Applied Microbiology and Biotechnology

, Volume 102, Issue 21, pp 9221–9230 | Cite as

Antioxidant activities of four superoxide dismutases in Metarhizium robertsii and their contributions to pest control potential

  • Xiao-Guan Zhu
  • Sen-Miao TongEmail author
  • Sheng-Hua Ying
  • Ming-Guang FengEmail author
Biotechnologically relevant enzymes and proteins


The superoxide dismutase (SOD) family of Metarhizium robertsii, a fungal insect pathogen, comprises six members functionally unknown yet, including Cu/ZnSODs (Sod1/5/6), MnSODs (Sod2/3), and FeSOD (Sod4). Here, we show a mitochondrial localization of Sod3 and Sod4 and a requirement of either sod4 or sod6 for the fungal life as suggested by an inability to be deleted. We found remarked roles of Sod1, Sod2, and Sod3 in sustaining antioxidant activity and the fungal potential against insect pests but no role of Sod5 in all examined phenotypes. Intracellular SOD activity decreased by 49% in Δsod1 and 22% in either Δsod2 or Δsod3. The decreased SOD activities concurred with altered enzymographs, in which one of two SOD-active bands in wild-type and rescued strains disappeared in Δsod1 rather than in Δsod2 and another band disappeared in Δsod3. Consequently, maximal cell sensitivity to superoxide anions generated by oxidant menadione occurred in Δsod1, followed sequentially by Δsod3 and Δsod2. The latter two mutants were more sensitive than Δsod1 to oxidant H2O2. Transcriptional analysis revealed partial compensation of one or two partner genes upregulated for the absence of sod1, sod2, or sod3 and full compensation of three partners largely upregulated for the absence of sod5, as well as differential expression of most catalase genes in each Δsod mutant. The three mutants also suffered defects in conidial thermotolerance, UVB resistance, and virulence. These findings unveil that, to adapt to different host spectra and habitats, some major SODs in M. roberstii are functionally differentiated from those known previously in Beauveria bassiana, a classic insect mycopathogen lacking Sod6.


Entomopathogenic fungi Antioxidant enzymes SOD activity Biological control potential 


Funding information

This work was financially supported by the Ministry of Science and Technology of the People’s Republic of China (Grant No.: 2017YFD0201202), the National Natural Science Foundation of China (Grant No.: 31772218), and the Fundamental Research Funds for the Central Universities (Grant No.: 2018FZA6003).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

This article does not contain any studies with human participants or animals other than Galleria mellonella moth larvae performed by any of the authors.

Supplementary material

253_2018_9302_MOESM1_ESM.pdf (1.7 mb)
ESM 1 (PDF 1744 kb)


  1. Abba S, Khouja HR, Martino E, Archer DB, Perotto S (2009) SOD1-targeted gene disruption in the ericoid mycorrhizal fungus Oidiodendron maius reduces conidiation and the capacity for mycorrhization. Mol Plant-Microbe Interact 22:1412–1421CrossRefGoogle Scholar
  2. Aguirre J, Ríos-Momberg M, Hewitt D, Hansberg W (2005) Reactive oxygen species and development in microbial eukaryotes. Trends Microbiol 13:111–118CrossRefGoogle Scholar
  3. Bai Z, Harvey LM, McNeil B (2003) Elevated temperature effects on the oxidant/antioxidant balance in submerged batch cultures of the filamentous fungus Aspergillus niger B1-D. Biotechnol Bioeng 83:772–779CrossRefGoogle Scholar
  4. Bischoff JF, Rehner SA, Humber RA (2009) A multilocus phylogeny of the Metarhizium anisopliae lineage. Mycologia 101:512–530CrossRefGoogle Scholar
  5. Borders CJ, Saunders JE, Blech DM, Fridovich I (1985) Essentiality of the active–site arginine residue for the normal catalytic activity of Cu/Zn superoxide dismutase. J Biol Chem 230:771–776Google Scholar
  6. Bordo D, Djinovic K, Bolognesi M (1994) Conserved patterns in the Cu/Zn superoxide dismutase family. J Mol Biol 238:366–386CrossRefGoogle Scholar
  7. Culotta VC, Yang M, O’Halloran TV (2006) Activation of superoxide dismutases: putting the metal to the pedal. Biochim Biophys Acta Mol Cell Res 1763:747–758CrossRefGoogle Scholar
  8. de Faria MR, Wraight SP (2007) Mycoinsecticides and mycoacaricides: a comprehensive list with worldwide coverage and international classification of formulation types. Biol Control 3:237–256CrossRefGoogle Scholar
  9. Fang GC, Hanau RM, Vaillancourt LJ (2002) The SOD2 gene, encoding a manganese-type superoxide dismutase, is upregulated during conidiogenesis in the plant-pathogenic fungus Colletotrichum graminicola. Fungal Genet Biol 36:155–165CrossRefGoogle Scholar
  10. Gao Q, Jin K, Ying SH, Zhang YJ, Xiao GH, Shang YF, Duan ZB, Hu X, Xie XQ, Zhou G, Peng GX, Luo ZB, Huang W, Wang B, Fang WG, Wang SB, Zhong Y, Ma LJ, St Leger RJ, Zhao GP, Pei Y, Feng MG, Xia YX, Wang CS (2011) Genome sequencing and comparative transcriptomics of the model entomopathogenic fungi Metarhizium anisopliae and M. acridum. PLoS Genet 7:e1001264CrossRefGoogle Scholar
  11. Huang BF, Feng MG (2009) Comparative tolerances of various Beauveria bassiana isolates to UV-B irradiation with a description of a modeling method to assess lethal dose. Mycopathologia 168:145–152CrossRefGoogle Scholar
  12. Hwang CS, Rhie GE, Oh JH, Huh WK, Yim HS (2002) Copper- and zinc-containing superoxide dismutase (Cu/ZnSOD) is required for the protection of Candida albicans against oxidative stresses and the expression of its full virulence. Microbiology-SGM 148:3705–3713CrossRefGoogle Scholar
  13. Hwang CS, Baek YU, Yim HS, Kang SO (2003) Protective roles of mitochondrial manganese–containing superoxide dismutase against various stresses in Candida albicans. Yeast 20:929–941CrossRefGoogle Scholar
  14. Kim JY, Na BK, Song KJ, Park MH, Park YK, Kim TS (2012) Functional expression and characterization of an iron-containing superoxide dismutase of Acanthamoeba castellanii. Parasitol Res 111:1673–1682CrossRefGoogle Scholar
  15. Lambou K, Lamarre C, Beau R, Dufour N, Latgé JP (2010) Functional analysis of the superoxide dismutase family in Aspergillus fumigatus. Mol Microbiol 75:910–923CrossRefGoogle Scholar
  16. Li J, Feng MG (2009) Intraspecific tolerance of Metarhizium anisopliae conidia to the upper thermal limits of summer with a description of a quantitative assay system. Mycol Res 113:93–99CrossRefGoogle Scholar
  17. Li F, Shi HQ, Ying SH, Feng MG (2015) Distinct contributions of one Fe- and two Cu/Zn-cofactored superoxide dismutases to antioxidation, UV tolerance and virulence of Beauveria bassiana. Fungal Genet Biol 81:160–171CrossRefGoogle Scholar
  18. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2–∆∆Ct method. Methods 25:402–408CrossRefGoogle Scholar
  19. Mari M, Morales A, Colell A, Garcia-Ruiz C, Fernandez-Checa JC (2009) Mitochondrial glutathione, a key survival antioxidant. Antioxid Redox Signal 11:2685–2700CrossRefGoogle Scholar
  20. Martchenko M, Alarco AM, Harcus D, Whiteway M (2004) Superoxide dismutases in Candida albicans: transcriptional regulation and functional characterization of the hyphal–induced SOD5 gene. Mol Cell Biol 15:456–467CrossRefGoogle Scholar
  21. Michielse CB, Hooykaas PJJ, van den Hondel CAMJJ, Ram AFJ (2008) Agrobacterium mediated transformation of the filamentous fungus Aspergillus awamori. Nat Protoc 3:1671–1678CrossRefGoogle Scholar
  22. Moore S, De Vries OMH, Tudzynski P (2002) The major Cu/ZnSOD of the phytopathogen Claviceps purpurea is not essential for pathogenicity. Mol Plant Pathol 3:9–22CrossRefGoogle Scholar
  23. Muniz-Paredes F, Miranda-Hernandez F, Loera O (2017) Production of conidia by entomopathogenic fungi: from inoculants to final quality tests. World J Microbiol Biotechnol 33:57CrossRefGoogle Scholar
  24. Narasipura SD, Chaturvedi V, Chaturvedi S (2005) Characterization of Cryptococcus neoformans variety gattii SOD2 reveals distinct roles of the two superoxide dismutases in fungal biology and virulence. Mol Microbiol 55:1782–1800CrossRefGoogle Scholar
  25. Roberts DW, St Leger RJ (2004) Metarhizium spp., cosmopolitan insect pathogenic fungi: mycological aspects. Adv Appl Microbiol 54:1–70CrossRefGoogle Scholar
  26. Schouten A, Tenberge KB, Vermeer J, Stewart J, Wagemakers L, Williamson B, van Kan JAL (2002) Functional analysis of an extracellular catalase of Botrytis cinerea. Mol Plant Pathol 3:227–238CrossRefGoogle Scholar
  27. Slade D, Radman M (2011) Oxidative stress resistance in Deinococcus radiodurans. Microbiol Mol Biol Rev 75:133–191CrossRefGoogle Scholar
  28. Wang CS, Feng MG (2014) Advances in fundamental and applied studies in China of fungal biocontrol agents for use against arthropod pests. Biol Control 68:128–135Google Scholar
  29. Wang ZL, Zhang LB, Ying SH, Feng MG (2013) Catalases play differentiated roles in the adaptation of a fungal entomopathogen to environmental stresses. Environ Microbiol 15:409–418CrossRefGoogle Scholar
  30. Xie XQ, Wang J, Huang BF, Ying SH, Feng MG (2010) A new manganese superoxide dismutase identified from Beauveria bassiana enhances virulences and stress tolerance when overexpressed in the fungal pathogen. Appl Microbiol Biotechnol 86:1543–1553CrossRefGoogle Scholar
  31. Xie XQ, Li F, Ying SH, Feng MG (2012) Additive contributions of two manganese-cored superoxide dismutases (MnSODs) to antioxidation, UV tolerance and virulence of Beauveria bassiana. PLoS One 7:e30298CrossRefGoogle Scholar
  32. Zhang LB, Feng MG (2018) Antioxidant enzymes and their contributions to biological control potential of fungal insect pathogens. Appl Microbiol Biotechnol 102:4995–5004CrossRefGoogle Scholar
  33. Zhang LB, Tang L, Ying SH, Feng MG (2016) Regulative roles of glutathione reductase and four glutaredoxins in glutathione redox, antioxidant activity and iron homeostasis of Beauveria bassiana. Appl Microbiol Biotechnol 100:5907–5917CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Institute of Microbiology, College of Life SciencesZhejiang UniversityHangzhouChina
  2. 2.College of Agricultural and Food ScienceZhejiang A&F UniversityLin’anChina

Personalised recommendations