Pathogenicity test with <i>Psn23</i> on oleander explants, following treatment with polyphenolic extracts VN, TV, FO or FC.

<p>Explants from adult oleander plants were inoculated with <i>P</i>. <i>savastanoi</i> pv. <i>nerii</i> strain <i>Psn23</i>, in the presence or absence of the VN, TV, FO or FC extracts (100 μM). As negative control the non pathogenic mutant ∆<i>hrpA</i> was used. (A) Development of hyperplastic knots at 21 dpi with (from left to right): <i>Psn23</i>, ∆<i>hrpA</i>, <i>Psn23+</i>VN, <i>Psn23+</i>TV, <i>Psn23+</i>FO, <i>Psn23+</i>FC. The symptoms are detectable as swelling at the inoculated end of oleander explants. (B) Normalized weight increase of oleander explants at 21 dpi inoculated with (from left to right): <i>Psn23</i>, ∆<i>hrpA</i>, <i>Psn23+</i>VN, <i>Psn23+</i>TV, <i>Psn23+</i>FO, <i>Psn23+</i>FC. Values are means ± SD of nine replicates for each treatment. Different letters indicate significant differences among means at <i>P</i> < 0.05, according to Tukey's test.</p

Current measurements on SR vesicles adsorbed on a SSM.

<p>Current signals induced by 100 μM ATP concentration jumps in the presence of 10 μM Ca²⁺_free and in the absence (black curve, control measurement) or in the presence of CuCl₂ (red curve) or of the polyphenolic compounds EGCG, catechin, oleuropein, hydroxytyrosol and chlorogenic acid (green curves).</p

Global Analysis of Type Three Secretion System and Quorum Sensing Inhibition of <i>Pseudomonas savastanoi</i> by Polyphenols Extracts from Vegetable Residues

<div><p>Protection of plants against bacterial diseases still mainly relies on the use of chemical pesticides, which in Europe correspond essentially to copper-based compounds. However, recently plant diseases control is oriented towards a rational use of molecules and extracts, generally with natural origin, with lower intrinsic toxicity and a reduced negative environmental impact. In this work, polyphenolic extracts from vegetable no food/feed residues of typical Mediterranean crops, as <i>Olea europaea</i>, <i>Cynara scolymus</i>, and <i>Vitis vinifera</i> were obtained and their inhibitory activity on the Type Three Secretion System (TTSS) and the Quorum Sensing (QS) of the Gram-negative phytopathogenic bacterium <i>Pseudomonas savastanoi</i> pv. <i>nerii</i> strain <i>Psn23</i> was assessed. Extract from green tea (<i>Camellia sinensis</i>) was used as a positive control. Collectively, the data obtained through <i>gfp</i>-promoter fusion system and real-time PCR show that all the polyphenolic extracts here studied have a high inhibitory activity on both the TTSS and QS of <i>Psn23</i>, without any depressing effect on bacterial viability. Extracts from green tea and grape seeds were shown to be the most active. Such activity was confirmed <i>in planta</i> by a strong reduction in the ability of <i>Psn23</i> to develop hyperplastic galls on explants from adult oleander plants, as well as to elicit hypersensitive response on tobacco. By using a newly developed Congo red assay and an ELISA test, we demonstrated that the TTSS-targeted activity of these polyphenolic extracts also affects the TTSS pilus assembly. In consideration of the potential application of polyphenolic extracts in plant protection, the absence of any toxicity of these polyphenolic compounds was also assessed. A widely and evolutionary conserved molecular target such as Ca²⁺-ATPase, essential for the survival of any living organism, was used for the toxicity assessment.</p></div

Relative gene expression analysis of key genes correlated to TTSS and QS of <i>Psn23</i>.

<p>Relative mRNA levels of <i>hrpA</i>, <i>hrpL</i>, <i>hrpV</i>, <i>hrpRS</i>, <i>rpoN</i>, <i>lon</i>, <i>psnI</i>, <i>psnR</i> genes of <i>Psn23</i>, grown in MM supplemented with the polyphenolic extracts TV, VN, FO or FC compared to levels in MM alone (untreated). The data are expressed as the average of three replicates ± SD. Asterisks indicate significant differences compared with the untreated sample at <i>P</i> <0.05.</p

Quali-quantitative HPLC/DAD/MS analysis of FO, FC, VN and TV extracts.

<p>Quali-quantitative HPLC/DAD/MS analysis of FO, FC, VN and TV extracts.</p

Effects on bacterial growth, and on the trans-activation of <i>hrpA</i> and <i>psnI</i> promoters of the polyphenolic extracts and their main constituents.

<p>Effects on bacterial growth, and on the trans-activation of <i>hrpA</i> and <i>psnI</i> promoters of the polyphenolic extracts and their main constituents.</p

Bacterial strains, mutant and plasmids used in this study.

<p>Bacterial strains, mutant and plasmids used in this study.</p

ELISA assay on <i>Psn23</i> bacterial supernatant amended with polyphenolic extracts.

<p>Quantification of HrpA protein by ELISA assay on bacterial supernatant of wild type <i>Psn23</i> grown on MM, or on MM amended with the polyphenolic extracts VN, TV, FO, or FC. As a negative control the ∆<i>hrpA</i> mutant was used. As a reference for quantification, a standard curve was established by a serial dilution of the <i>Psn23</i> HrpA recombinant protein (117 pg/ml– 40 ng/ml). The data represent the means ± SD of three replicates. Statistically significant differences are represented by different letters above the bars (ANOVA and Tukey’s test, <i>P</i> < 0.05).</p

Leishmanicidal activity of green tea leaves and pomegranate peel extracts on <i>L. infantum</i>

<p><i>Leishmania infantum</i> is responsible for the cutaneous and visceral form of this zoonotic disease, which is potentially lethal for humans and has dogs as natural reservoir. In the light of the antiparasitic properties displayed by several natural products, <i>L. infantum</i> promastigotes were exposed to green tea (<i>Camellia sinensis</i>) leaves extract (GTE) and pomegranate (<i>Punica granatum</i>) peel extract (PPE). Both extracts, characterized by NMR and HPLC analysis, inhibited parasite proliferation in a dose-dependent manner, as proved by IC₅₀ evaluation determined by MTT assay.Moreover, the reversibility assay showed that GTE and PPE have an aptotosis-mediated leishmanicidal effect, as evidenced by DNA degradation and confirmed by DNA fragmentation and real-time PCR analyses. Finally, for the first time morphological and ultrastructural alterations induced by a <i>P. granatum</i> extract on <i>Leishmania</i> were shown by the use of light, transmission and scanning electron microscopy.</p