Phytopathology. 2001 Jan;91(1):44-54. doi: 10.1094/PHYTO.2001.91.1.44.
ABSTRACT Pseudomonas species that produce 2,4-diacetylphloroglucinol (2,4-DAPG) play a significant role in the suppression of fungal root pathogens in the rhizosphere of crop plants. To characterize the abundance and diversity of these functionally important bacterial populations, we developed a rapid polymerase chain reaction (PCR)-based assay targeting phlD, an essential gene in the phloroglucinol biosynthetic pathway. The phlDgene is predicted to encode a polyketide synthase that synthesizes mono-acetylphloroglucinol, the immediate precursor to 2,4-DAPG. A major portion of the phlD open reading frame was cloned and sequenced from five genotypically distinct strains, and the sequences were screened for conserved regions that could be used as gene-specific priming sites for PCR amplification. Several new phlD-specific primers were designed and evaluated. Using the primers B2BF and BPR4, we developed a PCR-based assay that was robust enough to amplify the target gene from a diverse set of 2,4-DAPG producers and sensitive enough to detect as few as log 2.4 cells per sample when combined with enrichment from a selective medium. Restriction fragment length polymorphism analysis of the amplified phlD sequence allows for the direct determination of the genotype of the most abundant 2,4-DAPG producers in a sample. The method described was useful for characterizing both inoculant and indigenous phlD(+) pseudomonads inhabiting the rhizosphere of crop plants. The ability to rapidly characterize populations of 2,4-DAPG-producers will greatly enhance our understanding of their role in the suppression of root diseases.
摘要 能够产生 2,4-二乙酰基间苯三酚（2,4-DAPG）的假单胞菌在作物根际抑制真菌根病原体方面发挥着重要作用。为了描述这些具有重要功能的细菌群体的丰度和多样性，我们开发了一种基于聚合酶链反应（PCR）的快速检测方法，该方法靶向间苯三酚生物合成途径中的必需基因 phlD。预测 phlD 基因编码一种多酮合酶，可合成单乙酰基间苯三酚，这是 2,4-DAPG 的直接前体。从五个基因型不同的菌株中克隆和测序了 phlD 开放阅读框的大部分序列，并筛选了可作为 PCR 扩增基因特异性引物的保守区域。设计并评估了几个新的 phlD 特异性引物。使用引物 B2BF 和 BPR4，我们开发了一种基于 PCR 的检测方法，该方法足够稳健，可以从多种 2,4-DAPG 产生菌中扩增目标基因，并且与选择性培养基富集结合使用时，足够灵敏，可以检测到每样本低至 log 2.4 个细胞。扩增的 phlD 序列的限制性片段长度多态性分析允许直接确定样品中最丰富的 2,4-DAPG 产生菌的基因型。所描述的方法可用于表征定植于作物根际的接种菌和土著 phlD(+)假单胞菌。快速描述 2,4-DAPG 产生菌群体的能力将极大地增强我们对其在抑制根病中的作用的理解。
