Fungicide application

To create a fully factorial experimental design, half of the pots (7 pots) within each of the two soil origins were treated with fungicide to suppress soil fungal communities. The fungicide was applied in February 2009 before experimental setup to suppress the germination of fungal spores prior to sowing the seeds. The fungicide was then applied monthly throughout the growing season (i.e., from March to November) until termination of the experiment in September 2010 according to the manufacturer’s instructions [62]. Monthly application of fungicide in an established grassland was previously shown to have strong effects on soil fungal communities and plant community composition (e.g., [40]). In agreement with Dostálek et al. [40], we used the fungicide Karben Flo Stefes [62] in the experiment. This fungicide contains the same active ingredient (carbendazim) as the formerly used fungicide benomyl, which has not been manufactured since 2001 [63]. Benomyl was previously the fungicide most widely utilized to manipulate AMF communities in field experiments, although it is not specific to AMF and can also influence certain non-target organisms, such as soil pathogenic fungi ([54], [64]) and other organisms, such as bacteria (e.g., [65–68]), or other components of the soil biota (e.g., [69]). Similar to changes in the AMF community, changes in the communities of these organisms could have significant effects on the performances of the plants in our microcosms. To separate the effects of pathogen suppression and the suppression of symbiotic AMF, we conducted an additional experiment with the same fungicide dosage in sterilized soil and the addition of AMF and soil pathogens, as well as their combination. This experiment showed that although Karben also suppressed pathogenic fungi in the soil, the positive effects from this suppression were minimal in comparison with the importance of AMF (Pánková et al. in prep.). Similarly, Newsham et al. ([54]) showed that for overall plant fitness, the positive effects from symbiotic endophytes are more important than those from the suppression of soil pathogens. We thus assume that most of the changes observed are due to changes in AMF communities. To support this expectation, we also evaluated the presence of pathogens and Rhizobia in roots of the plants as described below. However, when interpreting the results it is important to keep in mind that we are, in fact, observing changes in overall soil biota.

A total of 100 ml of the fungicide Karben Flo Stefes was diluted in 3 liters of distilled water and applied to treated pots. The control pots obtained the same amount of water as the treated pots. The dosage used in this experiment corresponded to that used in our previous field study [40].