First Report of Phytophthora inundata Associated with Decline and Death of Walnut (Juglans regia L.) in Italy.

Persian walnut (Juglans regia L.) is an important nut crop in Italy. In recent years, incidence of walnut decline and death has increased in many Italian commercial orchards. In early summer 2020, we observed a serious decline in approximately 5% of trees in a waterlogged area of a Veneto-region walnut orchard (J. regia, cv Lara). Symptoms included extensive foliar wilt and canopy decline associated with collar and root rot. Symptomatic tissues excised from larger roots of affected trees were surface disinfested for 1 min in a 1% NaOCl solution, rinsed for 5 min in sterile distilled water, and placed onto P5ARPH selective medium (Jeffers and Martin 1986). A Phytophthora-like organism was consistently isolated. Pure cultures were obtained by single-hyphal transfers onto potato dextrose agar (PDA). Isolates were identified as Phytophthora inundata based on morphological characteristics (Brasier et al. 2003), sequences of internal transcribed spacer (ITS) amplicons from universal primers ITS6 (Cooke et al. 2000) and ITS4 (White et al. 1990) and sequences of cytochrome c oxidase, subunit II (Cox II) from Fm75 and Fm78 primer pair (Martin and Tooley 2003). On carrot agar (CA), colonies had a characteristic stellate to broad lobed patterns. On this medium, optimal growth was at 28-30 °C (7,3 mm/day) and the upper temperature limit for mycelial growth was 37°C. Mycelial disks of isolate CREADC-Om306, grown on CA, were floated in Petri plates with soil extract solution and incubated under continuous fluorescent light at room temperature (25+/-2 °C). Within 48 to 72 h, sporangia were produced that were persistent, non-papillate, ovoid or ovoid-obpyriform, measuring 55.0 to 80.7 (length) x 41.3 to 65.2 (width) µm (averages 64.3+/-10.2 x 47.9+/-9.7 µm). Oospores and chlamydospores were absent. BLAST analysis of the amplicons from CREADC-Om306 revealed ITS sequences (854-bp; GenBank accession no. OK342200) and Cox II sequences (568-bp; GenBank accession no. OK349677) that shared 100% identity with published P. inundata sequences available in GenBank (acc. n. AF266791 for ITS; MT458994 for Cox II). Pathogenicity tests were conducted in the greenhouse on six 2-year-old walnut (J. regia, cv Lara) plants. Four of the plants were inoculated with CREADC-Om306 on two opposite sides of each plant's stem at 1-2 cm above soil line. A cork borer was used to remove a 5-mm disk of bark that was replaced by a 5-mm diameter mycelial plug from 10-day-old cultures of the pathogen on PDA. Two control plants were treated in the same way except the bark wounds were inoculated with sterile PDA plugs. Plants were kept in greenhouse at 24 ± 2°C. After 3 months, lesions had developed from all points of inoculation with. P. inundata (mean lesion length 55,25+/-6,22 mm) and the pathogen was reisolated from the lesion margins of all inoculated plants. The control plants remained symptomless and did not yield the pathogen. P. inundata is widely distributed across the world as a plant pathogen on several native as well as horticultural crops, especially in riparian or other areas subject to flooding or waterlogging. This report is the first to document P. inundata as a pathogen on Persian walnut and adds it to the diverse list of known susceptible perennial native, ornamental, and agricultural hosts of this organism. In addition to P. inundata, which belongs to the major Phytophthora ITS Clade 6, other members of the clade including P. megasperma (Belisario et al. 2012) and P. gonapodyides (Belisario et al. 2016) have been described as walnut pathogens. References: Belisario, A., et al. 2012. Plant Dis. 96 (11):1695. https://doi.org/10.1094/PDIS-05-12-0470-PDN. Belisario, A., et al. 2016. Plant Dis. 100 (12):2537. https://doi.org/10.1094/PDIS-03-16-0394-PDN. Brasier, C.M., et al. 2003. Mycol. Res. 107 (4):477. DOI: 10.1017/S0953756203007548. Cooke, D. E. L., et al. 2000. Fungal Genet. Biol. 30:17. https://doi.org/10.1006/fgbi.2000.1202. Jeffers SN, Martin SB. (1986) Plant Dis70:1038. Martin, F. N., and Tooley, P. W. 2003. Mycologia 95:269. https://pubmed.ncbi.nlm.nih.gov/21156613/. Schena, L., et al. 2008. Plant Pathol. 57:64. https://doi.org/10.1111/j.1365-3059.2007.01689.x. White, T.J. et al. 1990. In PCR Protocols: A Guide to Methods and Applications; Innis, M.A., Gelfand, D.H., Sninsky, J.J., White, T.J., Eds.; Academic Press, (USA,) 18: 315-322.

First report of Colletotrichum fioriniae and C. nymphaeae as Causal Agents of anthracnose on walnut in Italy.

English walnut (Juglans regia L.) is species grown either for high quality wood or fruit production. In Italy walnut cultivation occupies an area of about 4600 ha (FAOSTAT, http://www.fao.org/faostat, 2020). In 2019-2020, walnut fruits (cv Lara) with anthracnose symptoms were collected from walnut orchards in Province of Venice (Northern Italy). Affected fruits showed necrotic and circular lesions with acervuli in the center causing the complete mummification of the fruit as described by Da Lio et al., 2018. Orange conidial masses appeared under wet conditions. The fungus was isolated from necrotic tissues and conidial masses were put on potato dextrose agar (PDA) medium. Plates were incubated at 25°C for 5 to 7 days. The colonies were white to pink on the upper side and pink with black spots on the reverse. Acervuli formed and produced conidial masses on PDA after 6 days. Culture and conidial morphology were in concordance with published descriptions of C. acutatum sensu lato (Damm et al., 2012). To confirm the identity, internal transcribed spacers (ITS), (glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT) and beta-tubulin (TUB2) genes were amplified and sequenced using the primer pairs ITS1/ITS4 (White at al. 1990), GDF1/GDR1 (Guerber et al., 2003), ACT512F/ACT783R and BT2Fd/BT4R primers (Da Lio et al., 2018). The isolates belonged to two different species of Colletotrichum acutatum complex: C. fioriniae (Marcelino & Gouli) and C. nymphaeae (Pass). Sequences of two representative isolates C. fioriniae CREADC-F2317 and C. nymphaeae CREADC-F2372 were deposited in GenBank with accession numbers MZ153170 and MZ191794 (ITS), MZ203522 and MZ224013 (GAPDH), MZ203521 and MZ224012 (ACT), and MZ203523 and MZ224014 (TUB2). For all the genes, isolates had a 100% similarity to multiple C. fioriniae and C. nymphaeae accessions, respectively. Maximum likelihood trees based on concatenated sequences of the four genes were constructed using MEGA 6.0 (Tamura et al., 2013). The phylogenetic analysis grouped the isolates in the C. fioriniae and nymphaeae clusters respectively. The two isolates CREADC-F2317 and CREADC-F2372 were used to confirm pathogenicity on walnut fruits. Fruits of cv Lara were surface disinfected by dipping in 3% NaOCl for 1 min, rinsed in sterile distilled water, and arranged in sterile humid chambers. Fruits were wounded with a sterile needle then inoculated with 20 µl of 106 conidia/ml suspensions of each isolate (one wound per fruit). Fruit treated with sterile distilled water served as a control. Inoculations were conducted on three fruits per replicate and three replicates per treatment arranged in a complete block randomized design. After 7 days incubation at 25 ± 1°C, all the inoculated fruits showed typical anthracnose symptoms and lesions with cream to salmon pink acervuli, whereas the controls remaied healthy. The species C. nymphaeae and C. fioriniae were reisolated from the rotted fruit. Pathogenicity tests were repeated twice with the same results. The morphology of the reisolated fungi was consistent with the inoculated one, fulfilling Koch's postulates. The species C. fioriniae and C. nymphaeae have been described affecting numerous species worldwide (Damm et al., 2012). C. fioriniae and C. nymphaeae have been previously reported to cause severe anthracnose on walnut, C. fioriniae in France (Da Lio et al., 2018) and Hungary (Varjas et al., 2019) and C. nymphaeae in France (Da Lio et al., 2018) and Brazil (Savian et al., 2019). To our knowledge, this is the first report of C. fioriniae and C. nymphaeae as causal agents of walnut anthracnose in Italy.

Characterization of bioactive compounds and antioxidant activity of fruit beers.

Total polyphenols and flavonoids content, phenolics profile by HPLC, and antioxidant activity of ten fruit beer produced adding fruits during the fermentation process were analyzed. The fruits were: cherry, raspberry, peach, apricot, grape, plum, orange and apple. Antioxidant activity, total polyphenols and flavonoids content were considerably higher in most of the fruit beers in respect to conventional, no-fruit beers. Cherries beers exhibit the highest values, followed by grape, plum and orange beers. An enrichment was observed in catechin and quercetin content in all fruit beers examined. Myricetin and resveratrol were also detected in most of the fruit beers. Among phenolic acids, an enrichment in chlorogenic, neochlorogenic, p-coumaric and caffeic acids was measured in most of the fruit beers in respect to conventional beers. Our findings show that fruits addition during the fermentation process considerably increased the antioxidant activity of beer and qualitatively and quantitatively improved its phenolics profile.

Redox Role of Lactobacillus casei Shirota Against the Cellular Damage Induced by 2,2'-Azobis (2-Amidinopropane) Dihydrochloride-Induced Oxidative and Inflammatory Stress in Enterocytes-Like Epithelial Cells.

In western societies where most of the day is spent in the postprandial state, the existence of oxidative and inflammatory stress conditions makes postprandial stress an important factor involved in the development of cardiovascular risk factors. A large body of evidence have been accumulated on the anti-inflammatory effects of probiotics, but no information is available on the mechanisms through which intestinal microbiota modulates redox unbalance associated with inflammatory stress. Here, we aimed to investigate the ability of Lactobacillus casei Shirota (LS) to induce an antioxidant response to counteract oxidative and inflammatory stress in an in vitro model of enterocytes. Our results show that pretreatment of enterocytes with LS prevents membrane barrier disruption and cellular reactive oxygen species (ROS) accumulation inside the cells, modulates the expression of the gastro-intestinal glutathione peroxidase (GPX2) antioxidant enzyme, and reduces p65 phosphorylation, supporting the involvement of the Nfr2 and nuclear factor kappa B pathways in the activation of antioxidant cellular defenses by probiotics. These results suggest, for the first time, a redox mechanism by LS in protecting intestinal cells from AAPH-induced oxidative and inflammatory stress.

Effect of Sulfites on Antioxidant Activity, Total Polyphenols, and Flavonoid Measurements in White Wine.

Polyphenols content and antioxidant activity are directly related to the quality of wine. Wine also contains sulfites, which are added during the winemaking process. The present study aimed to evaluate the effects of sulfites on the assays commonly used to measure the antioxidant activity and polyphenols and flavonoids content of white wines. The effects of sulfites were explored both in the standard assays and in white wine. The addition of sulfites (at 1-10 µg) in the standard assays resulted in a significant, positive interference in the Folin-Ciocalteu's assay used for polyphenols measurements and in both the Ferric Reducing Antioxidant Power and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radical cation decolorization assays, which were used for antioxidant activity evaluation. A negative interference of sulfites (at 1-20 µg) was observed for the colorimetric aluminium-chloride flavonoids assay. The addition of sulfites to organic white wines (at 25-200 mg/L wine) clearly resulted in a significant overestimation of antioxidant activity and polyphenols content, and in an underestimation of flavonoids concentration. To overcome sulfite interferences, white wines were treated with cross-linked polyvinylpyrrolidone. The total polyphenols content and antioxidant activity measurements obtained after polyvinylpyrrolidone treatment were significantly lower than those obtained in the untreated wines. Flavonoids were expected to be higher after polyvinylpyrrolidone treatment, but were instead found to be lower than for untreated wines, suggesting that in addition to sulfites, other non-phenolic reducing compounds were present in white wine and interfered with the flavonoid assay. In view of our results, we advise that a purification procedure should be applied in order to evaluate the quality of white wine.

The Potential Health Benefits of Polyphenol-Rich Extracts from Cichorium intybus L. Studied on Caco-2 Cells Model.

Phytochemicals can exert their bioactivity without reaching the systemic circulation; scarcely absorbed antioxidants might reach the large bowel contributing to protection from oxidative damage-induced gastrointestinal diseases. In the present work, we aimed to study the relationship between potential activity of polyphenol-rich extracts from Cichorium intybus L. and changes in morphological characteristics on Caco-2 cells. Phytochemicals content (carotenoids and flavonoids) and total antioxidant activity of Red Chicory of Treviso and Variegated Chicory of Castelfranco were evaluated. The bioactivity of polyphenol-rich extracts from chicories was studied in in vitro Caco-2 cell monolayers model. Morphological characteristics changes to test the antioxidant and/or prooxidant effect were verified by histological analysis and observed by Electronic Scansion Microscopy (SEM). On Caco-2 cell model, the polyphenols fractions from chicories have indicated a moderate antioxidant behavior until 17 µM concentration, while 70 µM and 34 µM exert cytotoxic effects for Treviso's and Castelfranco's Chicory, respectively, highlighted by TEER decreasing, increased permeability, and alteration of epithelium. Our findings support the beneficial effects of these products in counteracting the oxidative stress and cellular damage, induced in vitro on Caco-2 cell model, through interaction with the mucopolysaccharide complexes in the glycocalyx, maintaining in vivo a healthy and effective intestinal barrier.

Polyphenols content, phenolics profile and antioxidant activity of organic red wines produced without sulfur dioxide/sulfites addition in comparison to conventional red wines.

Wine exerts beneficial effects on human health when it is drunk with moderation. Nevertheless, wine may also contain components negatively affecting human health. Among these, sulfites may induce adverse effects after ingestion. We examined total polyphenols and flavonoids content, phenolics profile and antioxidant activity of eight organic red wines produced without sulfur dioxide/sulfites addition in comparison to those of eight conventional red wines. Polyphenols and flavonoids content were slightly higher in organic wines in respect to conventional wines, however differences did not reach statistical significance. The phenolic acids profile was quite similar in both groups of wines. Antioxidant activity was higher in organic wines compared to conventional wines, although differences were not statistically significant. Our results indicate that organic red wines produced without sulfur dioxide/sulfites addition are comparable to conventional red wines with regard to the total polyphenols and flavonoids content, the phenolics profile and the antioxidant activity.

Prevention of TNBS-induced colitis by different Lactobacillus and Bifidobacterium strains is associated with an expansion of gammadeltaT and regulatory T cells of intestinal intraepithelial lymphocytes.

BACKGROUND: Probiotics may protect against inflammatory bowel disease through regulation of lamina propria lymphocytes (LPLs) function. Data are lacking on possible involvement of intraepithelial lymphocytes (IELs). The aim of this study was to investigate whether different probiotic mixtures prevented gut inflammatory disease and the role of both IELs and LPLs. METHODS: BALB/c mice received 2 probiotic mixtures orally for 3 weeks, as Mix1 (Lactobacillus acidophilus and Bifidobacterium longum), or Mix2 (Lactobacillus plantarum, Streptococcus thermophilus, and Bifidobacterium animalis subsp. lactis). Colitis was induced by intrarectal administration of trinitrobenzene sulfonic acid (TNBS). Probiotics in stools were analyzed by real-time polymerase chain reaction (PCR). Colon subpopulations of IELs and LPLs were assayed by flow cytometry. Serum cytokines were measured by cytometric bead array (CBA). RESULTS: All probiotics colonized the intestine. The 2 mixtures prevented the TNBS-induced intestinal damage, and Mix1 was the most effective. The Mix1 protection was associated with a reduction in CD4(+) cells of IELs and LPLs, an increase in gammadeltaT cells of IELs, and a decrease in gammadeltaT cells of LPLs. An expansion of T regulatory (Treg) cells of IELs was induced by Mix1 and Mix2. Both probiotic mixtures inhibited tumor necrosis factor (TNF)-alpha and monocyte chemotactic protein (MCP)-1 production and upregulated interleukin (IL)-10. In addition, Mix1 prevented the TNBS-induced increase of IL-12 and interferon (IFN)-gamma. CONCLUSIONS: The 2 probiotic mixtures were able to prevent the TNBS-induced colitis; the L. acidophilus and B. longum mixture was the most effective. Other than an involvement of LPLs, our results report a novel importance of the IELs population in probiotic protection.

Zinc oxide protects cultured enterocytes from the damage induced by Escherichia coli.

There is some evidence that zinc oxide (ZnO) protects against intestinal diseases. However, despite the suggestions that ZnO may have an antibacterial effect, the mechanisms of this protective effect have not yet been elucidated. We investigated the potential benefits of ZnO in protecting intestinal cells from damage induced by enterotoxigenic Escherichia coli (ETEC, strain K88) and the related mechanisms, using human Caco-2 enterocytes. Cell permeability, measured as transepithelial electrical resistance (TEER), was unaffected by 0.01 and 1 mmol/L ZnO treatments and moderately increased by 5 mmol/L ZnO, compared with untreated cells. Transfer of (14)C-inulin was slightly increased by 5 mmol/L ZnO compared with untreated cells; transfer was unaffected by lower concentrations. The TEER and (14)C-inulin transfer were lower in ETEC-infected cells than in uninfected cells. Treatment of ETEC exposure with 0.2 mmol/L ZnO prevented disruption of membrane integrity. The ETEC was able to adhere to enterocytes and, to some extent, invade the cells. The ZnO treatment reduced bacterial adhesion and blocked bacterial invasion. The ETEC infection upregulated the expression of the inflammatory cytokines interleukin-8, growth-related oncogene-alpha and tumor necrosis factor-alpha, and reduced that of the anti-inflammatory cytokine transforming growth factor-beta, compared with uninfected cells. The addition of 0.2 or 1 mmol/L ZnO counteracted the alteration of cytokine mRNA levels caused by ETEC. The protective effects of ZnO were not due to any antibacterial activity, because the viability of ETEC grown in a medium containing ZnO was unaffected. In conclusion, ZnO may protect intestinal cells from ETEC infection by inhibiting the adhesion and internalization of bacteria, preventing the increase of tight junction permeability and modulating cytokine gene expression.