The role of melanin in the response of fungi to low-temperature plasma exposure

Conventional methods for plant disease control have often shown limitations in environmental impact, animal and human safety, as well as in economic costs and efficiency. Low-temperature plasma (LTP) is currently proposed as a new green technology for safe and sustainable food production raising considerable interest in recent years. Recent investigations on the inhibitory effect of plasma treatments against fungal plant pathogens revealed differences in survival rates of spores from different fungal species that require different treatment times to reach significant inhibition of spore germination, reduction of cell viability and morphological alterations of cell surface up to spore destruction. These differences could be related to the fungal cell wall thickness, structure and composition. In this work, the possible protective role of melanin against inactivation by plasma was investigated. Normally pigmented conidia of a wild-type strain of Botrytis cinerea and albino conidia from two UV-induced mutants were phenotypically and genetically characterized and compared for their in vitro responses to exposure to different plasma sources [Surface Dielectric Barrier Discharge (SDBD), Volume Dielectric Barrier Discharge (VDBD), and plasma-jet (Atmospheric-Pressure Plasma Jet, APPJ)]. B. cinerea mutants showed cream-coloured conidia and regularly pigmented sclerotia and carried a nonsense single-point mutation creating an in-frame stop codon in the bcpks13 gene, a key gene for conidial melanogenesis. Melanines from wild type pigmented conidia, based on their physio-chemical and spectral properties, were assigned to DHN melanins, and were absent or very little in the albino mutants. Mutants showed higher sensitivity to plasma treatments compared to the wild-type strain suggesting that melanin and other pigments could affect fungal response to plasma exposure. Albino conidia were significantly inhibited (> 81%) after only 1 min of SDBD exposure, while at least 10 min treatment were required for the wild-type one. All the strains were completely inhibited after 3 min of APPJ exposure, while significant differences among the strains were observed at shorter treatments times (5 sec). VDBD plasma caused the complete inhibition of conidia germination after just 10 sec or 30 sec of exposure in albino and wild-type strains, respectively.