Quantification of 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens strains in the plant rhizosphere by real-time PCR
Quantification of 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens strains in the plant rhizosphere by real-time PCR
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DOI：
10.1128/aem.00925-07
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发表时间：
2007-09-01
影响因子：
4.4
通讯作者：
Weller, David M.
中科院分区：
文献类型：
--
作者：
Mavrodi, Olga V.;Mavrodi, Dmitri V.;Weller, David M.
开发了一种实时 PCR SYBR green 测定法，用于量化土壤和根际中产生 2,4-二乙酰基间苯三酚 (2,4-DAPG) (phlD(+)) 的荧光假单胞菌菌株的数量。设计引物并优化 PCR 条件，以特异性扩增来自 phlD(+) 荧光假单胞菌四种不同基因型的 phlD 基因。使用纯化的基因组 DNA 和从掺有细菌的小麦根洗液中提取的基因组 DNA，为属于基因型 A (Pf-5)、B (Q2-87)、D (Q8rl-96 和 FTAD1 R34) 和 I (FTAD1R36) 的荧光假单胞菌菌株生成与阈值循环 (C(T)s) 和 phlD 基因拷贝相关的标准曲线。对于从荧光假单胞菌纯培养物中分离的基因组 DNA，优化的实时 PCR 测定的检测限为 60 至 600 fg（8 至 80 CFU）；对于从植物根洗液中提取的细菌 DNA，其检测限为 600 fg 至 6.0 pg（80 至 800 CFU，对应于 log 4 至 5phlD(+) 菌株 CFU/根际）。利用实时 PCR 测定法对小麦根际中的 phlD(+) 假单胞菌进行定量。通过实时 PCR 和之前描述的基于 phID 特异性 PCR 的稀释终点测定法检测到的群体密度的回归分析表明存在显着的线性关系（P = 0.0016，r(2) = 0.2）。使用两种不同的土壤对环境样品进行实时 PCR 检测验证，并证明在昆西全衰土壤中检测到了不止一种基因型。所开发的实时 PCR 的最大优点是培养独立性，可以直接从植物根际和土壤中确定 2,4-DAPG 生产者的群体密度和基因型组成。
A real-time PCR SYBR green assay was developed to quantify populations of 2,4-diacetylphloroglucinol (2,4-DAPG) -producing (phlD(+)) strains of Pseudomonas fluorescens in soil and the rhizosphere. Primers were designed and PCR conditions were optimized to specifically amplify the phlD gene from four different genotypes of phlD(+) P. fluorescens. Using purified genomic DNA and genomic DNA extracted from washes of wheat roots spiked with bacteria, standard curves relating the threshold cycles (C(T)s) and copies of the phlD gene were generated for P. fluorescens strains belonging to genotypes A (Pf-5), B (Q2-87), D (Q8rl-96 and FTAD1 R34), and I (FTAD1R36). The detection limits of the optimized real-time PCR assay were 60 to 600 fg (8 to 80 CFU) for genomic DNA isolated from pure cultures of P. fluorescens and 600 fg to 6.0 pg (80 to 800 CFU, corresponding to log 4 to 5phlD(+) strain CFU/rhizosphere) for bacterial DNA extracted from plant root washes. The real-time PCR assay was utilized to quantify phlD(+) pseudomonads in the wheat rhizosphere. Regression analysis of population densities detected by real-time PCR and by a previously described phID-specific PCR-based dilution endpoint assay indicated a significant linear relationship (P = 0.0016, r(2) = 0.2). Validation of real-time PCR assays with environmental samples was performed with two different soils and demonstrated the detection of more than one genotype in Quincy take-all decline soil. The greatest advantage of the developed real-time PCR is culture independence, which allows determination of population densities and the genotype composition of 2,4-DAPG producers directly from the plant rhizospheres and soil.