Nanoformulations with exopolysaccharides from cyanobacteria: enhancing the efficacy of bioactive molecules in the Mediterranean fruit fly control.

The increasing demand for food has required intensive use of pesticides which are hazardous to the ecosystem. A valid alternative is represented by biopesticides; however, these molecules are often insoluble in water, and poorly bioavailable. Nanopesticides can be engineered to reach a selected target with controlled release of the active principle. In this work, capsaicin, an irritant alkaloid from hot chili peppers, and hydroxytyrosol, a phenolic compound obtained from extra-virgin olive oil by-products, were loaded into innovative nanocarriers. These were designed ad hoc combining exopolysaccharides from the cyanobacteria Neocyanospira capsulata, and a lipid component, i.e., egg phosphatidylcholine. The polysaccharide was chosen for chemical affinity with the chitin of insect exoskeleton, while the lipids were introduced to modulate the carrier rigidity. The newly formed nanosystems were characterized by physico-chemical techniques and tested for their possible use in pest control programs. The Mediterranean Fruit Fly Ceratitis capitata Wiedemann, 1824 (Diptera, Tephriditae), a pest of the Mediterranean Region causing high economic losses, was used as a model insect. We found that the nanoformulations nanocarriers prepared in this work, were able to increase the ovicidal effect of hydroxytyrosol. Moreover, the formulation encapsulating either hydroxytyrosol or capsaicin were able to reduce the number of females landing on treated apricots.

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.

Tannins-lignin mixed nanoformulations for improving the potential of neem oil as fungicide agent.

Sustainability and circular economy are increasingly pushing for the search of natural materials to foster antiparasitic treatments, especially in the case of economically relevant agricultural cultivations, such as grapevine. In this work, we propose to deliver neem oil, a natural biopesticide loaded into novel nanovectors (nanocapsules) which were fabricated using a scalable procedure starting from Kraft lignin and grapeseed tannins. The obtained formulations were characterized in terms of size and Zeta potential, showing that almost all the nanocapsules had size in the suitable range for delivery purposes (mean diameter 150-300 nm), with low polydispersity and sufficient stability to ensure long shelf life. The target microorganisms were three reference fungal pathogens of grapevine (Botrytis cinerea, Phaeoacremonium minimum, Phaeomoniella chlamydospora), responsible for recurrent diseases on this crop: grey mold or berry rot by B. cinerea and diseases of grapevine wood within the Esca complex of diseases. Results showed that grapeseed tannins did not promote inhibitory effects, either alone or in combination with Kraft lignin. On the contrary, the efficacy of neem oil against P. minimum was boosted by more than 1-2 orders of magnitude and the parasite growth inhibition was higher with respect to a widely used commercial pesticide, while no additional activity was detected against P. chlamydospora and B. cinerea.

Fluorescent Labeling of Lignin Nanocapsules with Fluorol Yellow 088.

The microscopic visualization of nanoparticles in plants is crucial to elucidate the mechanisms of their uptake through the cell wall and plasma membrane and to localize the possible sites of their extracellular or intracellular accumulation. Lignin nanocarriers are polymeric hollow nanocapsules able to contain and transport several bioactive substances inside plant tissues. We describe here a method for the preparation of Fluorol Yellow 088-labeled lignin nanocapsules that allow their localization in plant organs and tissues by fluorescence microscopy.

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.

Plastic is in the air: Impact of micro-nanoplastics from airborne pollution on Tillandsia usneoides (L.) L. (Bromeliaceae) as a possible green sensor.

Due to the increasing evidence of widespread plastic pollution in the air, the impact on plants of airborne particles of polycarbonate (PC), polyethyleneterephthalate (PET), polyethylene (PE), and polyvinylchloride (PVC) was tested by administering pristine and aged airborne micro-nanoplastics (MNPs) to Tillandsia usneoides for two weeks. Here we showed that exposure to pristine MNPs, significantly reduced plant growth with respect to controls. Particularly, PVC almost halved plant development at the end of the treatment, while the other plastics exerted negative effects on growth only at the beginning of the exposure, with final stages comparable to those of controls. Plants exposed to aged MNPs showed significantly decreased growth at early stages with PC, later in the growth with PE, and even later with PET. Aged PVC did not exert a toxic effect on plants. When present, the plastic-mediated reduction in plant growth was coupled with a decrease in photosynthetic activity and alterations in the plant concentration of macro- and micronutrients. The plastic particles were showed to adhere to the plant surface and, preferentially, on the trichome wings. Our results reported, for the first time, evidence of negative effects of airborne plastic pollution on plant health, thus raising concerns for related environmental risks.

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.

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.

Characterization of ordered aggregates of cerato-platanin and their involvement in fungus-host interactions.

BACKGROUND: The "cerato-platanin family" consists of fungal-secreted proteins that are involved in various stages of the host-fungus interaction and act as phytotoxins, elicitors of defense responses and allergens. Cerato-platanin (CP) is a moderately hydrophobic protein secreted and localized in the cell wall of Ceratocystis platani, the causal agent of a severe disease of Platanus. These properties make CP like the hydrophobins: these are self-assembling proteins that form a surface coating which is involved in the formation of aerial hyphae and in adherence to surfaces. METHODS: CP aggregation was monitored by ThT, circular dichroism, and AFM. The eliciting activity of CP aggregates was assayed on leaves and cells. RESULTS: The CP self-assembles forming amyloid-like aggregates via a nucleated growth mechanism which is joined up with a cleavage of the N-terminus. The ovoidal shape and the lack of a clear transition toward an all-beta structure distinguish these aggregates from typical amyloid fibrils. Moreover, CP aggregates interact with hydrophobic surfaces and enhance the hypersensitive response of Platanus. CONCLUSION AND GENERAL SIGNIFICANCE: CP forms "ordered aggregates" for which the soluble prefibrillar structures are the end point of the aggregation process, and do not evolve to insoluble fibrils. An involvement in host-microbe interaction is also suggested.