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Molecular characterization and a multiplex allele-specific PCR method for detection of thiabendazole resistance in Penicillium expansum from apple

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Abstract

Thiabendazole (TBZ) is commonly used as a postharvest treatment for control of blue mold in apples caused by Penicillium expansum. Different point mutations in the β-tubulin gene conferring benzimidazole resistance have been reported in plant pathogens, but molecular mechanisms of TBZ resistance in P. expansum from apple in Washington State are unknown. Determination of TBZ resistance level showed that all 102 TBZ-resistant (TBZ-R) isolates were highly resistant. Sequencing of the majority of the β-tubulin gene showed that 76 TBZ-R isolates harboured the E198V mutation and 26 harboured the F167Y mutation, and all the sensitive isolates did not possess any of the mutations, indicating that these two point mutations in the β-tubulin gene were correlated with TBZ resistance in P. expansum from apple in Washington State. There was no association between levels of TBZ resistance and types of point mutations (E198V or F167Y) in the β-tubulin gene. A multiplex allele-specific PCR assay was developed to detect these two mutations simultaneously. Microsatellite-primed PCR derived presence-absence matrix used to assess the genetic relationship among 56 isolates suggested that the resistance mutations originated several times independently and that there was no correlation between the types of point mutation and the genetic background of the isolates.

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References

  • Avenot, H. F., Sellam, A., Karaoglanidis, G., & Michailides, T. J. (2008). Characterization of mutations in the iron-sulphur subunit of succinate dehydrogenase correlating with boscalid resistance in Alternaria alternata from California Pistachio. Phytopathology, 98, 736–742.

    Article  PubMed  CAS  Google Scholar 

  • Baraldi, E., Mari, M., Chierici, E., Pondrelli, M., Bertolini, P., & Pratella, G. C. (2003). Studies on thiabendazole resistance of Penicillium expansum of pears: pathogenic fitness and genetic characterization. Plant Pathology, 52, 362–370.

    Article  CAS  Google Scholar 

  • Boehm, E. W. A., Freeman, S., Shabi, E., & Michailides, T. J. (2003). Microsatellite primers indicate the presence of asexual populations of Venturia inaequalis in coastal Israeli apple orchards. Phytoparasitica, 31, 236–251.

    Article  CAS  Google Scholar 

  • Cabañas, R., Castellá, G., Abarca, M. L., Bragulat, M. R., & Cabañes, F. J. (2009). Thiabendazole resistance and mutations in the β-tubulin gene of Penicillium expansum strains isolated from apples and pears with blue mold decay. FEMS Microbiology Letters, 297, 189–195.

    Article  PubMed  Google Scholar 

  • Chen, C. J., Yu, J. J., Bi, C. W., Zhang, Y. N., Xu, J. Q., Wang, J. X., et al. (2009). Mutations in a β-tubulin confer resistance of Gibberella zeae to benzimidazole fungicides. Phytopathology, 99, 1403–1411.

    Article  PubMed  CAS  Google Scholar 

  • Chen, C. J., Zhao, W., Lu, Y., Wang, J., Chen, Y., Li, H., et al. (2009). High-throughput detection of highly benzimidazole-resistant allele E198A with mismatch primers in allele-specific real-time polymerase chain reaction. Pest Management Science, 65, 413–419.

    Article  PubMed  CAS  Google Scholar 

  • Drenkard, E., Richter, B. G., Rozen, S., Stutius, L. M., Angell, N. A., Mindrinos, M., et al. (2004). A simple procedure for the analysis of single nucleotide polymorphisms facilitates map-based cloning in Arabidopsis. Plant Physiology, 124, 1483–1492.

    Article  Google Scholar 

  • Eckert, J. W., & Ogawa, J. M. (1988). The chemical control of postharvest diseases: deciduous fruits, berries, vegetables and root/tuber crops. Annual Review of Phytopathology, 26, 433–469.

    Article  CAS  Google Scholar 

  • Errampalli, D., Brubacher, N. R., & DeEll, J. R. (2006). Sensitivity of Penicillium expansum to diphenylamine and thiabendazole and postharvest control of blue mold with fludioxonil in ‘McIntosh’ apples. Postharvest Biology and Technology, 39, 101–107.

    Article  CAS  Google Scholar 

  • Fraaije, B. A., Cools, H. J., Fountaine, J., Lovell, D. J., Motteram, J., West, J. S., et al. (2005). Role of ascospores in further spread of QoI-resistant cytochrome b alleles (G143A) in field populations of Mycosphaerella graminicola. Phytopathology, 95, 933–941.

    Article  PubMed  CAS  Google Scholar 

  • Gafur, A., Tanaka, C., Shimizu, K., Ouchi, S., & Tsuda, M. (1998). Molecular analysis and characterization of the Cochliobolus heterostrophus beta-tubulin gene and its possible role in conferring resistance to benomyl. The Journal of General and Applied Microbiology, 44, 217–223.

    Article  PubMed  CAS  Google Scholar 

  • Hayashi, K., Hashimoto, N., Daigen, M., & Ashikawa, I. (2004). Development of PCR-based SNP markers for rice blast resistance genes at the Piz locus. Theoretical and Applied Genetics, 108, 1212–1220.

    Article  PubMed  CAS  Google Scholar 

  • Jiang, J. H., Ding, L. S., Michailides, T. J., Li, H. Y., & Ma, Z. H. (2009). Molecular characterization of field azoxystrobin-resistant isolates of Botrytis cinerea. Pesticide Biochemistry and Physioloy, 93, 72–76.

    Article  CAS  Google Scholar 

  • Koenraadt, H., Somerville, S. C., & Jones, A. L. (1992). Characterization of mutations in the beta-tubulin gene of benomyl-resistant field strains of Venturia inaequalis and other plant pathogenic fungi. Phytopathology, 82, 1348–1354.

    Article  Google Scholar 

  • Lennox, C. L., Spotts, R. A., & Booyse, M. (2004). Incidence of postharvest decay of ‘d’Anjou’ pear and control with a thiabendazole drench. Plant Disease, 88, 474–478.

    Article  CAS  Google Scholar 

  • Leroux, P., Fritz, R., Debieu, D., Albertini, C., Lanen, C., Bach, J., et al. (2002). Mechanisms of resistance to fungicides in field strains of Botrytis cinerea. Pest Management Science, 58, 876–888.

    Article  PubMed  CAS  Google Scholar 

  • Li, H. X., & Xiao, C. L. (2008a). Baseline sensitivities to fludioxonil and pyrimethanil in Penicillium expansum populations from apple in Washington State. Postharvest Biology and Technology, 47, 239–245.

    Article  CAS  Google Scholar 

  • Li, H. X., & Xiao, C. L. (2008b). Characterization of fludioxonil-resistant and pyrimethanil-resistant phenotypes of Penicillium expansum from apple. Phytopathology, 98, 427–435.

    Article  PubMed  CAS  Google Scholar 

  • Liu, X., Yin, Y. N., Wu, J. B., Jiang, J. H., & Ma, Z. H. (2010). Identification and characterization of carbendazim-resistant isolates of Gibberella zeae. Plant Disease, 94, 1137–1142.

    Article  CAS  Google Scholar 

  • Ma, Z. H., & Michailides, T. J. (2005a). Advances in understanding molecular mechanisms of fungicide resistance and molecular detection of resistant genotypes in phytopathogenic fungi. Crop Protection, 24, 853–863.

    Article  CAS  Google Scholar 

  • Ma, Z. H., & Michailides, T. J. (2005b). Genetic population structure of Botrytis cinerea from different hosts in California. Plant Disease, 89, 1083–1089.

    Article  CAS  Google Scholar 

  • Ma, Z. H., Yoshimura, M., & Michailides, T. J. (2003). Identification and characterization of benzimidazole resistance in Monilinia fructicola from stone fruit orchards in California. Applied and Environmental Microbiology, 69, 7145–7152.

    Article  PubMed  CAS  Google Scholar 

  • Nakaune, R., Hamamoto, H., Imada, J., Akutsu, K., & Hibi, T. (2002). A novel ABC transporter gene, PMR5, is involved in multidrug resistance in the phytopathogenic fungus Pencillium digitatum. Molecular Genetics and Genomics, 267, 179–185.

    Article  PubMed  CAS  Google Scholar 

  • Richards, K. L., Anders, K. R., Nogales, E., Schwartz, K., Downing, K. H., & Botstein, D. (2000). Structure–function relationships in yeast tubulins. Molecular Biology of the Cell, 11, 1887–1903.

    PubMed  CAS  Google Scholar 

  • Rosenberger, D. A. (1990). Blue mold. In A. L. Jones & H. S. Aldwinckle (Eds.), Compendium of apple and pear diseases (pp. 54–55). St. Paul: American Phytopathological Society Press.

    Google Scholar 

  • Sholberg, P. L., Harlton, C., Haag, P., Lévesque, C. A., O’Gorman, D., & Seifert, K. (2005). Benzimidazole and diphenylamine sensitivity and identity of Penicillium spp. that cause postharvest blue mold of apples using β-tubulin gene sequences. Postharvest Biology and Technology, 36, 41–49.

    Article  CAS  Google Scholar 

  • Spotts, R. A., Seifert, K., Wallis, K. M., Sugar, D., Xiao, C. L., Serdani, M., et al. (2009). Description of Cryptosporiopsis kienholzii and species profiles of Neofabraea in major pome fruit growing districts in the Pacific Northwest USA. Mycological Research, 113, 1301–1311.

    Article  PubMed  CAS  Google Scholar 

  • Wong, F. P., De la Cerda, K. A., Hernandez-Martinez, R., & Midland, S. (2008). Detection and characterization of benzimidazole resistance in California populations of Colletotrichum cereale. Plant Disease, 92, 239–246.

    Article  CAS  Google Scholar 

  • Xiao, C. L. (2007). Management of fungicide resistance in postharvest pathogens. Proceedings of the Washington State Horticultural Association 102nd Annual Meeting, 136–139.

  • Xiao, C. L., & Kim, Y. K. (2010). Control of postharvest diseases in apples with reduced-risk fungicides. Stewart Postharvest Review. doi:10.2212/spr.2010.1.6.

    Google Scholar 

  • Xiao, C. L., Kim, Y. K., & Boal, R. J. (2011a). First report of occurrence of pyrimethanil resistance in Penicillium expansum from stored apples in Washington State. Plant Disease, 95, 72.

    Article  Google Scholar 

  • Xiao, C. L., Kim, Y. K., & Boal, R. J. (2011b). Control of Sphaeropsis rot in stored apple fruit caused by Sphaeropsis pyriputrescens with postharvest fungicides. Plant Disease, 95, 1075–1079.

    Article  Google Scholar 

  • Yan, L., Chen, J., Zhang, C., & Ma, Z. (2008). Molecular characterization of benzimidazole-resistant isolates of Cladosporium fulvum. FEMS Microbiology Letters, 278, 242–248.

    Article  PubMed  CAS  Google Scholar 

  • Yin, Y. N., Liu, X., Shi, Z. Q., & Ma, Z. H. (2010). A multiplex allele-specific PCR method for the detection of carbendazim-resistant Sclerotinia sclerotiorum. Pesticide Biochemistry and Physiology, 97, 36–42.

    Article  CAS  Google Scholar 

  • Zhao, H., Kim, Y. K., Huang, L., & Xiao, C. L. (2010). Resistance to thiabendazole and baseline sensitivity to fludioxonil and pyrimethanil in Botrytis cinerea populations from apple and pear in Washington State. Postharvest Biology and Technology, 56, 12–18.

    Article  CAS  Google Scholar 

  • Zhu, L. X., Zhang, Z. W., Liang, D., Jiang, D., Wang, C., Du, N., et al. (2007). Multiplex asymmetric PCR-based oligonucleotide microarray for detection of drug resistance genes containing single mutations in Enterobacteriaceae. Antimicrobial Agents and Chemotherapy, 51, 3707–3713.

    Article  PubMed  CAS  Google Scholar 

  • Zietkiewicz, E., Rafalski, A., & Labunda, D. (1994). Genome fingerprinting by simple sequence repeat (SSR) anchored polymerase chain reaction amplification. Genomics, 20, 178–183.

    Article  Google Scholar 

Download references

Acknowledgments

This research was supported in part by the Washington Tree Fruit Research Commission. We thank H. X. Li for technical assistance.

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Authors and Affiliations

  1. Department of Plant Pathology, Washington State University, Tree Fruit Research and Extension Center, Wenatchee, WA, 98801, USA

    Y. N. Yin

  2. Institute of Biotechnology, Zhejiang University, Hangzhou, China

    Y. N. Yin

  3. San Joaquin Valley Agricultural Sciences Center, United States Department of Agriculture - Agricultural Research Service, Parlier, CA, 93648, USA

    C. L. Xiao

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Correspondence to C. L. Xiao.

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Yin, Y.N., Xiao, C.L. Molecular characterization and a multiplex allele-specific PCR method for detection of thiabendazole resistance in Penicillium expansum from apple. Eur J Plant Pathol 136, 703–713 (2013). https://doi.org/10.1007/s10658-013-0199-2

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