Suitability of Quantitative Real-Time PCR To Estimate the Biomass of Fungal Root Endophytes

A nested single-copy locus-based quantitative PCR (qPCR) assay and a multicopy locus-based qPCR assay were developed to estimate endophytic biomass of fungal root symbionts belonging to the Phialocephala fortinii sensu lato-Acephala applanata species complex (PAC). Both assays were suitable for estimation of endophytic biomass, but the nested assay was more sensitive and specific for PAC. For mycelia grown in liquid cultures, the correlation between dry weight and DNA amount was strong and statistically significant for all three examined strains, allowing accurate prediction of fungal biomass by qPCR. For mycelia colonizing cellophane or Norway spruce roots, correlation between biomass estimated by qPCR and microscopy was strain dependent and was affected by the abundance of microsclerotia. Fungal biomass estimated by qPCR and microscopy correlated well for one strain with poor microsclerotia formation but not for two strains with high microsclerotia formation. The accuracy of qPCR measurement is constrained by the variability of cell volumes, while the accuracy of microscopy can be hampered by overlapping fungal structures and lack of specificity for PAC. Nevertheless, qPCR is preferable because it is highly specific for PAC and less time-consuming than quantification by microscopy. There is currently no better method than qPCR-based quantification using calibration curves obtained from pure mycelia to predict PAC biomass in substrates. In this study, the DNA amount of A. applanata extracted from 15 mm of Norway spruce fine root segments (mean diameter, 610 μm) varied between 0.3 and 45.5 ng, which corresponds to a PAC biomass of 5.1 ± 4.5 μg (estimate ± 95% prediction interval) and 418 ± 264 μg.Interactions between fungi and plants are very common in nature and range from mutualistic to pathogenic (41). The outcome of a plant-fungus interaction largely depends on the extent of colonization by the fungus, independent of whether pathogenic or mutualistic fungal species are involved (6, 8). This is also true for endophytic species. In contrast to infections by pathogenic fungi, where disease symptoms are expressed after a comparatively short period of incubation, infection by endophytic fungi does not cause disease symptoms for prolonged periods, because once inside the tissue, endophytes assume a quiescent state either for the whole lifetime of the infected plant tissue or until the host is adversely affected by the arrival of biotic or abiotic stress (34, 38, 42, 45). Therefore, switching endophyte behavior from neutral to pathogenic or mutualistic can depend on the predisposition of the host tissue, environmental factors, and the extent of colonization. For instance, in conifer needles, the biomass of endophytic Rhabdocline parkeri thalli increase over time (44). It has been postulated that the needles die as soon as the endophyte''s biomass exceeds a certain threshold value (42). Therefore, attainment of the threshold usually coincides with natural senescence. However, the threshold can either be lowered or reached prematurely if host resistance is reduced by adverse factors. Thus, the health status of plants depends on the density of colonization by the endophyte, and vice versa.Estimation of the extent of colonization is difficult, and there are certain conditions that must be met for techniques to determine fungal biomass. First, they should reproducibly combine target (i.e., species/genotype) specificity with accurate quantification of biomass. Traditionally, microscopy has been used to measure hyphal length or proportional colonization of host tissue (4, 28, 30). However, determination of fungal biomass by microscopy is very laborious, and results vary between investigators. Moreover, visual quantification is unspecific, as species designation is often difficult or impossible. Chemical methods measuring the amount of specific biomolecules stored inside fungal cells or released into the environment (e.g., the fatty acid ergosterol or the carbohydrate chitin) are also widely used (13, 49). Although these methods are much less laborious than microscopy, they are nonspecific and problems can arise if used for field samples (33), and the minimum sample size required is comparatively high (14, 31). Real-time quantitative PCR (qPCR) (23, 43) combines specificity at different taxonomic levels with accurate measurement of DNA copy number and allows quantification of DNA in very small samples. Different qPCR chemistries (TaqMan, SYBR green, or molecular beacons) and methods are available (22, 32). The choice of the locus used for qPCR assays largely depends on the aim of the study. While multicopy genes allow the detection of lower DNA amounts, single-copy genes give more precise measurements of DNA copy number, as the number of repeats of multicopy loci can differ between strains and even within a single individual strain (7, 24). In addition, sensitivity of qPCR can be increased by applying a nested approach, where the entire locus is initially amplified by conventional PCR, and the resulting product is then quantified with the specific primer-probe combination in a second step (11, 39). Diagnostic qPCR assays have been used for early and rapid detection of plant pathogens in the environment and in diseased tissues (9, 32, 50). However, little investigation has been done into the usefulness of qPCR to estimate fungal biomass, and considerable disagreement exists. For example, qPCR biomass estimates of multinucleate arbuscular mycorrhizal fungi (AMF) correlated only poorly with estimates obtained by visual assessment (12). This was proposed to be due to the multinucleate nature of Glomeromycota. On the other hand, a correlation between qPCR and mycelial dry weight was demonstrated for the Swiss needle cast-causing parasite Phaeocryptopus gaeumannii (52) and between qPCR and hyphal length for the ectomycorrhizal fungus (EMF) Piloderma croceum (36). Partial correspondence between ergosterol assays and qPCR has been demonstrated in needles infected with P. gaeumannii (51), and a high correlation between qPCR and ergosterol was found in tissues colonized by the conifer root pathogen Heterobasidion annosum (25).In the present study we tested the suitability of qPCR to estimate the biomass of a common group of ascomycetous root endophytes. Members of the Phialocephala fortinii sensu lato-Acephala applanata species complex (PAC) (17, 46) are ubiquitous root symbionts in woody plants, especially in conifers and heathland shrubs, where they are the most prominent endophytes (1). PAC form loose networks of hyphae running mostly parallel to the root axis on the root surface but also grow inter- and intracellularly within the root cortex (47). Inside cortical cells and under certain culture conditions, PAC species can form microsclerotia, which are tight complexes of more or less isodiametric, to irregular, thick-walled cells that can endure harsh conditions and may therefore serve as resting spores and units of dispersal (2, 37, 53). The ecological role of PAC members is still controversial, despite several studies (21). The effects of PAC on their hosts were described as being pathogenic in some studies but beneficial in others. This variability in behavior was mainly due to the use of different, undefined isolates and a multitude of experimental designs which either favored PAC members or the host plants (21). Recently, isolates characterized by specific molecular markers have become available, making PAC-host interaction studies more meaningful.In this study we aimed (i) to develop a specific qPCR method that allows detection of all PAC members, (ii) to test the method''s suitability for biomass estimation in three different experimental systems in vitro (liquid fungal cultures, cellophane culture, and colonized roots of Picea abies seedlings) by using biomass estimates obtained by microscopy as reference, and (iii) to compare the reproducibility, sensitivity, and specificity of a nested single-copy qPCR assay and a multicopy qPCR assay.