Effect of benomyl and diazinon on acquired azole resistance in Aspergillus flavus and expression of mdr1 and cyp51c genes


1 Div. of Molecular Biology, Dep. of Medical Mycology & Parasitology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.

2 Department of Medical Mycology and Parasitology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.

3 Department of Medical Mycology and Parasitology, School of Public Health, International Campus, Tehran University of Medical Sciences, Tehran, Iran

4 1 Div. of Molecular Biology, Dep. of Medical Mycology & Parasitology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. 2-Dep. of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran


Background and Purpose: Aspergillus flavus is an important pathogen in immunodeficient patients. Due to the abundance of this fungus in nature, fungicides are commonly used to preserve and maintain agricultural products. Long-term exposure to these pesticides can lead to the induction of drug resistance in this fungus.

Materials and Methods: For the purpose of the study, 10 strains of A. flavus ATCC 204304 were cultured in benomyl and diazinon pesticides at the concentrations of 62.5, 125, 250.500, 750, 1000, 1500, 2000, and 2500 mg/L in nine steps. Morphological changes and resistance to voriconazole, itraconazole, and amphotericin B were evaluated at the end of each step. Subsequently, changes in the expression of mdr1 and cyp51C genes were studied in the strains showing drug resistance.

Results: The results showed that during the nine stages of the adjacency of strains with benomyl and diazinon at different concentrations, resistance to voriconazole, itraconazole, and amphotericin B in these toxins increased by 30% and 10%, respectively. In addition, the microscopic examination of resistant strains revealed the absence of sporulation, and only mycelium was found. Macroscopically, the color of the colonies changed from green to white. Furthermore, the investigation of the expression of mdr1 and cyp51c genes in these strains showed a decrease and increase in adjacency with diazinon and benomyl, respectively.

Conclusion: As the findings indicated, exposure to agricultural pesticides can lead to the incidence of morphological changes and resistance to amphotericin B, itraconazole, and voriconazole in the sensitive species of A. flavus by altering the expression of genes involved in drug resistance.


1. Rocchi S, Reboux G, Millon L. Azole resistance with environmental origin: what alternatives for the future? J Mycol Med. 2015; 25(4):249-56. 
2. Zanganeh E, Zarrinfar H, Rezaeetalab F, Fata A, Tohidi M, Najafzadeh MJ, et al. Predominance of non-fumigatus Aspergillus species among patients suspected to pulmonary aspergillosis in a tropical and subtropical region of the Middle East. Microb Pathog. 2018; 116:296-300. 
3. Amaike S, Keller NP. Aspergillus flavus. Annu Rev Phytopathol. 2011; 49:107-33. 
4. Perrone G, Susca A, Cozzi G, Ehrlich K, Varga J, Frisvad JC, et al. Biodiversity of Aspergillus species in some important agricultural products. Stud Mycol. 2007; 59:53-66. 
5. Gonçalves SS, Souza AC, Chowdhary A, Meis JF, Colombo AL. Epidemiology and molecular mechanisms of antifungal resistance in Candida and Aspergillus. Mycoses. 2016; 59(4): 198-219. 
6. Maertens JA, Blennow O, Duarte RF, Muñoz P. The current management landscape: aspergillosis. J Antimicrob Chemother. 2016; 71(2):ii23-9. 
7. Tapwal A, Garg S, Gautam N, Kumar R. In vitro antifungal potency of plant extracts against five phytopathogens. Brazil Arch Biol Technol. 2011; 54(6):1093-8. 
8. Pfaller MA. Antifungal drug resistance: mechanisms, epidemiology, and consequences for treatment. Am J Med. 2012; 125(1):S3-13. 
9. Masiá Canuto M, Gutiérrez Rodero F. Antifungal drug resistance to azoles and polyenes. Lancet Infect Dis. 2002; 2(9):550-63. 
10. Razzaghi-Abyaneh M, Shams-Ghahfarokhi M, Allameh A, Kazeroon-Shiri A, Ranjbar-Bahadori S, Mirzahoseini H, et al. A survey on distribution of Aspergillus section Flavi in corn field soils in Iran: population patterns based on aflatoxins, cyclopiazonic acid and sclerotia production. Mycopathologia. 2006; 161(3):183-92. 
11. Astoreca AL, Dalcero AM, Pinto VF, Vaamonde G. A survey on distribution and toxigenicity of Aspergillus section Flavi in poultry feeds. Int J Food Microbiol. 2011; 146(1):38-43. 
12. Cardwell KF, Cotty PJ. Distribution of Aspergillus section Flavi among field soils from the four Agroecological zones of the Republic of Benin, West Africa. Plant Dis. 2002; 86(4):434-9. 
13. Espinel-Ingroff A, Diekema DJ, Fothergill A, Johnson E, Pelaez T, Pfaller MA, et al. Wild-type MIC distributions and epidemiological cutoff values for the triazoles and six Aspergillus spp. for the CLSI broth microdilution method (M38-A2 document). J Clin Microbiol. 2010; 48(9):3251-7. 
14. Chowdhary A, Sharma C, van den Boom M, Yntema JB, Hagen F, Verweij PE, et al. Multi-azole-resistant Aspergillus fumigatus in the environment in Tanzania. J Antimicrob Chemother. 2014; 69(11):2979-83. 
15. Burgos A, Zaoutis TE, Dvorak CC, Hoffman JA, Knapp KM, Nania JJ, et al. Pediatric invasive aspergillosis: a multicenter retrospective analysis of 139 contemporary cases. Pediatrics. 2008; 121(5):e1286-94. 
16. Van Der Linden JW, Warris A, Verweij PE. Aspergillus species intrinsically resistant to antifungal agents. Med Mycol. 2011; 49(Suppl 1):S82-9. 
17. Kontoyiannis DP, Lewis RE. Antifungal drug resistance of pathogenic fungi. Lancet. 2002; 359(9312):1135-44. 
18. Sanglard D, Kuchler K, Ischer F, Pagani JL, Monod M, Bille J. Mechanisms of resistance to azole antifungal agents in Candida albicans isolates from AIDS patients involve specific multidrug transporters. Antimicrob Agents Chemother. 1995; 39(11):2378-86. 
19. De Lucca AJ. Harmful fungi in both agriculture and medicine. Rev Iberoam Micol. 2007; 24(1):3-13. 
20. Arrus K, Blank G, Abramson D, Clear R, Holley RA. Aflatoxin production by Aspergillus flavus in Brazil nuts. J Stored Prod Res. 2005; 41(5):513-27. 
21. Dobolyi CS, SebÅ‘k F, Varga J, Kocsubé S, Szigeti G, Baranyi N, et al. Occurrence of aflatoxin producing Aspergillus flavus isolates in maize kernel in Hungary. Acta Alimentaria. 2013; 42(3):451-9. 
22. Snelders E1, Huis In 't Veld RA, Rijs AJ, Kema GH, Melchers WJ, Verweij PE. Possible environmental origin of resistance of Aspergillus fumigatus to medical triazoles. Appl Environ Microbiol. 2009; 75(12):4053-7. 
23. Escribano P, Recio S, Peláez T, Bouza E, Guinea J. Aspergillus fumigatus strains with mutations in the cyp51A gene do not always show phenotypic resistance to itraconazole, voriconazole, or posaconazole. Antimicrob Agents Chemother. 2011; 55(5):2460-2. 
24. Faria-Ramos I, Farinha S, Neves-Maia J, Tavares PR, Miranda IM, et al. Development of cross-resistance by Aspergillus fumigatus to clinical azoles following exposure to prochloraz, an agricultural azole. BMC Microbiol. 2014; 14(1):155. 
25. Faria-Ramos I, Tavares PR, Farinha S, Neves-Maia J, Miranda IM, Silva RM, et al. Environmental azole fungicide, prochloraz, can induce cross-resistance to medical triazoles in Candida glabrata. FEMS Yeast Res. 2014; 14(7):1119-23.
Volume 5, Issue 2
June 2019
Pages 27-32
  • Receive Date: 09 July 2019
  • Revise Date: 07 September 2020
  • Accept Date: 09 July 2019
  • First Publish Date: 09 July 2019