Zhang Zhenggang, Zhang Jingyu, Wang Yuanchao, Zheng Xiaobo
Department of Plant Pathology, Nanjing Agricultural University, Nanjing 210095, PR China.
FEMS Microbiol Lett. 2005 Aug 1;249(1):39-47. doi: 10.1016/j.femsle.2005.05.057.
We developed two species-specific PCR assays for rapid and accurate detection of the pathogenic fungi Fusarium oxysporum f. sp. niveum and Mycosphaerella melonis in diseased plant tissues and soil. Based on differences in internal transcribed spacer (ITS) sequences of Fusarium spp. and Mycosphaerella spp., two pairs of species-specific primers, Fn-1/Fn-2 and Mn-1/Mn-2, were synthesized. After screening 24 isolates of F. oxysporum f. sp. niveum, 22 isolates of M. melonis, and 72 isolates from the Ascomycota, Basidiomycota, Deuteromycota, and Oomycota, the Fn-1/Fn-2 primers amplified only a single PCR band of approximately 320 bp from F. oxysporum f. sp.niveum, and the Mn-1/Mn-2 primers yielded a PCR product of approximately 420 bp from M. melonis. The detection sensitivity with primers Fn-1/Fn-2 and Mn-1/Mn-2 was 1fg of genomic DNA. Using ITS1/ITS4 as the first-round primers, combined with either Fn-1/Fn-2 and or Mn-1/Mn-2, two nested PCR procedures were developed, and the detection sensitivity increased 1000-fold to 1ag. The detection sensitivity for the soil pathogens was 100-microconidia/g soil. A duplex PCR method, combining primers Fn-1/Fn-2 and Mn-1/Mn-2, was used to detect F. oxysporum f. sp. niveum and M. melonis in plant tissues infected by the pathogens. Real-time fluorescent quantitative PCR assays were developed to detect and monitor the pathogens directly in soil samples. The PCR-based methods developed here could simplify both plant disease diagnosis and pathogen monitoring as well as guide plant disease management.
我们开发了两种物种特异性PCR检测方法，用于快速、准确地检测患病植物组织和土壤中的致病真菌尖孢镰刀菌西瓜专化型（Fusarium oxysporum f. sp. niveum）和瓜球腔菌（Mycosphaerella melonis）。基于镰刀菌属（Fusarium spp.）和球腔菌属（Mycosphaerella spp.）内部转录间隔区（ITS）序列的差异，合成了两对物种特异性引物，即Fn-1/Fn-2和Mn-1/Mn-2。在对24株尖孢镰刀菌西瓜专化型分离株、22株瓜球腔菌分离株以及来自子囊菌门、担子菌门、半知菌门和卵菌门的72株分离株进行筛选后，Fn-1/Fn-2引物仅从尖孢镰刀菌西瓜专化型中扩增出一条约320 bp的单一PCR条带，而Mn-1/Mn-2引物从瓜球腔菌中产生了一条约420 bp的PCR产物。引物Fn-1/Fn-2和Mn-1/Mn-2的检测灵敏度为1 fg基因组DNA。以ITS1/ITS4作为第一轮引物，结合Fn-1/Fn-2或Mn-1/Mn-2，开发了两种巢式PCR程序，检测灵敏度提高了1000倍，达到1 ag。土壤病原菌的检测灵敏度为100个微分生孢子/克土壤。采用将引物Fn-1/Fn-2和Mn-1/Mn-2相结合的双重PCR方法，检测受病原菌感染的植物组织中的尖孢镰刀菌西瓜专化型和瓜球腔菌。开发了实时荧光定量PCR检测方法，用于直接检测和监测土壤样品中的病原菌。本文开发的基于PCR的方法可以简化植物病害诊断和病原菌监测，以及指导植物病害管理。
