Microsatellite-Based Quantification Method to Estimate Biomass of Endophytic Phialocephala Species in Strain Mixtures

Abstract

Fungi of the Phialocephala fortinii sensu lato-Acephala applanata species complex (PAC) are ubiquitous endophytic colonizers of tree roots in which they form genotypically diverse communities. Measurement of the colonization density of each of the fungal colonizers is a prerequisite to study the ecology of these communities. Up to now, there is no method readily available for the quantification of PAC strains co-colonizing the same root. The new DNA quantification method presented here is based on the amplification of microsatellites by competitive polymerase chain reaction (PCR). The method proved to be suitable to detect and quantify at least two strains within one single sample by the addition of a known amount of mycelium of a reference strain before DNA extraction. The method exploits the correlation between the reference/target ratio of light emitted during microsatellite detection (peak ratio) and the reference/target ratio of mycelial weights to determine the biomass of the target strain. Hence, calibration curves were obtained by linear regression of the peak ratios on the weight ratios for different mixtures of reference and target strains. The slopes of the calibration curves and the coefficients of determination were close to 1, indicating that peak ratios are good predictors of weight ratios. Estimates of fungal biomass in mycelial test mixtures of known composition laid within the 95% prediction interval and deviated on average by 16% (maximally 50%) from the true biomass. On average, 3-6% of the root biomass of Norway spruce seedlings consisted of mycelial biomass of either one of two inoculated PAC strains. Biomass estimates obtained by real-time quantitative PCR were correlated with the estimates obtained by the microsatellite-based method, but variation between the two estimates from the same root was high in some samples. The microsatellite-based DNA quantification method described here is currently the best method for strainwise estimation of endophytic biomass of PAC fungi in small root samples.

Mycorrhiza Reduces Adverse Effects of Dark Septate Endophytes (DSE) on Growth of Conifers

“…This contrasts with Reininger et al [43] who had more success using the microsatellite method. Apart from the different PAC inoculation method used by Reininger et al [43] differences to our results are mainly of stochastic nature. A total of 24 (21 for detection by microsatellites and three for detection by reisolation) 5-mm-long root segments, i.e.…”

“…The 21 segments from the three roots for DNA extraction were pooled, freeze dried and weighed. To estimate biomass of each PAC strain, 3 mg of freeze-dried reference mycelium was added before DNA extraction (mycelium of strain C was added as reference to root samples containing strain A, mycelium of strain A to root samples containing strains B, C and D; for details see Reininger et al [43]) and stored at −80°C until further processing (see ‘root-reference-mixtures’ below). The three fresh root segments for PAC reisolation were surface-sterilized in 30% H 2 O 2 for 30 s and 10 s in EtOH and incubated on terramycin-malt agar (20 g l −1 malt extract, 15 g l −1 agar, 50 mg l −1 terramycin®) at room temperature.…”

“…Biomass of PAC mycelia in and on roots was estimated using the method described in Reininger et al . [43].…”

A rapid and cost‐effective quantitative microsatellite genotyping protocol to estimate intraspecific competition in protist microcosm experiments

“…). However, stutter banding is neither limited to, nor an inherent problem of, dinucleotide microsatellite loci; indeed, Reininger, Grunig & Sieber () estimated fungal biomass from allele frequencies at dinucleotide microsatellite loci. Thus, other studies may use dinucleotide loci as a means of finding greater numbers of presumably more polymorphic loci.…”

“…Nonetheless, our study highlights an obvious time and cost benefit associated with using a protist model where the focus is on single cells. While there is no need to identify an appropriate reference strain for this method (Reininger, Grunig & Sieber ), it is advisable to initially culture and genotype individual strains so that standard pools of DNA (or mixed cells) can be used to calibrate and validate the assay. An additional advantage is that suitably polymorphic microsatellite loci are available for many protists.…”