Appropriate nonmycorrhizal controls in arbuscular mycorrhiza research: a microbiome perspective

Abstract

Establishment of nonmycorrhizal controls is a "classic and recurrent theme" in mycorrhizal research. For decades, authors reported mycorrhizal plant growth/nutrition as compared to various nonmycorrhizal controls. In such studies, uncertainties remain about which nonmycorrhizal controls are most appropriate and, in particular, what effects the control inoculations have on substrate and root microbiomes. Here, different types of control and mycorrhizal inoculations were compared with respect to plant growth and nutrition, as well as the structure of root and substrate microbiomes, assessed by next-generation sequencing. We compared uninoculated ("absolute") control to inoculation with blank pot culture lacking arbuscular mycorrhizal fungi, filtrate of that blank inoculum, and filtrate of complex pot-produced mycorrhizal inoculum. Those treatments were compared to a standard mycorrhizal treatment, where the previously sterilized substrate was inoculated with complex pot-produced inoculum containing Rhizophagus irregularis SYM5. Besides this, monoxenically produced inoculum of the same fungus was applied either alone or in combination with blank inoculum. The results indicate that the presence of mycorrhizal fungus always resulted in stimulation of Andropogon gerardii plant biomass as well as in elevated phosphorus content of the plants. The microbial (bacterial and fungal) communities developing in the differently inoculated treatments, however, differed substantially from each other and no control could be obtained comparable with the treatment inoculated with complex mycorrhizal inoculum. Soil microorganisms with significant biological competences that could potentially contribute to the effects of the various inoculants on the plants were detected in roots and in plant cultivation substrate in some of the treatments.

Sympatric diploid and tetraploid cytotypes ofCentaurea stoebes.l. do not differ in arbuscular mycorrhizal communities and mycorrhizal growth response

“…When interpreting the observed growth depression in the mycorrhizal treatment, we also need to remember that it is virtually impossible to produce nonmycorrhizal controls with identical composition of microbial communities as in the mycorrhizal treatment (Gryndler et al., ). We therefore cannot completely rule out two alternative explanations considering possible differences in the composition of non‐AMF microbial communities in both inoculation treatments.…”

“…The filtrate was prepared by filtration of a nonsterile soil suspension (1:10, w/v, 60 s homogenization by immersion blender, 1 h on a reciprocal shaker) through filter paper with a pore size of 15 μm to remove AMF propagules. We are aware that this approach does not allow separating organisms of similar or larger size than AMF spores such as representatives of micro‐ and mesofauna (Wagg et al., ); nevertheless, despite many efforts, there does not seem to be a way to establish a nonsterile cultivation experiment in which the treatments differ only by the presence of mycorrhizal fungi (Gryndler et al., ). Plants were cultivated in the greenhouse under natural daylight conditions, positioned randomly on the bench, and watered with distilled water to saturation as required.…”

Correlative evidence for co-regulation of phosphorus and carbon exchanges with symbiotic fungus in the arbuscular mycorrhizalMedicago truncatula

“…Mycorrhizal inoculum was obtained from pot culture of an AM fungus Rhizophagus irregularis strain SYM5 (described in Gryndler et al 2018), which was produced in the glasshouse for 8 months prior to the experiments described here, with leek ( Allium porrum ) as a host plant. Leek roots from the inoculum pots were harvested and cut mechanically into small pieces (< 1 cm) and then mixed back into the potting substrate from the inoculum production pots (which was the same as the potting substrate used in the experiments described here).…”

Interactions between functionally diverse fungal mutualists inconsistently affect plant performance and competition

“…Although previous studies have shown that the diversity of belowground mutualists can relax plant competition, these studies used fungal mutualists from the same functional group (AM fungi only) and plants of different functional groups (Wagg et al 2011a, b). We reiterate that our experimental design did not allow for disentangling the effects of other soil microorganisms that could have been present in our mycorrhizal inoculum, however, a recent study pointed that no proper control is feasible when using complex mycorrhizal inoculum (Gryndler et al 2018). Nevertheless, we highlight that the effect of fungal-fungal interactions on plant communities are likely to be moderated by the functional identity of mutualist fungi and how they form partnerships with the co-occurring closely-related plants.…”