Rapid quantitation by PCR of endomycorrhizal fungi colonizing roots.

The VANS1/NS21 primer pair is useful for specifically amplifying a S50bp ribosomal (r) DNA fragment from arbuscular endomycorrhizal fungi, directly from colonized root extracts. A procedure to quantitate these obligatory biotrophs rapidly, based on competitive PCR, was developed by constructing a suitable internal standard to be used with these primers. A 130-bp deletion in the Glomus mossae VANS1/NS21 amplified rDNA fragment was produced by amplifying separately external portions of that fragment, followed by ligation and amplification using the original external primers. When this deleted fragment was added to G. mossae rDNA, amplification using VANS1/ NS21 primers yielded the two expected products of 430 bp and $$0 bp, respectively, resolved by agarose electrophoresis. This fragment was cloned into the pCL1920 plasmid, a low-copy-number vector (five copies per cell), and mixed with the roots to be analyzed. This provides for a rapid quantitative assay because both steps-extraction of DNA from colonized roots and PCR amplif icationare taken into account by the same internal standard. Using this procedure, a sample of colonized leek roots (AIIium porum x Glomus vesicuIiferum) was shown to contain $ x 104 copies of arbuscular endomycorrhizal fungi rDNA genes per milligram of fresh weight. A simple way of getting quantitative information such as gene copy number from PCR amplification assays is by competitive PCR, (1-3) where a known amount of internal standard is added to the sample. This internal standard should be a DNA molecule that can be amplified by the same primer pair as the desired target, but yields a product that is easily distinguished from its counterpart. The DNA used as internal standard should be as similar as possible to the target DNA template to minimize bias during PCR assays. The ratio of amplified template versus amplified standard reflects the ratio of initial template versus initial standard, provided both were subjected to exactly the same PCR conditions and the ratios were close to 1. Hence, the determination of the initial concentration of template becomes a simple assessment of the concentration of a known DNA standard needed for equimolar production of both amplification products. Since the VANS1/NS21 primer pair was demonstrated to amplify a fragment of approximately 550 bp from the gene coding for the small subunit rRNA (SSU) of all arbuscular endomycorrhizal fungi tested, (4) a quantitative assay based on this system could find numerous applications. Furthermore, for the first time, the procedure developed and proposed takes into account variation in fungal DNA extraction yield from colonized roots.