Abstract
Tetra-primer ARMS-PCR (amplification refractory mutation system-PCR) was developed to detect isolates of Fusarium graminearum with resistance to carbendazim, a methyl benzimidazole carbamate-group fungicide. Resistance to carbendazim in F. graminearum is caused by point mutations at codon 167, 198, and 200 of the β 2-tubulin gene (FGSG_06611.3). According to single nucleotide polymorphisms on codon 167, 198, 200 of the resistance gene β 2-tubulin gene, two pairs of primer designed on each codon respectively, were used for amplification. SNP genotyping was made successfully based on the different patterns in agarose gels between point mutations and wild type strain. Tetra-primer ARMS-PCR protocol developed in this study is a simple and useful technique to identify genotypes and has a potential application in management of carbendazim resistance in F. graminearum. It will help researchers and growers select the best antifungal strategy to use in the field.
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References
Bu Y, Gu ZL, Zhang XD, Zhou GH (2004) A rapid single-tube SNP typing based on tetra-primer PCR amplification. Chin J Biochem Mol Biol 20:252–256
Chen CJ, Yu JJ, Bi CW, Zhang YN, Xu JQ, Wang JX, Zhou MG (2009) Mutations in a β-tubulin confer resistance of Gibberella zeae to benzimidazole fungicides. Phytopathology 99:1403–1411
Chen Y, Zhou MG (2009) Characterization of Fusarium graminearum isolates resistant to both carbendazim and a new fungicide JS399-19. Phytopathology 99:441–446
Chung WH, Ishii H, Nishimura K, Ohshima M, Iwama T, Yoshimatsu H (2008) Genetic analysis and PCR-based identification of major Fusarium species causing head blight on wheat in Japan. J Gen Plant Pathol 74:364–374
Eaves DJ, Liebana E, Woodward MJ, Piddock LJV (2002) Detection of gyrA mutations in quinolone-resistant Salmonella enterica by denaturing high-performance liquid chromatography. J Clin Microbiol 40:4121–4125
Etlik O, Koksal V, Arican-Baris ST, Baris I (2008) An improved tetra-primer PCR approach for the detection of the FGFR3 G380R mutation responsible for achondroplasia. Mol Cell Probes 22:71–75
Garcés-Claver A, Fellman SM, Gil-Ortega R, Jahn M, Arnedo-Andrés MS (2007) Identification, validation and survey of a single nucleotide polymorphism (SNP) associated with pungency in Capsicum spp. Theor Appl Genet 115:907–916
Guan F, Yang LG, An JT, Liu SR, Shi GQ (2005) Development of a rapid mismatch PCR method using tetra-primer ARMS for detection of BMPR-IB gene mutation in sheep. Hereditas 27:579–583
Hou YP, Luo QQ, Chen CJ, Zhou MG (2011) Application of cycleave PCR to the detection of a point mutation (F167Y) in the β2-tubulin gene of Fusarium graminearum. Pest Manag Sci 67:1124–1128
Luo QQ, Xu JQ, Hou YP, Chen CJ, Wang JX, Zhou MG (2009) PIRA-PCR for detection of Fusarium graminearum genotypes with moderate resistance to carbendazim. Plant Pathol 58:882–887
Ma ZH, Michailides TJ (2004) An allele-specific PCR assay for detecting azoxystrobin-resistant Alternaria isolates from pistachio in California. J Phytopathol 152:118–121
Ma ZH, Michailides TJ (2005) Advances in understanding molecular mechanisms of fungicide resistance and molecular detection of resistant genotypes in phytopathogenic fungi. Crop Prot 24:853–863
Ma ZH, Yoshimura MA, Holtz BA, Michailides TJ (2005) Characterization and PCR-based detection of benzimidazole-resistant isolates of Monilinia laxa in California. Pest Manag Sci 61:449–457
Muñoz C, Gómez Talquenca S, Volpe ML (2009) Tetra primer ARMS-PCR for identification of SNP in β-tubulin of Botrytis cinerea, responsible of resistance to benzimidazole. J Microbiol Methods 78:245–246
Nicholson P, Parry DW (1996) Development and use of a PCR assay to detect Rhizoctonia cerealis, the cause of sharp eyespot of wheat. Plant Pathol 45:872–883
Oshima M, Banno S, Okada K, Takeuchi T, Kimura M, Ichiishi A, Yamaguchi I, Fujimura M (2006) Survey of mutations of a histidine kinase gene BcOS1 in dicarboximide-resistant field isolates of Botrytis cinerea. J Gen Plant Pathol 72:65–73
Piddock LJ, Ricci V, McLaren I, Griggs DJ (1998) Role of mutations in the gyrA and parC gene of 196 nalidixic acid-resistant Salmonella serotypes isolated from animals. J Antimicrob Chemother 41:635–642
Saito K, Miyake S, Moriya H, Yamazaki M, Itoh F, Imai K, Kurosawa N, Owada E, Miyamoto A (2003) Detection of the four sequence variations of MDR1 gene using TaqMan-MGB probe based real-time PCR and haplotype analysis in healthy Japanese subjects. Clin Biochem 36:511–518
Saito S, Suzuki S, Takayanagi T (2009) Nested PCR-RFLP is a high-speed method to detect fungicide-resistant Botrytis cinerea at an early growth stage of grapes. Pest Manag Sci 65:197–204
Walker RA, Saunders N, Lawson AJ, Lindsay EA, Dassama M, Ward LR, Woodward MJ, Davies RH, Liebana E, Threlfall EJ (2001) Use of the LightCycler gyrA mutation assay for the rapid identification of mutations conferring decreased susceptibility to ciprofloxacin in multiresistant Salmonella enterica serotype typhimurium DT104 isolates. J Clin Microbiol 39:1443–1448
Yan ZQ, Yang SL, Gong Y (2003) Use of PCR related methods in detection of gene mutation. Hereditas 25:198–200
Yang YG, Kim JY, Park SJ, Kim SW, Jeon OH, Kim DS (2007) Apolipoprotein E genotyping by multiplex tetra-primer amplification refractory mutation system PCR in single reaction tube. J Biotechnol 131:106–110
Ye S, Dhillon S, Ke X, Collins AR, Day INM (2001) An efficient procedure for genotyping single nucleotide polymorphisms. Nucleic Acids Res 29:E88
Yoshimatsu H, Tomimura K, Ishii H, Ookubo H, Nakajima T, Hasama W (2006) Occurrence of resistant strains of Fusarium graminearum and baseline sensitivity of Fusarium avenaceum to thiophanate-methyl. Jpn J Phytopathol 72:32 (Abstract in Japanese)
Zhang YN, Fan PS, Chen CJ, Wang JX, Zhou MG (2009) Monitoring and evolvement of resistance to carbendazim of Fusarium graminearum. Agrochemicals 48:603–605+613
Zirnstein G, Li Y, Swaminathan B, Angulo F (1999) Ciprofloxacin resistance in Campylobacter jejuni isolates: detection of gyrA resistance mutations by mismatch amplification mutation assay PCR and DNA sequence analysis. J Clin Microbiol 37:3276–3280
Acknowledgments
This study was supported by: (1) The state “863” programs from the Ministry of Science and Technology of China (2012AA101502). (2) The National Science and Technology Support Program (2012BAD19B01). (3) National Science Foundation of China (31201543, 31171880). (4) Commonweal Specialized Research Fund of China agriculture (200903052, 201103016). (5) The Youth Science and Technology Innovation Fund of Nanjing Agricultural University (KJ2012005).
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Hou, Y., Luo, Q., Chen, C. et al. Application of tetra primer ARMS-PCR approach for detection of Fusarium graminearum genotypes with resistance to carbendazim. Australasian Plant Pathol. 42, 73–78 (2013). https://doi.org/10.1007/s13313-012-0162-2
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DOI: https://doi.org/10.1007/s13313-012-0162-2