Suga H, Hirayama Y, Morishima M, Suzuki T, Kageyama K, Hyakumachi M
Life Science Research Center, Gifu University, Gifu 501-1193, Japan.
Nara Prefectural Experiment Station, Nara 634-0813, Japan.
Plant Dis. 2013 May;97(5):619-625. doi: 10.1094/PDIS-07-12-0663-RE.
Fusarium oxysporum f. sp. fragariae is a fungal pathogen causing Fusarium wilt on strawberry. Polymerase chain reaction (PCR) primers that can discriminate F. oxysporum f. sp. fragariae from nonpathogenic F. oxysporum would greatly assist pathogen identification. In order to develop a molecular diagnostic tool for this pathogen, transposable elements in the pathogen were characterized and used for designing a specific set of PCR primers. Portions of the transposable elements Fot3, Han, Hop, Hornet1, and Skippy were detected in all 33 strains of F. oxysporum f. sp. fragariae tested by PCR, whereas Foxy was detected in 32 strains and Impala sequences were detected in 30 strains. Two types of sequences were detected for Hop, two types for Impala, and three types for Skippy. The genomic region between Han and Skippy was amplified by an inter-retrotransposon amplified polymorphism technique, and PCR primers (FofraF and FofraR) to specifically identify F. oxysporum f. sp. fragariae were designed from this region. The developed PCR primers discriminated F. oxysporum f. sp. fragariae strains from nonpathogenic F. oxysporum strains and five other formae speciales. Conidia of F. oxysporum f. sp. fragariae could be detected in brown lowland-type soil by PCR using the primers. After preculturing the soil sample on FoG2 medium, 1 × 10 conidia/g of soil could be detected; without preculturing, 1 × 10 conidia/g of soil were detected.
尖孢镰刀菌草莓专化型是一种引起草莓枯萎病的真菌病原体。能够区分尖孢镰刀菌草莓专化型与非致病性尖孢镰刀菌的聚合酶链反应（PCR）引物将极大地有助于病原体鉴定。为了开发针对这种病原体的分子诊断工具，对该病原体中的转座元件进行了表征，并用于设计一套特定的PCR引物。通过PCR检测，在所有33株尖孢镰刀菌草莓专化型菌株中均检测到转座元件Fot3、Han、Hop、Hornet1和Skippy的部分序列，而在32株菌株中检测到Foxy序列，在30株菌株中检测到Impala序列。Hop检测到两种类型的序列，Impala检测到两种类型，Skippy检测到三种类型。通过反转录转座子间扩增多态性技术扩增了Han和Skippy之间的基因组区域，并从该区域设计了用于特异性鉴定尖孢镰刀菌草莓专化型的PCR引物（FofraF和FofraR）。所开发的PCR引物能够区分尖孢镰刀菌草莓专化型菌株与非致病性尖孢镰刀菌菌株以及其他五个专化型。使用这些引物通过PCR可以在棕色低地型土壤中检测到尖孢镰刀菌草莓专化型的分生孢子。在FoG2培养基上对土壤样品进行预培养后，可检测到1×10个分生孢子/克土壤；未进行预培养时，检测到1×10个分生孢子/克土壤。
