Deletion of Cu/Zn Superoxide Dismutase Gene sodC Reduces Aspergillus niger Virulence on Chinese White Pear

in Journal of the American Society for Horticultural Science

Aspergillus niger is a common pathogenic fungus causing postharvest rot of fruit and vegetable, whereas the knowledge on virulence factors is very limited. Superoxide dismutase [SOD (EC 1.15.1.10)] is an important metal enzyme in fungal defense against oxidative damage. Thus, we try to study whether Cu/Zn-SOD is a virulence factor in A. niger. Cu/Zn-SOD encoding gene sodC was deleted in A. niger [MA70.15 (wild type)] by homologous recombination. The deletion of sodC led to decreased SOD activity in A. niger, suggesting that sodC did contribute to full enzyme activity. ΔsodC strain showed normal mycelia growth and sporulation compared with wild type. However, sodC deletion markedly increased the cell’s sensitivity to intracellular superoxide anion generator menadione. Besides, spore germination under menadione and H2O2 stresses were significantly retarded in ΔsodC mutant compared with wild type. Further results showed that sodC deletion induced higher superoxide anion production and higher content of H2O2 and malondialdehyde (MDA) compared with wild type, supporting the role of SOD in metabolism of reactive oxygen species (ROS). Furthermore, ΔsodC mutant had a reduced virulence on chinese white pear (Pyrus bretschneideri) as lesion development by ΔsodC was significantly less than wild type. The determination of superoxide anion, H2O2, and MDA in A. niger-infected pear showed that chinese white pear infected with ΔsodC accumulated less superoxide anion, H2O2, and MDA compared with that of wild type A. niger, implying that ΔsodC induced an attenuated response in chinese white pear during fruit–pathogen interaction. Our results indicate that sodC gene contributes to the full virulence of A. niger during infection on fruit. Aspergillus niger is one of the most common species found in fungal communities. It is an important fermentation industrial strain and is also known to cause the most severe symptoms in fruit during long-term storage (Pel et al., 2007). Meanwhile, plants activate their signaling pathways to trigger defense responses to limit pathogen expansion. One of the earliest host responses after pathogen attack is oxidative burst, during which large quantities of ROS are generated by different host enzyme systems, such as glucose oxidase (Govrin and Levine, 2000). ROS such as singlet oxygen, superoxide anion, hydroxyl (OH), and H2O2 are released to hinder the advance of pathogens (Gara et al., 2003). ROS can react with and damage cellular molecules, such as DNA, protein, and lipids, which will limit fungal propagation in the host plant (Apel and Hirt, 2004).

Contributor Notes

Funding for this work was provided by the National Natural Science Foundation of China (31670278, 31470013, 31300133), the earmarked fund for China Agriculture Research System CARS-10-B1, Anhui Provincial Science and Technology Major Project (6030701073) and Anhui Provincial Education Department (KJ2015ZD12).

These authors contributed equally to this work.

Corresponding author. E-mail: hzhanglab@hfut.edu.cn.

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    The identification of the deletion mutant ΔsodC and the assays of sodC gene expression and superoxide dismutases (SOD) activity in ΔsodC mutant. (A) Confirmation of sodC deletion mutant by polymerase chain reaction (PCR) analysis of genomic DNA from wild type Aspergillus niger MA 70.15 and ΔsodC. Lane 1 and 2, genomic PCR of A. niger MA 70.15 and ΔsodC with external primers respectively; lane 3 and 4, genomic PCR of A. niger MA 70.15, and ΔsodC with internal primers, respectively. (B) The expression of sodC gene in wild type A. niger MA 70.15 (WT, wild type) and ΔsodC. (C) The activity assay of SOD in wild type A. niger MA 70.15 (WT) and ΔsodC mutant. Values are mean ± sd (n = 3). The symbols * and ** in this figure and following ones stand for a significant difference between two data at P < 0.05 and P < 0.01, respectively.

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    Growth analysis of ΔsodC mutant on potato dextrose agar medium supplemented with uridine (PDA-Uri). (A) The mycelium growth of the wild type Aspergillus niger MA 70.15 (WT) and ΔsodC in the PDA-Uri solid medium for 96 h. (B) The colony diameter of wild type A. niger MA 70.15 (WT) and the ΔsodC in A. Values are mean ± sd (n = 9). (C) The biomass of the wild type A. niger MA 70.15 (WT) and the ΔsodC grown in PDA-Uri liquid culture medium for 96 h. Values are mean ± sd (n = 3).

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    The effect of menadione on the growth of the wild type Aspergillus niger MA 70.15 (WT) and ΔsodC mutant. (A) The mycelium growth of the wild type A. niger MA 70.15 (WT) and ΔsodC in potato dextrose agar medium supplemented with uridine (PDA-Uri) and PDA-Uri medium supplemented with 0.02 mm menadione for 4 d. (B) The colony diameter of wild type A. niger MA 70.15 (WT) and ΔsodC in A. Values are mean ± sd (n = 9).

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    The effect of menadione and H2O2 on the spore germination of ΔsodC. (A) The spore germination of the wild type Aspergillus niger MA 70.15 (WT) and ΔsodC mutant in potato dextrose agar medium supplemented with uridine (PDA-Uri) solid medium (16 h), PDA-Uri solid medium supplemented with 2 mm H2O2 (24 h), and supplemented with 0.02 mm menadione (30 h); bar = 100 μm. (B) Spore germination percentage of the wild type A. niger MA 70.15 (WT) and ΔsodC in A. Values are mean ± sd (n = 3).

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    The effect of menadione on the superoxide anion production (A) and content H2O2 (B) and malondialdehyde (MDA) (C) in Aspergillus niger MA 70.15 (WT) and ΔsodC were subjected to 0.02 mm menadione stress and sampled at 12 and 24 h in potato dextrose agar medium supplemented with uridine (PDA-Uri) liquid medium. Values are mean ± sd (n = 3).

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    Pathogenicity of ΔsodC mutant on Pyrus bretschneideri. (A) Photographs of P. bretschneideri fruit inoculated with the spores of wild type Aspergillus niger MA 70.15 (WT) and the ΔsodC for 5 d. (B) Lesion diameter caused by wild type A. niger MA 70.15 (WT) and ΔsodC on P. bretschneideri fruit as shown in A. Values are mean ± sd (n = 15).

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    Superoxide anion production (A) and content of H2O2 (B) and malondialdehyde (MDA) (C) in Pyrus bretschneideri inoculated with wild type Aspergillus niger MA 70.15 (WT) and ΔsodC. P. bretschneideri was inoculated with A. niger MA 70.15 (WT) and ΔsodC and were sampled immediately (Control) or on day 3 postinoculation. Values are mean ± sd (n = 3).

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