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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 with a scalable procedure starting from Kraft lignin and grapeseed tannins. The ensuing 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, nor alone nor 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.

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.

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.

First developed for biomedical and industrial applications, nanovectors have recently been extended to agriculture. Therefore, innovative plant growing procedures making use of nanoparticles should be adapted to sustainable processes and materials. This work aims at proposing newly synthetized polymeric nanocapsules (NCs) to be used as biocompatible vectors for delivery of bioactive compounds to plants. Nanoparticles were fabricated from lignin, which is the main byproduct of wood processing and is currently a waste material. Lignin can thus find a virtuous fate and be reused in the context of circular economy. Specifically, we loaded lignin NCs with Gibberellic Acid (GA), assessing that stable and reproducible nanoparticles could be obtained in a range of GA content that is relevant for delivery purposes, i.e. 0.5-1.5 mg ml-1. Plain and GA-loaded NCs were characterized by Dynamic Light Scattering (DLS) and Scanning Electron Microscopy (SEM). Assays of cytotoxicity and cargo release were carried out in two model plants, Eruca vesicaria and Solanum lycopersicum. These experiments, conducted both in vitro and in vivo, included the investigations of the percentage of germination, the stem and primary root lengths, as well as the fresh and dry weight of treated plants vs non-treated ones. Furthermore, NCs were loaded with Fluorol Yellow 088 to track their entrance and accumulation in seeds and seedlings.

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.

Spartium junceum L. is a typical species of Mediterranean shrubland areas, also grown in gardens and parks as an ornamental. In recent years in Europe, S. junceum has been recurrently found to be infected by different subspecies and genotypes of the quarantine regulated bacterium Xylella fastidiosa (Xf). This work presents for the first time the anatomy of S. junceum plants that we found, by means of genetic and immunochemistry analysis, to be naturally infected by Xf subsp. multiplex ST87 (XfmST87) in Monte Argentario (Grosseto, Tuscany, Italy), a new outbreak area within the EU. Our anatomical observations showed that bacteria colonized exclusively the xylem conductive elements and moved horizontally to adjacent vessels through pits. Interestingly, a pink/violet matrix was observed with Toluidine blue staining in infected conduits indicating a high content of acidic polysaccharides. In particular, when this pink-staining matrix was observed, bacterial cells were either absent or degenerated, suggesting that the matrix was produced by the host plant as a defense response against bacterial spread. In addition, a blue-staining phenolic material was found in the vessels and, at high concentration, in the pits and inter-vessels. SEM micrographs confirmed that polysaccharide and phenolic components showed different structures, which appear to be related to two different morphologies: fibrillary and granular, respectively. Moreover, our LM observations revealed bacterial infection in xylem conductive elements of green shoots and leaves only, and not in those of other plant organs such as roots and flowers.

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, s-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.

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.

Made available in DSpace on 2018-12-11T17:38:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-07-03 A general report on the use of the Allium test as cytotoxicological and genotoxicological assay is proposed, with particular emphasis about the standardization of the test in several common applications. The intraspecific variation in Allium cepa has been overlooked, as in most investigations no mention is made about origin and denomination of the onion cultivar used. A standardization of the used material would allow a better generalization of the results, since we cannot be sure that all cultivars would give the same response. A more frequent use of transmission electron microscopy (TEM) investigation is proposed. Even if relatively time consuming and not available in all laboratories, it may help to better understand the mechanism of cytotoxicity, since many morphological characters may appear similar but be arisen from different processes observable only with TEM. About statistical testing, tests other than chi-squared may be used in case of a lower amount of data. The most commonly used statistical tests are the parametric tests ANOVA and Student’s t, and the non-parametric tests Kruskal–Wallis and Mann–Whitney U, for analysis of variance. Tests should be used also to assess the minimal sample dimension for obtaining significance, since data collection (microscope observation) appears to be one of the main bottle necks of the test. Also the use of the Allium test for testing liposomes and other nanovectors for drug delivery is proposed, in order to assess the cytotoxicity of these types of medium and the possible increase in cytotoxicity of the associated drug. Department of Agricultural and Forestry Technology University of Craiova Private Laboratory for Plant Cytogenetics Department of Biology UNESP (Universidade Estadual Paulista) Tianjin Key Laboratory of Animal and Plant Resistance College of Life Sciences Tianjin Normal University Department of Biology Faculty of Science Istanbul University Department of Chemistry “Ugo Schiff” University of Florence Laboratory of Experimental Ecology Papanin Institute for Biology of Inland Waters Russian Academy of Sciences Department of Natural Sciences University of Pitesti Department of Biology University of Florence Department of Ecology and Environmental Protection Jan Kochanowski University Department of Botany University of Lucknow Department of Biology UNESP (Universidade Estadual Paulista)

Hairy roots were obtained after infection of Artemisia absinthium shoots with Agrobacterium rhizogenes strains 1855 and LBA 9402. The susceptibility to hairy root transformation varied between plant genotypes and bacterial strains. Hairy roots showed macroscopic differences from control root cultures. Southern blot hybridization confirmed the integration of T-DNA from both p1855 and pBin19, while polymerase chain reaction analysis indicated the presence of the neomycin phosphotransferase gene in the hairy root genome. Subcultured transformed root lines grew well in selective B5 agar-solidified medium containing kanamycin or rifampicin and without hormones. Shake-flask experiments with fast-growing root lines showed that 40 g l-1 was the best sucrose concentration for biomass production, yielding a 463-fold increase in dry weight after 28 days of culture. Great differences were found in the profiles of the essential oils isolated from normal and hairy roots. Gas chromatography/mass spectrometry analysis showed the oil produced by transformed cultures to be a mixture of 50 compounds with only one major component representing 37% of the oil content.

Gypsophilapaniculata L. is among the 10 best selling cut flower species globally and is considered a “target” plant for breeding varieties. The aim of this work was to make the genetic improvement of Gypsophila more rational and focused. The study of basic chromosome number and the reproductive system (potential barriers to intraspecific and interspecific incompatibilities) of the wild species as well as the commercial hybrids forms the basis of crossbreeding and selection of variety. A significant difference in ploidy was found between the species paniculata, its cultivar Flocon de Neige doubles, the species libanotica Boiss. (n = 34) and Pacifica Kom. (n = 68). A significant difference in the chromosome number was found among the commercial hybrids of Gypsophila under investigation, and this difference was observed among them as well as when compared with the wild type species. The flower structure of commercial hybrids can be remarkably complex. Some hybrids such as Biancanieves® and MeryG® showed empt...

A simple and efficient protocol for in vitro shoot regeneration from storage root explants of witloof chicory (Cichorium intybus L.) has been used to study the effect of colchicine at different concentrations, and colchi- cine in combination with kinetin or 2,4-D or with 2,4-D and kinetin, in comparison with kinetin and control (no treatment), upon this type of morphogenic event. The results demonstrated a strong stimulatory effect of colchicine at low concentration on shoot regeneration frequency when compared to the number of shoots formed in the con- trol. This effect was comparable to that of kinetin, a growth regulator that induces shoot differentiation. By contrast, the presence of 2,4-D completely inhibited shoot regeneration. Colchicine at high concentration did not induce shoots, and caused the complete death of the explants. A carefully histological analysis of the explants during the in vitro culture period showed the formation of numerous meristematic zones (precursors of adventitious bud and shoot development) in the parenchyma mass; these meris- tems were present only in the explants subjected to the colchicine and to the other treatments able to stimulate shoot differentiation. Nuclei of different sizes were observed in the explants treated with colchicine at low dose, the largest, probably, polyploids. Polyploid nuclei, in fact, were found in the regenerated plants.

Different NAA plus kinetin or BA combinations were tested on Francia Pernod fennel seedlings for callus induction and plant regeneration. Callogenesis from hypocotyls was obtained in all auxin/cytokinin-containing media. The organogenic response was observed especially in presence of NAA plus kinetin. The highest frequency of shoot regeneration was found when the auxin and kinetin were used at a 1:1 ratio. Moreover, a prolonged culture period increased shoot formation. Somatic embryogenesis was tested on several fennel populations. The results gave evidence of the genotypic importance. Two different protocols were used for somatic embryo induction. Using the first protocol among the different fennel genotypes tested, only Francia Pernod showed embryogenic capacity. In this case, from a primary non-embryogenic callus cultured for 12 months in presence of 2,4-D, an embryogenic secondary callus was produced. When transferred to the medium without 2,4-D (agarized or liquid), this gave embryogenic plants in high frequency. As far as the second embryogenic method is concerned, secondary embryogenic callus developed only in the presence of 2,4-D plus kinetin in Francia Pernod genotype. Thereafter, the replacement of those growth regulators by GA3 into the medium greatly increased the somatic embryo development, especially in `Francia Pernod', but also in `Aboca erbe' callus, a population with a very poor embryogenic capacity. In Francia Pernod, the primary and secondary (embryogenic) calli showed different morphological and histological responses, either when the secondary callus was induced by 2,4-D alone or by 2,4-D plus kinetin. Ontogenetic processes leading to somatic embryo formation are described in this context.

The action of several concentrations of an anionic synthetic surfactant alkylbenzene sulphonate (ABS) and a non-ionic surfactant arylalkylpolyglycol ether (Citowett) were studied using as test different in vitro culture systems (Pittosporum tobira Ait. and Nicotiana tabacum L. var. Bel W3). Three parameters were analyzed: i) callus or total biomass increase (P. tobira and N. tabacum, respectively); ii) shoot regeneration frequency (N. tabacum); iii) cell damage. Phytotoxic general effects occurred for ABS and Citowett in both species especially at higher concentrations; however some differences were observed between the two types of surfactants. A clear callus growth stimulation was found in P. tobira cultured in presence of ABS at lower doses whereas Citowett at high concentration induced, in N. tabacum, a light increase of the total number of regenerated shoots in comparison to control. Both surfactants at higher concentrations caused strong damages at cytological level. The effects of these surfactants on cytoplasm and nuclei were determined. The use of in vitro culture as a monitoring system is discussed.

Increasing manganese (Mn) concentrations to 5, 10, 15 and 20 mM caused a significant decrease in callus formation and shoot regeneration in comparison to 0.1 (control medium) and 2 mM in Nicotiana tabacum var. BEL W3 grown on Murashige and Skoog’s medium (MS) with 1 mg l−1 α-naphtaleneacetic acid and 1 mg l−1 kinetin. Concentrations of Mn higher than 5 mM also reduced callus growth in Nicotiana bigelovii bigelovii grown on MS solid medium. Callus cultures of N. tabacum with enhanced tolerance to Mn were selected by progressively elevating the level of MnSO4·4H2O in the culture medium. Selection of Mn-tolerant plants occurred only at 2 and 5 mM, because higher concentrations inhibited callus and shoot growth. Differences in mitotic phase indices in plants grown at 2 and 5 mM Mn were found in comparison to the control. In these plants, several mitotic alterations as well as damaged chloroplasts and a reduction in chloroplast number were also observed, especially for those treated with 5 mM Mn. Mn was also shown to affect the number of xylem elements and the degree of lignification, which differed between plants grown at 2 or 5 mM compared to the control.

In Nicotiana tabacum L. var. BEL W3 copper (Cu) at concentrations higher than 50 μM significantly inhibited callus growth and shoot regeneration. After 5–6 months of culture only a few morphogenic callus lines survived in the presence of 100 μM Cu. These calluses showed the capacity to grow and regenerate shoots through successive subcultures on medium containing 100 μM Cu. The 100 μM Cu-tolerant shoots, in comparison to regenerated control shoots, formed roots only when cultured in the presence of 100 μM Cu. From five independent Cu-tolerant callus lines in a culture period of 4–5 months more than 50 plants (defined ‘tolerant’) able to grow in presence of 100 μM Cu were obtained. These plants showed normal xylem tissue formation while control regenerated plants growing in normal Cu MS content (0.1 μM) had few xylem elements in the central cylinder. No difference as far chlorophyll content and chloroplast structure was found among Cu-tolerant and control plants. In Cu-tolerant plants, dry matter production was higher than in controls, particularly in roots.

A study has been carried out on several Amaranthus L species and breeding lines (A. caudatus, A. cruentus, A. hybridus, A. hypochondriacus), some of which have previously shown poor or no plant regeneration from callusing primary explants. Callus formation occurred from explants from almost all the lines tested. Significant differences in callus growth were observed among the lines depending on the growth regulator combinations used. Cultural conditions such as type and growth regulator combination, as well as age-dependent competence of the explant tissues, also influenced the frequency of shoot (and/or root) regeneration in Amaranthus species that had been shown difficult to regenerate in the past. The results obtained demonstrate that in Amaranthus the genotype, growth regulator dose and combination, the type and the physiological stage of explant are factors of great importance for in vitro plant regeneration.

Manganese toxicity to germination, callus induction and shoot regeneration was studied on three cultivars of Nicotiana tabacum: BEL W3, Burley 21, Bright NC 944. All materials were cultured on MS solid medium containing 0.1 (control), 2, 5, 10, 15 and 20 mM Mn, to which 5 µM NAA and 5 µM kinetin were added for callus induction and shoot regeneration. Mn toxicity to callus growth was tested using habituated callus of Nicotiana bigelovii var. bigelovii grown on MS medium without growth regulators. Mn concentrations higher than 2 mM were toxic for germination, and concentrations higher than 5 mM were toxic for callus induction, shoot regeneration and callus growth. Among the cultivars examined, Bright tobacco appeared more tolerant to high Mn concentrations during callus formation and shoot regeneration. However, many regenerated plants capable of growing in vitro in the presence of 2 and 5 mM Mn were obtained.

Callus cultures were initiated from micropropagated Artemisia absinthium plantlets on MS basal medium supplemented with different concentrations of BA, Kn, NAA, IAA and 2,4-d in combination or singly. Supplementing the medium with low doses of both BA in combination with NAA, and Kn in combination with NAA enhanced the growth rate of callus cultures. However, cultures grew slowly following the second subculture and the majority turned brown and died within the next month. Initiation of root and shoot primordia occured directly from leaf explants cultured on 1.81 μM 2,4-d, while adventitious shoot formation from callus was observed occasionally when BA was added to the medium in combination with IAA. Furthermore, medium containing 2.22 μM BA and 2.69 μM NAA stimulated both callus growth and organogenesis on some callus cultures derived from leaves and stems of young stock material. The best results were obtained with leaf explants. Cytological analysis of root meristems revealed that all regenerants were diploid (2n=18), as expected.

Variation in the capacity to regenerate shoots and morphogenetic potential were investigated by cyclic culture and regeneration of Nicotiana tabacum var. BEL W3. Explants were excised from different shoots formed by the same callus line. Differences in the organogenetic in vitro response among regenerated plantlets was observed in two subsequent regenerate ‘generations’. A progressive decrease of the regenerative capacity was observed across callus line ‘generations’.

Callus induction, callus growth and organogenetic processes were investigated in hypocotyl and stem cultures of four species of Amaranthus each of which comprised several varieties. Callus formation occurred in almost all the varieties in 100% of explants. The combinations of naphtaleneacetic acid plus benzylaminopurine or 2,4-dichlorophenoxyacetic acid plus Kinetin were very effective in causing callus formation; the results for callus growth in presence of the same growth regulator combinations revealed in some cases significant differences among the varieties. As far organogenesis-based on a few varieties-only A. caudatus and A. hypochondriacus responded well forming shoots from callus when cultured in presence of indoleacetic acid plus Kinetin and/or indoleacetic acid plus benzylaminopurine. Root regeneration was also observed in several varieties. Complete plantlets were obtained from rooted shoots. The results are discussed in relation to hormonal effect and the genotype importance.

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-β 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.

(1995). Characterization of Nicotiana tabacum var. BEL W3 plants regenerated from resistant to Mn2± callus. Giornale botanico italiano: Vol. 129, No. 4, pp. 1146-1147.

Callus induction and morphogenic response of several fennel populations were determined by genotype and hormonal treatment. 100% callus formation occurred only in the Francia Pernod population under the action of 2, 4-dichlorophenoxyacetic acid or α-naphthaleneacetic acid and kinetin. Only this last hormonal treatment induced shoot regeneration. Plant regeneration was observed especially in Francia Pernod population. Ths calluses grown in presence of 2,4-dichlorophenoxyacetic acid or α-naphthaleneacetic acid plus kinetin, showed considerable differences at cytological and histological level which were correlated to their different morphogenic capability.

(1994). In Vitro Tests to Select Plants of Foeniculum Vulgare Miller (Umbelliferae) for Resistance to Phomopsis Foeniculi du Manoir et Vegh (Sphaeropsidaceae) Giornale botanico italiano: Vol. 128, No. 1, pp. 325-325.