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Cytochrome b Gene-Based Assay for Monitoring the Resistance of Colletotrichum spp. to Pyraclostrobin
Dalha Abdulkadir Isa(Dalha Abdulkadir Isa ),Heung Tae Kim(Heung Tae Kim) 한국식물병리학회 2022 Plant Pathology Journal Vol.38 No.6
Resistance to pyraclostrobin due to a single nucleotide polymorphism at 143rd amino acid position on the cytochrome b gene has been a major source of concern in red pepper field infected by anthracnose in Korea. Therefore, this study investigated the response of 24 isolates of C. acutatum and C. gloeosporioides isolated from anthracnose infected red pepper fruits using agar dilution method and other molecular techniques such as cytochrome b gene sequencing, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and allele-specific polymerase chain reaction (PCR). The result showed that four isolates were resistant to pyraclostrobin on agar dilution method and possessed GCT (alanine) codon at 143rd amino acid position, whereas the sensitive isolates possessed GGT (glycine). Furthermore, this study illustrated the difference in the cytochrome b gene structure of C. acutatum and C. gloeosporioides. The use of cDNA in this study suggested that the primer Cacytb-P2 can amplify the cytochrome b gene of both C. acutatum and C. gloeosporioides despite the presence of various introns in the cytochrome b gene structure of C. gloeosporioides. The use of allele-specific PCR and PCR-RFLP provided clear difference between the resistant and sensitive isolates. The application of molecular technique in the evaluation of the resistance status of anthracnose pathogen in red pepper provided rapid, reliable, and accurate results that can be helpful in the early adoption of fungicide-resistant management strategies for the strobilurins in the field.
Detection of Colletotrichum spp. Resistant to Benomyl by Using Molecular Techniques
Dalha Abdulkadir Isa(Dalha Abdulkadir Isa ),Heung Tae Kim(Heung Tae Kim) 한국식물병리학회 2022 Plant Pathology Journal Vol.38 No.6
Colletotrichum species is known as the major causal pathogen of red pepper anthracnose in Korea and various groups of fungicides are registered for the management of the disease. However, the consistent use of fungicides has resulted in the development of resistance in many red pepper-growing areas of Korea. Effective management of the occurrence of fungicide resistance depends on constant monitoring and early detection. Thus, in this study, various methods such as agar dilution method (ADM), gene sequencing, allele-specific polymerase chain reaction (PCR), and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were applied for the detection of benzimidazole resistance among 24 isolates of Colletotrichum acutatum s. lat. and Colletotrichum gloeosporioides s. lat. The result of the ADM showed that C. gloeosporioides s. lat. was classified into sensitive and resistant isolates to benomyl while C. acutatum s. lat. was insensitive at ≥1 μg/ml of benomyl. The sequence analysis of the β-tubulin gene showed the presence of a single nucleotide mutation at the 198th amino acid position of five isolates (16CACY14, 16CAYY19, 15HN5, 15KJ1, and 16CAYY7) of C. gloeosporioides s. lat. Allele-specific PCR and PCR-RFLP were used to detect point mutation at 198th amino acid position and this was done within a day unlike ADM which usually takes more than one week and thus saving time and resources that are essential in the fungicide resistance management in the field. Therefore, the molecular techniques established in this study can warrant early detection of benzimidazole fungicide resistance for the adoption of management strategies that can prevent yield losses among farmers.
청양 지역 고추와 구기자에서 분리한 탄저병균의 기주 교차 감염과 유전적 다양성
Dalha Abdulkadir Isa,김가혜,손승완,김흥태 한국농약과학회 2022 농약과학회지 Vol.26 No.1
탄저병이 발생한 구기자와 고추에서 단포자 분리하여 얻은 34균주와 11균주를 사용하여 , 구기자와 고추 열 매에 대한 병원성 실험과 병원균의 유전적 다양성을 조사하였다 . 구기자에서 분리한 34개 균주 중에서 14균주는 C. gloeosporioides로, 20균주는 C. acutatum으로 동정되었으며 , 고추에서 분리한 11균주는 모두 C. acutatum으로 동정 되었다 . 구기자에서 분리한 C. gloeosporioides와 C. acutatum 균주를 구기자 열매에 상처 또는 무상처 접종하였을 때, 모든 균주가 병원성을 나타내었지만 , 고추 열매에 접종하였을 때는 병원성이 나타나지 않거나 , 아주 약한 병원성 을 나타내었다 . 하지만 고추에서 분리한 C. acutatum 균주는 상처 접종 시 고추와 구기자 열매 모두에서 강한 병원성 을 보였다 . 하지만 구기자 열매에 무상처 접종하였을 때에는 대부분의 균주가 병원성을 나타내지 않았다 . PCR- RAPD법으로 병원균 집단의 유전적 다양성을 분석한 결과 , 구기자와 고추에서 분리된 C. acutatum은 서로 다른 그 룹으로 구분되었다 .