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PdCYP51B, a new putative sterol 14α-demethylase gene of Penicillium digitatum involved in resistance to imazalil and other fungicides inhibiting ergosterol synthesis

  • Applied Genetics and Molecular Biotechnology
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Abstract

Penicillium digitatum, causing green mold decay, is the most destructive postharvest pathogen of citrus fruits worldwide. The phenotypes and genotypes of 403 isolates of P. digitatum, collected from packing houses and supermarkets in Zhejiang, China, during 2000 to 2010, were characterized in terms of their imazalil sensitivity. The frequency of detected imazalil-resistant (IMZ-R) isolates increased from 2.1% in 2000 to 60–84% during 2005–2010. Only 6.5% and 4.5% of the collected IMZ-R isolates belong to the previously described IMZ-R1 and IMZ-R2 genotypes, respectively. To determine the resistance mechanism of the predominant and novel IMZ-R isolates of P. digitatum (termed IMZ-R3), genes PdCYP51B and PdCYP51C, homologous to the sterol 14α-demethylase encoded gene PdCYP51, were cloned from six IMZ-R3 and eight imazalil-sensitive (IMZ-S) isolates of P. digitatum. A unique 199-bp insertion was observed in the promoter region of PdCYP51B in all IMZ-R3 isolates examined but in none of the tested IMZ-S isolates. Further analysis by PCR confirmed that this insertion was present in all IMZ-R3 isolates but absent in IMZ-S, IMZ-R1, and IMZ-R2 isolates. Transcription levels of PdCYP51B in three IMZ-R3 isolates were found to be 7.5- to 13.6-fold higher than that in two IMZ-S isolates of P. digitatum. Introduction of another copy of PdCYP51B s (from IMZ-S) into an IMZ-S isolate decreased the sensitivity of P. digitatum to 14α-demethylation inhibitors (DMIs) only to a small extent, but introduction of a copy of PdCYP51B R (from IMZ-R3) dramatically increased the resistance level of P. digitatum to DMIs. Regarding PdCYP51C, no consistent changes in either nucleotide sequence or expression level were correlated with imazalil resistance among IMZ-R and IMZ-S isolates. Based on these results, we concluded that (1) the CYP51 family of P. digitatum contains the PdCYP51B and PdCYP51C genes, in addition to the known gene PdCYP51A (previously PdCYP51); (2) PdCYP51B is involved in DMI fungicide resistance; and (3) overexpression of PdCYP51B resulting from a 199-bp insertion mutation in the promoter region of PdCYP51B is responsible for the IMZ-R3 type of DMI resistance in P. digitatum.

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Acknowledgments

Special thanks go to Dr. Xiaoguang Liu (School of Bioengineering, Tianjin University of Science and Technology, China) and Dr. Matthias Hahn (Department of Biology, University of Kaiserslautern, Germany) for their helpful suggestions and critical reviews of the manuscript. This work was supported by the National Foundation of Natural Science of China (30571236 & 31071649) and the earmarked fund for Modern Agro-industry Technology Research System (MATRS).

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Authors and Affiliations

  1. Institute of Biotechnology, Zhejiang University, Hangzhou, Zhejiang, 310029, China

    Xuepeng Sun, Jiye Wang, Dan Feng, Zhonghua Ma & Hongye Li

  2. Key Laboratory of Molecular Biology for Crop Pathogens and Insect Pests of Ministry of Agriculture, Zhejiang University, Hangzhou, Zhejiang, 310029, China

    Xuepeng Sun, Jiye Wang, Dan Feng, Zhonghua Ma & Hongye Li

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  1. Xuepeng Sun
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Correspondence to Hongye Li.

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Sun, X., Wang, J., Feng, D. et al. PdCYP51B, a new putative sterol 14α-demethylase gene of Penicillium digitatum involved in resistance to imazalil and other fungicides inhibiting ergosterol synthesis. Appl Microbiol Biotechnol 91, 1107–1119 (2011). https://doi.org/10.1007/s00253-011-3355-7

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