| 用Real-Time PCR评价花生田间金龟子绿僵菌的存活能力 |
| Evaluating survival ability of Metarhizium anisopliae in the peanut field by Real-Time PCR |
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| 中文关键词:金龟子绿僵菌，种群动态，定量分析 |
| 英文关键词:Metarhizium anisopliae, population dynamic, quantitative analysis |
| 基金项目:公益性（农业）行业专项（No. 201003025）；农业部948计划（No. 2011-G4）；现代农业产业体系（No. CARS-35） |
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| 中文摘要: |
| 为了能够对金龟子绿僵菌在花生田间的存活能力进行精确的定量研究，通过比对真菌ITS序列，设计出针对金龟子绿僵菌的特异性引物，并建立金龟子绿僵菌Real-Time PCR反应体系。结果表明，标准品在拷贝数为8.49×103–8.49×109copies/μL之间，线性关系良好，线性方程为y=-3.257x+6.969，相关系数为R=0.9980，扩增效率E=102.8%，检出痕量为20个孢子/g土壤。以Real-Time PCR方法和平板稀释法，分别对花生根部施用金龟子绿僵菌后不同时期的土壤进行定量分析，结果表明两种方法检测的金龟子绿僵菌数量变化趋势相似，施用的金龟子绿僵菌在根围与根际都呈现先快速下降后缓慢回升的趋势，90d时金龟子绿僵菌DNA量和CFU值均下降至初始的10%以下，之后出现回升；根际的金龟子绿僵菌相对地下降较慢而回升较快，在120d时可恢复到初始的52.17%和38.65%，显著高于根围的数量。试验建立的Real-Time PCR体系可用于金龟子绿僵菌土壤宿存的定量检测，而且适用于低含量的检测。 |
| 英文摘要: |
| Metarhizium anisopliae viability in the peanut field was studied based on accurately quantitative monitoring by Real-Time PCR reaction. At first, specific primers for Metarhizium anisopliae were designed after comparing the internal transcribed spacer (ITS) sequences of fungi, and an optimal reaction system for Real-Time PCR was established. The results showed that the copy number of standard ranged from 8.49×103copies/μL to 8.49×109copies/μL with a good linear relationship. The linear equation was y=-3.257x+6.969 and correlation coefficient R=0.9980. The PCR amplification efficiency was E=102.8% and the detection of trace to 20spores/g soil. Two analysis methods, Real-Time PCR and classic dilution plates, were used to analyze the survival quantitaties of Metarhizium anisopliae applied in peanut field. The results indicated that the tendency of the fungal population detected by the two methods was similar during different periods. Metarhizium anisopliae around the root, either in rhizosphere soil or in bulk soil, would all decline rapidly at early stage, followed by a gradual recovery. For 90d, the fungal quantity dropped to below 10% of the initial level based on the DNA content or CFU value, and rebound after. Relatively, the fungal quantity in rhizosphere soil dropped slower and recovered faster than that in bulk soil. For 120d, the DNA content and CFU value in rhizosphere could recover to the initial 52.17% and 38.65% respectively, significantly higher than the number in bulk. The tests proved that the establishment of the Real-Time PCR system can be used for the quantitative detection of Metarhizium anisopliae persistence in soil, even suitable for the detection of low levels. |
| 李兴佳,农向群,曹广春,刘迅,王广君,张泽华. 用Real-Time PCR评价花生田间金龟子绿僵菌的存活能力[J]. 菌物学报, 2013, 32(4): 710-720 |
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