Endophytic Fungi on Coriander Crop: Influence in Plant Development and Essential Oil Profile

1. Introduction

Coriander has been cultivated primarily for its culinary uses and aromatic properties, both fresh herb and its seeds, and is also used in pickles and brines. Furthermore, it is valued in traditional medicine and the cosmetics industry, among many other uses[1,2]. Although less well-known, coriander is also nutritionally important, containing significant amounts of vitamins A, B1, B2, and C, riboflavin, carotenes, calcium, and iron [3].

The composition of coriander essential oil (EO) varies depending on the part of the plant (leaves, seeds…), the harvest time (phenological stage), the cultivated genotypes, and the environmental conditions, soil type, altitude, irrigation, among other factors [4,5]. Authors such as Burdock and Carabin [6] report that coriander EO contains more than 200 components, with 18 major components contributing to 97% of its total composition. Especially important are the aromatic compounds, which give coriander leaves and fruits their characteristic odor—pleasant and acidic in ripe fruits and unpleasant in unripe green fruits [6]. Among these aromatic compounds, coriander EO contains mainly terpene alcohols (linalool), terpene hydrocarbons, and the remainder are grouped into ketones, esters, and aromatic acids. The composition of the volatile components of the leaves differs significantly from that of the fruits, with the leaves being primarily composed of short-chain aldehydes [7]. Within the pharmaceutical and medical fields, the most common use of coriander is the essential oil (EO) extracted primarily from fresh whole plants, given its functional importance related to its anticancer, anti-inflammatory, antioxidant, and antimicrobial properties [5]. Thus, different chemotypes have been found, sowing slight variations in composition—mainly in linalool content and the presence of trace components [5]—as well as different EO yield.

Currently, due to the numerous applications of coriander essential oil (EO), there is a need for its large-scale production, which would require either increased production, genetic selection of elite germplasm with high EO production capacity and resistance to biotic and abiotic stress, or the use of technology that improves the efficiency of EO extraction or the extraction of some of its main phytochemicals, such as linalool, as well as improving its preservation by preventing oxidation [8]. In the case of its use in agricultural production, the possibility of more direct application in the field is being considered, either of the EO itself (extraction and application) or of some of its derivatives (as dried seed powder) or metabolites with greater functionality for phytoactive or biocidal purposes [9,10]. Given that coriander or its essential oil (EO) can be used in the food, pharmaceutical, and cosmetic industries, it is crucial to obtain safe, high-quality EOs with a high content of compounds of interest. The economic impact of this approach requires quality control and the development of standardized products safe for human consumption [1].

It is likely that a significant portion of the production of bioactive secondary metabolites of biological interest to both the plant and humans is due to the potential of the microbial communities hosted by the plant. Among these communities, endophytic fungi play a very important role, as demonstrated by authors such as Zhao et al. [11], Rodrigo et al. [12], and Iglesias et al. [13], as well as George and Singh [14], who worked with coriander plants. It is therefore interesting to study the potential of endophytic fungi to produce better and higher-quality crops; however, there are factors that can regulate endophyte colonization within a plant, such as the plant's genotype, growth stage, type of plant tissue, and environmental conditions, among others [15].

The coevolution between endophytic fungi and their host plants results in the production of bioactive compounds by the fungi themselves, which contribute in various ways to plant-microbe interactions: stimulating plant growth, inducing defense mechanisms against pathogens and abiotic stresses, and providing adaptive benefits to the host plant through metabolic interactions [16]. Thus, biotransformation processes mediated by endophytic fungi, either as enzymes or as whole-cell catalysis, result in novel pharmaceutically active molecules [17]. Most research in this field has focused on endophytic fungi of medicinal plants that produce molecules with anticancer, anti-inflammatory, antimicrobial, antioxidant, cardioprotective, and immunomodulatory properties. Over the past three decades, compounds such as taxol, podophyllotoxin, huperzine, camptothecin, and resveratrol have been effectively isolated and characterized after extraction from endophytic fungi [16,18]. Furthermore, the use of fungal endophytes in the biotransformation of whole molecules is an emerging biotechnological field for the production of novel modified compounds with enhanced biological activity. This technology allows for the modification of precursor compounds to produce new molecules with greater bioactivity, leveraging the natural enzymatic machinery of these microorganisms [17]. Within this field of work is the use of the endophytic fungus Penicillium oxalicum B4 in the biotransformation of artemisinic acid, which yielded eight metabolites with stronger cytotoxic and anti-inflammatory potential than the original fraction [19]. Similarly, the participation of another endophytic fungus, Camarosporium laburnicola, isolated from Astragalus species, has been described in the biotransformation of Astragalus sapogenins, leading to the production of cycloastragenol derivatives with increased telomerase activity [20].

The fungal endophytic community constitutes an inexhaustible source of biocatalysts for a wide range of substances [17]. In this context, the food industry has found in endophytic fungi a source of enzyme supply [21]: pectinases as clarifying agents in the juice and wine industry [22], fungal amylases in the hydrolysis of starch and the production of simple sugars and syrups, as well as in the optimization of the beer mashing process [23]; microbial proteases and lipases, for the stabilization of dry products in the dairy industry and to prolong the shelf life of bakery products [22]. Other fungal enzymes used in the cosmetics industry include proteases, as components of creams for removing dead skin cells [24]; lipases, for obtaining fatty amides [25]; and laccases, used in the preparation of hair dyes [26].

The potential of endophytic fungi as plant growth protectors or promoters, as well as the different species studied to date, has been described by numerous authors in various plant species. Zheng et al. [27] indicated that chitinases generated by endophytes could be used for the biological control of phytopathogens by degrading the chitin in their cell w[28]alls, while Jordaan et al. [28] concluded that lignocellulolytic enzymes from endophytic fungi could accelerate pod dehiscence and allow for effective seed germination in arid environments under favorable conditions. Furthermore, several studies have demonstrated that endophytes can act as plant growth promoters by producing phytohormones [29], producing siderophores that enhance iron uptake by the plant [30], or increasing nitrogen fixation [31] and phosphorus solubilization [32]. Finally, the literature indicates that endophytic fungi are agents for bioremediation/biodegradation, possessing numerous systems that can decompose complex compounds, degrade chemical pollutants, and bioadsorb heavy metals [33,34].

Currently, a large amount of evidence supports the idea that some of the limitations related to large-scale field inoculation mentioned above could be overcome through the use of fungal filtrates. Their composition, rich in phytohormones, siderophores, proteins, peptides, amino acids, organic acids, etc., could have multiple applications in agricultural production [13,35,36]. It is now possible to obtain these compounds in the laboratory and subsequently apply them in the field, without resorting to inoculation [37]. Given the enormous potential of endophytic fungi for more efficient agriculture, there is a need to further understand the role of these fungi in the production of both coriander biomass and certain bioactive components of the EOs from its aerial parts, so in this work, authors tried to determine the influence of using fungal endophytes, inoculated, or using their filtrates, in the development of coriander plants and the metabolic profile of their EOs.

2. Materials and Methods

2.1. Fungal and Plant Material

To obtain sufficient inoculum for plant inoculations and filtrate to spray the plants, Byssochlamys spectabilis (Genbank accession number: KP899436), endophytic fungus isolated from Biserrula pelecinus L. plants in Extremadura (Spain) [12], and one endophytic fungus isolated from Ornithopus compressus L. belonging to Xylariaceae family [21], were grown in a thermostirrer (Optic Ivymen System. COMECTA), at 23 °C in darkness and 140 rpm in PDB (Potato Dextrose Broth) culture medium.

Before sowing, coriander seeds cv. Campo Maior (https://www.dgav.pt/wp-content/uploads/2025/06/DGAV_CNV_-1.a-Edicao-de-2025.pdf) were surface disinfected by immersion for 2 min in 2.5% NaClO, to be then washed three times with sterilized distilled water. After that, five seeds per pot were sown in 22x22x27 cm plastic pots containing a mixture of 5:1 (vol/vol) universal substrate (25% organic matter, pH 6.1, 2 KCl/L regarding salinity, and available nutrients of 240 mg/L of N and P₂O₅, 340 mg/L K₂O and 120 mg/L Mg) and sand. Because plant age affects fungal endophyte colonization [38], two different planting dates were established; the first replicate was sown at early November and emerged 11 days after seeds sowing, while second replicate were sown at late November with 14 days from sowing to emergence. Pots were placed in a greenhouse at room conditions and watered to field capacity when necessary.

Once the seedlings emerged and showed true leaves, they were thinned, leaving the most vigorous plant in each pot. As fertilizer, 2 L/ha of Efisoil Superbia was applied—a fortifying plant activator with a pH of 5, based on free amino acids (55%) and total nitrogen (N) of 10%, of which 9% is organic and 1% ammoniacal.

2.2. Inoculations and Experimental Design

The growing of the two fungi was monitored and two days after total glucose depletion, mycelia were separated from spent broth (hereinafter Filtrate) through sterile paper discs (ø = 0.2 μm) to separate both parts. Thus, the experiment determined the influence of the fungus (control treatment, B. spectabilis and Xylariaceae), as well as the type of treatment (inoculation -mycelium- or application of the filtrate) and the age of the plant in the moment of the treatment application (old and young plants according to the sowing date); all treatments had 5 replicates (pots or plants).

Inoculation took place on January with a dose of 50 mg/L and 100 mg/L for B. spectabiliys and Xylariaceae fungus due to the lower infection ability of the later observed previously by our group. Prior to inoculation, with the aim of favoring colonization, abundant watering was performed to create a humid environment in the plant and substrate. Before the mycelium application by spraying, the plant leaves were slightly and superficially damaged with a pointed object to facilitate fungal penetration. Mycelium was homogenized by a blender before spraying to all 10 plants in the old plant trial (first replicate) and to the 10 young plants trial (second replicate). This treatment was repeated two weeks later, with the same doses, to ensure fungal colonization of the plants. Subsequently, colonization was confirmed by re-isolating surface-sterilized plant portions (5 mm square leaf portions immersed for 30 seconds in 70% ethanol, followed by 2 minutes in 2% NaCl and three 30 s washes in autoclaved distilled water, then grown on PDA (potato dextrose agar- plates). Another 10 /pots (1 plant/pot) in the trial were not inoculated with the fungi but sprayed with the fungal filtrates, using the filtrate at the initial concentration after mycelium separation, i.e., undiluted. A control treatment (10 plants with 1plant/pot) was also performed, which consisted of the application of sterilized autoclaved water. All pots were placed on greenhouse benches separated enough to avoid secondary infections and randomly arranged. Light, temperature and irrigation conditions were the same for all pots.

2.3. Response-Variables Determinations

2.3.1. Height and Phenological Stage

Plant height and stage of plant development were recorded in plants before harvest. Thus, plant height was observed twice (February and March), measuring from plant base to the last two completely developed leaves of the plant, classifying them by size. Categories were: L (large, >35 cm), M-L (medium-large, between 30 and 35 cm), M (medium, between 25 and 30 cm), S-M (small-medium, between 15 and 25 cm), S (small, <15 cm). Regarding plant phenology, one week before harvest, data was recorded based on a scale of 1 to 5, the stages being as follows: 1- basal leaves (BBCH 19); 2- stem emergence (BBCH 31); 3- beginning of flowering (BBCH 51); 4- full flowering (BBCH 69); 5- fruiting (BBCH 79). Treatment was considered in one specific stage when 3 of the 5 plants in the treatment presented the same stage.

2.3.2. Dry Matter, Antioxidants and Mineral Element Profile

At the end of March, plants were harvested by cutting aerial part of the plants leaving enough material for the re-growth; regrowth took place, but unfortunately, at the end of cycle most of plants died. Biomass harvested was air dried in the dark and weighed. Harvest took place before the umbels appeared.

For antioxidant analysis, leaf aliquots were immediately frozen in liquid nitrogen and stored at -80 ºC. Samples were extracted to determine antioxidant parameters by following the method described by López-Hidalgo et al.[39]. Thus, 40 mg of each frozen coriander sample were ground in a mortar using liquid nitrogen until fine powder was obtained. After grinding, methyl extraction was performed with 1.5 mL of 70% MeOH, followed by vortexing and thermal agitation (25 °C at 700 rpm for 1 h). Subsequently, each sample was vortexed and centrifuged at 10,444 rpm and 4 °C for 15 min. Once the supernatant was collected and stored at -20 °C, the process was repeated until 6 mL of supernatant was obtained. Each sample was extracted in triplicate.

The antioxidant activity was evaluated by the means of determining the radical scavenging activity by using the DPPH (2,2-diphenyl-1-picrylhydrazyl) method described by Liu et al.[40], where 200 µL of DPPH 120 µM was added to 22 µL of sample and left in the dark for 30 min at room temperature. Absorbance was read on an 800TS microplate reader (BioTek Instruments, USA) at 490 nm. A Trolox standard curve was prepared (Trolox standards: 0, 0.008, 0.016, 0.032, 0.063, 0.125, 0.250, 0.5, and 1 mg/mL), and the antioxidant activity was calculated using a linear regression line (y = -0.4664x + 0.3744 with r = 0.92). Both samples and standards were read in duplicate.

Following the method proposed by Yadav et al.[41], the total polyphenol content (TPC) was evaluated by adding 20 µL of sample, 90 µL of distilled water, and 10 µL of Folin-Ciocalteu reagent to each cell of a multiwell plate. After keeping the mixture for 6 min in the dark, 80 µL of 7% Na₂CO₃ were added, and the mixture was left for 2 h in the dark. The sample was then read at 750 nm. Gallic acid was used as the standard for preparing the standard curve, with concentrations ranging from 0 to 1 mg/mL. The TPC was calculated based on the obtained linear regression (y = 2.0632x + 0.1039 with r = 0.98), with each sample and standard being duplicated before taking the readings.

Finally, to evaluate the flavonoid concentration, a catechin standard curve was prepared using standards with concentrations of 0–0.5 mg/mL. Here 60 µL of sample and 28 µL of 5% NaNO₂ was added to each well of a multiwell plate and allowed to react for 6 min in the dark. Then, 28 µL of 10% AlCl3 was added, and the reaction was allowed to proceed again in the dark for another 6 min. Finally, 120 µL 4% NaOH was added, and the microplate was shaken for 20 s. Absorbance was measured at a wavelength of 490 nm [42]. The total flavonoid content was calculated based on the linear regression line obtained for the catechin standards (y = 3.443x + 0.0663 with r = 0.98). Absorbance measurements were performed in duplicate for both each sample and the standards.

Milled dry leaves were subjected to a XRF analysis to determine their mineral profile. X-Ray fluorescent analyses were carried out by means of an Olympus Vanta XRF Analyzer (Olympus Corporation, Japan) equipped with sensitive large-area silicon drift detectors and 50 kV X-ray tubes with a rhodium (Rh) anode.

2.3.3. Essential Oils Isolation and Analysis

According to the European Pharmacopoeia[43], the essential oils (EOs) were extracted by hydrodistillation (HD) using a Clevenger device. Given the EO low yield, after the distillation process was complete and letting it cool for 10-15 min, the EOs were collected from the Clevenger apparatus graduated tube by rinsing with laboratory-distilled n-pentane. To achieve this, a portion of the hydrolate from the connection tube was flowed out until it was just below the filling funnel, and then the distilled n-pentane was added to the filling funnel. The distilled pentane condensed and dissolved the volatiles above the aqueous phase in the graduated tube after being evaporated by the distillation flask's residual heat. Then, the distilled n-pentane and volatiles mixture was collected in a vial and concentrated to around 100 µl at room temperature under nitrogen flux using a blow-down evaporator system. Until analysis, the volatiles were stored in the dark at -20°C.

The EOS obtained from hydrodistillation were analyzed by Gas Chromatography with Flame Ionisation Detection (GC-FID) for quantification and by Gas chromatography-Mass spectrometry (GC-MS) for component identification as in Póvoa et al. [2].

Gas chromatographic analyses were performed using a Clarus 400 gas chromatograph (Perkin-Elmer, Shelton, CT, USA), equipped with two flame ionization detectors, with a data handling system and a split-splitless injector port into which two columns of different polarities were inserted: a DB-1 fused-silica column (polydimethylsiloxane, 30 m x 0.25 mm i.d., film thickness 0.25 µm; J & W Scientific Inc., Rancho Cordova, CA, USA) and a DB-17HT fused-silica column [(50% phenyl)-methylpolysiloxane, 30 m x 0.25 mm i.d., film thickness 0.15 µm; J & W Scientific Inc.]. The oven temperature was programmed from 45 to 175 °C at 3 °C/min, subsequently at 15 °C/min up to 300 °C, and then held isothermal for 10 min. The injector and detector temperatures were 280 °C and 300 °C, respectively. Hydrogen was the carrier gas adjusted to a linear velocity of 30 cm/s. The split sampling technique ratio was 1:50. The injection volume was 0.1 µL of a 1:1 distilled n-pentane-essential oil solution. The EOs percentage composition was determined using the normalization method for the GC peak areas, calculated as the average of two injections per sample, without using the response factors, according to ISO 7609 [44]

GC-MS analyses were performed on a Perkin Elmer Clarus 600 gas chromatograph, equipped with a DB-1 fused-silica column as described above, and interfaced with a Perkin-Elmer 600T mass spectrometer (software version 5.4.2.1617, Perkin Elmer, Shelton, CT, USA). Oven and injector temperatures were the same as in GC analyses. Transfer line at 280 °C. Carrier gas, helium (30 cm/s). Ion source at 220 °C. Split ratio, 1:40. Ionization energy, 70 eV. Scan range at 40-300 u and scan time of 1 s. Compounds were identified by calculation of their retention indices (RI) relative to a C₇-C₂₉ n-alkane ladder, and from mass spectra from a custom-made library, created as fully detailed in the supplementary material of Póvoa et al. [2], based upon the analyses of reference essential oils, laboratory-synthesized components, and commercially available standards.

2.4. Statistical Analysis

Data was analyzed using the statistical software package XLStat (Addinsoft, 2024) as a Microsoft Excel add-in. Analysis of variance (ANOVA) and Tukey's test were applied to compare means for the parameters of dry weight, and antioxidants and minerals. The significance level was set at 0.05. The chi-square test was applied for the parameters of size and phenological stage.

3. Results

3.1. Dry Matter

Regarding dry matter, a significant interaction between plant age and endophyte was recorded when the fungus was inoculated into the plant (hereafter treatment mycelium). Plants inoculated with Xylariaceae fungus had a higher dry weight than the control (E-) (7.28 g vs. 5.05 g). Inoculation with B. spectabilis showed no significant difference in dry weight compared to E0 (5.92 g vs. 5.05 g) (Table 1). Regarding to plant age, younger plants showed significantly higher weights than older plants—those sown in the first replicate of the trial—with mean values greater than 6 g compared to less than 5 g in older plants. When filtrates were sprayed (hereafter treatment filtrate), there were no significant differences, either for the main variables or for the interaction, with mean values ranged from 4.45 to 5.32 g/plant.

3.2. Plant Height and Phenological Stage

After performing the chi-square test, significant differences in plant size/height were observed with reference to the plant age in the mycelium treatment in the first data recorded; younger coriander plants were significantly larger. Regarding the filtrate treatment, no significant differences were found indicating the absence of any effect of the filtrate on plant (Table 2). When studying the fungal treatment instead of the plant age, the data showed that no significant differences were found, neither analyzing plant age nor fungus used in the experiment (Table 4).

Second set of data also showed a total absence of significant differences in plant height, no matter what plant age, fugus used or treatment applied (mycelium or filtrate) (Table 2 and Table 3).

The chi-square tests for the phenological stage of coriander, with data recorded a few days before harvest, are presented in Table 4 (scale indicated in Materials section). Thus, the upper part of the table shows the test regarding the plant age, and the lower part, the test regarding the fungus used. In any case, no significant differences were found. However, it is interesting to note that the coriander plants inoculated with the mycelium of the Xylariaceae showed a tendency to extend the juvenile stage (rosette stage), unlike that inoculated with B. spectabilis which showed a tendency to the opposite effect (in this case all plants were in the imminent stem stage in both mycelium and filtrate treatments).

3.3. Antioxidants and Mineral Profile

The analysis of antioxidant activity, determined by the DPPH free radical scavenging capacity, showed significant differences in the mycelium treatment for both main factors and their interaction (Table 5). However, in filtrate treatment, significant differences were only observed for the main factors, plant age and endophyte. Thus, in the mycelium treatment, both young and old plants showed significantly higher antioxidant activity when inoculated, compared to uninoculated plants (E-), with the highest value recorded in young plants inoculated with Xylariaceae (39.21 mg Trolox/g), and in old plants inoculated with B. spectabilis (36.94 mg Trolox/g). Plant age showed to have a significant influence in this parameter, with higher antioxidant activity observed in older plants compared to younger ones (27.16 vs. 20.57 mg Trolox/g); these results were consistent in the filtrate treatment, where older plants showed also greater antioxidant activity than younger plants (Table 5). Regarding the endophyte, plants treated with B. spectabilis exhibited more than double antioxidant activity than the control plants, exceeding 25 mg Trolox/g compared to approximately 11 mg Trolox/g recorded in the control.

No significant differences for plant age, in terms of the total polyphenol content (TPC), were found in the experiment (Table 5). However, the inoculation with the fungi, regardless of the endophyte species, showed higher TPC than the control plants, almost tripling the values of mg Trolox per gram of sample. In case of filtrate application, the TPC of plants using B. spectabilis filtrate showed higher values than those of plants inoculated with Xylariaceae and E-, (8.04 and 8.05 versus 21.43 mg gallic acid). Regarding the interaction, Table 5 shows how B. spectabilis inoculation prompted higher TPC in older plants, while Xylariaceae did it in younger ones; in case of filtrate application, B. spectabilis filtrate promoted the plants with the highest TPC values, regardless of the age of the plant. In case of total flavonoid content (TFC), significances were only found when B. spectabilis was inoculated, doubling the TFC values of the control plants (Table 5).

Both the application of mycelium and the fungal filtrate to the plants influenced their mineral nutrition; however, the magnitude of the effect differed significantly between treatments. Inoculation with mycelium from fungi Xylariaceae and B. spectabilis modified the mineral content of coriander, with effects varying depending on the mineral, age, and fungus used. Thus, according to Table 6, there were no significant differences for zirconium (Zr) and scandium (Sc) in the mycelium treatment. In young plants, the control showed higher levels of potassium (K) and rubidium (Rb) (118,900.92 ppm and 9.54 ppm, respectively); in older plants, calcium (Ca) and potassium (K) showed higher levels in the control (29,668.51 ppm and 119,440.24 ppm, respectively) compared to treatments with fungi. In the case of plants inoculated with Xylariaceae, they showed lower Fe content and higher K, Sr and Rb content than the control, while when inoculating B. spectabilis, the K in these plants was lower than the control (107149.45 ppm in young plants and 112316.57 ppm in old plants). According to what was observed in Table 6 considering the age × fungus interaction for the filtrate treatment, control young plants showed higher levels of K, S, and Rb, and lower levels of Zr, while regardless of plant age, E- plants showed higher Sr levels. Plants treated with Xylariaceae filtrate showed reduced concentrations of K, S, Sr, and Rb, compared with the control, and with no differences observed between E- and Xylariaceae in Fe and Mo. Mineral concentrations in plants treated with the B. spectabilis filtrate showed greater differences between age groups than with respect to the fungus itself. Concentrations of Ca, K, S, Zn, and Sc were higher in older plants than in younger ones. Iron and Rb levels were lower in older plants (Fe ranging from 64.95 to 69.76 ppm in young plants and from 52.25 to 74.51 ppm in older plants; and Rb ranging from 5.57 to 7.47 ppm in young plants and from 3.63 to 4.53 ppm in older plants).

3.4. Essential Oil Profile

Thrity-one compounds were identified in coriander EOs, with varying percentages depending on the sample analyzed (Table 7). The relative amounts of each of the compounds identified are listed in Table 7, following the elution order on the DB-1 column.

A higher number of compounds present in above trace amounts (> 0.05%) were found in young plants than in older ones. Untreated young plants (E-) showed 12 compounds above trace amounts, while in plants inoculated with Xylariaceae, 14 different compounds were found. Conversely, when B. spectabilis was used (mycelium or filtrate) the chromatogram was simplified compared to the control plants, showing only 8 compounds. In older plants, the number of compounds present in above trace amounts in the essential oils of the control plants (E-) was 9, while 6 compounds were detected in filtrate plants and 10 when plants were inoculated (mycelium) with Xylariaceae. In the case of applying B. spectabilis filtrate, volatiles profile showed 12 compounds >0.05%, reducing to 6 compounds in mycelium plants essential oil (Table 7).

Specifically, it was observed that, regardless of plant age, applying B. spectabilis filtrate increased the percentage of myristic acid compared to the control (from 1.9% to 2.6% in young plants and from <0.05% to 0.7% in old plants), while inoculation with the fungus Xylariaceae increased the percentage of n-decanal and n-undecanal, rising from 1% to 2.6% and from 1.1% to 1.8% respectively in young plants, with the differences being less significant in old plants (Table 7). On the contrary, n-undecanal was undetectable in young plants inoculated or treated with B. spectabilis filtrate, and old plants from mycelium treatment. In young plants, a positive effect was observed on the percentage of n-undecanal, increasing from 1.1% in control plants to 2% and 1.8%, in filtrate and mycelium plants, respectively. This resulted in a decrease in the percentage of oleic and linolenic acids, an effect contrary to that shown by the fungus B. spectabilis. The application of filtrate or inoculation with B. spectabilis increased linolenic acid, as well as palmitic acid. In older plants, the application of filtrate or inoculation of either fungus (Xylariaceae or B. spectabilis) caused a decrease in the main fatty acids (oleic, linolenic, and palmitic) as well as in n-docosanol content, while increased phytol acetate and hexahydrofarnesyl acetone.

Focusing on the compound groups, the presence of the fungus Xylariaceae or its filtrate causes an increase in oxygenated sesquiterpenes in the EOs compared to the essential oils of the control plants, while fatty acids content decreased. Similarly, the application of B. spectabilis filtrate or its inoculation into the plants also increased the percentage of oxygenated sesquiterpenes, raising the proportion of fatty acids in young plants, but decreasing it compared to the control in older plants. In addition, B. spectabilis reduces the percentage of alkanes to undetectable levels (except when the filtrate is applied to older plants) (Table 7).

4. Discussion

Previous studies such as that by Haidar et al. [45] demonstrated the positive effect of some endophytic fungi on increased growth and fresh and dry weight of jute plants. AlKahtani et al. [46] observed higher dry weights in inoculated corn plants compared to control plants. Similar results were shown by Toppo et al. [47], who observed the influence of endophytic fungal application on tomato plants, specifically on parameters such as root and shoot growth, and fresh and dry weight. Furthermore, the present results align with other studies where the application of endophytic fungi such as Xylariaceae sp. resulted in higher dry weight compared to controls, which could be due to the production of phytohormone-like substances by these endophytes [21] or the activation of hormonal signaling networks [48]. In addition, several studies have demonstrated that endophytic fungi can influence plant growth by modulating photosynthetic parameters [49], contributing to a better photosynthetic rate, and thus to a higher biomass [50]. Endophytic fungi have demonstrate to enhance photosynthetic capacity by increasing chlorophyll content, optimizing chloroplast metabolism, and supplying the NADPH and ATP necessary for carbon assimilation [51], which would support the effects observed in this study. Thus, understanding the functional adaptability and temporal dynamics of the endophytic microbiome throughout plant development is key to designing microbiome-based production strategies [52]. In this context, the influence of plant growth stage —here plant age— has been addressed by Xiong et al. [53] and Gao et al. [54], who highlight the contribution of plant developmental stages to the assembly of the plant microbiome, and indicated that, in initial growth stages, greater functional diversity of the microbiome, particularly in the phylloplane, along with the presence of genes related to nutrient supply, favors a more intense plant response [53].

Regarding the phenological stage, the differences observed in inoculated plants with the mycelium of fungi, although not statistically significant, were consistent with the possible differential production of compounds with hormonal activity rather than a nutritional effect. While the mycelium of fungus Xylariaceae appeared to induce or maintain a hormonal balance associated with prolonging the juvenile stage, the mycelium of B. spectabilis could produce diffusible metabolites with growth-promoting activity, supporting a possible physiological basis for the phenotypic differences observed between treatments in coriander. However, although the synthesis of phytohormones by a wide range of endophytes is now widely known[55], this hypothesis requires confirmation through other types of analysis.

In the same way that endophytes can synthesize phytohormones, as stated above, several authors have confirmed the hypothesis that endophytes can modulate the antioxidant physiology of plants [56,57]. The greater effectiveness of the fungus B. spectabilis in older plants could be related to greater accumulated oxidative stress in aged tissues. Although some authors indicate that older tissues can produce more antioxidants [58], most studies focus on plants subjected to environmental stress and do not compare young and old plants. The greater antioxidant activity using B. spectabilis filtrate in older plants showed a high antioxidant potential; in any case, it would be important to also consider the possibility of interaction with phytohormones such as abscisic and jasmonic acid or ethylene, among others, that can also give information about oxidative stress [59]. However, most of the studies consulted were conducted in vitro or under stress conditions, so the results of our study, demonstrating that fungal filtrate applied to the plant can increase antioxidant capacity in in vivo application represents another step forward for further research.

Our results suggest that the use of fungal endophytes is also beneficial for polyphenol accumulation, but selectively, depending on the physiological state of the plant and the fungal species [60]. Under the current conditions, Xylariaceae fungus appeared to be more effective in early developmental stages to increase TPC when inoculated, while B. spectabilis showed greater effectiveness in more developed plants. The filtrate treatment only increased TPC when the filtrate of B. spectabilis was used, showing higher values than the control and Xylariaceae; these results indicate that inoculation with the live fungus and its direct interaction with the plant tissue is key to activating phenolic metabolism [61] in young and old plants, while the metabolites present in the filtrate are able to induce a significant response on their own, increasing the production of polyphenols regardless of the age of the plant, but being more depending on the fugal species. In any case, the increase in TPC in inoculated plants contributes to the explanation of the previously observed increase in antioxidant activity.

Despite the inoculated/treated plants showing higher antioxidant capacity, the flavonoid content was only higher than the control, as mean value, when B. spectabilis was inoculated (mycelium) but no significance in the interaction age × fungus or the filtrate treatments were found. This apparent discrepancy can be explained by the fact that antioxidant activity results from the combined action of various antioxidant compounds, not just flavonoids; there is not always a direct correlation between flavonoids and total antioxidant capacity [62], and numerous bioactive compounds can confer antioxidant activity to the plant. Endophytic fungi can modulate the expression of genes involved in the biosynthetic pathways of secondary metabolites, affecting not only flavonoids but also multiple bioactive compounds with antioxidant potential[61]. In general, it can be seen in our results that inoculation with endophytic fungal mycelium can positively modulate phenolic metabolism and the plant's total antioxidant capacity, without necessarily increasing flavonoid content; the response appears to depend on differential regulation of the biosynthesis pathways of polyphenols or other antioxidant compounds, modulated by the inoculated fungus and the plant's age [63], reinforcing the importance of endophyte selection and application timing.

The present findings align with recent research demonstrating that endophytic fungi can enhance nutrient uptake in plants, even under nutritional stress, by improving physiological processes such as the acquisition of iron and other important minerals [64]. Furthermore, fungal secondary metabolites contained in filtrates and extracts have shown stimulatory activity in mineral accumulation, suggesting mechanisms of action that do not depend on active mycelial colonization [65,66], but rather on biochemical mechanisms activated by the secondary metabolites present in fungal filtrates, which act as elicitors of certain metabolic processes. Regarding the nutritional profile of coriander, Ciorcalam et al.[67] and Agarwal et al. [68] have shown high variability in the results, which translates into wide ranges, especially for macroelements, with our results falling within these average values.

Previous studies have shown that the distinctive aroma of coriander is determined by a combination of volatiles present on its essential oils, most notably aliphatic aldehydes [69]. The composition obtained in this study largely agrees with profiles obtained for essential oils from the leaves and stems of Coriandrum sativum, characterized by a high proportion of medium-chain (C9–C13) aliphatic aldehydes and linear alkanes [70], as well as alcohols and diterpenes such as phytol [71]. This distinguishes this EO from that obtained from seeds, which is dominated by monoterpenes [5].There is some evidence, albeit limited in coriander, that inoculation with endophytic fungi in aromatic plants can alter the essential oil profile by activating secondary metabolic pathways and modulating the number and content of compounds [72,73]. In the present case, results demonstrate that the essential oil profile of coriander varies when plants were inoculated with endophytes, and also depending on the plant's age. Thus, the consulted literature indicates that plant age is a relevant physiological indicator [74] that can influence the plant-endophyte interaction [75] and the variability of essential oil composition [76]. Our results confirm that fungal filtrate also exerts external elicitor activity without the need for endophytic colonization. Specifically, treatment with filtrate increases the number of volatile compounds compared to the control, varying according to the fungus used, as observed in Table 7. These differences could be explained by the distinct nature of the elicitors produced by each fungus, as well as by the sensitivity of the plant tissue, its developmental stage, and the complexity of hormonal interactions during different plant growth stages [77].

Detailed analysis of the chemical profile confirmed subtle changes in the groups of compounds detected (Table 7). Taking into account the obtained data, and assuming that the results are almost preliminary, it appears that hexahydrofarnesyl acetone, belonging to the oxygenated sesquiterpenes, could play a role in inducing antioxidant bioactivity in the plant, rather than contributing to the aromatic profile of the essential oil. Similarly, the presence of phytol acetate, an oxygenated diterpene derived from phytol, serves a physiological rather than an aromatic function. Since phytol constitutes the side chain of chlorophyll, its release during the degradation of this pigment—associated with leaf aging or stress—can lead to its metabolism or re-esterification into compounds such as phytol acetate, or its incorporation into other metabolic pathways [78]. In our results, we observed greater activation of oxygenated sesquiterpene production in the filtered treatments, which could confirm the elicitor function of the filtrate against the mycelium for these types of compounds. The filtrate would induce a change in the plant's defensive/metabolic physiology [78] rather than a modification in the volatile aromatic profile. Supporting this eliciting effect of the filtrates on the generation of protective metabolites, we observed in the studied phenols that the application of B. spectabilis filtrate resulted in a greater quantity of phenols in the aerial parts of the plant. Regarding oxygenated diterpenes, we found higher values in older plants. Fatty acid metabolism plays a crucial role in plant defense mechanisms and is modulated by biotic and abiotic stress. It is known that unsaturated fatty acids (oleic, linoleic, α-linolenic) are key signaling molecules in defense, including the production of jasmonates and the modulation of redox status—via the oxylipin pathway [79]. Saturated fatty acids—in this case, palmitic acid—are essential for membrane stability, and their production is also stimulated by biotic and abiotic stress, combating oxidative stress [80]. Its presence and increase in leaves is explained by the defensive behavior of leaf tissues [81]. The increase in fatty acids in young plants treated with B. spectabilis could suggest the bioactivation of early defense pathways and lipid signaling; in the case of the mycelium, this is possibly due to the physiological response produced by the early direct interaction of the fungal mycelium with the tissue [82]. We found an identical relationship with the increase in palmitic acid levels in young plants treated with B. spectabilis, reflecting its structural and functional role in stress. In the case of older plants, in relation to palmitic acid, is Xylariaceae fungus that stands out as this acid promoter, both using the filtrate and the mycelium.

As can be seen, the modification of the metabolite profile in essential oils depends not only on the age of the plant, but also on the presence of endophytic fungi or the application of their secondary metabolites through filtrate application. The fungi act as elicitors, promoting the synthesis of compounds that protect against stress, especially antioxidants and unsaturated fatty acids. Furthermore, from an applied perspective, obtaining coriander leaves enriched with antioxidants and healthy fatty acids, such as oleic and linoleic acids, is of interest to the food and cosmetic industries, as it would improve extraction yields with smaller quantities of raw material.

On the other hand, the application of certain fungi or their filtrates to coriander significantly increased the concentration of compounds of high industrial interest in its essential oil, notably the increase in α-linolenic acid after inoculating B. spectabilis. This is relevant to the pharmaceutical industry because α-linolenic acid is a precursor of the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are involved in neurological and visual development [83,84] and have the potential to improve the response to chemotherapy treatments [85]. The use of its filtrate also increased the levels of myristic acid, a compound with demonstrated anti-inflammatory activity [86] and efficacy against cutaneous inflammatory processes [84]. Furthermore, this acid is of great interest to the pharmaceutical and cosmetic industries due to its protective, lubricating, and stabilizing properties, as well as its ability to facilitate skin penetration [87]. Regarding the fungus Xylariaceae, inoculation increased the concentration of decanal, an aldehyde used as a food flavoring [88] for its citrus notes [89] and antioxidant activity [90]. Decanal is also of interest to the agri-food sector due to its effectiveness in controlling Penicillium expansum, principal agent of post-harvest fruit rot, by affecting fungal growth and the biosynthesis of the neurotoxic mycotoxin patulin [91]. This compound also possesses bactericidal activity against human pathogens such as Staphylococcus aureus and Escherichia coli [89]. Recent studies also indicate its potential as an antitumor agent, with antiproliferative activity against lung cancer cell lines and possible applications in breast cancer [88,92].

5. Conclusions

Xylariaceae fungus can increase coriander plant biomass more than 30% compared to uninoculated plants, demonstrating its potential as a plant growth promoter, and significantly increase antioxidant activity in the infected plants. The use of the filtrate of endophytic fungus Byssochlamys spectabilis can act as elicitor in coriander plants and increases the antioxidant capacity and phenol content of the aerial biomass of coriander plants by more than one-third. Both fungi generated interesting modifications in the profile of coriander essential oil, notably an increase in compounds with antioxidant activity and a protective profile against abiotic stresses. Thus, the application of fungal mycelia and filtrates presents high potential as a sustainable and environmentally friendly strategy to improve coriander crop productivity with potential applications in the agricultural, food, cosmetic, and pharmaceutical sectors.