基因宏阵列和荧光定量PCR方法对西瓜枯萎病害土壤中尖孢镰刀菌的快速检测和定量

QUICK CHECK AND QUANTIFICATION OF FUSARIUM OXYSPORUM IN SOIL WITH MACROARRAY AND REAL-TIME PCR METHOD

采用基因宏阵列(Macroarray)和荧光定量PCR(Real-time PCR)的方法,对引起西瓜枯萎病害的病原菌尖孢镰刀菌(Fusarium oxysporum)进行了快速监测和快速绝对定量,并且对实验条件进行了优化和摸索。结果显示,在西瓜枯萎病害发病较为严重的处理N-+P-和N-+P+的根际土壤中,Macroarray检测到强烈的阳性信号,表明尖孢镰刀菌的大量存在,同时荧光定量PCR的结果也表明在这两个处理的根际土壤中尖孢镰刀菌数量最多,分别为每g土壤8.89×105和2.24×105个拷贝数;而在未发生枯萎病害的处理N++P-和N++P+中,Macroarray未检测到阳性信号,根际土壤中尖孢镰刀菌数量分别为每g土壤6.23×103和3.28×103个拷贝数;而四个处理的土体土壤中尖孢镰刀菌的数量均维持在每g土壤102～103个拷贝数,Macroarray未检测到阳性信号。与传统检测方法如病原菌的分离培养、平板稀释计数等相比,上述分子生物学方法对病原真菌的检测和定量更为准确快速、省时省力。

Macroarray and Real-time quantitative polymerase chain reaction （Real-time PCR） methods were used to perform quick check and quick quantification of Fusarium oxysporum in soils infected with watermelon wilt disease, and their usage was optimized and studied in lab. Results show that in rhizospheric soils of Treatments N- ＋ P-and N- ＋ P ＋ severely infected with the disease, the Macroarray System detected strong positive signals, indicating that Fusarium oxysporum existed in these soils in large quantity. The real-time PCR method also found that the number of Fusarium oxysporum in treatments N- ＋ P-and N- ＋ P＋was the largest, being up to 8.89 ×10^5 dRn g^-1soil and 2.24 ×10^5dRn g^-1 soil, respectively, while in other two treatments not infected with the disease, the number was 6.23 ×10^3 dRn g^-1 soil and 3.28 ×10^3 dRn g^-1 soil. While in bulk soils of all the four treatments, the number of Fusarium oxysporum varied between 10^2 - 10^3dRn g^-1 soil, and the Macroarray System didn＇t detect any strong positive signals. Compared to the traditional isolation, culture, plate screening and counting method for identification and detection of pathogens, the two abovedescribed methods are accurate, instant, and time-and-labor saving.