Fig. 1. Details of mutations and ARMS-PCR primers (A: SdhBP225F; B: SdhBH272R). Mutations in genomic sequences are indicated by dotted box. Bold characters in primers are the positions of point mutations. Lower italic letters are the extra mismatch bases. Direction of primers are indicated by arrows.
Fig. 2. Electrophoretogram of tetra-primer ARMS-PCR system for detection of the Sdh-BP225F and SdhBH272R mutation associated with boscalid resistance. (A) The 426 bp and 330 bp fragments are directed to the P225F mutant (B1~B3). The 426 bp and 142 bp fragments on agarose gel are directed to the native Pro225 codon (B4~B9). The mixture of the mutant and the native were shown behind, with three fragments (B1+B4 and B1+ B7). (B) The 540 bp and 192 bp fragments are directed to the H272R mutant (B4~B6). The 540 bp and 394 bp fragments are directed to the native His272 codon (B1~B3, B7~B9). The mixture of the mutant and the native were shown behind, with three fragments (B1+B4 and B4+ B7).
Table 1. Botrytis cinerea strains for ARMS-PCR validation
Table 2. Primers used in this study
Table 3. Boscalid resistance monitoring by tetra-primer ARMS-PCR, conidial germination, and sequencing
참고문헌
- Angelini, R. M. D. M., Habib, W., Rotolo, C., Pollastro, S. and Faretra, F. 2010. Selection, characterization and genetic analysis of laboratory mutants of Botryotinia fuckeliana (Botrytis cinerea) resistant to the fungicide boscalid. Eur. J. Plant Pathol. 128:185-199. https://doi.org/10.1007/s10658-010-9643-8
- Banno, S., Fukumori, F., Ichiishi, A., Okada, K., Uekusa, H., Kimura, M. and Fujimura, M. 2008. Genotyping of benzimidazole-resistant and dicarboximide-resistant mutations in Botrytis cinerea using real-time polymerase chain reaction assays. Phytopathology 98:397-404. https://doi.org/10.1094/PHYTO-98-4-0397
- Chatzidimopoulos, M., Papaevaggelou, D. and Pappas, A. C. 2013. Detection and characterization of fungicide resistant phenotypes of Botrytis cinerea in lettuce crops in Greece. Eur. J. Plant Pathol. 137:363-376. https://doi.org/10.1007/s10658-013-0248-x
- Chatzidimopoulos, M., Ganopoulos, I., Madesis, P., Vellios, E., Tsaftaris, A. and Pappas, A. C. 2014. High-resolution melting analysis for rapid detection and characterization of Botrytis cinerea phenotypes resistant to fenhexamid and boscalid. Plant Pathol. 63:1336-1343. https://doi.org/10.1111/ppa.12210
- Fernandez-Ortuno, D., Chen, F. and Schnabel, G. 2012. Resistance to pyraclostrobin and boscalid in Botrytis cinerea isolates from strawberry fields in the Carolinas. Plant Dis. 96:1198-1203. https://doi.org/10.1094/PDIS-12-11-1049-RE
- Harju, S., Fedosyuk, H. and Peterson, K. R. 2004. Rapid isolation of yeast genomic DNA: Bust n' Grab. BMC Biotechnol. 4:8. https://doi.org/10.1186/1472-6750-4-8
- Kim, Y. K. and Xiao, C. L. 2010. Resistance to pyraclostrobin and boscalid in populations of Botrytis cinerea from stored apples in Washington State. Plant Dis. 94:604-612. https://doi.org/10.1094/PDIS-94-5-0604
- Leroux, P., Gredt, M., Leroch, M. and Walker, A. S. 2010. Exploring mechanisms of resistance to respiratory inhibitors in field strains of Botrytis cinerea, the causal agent of gray mold. Appl. Environ. Microbiol. 76:6615-6630. https://doi.org/10.1128/AEM.00931-10
- Liu, Q., Thorland, E. C., Heit, J. A. and Sommer, S. S. 1997. Overlapping PCR for bidirectional PCR amplification of specific alleles: a rapid one-tube method for simultaneously differentiating homozygotes and heterozygotes. Genome Res. 7:389-398. https://doi.org/10.1101/gr.7.4.389
- Luck, J. E. and Gillings, M. R. 1995. Rapid identification of benomyl resistant strains of Botrytis cinerea using the polymerase chain reaction. Mycol. Res. 99:1483-1488. https://doi.org/10.1016/S0953-7562(09)80797-1
- Munoz, C., Gomez Talquenca, S. and Volpe, M. L. 2009. Tetra primer ARMS-PCR for identification of SNP in beta-tubulin of Botrytis cinerea, responsible of resistance to benzimidazole. J. Microbiol. Methods 78:245-246. https://doi.org/10.1016/j.mimet.2009.06.007
- Stammler, G., Brix, H. D., Nave, B., Gold, R. and Schoefl, U. 2008. Studies on the biological performance of boscalid and its mode of action. In: Modern fungicides and antifungal compounds V, eds. by H. W. Dehne, H. B. Deising, U. Gisi, K. H. Kuck, P. E. Russell and H. Lyr, pp. 45-51. DPG Spectrum Phytomedizin, Friedrichroda, Germany.
- Williamson, B., Tudzynski, B., Tudzynski, P. and van Kan, J. A. 2007. Botrytis cinerea: the cause of grey mould disease. Mol. Plant Pathol. 8:561-580. https://doi.org/10.1111/j.1364-3703.2007.00417.x
- Ye, S., Humphries, S. and Green, F. 1992. Allele specific amplification by tetra-primer PCR. Nucleic Acids Res. 20:1152. https://doi.org/10.1093/nar/20.5.1152
- Yin, Y. N., Kim, Y. K. and Xiao, C. L. 2011. Molecular characterization of boscalid resistance in field isolates of Botrytis cinerea from apple. Phytopathology 101:986-995. https://doi.org/10.1094/PHYTO-01-11-0016
- Zhang, H., Kong, F., Wang, X., Liang, L., Schoen, C. D., Feng, J. and Wang, Z. 2017. Tetra-primer ARMS PCR for rapid detection and characterisation of Plasmopara viticola phenotypes resistant to carboxylic acid amide fungicides. Pest Manag. Sci. 73:1655-1660. https://doi.org/10.1002/ps.4506