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Article

Isolation, Identification, and Application of Endophytic Fungi from Lavandula stoechas L.: Mitigating Salinity Stress in Hydroponic Winter Cereal Fodder

by
Carlos García-Latorre
* and
María José Poblaciones
*
Department of Agronomy and Forest Environment Engineering, University of Extremadura, Avenida Adolfo, Suárez s/n, 06007 Badajoz, Spain
*
Authors to whom correspondence should be addressed.
Agronomy 2024, 14(11), 2501; https://doi.org/10.3390/agronomy14112501
Submission received: 10 August 2024 / Revised: 21 October 2024 / Accepted: 21 October 2024 / Published: 25 October 2024
(This article belongs to the Special Issue Advances in Agricultural Engineering for a Sustainable Tomorrow)
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Abstract

:
Soil and irrigation water salinity is a growing global problem affecting farmland, due to poor agricultural practices and climate change, leading to reduced crop yields. Given the limited amount of arable land and the need to boost production, hydroponic systems offer a viable solution. Additionally, endophytic fungi have been shown to mitigate salinity effects through symbiosis with plants. This study evaluated three endophytic fungi isolated from Lavandula stoechas L. in the grasslands of Extremadura (i.e., Diplodia corticola L11, Leptobacillium leptobactrum L15, and Cladosporium cladosporioides L16) for their ability to improve hydroponic forage production under saline conditions. In vitro experiments were conducted assessing plant growth promotion and fungal growth under salinity, followed by research evaluating the impact of fungal inoculation on hydroponic wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) forages irrigated with NaCl solutions (0, 100, and 200 mM). The results showed improved fungal growth and production of plant growth-promoting substances, which could explain the improved plant germination, shoot and root length, fresh and dry weight, and yield of inoculated plants growing under salinity. Plants inoculated with L15 or L16 showed the best performance overall. L15 demonstrated broader bioactivity in vitro, potentially explaining its superior performance in both wheat and barley growth. Conversely, L16 was more effective in barley, while L11 was beneficial in wheat but detrimental in barley. This study provides a preliminary exploration of the capabilities of these fungi and their optimization for hydroponic forage production.

1. Introduction

The global population is projected to reach approximately 9.7 billion by 2050, which will result in a significant increase in food demand [1]. It is, therefore, imperative to address this situation, in conjunction with the impacts of climate change [2,3] and agricultural malpractices [4], in order to implement more sustainable practices of production for staple crops and food security [5,6,7]. A critical challenge in this context is the need to manage the effects of water mismanagement and soil salinity, which significantly hamper crop productivity and food security on a global scale [8,9].
The availability and quality of water in agriculture is a key factor that significantly impacts the latter’s productivity [10,11]. The long-term use of poor-quality irrigation water has been identified as a significant factor in intensifying salinity issues, further challenging sustainable agriculture [10]. Salinity is a naturally occurring phenomenon in arid and semi-arid regions with low rainfall, but it is exacerbated by human activities, and particularly by improper irrigation practices [12,13]. Soil salinity causes substantial annual production losses [14] because of the disruption of plant growth caused by the reduction in chlorophyll content, which hinders photosynthesis and compromises cell membrane integrity [15,16]. Furthermore, salinity stress leads to osmotic stress, impaired water absorption, dehydration, and ion toxicity due to salt accumulation [17]. Thus, unless it is managed effectively, irrigation with saline water can lead to progressive accumulation of salt in soil, significantly altering soil properties and impacting crop yields.
Various strategies have been explored to mitigate salinity stress in plants, including the use of organic and inorganic amendments [18], the optimization of irrigation practices [8], and the development of resistant cultivars and varieties [19,20]. Another alternative that has recently gained importance is the use of beneficial microorganisms which can establish symbiotic relationships with plants and promote the development of the plants, while also protecting against biotic and abiotic stresses [21,22,23]. Studies have shown that by colonizing plants, fungal endophytes can protect their hosts against biotic and abiotic stresses by regulating root architecture [24], inducing systemic resistance [25,26], increasing levels of protective metabolites [27,28], modulating the phytohormone profile [29] and the levels of antioxidant enzymes [30], and solubilizing nutrients [31].
Under saline conditions, wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) exhibit reduced primary root growth due to salt-induced inhibition [32,33]. Endophytic fungi can influence the architecture and branching of plant roots, which helps in the regulation of salt acquisition and translocation [34]. Endophytes such as Alternaria chlamydospora and Fusarium equiseti have been observed to increase root growth in grains like wheat, thereby improving ion homeostasis and osmoregulation [35]. Salinity stress also induces oxidative stress, which is marked by increased reactive oxygen species (ROS) production, which can damage cellular components [36]. Endophytic fungi can modify the osmolyte concentration and profile [34,37], thereby enhancing plant resilience to salinity, as observed in wild barley [38]. Endophyte-inoculated plants also exhibit enhanced activity of antioxidant enzymes such as superoxide dismutase, catalase, and peroxidase, which mitigate oxidative damage [39]. For example, increased activity of these enzymes in barley [40] and wheat [41] under saline conditions aids in ROS detoxification.
Salinity stress reduces photosynthesis by decreasing chlorophyll content and stomatal aperture, limiting CO2 assimilation and productivity [42,43]. However, plants inoculated with endophytes show improved water status, increased leaf area, and enhanced photosynthetic efficiency [44]. Finally, endophytic fungi can produce phytohormone analogs or induce their production in plants, thereby promoting growth under stress conditions [45,46,47]. For instance, Bipolaris sp. CSL-1 inoculation in soy plants (Glycine max. L.) under saline stress modified phytohormone levels, enhancing growth and stress tolerance [48].
The Lavandula genus, including L. stoechas, is a key medicinal and aromatic plant in the Mediterranean basin, well-adapted to harsh natural conditions [49]. L. stoechas essential oils and extracts, rich in bioactive molecules such as phenolics, flavonoids, and monoterpenoids, exhibit antioxidant and biocontrol activities [49,50]. Carrasco et al. [51] identified antioxidant compounds such as linalool and thymol in L. stoechas essential oils from Spain. This activity was mainly attributed to the plant’s ability to eliminate free radicals. Consequently, this plant may serve as a significant source of fungal endophytes with beneficial bioactivities in the context of abiotic stress mitigation. This is particularly relevant, given that many endophytic fungi are often capable of producing secondary metabolites analogous or identical to those of their host plant [52].
In the face of diminishing arable land and increasing soil and water salinization, hydroponic systems present a sustainable alternative for producing animal feed under controlled conditions [53]. These systems enable soilless cultivation, reducing the need for agrochemical inputs, minimizing environmental pollution, and even allowing the treatment of wastewater [54]. The systems also optimize space with vertical trays, increasing productivity and ensuring consistent high-quality harvests in shorter growing cycles [55]. Hydroponics also promotes sustainability by efficiently recycling water and nutrients and using only 10% of the water required by traditional methods [56]. These systems enable the cultivation of fresh forage, typically grown from cereal seeds over a short period of six to eight days, which is more palatable and easily digestible by cattle, in an environmentally controlled system [57,58]. These short production cycles allow for forage production without the need for fertilization, as it utilizes the nutrients naturally present in the seeds, which helps to improve even more the use of resources [59].
Integrating endophytic fungi in hydroponic systems would further strengthen plant resistance to salinity stress, allowing the recycling of salinized water to produce key crops such as wheat or barley, grown as hydroponic fodder, based on the premise that inoculation with endophytic fungi isolated from L. stoechas could provide a protective effect. Therefore, it was hypothesized that the combination of hydroponics with plant-endophyte symbiosis could enhance the production of winter cereals (wheat and barley) as hydroponic fodder and facilitate the recycling of salinized water, leading to more sustainable and efficient agricultural practices.

2. Materials and Methods

2.1. Plant Material

Two of the most used winter forage cereals, namely, bread wheat (T. aestivum L.) and barley (H. vulgare L.), were selected for the study. A germination test had been previously carried out to assess the viability of those seeds under the same conditions as those of the experiments, without salt application, using 100 seeds per plant, and with 3 replications. The germination rates were 96% for bread wheat and 97.2% for barley. The plant material was sterilized in accordance with the procedure described by Zabalgogeazcoa et al. [60]. Initially, seeds were examined to select only those that were intact, thereby ensuring the integrity of the results. They were then washed with abundant tap water to remove any larger particles and adhering dust. Subsequently, the seeds were first immersed in a 70% ethanol solution (Panreac, Barcelona, Spain) for two minutes, and later in a 2% sodium hypochlorite solution (Panreac, Barcelona, Spain) for an additional two minutes. Finally, the seeds underwent three consecutive washes with sterile distilled water to ensure the complete removal of any residue that could affect germination.

2.2. Fungal Material

2.2.1. Isolation of Fungi

Samples of apparently healthy Spanish lavender (Lavandula stoechas L.) plants were collected in June 2023 from the practice fields of the School of Agricultural Engineering in Badajoz (38°53′49.26″ N; 6°58′4.88″ W) and transported immediately to the laboratory for the isolation of endophytic fungi within four hours. Initially, the samples were meticulously rinsed with tap water to eliminate coarse particles and dust, and then separated into leaves and stem subsamples. These subsamples were subjected to surface sterilization in accordance with the methodology described for the seeds [60]. Following the process previously outlined [61,62], the samples were placed on sterile filter paper in a laminar air-flow chamber (Aeolus Huntil, Telstar, Barcelona, Spain) they were dry and then cut into 5 mm pieces. Four pieces from each subsample (leaf and stem) were sown on Petri dishes containing potato dextrose agar (PDA, 39 g L−1, Panreac, Barcelona, Spain). The Petri dishes were stored in a growth chamber (Radiber Ger-500, Radiber, S.A., Barcelona, Spain) at 24 °C under dark conditions. During the following four weeks, as the fungi grew, they were continually transferred to new PDA plates until an axenic culture was obtained in each plate. To obtain a representative sample of the plant’s endophytic fungi, this process was repeated with up to 20 plates, 10 of which were inoculated with leaf samples and 10 with stem samples. Additionally, five surface-sterilized stems and five surface-sterilized leaves were directly sown on PDA plates without cutting. The absence of fungal growth in these plates guaranteed the efficacy of the sterilization method.

2.2.2. Identification of Fungi

The fungal isolates were initially grouped into morphotypes according to their mycelium color, texture and form, diffusion of visible exudates into the PDA medium, and growth rate, reducing the initial 37 isolates into 16 morphotypes. Among these, three were selected for use in this experiment, based on parameters such as the frequency of isolation and growth characteristics. For the molecular procedure identification, DNA was directly extracted and purified from colonies growing on PDA in all cases. Briefly, the DNA extraction phase was carried out using the “Quick-DNA Fungal/Bacterial Miniprep” kit (Zymo Research, Irvine, CA, USA), following the manufacturer’s instructions. PCR amplification was performed using the necessary primers for the chosen region (ITS) in an thermocycler (iCycler Thermal Cycler, BioRad, Hercules, CA, USA) using, which included a central part of 35 cycles at 94 °C, 55 °C, and 72 °C for one minute each. The success of the extraction was verified by electrophoresis, allowing the DNA purification process to be carried out with a specific kit, in this case, “NucleoSpin Gel and PCR Clean-up” (VWR, Vienna, Austria). Once purified, the DNA samples were sent to the Central Services department of the University of Extremadura to complete the identification process. The obtained sequences were compared with sequences from the GenBank database (www.NCBI.nlm.nih.gov, accessed on 12 February 2024) using a BLAST search.

2.2.3. Growth in Liquid Medium and Preparation of Inoculum

In order to prepare the fungal inoculum for the tests, the selected isolates were cultivated in autoclaved potato dextrose broth (PDB, 26.5 g L−1, Panreac, Barcelona, Spain). For this purpose, three 5 mm diameter fragments of each isolate were inoculated into a 1 L Erlenmeyer flask containing 0.5 L of sterilized potato dextrose broth (PDB, 26.5 g L−1) medium. The cultures were incubated in a growth chamber at 23 °C and 140 rpm under dark conditions for eight days. Subsequently, the mycelium was aseptically filtered through a 0.22 μm filter paper (Sigma-Aldrich, St. Louis, MO, USA) to separate it from the growth medium, thus obtaining the inoculum for seed treatment. The resulting growth medium, known as fungal filtrate, was utilized to ascertain in vitro auxin production. Each isolate was cultured in triplicate.

2.2.4. Determination of Plant Growth Promotion Traits and Stress Defense of Selected Fungi

To assess the bioactivities of the selected fungi, a series of in vitro assays were conducted. These assays evaluated the fungi’s varying potential for auxin production (with and without tryptophan as a precursor) and ammonia production, and their abilities to solubilize phosphate, phytic acid, and potassium. Additionally, fungal tolerance to salinity was tested by evaluating their growth in media with increasing NaCl concentrations. These tests aimed to provide a basis for interpreting the results of the in vivo tests.

Auxin Production (Indole-3-Acetic Acid, IAA)

The indole-3-acetic acid (IAA; Sigma-Aldrich, St. Louis, MO, USA) content in the fungal filtrate was determined using the method by Devi et al. [63], as modified from Gordon and Weber [64]. The Salkowski reagent (1 mL ferric chloride [0.5 M] and 50 mL 35% perchloric acid; Sigma-Aldrich, St. Louis, MO, USA) was mixed with the fungal filtrate (1 mL) at a 2:1 ratio. After vortexing for 30 s, the mixtures were incubated in the dark for 30 min. Absorbance was measured at 530 nm using a spectrophotometer (UV-3100, JP Selecta, Barcelona, Spain), and a standard curve of IAA (0–150 µg mL−1) was used to express the results as IAA equivalents. For tryptophan-supplemented cultures, the fungus was grown as described before, supplemented with 0.01% L-tryptophan (Sigma-Aldrich, St. Louis, MO, USA). The IAA content of this filtrate was also analyzed using the same method. Both tests were conducted in triplicate.

Ammonia Synthesis

The ability of the fungi to synthesize ammonia was assessed using the method by Fouda et al. [65]. A 5 mm diameter piece of mycelium was inoculated into 30 mL of liquid peptone water (10 g L−1 peptone, 5 g L−1 NaCl, 0.1 g L−1 CaCl2·H2O; Panreac, Barcelona, Spain) and incubated at 28 °C, with shaking at 140 rpm, in the dark for 72 h. After incubation, 1 mL of Nessler reagent was added. The resulting color change indicated ammonia production: pale yellow for limited production and dark yellow or orange for maximum concentration. The test was conducted in triplicate.

Phosphate and Phytic Acid Solubilization

The fungi’s ability to solubilize phosphates and phytic acid was determined using the method developed by Nautiyal [66] with NBRIP medium (NBRIP, in g L−1: glucose, 10.00; MgCl2·6H2O, 5.00; MgSO4·7H2O, 0.25; KCl, 0.20; (NH4)2SO4, 0.10; agar, 20.00; pH 7; Panreac, Barcelona, Spain). For phosphate solubilization, the medium was supplemented with 5 g L−1 tricalcium phosphate, Ca3(PO4)2 (Sigma-Aldrich, St. Louis, MO, USA), and for phytic acid solubilization, 5 g L−1 phytic acid, C6H18O24P6 (Sigma-Aldrich, St. Louis, MO, USA), was added. The media were autoclaved, poured into Petri dishes, and inoculated with 5 mm diameter pieces of mycelium. The dishes were incubated at 23 °C in darkness for seven days. Solubilizing activity was indicated by the formation of a halo around the colony. Both tests were conducted in triplicate.

Potassium (K) Solubilization

The potential of the fungi to solubilize K was assessed using Alexandrov medium, supplemented with 0.3% feldspar as the K source (Alexandrov, in g L−1: glucose, 5.00; MgSO4·7H2O, 0.50; FeCl3, 0.006; CaCO3, 0.60; CaPO4, 2.00; agar, 30.00; pH 7; Panreac, Barcelona, Spain) [67]. The medium was autoclaved and poured into Petri dishes. A 5 mm diameter piece of mycelium was placed in the center of each dish, and the plates were incubated at 23 °C in darkness for seven days. Solubilizing activity was indicated by the formation of a halo around the colony. The assay was conducted in triplicate.

Fungal Growth on PDA with Salt

Following the method of Hammami et al. [68], PDA medium was prepared and amended with different concentrations of NaCl (Panreac, Barcelona, Spain): 0 mM (Control), 100 mM, 200 mM, and 500 mM. Petri dishes were divided into quadrants, and a 5 mm diameter fungal plug was inoculated in the center of each dish. Three replicates were prepared for each fungus and treatment, and the plates were incubated in the dark at 23 °C. Colony growth was measured along each radius every three days until the ninth day.

2.3. Hydroponic Forage Assays: Treatments and Experimental Design

An experiment was conducted to study the responses of two plant species, common wheat and barley, to the combined effect of three fungal isolates and three levels of salinity (0 mM, 100 mM, and 200 mM of NaCl) under hydroponic conditions. Conducted in a growth chamber with a 16 h light/8 h dark photoperiod at 20 °C, the assays for each plant species were performed separately and consecutively. Each trial used 36 perforated trays (16 cm × 11 cm × 5 cm) containing seeds inoculated with four fungal treatments (the three endophytic fungi and a negative Control, L0, with sterile distilled water) across the three salinity levels, with three replicates per treatment combination (Table 1). The entire experiment was repeated 15 days after the first trial to ensure reproducibility. Pictures from the assays are presented as Supplementary Material for bread wheat (Figure S1) and barley (Figure S2).
The mycelium inoculum, once obtained and filtered as explained before, was resuspended in sterile distilled water and homogenized to 50 mg mycelium per mL. Sterilized seeds were submerged in each treatment (or sterile distilled water for the Control, L0) for 6 h at 23 °C in darkness. After treatment, 20 g of seeds were placed in each sterilized tray and incubated in the growth chamber at 20 °C with a 16 h light/8 h dark cycle, on a 3% incline. Trays were irrigated every 12 h with 10 mL of the corresponding saline solution. Seedling growth was recorded daily. After this measurement, the trays were randomly distributed within the growth chamber to minimize variability within treatment conditions.
After eight days of growth, the plants were harvested, and measurements were taken for shoot and root heights and fresh weight. These data were used to determine the green fodder system yield, calculated as the ratio of fresh weight (in grams) produced per gram of seed used. Subsequently, plants were dried in an oven at 50 °C until a constant weight was achieved, allowing for the subsequent determination of dry weight.

2.4. Re-Isolation of the Fungal Endophytes

To confirm that the fungal isolates had colonized the plants, leaf samples from each treatment were surface-sterilized and placed in Petri dishes containing PDA medium. The dishes were stored in a growth chamber at 24 °C in the dark and observed daily until the isolates were recovered. For the negative Control (L0), the plates were observed for fifteen days to confirm the absence of fungal colonization.

2.5. Statistical Analysis

The effects of endophytic fungal inoculation and salt stress on each plant species, as well as their interaction, were assessed using a split-plot analysis of variance (ANOVA), with salt stress as the main factor and fungal inoculation as the secondary factor. Significant differences among mean values were determined using Fisher’s least significant difference (LSD) test at a 95% confidence level. Assumptions of normality and homoscedasticity were verified using the Shapiro–Wilk test and Levene’s test, respectively. Statistical analyses were conducted using STATISTIX 8.1 software (Analytical Software, Tallahassee, 2005; https://www.statistix.com, accessed on 20 October 2024). Additionally, Pearson’s correlation coefficient was calculated to assess relationships between different parameters using R 4.4.1 (R Core Team, Vienna, Austria, 2024; https://www.r-project.org, accessed on 20 October 2024) [69]. The correlation matrixes for the main parameters for bread wheat and barley are shown as correlograms in Appendix A, as Figure A3 and Figure A4, respectively.

3. Results

3.1. Identification of the Fungal Isolates and Evaluation of Their Plant Growth-Promoting Traits

Forty-two fungal isolates were obtained from the twenty samples of leaves and stems of Spanish lavender (L. stoechas) and then grouped into sixteen morphotypes. The three morphotypes selected for this study were identified through ITS sequencing as Diplodia corticola (L11), Leptobacillium leptobactrum (L15), and Cladosporium cladosporioides (L16). The GenBank identity for these isolates was greater than 99%, confirming accurate taxonomic identification (Table 2).
The potential of the endophytic fungi to promote plant growth was assessed through a series of biochemical assays, as detailed in Table 3. The parameters studied included the production of auxins and ammonia, as well as the enzymatic activity on phosphate, phytic acid, and potassium. All three isolates demonstrated the capacity to synthesize IAA, both in standard PDB medium and in PDB supplemented with L-tryptophan. The concentration of IAA was approximately two-fold higher when L-tryptophan was added to the medium, increasing from 7.63 µg mL−1, 9.35 µg mL−1, and 8.65 µg mL−1 to 16.39 µg mL−1, 19.23 µg mL−1, and 17.43 µg mL−1 for L11, L15, and L16, respectively (Table 3). Isolate L15 (L. leptobactrum) demonstrated the highest auxin production, in both the presence and absence of L-tryptophan as precursor. Additionally, isolate L15 exhibited moderate-to-high activity in ammonia production, phosphate solubilization, phytic acid solubilization, and potassium solubilization, while isolate L16 (C. cladosporioides) showed high ammonia production and moderate potassium solubilization. In contrast, the isolate L11 (D. corticola) did not exhibit any of the aforementioned activities, with the exception of IAA production.

3.2. Effect of Salinity on Fungal Growth

The evaluation of the potential of the fungal isolates to grow under salt conditions revealed that the salt concentration in the culture medium significantly affected the diametral growth of selected fungi nine days after sowing (df = 3; F = 21.25; p ≤ 0.01). Similarly, fungal growth also varied significantly depending on the isolate (df = 2; F = 15.71; p ≤ 0.01). Furthermore, Figure 1 illustrates the significant effect of the interaction between the salt concentration and the fungal isolate on the diametral growth after nine days (df = 6; F = 10.46; p ≤ 0.01). The results indicate varying responses to salt stress among the fungal isolates. Specifically, isolate L11 (D. corticola) demonstrated increased growth with higher NaCl concentrations, peaking at 500 mM NaCl, which was not significantly different from the growth at 200 mM. However, both concentrations exhibited approximately double the growth observed in the absence of salt. Isolate L15 (L. leptobactrum) exhibited optimal growth at 100 mM and 200 mM NaCl, with significantly higher growth at 200 mM (a 47.7% increase compared to growth under standard conditions), followed by a decline in growth at 500 mM NaCl. Isolate L16 (C. cladosporioides) showed minimal variation in growth across the different NaCl concentrations, with only slight, although not significant, increases observed at higher salt levels.

3.3. Hydroponic Forage Assays: Impact on Seedling Daily Growth Under Salinity Stress

Table 4 presents a summary of the ANOVA results for the effects of different salinity stress levels and the inoculation with the selected fungal isolate on both plant species, from the third day after sowing until the eighth day, when the seedlings were harvested. For both bread wheat and barley, the effects of salt and endophyte, as well as their interactions, were significant (p ≤ 0.001) across all days measured. The complete results for the daily growth of seedlings are shown in Appendix A, for both bread wheat (Figure A1) and barley (Figure A2).
The daily growth of wheat seedlings from day 3 to day 8 shows significant variation based on salinity level and fungal treatment (Figure S1). Focusing on the final growth, on day 8 and at 0 mM NaCl (S0), L15 (L. leptobactrum) resulted in the highest growth, with samples reaching 16.63 cm by day 8, followed by L11 (D. corticola) with 14.03 cm, and L16 (C. cladosporioides) with 13.77 cm, all outperforming the Control (L0) with 11.73 cm (Figure 2). At 100 mM NaCl (S100), although the Control plants (L0) showed stunted growth, the application of fungal treatments resulted in the mitigation of some of the adverse effects associated with salt stress. L16 (C. cladosporioides) achieved the highest growth (10.53 cm), followed by L15 (L. leptobactrum) (10.12 cm) and L11 (D. corticola) (8.47 cm), while the Control (L0) only reached 1.00 cm. At the highest salinity level, 200 mM NaCl (S200), fungal inoculations still significantly improved growth, with L15 (L. leptobactrum) growing up to 1.50 cm, L11 (D. corticola) to 1.40 cm, and L16 (C. cladosporioides) to 1.22 cm, compared to the Control (L0) with 0.50 cm.
The growth patterns observed in barley seedlings were comparable to those observed in wheat seedlings for seeds treated with L15 (L. leptobactrum) and L16 (C. cladosporioides). However, the inoculation of barley seeds with L11 (D. corticola) resulted in a significant decline in plant growth compared to the Control group (Figure S2). At 0 mM NaCl, L15 (L. leptobactrum) and L16 (C. cladosporioides) showed significantly enhanced growth compared to the Control (L0), with heights of 4.87 cm and 5.08 cm on day 3 and reaching 13.00 cm and 12.50 cm by day 8, respectively (Figure 3). In contrast, the final length of plants treated with L11 (D. corticola) showed a significant decrease of 12%, in comparison to the Control plants (L0). Under 100 mM NaCl, L16 (C. cladosporioides) and L15 (L. leptobactrum) treatments resulted in increased growth (10.27 cm and 8.95 cm by day 8), compared to the Control (7.55 cm). At 200 mM NaCl, L15 (L. leptobactrum) and L16 (C. cladosporioides) also showed better growth (6.70 cm and 6.55 cm by day 8) than the Control (5.08 cm). On the other hand, inoculation with L11 (D. corticola) led to a notable decline in growth, with reductions of 11.5% and 14.4% observed at 100 mM and 200 mM NaCl, respectively, in comparison to the respective Control treatments

3.4. Hydroponic Forage Assays: Impact on Seedling Yield Parameters Under Salinity Stress

Eight days after sowing, the seedlings were harvested from each tray and a series of yield parameters were measured. These parameters included fresh and dry weight, plant yield (expressed as g FW (grams of fresh weight) obtained per gram of seeds used for each tray), and seedling root length. As illustrated in Table 5, these parameters were significantly affected by the salt solution and the fungal inoculation treatments, either separately or as to their interaction, for both plant species.
Fresh weight was found to be significantly increased by all fungal treatments at 0 mM NaCl when compared to the Control (L0) for wheat seedlings (Figure 4a). The highest increase was observed in plants treated with L15 (L. leptobactrum), which demonstrated a 41.7% rise in fresh weight compared to the Control group (156 g vs. 110 g). In the presence of 100 mM NaCl, plants treated with L15 (L. leptobactrum) and L16 (C. cladosporioides) exhibited fresh weights which, although significantly lower than those of plants inoculated with any of the isolates under standard conditions (S0), remained significantly higher than those of any Control group, including the negative Control (L0 + S0). At 200 mM NaCl, a reduction in fresh weight was observed across all treatments. However, inoculation with L15 (L. leptobactrum) and L11 (D. corticola) resulted in a significant improvement in plant performance compared to values for the untreated plants grown under the same conditions (L0 + S200). Once again, L15 (L. leptobactrum) demonstrated the least significant reduction in fresh weight (107 g vs. 91 g).
As can be seen in Figure 4b, plant dry weight followed a pattern similar to that of the fresh weight (R2 = 0.94; p < 0.001). At 0 mM NaCl, inoculation with L11 (D. corticola) and L15 (L. leptobactrum) significantly increased dry weight (29.6 g and 29.1 g, respectively). Inoculation with L16 (C. cladosporioides) increased weight to 27.9 g compared to the Control (23.4 g), but the value was significantly lower than those of the other two isolates. Under 100 mM NaCl, all fungal treatments maintained significantly higher dry weights compared to the Control L0, with L16 (C. cladosporioides) leading in this case (25.6 g). At 200 mM NaCl, decreases were observed, yet L11 (D. corticola) and L15 (L. leptobactrum) still performed better than Control L0.
Wheat yield (fresh weight/seed weight ratio) was significantly higher for plants inoculated with L15 (L. leptobactrum) when compared to the Control group (L0) across all salinity levels, particularly at 0 mM NaCl (7.8 g/g vs. 5.5 g/g, respectively) (Figure 4c). L16 (C. cladosporioides) also showed enhanced yield compared to the Control (L0), but only at 0 mM (7.1 g/g) and 100 mM NaCl (5.9 g/g). At 200 mM NaCl, yields decreased, while L15 maintained the highest value (5.3 g/g), significantly higher than the respective Control L0 (4.6 g/g), as was also the case with plants treated with D. corticola L11 (5.0 g/g).
Root length (Figure 4d) was similarly affected, with L15 (L. leptobactrum) again showing the greatest root-length at 0 mM NaCl (2.08 cm). Under 100 mM NaCl, L15 (L. leptobactrum) and L16 (C. cladosporioides) exhibited longer roots compared to the Control L0. However, at 200 mM NaCl, all treatments showed significant reductions, though L15 (L. leptobactrum) still had the longest roots (0.50 cm), with a value significantly higher than the rest of treatments under the same salt stress.
For barley seedlings, fresh weight was significantly increased by all fungal treatments at 0 mM NaCl, with L15 (L. leptobactrum) showing the greatest increase (199.7 g vs. 130.9 g for L0) (Figure 5a). At 100 mM NaCl, L15 again had the highest fresh weight (166.5 g), while L16 (C. cladosporioides) also performed well (154.5 g). At 200 mM NaCl, fresh-weight reductions were observed across all treatments, yet L15 maintained the highest value (149.4 g), significantly higher than the fresh weights of the untreated plants at any salt-stress level. The same trends were observed for the dry weight (Figure 5b) and yield of the barley seedlings. Thus, these parameters were significantly higher for plants inoculated with L15 (L. leptobactrum) at 0 mM NaCl (29.2 g) and remained higher than the Control L0 under both salinity stress levels. Root length in barley seedlings (Figure 5d) was longest for L15 (L. leptobactrum) at 0 mM NaCl (2.0 cm). Under 100 mM NaCl, L15 and L16 exhibited longer roots compared to the Control L0. At 200 mM NaCl, all treatments showed significant reductions, with L15 (L. leptobactrum) having the longest roots (1.1 cm).

4. Discussion

The study identified and evaluated three endophytic fungal isolates from Spanish lavender (Lavandula stoechas)—Diplodia corticola (L11), Leptobacillium leptobactrum (L15), and Cladosporium cladosporioides (L16)—for different plant growth-promoting (PGP) traits. The significant performance increase in inoculated barley and wheat seedlings in the laboratory assays reinforces the potential of these isolates to mitigate salt stress and influence plant growth in species beyond their initial isolation hosts.
The viability of the fungal treatments was validated at harvest through the successful re-isolation of the three fungal isolates from plant tissues, confirming effective seed inoculation and persistence under varying salinity conditions. As previously outlined [70], this outcome is crucial for linking the effects registered in the plants to the fungi’s activity, highlighting the method’s robustness and potential for future applications. L11 (D. corticola), L15 (L. leptobactrum), and L16 (C. cladosporioides) efficiently established in Hordeum vulgare and Triticum aestivum plants and colonized the internal tissues of the plants without causing apparent harm. However, the variable effect observed for L11 (D. corticola) suggests that endophyte–host interactions can vary significantly, underscoring the need to explore these affinities further for optimized results [71]. Additional limitations include the need to replicate complex environmental conditions, evaluate their persistence and long-term effects throughout the full growth cycles of wheat and barley, and assess the responses to additional stress factors. Understanding the specific mechanisms of action will also be crucial for optimizing the uses of these endophytes in enhancing crop resilience and productivity.
The significant performance improvements in inoculated barley and wheat seedlings reinforce the potential of the selected fungal isolates as growth promoters, particularly for L15 (L. leptobactrum). This aligns with previous studies [72,73,74] showing that endophytes, such as Piriformospora indica or Epichloë bromicola, can improve cereal growth by releasing growth-promoting compounds like IAA (indole-3-acetic acid). In this context, all three isolates produced IAA with amounts below 10 µg per ml of filtrate, values which doubled in the presence of tryptophan. This also supports the fact that they can produce IAA via both tryptophan-dependent and independent pathways [75].
Notably, L15 (L. leptobactrum) showed the highest plant growth-promoting (PGP) activity, including IAA and ammonia production, and phosphate, phytic acid, and potassium solubilization. L16 (C. cladosporioides) showed ammonia production and potassium solubilization. These PGP traits are of critical importance for enhancing nutrient availability and promoting plant growth during the initial stages of the seedling [28,76,77,78,79,80,81], which is crucial for the goal of minimizing the growing period of forage fodder.
The differential responses of the isolates to salt stress, considering the significant variability in their growth under varying NaCl concentrations, underscore again these isolates’ varied effectiveness. L11 (D. corticola) thrived in high salinity, with increased growth at 200 mM and 500 mM NaCl, suggesting mechanisms of osmotic adjustment or ion sequestration of the excessive Na+ [82]. This strong response indicated the potential of L11 (D. corticola) for promoting plant growth in saline conditions, even if this isolate was only effective in IAA production. L15 (L. leptobactrum) exhibited optimal growth at 200 mM NaCl but declined at higher concentrations, indicating a certain threshold for salt tolerance. L16 (C. cladosporioides) maintained relatively stable growth across different salt concentrations, suggesting adaptability to various environments. Despite their different responses, all three isolates could effectively grow in the presence of NaCl, which may benefit plants under saline conditions, considering a previous study that found that fungal isolates that kept growing under salt stress (at 500 mM NaCl) also retained their ability to produce bioactive substances, particularly extracellular enzymes [83].
The hydroponic forage assays revealed significant effects of salinity and fungal inoculation on wheat and barley daily growth. L15 (L. leptobactrum) consistently promoted the highest growth in wheat and barley, especially under standard conditions (0 mM NaCl) and at moderate salinity (100 mM NaCl), followed by L16 (C. cladosporioides), which also provided consistent growth benefits. On the other hand, L11 (D. corticola) was effective for wheat under lower salinity, but had a species-specific negative impact on barley. These results are in line with previous studies, showing that fungal inoculation can significantly promote plant growth and mitigate the effects of salt stress under various conditions, including hydroponics systems [35,84,85,86].
Additionally, wheat yields increased to 6.81 g g−1, 7.80 g g−1, and 7.11 g g−1 with L11 (D. corticola), L15 (L. leptobactrum), and L16 (C. cladosporioides) inoculation, respectively, reflecting increases of 23.6%, 41.6%, and 29.0% over the Control L0, while barley yields were improved to 7.2 g g−1, 10.0 g g−1, and 8.9 g g−1, translating to increases of 9.3%, 52.5%, and 35.4%. These results align with and exceed previous hydroponic trials [53,87,88], suggesting that fungal inoculation can significantly boost yields under controlled conditions.
A significant yield increase was also observed in plants treated with 100 mM NaCl, with increases for bread wheat of 18.5%, 23.0%, and 20.7% for L11 (D. corticola), L15 (L. leptobactrum), and L16 (C. cladosporioides) applications, respectively, in comparison with the Control plants (L0). In the case of barley, only the application of L15 (L. leptobactrum) or L16 (C. cladosporioides) improved yields significantly, by 24.8% and 15.8%, respectively, in comparison with the Control group (L0). For all cases, the fungal inoculation implied yields at 100 mM of between 5.88 g g−1 and 6.11 g g−1 for bread wheat and between 6.33 g g−1 and 8.33 g g−1 for barley, which surpassed the cited reference values, of approximately 5 g of green-weight per gram of wheat seeds [53,87] and of 5.2 g of fresh-weight per gram of barley seeds [88]. Particularly, L15 (L. leptobactrum) inoculation on plants treated with 200 mM NaCl showed no significant differences compared to the Control (L0 S0) in wheat seedlings, while differences were significantly higher for barley, while L16 (C. cladosporioides) performed better under 100 mM NaCl, suggesting that both nutrient absorption and stress resistance are crucial for seedling development. Further research is needed to explore how these isolates induce physiological and biochemical changes in plants, such as the production of osmoprotectants and antioxidant compounds, investigations which could further enhance the efficacy of these isolates under saline conditions [34].

5. Conclusions

The evaluation of the three endophytic fungal isolates—Diplodia corticola L11, Leptobacillium leptobactrum L15, and Cladosporium cladosporioides L16—derived from Spanish lavender demonstrated their significant potential as plant growth promoters, particularly under saline conditions. L15 (L. leptobactrum) emerged as the most effective isolate, showing superior plant growth-promoting traits and resilience under moderate salinity stress, while L16 (C. cladosporioides) exhibited consistent performance across varying salt concentrations. Although L11 (D. corticola) thrived in high salinity, its variable effects suggest a need for more nuanced application strategies. These findings underscore the importance of selecting specific fungal isolates for targeted applications to optimize crop growth and resilience. Using these natural symbionts could reduce reliance on chemical treatments, leading to more sustainable and cost-effective farming. The significant yield improvements observed showed that these fungi could be particularly valuable for greenhouse, vertical farming, and hydroponic fodder production, even in areas with saline water resources. Future research should focus on developing user-friendly inoculation methods, optimizing application rates, assessing endophyte persistence throughout plant cycles, and profiling secondary metabolites to understand stress mitigation mechanisms. Additionally, the development of a non-invasive method, such as overhead imaging and image analysis, to assess germination rates early in the experiment would provide deeper insight into the results in future studies. These insights offer promising solutions for farmers facing soil and water salinity challenges in various agroecosystems.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy14112501/s1, Figure S1: Comparative photos showing the effect of fungal inoculation, with Control (L0), D. corticola (L11), L. leptobactrum (L15), and C. cladosporioides (L16), on the seedling growth under varying salinity conditions (0 mM, 100 mM, and 1200 mM), for bread wheat harvested after 8 days; Figure S2: Comparative photos showing the effect of fungal inoculation, with Control (L0), D. corticola (L11), L. leptobactrum (L15), and C. cladosporioides (L16), on the seedling growth under varying salinity conditions (0 mM, 100 mM, and 1200 mM), for barley harvested after 8 days.

Author Contributions

Conceptualization, C.G.-L.; Data curation, C.G.-L. and M.J.P.; Formal analysis, C.G.-L. and M.J.P.; Funding acquisition, C.G.-L. and M.J.P.; Investigation, C.G.-L. and M.J.P.; Methodology, C.G.-L.; Project administration, C.G.-L.; Resources, C.G.-L. and M.J.P.; Software, C.G.-L. and M.J.P.; Supervision, M.J.P.; Validation, M.J.P.; Visualization, C.G.-L. and M.J.P.; Writing—original draft, C.G.-L.; Writing—review and editing, C.G.-L. and M.J.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Materials; further inquiries can be directed to the corresponding authors.

Acknowledgments

The authors would like to thank Teodoro Garcia and Francisco Barroso for their invaluable help in the laboratory work.

Conflicts of Interest

The authors declare no conflicts of interest.

Appendix A

The daily growth measurements of bread wheat and barley seedlings from day 3 to day 8 showed significant variations depending on the salinity level and the fungal treatment. The complete information is offered here as a complement (Figure A1 and Figure A2) for the information presented in Figure 2 and Figure 3.
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Figure A1. Evaluation of the effect of fungal inoculation with (a) L0, Control; (b) L11, D. corticola; (c) L15, L. leptobactrum; and (d) L16, C. cladosporioides, as to the daily growth of bread wheat seedlings from day 3 to day 8 under varying salinity conditions (0 mM, 100 mM, and 1200 mM). Lines represent the mean values, and error bars indicate the standard error. Different letters indicate significant differences according to LSD (p ≤ 0.05).
Figure A1. Evaluation of the effect of fungal inoculation with (a) L0, Control; (b) L11, D. corticola; (c) L15, L. leptobactrum; and (d) L16, C. cladosporioides, as to the daily growth of bread wheat seedlings from day 3 to day 8 under varying salinity conditions (0 mM, 100 mM, and 1200 mM). Lines represent the mean values, and error bars indicate the standard error. Different letters indicate significant differences according to LSD (p ≤ 0.05).
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Figure A2. Evaluation of the effect of fungal inoculation with (a) L0, Control; (b) L11, D. corticola; (c) L15, L. leptobactrum; and (d) L16, C. cladosporioides as to the daily growth of barley seedlings from day 3 to day 8, compared to the Control, under varying salinity conditions (0 mM, 100 mM, and 1200 mM). Lines represent the mean values, and error bars indicate the standard error. Different letters indicate significant differences according to LSD (p ≤ 0.05).
Figure A2. Evaluation of the effect of fungal inoculation with (a) L0, Control; (b) L11, D. corticola; (c) L15, L. leptobactrum; and (d) L16, C. cladosporioides as to the daily growth of barley seedlings from day 3 to day 8, compared to the Control, under varying salinity conditions (0 mM, 100 mM, and 1200 mM). Lines represent the mean values, and error bars indicate the standard error. Different letters indicate significant differences according to LSD (p ≤ 0.05).
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Figure A3. Correlation matrix showing the significant Pearson correlations (p ≤ 0.05) between parameters investigated in the study for bread wheat. The robustness of the correlations is depicted using a gradation of colors, with darker colors indicating stronger correlations.
Figure A3. Correlation matrix showing the significant Pearson correlations (p ≤ 0.05) between parameters investigated in the study for bread wheat. The robustness of the correlations is depicted using a gradation of colors, with darker colors indicating stronger correlations.
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Figure A4. Correlation matrix showing the significant Pearson correlations (p ≤ 0.05) between parameters investigated in the study for barley. The robustness of the correlations is depicted using a gradation of colors, with darker colors indicating stronger correlations.
Figure A4. Correlation matrix showing the significant Pearson correlations (p ≤ 0.05) between parameters investigated in the study for barley. The robustness of the correlations is depicted using a gradation of colors, with darker colors indicating stronger correlations.
Agronomy 14 02501 g0a4

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Agronomy 14 02501 g001
Figure 1. Effects of different salinity levels (S0: 0 mM; S100: 100 mM; S200: 200 mM; S500: 500 mM of NaCl) on the in vitro diametral growth of selected fungi (L11: D. corticola; L15: L. leptobactrum; L16: C. cladosporioides) measured nine days after inoculation on Petri dishes. The S500 treatment was only included in this experiment for determinations previous to plant inoculation. The bars show the mean values, and the error bars, the standard error. Different letters indicate significant differences according to LSD (p ≤ 0.05).
Figure 1. Effects of different salinity levels (S0: 0 mM; S100: 100 mM; S200: 200 mM; S500: 500 mM of NaCl) on the in vitro diametral growth of selected fungi (L11: D. corticola; L15: L. leptobactrum; L16: C. cladosporioides) measured nine days after inoculation on Petri dishes. The S500 treatment was only included in this experiment for determinations previous to plant inoculation. The bars show the mean values, and the error bars, the standard error. Different letters indicate significant differences according to LSD (p ≤ 0.05).
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Figure 2. Evaluation of the effects of fungal inoculation, with Control (L0), D. corticola (L11), L. leptobactrum (L15), and C. cladosporioides (L16), as to seedling growth under varying salinity conditions (0 mM, 100 mM, and 1200 mM) for bread wheat, harvested after 8 days. Bars represent the mean, and error bars indicate the standard error. Different letters indicate significant differences according to LSD (p ≤ 0.05).
Figure 2. Evaluation of the effects of fungal inoculation, with Control (L0), D. corticola (L11), L. leptobactrum (L15), and C. cladosporioides (L16), as to seedling growth under varying salinity conditions (0 mM, 100 mM, and 1200 mM) for bread wheat, harvested after 8 days. Bars represent the mean, and error bars indicate the standard error. Different letters indicate significant differences according to LSD (p ≤ 0.05).
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Agronomy 14 02501 g003
Figure 3. Evaluation of the effect of fungal inoculation, with Control (L0), D. corticola (L11), L. leptobactrum (L15), and C. cladosporioides (L16), as to seedling growth under varying salinity conditions (0 mM, 100 mM, and 1200 mM) for barley, harvested after 8 days. Bars represent the mean, and error bars indicate the standard error. Different letters indicate significant differences according to LSD (p ≤ 0.05).
Figure 3. Evaluation of the effect of fungal inoculation, with Control (L0), D. corticola (L11), L. leptobactrum (L15), and C. cladosporioides (L16), as to seedling growth under varying salinity conditions (0 mM, 100 mM, and 1200 mM) for barley, harvested after 8 days. Bars represent the mean, and error bars indicate the standard error. Different letters indicate significant differences according to LSD (p ≤ 0.05).
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Agronomy 14 02501 g004
Figure 4. Effects of selected fungi (L0, Control; L11, D. corticola; L15, L. leptobactrum; and L16, C. cladosporioides) on (a) fresh weight, (b) dry weight, (c) yield, and (d) root length of bread wheat seedlings grown in different salt concentrations (0 mM, S0; 100 mM, S100; and 200 mM, S200) and harvested after 8 days. Bars represent the mean, and error bars indicate the standard error. Different letters indicate significant differences according to LSD (p ≤ 0.05).
Figure 4. Effects of selected fungi (L0, Control; L11, D. corticola; L15, L. leptobactrum; and L16, C. cladosporioides) on (a) fresh weight, (b) dry weight, (c) yield, and (d) root length of bread wheat seedlings grown in different salt concentrations (0 mM, S0; 100 mM, S100; and 200 mM, S200) and harvested after 8 days. Bars represent the mean, and error bars indicate the standard error. Different letters indicate significant differences according to LSD (p ≤ 0.05).
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Agronomy 14 02501 g005
Figure 5. Effects of selected fungi (L0, Control; L11, D. corticola; L15, L. leptobactrum; and L16, C. cladosporioides) on (a) fresh weight, (b) dry weight, (c) yield, and (d) root length of barley seedlings grown at different salt concentrations (0 mM, S0; 100 mM, S100; and 200 mM, S200) and harvested after 8 days. Bars represent the mean, and error bars indicate the standard error. Different letters indicate significant differences according to LSD (p ≤ 0.05).
Figure 5. Effects of selected fungi (L0, Control; L11, D. corticola; L15, L. leptobactrum; and L16, C. cladosporioides) on (a) fresh weight, (b) dry weight, (c) yield, and (d) root length of barley seedlings grown at different salt concentrations (0 mM, S0; 100 mM, S100; and 200 mM, S200) and harvested after 8 days. Bars represent the mean, and error bars indicate the standard error. Different letters indicate significant differences according to LSD (p ≤ 0.05).
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Table 1. Schematic of the split-plot experimental design of the study. For the main plot, three levels salt stress were stablished (0 mM, 100 mM, and 200 mM of NaCl), and in the subplots, in the inoculation with the fungal isolates, there were four levels (L0: Control with distilled water; L11: Diplodia corticola; L15: Leptobacillium leptobactrum; and L16: Cladosporium cladosporioides).
Table 1. Schematic of the split-plot experimental design of the study. For the main plot, three levels salt stress were stablished (0 mM, 100 mM, and 200 mM of NaCl), and in the subplots, in the inoculation with the fungal isolates, there were four levels (L0: Control with distilled water; L11: Diplodia corticola; L15: Leptobacillium leptobactrum; and L16: Cladosporium cladosporioides).
Salt Stress (Main Plot)
S0 (0 mM)S100 (100 mM)S200 (200 mM)
Fungal isolateL0L0 S0 IL0 S100 IL0 S200 I
L0 S0 IIL0 S100 IIL0 S200 II
L0 S0 IIIL0 S100 IIIL0 S200 III
L11L11 S0 IL11 S100 IL11 S200 I
L11 S0 IIL11 S100 IIL11 S200 II
L11 S0 IIIL11 S100 IIIL11 S200 III
L15L15 S0 IL15 S100 IL15 S200 I
L15 S0 IIL15 S100 IIL15 S200 II
L15 S0 IIIL15 S100 IIIL15 S200 III
L16L16 S0 IL16 S100 IL16 S200 I
L16 S0 IIL16 S100 IIL16 S200 II
L16 S0 IIIL16 S100 IIIL16 S200 III
This scheme is a representation of the treatments, their combinations, and the number of repetitions. Each repetition (marked as I, II, and III) was randomly distributed within the growth chamber.
Table 2. Taxonomic identification of the fungi used in the experiments.
Table 2. Taxonomic identification of the fungi used in the experiments.
CodeIdentification 1GenBank Accession NumberGenBank
Identity (%)
L11Diplodia corticolaMN698983.1100.00
L15Leptobacillium leptobactrumOW983854.199.79
L16Cladosporium cladosporioidesMF475952.1100.00
1 Based on comparison to ITS sequences in GenBank with similarity values ≥ 99%.
Table 3. Summary of the potential of endophytic fungi isolated from Lavandula stoechas (L11, D. corticola; L15, L. leptobactrum, and L16, C. cladosporioides) related to plant growth promotion.
Table 3. Summary of the potential of endophytic fungi isolated from Lavandula stoechas (L11, D. corticola; L15, L. leptobactrum, and L16, C. cladosporioides) related to plant growth promotion.
EndophyteL11L15L16
IAA-Trp (µg mL−1)7.63 ± 0.119.35 ± 0.128.65 ± 0.13
IAA+Trp (µg mL−1)16.39 ± 0.2119.23 ± 0.1717.43 ± 0.13
NH3−+++
Phosphate−+−
Phytic acid−+−
Potassium−++
IAA-Trp: auxin production in the absence of tryptophan; IAA+Trp: auxin production in the presence of tryptophan; NH3: ammonia production; solubilization of phosphates, phytic acid, and potassium. For qualitative assays: − no activity; + moderate activity; ++ high activity.
Table 4. Summary of ANOVA evaluating the effect of selected fungi on the daily growth of wheat and barley seedlings under varying salinity conditions. Results are presented as mean ± standard error for daily measured wheat and barley plant-length in centimeters.
Table 4. Summary of ANOVA evaluating the effect of selected fungi on the daily growth of wheat and barley seedlings under varying salinity conditions. Results are presented as mean ± standard error for daily measured wheat and barley plant-length in centimeters.
Bread WheatBarley
Salt (S)Endophyte (E)S×ESalt (S)Endophyte (E)S×E
Df236236
Day 3226.06 ***168.08 ***16.12 ***414.08 ***58.54 ***5.64 **
Day 44996.01 ***249.91 ***33.43 ***2812.88 ***55.57 ***5.79 **
Day 58453.43 ***443.27 ***97.14 ***2278.09 ***478.55 ***61.54 ***
Day 66901.19 ***934.38 ***224.69 ***2789.45 ***405.86 ***49.03 ***
Day 77179.88 ***283.34 ***120.41 ***3760.16 ***191.02 ***20.58 ***
Day 815998.3 ***1141.72 ***378.67 ***17027.2 ***353.07 ***15.99 ***
Degrees of freedom (df) are reported, along with F-values and their significance (** p ≤ 0.01, and *** p ≤ 0.001).
Table 5. Summary of ANOVA for evaluating the effects of selected fungi on the growth parameters of wheat seedlings harvested after 8 days.
Table 5. Summary of ANOVA for evaluating the effects of selected fungi on the growth parameters of wheat seedlings harvested after 8 days.
Plant Salt (S)Endophyte (E)S×E
gl236
Bread wheatFresh weight (g)219.19 ***62.1 ***13.72 ***
Dry weight (g)715.49 ***100.73 ***19.56 ***
Yield (g g−1)217.18 ***61.76 ***13.69 ***
Root length (cm)1969.49 ***218.3 ***61.17 ***
BarleyFresh weight (g)211.97 ***223.45 ***13.75 ***
Dry weight (g)72.14 ***162.37 ***30.48 ***
Yield (g g−1)212.17 ***224.99 ***13.82 ***
Root length (cm)1539.86 ***170.43 ***8.66 ***
Degrees of freedom (df) are reported, along with F-values and their significance (*** p ≤ 0.001).
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García-Latorre, C.; Poblaciones, M.J. Isolation, Identification, and Application of Endophytic Fungi from Lavandula stoechas L.: Mitigating Salinity Stress in Hydroponic Winter Cereal Fodder. Agronomy 2024, 14, 2501. https://doi.org/10.3390/agronomy14112501

AMA Style

García-Latorre C, Poblaciones MJ. Isolation, Identification, and Application of Endophytic Fungi from Lavandula stoechas L.: Mitigating Salinity Stress in Hydroponic Winter Cereal Fodder. Agronomy. 2024; 14(11):2501. https://doi.org/10.3390/agronomy14112501

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García-Latorre, Carlos, and María José Poblaciones. 2024. "Isolation, Identification, and Application of Endophytic Fungi from Lavandula stoechas L.: Mitigating Salinity Stress in Hydroponic Winter Cereal Fodder" Agronomy 14, no. 11: 2501. https://doi.org/10.3390/agronomy14112501

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