Our motivation for this study was to develop a qPCR method to quantify AMF root colonization in a more high-throughput and less operator-dependent manner than classical microscopy. Our starting point was the qPCR primers developed by Hewins et al. (2015). First, we confirmed in silico their specificity to most AMF species (Fig. 1). We then validated their suitability to discriminate quantitative differences of AMF root colonization in a real experiment with Petunia plants. We demonstrated that AMF quantification with these qPCR primers is consistent with traditional microscopic analysis (Fig. 2). Of note, we normalized the quantification of the AMF qPCR signal relative to a plant qPCR signal, revealing that relative qPCR quantification works for quantification of root colonization. Finally, we validated using MiSeq sequencing that the qPCR primers produce AMF amplicons for mycorrhizal Petunia plants (Fig. 3). Our approach of relative qPCR with the primer pair AMG1F and AM1 presents an alternative molecular method to quantify AMF root colonization compared with traditional microscopy.
The advantage of the primer pair AMG1F and AM1 is that they amplify a wide range of species of the Glomeromycota. Our in silico analysis suggests this primer pair covers the Acaulosporaceae, Gigasporaceae, Pacisporaceae, most Glomeraceae, and probably also the Diversisporaceae but not the ancestral AMF lineages of the Claroideoglomeraceae, Archeosporales, and Paraglomerales (Fig. 1). Note that these ancestral AMF lineages are poorly visible under the microscope and as such also cannot be quantified using microscopy (Oehl et al. 2011). Broad taxonomic coverage of a wide range of AMF is a prerequisite to quantify the whole mycorrhizal community in different root samples, for example, from plants growing under different treatments or environments. These primers are different from qPCR primer pairs that were designed to specifically amplify a single AMF species (Alkan et al. 2004, 2006; Isayenkov et al. 2004; Jansa et al. 2008; Gamper et al. 2008; Thonar et al. 2012; Knegt et al. 2016), which are useful for research questions that require the quantification of colonization after inoculation with that single species.
In addition to covering a broad range of AMF species of the Glomeromycota, the primer pair AMG1F and AM1 is expected to avoid amplification of DNA from Petunia, as suggested by our in silico analysis (Fig. 1). We confirmed this prediction by sequencing qPCR products (Fig. 3). Our work with Petunia is consistent with an earlier validation by Hewins et al. (2015) that used Sanger sequencing to sequence qPCR amplicons from colonized wild leek. They sequenced 47 clones and found that all of them matched AMF, while none of them matched the plant host. The inclusion of non-mycorrhizal Arabidopsis as a negative control in our primer tests revealed that the PCR amplicon is formed in “late” PCR cycles (Fig. S2) and that it comprises mainly non-Glomeromycota fungi (Fig. 3). The PCR product of Petunia plants that were little colonized by AMF (high P supply) also forms at similarly “late” PCR cycles, but in that case this primer pair amplified mostly Glomeromycota sequences. Together this suggests for mycorrhizal plant species that AMG1F and AM1 primers specifically amplify Glomeromycota members even at low levels of AMF colonization. This observation is supported by the in silico analysis with the SILVA database which reveals that specific amplification is expected up to two mismatches to these primers (Fig. S4). Therefore, we think that the high level of detected Chytridiomycetes in the non-mycorrhizal Arabidopsis samples is likely because of abundant Olpidium brassicae (classified as Chytridium depending on the taxonomy and database, Lay et al. 2018) as previously reported in these roots (Bodenhausen et al. 2019).
In summary, the primer pair AMG1F and AM1 satisfies the requirement of covering the majority of AMF without amplifying host DNA which is necessary for a molecular method that quantifies total AMF root colonization.
Independent method validation
A further requirement of a molecular method that quantifies AMF root colonization is that it should recapitulate the observations and findings from classical microscopy. In this study, we compared the relative qPCR approach to the traditional microscopy method, and we discussed the strengths and weaknesses of both methods (Table 1). We found good agreement in sample-to-sample comparisons of the microscopy quantifications with the results from relative qPCR (Fig. 2). The major difference was that qPCR showed less agreement at low root colonization levels than at high root colonization. This could be due to stochastic amplification during PCR at low DNA concentration. On the other hand, it could be due to enhanced resolution of qPCR at low colonization. For this reason, qPCR potentially can differentiate better between different intensities of colonization (as quantified by Trouvelot et al. 1986) compared with the classical microscopy method (as quantified by McGonigle et al. 1990). Independent method validations were successful for qPCR approaches that target single AMF species (Alkan et al. 2004; Isayenkov et al. 2004; Gamper et al. 2008; Werner and Kiers 2015), and here we show this is also true for the primer pair AMG1F and AM1, which depicts almost the entire AMF community.
The main advantage of the qPCR method compared with the traditional microscopy method is that results are not biased by observers. Quantitative PCR permits determination of the overall level of AMF root colonization, but it does not permit quantification of different fungal structures (e.g., arbuscules, vesicles, or hyphae), which give insights into fungal physiology, as can be done with microscopy, nor does it permit distinction between frequency and intensity of colonization (Trouvelot et al. 1986). On the other hand, beginners or experienced researchers may differ in their recognition of different fungal structures. Even in the original article describing the magnified intersection method (McGonigle et al. 1990), the authors advised caution by stating “Most observers either overestimated or underestimated both proportions. By being consistent in this way an observer will correctly detect relative levels of colonization across experimental treatments, but these data will be observer-dependent and so should not be directly compared across experiments conducted by different researchers”. Because no individual enumeration of fungal structures is performed, qPCR results can be compared among different experiments even if different people processed the samples.
We see the scalability of the qPCR approach as a further advantage because many samples can be analyzed in relatively little time. We illustrate the time needs of both methods with the example of the pot experiment we analyzed (Table S2): For microscopy, samples need to be stained (ca. 3 h for 20 samples), then tiny root fragments need to be carefully mounted on a microscope slide (ca. 10 min per sample), finally at least 100 intersections need to be counted for each sample (ca. 15 min per sample). For the qPCR method, manual DNA extraction for 20 samples takes maximally 2 h, and 15 samples can be analyzed in one qPCR run, using a 96-well plate system and two primer pairs in triplicates, leading to ca. 3 h/run for preparation and analysis. Based on these rough estimates, the qPCR method takes about 2 times less time than the traditional microscopy method (Table S2).
In conclusion, a qPCR method that quantifies AMF root colonization is attractive compared with classical microscopy because the results are not observer dependent and because it saves time.
Advantages of relative normalization
The qPCR primer pair AMG1F and AM1 was previously utilized for absolute quantification based on a standard curve (Hewins et al. 2015). In this study, we show that these primers also function to quantify AMF in Petunia roots with relative data normalization. We do not aim to directly compare absolute vs. relative data normalizations because both approaches have advantages and disadvantages depending on the research question and needs, so the choice of method depends on pragmatic reasons. For example, absolute quantification is appropriate for single AMF species analyses or when comparing colonization across different plant species. However, for multi-species quantification and comparative research questions—e.g., comparisons between treatments or plant genotypes—relative quantification has advantages related to the normalization of starting material and the nature of the DNA standard which are described in the following paragraphs.
The first advantage of the relative qPCR method is that there is no need of normalization with the starting material. Absolute quantification relies on highly accurate sample normalization, for example, of the amount of starting root material. Moreover, calculations can be affected by down-stream variation as, for instance, introduced by variable DNA extraction efficiencies of different sample types. A solution to this problem is to spike the sample with foreign DNA before DNA extraction and to perform a separate qPCR with a primer pair targeting this foreign DNA in order to quantity the DNA recovery factor (Thonar et al. 2012). Therefore, an advantage of relative quantification is that there is no need for normalization of the starting material or performing corrections for extraction recovery.
The second advantage of the relative qPCR method is that no DNA standard is required. A representative standard DNA is virtually impossible when quantifying mixtures of unknown microbes in environmental samples (Brankatschk et al. 2012). Standard curves are typically derived from a single cloned AMF sequence, and this permits the accurate quantification of the single AMF species from which the sequence clone was derived. However, the qPCR signal of such a standard curve might differ greatly from the signal of AMF mixtures present in environmental samples. On the other hand, relative quantification requires twice as many qPCR reactions, which will become costly with many samples. Each DNA extract needs to be assessed for both the AMF and the plant targets so that half a qPCR run will be filled with the AMF and the other half with the plant gene reactions. Moreover, if quantifying AMF root colonization across several plant genotypes, one first needs to establish that the primers for the plant gene perfectly match the genomic sequences of the locus in all compared genotypes so that they will amplify all genotypes with the same efficiency. In our case, a first version of the designed primer of the pair targeting the plant had a mismatch to the target sequence of one of the Petunia genotypes, so we had to design a second version to amplify the GAPDH gene in both Petunia lines with the same efficiency. Similarly, if the method were to be used for other plant species, we recommend validating the “non-specificity” of the PCR primers AMG1F and AM1 to the rRNA gene sequences of the plant species of interest by sequencing PCR products.