Photodynamic inactivation of conidia of the fungus Colletotrichum abscissum on Citrus sinensis plants with methylene blue under solar radiation.

Antimicrobial photodynamic treatment (APDT) is a promising light based approach to control diseases caused by plant-pathogenic fungi. In the present study, we evaluated the effects of APDT with the phenothiazinium photosensitizer methylene blue (MB) under solar radiation on the germination and viability of conidia of the pathogenic fungus Colletotricum abscissum (former Colletotrichum acutatum sensu lato). Experiments were performed both on petals and leaves of sweet orange (Citrus sinensis) in different seasons and weather conditions. Conidial suspensions were deposited on the leaves and petals surface, treated with the PS (25 or 50µM) and exposed to solar radiation for only 30min. The effects of APDT on conidia were evaluated by counting the colony forming units recovered from leaves and petals and by direct evaluating conidial germination on the surface of these plant organs after the treatment. To better understand the mechanistic of conidial photodynamic inactivation, the effect of APDT on the permeability of the conidial plasma membrane was assessed using the fluorescent probe propidium iodide (PI) together with flow cytometry and fluorescence microscopy. APDT with MB and solar exposure killed C. abscissum conidia and prevented their germination on both leaves and petals of citrus. Reduction of conidial viability was up to three orders of magnitude and a complete photodynamic inactivation was achieved in some of the treatments. APDT damaged the conidial plasma membrane and increased its permeability to PI. No damage to sweet orange flowers or leaves was observed after APDT. The demonstration of the efficacy of APDT on the plant host represents a further step towards the use of the method for control phytopathogens in the field.

Inactivation of plant-pathogenic fungus Colletotrichum acutatum with natural plant-produced photosensitizers under solar radiation.

The increasing tolerance to currently used fungicides and the need for environmentally friendly antimicrobial approaches have stimulated the development of novel strategies to control plant-pathogenic fungi such as antimicrobial phototreatment (APT). We investigated the in vitro APT of the plant-pathogenic fungus Colletotrichum acutatum with furocoumarins and coumarins and solar radiation. The compounds used were: furocoumarins 8-methoxypsoralen (8-MOP) and 5,8-dimethoxypsoralen (isopimpinellin), coumarins 2H-chromen-2-one (coumarin), 7-hydroxycoumarin, 5,7-dimethoxycoumarin (citropten) and a mixture (3:1) of 7-methoxycoumarin and 5,7-dimethoxycoumarin. APT of conidia with crude extracts from 'Tahiti' acid lime, red and white grapefruit were also performed. Pure compounds were tested at 50µM concentration and mixtures and extracts at 12.5mgL(-1). The C. acutatum conidia suspension with or without the compounds was exposed to solar radiation for 1h. In addition, the effects of APT on the leaves of the plant host Citrus sinensis were determined. APT with 8-MOP was the most effective treatment, killing 100% of the conidia followed by the mixture of two coumarins and isopimpinellin that killed 99% and 64% of the conidia, respectively. APT with the extracts killed from 20% to 70% of the conidia, and the extract from 'Tahiti' lime was the most effective. No damage to sweet orange leaves was observed after APT with any of the compounds or extracts.

In vitro photodynamic inactivation of plant-pathogenic fungi Colletotrichum acutatum and Colletotrichum gloeosporioides with Novel Phenothiazinium photosensitizers.

The increasing tolerance to currently used fungicides in both clinical and agricultural areas is of great concern. The nonconventional light-based approach of antimicrobial photodynamic treatment (APDT) is a promising alternative to conventional fungicides. We evaluated the effects of APDT with four phenothiazinium derivatives (methylene blue [MB], new methylene blue N [NMBN], toluidine blue O [TBO], and the novel pentacyclic phenothiazinium photosensitizer [PS] S137) on conidia of three fungal species (Colletotrichum acutatum, Colletotrichum gloeosporioides, and Aspergillus nidulans). The efficacy of APDT with each PS was determined, initially, based on photosensitizer MICs. Additionally, the effects of APDT with two selected PSs (NMBN and S137) on survival of conidia were evaluated. The subcellular localization of the PS in C. acutatum conidia was determined. The effects of photodynamic treatments on leaves of the plant host Citrus sinensis were also investigated. APDT with S137 showed the lowest MIC. MICs for S137 were 5 µM for the three fungal species when a fluence of 25 J cm(-2) was used. APDT with NMBN (50 µM) and S137 (10 µM) resulted in a reduction in the survival of the conidia of all species of approximately 5 logs with fluences of ≥15 J cm(-2). Washing of the conidia before light exposure did not prevent photodynamic inactivation. Both NMBN and S137 accumulated in cytoplasmic structures, such as lipid bodies, of C. acutatum conidia. No damage to orange tree leaves was observed after APDT.

In vitro photodynamic inactivation of Candida species and mouse fibroblasts with phenothiazinium photosensitisers and red light.

In the present study, the in vitro susceptibilities of five Candida spp. to photodynamic antimicrobial chemotherapy (PACT) with four phenothiazinium derivatives, methylene blue (MB), new methylene blue N (NMBN), toluidine blue O (TBO) and the novel pentacyclic phenothiazinium photosensitiser S137, in combination with red light were investigated. The efficacy of each PS was determined, initially, based on its minimal inhibitory concentration (MIC). Additionally, we evaluated the effect of the photodynamic treatment with NMBN and S137 on Candida survival and on the mouse fibroblast cell line L929. MICs varied both among PS and species and decreased with light dose increase. For most treatments (species and fluences) NMBN and S137 showed the lowest MICs. MICs for NMBN and S137 were <2.5 µM for all the Candida species when a fluence of 25 J cm⁻² was used. PACT with NMBN (fluence of 15 J cm⁻²) resulted in reductions in survival from 0.3 log (Candida krusei) to 3 logs (C. parapsilosis). PACT with S137 was more effective than with NMBN. Fluence of 15 J cm⁻² resulted in reductions in survival from 1 log (C. krusei) to 3 logs (C. parapsilosis) and fluence of 25 J cm⁻² resulted in a reduction of approximately 2 logs (C. krusei) and between 3 and 4 logs in survival of the other 4 species of Candida. In vitro relative toxicities of the phenothiazinium PS to mammalian cells exhibited a similar trend to the antifungal data, i.e. greater toxicity and phototoxicity with NMBN and S137 compared to the established PS.

Susceptibilities of the dermatophytes Trichophyton mentagrophytes and T. rubrum microconidia to photodynamic antimicrobial chemotherapy with novel phenothiazinium photosensitizers and red light.

Photodynamic antimicrobial chemotherapy (PACT) is a promising alternative to conventional chemotherapy that can be used to treat localized mycosis. The development of PACT depends on identifying effective and selective PS for the different pathogenic species. The in vitro susceptibilities of Trichophyton mentagrophytes and Trichophyton rubrum microconidia to PACT with methylene blue (MB), toluidine blue O (TBO), new methylene blue N (NMBN), and the novel pentacyclic phenothiazinium photosensitizer S137 were investigated. The efficacy of each PS was determined based on its minimal inhibitory concentration (MIC). Additionally, we evaluated the effect of PACT with NMBN and S137 on the survival of the microconidia of both species. S137 showed the lowest MIC. MIC for S137 was 2.5 µM both for T. mentagrophytes and T. rubrum, when a light dose of 5 J cm(-2) was used. PACT with NMBN (10 µM and 20 J cm(-2)) resulted in a reduction of 4 logs in the survival of the T. rubrum and no survivor of T. mentagrophytes was observed. PACT with S137 at 1 µM and 20 J cm(-2) resulted in a reduction of approximately 3 logs in the survival of both species. When a S137 concentration of 10 µM was used, no survivor was observed for both species at all light doses (5, 10 and 20 J cm(-2)).

In vitro photodynamic inactivation of Cryptococcus neoformans melanized cells with chloroaluminum phthalocyanine nanoemulsion.

The selection of fungi resistant to currently used fungicides and the emergence of new pathogenic species make the development of alternative fungus-control techniques highly desirable. Photodynamic antimicrobial chemotherapy (PACT) is a promising method which combines a nontoxic photosensitizer (PS) with visible light to cause selective killing of microbial cells. The development of PACT to treat mycoses or kill fungi in the environment depends on identifying effective PS for the different pathogenic species and delivery systems able to expand and optimize their use. In the present study, the in vitro susceptibility of Cryptococcus neoformans melanized cells to the photodynamic effects of the PS agent ClAlPc in nanoemulsion (ClAlPc/NE) was examined. Cells were killed in a PS concentration- and light dose-dependent manner. Treatment with ClAlPc/NE, using PS concentrations (e.g. 4.5 µm) and light doses (e.g. 10 J cm(-2)) compatible with PACT, resulted in a reduction of up to 6 logs in survival. Washing the cells to remove unbound PS before light exposure did not inhibit fungal photodynamic inactivation. Internalization of ClAlPc by C. neoformans was confirmed by confocal fluorescence microscopy, and the degree of uptake was dependent on PS concentration.