An antifungal role of hydrogen sulfide on the postharvest pathogens Aspergillus niger and Penicillium italicum

Abstract

In this research, the antifungal role of hydrogen sulfide (H2S) on the postharvest pathogens Aspergillus niger and Penicillium italicum growing on fruits and under culture conditions on defined media was investigated. Our results show that H2S, released by sodium hydrosulfide (NaHS) effectively reduced the postharvest decay of fruits induced by A. niger and P. italicum. Furthermore, H2S inhibited spore germination, germ tube elongation, mycelial growth, and produced abnormal mycelial contractions when the fungi were grown on defined media in Petri plates. Further studies showed that H2S could cause an increase in intracellular reactive oxygen species (ROS) in A. niger. In accordance with this observation we show that enzyme activities and the expression of superoxide dismutase (SOD) and catalase (CAT) genes in A. niger treated with H2S were lower than those in control. Moreover, H2S also significantly inhibited the growth of Saccharomyces cerevisiae, Rhizopus oryzae, the human pathogen Candida albicans, and several food-borne bacteria. We also found that short time exposure of H2S showed a microbicidal role rather than just inhibiting the growth of microbes. Taken together, this study suggests the potential value of H2S in reducing postharvest loss and food spoilage caused by microbe propagation.

Figure 1. Effect of H₂S on A. niger and P. italicum growth in inoculated fruits and on defined media.

A, photographs of six kinds of postharvest fruits inoculated with A. niger and P. italicum when fumigated with 0.0 mM (control) or 0.5 mM NaHS solution for 9 d at 25°C; B, photographs of A. niger and P. italicum grown on Petri plates on defined media after fumigation with 0.00, 0.01, 0.05, 0.10, 0.50 and 2.50 mM NaHS solutions for 2 d at 25°C; C, photographs of A. niger and P. italicum after 1 d of 2.5 mM NaHS treatment and recovered in water fumigation for another 8 d at 25°C.

Figure 2. Effect of H₂S fumigation on mycelial growth and micro-morphology of A. niger and P. italicum grown on defined media.

Colonies were cultured for 3°C, and then exposed to H₂S released from 0.5 mM and 2.5 mM NaHS for 1 d. A and B, mycelial growth and micro-morphology of A. niger; C and D, mycelial growth and micro-morphology of P. italicum.

Figure 3. Effect of H₂S fumigation on ROS level in A. niger.

A. niger cells in Figure 2 were used for ROS detection. A, ROS staining in sporangia and sporangiophores; B, ROS staining in A. niger spores. Left parts of A and B shows the bright field images, and right parts fluorescence images.

Figure 4. Effect of H₂S fumigation on enzyme activities and gene expression of SOD and CAT in A. niger.

Colonies were cultured for 3°C, and then exposed to H₂S released from 0.5 mM and 2.5 mM NaHS for 1 d. A, SOD activity in A. niger; B, CAT activity in A. niger; C, RT-PCR results to analyze gene expressions of SOD and CAT in A. niger. D, relative expression levels by quantifying RT-PCR results in C (with the actin-encoding gene value [ANI_1_106134] set as 1.0) with Image J software. Different letters mean significance of difference between the treatments (P<0.01, ANOVA, LSD).

Figure 5. Effect of H₂S fumigation on grown of baker's yeast, C. albicans, R. oryzae, and some food-borne bacteria growth on defined media.

A–F, photographs of S. cerevisiae, S. aureus, S. typhimurium, L. monocytogenes, B. subtilis and B. thuringiensis after 3 d exposure to H₂S released from different concentrations of NaHS at 25°C; G–H, photographs of E. coli and E. aerogenes after exposure to H₂S for 1 d; I–J, photographs of C. albicans and R. oryzae after exposure to H₂S for 4 d and 7 d, respectively.

Figure 6. Effect of short term H₂S exposure to baker's yeast, C. albicans, R. oryzae, and some food-borne bacteria grown on defined media.

After 1°C, the photographs were taken. A–H show photographs of S. cerevisiae, S. aureus, S. typhimurium, L. monocytogenes, B. subtilis, B. thuringiensis, E. coli and E. aerogenes, respectively; I–J show photographs of C. albicans and R. oryzae recovered in water atmosphere for 2 and 4 d after 2.5 mM NaHS treatment for 1 d.