Effect of Fungal Endophytes on Plant Growth and Nutrient Uptake in Trifolium Subterraneum and Poa Pratensis as Affected by Plant Host Specicity

The introduction of well-adapted species, such as Trifolium subterraneum and Poa pratensis, might enhance the forage yield and quality of dehesas pastures for feeding livestock. However, the climatic hardness and poor soils in these agrosystems may limit plant establishment and development. Since fungal endophytes have been found to alleviate the environmental stresses of their host, the aim of this study was to assess the effect of ve isolates on forage yield, nutritive value and plant mineral uptake after their inoculation in the two above-mentioned plant species. Two experiments were established (under greenhouse and eld conditions) using plants inoculated with two isolates in 2012/2013 (Epicoccum nigrum, Sporormiella intermedia) and three isolates in 2013/2014 (Mucor hiemalis, Fusarium equiseti, Byssochlamys spectabilis). Thus, F. equiseti (E346) increased the herbage yield of T. subterraneum under greenhouse conditions, B. spectabilis was found to improve the forage quality of T. subterraneum by reducing bre content and of P. pratensis by increasing crude protein. Also, S. intermedia increased the mineral uptake of Ca, Cu, Mn, Pb, Tl and Zn in subclover and M. hiemalis, the uptake of K and Sr in Kentucky bluegrass. These results evidenced the potential of the studied fungal endophytes to enhance herbage yield and the forage nutritional value, although further studies should include all of the intended forage species as certain host specicity in the effect was observed.


Introduction
Dehesas are Mediterranean agrosilvopastoral systems located mainly in the southwest of the Iberian Peninsula, characterized by a steadiness between production and conservation [38,62]. Extensive livestock grazing is usually the main activity and it plays a key role in Correctly evaluated, the optimization of the plant-endophyte interaction may become an important pathway to increase productivity, since these effects could be obtained in a wide variety of plant species [49,52]. However, the effect of an endophyte on a particular plant host may be variable [16,39] depending on the endophytic species, the host genotype and environmental conditions [1]. Consequently, it should be conveniently outlined the particular outcomes of the association between speci c fungal symbionts and plants [7] in order to get the widest range of application. This would be particularly important in natural grasslands, such as dehesas, and other polyculture systems, where the speci c variability of plants is quite high, and where not very closely related species, such as grasses and leguminous species, co-occur.
Several studies concerning the effect of endophytes on a single pasture host under the same environmental conditions have already been conducted in Poa pratensis [31], Trifolium subterraneum [32] and another legume species such as Ornithopus compressus [56], but they were carried out with a different set of fungal endophytes and the effect of the host was not considered. Therefore, in order to nd a wider number of fungal endophytes with positive effects on the general performance of plant hosts and at the same time to evaluate the speci city of these eventual effects, the objective of the present study was to assess the effect of the arti cial inoculation with each of ve different species of non-clavicipitaceous endophytes on the forage yield (herbage and root biomass), quality traits and nutrient uptake in two important and not very taxonomically related forage crops, such as a leguminous species (Trifolium subterraneum) and a gramineous species (Poa pratensis).

Fungal and plant material
For the inoculations, ve fungal endophytes (Table 1), previously isolated from different pastures species of dehesas from Extremadura (Southwest of Spain), were selected either, because they had been quite frequently isolated from several pasture hosts, a fact which could re ect an eventual important ecological role, or based on the observation of some interesting properties related to plant protection in previous experiments carried out in our laboratory, such as antagonism against fungal pathogens or the production of several secondary metabolites (data not shown). These endophytes had been previously identi ed by morphological characteristics, when possible, and by the comparison of the ITS region sequence with sequences from EMBL/GenBank (www.NCBI.nlm.nih.gov) and UNITE (https://unite.ut.ee. Version 8.3) [43] databases using a BLAST search. A more detailed explanation about how the species assignation was made when multiple accessions or ambiguous results were found, and other aspects about identi cation, can be obtained from Lledó et al. [33] and Santamaría et al. [57]. Table 1 Origin and identi cation of the endophytes used in the experiments. For each endophyte, the plant host from which the fungal strain was originally isolated and the Genbank identi cation data are shown. each fungus was grown at 25°C in 1.5 L asks containing 1 L of Potato Dextrose Broth (PDB). The asks were kept in the dark during two months before the inoculation, being shaken manually for 5 minutes every 3 days.
Inoculations were carried out in two-month old seedlings of T. subterraneum cv 'Valmoreno' and P. pratensis cv 'Sobra'. In order to obtain the plant seedlings, seeds of each species were rst surface disinfected by immersion for 5 and 2 minutes respectively in 2.5 % NaClO, followed by three washes with sterilized distilled water. Five individual surface sterilized seeds per plant species (N = 10 total samples) were randomly selected and imprinted onto fresh potato dextrose agar (PDA, Scharlau, Spain) as a way to verify the surface sterilization e ciency, and no outgrowing fungal colonies were observed. After that, ve sterilized seeds per pot for T. subterraneum and ten seeds for P. pratensis were sown in 7x7x6 cm plastic pots containing autoclaved (twice during 1 hour at 121º C) soil substrate consisting of a 1:  The sowing date was in early December in both study years. After that, pots were placed in a greenhouse and watered to eld capacity every 2 to 3 days. Environmental conditions in the greenhouse, such as maximum and minimum temperatures and relative humidity, were monitored throughout the entire length of the experiment (Fig. 1).
Before the endophyte inoculation, plants were treated three times with a systemic fungicide, Amistar Xtra® (20 g of Azoxystrobin and 8 g of cyproconazole each 5 L; Syngenta, Madrid, Spain). The aim of this treatment was to remove any pre-existing fungus within the plant that could limit the colonization of our selected strains or interact somehow with them, altering the results. Thus, the rst treatment was made when plants were 1 month old, and then again, every 10 days as a foliar spray (approx. 1 mL per pot of a dilution of 1 mL fungicide in 1 L distilled water). With the third application, 1 mL of fungicide solution was also added to the soil substrate in each pot. The effectiveness of the fungicide treatment was evaluated just before inoculations by taking randomly four plants and analysed in the laboratory. Five mm-long segments were cut from different parts of each plant, surface disinfected and placed on PDA amended with 50 mg L -1 chloramphenicol (to avoid bacteria development) in a Petri dish. After two months of incubation at 25ºC in the dark, no colonies of fungi were observed growing out of any of the plant segments.

Inoculations
Inoculations were performed ve weeks after the last fungicide application. Just before inoculations, plants were wounded by using a home-made tool that allowed puncturing their leaves and stems to facilitate fungal infection without serious plant damage. Then, a homogenized blended mix of inoculum was applied by means of a hand sprayer to all the plants in two doses: one dose just after plant wounding, and the second one 3 days later. Then the inoculated seedlings were maintained in a high humidity atmosphere during the 48h after the application in order to favour infections. The viability of the mycelium after blending was evaluated in vitro, by verifying the growth of new colonies on Petri dishes containing PDA sprayed with the blended mix. In order to check the effectiveness of the inoculations, approximately 1 month after the inoculations, plant samples of each treatment were taken to the laboratory for re-isolation, which was performed by using the same procedure followed to evaluate the effectiveness of the systemic fungicide described above. After the re-isolation process, the ve endophytes were positively re-isolated and identi ed in culture medium. This might mean that the inoculation method was su ciently effective to cause infection, and consequently the differences observed between treatments could be attributed to the inoculation.
Each year, one half of the pots containing inoculated plants were kept in the greenhouse (i.e. a total of 60 pots for each plant host), arranged on benches by following a completely randomized design, separated at least 5 cm apart to avoid cross-contamination. Plants were watered to eld capacity every 2 to 3 days. In order to evaluate eventual pathogenic behaviour of the endophytes used in the present study on the studied hosts, these plants were visually examined ve times every three weeks (starting three weeks after inoculation) to check for disease symptoms (yellowing, drying, rotting leaves, blackish spots, etc.). According to the severity of these symptoms, on average the plants in each pot were assigned to one category on the following scale: 1 = healthy, 2 = slightly affected, 3 = moderately affected, 4 = severely affected and 5 = dead. Disease progress curves for each pot were constructed by plotting the values of disease severity over time. The area under the disease progress curve (AUDPC) was calculated as the sum of the area of the corresponding trapezoids, considering one unit per period between two consecutive measurements. The AUDPC was used as response variable to evaluate disease severity. Three months after inoculations, herbage and roots were harvested and taken to the laboratory for processing.
To evaluate the consistency of the eventual effects caused by the endophytes on the host plants, as they have been shown to be dependent on the environmental conditions [1], the second half of the pots (the remaining ve repetitions) were transported to the eld approximately one month after the inoculations. In 2012/2013, only plants of T. subterraneum could be transplanted in the eld, while in 2013/2014, plants of both species were used for this part of the assay. In both years, the experimental area was in the Dehesa Valdesequera, owned by the regional government of Extremadura and located in Badajoz, south-west Spain (UTM Coordinates, Zone 29 North Datum: X = 685,365 m; Y = 4,325,603). Four soil samples 30-cm depth of the experimental area were taken to the laboratory to determine their properties by using the same procedures followed for the substrate. The values can be observed in Table 2. Climatic data during the experiment, which were taken from a weather station located close to the study site, are shown in Fig. 1. Before transplanting, in late February of each year, conventional tillage was applied to prepare an appropriate environment for plants. The experimental units (a set of ve plants) were arranged by using a randomized design with a planting layout of 50 cm×50 cm. After transplanting, seedlings were maintained without further fertilization or irrigation. Two months and a half after transplanting, herbage was harvested and taken to the laboratory for processing.

Yield and quality determinations
After harvesting, plant samples were oven dried at 70°C until constant weight for dry matter (DM) of herbage and root biomass (from greenhouse) determination. The dried samples, after grinding, were also used to determine the main quality parameters. Thus, total N content was analysed by the Kjeldahl method (Kjeltec™ 8200 Auto Distillation Unit. FOSS Analytical. Hilleroed, Denmark) and used to estimate crude protein (CP) by multiplying the biomass N by 6.25 [64]. O cial procedures [3] were followed to determine neutral detergent bre (NDF), acid detergent bre (ADF), and acid detergent lignin (ADL) by means of a bre analyzer (ANKOM8-98, ANKOM Technology, Macedon, NY). Total ash content was determined by ignition of the sample in a mu e furnace at 600°C, according to the o cial procedure [3].

Results
Effects of endophytes on disease severity, biomass yield, quality parameters and mineral content in the greenhouse experiment Although the area under the disease progress curve (AUDCP) was signi cantly affected by the endophyte treatment in 2012/2013 (  Table 3   When the interaction 'inoculated endophyte*host' was analysed (Fig. 2b), signi cant differences (P ≤ 0.05) in the herbage and root biomass yield of the host caused by the inoculation were only found on Trifolium subterraneum. In 2012/203 for that host, in relation to HDM, such differences were negative, as the inoculation with endophyte E636 caused a decrease of around 36% in the herbage dry matter in comparison with control plants (Fig. 2b). During 2013/2014, however, the inoculation of subclover with E346 did increase the herbage biomass with respect to their control. In relation to RDM, the inoculation with endophyte E179 increased the root biomass yield in plants of T. subterraneum when comparing with non-inoculated plants (Fig. 2b).
In general, the in uence of the endophyte inoculation on the nutritional quality traits was quite limited. In 2012/2013 a signi cant in uence of the endophyte, in terms of either main effect or the interaction with the host, was only found for the acid detergent bre (ADF) and the ash content in the herbage, respectively (Table 3). In such a case, the endophyte E636 caused a decrease of 9.3% in the ADF percentage in comparison with control (Fig. 3c). Regarding the ash content, the inoculation with either, endophyte E179 or E636, produced plants with signi cantly lower values for Trifolium subterraneum, 0.26 and 0.27% respectively, in comparison with controls, which accounted 0.43%. No differences were observed for Poa pratensis for this parameter. In 2013/2014, the interaction between the endophyte inoculation and the plant host species affected signi cantly the neutral detergent bre (NDF). Thus, the inoculation with E408 (B. spectabilis) caused a decrement of a 10% in the plants of Trifolium subterraneum with respect to the uninoculated ones (Fig. 3b).
Regarding the nutrient uptake, only the main signi cant effects of the endophyte inoculation on the concentration of minerals in herbage are shown in Table 4 (the whole set of results can be found in Appendix A, Table S1). Thus, results in the greenhouse showed that the fungal inoculation affected the plant uptake of Ca, Cr, Mo, Na, Pb and Tl, when considered as main effect, and Ca, Cr, Cu, Mn, Na, P, Pb, S, Ti, Tl and Zn, when considering its interaction with the plant host. The inoculation with Sporormiella intermedia (E636) caused a signi cant increase in the uptake of Ca, Cu, Mn, Pb, Tl and Zn by the plants of T. subterraneum and a decrease in the uptake of Cr in this same host plants (

Effects of endophytes on biomass yield, quality parameters and mineral content in the eld experiment
Results from the eld experiment showed no signi cant effect of the endophyte inoculation in the herbage yield in any of the years (Table 2). Field dry matter (FDM) for T. subterraneum ranged from 4.18 g unit − 1 in the control to 5.23 g unit − 1 when E179 was inoculated in 2012/2013, and from 15.72 g unit − 1 to 17.88 g unit − 1 when E179 and E346 were inoculated in 2013/2014, respectively. In the case of Poa pratensis, FDM ranged from 1.59 g unit − 1 to 2.27 g unit − 1 when E346 and E408 were inoculated in 2013/2014 (Fig. 1a).
Regarding the in uence on the quality traits, the fungal inoculation, as main effect, affected signi cantly (P < 0.05) ADF and ADL in the rst year (2013/2014). The crude protein (CP) was also affected by the fungal inoculation in 2013/2014, but only considering the interaction with the host. In 2012/2013, the inoculation with E636 increased the ADF content of the herbage by around a 15% and the ADL by around a 79.06% with respect to the control ( Fig. 3c; 3d). On the other hand, in 2013/2014, when the interaction 'inoculated endophyte*plant host' was analysed, it was observed that the inoculation with the endophyte E346 (Fusarium equiseti) caused a decrease in the protein content from 17.75-15.70%, but only in T. subterraneum. The same effect was observed when Byssochlamys spectabilis (E408) was inoculated in the same host, although such a response was completely opposite when it was inoculated in P. pratensis, increasing the crude protein content around a 9% (Fig. 3a).
As in the greenhouse experiment description, in relation to the in uence of the endophyte inoculation on the mineral accumulation in herbage, here also the main signi cant effects are only shown in this case in Table 5 (the whole set of results can be found in Appendix A, Table S2). In 2012/2013, the uptake of Cu, P, Tl and Zn by T. subterraneum (only species in the eld experiment during that year) was signi cantly affected by the inoculation with the endophyte E636, causing a lower uptake of P (-33.33%) and Zn (-23.51%) but increasing the uptake of Tl in a 45% (Appendix A; Table S2). In 2013/2014, the uptake of B, Cu, Na, S and Zn was signi cantly affected by the endophyte inoculation when it was considered as main effect (  Cu (mg kg − 1 ) K (g 100 g − 1 ) Mg (g 100 g − 1 ) Na (g 100 g − 1 ) S (g 100 g − 1 ) Considering the interaction 'inoculated endophyte*plant host', the uptake of B was signi cantly increased by the inoculation with E063 (+ 26.73%) and E408 (+ 19.74%) only in T. subterraneum ( Table 5). The inoculation with E063 also increased the Trifolium uptake of Cr by a 75%, while the inoculation with E408 caused an increase in the uptake of Cr, K, Mg and Sr (33.71%, 12%, 16.67% and 18.82%) in P. pratensis. Finally, plants of Poa pratensis inoculated with E346 presented a higher concentration of K (+ 10%) and Sr (+ 18.82%) in relation with controls (Table 5).

Discussion
The effectiveness of the arti cial inoculations was con rmed by the fact that the ve fungi used were successfully re-isolated from the inner tissues of host plants. Therefore, it may be reasonable to suggest that the selected endophytes colonized the internal tissues of both pasture species, T. subterraneum and P. pratensis, and consequently the different effects found on the parameters analysed after inoculations could be mainly attributed to the endophyte inoculated. In addition, no other colony was re-isolated from any of the plant samples after the fungicide application. Consequently, the in uence of the naturally occurring microbiome in the plant could be considered as mostly removed, eliminating then any interaction with the inoculated organisms or cross-effects which could bias the results. The eventual in uence of other organisms, such as endophytic bacteria, was not considered in the present study with the hypothesis that the interaction degree between those two unrelated groups might be low. In fact, chloramphenicol was used in culture media to limit the bacteria development, but their occurrence into plants might be of course very plausible. Nevertheless, further studies should include this aspect to establish the real in uence of these other microorganisms groups in the response given by fungal endophytes.
It is also important to note that the experiment was rst performed in the greenhouse in order to assess the endophytes behaviour under the most standard and controlled conditions as possible, reducing the number of variables that could affect their performance. Nevertheless, once the in uence of the endophyte inoculation on the studied parameters can be demonstrated under those conditions, it might be also important to evaluate it under eld conditions in order to contrast their consistency, as it is known that endophytes performance is clearly dependent on the environmental conditions [1]. In this case, during the study years, in the eld, the relatively high temperatures and low rainfall during April and May might introduce stress factors for plants. This should be taken into account in the interpretation of the results as it is known that fungal endophytes often improve the performance to the plants when those are subjected to biotic or abiotic stresses [4].
According to the evaluation of the disease incidence showed by the endophytes studied, none of the isolates produced any external signs of disease, at least during the experiment duration. Therefore, their potential to act as pathogens could be discarded, validating the premise of their role as endophytes. This aspect was supported by the fact that all of them have been previously described as endophytes before [30,36,45,61,70]. This is an important issue in order to use the studied endophytes in eventual future applications as plant growth promoters or as biological agents to improve the performance of forage crops or the nutritional quality of their herbage. Nevertheless, further studies including the evaluation of the production by the endophytes of eventual toxic compounds for livestock should be performed before a commercial use of endophyte-based products.
Regarding herbage yield, although inoculations did not have effect on the herbage biomass in P. pratensis when compared to the control, the inoculation with the endophyte E636 (identi ed as Sporormiella intermedia) produced a decrease in the herbage yield in T. Subterraneum in relation to controls under greenhouse conditions. Similar results were found by Newcombe et al. [41] with different Sporormiella species reducing the herbage yield of Bromus tectorum L. The inoculation with endophyte E408 (Byssochlamys spectabilis) also caused a signi cant decrement in the biomass yield (in both HDM and RDM) in comparison with controls of about a 11% and a 14%, respectively. This result was completely opposite to that obtained in the study conducted by Santamaria et al. (2018) where, the inoculation with the same strain of this endophyte, caused an increment of ~ 42% in the herbage yield of Ornithopus compressus with respect to the control under the exact same in-eld conditions. This fact might evidence that the effect of endophytes on plant growth might not be only dependent on the fungal species inoculated, as it has been previously stated [24,29] but also on the plant host where it is inoculated and the interaction that it establishes with the fungus. This inconsistency in the observed effect could be due to a different nature of the interaction fungus*plant host, playing a role as endophyte in some cases or as pathogen in others. Although the pathogenic role might be quite secondary in this case as no disease symptoms were observed during the experiment, it could have been the responsible of such a decrease in the biomass yield. This different behaviour of fungi, playing a role of endophyte or pathogen depending on plant host, has been already stated in several cases [21,23]. Further studies should be developed to understand the mechanisms involved in this different behaviour.
Conversely, the inoculation with Fusarium equiseti (E346) produced an increase of about 13% in the herbage yield of T. subterraneum under greenhouse conditions, and a positive trend (although no signi cant) in this sense in the eld experiment. This fungus has already been considered as a plant growth promoter by Hyakumachi and Kubota [23]. In such a case, its mechanism of action may be related to its capacity to induce resistance to host plants against diseases [21]. By improving the general health status of the plant, the endophyte could confer a better performance, being able thus to increase biomass yield. Since in our experiments no symptoms of disease were observed in any of the plants, other mechanisms might have been acting to explain this increase in the plant growth. Further experiments should be performed to elucidate such mechanisms. The endophyte Epicoccum nigrum (E179) had a positive effect on the root biomass of T. subterraneum. Although this higher root development did not produce a higher herbage yield, it could be the responsible of the healthier aspects of the plants inoculated with this endophyte in the disease severity experiment.
Therefore, it seems clear that the expression of the positive effect of the endophyte might be in uenced by its host preference. The metaanalysis made by Mayerhofer et al. [37] showed that the effect of an endophyte inoculation on root biomass tends to be positive when the fungus had been isolated from the same host species and negative or neutral otherwise. This analysis may be corroborated in our study by several cases. For example, Mucor hiemalis (E063), which had a negative effect on the root biomass, much more pronounced in T subterraneum, had originally been isolated from Poa annua, a very much taxonomically related species to the Poa pratensis used in the experiments.
Regarding the quality parameters of the forage, in the greenhouse, none of the endophytes affected signi cantly the protein level. The neutral and acidic detergent bre (NDF and ADF) contents decreased in plants of T. subterraneum inoculated with Byssochlamys spectabilis (E408) and Sporormiella intermedia (E636), respectively. The NDF and ADF contain mostly cellulose and lignin, which are mostly indigestible by non-ruminants [42]. Consequently, a decrease in the bre content caused by the endophyte could be considered as positive, since an animal nutritive value point of view, as it may imply an increase of the forage digestibility. Soto-Barajas et al.
[65] found signi cant variations on NDF and lignin content for Lolium perenne when Epichloë endophytes were inoculated, but not on ADF. However, Rodrigo et al. [51] did not nd differences in the bre content of Lolium rigidum forage when plants were inoculated with the endophyte E408. Once again, the in uence of the host preference, and the speci c interaction endophyte-plant host, might determine the observed response.
Rasmussen et al. [47] stated that the infection of Lolium perenne with a Neotyphodium lolii entailed an "up-regulation of fungal cell wall hydrolases", which could explain the reduction of the bre content. Also, some endophytes can synthetize 1-aminocyclopropane-1carboxylate (ACC) deaminase [2,11,69] the immediate precursor of ethylene in plants. These molecules could delay plant maturity, prolonging its vegetative growth stage [57]. Thus, endophytes could act as a plant-growth cycle regulator. Considering that bre content increase with the growth stage of the plant [55], such a delay caused by the endophyte may produce a lower bre content, improving thus forage digestibility.
The interpretation of this effect becomes more complicated when the results of the in-eld experiment are also considered, because in such a case the inoculation with E636 caused the opposite effect regarding ADF, increasing also ADL in relation to controls. This could be explained if the production of the phytohormone-like substances by endophytes with effects on the life cycle of the plant indicated above, might be modulated by the environmental conditions. Thus, considering that plants and their endophytes prioritize their survival over growth [40], under the favourable environmental conditions of the greenhouse, the endophyte might respond by producing substances which may enlarge the cycle length. However in the eld, the endophyte might detect somehow the high temperatures registered during the 2012/2013 campaign, and might respond by producing phytohormone-like substances which might shorten the vegetative stage of the plant, increasing thus the cellulose and lignin content and decreasing the protein content in the forage, such as it has been indicated previously [55]. The same fact might explain the lower protein content of the plants of T. subterraneum inoculated with endophytes E346 and E408, during 2013/2014.
These facts may impact negatively the suitability of an eventual application of these fungi in this forage crop, especially when the aim is oriented to the cattle feeding, as the crude protein is one of the main nutritive quality parameters of forage. However once again, the effect depended on the combination 'endophyte*host*environment', as the inoculation with the endophyte E408 (Byssochlamys spectabilis) caused the opposite effect on P. pratensis, increasing the crude protein of the forage under in-eld conditions. This lack of consistency on the effect of endophytes in the crude protein content of the forage has been evidenced in many studies where the results were different in each case, such as those of Santamaría et al. [57] or Rasmussen et al. [48].
Regarding the nutrient uptake, the inoculation with Epicoccum nigrum (E179) caused a lower accumulation of Cr, as well as S in the forage of P. pratensis and Ti in the forage of T. subterraneum in the greenhouse experiment. This effect can be considered negative as Cr and S are essential nutrients for plants and animals. The role of Ti is perhaps less clear, since it may improve plant growth, but higher concentrations might negatively affect the uptake of Fe [12,35], although such a situation did not happen here. However, the inoculation with this endophyte did not alter the mineral content of T. subterraneum in the eld experiment, probably because the effect on the plant uptake may be related to the concentrations in the soil. Thus, in the cases where the mineral concentration in the soil/substrate is not a limiting factor, the effect may go unnoticed.
The effect of the inoculation with the endophyte Sporormiella intermedia (E636) in the greenhouse trial was heterogeneous, as it increased the content of essential nutrients such as the overall uptake of Ca and Mn and Zn in T. subterraneum, but also reduced the overall Cr, Mo and Na, and the Ti content in P. pratensis. However, the main problem found with the inoculation with this endophyte was the increase of the Pb uptake, especially important in the case of the subterranean clover, as it could inhibit the uptake and translocation of other mineral ions [28]. Nevertheless, although this situation might imply the unsuitability of this fungus for its use in a livestock feeding system, it could be successfully used for other applications such as phytoremediation of lead in polluted soils due to the mining activity for instance. In any case, further studies should be made to assess the viability of this option since this effect was not observed in the eld experiment.
During 2013/2014, the effect on the accumulation of mineral content in the forage was in general more positive than in the previous year.
Thus, M. hiemalis (E063) increased the overall uptake of B, Na and Cr in the case of T. subterraneum. Tewari et al.
[66] have already described the capacity of this endophyte for the extraction of Cr(VI) from substrate by biosorption. Thus, this endophyte may increase the concentration of an essential nutrient for both plants and animals in the forage by binding it to the Trifolium subterraneum biomass. Finally, the inoculation with the endophyte E408 increased signi cantly the general concentration of B, Cu, Na, S and Zn in the forage of both host species, as well as the accumulation of Cr, K, Mg and Sr in the forage of Poa pratensis. Although all of these minerals are essential nutrients for plants and animals, the increase of the Zinc is of special relevance since Zn de ciency affects more than 20% of the world's population, being Zn de ciency one of the most important factors causing disease or death in the world [58]. Such a de ciency is mainly due to the poor Zinc concentration in many soils of the world, including those of the present study, which limits adequate Zn levels in the food, main route of Zn supply in humans and animals [22]. Thus, both endophytes, E346 and E408, which caused an increase of the Zinc uptake and later accumulation in the forage, could be further studied to act as Zn accumulators in plants once inoculated, to be used in bioforti cation programs of crops growing in Zn de cient soils.
In conclusion, the results of this study showed the capacity of endophytes to affect the yield and quality parameters of pasture species.
Thus, Fusarium equiseti (E346) increased the herbage yield and Byssochlamys spectabilis (E408) improved forage quality of T. subterraneum. On the other hand, Sporormiella intermedia (E636) caused the increase in the uptake of minerals such as Ca, Cu, Mn, Pb, Tl and Zn together with the total ashes content. Nevertheless, the great in uence of the interaction between a fungal strain and its host species has been then clearly evidenced as the results obtained were completely different for either T. subterraneum or P. pratensis. However, further research is still needed to clarify the mechanisms that affect this interaction in order to optimize its agricultural application.

Declarations
Funding: This research was funded by Project AGL2011-27454, granted by the Ministry of Economy and Competitiveness of Spain (the former Ministry of Science and Innovation) and by the European Regional Development Fund (ERDF).
Con icts of interest: The authors declare no con ict of interest.  Effect of a) endophyte inoculation and b) endophyte x host interaction on biomass yield (herbage dry matter in the eld: FDM; herbage dry matter in the greenhouse: HDM and root dry matter in the greenhouse: RDM) in both years of study. Untransformed data is shown.
Charts indicate means (n = 5) and error bars indicate standard error. Within each analysis, parameter and year, different letters mean signi cant differences between means according to LSD test (p ≤ 0.05). In order to make the differences clearer, a different set of letters was assigned to each case (lowercase letters for HDM in 2012/13, Greek letters for HDM in 2013/14 and uppercase letters for RDM in both years. E0 (control), E063 (Mucor hiemalis), E179 (Epicoccum nigrum), E346 (Fusarium equiseti), E408 (Byssochlamys spectabilis and E636 (Sporormiella intermedia).