Luciana Cerioni

Cellular damage induced by a sequential oxidative treatment on Penicillium digitatum

Aim:  To investigate the cellular damage on Penicillium digitatum produced by a sequential oxidative treatment (SOT), previously standardized in our laboratory, to prevent the conidia growth. Lethal SOT consists of 2‐min preincubation with 10 ppm NaClO followed by 2‐min incubation with 6 mmol l−1 CuSO4 and 100 mmol l−1 H2O2 at 25°C.

Synergistic Antifungal Activity of Sodium Hypochlorite, Hydrogen Peroxide, and Cupric Sulfate against Penicillium digitatum

Oxidizing compounds such as sodium hypochlorite (NaCIO) and hydrogen peroxide (H2O2) are widely used in food sanitization because of their antimicrobial effects. We applied these compounds and metals to analyze their antifungal activity against Penicillium digitatum, the causal agent of citrus green mold. The MICs were 300 ppm for NaClO and 300 mM for H2O2 when these compounds were individually applied for 2 min to conidia suspensions. To minimize the concentration of these compounds, we developed and standardized a sequential treatment for conidia that resulted in loss of viability on growth plates and loss of infectivity on lemons. The in vitro treatment consists of preincubation with 10 ppm of NaClO followed by incubation with 100 mM H2O2 and 6 mM CuSO4 (cupric sulfate). The combination of NaClO and H2O2 in the presence of CuSO4 produces a synergistic effect (fractional inhibitory concentration index of 0.36). The sequential treatment applied in situ on lemon peel 24 h after the fruit was inoculated with conidia produced a significant delay in the fungal infection. The in vitro treatment was effective on both imazalil-sensitive and imazalil-resistant strains of P. digitatum and Geotrichum candidum, the causal agent of citrus sour rot. However, this treatment inhibited 90% of mycelial growth for Penicillium italicum (citrus blue mold). These results indicate that sequential treatment may be useful for postharvest control of citrus fruit diseases.

Inhibition of Penicillium expansum by an oxidative treatment

Several oxidizing compounds such as sodium hypochlorite (NaClO) and hydrogen peroxide (H(2)O(2)) are used to control postharvest decay in fresh fruit due to their antimicrobial effects. Here, we applied these compounds in vitro, in the presence of CuSO(4), against Penicillium expansum, causal agent of apple blue mold. MICs were 50 mg L(-1) and 400 mmol L(-1) for NaClO and H(2)O(2), respectively, when these compounds were individually applied to conidia suspensions during 2 min. A combined oxidative treatment (OT) consisting on an incubation with 1 mg L(-1) NaClO and 200 mmol L(-1) H(2)O(2), in the presence of 6 mmol L(-1) CuSO(4), inhibited growth, conidial germination and fungal infectivity on apple. The fractional inhibitory concentration index for the interaction between NaClO and H(2)O(2) in the OT was 0.52 indicating a synergistic effect of the oxidizing compounds. These results suggest that the OT could be an interesting alternative for apple diseases postharvest control.

UVA Photoactivation of Harmol Enhances Its Antifungal Activity against the Phytopathogens Penicillium digitatum and Botrytis cinerea

Phytopathogenic fungi responsible for post-harvest diseases on fruit and vegetables cause important economic losses. We have previously reported that harmol (1-methyl-9H-pyrido[3,4-b]indol-7-ol) is active against the causal agents of green and gray molds Penicillium digitatum and Botrytis cinerea, respectively. Here, antifungal activity of harmol was characterized in terms of pH dependency and conidial targets; also photodynamic effects of UVA irradiation on the antimicrobial action were evaluated. Harmol was able to inhibit the growth of both post-harvest fungal disease agents only in acidic conditions (pH 5), when it was found in its protonated form. Conidia treated with harmol exhibited membrane integrity loss, cell wall disruption, and cytoplasm disorganization. All these deleterious effects were more evident for B. cinerea in comparison to P. digitatum. When conidial suspensions were irradiated with UVA in the presence of harmol, antimicrobial activity against both pathogens was enhanced, compared to non-irradiated conditions. B. cinerea exhibited a high intracellular production of reactive oxygen species (ROS) when was incubated with harmol in irradiated and non-irradiated treatments. P. digitatum showed a significant increase in ROS accumulation only when treated with photoexcited harmol. The present work contributes to unravel the antifungal activity of harmol and its photoexcited counterpart against phytopathogenic conidia, focusing on ROS accumulation which could account for damage on different cellular targets.

Short-term UV-B exposure induces metabolic and anatomical changes in peel of harvested lemons contributing in fruit protection against green mold.

UV-B radiation (UVBR) is a small fraction of the solar spectrum from 280 to 315nm. UVBR produces photomorphogenic acclimation responses in plants, modulating their cellular structure and physiology. Here, changes in the peel of harvested lemons after short time exposure to UVBR were analyzed and its potential effects against fungal infection were studied. In the flavedo, UVBR treatment induced variations in the respiratory profiles and increased the phenolic compound contents. Final products of the flavonoid pathway (flavones, flavonols and anthocyanins) increased more markedly than their precursors (flavanones and dihydroflavonols). The increased accumulation of soluble phenolics in the flavedo of treated lemons is associated with the high antioxidant activity found in the flavedo of these samples. Supporting the biochemical determinations, anatomical observations showed abundant intravacuolar deposits of phenolic compounds and an increase in the cell wall thickness in UVBR-treated samples. Metabolic and anatomical modifications associated to UVBR improved natural defenses against Penicillium digitatum, the causal agent of green mold disease. Our results suggest that mature postharvest lemons exposed to the artificial radiation showed phenotypic plasticity, allowing an acclimation response to UVBR which confers fruit resistance to pathogens. Thus, combination of UVBR with other treatments could represent an important improvement to control postharvest diseases on citrus.

Removal of pathogenic bacterial biofilms by combinations of oxidizing compounds.

Bacterial biofilms are commonly formed on medical devices and food processing surfaces. The antimicrobials used have limited efficacy against the biofilms; therefore, new strategies to prevent and remove these structures are needed. Here, the effectiveness of brief oxidative treatments, based on the combination of sodium hypochlorite (NaClO) and hydrogen peroxide (H2O2) in the presence of copper sulfate (CuSO4), were evaluated against bacterial laboratory strains and clinical isolates, both in planktonic and biofilm states. Simultaneous application of oxidants synergistically inactivated planktonic cells and prevented biofilm formation of laboratory Escherichia coli, Salmonella enterica serovar Typhimurium, Klebsiella pneumoniae, and Staphylococcus aureus strains, as well as clinical isolates of Salmonella enterica subsp. enterica, Klebsiella oxytoca, and uropathogenic E. coli. In addition, preformed biofilms of E. coli C, Salmonella Typhimurium, K. pneumoniae, and Salmonella enterica exposed to treatments were removed by applying 12 mg/L NaClO, 0.1 mmol/L CuSO4, and 350 mmol/L H2O2 for 5 min. Klebsiella oxytoca and Staphylococcus aureus required a 5-fold increase in NaClO concentration, and the E. coli clinical isolate remained unremovable unless treatments were applied on biofilms formed within 24 h instead of 48 h. The application of treatments that last a few minutes using oxidizing compounds at low concentrations represents an interesting disinfection strategy against pathogens associated with medical and industrial settings.