Assessment of pulsed light induced damage on Botrytis cinerea conidia by flow cytometry and transmission electron microscopy

ROMERO-BERNAL, ANGELA; ALZAMORA, STELLA M.; GÓMEZ, PAULA L.; GONZALEZ, HÉCTOR L.; RAFFELLINI, SILVIA

Chicago

Congreso; Institute of Food Technology Annual Meeting -IFT 2018; 2018

Institute of Food Technology (IFT)

Botrytis cinerea causes post-harvest spoilage in important crops such as strawberry and other berries. The application of chemical fungicides for its control may induce resistance and causes environmental pollution. Pulsed light (PL) treatment of berries is being studied as an environmental friendly alternative to synthetic fungicides.This work was aimed to analyze the injury induced by PL on Botrytis cinerea conidia using flow cytometry analysis (FCM) and transmission electron microscopy (TEM). Conidia suspensions of Botrytis cinereaBAFC 3003in peptone water (0.1 % w/v) + Tween 80 (0.05 % v/v) (105 conidia/mL) were treated in a Xenon RS-3000B Steripulse-XL equipment, which generated high intensity pulsed light of polychromatic radiation (200 to 1100 nm; 3 pulses/s, 360 μs pulse width).Treatments were performed by duplicate in Petri dishes (5 cm diameter) containing 3 mL of conidia suspension and located at 10 cm from the lamp.Samples were exposed to irradiation for 5 to 40 s (fluence: 6.0 - 47.8 J/cm2; temperature build-up < 35 ºC). For FCM analysis, PL-treated and untreated cells were labeled with fluorescein diacetate (FDA) for detecting esterase activity, and with propidium iodide (PI) for monitoring membrane integrity. Samples for TEM observations were prepared according to conventional techniques. FCM revealed that 22 to 70 % of conidia population irradiated during10 to 40 swere stained with PI respectively, indicating rupture of plasmalemma. Increase of a subpopulation of viable cells which maintained metabolic activity but were non-culturable due to membrane rupture was also detected. Such injured cells might be critical in terms of their potential spoilage activity.TEM showed that PL attacked multiple targets in B. cinerea; however, ultrastructural changes varied within the conidia population and supported FCM results. Damage was dose-dependent and included plasmalemma dissociation from the cell wall, cytoplasm collapse, disruption of cell wall and plasmalemma with massive loss of cytoplasm and/or disruption and degeneration of various organelles. Results showed that PL induced cellular damage in B. cinereaand could be an alternative strategy in combination with other preservation factors to control this fungus in the post-harvest.