In vitro and in planta antimicrobial photodynamic treatment of Colletotrichum acutatum with phenothiazinium photosensitizers

Author(s)

Júlia Cunha Gonzales

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2016-06-14

Examining board members

Gilberto Ubida Leite Braga; Nilce Maria Martinez Rossi; Geraldo José da Silva Junior

• Advisor: Gilberto Ubida Leite Braga

Abstract

The increasing tolerance to conventional fungicides observed in several genera of pathogenic fungi and the growing concern about the environment have stimulated the development of new alternatives to control of plant pathogens. Among the new antimicrobial strategies that have been evaluated, antimicrobial photodynamic treatment (APDT) is one of the most promising. APDT is based on the use of a photosensitizer (PS) that selectively accumulates in the target microbial cells. Activation of the PS by light in the presence of molecular oxygen induces the formation of reactive oxygen species, such as singlet oxygen, which cause damages to the target microorganism without significant harm to the host. Although very promising, APDT has not yet been used to control plant-pathogenic fungi. In the present study, the effectiveness of the APDT with the photosensitizer methylene blue (MB) and solar radiation on conidia of the plant-pathogenic fungus Colletotricum acutatum was evaluated in planta. The experiments were conducted with MB from different brands, in two concentrations (25 and 50 ?M) and different formulations, throughout the different seasons of the year, on cloudy and sunny days and at different temperatures. APDT with MB and solar radiation was extremely effective, killing around 100% of conidia in all conditions evaluated. The effects of the MB on the host plant Citrus sinensis were also evaluated. The PS was applied repeatedly on the leaves and flowers of the plants. No damage was observed on the leaves or the petals after treatment with MB. For the first time the mechanistic study evaluating the effects of APDT on C. acutatum conidia was carried out. The effects of APDT on the permeability of the conidial plasma membrane and on the production of secondary reactive oxygen species were evaluated by using the fluorescent probes propidium iodide (PI) and dihydroethidium (DHE), respectively. APDT with MB damaged the plasma membrane, reducing its selective permeability and induced the formation of secondary reactive oxygen species in conidia. It was also conducted a long term study which assessed the tolerance of conidia of C. acutatum to APDT, after each of 45 successive cycles of APDT with new methylene blue followed by the selection of the survivors. A gradual increase in APDT tolerance was observed from the 21st cycle. Besides the increase in tolerance to APDT, it was also observed an increase in the pigmentation of conidia probably due to the selection of mutants that accumulate more carotenoids. (AU)

• FAPESP's process: 14/04162-8 - Mechanistic study of the photodynamic inactivation of pathogenic fungi with novel phenothiazinium photosensitizer
• Grantee: Júlia Cunha Gonzales
• Support Opportunities: Scholarships in Brazil - Master