Competitive PCR for quantitation of a Cytophaga-Flexibacter-Bacteroides phylum bacterium associated with the Tuber borchii Vittad. mycelium

Abstract

An uncultured bacterium associated with the ectomycorrhizal fungus Tuber borchii Vittad. was identified as a novel member of the Cytophaga-Flexibacter-Bacteroides group. Utilizing a quantitative PCR targeting the 16S rRNA gene, we relatively quantified this bacterium in the host. The estimated number of bacteria was found to be approximately 10(6) cells per 30-day-old T. borchii mycelium culture. This represents the first molecular attempt to enumerate an uncultured bacterium associated with a mycorrhizal fungus.

FIG. 1.

QPCR of pb-17BO and competitor. Increasing numbers of molecules (from 100 to 10⁷) of pb-17BO target (•) were coamplified with a fixed amount (10⁴ molecules) of competitor, pb-17BO-c (○). The intensity value incorporated the band area for each PCR band visualized in the agarose gel was plotted as a function of the number of pb-17BO molecules. The intersection of the two curves shows the point of equivalence. The determined value of (1.2 ± 0.122) × 10⁴ molecules for the input pb-17BO sequence corresponds with the actual input of 1.2 × 10⁴ molecules of the competitor pb-17BO-c. Three independent QPCRs were carried out, and some points in the figure are hidden.

FIG. 2.

Amplification efficiency of the 16S rDNA from the 17BO T. borchii mycelium (▪) and the competitor pb-17BOc (○). Band intensities were calculated as described for Fig. 1. Efficiency was calculated from the slope of each regression.

FIG. 3.

cPCR for 16S rRNA gene from the uncultured CFB bacterium in the host T. borchii mycelium. DNAs extracted from T. borchii mycelium (strain 17BO) with masses ranging from 50 to 1.5 ng were coamplified with a fixed amount of competitor, 0.025 pg (1.20 × 10⁴ molecules). The relative intensities of the PCR bands, corresponding to the target and the competitor of the agarose gel (A), were used for the determination of the competition equivalence point (B). Three determinations are plotted.