Honokiol suppresses mycelial growth and reduces virulence of Botrytis cinerea by inducing autophagic activities and apoptosis
Introduction
Fruit provide numerous health benefits for mankind and thus are favored by consumers worldwide (Romanazzi and Feliziani, 2014; Tian et al., 2016). However, their quality deterioration and decay can lead to tremendous yield losses at the postharvest stage, becoming an urgent problem that must be tackled (Droby et al., 2016; FAO, 2011). Among the factors influencing postharvest decay, pathogenic fungi are above all the principal trouble, for example, Botrytis cinerea, a notorious pathogen, is the causal agent of gray mold on more than 1400 species of cultivated plants, ranked second only to Magnaporthe oryzae in scientific and economic importance among the top 10 fungal pathogens (Dean et al., 2012; Elad et al., 2016).
Given its exceptionally wide host range, cryptic infection and high genetic variability (Elad et al., 2016; Romanazzi and Feliziani, 2014), B. cinerea is among the most important fungal pathogens having a substantial impact on our daily life, and the global expenses for controlling gray mold now exceeds 1 billion Euros per year worldwide. Up till now, control measures based on synthetic fungicides have been and still are the most economical and effective under most circumstances, but public concern is growing over environmental pollution and food security after the application of synthetic fungicides at high doses. Hence, there is now great demand to find and test plant-derived alternatives with new cellular targets and to unravel their mechanisms of action (Droby et al., 2016; Li et al., 2018). Autophagy is required for several key physiological functions in filamentous fungi, such as asexual and sexual differentiation, regulations on pathogenicity, starvation responses and programmed cell death (Liu et al., 2012; Ren et al., 2017; Veneault-Fourrey et al., 2006). To date, few agents able to target autophagic activities have been documented, and no result has been reported yet for plant-derived substances that can induce autophagic activity for controlling gray mold.
In the present study, utilizing in vitro and in vivo assays for antifungal efficacy, we found that honokiol significantly suppressed the mycelial growth and pathogenicity of B. cinerea on harvested apple fruit and grapes by inducing autophagic activities and cell apoptosis. Moreover, honokiol also induced the cytosolic accumulation of reactive oxygen species (ROS) in this fungus, which further drove the loss of mitochondrial membrane potential and impairments to membrane integrity. These results may further enrich our understanding of antimicrobial mechanisms of plant-derived substances and provide alternatives for postharvest handling of fresh products in the food industry.
Section snippets
Chemicals
Honokiol (5,5′-diallyl-2,4′-dihydroxybiphenyl, Sigma-Aldrich Co., Ltd. [St. Louis, MO]) was prepared as a stock solution at 500 g/L and stored at 4 °C.
Strain used in this study
The B. cinerea strain B05.10 was activated on potato dextrose agar (PDA) as previously described (Ji et al., 2018). Conidia were filtered through sterile gauze and were counted under a microscope.
In vitro inhibition of mycelial growth
Honokiol was added to the PDA to generate a concentration gradient (0, 10, 50, 100 and 200 mg/L), for which 0.04% DMSO served as the control group. Agar
Honokiol inhibited mycelial growth of B. cinerea and impaired its pathogenicity on harvested fruit
As shown in Fig. 1, honokiol suppressed mycelial growth of B. cinerea on PDA in a concentration-dependent manner. After incubation for 72 h, the colony diameter for the treatment with 10 mg/L of honokiol was about 60% that of the control group, whereas mycelial growth was completely arrested when the honokiol concentration was increased to 200 mg/L. Similarly, conidial germination and germ tube elongation were both suppressed showing similar patterns, in the presence of honokiol (Fig. S1).
Discussion
B. cinerea ranks second among the top 10 fungal pathogens in terms of scientific value and economic importance, and it has become a model strain for investigating pathogen–host interactions (Dean et al., 2012; Spanu et al., 2012). Over the past several decades, due to the high variability of B. cinerea genome and the greater dependence on commercial fungicide applications, the practical situation in controlling gray mold has gradually become aggravated, and fungicide-resistant strains have even
Conclusions
Honokiol triggered multifaceted responses in B. cinerea by simultaneously activating autophagic activities, inducing ROS over-production and disrupting mitochondrial functioning, eventually giving rise to the loss of membrane integrity and abolishment of cell viability. The elucidation of this antimicrobial efficacy provided by honokiol and its possible mechanisms may further enrich the toolbox for plant-derived fungicidal substances, which is promising for exploring alternatives with new
Acknowledgements
The authors sincerely thank Mrs. Fengqin Dong and Dr. Suhua Yang for their help in transmission electron microscopy and flow cytometry analysis. This work was supported by the projects from National Key Research and Development Program of China (2017YFD0401301), National Natural Science Foundation of China (31930086, 30672210).
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