The efficacy of essential oils and Bacillus spp. against Lecanicillium fungicola, the causal agent of dry bubble disease of the button mushroom

Abstract

The white button mushroom (Agaricus bisporus) stands as one of the most consumed mushrooms in the world. In addition to being a food with nutritional value, mushroom production makes it possible to recycle agricultural and agro-industrial waste. The button mushroom cultivation can suffer injuries from insects, bacteria, viruses and fungi. However, the most relevant disease in this culture is the dry bubble disease caused by the fungus Lecanicillium fungicola, which causes necrotic lesions to total deformity of the mushrooms and results in significant losses of production. The control of pests in mushroom cultivation is done through the use of pesticides, however in Brazil there is no legislation in force for this practice or a registered and safe commercial product. Therefore, natural alternatives that do not cause environmental and health damage have been sought. Essential oils are natural products, extracted from aromatic plants and with known antimicrobial properties. Within this context, the aim of this work was to evaluate the antifungal properties of the essential oils of Cymbopogon winterianus (citronella), Eugenia caryophyllus (clove leaf) and Litsea cubeba (Chinese pepper) in vitro and in vivo for the control of L. fungicola strains. var. fungicola, as well as their synergistic effect. The effect of EO on the morphology of L. fungicola was studied by scanning electron microscopy (SEM). EO were characterized by the presence of citronellal (35.57%), eugenol (88.90%) and geranial (42.25%), respectively. All EO showed 100% inhibition on the mycelial growth of L. fungicola at concentrations of 1% and 0.1%. Litsea cubeba showed minimum inhibitory concentration (MIC) at lower concentrations (≥ 0.0156%). Based on the results of the minimum fungicidal concentrations (CFM ≥ 0.0312%), the inhibitory effect of Litsea cubeba EO was more efficient. A synergistic effect was observed from the combination of the L. Cubeba EO with C. winterianus and E. caryophyllus with L. cubeba at a much lower concentration. The SEM observation revealed an aspect of roughness of hyphae and spores in samples treated with EO. In the in vivo test, EO were efficient in controlling the incidence of dry bubble disease without impairing the production of A. bisporus. Thus, the synergistic antimicrobial effect and effectiveness observed in this study open new perspectives on the development of a new natural and potent antifungal mixture against L. fungicola in the mushroom industry. For the antagonism test, two strains of bacteria isolated from mushrooms, Bacillus sp. (M17) and Bacillus sp. (M18), were evaluated by four different methods on four different strains of L. fungicola. the results showed that the two bacterial strains exert antagonistic activity and inhibit the growth of the fungus. In addition to preparing the spore suspension, surfactants are used in the solubilization or preparation of essential oil emulsions tested in the control of L. fungicola. However, as these products may also have an inhibitory effect, DMSO (dimethylsulfoxide), Tween 80 and Triton X-100 were also evaluated at the concentrations of 0.5 and 1% on the mycelial growth of L. fungicola and A. bisporus. For DMSO, no visual changes were observed in the colonies for both species. Tween 80 affected mycelial growth, causing colonies to grow less when compared to control, in addition to changing the appearance of the colony for both species. Triton X-100 showed an even greater inhibitory effect on the growth of the two fungal species, considerably affecting both the size and the morphology of the colony. Scanning electron microscopy analyzes showed that surfactants alter the structures and aspects of the hyphae of both fungi and in the case of L. fungicola they also affect the quantity and aspect of the spores.