Role of methyl jasmonate and salicylic acid in kiwifruit plants further subjected to Psa infection: biochemical and genetic responses.

The use of plant elicitors for controlling Pseudomonas syringae pv. actinidiae (Psa), the etiological agent of the kiwifruit bacterial canker (KBC), has been analysed in the past and, while salicylic acid (SA) seems to decrease disease susceptibility, methyl jasmonate (MJ) shows an opposite effect. However, the metabolic and genomic responses of Psa-infected plants following elicitation with these two compounds, as compared with non-elicited Psa-inoculated plants, are poorly understood, being the focus of this study. Micropropagated A. chinensis 'Hayward' plants were elicited with MJ or SA, and further inoculated with Psa. Fifteen days post-inoculation, Psa population in MJ-treated plants was increased by 7.4-fold, whereas SA elicitation led to decreased Psa colonization (0.5-fold), as compared with non-elicited inoculated plants. Additionally, elicitation with MJ or SA generally decreased polyphenols and lignin concentrations (by at least 20%) and increased total proteins (by at least 50%). MJ led to the upregulation of SOD, involved in plant antioxidant system, and reporter genes for the jasmonic acid (JA) (JIH and LOX1), abscisic acid (SnRK), SA (ICS1), and ethylene (ACAS1, ETR1 and SAM) pathways. Moreover, it increased ABA (40%) and decreased carotenoids (30%) concentrations. Contrastingly, comparing with non-elicited Psa-inoculated plants, SA application resulted in the downregulation of antioxidant system-related genes (SOD and APX) and of reporter genes for ethylene (ETR1) and JA (JIH and ETR1). This study contributes to the understanding of potential mechanisms involved in kiwifruit plant defences against Psa, highlighting the role of the JA, ABA and ethylene in plant susceptibility to the pathogen.

Early Pathogen Recognition and Antioxidant System Activation Contributes to Actinidia arguta Tolerance Against Pseudomonas syringae Pathovars actinidiae and actinidifoliorum.

Actinidia chinensis and A. arguta have distinct tolerances to Pseudomonas syringae pv. actinidiae (Psa), but the reasons underlying the inter-specific variation remain unclear. This study aimed to integrate the metabolic and molecular responses of these two kiwifruit species against the highly pathogenic Psa and the less pathogenic P. syringae pv. actinidifoliorum (Pfm) bacterial strains. Disease development was monitored weekly till 21 days post inoculation (dpi), analysing a broad number and variety of parameters including: colony forming units (CFU), foliar symptoms, total chlorophylls, lipid peroxidation, soluble polyphenols, lignin and defense-related gene expression. At the end of the experimental period A. chinensis inoculated with Psa presented the highest endophytic bacterial population, whereas A. arguta inoculated with Pfm showed the lowest values, also resulting in a lower extent of leaf symptoms. Metabolic responses to infection were also more pronounced in A. chinensis with decreased total chlorophylls (up to 55%) and increased lipid peroxidation (up to 53%), compared with non-inoculated plants. Moreover, at 14 dpi soluble polyphenols and lignin concentrations were significantly higher (112 and 26%, respectively) in Psa-inoculated plants than in controls, while in A. arguta no significant changes were observed in those metabolic responses, except for lignin concentration which was, in general, significantly higher in Psa-inoculated plants (by at least 22%), comparing with control and Pfm-inoculated plants. Genes encoding antioxidant enzymes (SOD, APX and CAT) were upregulated at an earlier stage in Psa-inoculated A. arguta than in A. chinensis. In contrast, genes related with phenylpropanoids (LOX1) and ethylene (SAM) pathways were downregulated in A. arguta, but upregulated in A. chinensis in the later phases of infection. Expression of Pto3, responsible for pathogen recognition, occurred 2 dpi in A. arguta, but only 14 dpi in A. chinensis. In conclusion, we found that A. arguta is more tolerant to Psa and Pfm infection than A. chinensis and its primary and secondary metabolism is less impacted. A. arguta higher tolerance seems to be related with early pathogen recognition, the activation of plant antioxidant system, and to the suppression of ET and JA pathways from an earlier moment after infection.

A physico-chemical study on amphiphilic cyclodextrin/liposomes nanoassemblies with drug carrier potential.

In this paper, two medusa-like ACyDs, modified at the primary rim bearing four (ACyD4) and eight carbons (ACyD8) acyl chain length, and one bouquet-like CyD, modified at primary side with thiohexyl and at secondary one with oligoethylene moiety (SC6OH), were investigated for their ability to assemble in nanostructures or to form hybrid dipalmitoylphosphatidylcholine (DPPC)/ACyDs systems. The lipophilicity of these molecules and the different preparation methods used in this study (thin layer evaporation and nanoprecipitation method) significantly affect the aggregation behaviour in aqueous medium. Except for the shortest medusa-like ACyD4, the other ACyDs formed stable nanoaggregates for at least 45 days. The effect of ACyDs on the thermotropic behaviour of DPPC liposomes was also studied by differential scanning calorimetry analysis, thus elucidating their interaction with liposomes to afford hybrid liposome/ACyDs systems. The medusa-like ACyD4 cannot be used to realize nanosystems because it quickly aggregates or it induces a complete destabilization of the liposomes. At the highest concentration investigated (0.01 molar fraction), both ACyD8 and SC6OH interacted with DPPC liposomes, forming ACyD/DPPC or SC6OH/DPPC hybrid vesicular carriers.

Tailored-BODIPY/Amphiphilic Cyclodextrin Nanoassemblies with PDT Effectiveness.

Amphiphilic cyclodextrins (aCDs) are an intriguing class of carrier systems which, recently, have been proposed to deliver porphyrinoids and anticancer drugs or combined dose of both for dual therapeutic applications. The design of nanoassemblies based on aCD and photosensitizers (PSs) aims to preserve the photodynamic therapy (PDT) efficacy of PS, reducing the tendency of PS to self-aggregate, without affecting the quantum yield of singlet oxygen (1O2) production, and, not less importantly, minimizing dark toxicity and reducing photosensitization effects. With this idea in mind, in this paper, we focus on nanoassemblies between a non-ionic aCD (SC6OH) and halo-alkyl tailored iodinated boron-dipyrromethenes (BODIPY) dye, a class of molecules which recently have been successfully proposed as a stimulating alternative to porphyrinoids for their high photodynamic efficacy. Nanoassemblies of BODIPY/aCD (BL01I@SC6OH) were prepared in different aqueous media by evaporation of mixed organic film of aCD and BODIPY, hydration, and sonication. The nanostructures were characterized, measuring their hydrodynamic diameter and ξ-potential and also evaluating their time-stability in biological relevant media. Taking advantage of emissive properties of the not-iodinated BODIPY analogue (BL01), nanoassemblies based on aCD and BL01 were investigated as model system to get insight on entanglement of BODIPY in the amphiphile in aqueous dispersion, pointing out that BODIPY is well-entrapped in monomeric form (τ ≅ 6.5 ns) within the colloidal carriers. Also morphology and fluorescence emission properties were elucidated after casting the solution on glass. BL01@SC6OH is easily detectable in cytoplasm of HCT116 cell lines, evidencing the remarkable intracellular penetration of this nanoassembly similar to free BODIPY. On the same cell lines, the photodynamically active assembly BL01I/aCD shows toxicity upon irradiation. Despite the fact that free BL01I is more PDT active than its assembly, aCD can modulate the cell uptake of BODIPY, pointing out the potential of this system for in vivo PDT application.

Pomological Traits, Sensory Profile and Nutraceutical Properties of Nine Cultivars of Loquat (Eriobotrya japonica Lindl.) Fruits Grown in Mediterranean Area.

In this paper the diversity of fruit quality within nine loquat cultivars, including five international affirmed cultivars (Algerie, Golden Nugget, Peluche, Bueno, El Buenet) and four local cultivars (Sanfilippara, Nespolone di Trabia, BRT20 and Claudia), were investigated in order to discriminate the variation in pomological characteristics, sensory profile, and antioxidant properties. Finally, to evaluate potential bioactivity, antiproliferative activity of hydrophilic extracts from loquat fruits was assessed, at dietary relevant concentrations, against three human epithelial cell lines. Even though the international cultivars confirmed an appropriate level of commercial qualities in association to high levels in antioxidant compounds, the local cultivars revealed the best performances in a wide range of chemical-physical and sensory characteristics. Concerning bioactivity, our results indicate that hydrophilic extracts from all tested cultivars showed concentration-dependent antiproliferative activity with a significant variability of effects between different cell lines and between different cultivars. HeLa cells, the most sensitive and hydrophilic extracts from Peluche, showed the highest inhibitory effect followed by Nespolone di Trabia and Claudia. The results of this trial provide useful information on the pomological traits and the not yet known specific nutritional and functional properties of loquat fruits. Our data, besides helping to promote specific local cultivars, could serve to establish a database that will permit to improve the utilization of specific genetic resources in breeding programs.

Effect of cellulose and lignin on disintegration, antimicrobial and antioxidant properties of PLA active films.

This study reports the effects on antimicrobial, antioxidant, migration and disintegrability activities of ternary nanocomposite films based on poly(lactic acid) incorporating two biobased nanofillers, (cellulose nanocrystals (CNC) and lignin nanoparticles (LNP)), in two different amounts (1 and 3% wt.). Results from antimicrobial tests revealed a capacity to inhibit the Gram negative bacterial growth of Xanthomonas axonopodis pv. vesicatoria and Xanthomonas arboricola pv. pruni along the time, offering innovative opportunities against dangerous bacterial plant pathogens. LNP proved to be highly efficient in antioxidation activity, based on the disappearance of the absorption band at 517nm of the free radical, 2,2-diphenyl-1-picrylhydrazyl (DPPH) upon reduction by an antiradical compound; moreover the combination of LNP and CNC generates a synergistic positive effect in the antioxidation response of PLA ternary films. Furthermore, all the studied formulations showed a disintegrability value up to 90% after 15days of incubation in composting conditions. Migration results showed that the films can be considered suitable for application in food packaging field.

Engineering of carbon based nanomaterials by ring-opening reactions of a reactive azlactone graphene platform.

A reactive azlactone-based graphene nanoplatform was successfully synthesized by the ligation of azido-azlactone with alkyne-terminated graphene via Cu(I)-catalyzed cycloaddition. The reactive azlactone rings, grafted on graphene sheets, were subjected to highly efficient ring-opening reactions with functionalized primary amine derivatives incorporating an aminosilane coupling agent or a biological fragment.

Biodegradable core-shell nanoassemblies for the delivery of docetaxel and Zn(II)-phthalocyanine inspired by combination therapy for cancer.

Combination therapies for cancer aim to exploit either additive or synergistic effects arising from the action of two species with the final goal to maximize the therapeutic efficacy. In this work, we develop multifunctional nanoparticles (NPs) for co-delivery of the conventional anticancer drug docetaxel (DTX) and the second generation photosensitizer zinc-phthalocyanine (ZnPc) as potential dual carrier system for the combination of chemotherapy and photodynamic therapy (PDT). Biodegradable and amphiphilic block copolymers based on poly(ε-caprolactone) (PCL=B) and poly(ethylene oxide) (PEO=A), with AB and ABA architectures, were assembled in "core-shell" NPs and loaded with both DTX and ZnPc employing the melting/sonication method. Hydrodynamic diameters within the range 60-100nm and low polydispersity indexes were obtained. Zeta potential was negative for all the formulations and unaffected by drug encapsulation. Concerning drug loading ability of NPs, the entrapment efficiency was related to initial ZnPc/DTX ratio. Steady-stationary and time-resolved emission fluorescence measurements pointed out the embedding of monomeric ZnPc in the NPs, excluding the presence of ZnPc self-supramolecular oligomers. The release of DTX was biphasic whereas ZnPc remained mainly associated with NPs. Singlet oxygen generation was observed when ZnPc-loaded NPs were irradiated at 610nm within a 45min time range, despite that ZnPc was not released in the medium. Stability of NPs in the presence of serum proteins and plasma was excellent and no toxicity toward red blood cells was found. NPs cytotoxicity was evaluated in HeLa cells irradiated for 30min with a halogen lamp. After 72h, viability of cells treated with ZnPc/DTX-loaded NPs strongly decreased as compared to NPs loaded only with DTX, thus showing a combined effect of both DTX and ZnPc. Superior antitumor activity of ZnPc/DTX-loaded NPs as compared to DTX-loaded NPs was confirmed in an animal model of orthotopic amelanotic melanoma, thus pointing to the application of PEO-PCL NPs in the combined chemo-photodynamic therapy of cancer.

A Multiplex PCR Assay for Detection of Pseudomonas syringae pv. actinidiae and Differentiation of Populations with Different Geographic Origin.

Pseudomonas syringae pv. actinidiae is responsible for severe outbreaks of bacterial canker of kiwifruit currently occurring around the world. Although molecular detection methods have been reported, none provide complete selectivity for this pathovar or discriminate among pathogen haplotypes. Therefore, a new multiplex polymerase chain reaction (PCR) assay was developed and validated. The assay was tested on 32 P. syringae pv. actinidiae isolates and 15 non-P. syringae pv. actinidiae strains and correctly assigned P. syringae pv. actinidiae strains to three different haplotypes: a Japanese/Korean group, a European group, and a Chinese group. Two P. syringae pv. actinidiae isolates from New Zealand were found to belong to the Chinese group whereas two other isolates from New Zealand, which were isolated from kiwifruit plants but which do not cause bacterial canker, tested negative. The described PCR assays has a limit of detection of approximately 5 to 50 pg of purified DNA or of 5 × 102 bacteria/PCR and were shown to work with both artificially and naturally infected plant tissues. Thus, the described method represents a suitable tool for detection of P. syringae pv. actinidiae and haplotype attribution, in particular, when testing a high number of samples during surveillance and prevention activities.

Physico-chemical characterization of an amphiphilic cyclodextrin/genistein complex.

Specific recognition of cell-targeting systems as host-carriers modified with receptor targeting groups, is a major ambition in the application of supramolecular science to medicine and life science. Genistein (Gen), an isoflavone belonging to the class of phytoestrogens, is of great interest because it has been considered as potential remedy for many kinds of disease. In this work, genistein in aqueous medium and in the presence of an host nanocarrier as amphiphilic cyclodextrin (CyD) modified in the upper rim with oligoethylene hydroxyl groups [(2-oligo(ethyleneoxide)-6-hexylthio)-beta-CyD, SC6OH] at 1:1 molar ratio, has been firstly investigated by UV-vis measurements coupled with circular dichroism data, in order to characterize the drug/macrocycle binding affinity through the formation of the complex. Furthermore, FTIR-ATR technique has been used to detect the complex formation in solid phase and to characterize the functional groups responsible of the solid Gen/SC6OH complex stability. The infrared absorbance spectra of the complex, collected in a wide range of wavenumber and around the physiological temperature, have been analysed and compared with the spectra of the pure compounds and their physical mixture. By monitoring the most significant changes in the shape and position of the absorbance bands of the Gen functional groups, we showed that the formation and/or modification of polar bonds play the main role in the interaction of the drug with the amphiphilic CyD. From the results, Gen is shown to be entangled in SC6OH nanoaggregates, establishing hydrogen bonding with the hydrophilic PEG chains.

Outbreak of Bacterial Blossom Blight Caused by Pseudomonas viridiflava on Actinidia chinensis Kiwifruit Plants in Italy.

During the springs from 2006 to 2008, a new disease was observed on 4- to 5-year-old Actinidia chinensis (cv. Jin Tao) trees in different commercial kiwifruit-production areas in northern Italy (Lombardy). Initially, disease occurrence was sporadic but later became widespread. Symptoms on flowers appeared as a dark brown rot of anthers, filaments, sepals, and whole buds. Blossoms abscised prematurely from buds. Symptoms appeared as confluent brown spots often present on rolled margins. Bacteria were isolated from symptomatic tissue on nutrient agar medium supplemented with 5% sucrose. The isolated bacteria were aerobic; produced a diffusible fluorescent pigment on King's B medium; levan, oxidase, and arginine dihydrydrolase negative, and catalase positive; rotted potato tuber tissue; caused a hypersensitive response on tobacco; and failed to reduce nitrate or utilize sucrose and were ice nuclease-positive at -5°C, suggesting the organism was P. viridiflava (1,3). Inoculation of 2-year-old A. chinensis cv. Jin Tao plants were carried out in the greenhouse under controlled environmental conditions (15 to 27°C, night/day; relative humidity up to 70%) by spraying five plants in bloom with a suspension (1 × 108 CFU/ml) of isolated bacteria with a hand-held sprayer that produced large spray droplets. Symptoms, similar to those in nature, were observed on flowers and buds 3 to 5 days after inoculation and on leaves after 7 to 10 days. Using the same tests described above with the original strains, the strains that were isolated from symptomatic tissue were identified as Pseudomonas viridiflava. Seven bacterial strains (PV508-PV1108) were identified by sequencing 1,481 bp of their 16S rDNA region (2) and using BlastN (4) for the most similar sequences in the INSD (GenBank, EMBL, and DDBJ). Our sequences shared 99.53% (1,474 of 1,481 bp) to 99.9% (1480 of 1,481 bp) identity with the analogous sequences of P. viridiflava available in the database. To our knowledge, this is the first report of an outbreak of blossom blight caused by P. viridiflava on A. chinensis cv. Jin Tao kiwifruit plants in Italy. Previously, it was reported on A. deliciosa cv. Hayward (3). Because of the risk of bacterial contamination among the different cultivars of kiwifruit, further investigation and development of control measures are in progress. References: (1) R. A. Lelliott and D. E. Stead. Methods for the Diagnosis of Bacterial Diseases of Plants. Blackwell Scientific, Oxford, UK, 1987. (2) E. R. B. Moore et al. Syst. Appl. Microbiol. 19:478, 1996. (3) L. Varvaro et al. Inf. Fitopatol. 6:49, 1990. (4) Z. Zhang et al. J. Comput. Biol. 7(1-2):203, 2000.

The enhancement of isoflavones water solubility by complexation with modified cyclodextrins: a spectroscopic investigation with implications in the pharmaceutical analysis.

The improvement of isoflavones bioavailability by complexation with chemically modified cyclodextrins (CyDs) has been exploited to analyse the drug/macrocycle binding affinity by a conventional method with new useful measures. Genistein (Gen) and daidzein (Daidz) were investigated in aqueous medium and in presence an amount of (2-hydroxypropyl)-beta-cyclodextrin (HP-beta-CyD) at different host/guest molar ratios. The solubility in pure water, approximately 3 x 10(-6)M for Gen and approximately 10 x 10(-6)M for Daidz, was obtained by distributing the of guest molecule between water and the organic solvent. The stoichiometric ratios and stability constants describing the extent of formation of the complexes have been determined by phase-solubility UV-vis measurements and confirmed by circular dichroism data. These results have implications in the determination of the carrier's capacity for the complexation of the drug in water solution.

New cyclodextrin derivatives as chiral selectors in capillary electrophoresis.

The separation of three pairs of enantiomeric herbicides has been successfully achieved by capillary electrophoresis at two different pH values in the presence of cyclodextrin derivatives previously synthesized in our laboratory. Two of these derivatives constitute a new class of receptor, the hemispherodextrins, in which a trehalose capping moiety is bonded to beta-cyclodextrin. Because of their peculiar structure hemispherodextrins have very promising characteristics and the low receptor concentration required to achieve separation deserves particular interest.