber byproduct, as a source of phenolic compounds with antioxidant properties for potential applications in forage, nutraceutical and pharmacological fields. Two accessions (TKS016, TKS018) grown under Mediterranean conditions of Sardinia were evaluated at vegetative and flowering stages. The leaves of TKS018 had the highest antioxidant capacity (19.6 mmol trolox equivalent antioxidant capacity 100 g-1 ), total phenolic (106.4 g gallic acid equivalent kg-1 ), tannic phenolics (58.5 g gallic acid equivalent kg-1 ) and total flavonoid contents (22.9 g catechin equivalent kg-1 ). At both phenological stages, TKS016 showed significantly lower values than TKS018 in 1,1-diphenyl-2-picrylhydrazyl (DPPH), total phenolic and tannic phenolics. Six individual molecules were identified, namely chlorogenic, cryptochlorogenic, caffeic, sinapic, chicoric and 3,4-dimethoxycinnamic acids. Chicoric (8.53-10.68 g kg-1 DW) and chlorogenic acids (4.18-7.04 g kg-1 DW) were the most abundant. TKS leaves represent a valuable source of chicoric acid with potential application as antioxidant to be used as herbal medicine and nutrition for production of healthy food/feed.

Nodule-associated microbiome diversity in wild populations of Sulla coronaria reveals clues on the relative importance of culturable rhizobial symbionts and co-infecting endophytes.

The culturable bacteria from root nodules of Sulla coronaria growing in spontaneous conditions in Sardinia were characterized. This plant's peculiarity is to represent a legume still found in both wild and cropped statuses. We tested whether culturable bacteria would differ from those commonly isolated from its field-cropped varieties, to date exclusively represented by Rhizobium sullae. 63 isolates from 60 surface-sterilized nodules were analyzed by ARDRA and 16S rDNA sequencing. The official nitrogen-fixing symbiont Rhizobium sullae was found only in 25 nodules out of 60. The remaining nodules did not yield culturable rhizobia but a number of different endophytic genera including Pseudomonas sp. (17 nodules), Microbacterium sp. (15 nodules), Pantoea agglomerans (5 nodules). The situation appears therefore a hybrid between what is commonly observed in other Mediterranean legumes occurring only in wild status (featuring non-culturable rhizobia and arrays of culturable endophytes within nodules), as opposed to cropped legumes (endowed with fully culturable rhizobia and minimal endophytic occurrence). These findings, within a species bridging the ecology between native and cropped conditions, suggest insights on the relative importance of endophytic co-occupancy vs. true N-fixing symbiont culturability within nodules. The latter trait thus appears to accompany the domestication path of plants with a main trade-off of renouncing to interactions with a diversity of endophytic co-invaders; the relationships with those being critical in the non-cropped status. In fact, endophytes are known to promote plant growth in harsh conditions, which can be particularly stressful in the Mediterranean due to drought, highly calcareous soils, and pathogens outbreaks.

Polyphenolic composition and antioxidant capacity of legume-based swards are affected by light intensity in a Mediterranean agroforestry system.

BACKGROUND: In Mediterranean grazed woodlands, microclimate changes induced by trees influence the growth and development of the understory, although very little is known about its polyphenolic composition in relation to light intensity. We investigated the bioactive compounds and antioxidant capacity of different legume-based swards and variations as a result of full sunlight and partial shade. The research was carried out in a cork oak agrosilvopastoral system in Sardinia. RESULTS: The highest values of (1,1-diphenyl-2-picrylhydrazyl) (DPPH) reached 7 mmol Trolox equivalent antioxidant capacity 100 g-1 dry weight (DW), total phenolics 67.1 g gallic acid equivalent kg-1 DW and total flavonoids 7.5 g catechin equivalent kg-1 DW. Compared to full sunlight, partial shade reduced DPPH values by 29% and 42%, and the total phenolic content by 23% and 53% in 100% legume mixture and semi-natural pasture, respectively. Twelve phenolic compounds were detected: chlorogenic acid in 80% legume mixture (partial shade) and verbascoside in pure sward of bladder clover (full sunlight) were the most abundant. CONCLUSION: Light intensity significantly affected antioxidant capacity, composition and levels of phenolic compounds. The results of the present study provide new insights into the effects of light intensity on plant secondary metabolites from legume-based swards, highlighting the important functions provided by agroforestry systems. © 2018 Society of Chemical Industry.

Bioactive compounds and antioxidants from a Mediterranean garland harvested at two stages of maturity.

Chrysanthemum coronarium L. (garland) is an herbaceous plant rich in bioactive compounds. The chemical composition, bioactive compounds and antioxidant properties of a Mediterranean garland population were investigated in different organs at two phenological stages. Antioxidant capacity varied from 7.9 (vegetative) to 14.4 (flowering) mmol Trolox equivalent antioxidant capacity 100 g-1 dry weight (DW). A significant correlation between antioxidant capacity and total phenolics and total flavonoids was found at flowering stage. LC-MS/MS analysis revealed that chlorogenic acid reached a maximum of 4.7 µg g-1 DW in leaves; flowers were high in luteolin (2.37 µg g-1 DW), whereas leaves showed a remarkable content of rutin (1.78 µg g-1 DW). Results highlight differences in bioactive compound levels and antioxidant capacity related to plant stages and organs. This research provides new insights into antioxidant activities and chemistry of garland, in view of its exploitation in areas of fodder resources, functional foods and natural antioxidants.

High-quality permanent draft genome sequence of Rhizobium sullae strain WSM1592; a Hedysarum coronarium microsymbiont from Sassari, Italy.

Rhizobium sullae strain WSM1592 is an aerobic, Gram-negative, non-spore-forming rod that was isolated from an effective nitrogen (N2) fixing root nodule formed on the short-lived perennial legume Hedysarum coronarium (also known as Sulla coronaria or Sulla). WSM1592 was isolated from a nodule recovered from H. coronarium roots located in Ottava, bordering Sassari, Sardinia in 1995. WSM1592 is highly effective at fixing nitrogen with H. coronarium, and is currently the commercial Sulla inoculant strain in Australia. Here we describe the features of R. sullae strain WSM1592, together with genome sequence information and its annotation. The 7,530,820 bp high-quality permanent draft genome is arranged into 118 scaffolds of 118 contigs containing 7.453 protein-coding genes and 73 RNA-only encoding genes. This rhizobial genome is sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.

Condensed tannin accumulation and nitrogen fixation potential of Onobrychis viciifolia Scop. grown in a Mediterranean environment.

BACKGROUND: Sainfoin (Onobrychis viciifolia Scop.) is a forage legume found in temperate areas but is less widespread in Mediterranean environments. Compared with other perennial legumes, it has the advantage of containing condensed tannins (CT) that can be important for their implications on ruminant nutrition and health. Data on nitrogen (N) fixation by sainfoin in the literature originate from very different environments and only a few field data are available, so it is important to improve knowledge on the N fixation potential of this species, particularly under a Mediterranean climate. Here the accumulation pattern of polyphenolic compounds (total, non-tannic polyphenols and CT) and the N fixation potential of sainfoin were studied in order to contribute to its valorisation for sustainable farming management in Mediterranean environments. RESULTS: CT concentrations were always in the range considered beneficial for animals, not exceeding 50 g delphinidin equivalent kg⁻¹ dry matter (DM). The regression of aerial fixed N on aerial DM showed a relationship of 22 kg fixed N t⁻¹ aerial DM in a Mediterranean environment. CONCLUSION: A wider exploitation of sainfoin is suggested for production under rain-fed conditions, thus enlarging the limited set of available perennial legumes suitable for Mediterranean environments.

Antioxidant treatments counteract the non-culturability of bacterial endophytes isolated from legume nodules.

In many wild legumes, attempts to cultivate nodule bacteria fail. We hypothesized that the limited culturability could be related to injury from oxidative stress caused by disruption of plant tissues during isolation. To test that, we isolated bacteria from nodules of Hedysarum spinosissimum and Tetragonolobus purpureus using buffers supplemented with scavenging systems to prevent damage from reactive oxygen species (ROS). Treatments included the following: antioxidants (glutathione, ascorbate, EDTA) or enzymes (catalase, peroxidase, superoxide dismutase), tested either as modified squashing buffers or added in plates. Some combinations yielded dramatic increases of culturability. Different endophytes were found, including additional Rhizobiaceae that were not the primary symbiont and were unable to nodulate. Their H2O2 tolerance in broth culture showed differences consistent with the unequal culturability observed. In wild legumes species, ROS generation during extraction appears to be a major factor limiting microbiota isolation, and protocols presented here significantly improve the recovery of culturable bacterial endophytes from plants.

Coexistence of predominantly nonculturable rhizobia with diverse, endophytic bacterial taxa within nodules of wild legumes.

A previous analysis showed that Gammaproteobacteria could be the sole recoverable bacteria from surface-sterilized nodules of three wild species of Hedysarum. In this study we extended the analysis to eight Mediterranean native, uninoculated legumes never previously investigated regarding their root-nodule microsymbionts. The structural organization of the nodules was studied by light and electron microscopy, and their bacterial occupants were assessed by combined cultural and molecular approaches. On examination of 100 field-collected nodules, culturable isolates of rhizobia were hardly ever found, whereas over 24 other bacterial taxa were isolated from nodules. None of these nonrhizobial isolates could nodulate the original host when reinoculated in gnotobiotic culture. Despite the inability to culture rhizobial endosymbionts from within the nodules using standard culture media, a direct 16S rRNA gene PCR analysis revealed that most of these nodules contained rhizobia as the predominant population. The presence of nodular endophytes colocalized with rhizobia was verified by immunofluorescence microscopy of nodule sections using an Enterobacter-specific antibody. Hypotheses to explain the nonculturability of rhizobia are presented, and pertinent literature on legume endophytes is discussed.

PCR primers based on different portions of insertion elements can assist genetic relatedness studies, strain fingerprinting and species identification in rhizobia.

Using the sequence of an insertion element originally found in Rhizobium sullae, the nitrogen-fixing bacterial symbiont of the legume Hedysarum coronarium, we devised three primer pairs (inbound, outbound and internal primers) for the following applications: (a) tracing genetic relatedness within rhizobia using a method independent of ribosomal inheritance, based on the presence and conservation of IS elements; (b) achieve sensitive and reproducible bacterial fingerprinting; (c) enable a fast and unambiguous detection of rhizobia at the species level. In terms of taxonomy, while in line with part of the 16S rRNA gene- and glutamine synthetase I-based clustering, the tools appeared nonetheless more coherent with the actual geographical ranges of origin of rhizobial species, strengthening the European-Mediterranean connections and discerning them from the asian and american taxa. The fingerprinting performance of the outward-pointing primers, designed upon the inverted repeats, was shown to be at least as sensitive as BOX PCR, and to be functional on a universal basis with all 13 bacterial species tested. The primers designed on the internal part of the transposase gene instead proved highly species-specific for R. sullae, enabling selective distinction from its most related species, and testing positive on every R. sullae strain examined, fulfilling the need of PCR-mediated species identification. A general use of other IS elements for a combined approach to rhizobial taxonomy and ecology is proposed.