Potential of Fungal Endophytes Isolated from Pasture Species in Spanish Dehesas to Produce Enzymes under Salt Conditions

Endophytic fungi have been found to produce a wide range of extracellular enzymes, which are increasingly in demand for their industrial applications. Different by-products from the agrifood industry could be used as fungal growth substrates for the massive production of these enzymes, specifically as a way to revalorize them. However, such by-products often present unfavorable conditions for the microorganism’s growth, such as high salt concentrations. Therefore, the objective of the present study was to evaluate the potential of eleven endophytic fungi—which were isolated from plants growing in a harsh environment, specifically, from the Spanish dehesas—for the purposes of the in vitro production of six enzymes (i.e., amylase, lipase, protease, cellulase, pectinase and laccase) under both standard and salt-amended conditions. Under standard conditions, the studied endophytes produced between two and four of the six enzymes evaluated. In most of the producer fungal species, this enzymatic activity was relatively maintained when NaCl was added to the medium. Among the isolates evaluated, Sarocladium terricola (E025), Acremonium implicatum (E178), Microdiplodia hawaiiensis (E198), and an unidentified species (E586) were the most suitable candidates for the massive production of enzymes by using growth substrates with saline properties (such as those found in the many by-products from the agrifood industry). This study should be considered an initial approach by which to further study the identification of these compounds as well as to develop the optimization of their production by directly using those residues.


Introduction
The term endophyte includes microorganisms, mainly fungi and bacteria, which colonize the internal tissues of plants without causing any visible disease symptoms [1]. Although many of these endophytes have been described to have a symbiotic role [2], many others are opportunistic species that are waiting for plant senescence in order to take advantage of the plant tissues' colonization [3]. In the case of endophytic fungi, this colonization is carried out through extracellular enzymes that, together with the production of many other metabolites, are capable of degrading the cell wall of the plant [4] and counteracting its chemical defenses [5,6]. Different groups of these enzymes, such as pectinases, xylanases, cellulases, lipases, proteases, or phenol oxidases have been described to be involved in this process [7]. These compounds have been found to be also involved in the plant immune system by eliciting defense mechanisms against pathogens [8], as well as in the growth status of the host, whereby nutrient uptake through the roots is enhanced [9].
Besides their ecological roles, all these enzymes are valuable supplies that are used in numerous food, pharmaceutical, or paper industries [10][11][12]. For example, pectinases play an important role as fining agents in the juice and wine industries by enhancing the

Fungal Material
Eleven fungal strains, previously isolated from different healthy plants collected from the dehesas of Extremadura (in the southwest of Spain), were selected for the study ( Table 1). The identification of these eleven fungi was first attempted morphologically by means of their reproductive structures, and then at a molecular level through the comparison of their ITS region sequence with those included in two databases, GenBank (www.NCBI. nlm.nih.gov, accessed on 13 December 2022) and UNITE (https://unite.ut.ee, accessed on 13 December 2022), while using a BLAST search [31]. A more exhaustive explanation regarding the identification and the species assignation process can be consulted in the works of Lledó et al. [32] and Santamaría et al. [33]. The endophytic strains were selected according to their frequency of isolation from the original plant hosts or through the observation of bioactivity in preliminary assays [34,35].

Evaluation of Extracellular Enzymatic Activity
The six most used enzymes by industry (i.e., amylase, cellulase, laccase, lipase, pectinase, and protease) were chosen in order to qualitatively evaluate the extracellular enzymatic activity of the selected fungi [36]. For that purpose, a 5 mm diameter plug of mycelia (obtained from an actively growing 7-day-old colony on potato dextrose agar medium; PDA) was placed in the center of a Petri dish containing the specific culture medium necessary to assess the production of each enzyme, as indicated below. Agar plugs without mycelia were placed in Petri dishes with the specific media to be used as a negative control. Once inoculated, the plates, prepared in triplicate, were later incubated for 7 days at 23 • C, as this is considered the optimal growth temperature for the selected fungi. After the incubation period, the formation of a hydrolysis halo around the colony was considered an indicator of enzymatic activity ( Figure 1). In positive cases, the extension of both the colony and the clear area around it were measured to calculate the solubilization index (SI) as SI = (colony diameter + halo zone diameter)/colony diameter [37]. The specific media for the identification of each enzyme activity is described as follows.
fungi. After the incubation period, the formation of a hydrolysis halo around the colony was considered an indicator of enzymatic activity (Figure 1). In positive cases, the extension of both the colony and the clear area around it were measured to calculate the solubilization index (SI) as SI = (colony diameter + halo zone diameter)/colony diameter [37]. The specific media for the identification of each enzyme activity is described as follows. Amylase activity was assessed by using a yeast malt agar medium (malt extract 10.0 g; yeast extract 6.0 g; D-glucose 4.0 g; agar 20 g; in 1 L of distilled water; pH 6.3), which was amended with a 1% soluble starch (Panreac Química SLU, Castellar del Vallès, Barcelona, Spain). After the incubation time, plates were flooded with a 1% iodine solution, which allowed us to identify the clear halo surrounding the colony in the case of positive activity [38].
In the case of cellulase activity, Petri dishes with a yeast malt agar medium were supplemented with 0.5% Na-carboxy-methylcellulose (Sigma-Aldrich, San Luis, MO, USA). After the growing period, plates were first flooded with 0.2% Congo Red (Merck KGaA, Darmstadt, Germany) and then with a 1 M NaCl solution, which allowed us to identify positive cellulase activity through the hydrolysis halo [35,38,39].
Laccase activity was detected by using glucose yeast extract peptone agar medium (glucose 5.0 g; peptone 5 g; yeast extract 3.0 g; agar 20.0 g; in 1 L of distilled water; pH 6.8) with 0.05g 1-napthol L-1 (pH 6.0) (Sigma-Aldrich, San Luis, MO, USA). As the fungus produced the enzyme, the colorless medium turned blue due to oxidation of the substrate [20].
For lipase activity, the endophytes were grown in a peptone agar medium (peptone 10.0 g; NaCl 5.0 g; CaCl2·2H2O 0.1 g; agar 16.0 g; in 1 L of distilled water; pH 6.0) supplemented with 1% Tween 20 (Merck KGaA, Darmstadt, Germany) which was sterilized separately and added before pouring onto the plates. The hydrolysis halo was directly visible as the fungi grew if they exhibited lipase activity [36].
Pectinolytic activity was determined by growing the fungi in a pectin agar medium (pectin 5 g; yeast extract 1 g; agar 20 g; in 1 L of distilled water; pH 5.0). After the incubation period, the plates were flooded with a 1% aqueous solution of hexadecyl trimethylammonium bromide (Panreac Química SLU, Castellar del Vallès, Barcelona, Spain) in order to detect the clear zone that formed around the fungal colony in the case of positive activity [20].
Finally, protease activity was evaluated by using a casein hydrolysis medium (skimmed milk powder 28.0 g; peptone 5.0 g; yeast extract 2.5 g; glucose 1.0 g; agar 20 g; Amylase activity was assessed by using a yeast malt agar medium (malt extract 10.0 g; yeast extract 6.0 g; D-glucose 4.0 g; agar 20 g; in 1 L of distilled water; pH 6.3), which was amended with a 1% soluble starch (Panreac Química SLU, Castellar del Vallès, Barcelona, Spain). After the incubation time, plates were flooded with a 1% iodine solution, which allowed us to identify the clear halo surrounding the colony in the case of positive activity [38].
In the case of cellulase activity, Petri dishes with a yeast malt agar medium were supplemented with 0.5% Na-carboxy-methylcellulose (Sigma-Aldrich, San Luis, MO, USA). After the growing period, plates were first flooded with 0.2% Congo Red (Merck KGaA, Darmstadt, Germany) and then with a 1 M NaCl solution, which allowed us to identify positive cellulase activity through the hydrolysis halo [35,38,39].
Laccase activity was detected by using glucose yeast extract peptone agar medium (glucose 5.0 g; peptone 5 g; yeast extract 3.0 g; agar 20.0 g; in 1 L of distilled water; pH 6.8) with 0.05g 1-napthol L-1 (pH 6.0) (Sigma-Aldrich, San Luis, MO, USA). As the fungus produced the enzyme, the colorless medium turned blue due to oxidation of the substrate [20].
For lipase activity, the endophytes were grown in a peptone agar medium (peptone 10.0 g; NaCl 5.0 g; CaCl 2 ·2H 2 O 0.1 g; agar 16.0 g; in 1 L of distilled water; pH 6.0) supplemented with 1% Tween 20 (Merck KGaA, Darmstadt, Germany) which was sterilized separately and added before pouring onto the plates. The hydrolysis halo was directly visible as the fungi grew if they exhibited lipase activity [36].
Pectinolytic activity was determined by growing the fungi in a pectin agar medium (pectin 5 g; yeast extract 1 g; agar 20 g; in 1 L of distilled water; pH 5.0). After the incubation period, the plates were flooded with a 1% aqueous solution of hexadecyl trimethylammonium bromide (Panreac Química SLU, Castellar del Vallès, Barcelona, Spain) in order to detect the clear zone that formed around the fungal colony in the case of positive activity [20].
Finally, protease activity was evaluated by using a casein hydrolysis medium (skimmed milk powder 28.0 g; peptone 5.0 g; yeast extract 2.5 g; glucose 1.0 g; agar 20 g; in 1 L of distilled water; pH 7.0). A clear zone, directly visible around the colony, confirmed positive activity [39].

Halotolerance Test
To assess the capacity of the fungi to grow under salt stress conditions, Petri dishes were prepared with a PDA medium adjusted to different concentrations of NaCl (100, 200, and 500 mM) [40]. Likewise, plates containing the same medium but without NaCl (0 mM) were introduced as the control. After placing an actively growing plug (ø = 5 mm) of mycelia from each of the endophytes on the center of the plate, its radial growth was measured 9 days later in order to assess the maximum saline concentration that they were able to tolerate. All samples were analyzed in triplicate, and the results were expressed as the cm of radial growth.

Evaluation of Enzymatic Activity under Salt Stress Conditions
Once both the qualitative enzymatic activity and the halotolerance of fungal strains were assessed, another test was conducted in order to evaluate their potential to produce the different enzymes under salt stress. The selected endophytes were placed again in the basic agar media with the specific substrate for the corresponding enzyme, but in this case, they were adjusted to a salt concentration of 500 mM. After the incubation period, the extension of both the colony and the clear area around it were measured to calculate the solubilization index, as indicated above. The test was conducted in triplicate and plates with the specific substrate but without NaCl were used as the negative control.

Statistical Analysis
The results of all the experiments were analyzed with the Statistix v. 8.10 package (Analytical Software, USA). The effect of the endophytic strain on the enzymatic production was evaluated through a one-way ANOVA. The effect of the salt concentration on mycelial growth and the evaluation of the enzyme production under salt conditions were analyzed through a mixed-design analysis of variance (split plot ANOVA). Furthermore, this was achieved by considering the NaCl content and the fungal strain as the main and subplot factors, respectively. A Fisher's protected least significant difference (LSD) test for multiple comparison was used when significant differences (p < 0.05) were found in the tests.

Evaluation of Extracellular Enzymatic Activity
The selection of endophytes included strains of some of the most representative genera of fungi, such as Acremonium, Didymella, Drechslera, Fusarium, Penicillium or Podospora, among others. The screening of their extracellular enzymatic activity showed that all the isolates produced at least two of the enzymes ( Table 2). The endophytes that produced a wider range of enzymes were E064 (Drechslera biseptata), E198 (Microdiplodia hawaiiensis), and E635 (Penicillium chrysogenum), which produced four types of enzymes, three of them being in common: amylases, cellulases, and lipases.

Halotolerance Test
The growth response to saline treatments varied widely depending on the fungal species, as can be observed in Figure 2. Thus, after nine days, two of the eleven strains, E178 (Acremonium implicatum) and E198 (Microdiplodia hawaiiensis), showed a significantly higher radial growth in 500 mM of NaCl than those which were found when grown without salt stress. At the same time, two of the fungi, E168 (Fusarium avenaceum) and E224 (Colletotrichum cereale), showed higher growth under saline conditions, but only at the lower values of NaCl concentrations. On the other hand, six of the isolates (E064, E138, E528, E532, E586, and E635) showed reduced growth when they were incubated in the medium with the highest concentrations of NaCl. In any case, the eleven strains were able to grow, even under harsh saline conditions.

Evaluation of Enzymatic Activity under Salt Stress Conditions
This experiment showed that the solubilization index significantly changed with the salinity of the culture medium for all the parameters, except with respect to the amyloly-

Evaluation of Enzymatic Activity under Salt Stress Conditions
This experiment showed that the solubilization index significantly changed with the salinity of the culture medium for all the parameters, except with respect to the amylolytic activity ( Table 3). The production of amylase was not significantly affected by the salt concentration in any of the isolates (as the salt concentration*endophyte interaction did not significantly affect the production either), with values that ranged between 1.65, for the strain E635 (Penicillium chrysogenum) growing in the non-saline medium, and 2.72, for the strain E064 (Drechslera biseptata). Table 3. Enzymatic production under different salt conditions by the selected strains. The positive results (mean ± se) are shown as the solubilization index ((colony + halo zone diameters)/colony diameter). A summary of the ANOVA (DF: degree of freedom; F values; and level of significance (** p ≤ 0.01, *** p ≤ 0.001)), showing the effect of the endophyte, the salt concentration, and their interaction, is shown for each parameter. Regarding the other enzymes, the effect of salt concentration on the solubilization index changed significantly depending on the fungal strain. In the case of cellulase, four of the eight bioactive strains that produced it (E198, E224, E586 and E635) did not reduce such activity when growing in a saline medium. A better trend was found for the lipolytic activity, where 89% of the strains, i.e., all of them other than Didymella phacae (E532), maintained the production of this enzyme under salt conditions. In the case of pectinase production, the result for the fungus E198 (Microdiplodia hawaiiensis) stood out, since it was able to significantly increase its activity under saline growth conditions (with solubilization indexes of 1.33 and 1.83 for the control and salt-amended media, respectively). The same result was found in the case of proteolytic activity for the endophytes E025 (Sarocladium terricola) and E064 (Drechslera biseptata), with significant increases of 23% and 15%, respectively, when compared with the corresponding controls without salt added. Only in regard to laccases did the salt conditions significantly reduce the enzymatic activity for the three strains. However, even in this case, the three endophytes maintained their capacity to produce such compounds.

Discussion
All the studied fungi produced at least two of the six different enzymes that were analyzed. From them, ≈27% of the strains (3 out of 11) produced three of them, and the same percentage produced four of these compounds. The fact that none of the isolates showed the potential to produce all the tested enzymes is supported by the literature, where it is reported that this outcome is very rare [7,26]. Regarding the frequency of enzyme occurrence, lipases and cellulases were the most frequent enzymes since they were found in ≈82% and ≈72%, respectively, of the selected strains.
The high frequency of lipolytic activity may be considered an expected outcome, as endophytic fungi usually produce these compounds in order to overcome the defense mechanisms of their hosts [41]. This group of enzymes, together with proteases, facilitates the hyphal penetration of endophytes through the plant cell wall [42]. In this sense, three strains could produce both lipases and proteases, i.e., Sarocladium terricola (E025), Drechslera biseptata (E064), and Acremonium implicatum (E178). In addition, ten out of eleven could produce at least one of both groups of compounds. The solubilization index for lipases ranged from 1.10 for E138 (Embellisia leptinellae) to 2.07 for the unidentified strain E586, which are values that are quite similar to those found by other authors who worked with other fungal species [39] and who produced these kinds of enzymes. Therefore, the results obtained in the present study are promising enough to justify being further tested in other different conditions in order to maximize such a type of enzyme production.
Cellulases were produced by 80% of the isolates studied. It is known that cellulolytic activity is widespread among pathogenic and saprophytic microorganisms [43]. Endophytes can behave as both pathogens or as saprophytes at times during their biological cycle. This fact could explain the high proportion of endophytes which produced cellulases in the present study, although the type of relationship established between our fungi and plant host should be further investigated in order to confirm this. This is supported by the results of other studies, where a similar proportion of fungal endophytes producing cellulases was found [27]. Nevertheless, further studies should be performed to confirm this fact as other quite different results have also been found when analyzing other hosts, such as those recorded by Uzma et al. [44], where only ≈28% of the endophytes that were studied were capable of producing cellulase.
Amylolytic activity was found in ≈36% of the isolates, which is a lower frequency with respect to other articles in which the percentage of occurrence of this enzyme ranged from 78% to 100% [8,37,38,45]. This outcome may be related to the fact that fungal amylases occur more commonly in saprophytic genera, such as those found in Aspergillus and Rhizopus [46], which were not present in our study. Among our selection, Penicillium chrysogenum (E635), belonging to a genus of known saprotrophs, was the endophyte which presented a significantly higher amylase concentration, as was also observed by Fouda et al. [38]. This isolate (E635) was one of the most prolific enzyme producers in the present paper, producing four different enzymes. However, no pectinolytic ability was observed, which is in contrast to that which was observed in the aforementioned article. The proportion of isolates with pectinolytic activity in our study (≈27%) was in agreement with the results obtained by Shubba and Srinivas [27], albeit a little bit higher than the 19% that was observed in the endophytes isolated from different medicinal plants of India [44]; moreover, it was also lower than the 49% detected in the fungi that were isolated from Thai orchids [7].
Regarding laccases, only three of our isolates (≈27% of the total) produced them. This is in agreement with the common trend found in other similar studies, where the frequency of fungal endophytes producing this enzyme was also very low [27]. As pointed out by Uzma et al. [44], laccases are able to degrade lignin, which might have a detrimental effect on the plant host in terms of limiting their inter-relationships. Such a feature might be more common in saprophytic species. Therefore, the occurrence frequency of the production of laccases in endophytic species may be very low, appearing only in species that can also act as saprophytes at a specific moment of their life cycle. Therefore, the identification of three different isolates, E138 (Embellisia leptinellae), E528 (Dydimella exitialis), and E635 (Penicillium chrysogenum) with the potential to produce this scarce enzyme may also allow further development of its production for industrial application. In the same way, the results obtained by fungi E064 (Drechslera biseptata) and E198 (Microdiplodia hawaiiensis) should be highlighted, along with E635, due to their great versatility, as these three endophytes were able to produce four of the enzymatic groups that have been mentioned.
Regarding the halotolerance tests, the preliminary hypothesis that plants from the dehesa would be suitable for the identification of endophytes that are capable of growing in relatively harsh environments has been supported by the evidence. In this regard, ≈45% (five out of eleven) of the isolates showed greater mycelial growth in plates that were supplemented with 500 mM NaCl than in those which were in a non-saline PDA medium after nine days. In addition, and more importantly, none of the isolates ceased their growth under saline conditions. These data may also explain the higher enzymatic activity of several endophytes under high salinity conditions. This was the case of E198 (Microdiplodia hawaiiensis) with respect to pectinolytic activity, as well as in E025 (Sarocladium terricola) and E064 (Drechslera biseptata) for protease production. These endophytes may be the most suitable candidates in terms of producing the involved enzymes via by-products generated by other industries with saline properties, such as grape by-products, which are well-known for their salinity and sodicity problems [47], or olive oil mill waste, which may contain up to 2% of salt [48]. This enzymatic activity under saline conditions has also been observed in other endophytes, such as Microsphaeropsis arundinis that are isolated from mangrove trees and which showed a higher ligninolytic activity under saline conditions [49]. For the massive production of such enzymes with these endophytes, the following step may be the evaluation of their production by directly using different by-products as the growing substrate medium in order to confirm their suitability and their optimization of the growth conditions. The specific hydrolytic compounds produced and their quantification may also require further investigation. Other enzymatic activities, such as the production of amylases, although not increased, were not affected by the salinity of the media. Additionally, even in the cases where the production of enzymes was significantly reduced, such as the production of laccases in the three tested isolates, only one endophyte, Fusarium avenaceum (E168), lost its enzymatic activity under saline conditions. Therefore, these endophytes, although not so promising, could also be considered in further steps.

Conclusions
The experimental results evidenced that the fungal endophytes that were isolated from plants naturally present in Spanish dehesas can produce a wide range of enzymes that are greatly valued for numerous industries. Among them, some of the strains-especially Sarocladium terricola (E025), Acremonium implicatum (E178), Microdiplodia hawaiiensis (E198), and an unidentified species (E586)-could be suitable for the production of such enzymes under saline conditions, which may allow the utilization of by-products as growth substrates for large-scale production. However, since the enzymatic activity is somehow modulated by the substrate, this study should only be considered an initial approach, which was conducted in order to continue working on the identification of the compounds and to develop the optimization of their production by directly using those residues. Funding: Carlos García-Latorre has been financed by a pre-doctoral grant (PD18037) from the Regional Government of Extremadura (Spain) and by the European Social Fund (ESF).