Mycorrhizal fungi are central to soil fertility and can affect both crop productivity and cropping security (Rooney et al., 2009). Both arbuscular mycorrhizal (AM) and ectomycorrhizal (EM) fungi are known to increase the uptake of nutrients like phosphorus (P) and nitrogen (N) by the host plants, especially in infertile soils. However, their functions and benefits for the host plants might not be equivalent (Jones et al., 1998). For this reason, a change in the proportions of mycorrhizal colonization, or type of mycorrhizal association when the host plant can form dual mycorrhiza, might affect the mycorrhizal benefit of the host plant. The mycorrhizal colonization of plants can be affected by soil nutrient level and water content, site management as well as the diversity of the vegetation composition. An increased host plant diversity is known to influence the nutrient fluxes and the interactions with soil microorganisms (Courty et al., 2011). Endophytic fungi and mycorrhizal fungi can interact in their impact on the plant growth (Rillig et al., 2014). For example, AM fungi were revealed to be able to modulate the impact of endophytic fungi beneficially for the plant growth (Wężowicz et al., 2017).

Although the majority of higher plants form mycorrhizal associations, only a low number is dual mycorrhizal, forming both AM and EM associations (Smith and Read, 2008). Salix belongs to the dual mycorrhizal plant genera, and can also harbor diverse endophytic fungi, which may affect the hosts growth and ecophysiological traits (An et al., 2015). Although Salix spp. were mainly shown to be pre-dominantly EM (Püttsepp et al., 2004; Hrynkiewicz et al., 2010b), the opposite has also been shown with a dominance of AM colonization, e.g., in response to flooding (Lodge, 1989). The ability to change their mycorrhizal association in response to the environmental conditions makes them well-suitable model plants to investigate the changes in the mycorrhizosphere in response to a changed host diversity.

Fast growing Salix genotypes have been cultivated successfully in short rotation coppice (SRC) for a long time, mainly for biomass production for energy purposes in boreal climates (Weih, 2004). They are viewed as a sustainable source of biomass with a positive greenhouse gas balance due to their potential to fix and accumulate carbon (Cunniff et al., 2015). Furthermore, compared to annual systems grown on arable land, the management of perennial Salix stands in SRC leads to decreased mechanical disturbance of the soil and changed biochemical soil properties; along with changes in the abundance and diversity of soil organisms including mycorrhizal fungi (Baum et al., 2009a; Hrynkiewicz et al., 2010a; Fransson et al., 2013; Toju et al., 2013a; Goldmann et al., 2015). In addition, Salix genotype identity can significantly affect the soil enzyme activities at the same site (Baum and Hrynkiewicz, 2006), and Salix stands grown in a floodplain revealed higher activities of the soil enzyme β-glucosidase compared to perennial grassland (Zimmer et al., 2012). Changed soil enzyme activities are considered to be a direct expression of the soil community to metabolic requirements and available nutrients, and can therefore be used as indicators of soil functional diversity. Enzyme activities might be more indicative for ecosystem productivity and stability than the taxonomic diversity of soil microorganisms (Caldwell, 2005). Acid phosphatases and β-glucosidases are hydrolytic soil enzymes involved in P- and C-cycling of the soil and originate from plant roots and microorganisms. Mycorrhizal fungi can significantly increase the activities of these enzymes in the soil (Burke et al., 2011).

In SRC, Salix is usually planted with single genotypes, but this could possibly make them less resistant to pest organisms, diseases and abiotic stress. This is a major concern as a SRC must remain viable for up to 20 years to be profitable (Aravanopoulos et al., 1999). For this reason, an increased host plant diversity within Salix stands would be advantageous, but it will also change the below-ground plant–plant interactions within the stand. The soil ecological consequences of mixed Salix stands on former arable land are scarcely known so far (Hoeber et al., 2017).

We hypothesize that the mixture of genotypes with strong eco-physiological differences changes the mycorrhiza formation and activities in the mycorrhizosphere, based on different root characteristics of the genotypes involved. To test this hypothesis we have studied the mixture of two fundamental eco-physiological different Salix genotypes (S. schwerinii × S. viminalis cv. ‘Tora’ and S. dasyclados cv. ‘Loden’) grown in pure and mixed cultures in a field experiment in Northern Germany. Specifically, we expected (1) a higher diversity of root-associated fungi under mixed culture than in culture with the pure genotypes; and (2) changed fine root growth, abundance of root-associated fungi and enzymatic activities in the mycorrhizosphere under host genotype mixtures resulting from changed competitive conditions for the individual genotypes. Such insights into the overall structure of belowground plant–fungal associations will help to understand mechanisms that regulate the coexistence of eco-physiological diverse dual mycorrhizal plant species.

The present results confirm the significant impact of the Salix genotype on its phenotypical traits (Cunniff et al., 2015) including the mycorrhizal (see Figures 2, 3A) and endophytic (see Figure 3B) fungal colonization and host-specific differences in the fungal root colonization within a plant community as described by Toju et al. (2013b). The soil properties of the test site indicated P-deficiency (see section “Study Site and Test Plants”), which increases the importance of mycorrhizal fungi for the P-supply of the host plants (Smith et al., 2011). Assuming that plant neighbors with a faster growth rate can increase the competition for plants with slower growth, in the present mixture ‘Tora M’ had an advantage over ‘Loden M.’ Both, decreased fine root biomass under ‘Loden M’ grown in a mixture (see Figure 1) and greater similarity of the root traits and soil enzyme activities of the mixture compared with pure cultures ‘Tora P’ or ‘Loden P’ support this assumption (see Figure 5). These results are in line with our hypothesis that fine root growth, fungal abundance and activities are changed under host genotype mixtures, caused by changed competitive conditions for the individual genotypes and thereby changed interactions between them. However, the genotype-effect on the mycorrhizal colonization exceeds the impact of the plant design (pure or mixture of genotypes) on the mycorrhizal abundance (see Figures 2, 3A and Table 2).

We predicted that the increased diversity of host plants in the genotype mixture will increase the diversity of root associated fungi too, since aboveground and belowground diversity can be linked (De Deyn and Van der Putten, 2005). However, we found increased fungal diversity only in endophytic fungi, not in mycorrhizal ones. This increased diversity was associated with increased colonization of the roots with DSE. Generally, low mycorrhizal diversity on Salix found here by only one dominating EM fungal species (Laccaria tortilis; see Table 1) causes problems when interpreting the results. However, the low EM diversity is in line with the results on other genotypes of Salix (Püttsepp et al., 2004; Hrynkiewicz et al., 2012). It might be caused by the genotypic-specificity of this plant genus and additionally by the former arable site-conditions with lack of EM host plants. Therefore, we have focused rather on the ratios of root associations than on total mycorrhizal and endophytic diversity.

For the first time, we disclosed that increased host diversity of Salix genotypes can promote endophytic root colonization and increase soil enzyme activities involved in P-mobilization (see Figures 2–4). These results of a Continental SRC support the assumption of Pérez-Izquierdo et al. (2017) taken for Mediterranean forests, who highlighted the importance of structural shifts in fungal communities for their possible functional consequences. Functional consequences were revealed in an increased enzymatic P mobilization (see Figure 4) in the mycorrhizosphere with increased host plant diversity (mixture). These increased enzymatic activities can be caused directly by fungal impact, but also by the plant impact or most probably by a combination of both.

Dark septate endophytes were previously described to be the dominating root associated fungi under S. caprea on heavy metal contaminated sites (Likar and Regvar, 2013). In agreement with the results of these authors, we also found Cadophora spp. as a very common DSE in the roots of the two tested Salix genotypes at the non-contaminated arable site. This endophyte revealed plant growth promotion in heavy metal contaminated soils (Likar and Regvar, 2013). Also the second most common DSE in the present study (Paraphaeosphaeria sp., see Table 1), revealed plant growth promoting traits on another Salix sp. in heavy metal contaminated soil (An et al., 2015). The endophytic yeast Rhodotorula mucilaginosa, found under mixed Salix growth in the present study, was revealed to be plant growth promoting in Populus (Xin et al., 2009). However, from the present data no statement can be made on the growth response of Salix on the different fungal root colonization and the impact of DSE can vary from growth promotion to growth suppression of host plants (Aguilar-Trigueros and Rillig, 2016).

Increased colonization of the roots with DSE combined with consistent colonization with AM fungi in the mixture (see Figures 1, 3B) were accompanied by increased soil enzyme activities involved in P-mobilization (see Figure 4). Della Monica et al. (2015) assumed, that DSE can be more effective to mobilize organic P, than AM fungi. However, in the present study increased enzyme activities might be stronger affected directly by the changed nutrient competition of the host plants in the mixture. Increased abundance of DSE without suppression of mycorrhiza formation supports that these fungal groups can interact beneficially in a joint host plant (Wężowicz et al., 2017). This might be caused by exudates of DSE, which can even stimulate the growth of mycorrhizal fungi (Scervino et al., 2009). Oppositely to the promotion of DSE in the mixture of different Salix genotypes, Becklin et al. (2012) revealed promoted of EM fungi on Salix in increased plant diversity by combination with herbaceous plants in an alpine meadow. However, these results have in common that host plant diversity changes the fungal root colonization of Salix.

The present data on dual mycorrhizal colonization of AM and EM fungi on Salix clearly confirmed the assumption of Lodge and Wentworth (1990), that these types of mycorrhizal fungi colonize roots with an antagonistic behavior. Interestingly, Chen et al. (2000) found that antagonistic behavior of AM and EM fungi was most severe when the EM fungus Laccaria was present, which was the dominating EM fungus in the present study. Low fungal diversity was observed also in the sporocarp production in other SRCs (Baum et al., 2002).

The slow-growing genotype ‘Loden’ seems to be rather EM dominated, whereas the fast-growing genotype ‘Tora’ seems to promote AM formation (see Figures 2, 3). This might be affected by the different litter quality of these genotypes, since the litter quality affects mycorrhizal communities (Lambers et al., 1998). In a pot experiment, leaves of ‘Loden’ revealed higher phenolic concentrations in the later growth than ‘Tora’ under the same environmental conditions (Baum et al., 2009b). Leaf litter with higher phenolic concentrations was described to support EM formation, whereas low phenolic concentrations were found to promote AM colonization of the host plants (Lambers et al., 1998).

Interestingly, the colonization of the roots with DSE was stronger affected by the host plant diversity than by the genotype-specific differences, which was revealed by an increased colonization of the roots (see Figure 3B) combined with increased species richness (see Table 1) in the mixture. Increased stress tolerance of Salix spp. was previously linked to colonization by several DSE (An et al., 2015). However, root endophytes can also include potential pathogens and each plant species and combination with different fungal strains might respond differently in plant growth (Aguilar-Trigueros and Rillig, 2016). Furthermore, the colonization of roots with DSE is also affected by edaphic properties of the site (Xie et al., 2017). The impact of edaphic differences within the present test site was initially balanced between the treatments by the randomized plot design in the present study. However, the increased colonization by DSE might also lead back to changed nutrient mobilization caused by the changed root growth of at least one of the mixed genotypes (see Figure 1) and indicated by changed enzymatic P mobilization under the mixture (see Figure 4).

Based on our results, it can be concluded that increased genotype diversity in Salix cultivation can lead to changed root competition, fungal association and enzymatic P-mobilization in the mycorrhizosphere. Subsequent investigations should assess the generality of promotion of DSE and P-mobilization in increased host diversity.

MW conceived and designed the field experiment within the frame of ECOLINK-Salix. CB managed the field site Rostock, collected the samples, did soil and root analyses and statistical analyses and wrote the first draft of the manuscript; did all soil chemical and biochemical analyses and wrote part of Section “Materials and Methods.” NV and KH isolated the ectomycorrhizal fungi, created figures and tables and did statistical analyses. KH and SS identified the fungal species. NV analyzed the colonization density of mycorrhizal fungi and analyzed these parts of the results. SH and PF evaluated and discussed the mycorrhizal and endophytic results. All authors edited and revised the manuscript and approved the publication.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.