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番石榴焦腐病是台湾入境大陆水果的重要植物病害,由葡萄座腔菌Botryosphaeria rhodina引起。为建立该病原菌快速、灵敏的检测技术,比较分析了葡萄座腔菌和葡萄座腔菌属其它种的ITS序列,在此基础上设计了1对检测番石榴焦腐病菌的特异性引物BF1/BR1,利用此引物从葡萄座腔菌中特异性扩增出287 bp条带,而其余参试的菌株未能获得扩增条带。将真菌通用引物ITS1/ITS4和BF1/BR1进行巢式PCR扩增后,检测灵敏度提高1 000倍,可检测到葡萄座腔菌1 pg的基因组DNA。结合快速碱裂解法提取发病组织的DNA,采用该PCR检测技术可从自然感染焦腐病果实中检测到葡萄座腔菌。
Guava scorch is an important plant disease of Taiwan’s inbound mainland fruits and is caused by Botryosphaeria rhodina. In order to establish a rapid and sensitive detection technique for this pathogen, the ITS sequences of other species of the genera Bacteroides and other strains of Humicola in this study were compared. Based on these results, a pair of specific primers BF1 / BR1. The 287 bp band was amplified specifically from Bacteroides spp., While the rest of the tested strains failed to obtain the amplified bands. After nested PCR amplification of common fungal primers ITS1 / ITS4 and BF1 / BR1, the detection sensitivity was increased by 1 000 times, and 1 pg of genomic DNA of V. graminis was detected. Combined with rapid alkaline lysis method to extract the DNA of the diseased tissue, this PCR detection technique can be detected naturally in the fruit of Pyricularia graminis.