The Plant Pathology Journal

The Korean Society of Plant Pathology (한국식물병리학회)
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Rapid Detection of SdhBP225F and SdhBH272R Mutations in Boscalid Resistant Botrytis cinerea Strains by ARMS-PCR

  • Liu, Xin (Institute of Eco-Environment and Plant Protection, Shanghai Key Laboratory of Protection Horticultural Technology, Shanghai Academy of Agricultural Sciences) ;
  • Zeng, Rong (Institute of Eco-Environment and Plant Protection, Shanghai Key Laboratory of Protection Horticultural Technology, Shanghai Academy of Agricultural Sciences) ;
  • Gao, Shigang (Institute of Eco-Environment and Plant Protection, Shanghai Key Laboratory of Protection Horticultural Technology, Shanghai Academy of Agricultural Sciences) ;
  • Xu, Lihui (Institute of Eco-Environment and Plant Protection, Shanghai Key Laboratory of Protection Horticultural Technology, Shanghai Academy of Agricultural Sciences) ;
  • Dai, Fuming (Institute of Eco-Environment and Plant Protection, Shanghai Key Laboratory of Protection Horticultural Technology, Shanghai Academy of Agricultural Sciences)
  • Received : 2018.08.06
  • Accepted : 2018.12.03
  • Published : 2019.02.01
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Abstract

$SdhB^{P225F}$ and $SdhB^{H272R}$ mutations have been found associated with boscalid resistance in Botrytis cinerea from strawberry in Shanghai, China. For rapid detection of two mutations, tetra-primers were designed and optimized to gain the relatively high accuracy and specificity based on the ARMS-PCR technique, by which resistance can be identified with different lengths of products on agarose gels. The tetra-primer ARMS-PCR systems for $SdhB^{P225F}$ and $SdhB^{H272R}$ were validated by 9 SdhB-squenced strains repeatedly. Then, sensitivity of 30 more strains were also tested by the methods, which were accordant with genotypes by sequencing and the sensitivity of conidial germination to boscalid by 100%. Thus, the methods developed in this study are proved to be rapid, inexpensive, accurate and practical for resistance detection of Botrytis cinerea caused by $SdhB^{P225F}$ and $SdhB^{H272R}$ mutations.

Keywords

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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.

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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

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Table 2. Primers used in this study

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Table 3. Boscalid resistance monitoring by tetra-primer ARMS-PCR, conidial germination, and sequencing

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