The effectiveness of the artificial inoculations was confirmed by the fact that the five 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 influence 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 influence 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 influence of these other microorganisms groups in the response given by fungal endophytes.
It is also important to note that the experiment was first 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 influence of the endophyte inoculation on the studied parameters can be demonstrated under those conditions, it might be also important to evaluate it under field conditions in order to contrast their consistency, as it is known that endophytes performance is clearly dependent on the environmental conditions (Ahlholm et al. 2002). In this case, during the study years, in the field, 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 (Assuero et al. 2006).
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 (Maciá-Vicente et al. 2009; Oliveira Silva et al. 2009; Leyte-Lugo et al. 2013; Perveen et al. 2017; Zheng et al. 2017). 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 (identified 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. (2016) with different Sporormiella species reducing the herbage yield of Bromus tectorum L. The inoculation with endophyte E408 (Byssochlamys spectabilis) also caused a significant 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-field 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 (Ismail et al., 2018) 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 (Brader et al. 2017). 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 significant) in this sense in the field experiment. This fungus has already been considered as a plant growth promoter by Hyakumachi and Kubota (2003). In such a case, its mechanism of action may be related to its capacity to induce resistance to host plants against diseases (Horinouchi et al. 2008). 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 influenced by its host preference. The meta-analysis made by Mayerhofer et al. (2013) 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 significantly the protein level. The neutral and acidic detergent fibre (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 (Newman and Newman 1992). Consequently, a decrease in the fibre 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. (2015) found significant variations on NDF and lignin content for Lolium perenne when Epichloë endophytes were inoculated, but not on ADF. However, Rodrigo et al. (2017) did not find differences in the fibre content of Lolium rigidum forage when plants were inoculated with the endophyte E408. Once again, the influence of the host preference, and the specific interaction endophyte-plant host, might determine the observed response.
Rasmussen et al. (2012) 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 fibre content. Also, some endophytes can synthetize 1-aminocyclopropane-1-carboxylate (ACC) deaminase (Zabalgogeazcoa et al. 2006; Chaturvedi and Singh 2016; Ali et al. 2019) the immediate precursor of ethylene in plants. These molecules could delay plant maturity, prolonging its vegetative growth stage (Santamaria et al. 2018). Thus, endophytes could act as a plant-growth cycle regulator. Considering that fibre content increase with the growth stage of the plant (Santamaria et al. 2014), such a delay caused by the endophyte may produce a lower fibre content, improving thus forage digestibility.
The interpretation of this effect becomes more complicated when the results of the in-field 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 (Nanda et al. 2019), under the favourable environmental conditions of the greenhouse, the endophyte might respond by producing substances which may enlarge the cycle length. However in the field, 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 (Santamaria et al., 2014). 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-field 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. (2018) or Rasmussen et al. (2007).
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 (Cigler et al. 2010; Lyu et al. 2017), 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 field 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 (Lamhamdi et al. 2013). 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 field 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. (2005) 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 significantly 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 deficiency affects more than 20% of the world’s population, being Zn deficiency one of the most important factors causing disease or death in the world (Sauer et al. 2016). Such a deficiency 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 (Hotz and Brown 2004). 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 biofortification programs of crops growing in Zn deficient 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 influence 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.