The cypsela (achene) of Echinacea purpurea as a diffusion unit of a community of microorganisms.

Echinacea purpurea is a plant cultivated worldwide for its pharmaceutical properties, mainly related to the stimulation of the immune system in the treatment of respiratory infections. The cypselas (fruits) of E. purpurea were examined in order to investigate the presence, localization and potential function(s) of endophytic microorganisms. Electron and confocal microscopy observations showed that three different components of microorganisms were associated to cypselas of E. purpurea: (i) one endocellular bacterial component in the cotyledons, enclosed within the host membrane; (ii) another more generic bacterial component adhering to the external side of the perianth; and (iii) a fungal component inside the porous layer of the perianth, the woody and porous modified residual of the flower, in the form of numerous hyphae able to cross the wall between adjacent cells. Isolated bacteria were affiliated to the genera Paenibacillus, Pantoea, and Sanguibacter. Plate tests showed a general resistance to six different antibiotics and also to an antimicrobial-producing Rheinheimera sp. test strain. Finally, microbiome-deprived E. purpurea seeds showed a reduced ability to germinate, suggesting an active role of the microbiome in the plant vitality. Our results suggest that the endophytic bacterial community of E. purpurea, previously found in roots and stem/leaves, might be already carried at the seed stage, hosted by the cotyledons. A further microbial fungal component is transported together with the seed in the perianth of the cypsela, whose remarkable structure may be considered as an adaptation for fungal transportation, and could influence the capability of the seed to germinate in the soil.Key Points• The fruit of Echinacea purpurea contains fungi not causing any damage to the plant.• The seed cotyledons contain endocellular bacteria.• Seed/fruit deprived of the microbiome showed a reduced ability to germinate.

Anatomical Investigation and GC/MS Analysis of 'Coco de Mer', Lodoicea maldivica (Arecaceae).

Lodoicea maldivica (J.F.Gmel.) Pers. (Arecaceae), 'Coco de Mer', is a palm, growing as endemic in the Seychelles islands. Its fruit weighs up to 20 kg and is characterized by a fleshy and fibrous envelope surrounding the nutlike portion. The present work combines a morpho-anatomical and a phytochemical analysis of the fruit exocarp and mesocarp. The exocarp is composed by a layer of palisade cells. The mesocarp is characterized by vascular bundles and by sclereids. In the aerenchyma, the internal zone of the mesocarp, cells aggregates were positive to phenols, while idioblasts were positive to terpenes. We performed a GC/MS analysis with a semi-quantitative relative amount calculation of the recorded compounds. The GC/MS essential oil profile revealed the dominance of acyclic sesquiterpenoids (53.95 %), followed by bicyclic sesquiterpenoids (31.69 %), monoterpenes (11.89 %) and monocyclic sesquiterpenoids (2.44 %). The terpenes detected in higher amounts, ß-caryophyllene and bicyclogermacrene, are known for activity against insect larvae, but have been proposed as antiviral candidates against SARS-CoV-2. The third compound in amount, aromadendrene, is active against bacteria and, again, known to possess insecticidal properties.

Saline Stress Impairs Lipid Storage Mobilization during Germination in Eruca sativa.

Soil salinization become worse in the last decades, leading to reduced crop yields, especially in the Mediterranean basin. Eruca sativa is a common species cultivated in this area with remarkable economic importance. This study aimed at investigating the effect of salinity on this plant, focusing on (i) seedling development in terms of variations in germination and growth parameters and (ii) anatomical and ultra-structural changes in the morphology of cotyledons. For this reason, seeds were treated with different salinity levels ranging from 137 to 548 mM NaCl. Seed germination was delayed by all the concentrations tested, but only above 137 mM seedling growth was impaired. Results showed a high occurrence of lipid bodies within the mesophyll cells of cotyledons of seedlings exposed to salt concentrations above 137 mM, suggesting an impairment in lipid mobilization caused by salinity during plant development. The cotyledons of treated seedlings showed reduced intercellular spaces and ultrastructural changes in chloroplasts and peroxisomes. Moreover, salt-induced autophagic processes were present in samples grown at the highest NaCl levels. Interestingly, at 137 mM NaCl, seedlings showed the highest values of mesophyll thickness and fresh weight, implying a possible mechanism of salt adaptation during germination.

Ultrastructure of Terpene and Polyphenol Synthesis in the Bark of Cupressus sempervirens After Seiridium cardinale Infection.

Cypress Canker Disease (CCD) pandemic caused by Seiridium cardinale is the major constraint of many Cupressaceae worldwide. One of the main symptoms of the disease is the flow of resin from the cankered barks. While inducible phloem axial resin duct-like structures (PARDs) have recently been characterized from an anatomical point of view, their actual resin production is still being debated and has never been demonstrated. Although the involvement of polyphenolic parenchyma cells (PP cells) in the bark of Cupressus sempervirens after S. cardinale infection was revealed in one of our previous studies using light microscopy, their evolution from the phloem parenchyma cells is yet to be clarified. This study investigated functional and ultrastructural aspects of both PARD-like structures and PP cells by means of more in-depth light (LM) and fluorescence microscopy (FM) combined with histochemical staining (using Sudan red, Fluorol Yellow, NADI Aniline blue black, and Toluidine blue staining), in addition to Transmission Electron Microscope (TEM). Two-year-old stem sections of a C. sempervirens canker-resistant clone (var. "Bolgheri"), artificially inoculated with S. cardinale, were sampled 5, 7, 14, 21, and 45 days after inoculation, for time-course observations. FM observation using Fluorol yellow dye clearly showed the presence of lipid material in PARD-like structures lining cells of the cavity and during their secretion into the duct space/cavity. The same tissues were also positive for NADI staining, revealing the presence of terpenoids. The cytoplasm of the ducts' lining cells was also positive for Sudan red. TEM observation highlighted the involvement of plastids and endoplasmic reticulum in the production of terpenoids and the consequent secretion of terpenoids directly through the plasma membrane, without exhibiting vesicle formation. The presence of a high number of mitochondria around the area of terpenoid production suggests that this process is active and consumes ATP. The LM observations showed that PP cells originated from the phloem parenchyma cells (and possibly albuminous cells) through the accumulation of phenolic substances in the vacuole. Here, plastids were again involved in their production. Thus, the findings of this work suggest that the PARD-like structures can actually be considered PARDs or even bark traumatic resin ducts (BTRD).

Xylem anatomy and hydraulic traits in Vitis grafted cuttings in view of their impact on the young grapevine decline.

Grapevine grafting is an essential practice in viticulture and over the years, various bench grafting techniques have been developed to mechanize the nursery process and to increase the yield in number of viable cuttings. Bench grafting is a fundamental nursery practice that can potentially affect the quality of propagation material also in young decline associated to grapevine trunk diseases and has been recently reported to influence leaf symptoms development associated with diseases of Esca complex. The study aimed to investigate how three bench grafting methods [i.e., (i) Omega graft as mechanical technique, (ii) Whip and Tongue graft as manual technique and (iii) Full Cleft graft as semi-mechanical technique] can influence these phenomena. Specifically, the different methods were compared for their effect on the anatomical development of the grafting point and the functionality of the xylem, also considering two factors: the grapevine cultivar (Cabernet Sauvignon, Glera and Teroldego) and the scion/rootstock diameter (thin and large). Observations by light microscopy on the anatomical evolution and measurements on the xylem morphology and hydraulic traits were correlated with the grafting methods and the investigated varieties. The anatomical observations revealed that the mechanical (Omega) and semi-mechanical (Full Cleft) grafting methods have a faster callusing response while the manual technique (Whip and Tongue) has a slower but greater vascularization of the differentiated callus. Significant differences between cultivars and/or grafting types were also detected in necrotic area on the grafted tissues. Statistical analysis of the grapevine vessels suggested differences in xylem parameters between cultivars, while grafting type had no significant effects. On the other hand, the grafting type significantly affected the intrinsic growth rate. The study confirms the potential incidence of lesions and dysfunctionalities correlated with the grafting method applied, which can potentially induce grafted vine declines in vineyards due to the necrotic area detected on the grafted tissues.

A new method for the direct tracking of in vivo lignin nanocapsules in Eragrostis tef (Poaceae) tissues.

Environmental concerns have driven scientists to research new eco-friendly approaches for the preparation of nanosystems. For this purpose, novel bio-polymers have been selected. Among these, one of the most promising is lignin, which is biodegradable and biocompatible. Additionally, lignin is one of the main by-products of the paper industry and can be re-used in nanosystems building. Lignin-based nanosystems could be used in agriculture, to improve the uptake of bioactive compounds, thus avoiding soil pollution. However, the mechanism of penetration in the plant and the route of transportation within the internal plant tissues are unknown and need to be clearly elucidated. Here we present a method of lignin nanocapsules staining and tracking by fluorochrome: Fluoral Yellow 088, which is a well-suited dye for the tracking of lipids and other oil phases. Two different applications were applied: in the first one fourteen-day plants were soaked with fluorescent nanocapsules (fNCs) pure solution and in the second one, Eragrostis tef plants were laid down on blotting paper and soaked with diluted fNCs solution. Wetting the roots of Teff plantlets with the pure fNCs solution resulted in the most efficient way of nanocapsule entrance. The dyeing of lignin nanocapsules allowed us to track them in Eragrostis tef plant tissues through microscopic observations. In particular, fNCs were proven to be able to permeate roots, reaching xylem vessels where, through water pressure, they reached the leaf.

Altered feeding behavior and immune competence in paper wasps: A case of parasite manipulation?

Paper wasps (Polistes dominula), parasitized by the strepsipteran Xenos vesparum, are castrated and desert the colony to gather on plants where the parasite mates and releases primary larvae, thus completing its lifecycle. One of these plants is the trumpet creeper Campsis radicans: in a previous study the majority of all wasps collected from this plant were parasitized and focused their foraging activity on C. radicans buds. The unexpected prevalence and unusual feeding strategy prompted us to investigate the influence of this plant on wasp behavior and physiology through a multidisciplinary approach. First, in a series of laboratory bioassays, we observed that parasitized wasps spent more time than non-parasitized ones on fresh C. radicans buds, rich of extra-floral nectaries (EFNs), while the same wasps ignored treated buds that lacked nectar drops. Then, we described the structure and ultra-structure of EFNs secreting cells, compatible with the synthesis of phenolic compounds. Subsequently, we analysed extracts from different bud tissues by HPLC-DAD-MS and found that verbascoside was the most abundant bioactive molecule in those tissues rich in EFNs. Finally, we tested the immune-stimulant properties of verbascoside, as the biochemical nature of this compound indicates it might function as an antibacterial and antioxidant. We measured bacterial clearance in wasps, as a proxy for overall immune competence, and observed that it was enhanced after administration of verbascoside-even more so if the wasp was parasitized. We hypothesize that the parasite manipulates wasp behavior to preferentially feed on C. radicans EFNs, since the bioactive properties of verbascoside likely increase host survival and thus the parasite own fitness.

The multi-purpose role of hairiness in the lichens of coastal environments: Insights from Seirophora villosa (Ach.) Frödén.

The fruticose epiphytic lichen Seirophora villosa, strictly associated with Juniperus shrublands in the Mediterranean basin, was used to investigate the role of hairiness on a lichen thallus, as a characteristic morphological trait. We evaluated the effect of hair removal on the physiological parameters of a set of samples, during desiccation and on exposure to different salt concentrations. Hairy thalli were less affected by salt, suggesting that during dehydration, the presence of hair protects the thallus from light irradiance, oxidative stresses and the lipid peroxidation generated by free radicals, and could offer passive, but selective, water control. Our results showed that hair could not only increase thallus surface and promote water absorption when availability is low, but could also repel the salt dissolved in water by activating a passive resistance mechanism, by preventing salt entering.

Localization of phenolic compounds in the fruits of Silybum marianum characterized by different silymarin chemotype and altered colour.

Silybum marianum (L.) Gaertn (Asteraceae) is a valuable medicinal plant utilized for silymarin production. However, only fragmentary and contradictory information about silymarin localization within S. marianum fruit are available. In this work, a twofold research approach was adopted in order to investigate the distribution and quantification of silymarin and of other phenolic compounds within the different fruit regions (pericarp, seed integument, cotyledon). Two S. marianum wild accessions with contrasting silymarin chemotype (A and B) and a mutant line (C) with an altered fruit colour were analysed. Fruits of Cynara cardunculus were studied as a reference. Firstly, the fruit morpho-anatomy was reviewed by means of light microscopy digital imaging and, secondly, a comprehensive histolocalization of the different classes of polyphenols within the fruit was carried out. The experimental evidences confirmed that silymarin, and its precursor taxifolin, are only accumulated in the seed integuments. The dark colour of fully-ripened fruits is due to the accumulation of condensed tannins in the pericarp subepidermal cell layer. On the contrary, the studied mutant line shows reduced condensed tannin content that probably result from impairment at the level of flavonoid biosynthetic pathway. Condensed tannins content is comparatively low in S. marianum fruits and very low in the identified mutant line. This could represent an advantage for the possible employment of S. marianum fruits and of silymarin extraction by-products in the feed and food sector.

Secretory structures and essential oil composition in Stachys officinalis (L.) Trevisan subsp. officinalis (Lamiaceae) from Italy.

The secretory structures and the volatile fraction of Stachys officinalis (L.) Trevisan subsp. officinalis (Lamiaceae) from Italy were studied for the first time. Peltate and small capitate trichomes were observed on the whole plant (leaves and inflorescences). In the peltate trichomes, an unusual polyphenols content was evidenced by the histochemical methods. The volatile fraction was obtained by a solvent extract from the distillation water of leaves and inflorescences and analysed by GC-MS. Forty-four constituents for leaves, representing 94.1% of the total volatiles, and 57 compounds for flowers, accounting for 90.1% of the total volatiles, were identified. (E)-caryophyllene (20.1%), (E)-nerolidol (14.3%), caryophyllene oxide (6.1%) and γ-cadinene (5.7%) were recognised as the main constituents for the leaf volatile fraction, while caryophyllene oxide (16.5%), (E)-nerolidol (15.4%), humulene epoxide II (9.2%) and α-pinene (7.0%) were the main compounds for the flower volatile fraction.

History vs. legend: Retracing invasion and spread of Oxalis pes-caprae L. in Europe and the Mediterranean area.

Oxalis pes-caprae L. is a South African geophyte that behaves as an invasive in the eurimediterranean area. According to a long-established hypothesis, O. pes-caprae may have invaded Europe and the Mediterranean area starting from a single plant introduced in the Botanical Garden of Malta at the beginning of the 19th century. The aim of this work was to test this hypothesis, to track the arrival of O. pes-caprae in different countries of the Euro-Mediterranean area and to understand the pathways of spreading and particularly its starting point(s). Historical data attesting the presence of the plant in the whole Euro-Mediterranean region were collected from different sources: herbarium specimens, Floras and other botanical papers, plant lists of gardens, catalogs of plant nurseries and plant dealers. First records of the plant (both cultivated and wild) for each Territorial Unit (3rd level of NUTS) were selected and used to draw up a diachronic map and an animated graphic. Both documents clearly show that oldest records are scattered throughout the whole area, proving that the plant arrived in Europe and in the Mediterranean region more times independently and that its spreading started in different times from several different centers of invasion. Botanical gardens and other public or private gardens, nurseries and plant dealers, and above all seaside towns and harbors seemingly played a strategic role as a source of either intentional and unintentional introduction or spread. A geographic profiling analysis was performed to analyse the data. We used also techniques (Silhouette, Kmeans and Voronoi tessellation) capable of verifying the presence of more than one independent clusters of data on the basis of their geographical distribution. Microsatellites were employed for a preliminary analysis of genetic variation in the Mediterranean. Even if the sampling was insufficient, particularly among the populations of the original area, our data supported three main groups of populations, one of them corresponding to the central group of populations identified by GP analysis, and the other two corresponding, respectively, to the western and the eastern cluster of data. The most probable areas of origin of the invasion in the three clusters of observations are characterized by the presence of localities where the invasive plant was cultivated, with the exception of the Iberian cluster of observation where the observations in the field predate the data about known cultivation localities. Alternative possible reasons are also suggested, to explain the current prevalence of pentaploid short-styled plants in the Euro-Mediterranean area.

Foliar response of an Ailanthus altissima clone in two sites with different levels of ozone-pollution.

Potted plants of Ailanthus altissima, produced by root suckers coming from a single symptomatic mother tree, were placed in two sites in the vicinity of Florence (central Italy), with different levels of ozone pollution. These plants were kept in well watered conditions during the period May-September 1999. In the high pollution site (Settignano-SET) the level of ozone exposure (AOT40) reached at the end of the season a value of 31 ppm h, whereas in the "low pollution" site (Cascine-CAS) the exposure to ozone was 11 ppm h. A. altissima showed foliar symptoms in early July at SET and in the second half of July at CAS when exposure values reached 5 ppm h at both sites. However, at the end of August the conditions of the plantlets were rather similar in both sites. Microscopic and ultrastructural analysis were performed at the first onset of symptoms at SET (the CAS leaflets were asymptomatic). Observing the upper leaf surface where the brown stipples were visible, it was found that the cells of the palisade mesophyll displayed loss of chlorophyll and the organelles in the cytoplasm were damaged. Swelling of thylacoids was observed in the CAS leaflets, thus indicating the possible onset of a pre-visual damage. The injured cells were separated from the healthy ones by a layer of callose. We conclude that the sensitivity to ozone of A. altissima leaves is related to its leaf structure, with low leaf density and large intercellular spaces. Cell walls, as well as acting as mechanical barriers against the spread of ozone within the cell, also provide important detoxifying processes.

Ozone foliar symptoms in woody plant species assessed with ultrastructural and fluorescence analysis.

This paper compares the responses to ozone in five woody species: Fagus sylvatica (FS), Acer pseudoplatanus (AP), Fraxinus excelsior (FE), Viburnum lantana (VL) and Ailanthus altissima (AA). The hypothesis being tested was that the strategies that plants adopt to resist oxidative pressure are species-specific. The study was carried out on field grown plants in an area in Northern Italy characterized by elevated levels of ozone pollution. The observations were made both at ultrastructural (using light and electronic microscopy) and physiological (using chlorophyll a transient fluorescence and microspectral fluorometry) level. Common responses were: the hypersensitive response (i.e. the death of palisade mesophyll cells) and the formation of callose layers separating injured from healthy cells. FS and AP were capable of thickening the palisade mesophyll cell walls. This thickening process involved changes in cell wall chemical structure, evidenced by the accumulation of yellow autofluorescence compounds. Species-specific behaviours were observed with the fluorescence analysis, with special reference to the photochemical de-excitation constant (Kp). This value increased in FE and AP, and decreased in AA. The observed responses are interpreted as adaptative strategies against the ozone stress. The increase of Kp indicates that the reaction centres were working as more effective quenchers.