5-Aminolevulinic Acid and Soil Fertility Enhance the Resistance of Rosemary to Alternaria dauci and Rhizoctonia solani and Modulate Plant Biochemistry

Fungal infection of horticultural and cereal crops by Alternaria dauci and Rhizoctonia solani represents an important biotic stress that could be alleviated by application of 5-aminolevulinic acid (ALA) to fertile and poor soils. Therefore, in this study, the morphological, physiological, biochemical, and genetic effects of ALA application (eight weekly applications at 3–10 ppm) to A. dauci- and R. solani-infected Salvia rosmarinus (rosemary) in fertile and poor soils were investigated. ALA-treated plants produced the longest and highest number of branches and had higher fresh and dry weights. There were increases in the major essential oil constituents (1,8-cineole, linalool, camphor, and borneol), as shown by Gas chromatography–mass spectrometry (GC-MS); higher antioxidant activities in DPPH and β-carotene-bleaching assays; upregulated superoxide dismutase (SOD) and catalase (CAT) antioxidant enzyme activities; increased total phenolics, chlorophyll, soluble sugars, and proline; increased gas exchange parameters; enhanced leaf water potential and relative water content (RWC); and upregulated expression of DREB2 and ERF3 (stress-related genes) and FeSOD, Cu/ZnSOD, and MnSOD (antioxidant genes). Several mechanisms were involved, including stress tolerance, antioxidative, and transcription regulation mechanisms. Furthermore, ALA performance was increased in higher-quality soils with higher nutrient content. This study demonstrated the novel application of ALA as a biotic stress ameliorant with enhanced performance in fertile soils.


Introduction
5-Aminolevulinic acid (ALA) is a natural compound found in plants that is used internally as a precursor of chlorophylls and phytochromes [1]. ALA has proven effects as an abiotic stress ameliorant against salinity, drought, cold, and heat [2][3][4], with other applications in agriculture, such as fruit coloring [5], fruit thinning [6], and ion uptake [7]. This natural compound, when applied externally to plant leaves, stimulates the photosynthetic apparatus and gas exchange, leading to enhanced stress tolerance during abiotic stress. Some mechanisms have been indicated in these effects, including the osmotic adjustment and antioxidative mechanisms. However, a complete picture of the different mechanisms involved and an in-depth understanding of the interactions of the known mechanisms are

Vegetative Growth
The effects of ALA, soil type, and fungal infection on the rosemary plant's vegetative growth are shown in Figure 1. Two different media were used: A (30% sand + 70% loam) and B (100% loam soil). The biotic stress produced shorter plants with a lower number of leaves and caused reductions in the fresh and dry weights. Plants infected with A. dauci/R. solani and treated with high doses of ALA (5 and 10 ppm) produced the longest and highest number of branches under different soil mixes. The fresh and dry weights were the highest in infected and healthy plants treated with high doses of ALA (5 and 10 ppm). Healthy plants showed the highest vegetative growth among plants treated with ALA. Soil mix B plants showed higher performance than mix A under all growing conditions.

Essential Oil Composition
Four major essential oil constituents were identified: 1,8-cineole, linalool, camphor, and borneol, as shown in Table 1. The biotic stress produced higher amounts of linalool, camphor, and borneol compared to the control. In the control treatments, the application of ALA increased the content of these four major constituents, and soil mix B showed no significant difference from mix A. Under fungal infection, the application of higher doses of ALA yielded significant enhancement in the

Essential Oil Composition
Four major essential oil constituents were identified: 1,8-cineole, linalool, camphor, and borneol, as shown in Table 1. The biotic stress produced higher amounts of linalool, camphor, and borneol compared to the control. In the control treatments, the application of ALA increased the content of these four major constituents, and soil mix B showed no significant difference from mix A. Under fungal infection, the application of higher doses of ALA yielded significant enhancement in the content of the major essential oil constituents.

Antioxidant Activities
The essential oil and leaf extract antioxidant activities are shown in Figure 2. Under control conditions, ALA treatment and soil mix B showed the highest antioxidant activities (lower values of IC 50 ) in both essential oils and leaf extracts in the DPPH and β-carotene-linoleic acid assay. The fungus-infected plant essential oils and leaf extracts showed lower total antioxidant activities (higher values of IC 50 ) compared to infected plants treated with ALA. Superoxide dismutase (SOD) and catalase (CAT) enzyme activities were higher in the leaves of plants treated with ALA for both healthy and infected plants, as shown in Figure 3. Soil mix B showed higher SOD activity in leaf extracts as compared to soil mix A. IC50) in both essential oils and leaf extracts in the DPPH and β-carotene-linoleic acid assay. The fungus-infected plant essential oils and leaf extracts showed lower total antioxidant activities (higher values of IC50) compared to infected plants treated with ALA. Superoxide dismutase (SOD) and catalase (CAT) enzyme activities were higher in the leaves of plants treated with ALA for both healthy and infected plants, as shown in Figure 3. Soil mix B showed higher SOD activity in leaf extracts as compared to soil mix A.

Chlorophyll, Soluble Sugars, and Proline Compositions
Total phenolics, chlorophyll, carbohydrates, and proline were determined in the leaves, as shown in Figure 4. Under control conditions, ALA caused increases in phenolics as compared to untreated plants. Plants infected with A. dauci/R. solani and treated with ALA showed higher values of total phenolics compared to ALA-untreated plants. Total chlorophyll was higher in ALA-treated plants in the control as well as in the infected plants. Carbohydrates were higher in infected plants

Chlorophyll, Soluble Sugars, and Proline Compositions
Total phenolics, chlorophyll, carbohydrates, and proline were determined in the leaves, as shown in Figure 4. Under control conditions, ALA caused increases in phenolics as compared to untreated plants. Plants infected with A. dauci/R. solani and treated with ALA showed higher values of total phenolics compared to ALA-untreated plants. Total chlorophyll was higher in ALA-treated plants in the control as well as in the infected plants. Carbohydrates were higher in infected plants treated with ALA as compared to untreated plants. Proline accumulated in larger amounts in the leaves of infected plants treated with ALA as compared to the control.

Gas Exchange and Water Measures
The gas exchange parameters of the experiment are shown in Figure 5. The photosynthetic and transpiration rates and the conductance were higher in plants subjected to higher doses of ALA under normal growing conditions. Fungus-infected plants showed higher levels of photosynthesis and transpiration as well as stomatal conductance when subjected to ALA, as compared to untreated plants. Leaf water potential and relative water content (RWC) were higher also in ALA-treated plants under control conditions and in plants subjected to fungal infections ( Figure 6). Soil mix B showed higher patterns of gas exchange as compared to soil mix A.

Gas Exchange and Water Measures
The gas exchange parameters of the experiment are shown in Figure 5. The photosynthetic and transpiration rates and the conductance were higher in plants subjected to higher doses of ALA under normal growing conditions. Fungus-infected plants showed higher levels of photosynthesis and transpiration as well as stomatal conductance when subjected to ALA, as compared to untreated plants. Leaf water potential and relative water content (RWC) were higher also in ALA-treated plants under control conditions and in plants subjected to fungal infections ( Figure 6). Soil mix B showed higher patterns of gas exchange as compared to soil mix A.

Gene Expression
The gene expression of control plants, as well as infected plants, during ALA treatment is shown in Figures 7 and 8. Both DREB2 and ERF3 stress-related gene expression patterns showed significant increases under ALA treatment in both control and infected plants. The highest values were achieved when applying higher doses of ALA. Expression of antioxidant genes FeSOD, Cu/ZnSOD, and MnSOD was higher in ALA-treated plants under all conditions, and this expression was elevated in infected plants.

Discussion
Several reports have indicated that ALA might increase the fresh and dry weight of plants during stress conditions [2,4]. However, other reports indicate that ALA might act as a vegetative growth retardant with herbicidal activity and cause leaf necrosis [15,16]. The present study found stimulatory morphological effects of ALA in rosemary plants, including increased length, number of branches, and fresh and dry weights, which have not been previously reported in this species. The

Discussion
Several reports have indicated that ALA might increase the fresh and dry weight of plants during stress conditions [2,4]. However, other reports indicate that ALA might act as a vegetative growth retardant with herbicidal activity and cause leaf necrosis [15,16]. The present study found stimulatory morphological effects of ALA in rosemary plants, including increased length, number of branches, and fresh and dry weights, which have not been previously reported in this species. The improved vegetative growth of plants infected with fungi as a biotic stressor and the higher ALA activity with increasing soil fertility or improvement in physiochemical properties (as in soil mix B) are novel findings. These could be attributed to the increased photosynthetic rates in plants treated with high doses of ALA. ALA stimulates vegetative growth by stimulating photosynthesis and photosynthetic rates [3]. Fungal infection by A. dauci and R. solani reduced the growth of infected plants that were not treated with ALA ( Figure 1). The application of ALA alleviated this biotic stress and enhanced vegetative growth; to our knowledge, the present study is the first to report this finding. Furthermore, the availability of macro-and micronutrients in the soil increased the performance of ALA, as expressed in increased fresh and dry weights [8].
Rosemary plants are characterized by the presence of 1,8-cineole, linalool, camphor, and borneol in the essential oil of the leaves [12], as found in this study. Treatment with high doses of ALA significantly increased the ratio of the main components in the essential oil of the leaves in plants showing increased vegetative growth due to alleviated biotic stress. The change in major components of the essential oil could be attributed to the regulatory effects of ALA on plant metabolism, identified here as increased gas exchange and leaf water potential and relative water content, soluble sugars, and phenols. Previous investigations have found that ALA can modulate leaf water potential and gas exchange under abiotic stress conditions [4]. The shifts in essential oil composition of the leaves help to control fungal infections and increase the resistance of infected plants. Most of these main constituents have shown antifungal activities against A. dauci and R. solani [17][18][19].
ALA is known to influence the antioxidant mechanism of treated plants during specific stress conditions, such as drought, salinity, and cold, by increasing the photosynthetic rate, reducing damage to the photosynthetic apparatus, enhancing gas exchange, and increasing chlorophyll content [20][21][22]. The antioxidant activities of the essential oils and leaf extracts were higher in ALA-treated plants than in the control because of the increased SOD and CAT enzyme activities in the leaves, as well as the increase in primary essential oil constituents exhibiting strong antioxidant activities, such as cineol and linalool [23,24]. Such increases in the antioxidant defense mechanism associated with increased growth of plants and alleviation of fungal infection stress have not been previously reported. In addition, there was an accumulation of proline, which is known to accumulate under stress conditions in most plants. Proline is the antioxidant defense mechanism molecule that maintains turgor pressure and membrane stability.
Increases in the total phenolic composition of the leaves were found, and this accumulation played a major role in increasing the antioxidant activity of the leaves and resistance to biotic and abiotic stresses. Previous reports have indicated that ALA application can increase the phenolic composition of treated plants [4,25]. The present study showed that soil fertility might increase phenolic and antioxidant accumulation in the leaves of ALA-treated plants under biotic stress. Previous studies found that ALA application enhances nutrient uptake under normal and abiotic stress conditions [7,26]. The present study confirmed that this occurs under biotic stress conditions as well. There were increases in the chlorophyll composition of leaves in plants treated with ALA, which is the precursor of chlorophyll and other important molecules in plants [1]. The accumulation of soluble sugars found here under biotic stress might be considered a stress tolerance mechanism because this accumulation enhanced the osmotic adjustment and performance of the plants subjected to fungal infection. The accumulation of sugars was higher in loamy soil subjected to ALA, owing to the higher rate of photosynthesis in the leaves and levels of macro-and micronutrients in the soil. The increases in gas exchange parameters in ALA-treated plants were associated with increased leaf water potential and RWC under fungal infection, which play a major role in the improvement of vegetative growth of the plants and are considered a stress tolerance mechanism [27]. The treatment with ALA can recover the transpiration rate and the stomatal conductance when the plants are attacked by Alternaria, with respect to the non-attacked plants.
The present study also showed increases in the gene expression of stress-related (DREB2 and ERF3) and antioxidant genes (FeSOD, Cu/ZnSOD, and MnSOD) in plants treated with ALA under biotic stress conditions as compared to the control. Previous studies have indicated that ALA upregulates the expression of POD, CAT, and ascorbate peroxidase (APX) in Brassica napus subjected to salt stress conditions [21]. However, the stimulatory activity of these genes had not previously been reported under biotic stress conditions, as in this study. Furthermore, these activities explain the enhanced antioxidant properties of ALA-treated plants and indicate that the antioxidant mechanism might be considered the main function of ALA as a biotic stress ameliorant. It was clear that soil mix B (100% loam) yielded better morphological and physiological performance than soil mix A (70% loam + 30% sand).
In the current study, we demonstrated the novel finding that ALA application alleviates plant biotic stress conditions via several mechanisms, including stress tolerance and the antioxidative mechanism, by enhancing relative water content gas exchange and osmotic adjustment, activating antioxidant enzymes, accumulating antioxidants, and stimulating stress and antioxidant genes. Furthermore, ALA performance was increased in higher-quality soils with higher nutrient levels.

Plant Material, Soil Mixes, ALA Treatment, and Inoculum Preparation
Young Salvia rosmarinus (L.) Schleid. plantlets (10 cm) containing 4-5 buds were obtained from local commercial nurseries in two successive seasons (January 2018 and 2019). The plants were identified, and voucher specimens were maintained at Alexandria University, Egypt. Plants were transplanted into larger 3.1 L plastic pots containing two different media: A (30% sand + 70% loam) and B (100% loam soil). The physiochemical properties of both soils are shown in Table 2. Organic carbon was determined, following Reference [28]. Fe, Cu, Zn, and Mn were determined by atomic absorption spectrophotometry (AASVario 6, Jena, Germany) [29]. Nitrogen was determined using the Kjeldahl method [30] and phosphorus was determined via the phosphomolybdic acid method [31]. Experiments were conducted in a greenhouse and repeated during the years 2018 and 2019. Growing media were supported by 2g L −1 fertilizer (Crystalon ® , 19% N: 19% P: 19% K). The mean growing temperatures were 15.1 • C at night a 24.5 • C during the day, relative humidity ranged between 68% and 82%, and photosynthetic active radiation was 900 W m −2 at 12:00. The plants were watered every 48 h until drop-off. A foliar spray of 5-aminolevulinic acid (ALA, Sigma-Aldrich, Berlin, Germany) at 3, 5, and 10 ppm was applied weekly until drop-off. Untreated plants were considered the control. A. dauci and R. solani fungi were isolated from infected rosemary plants showing symptoms of blight and crown rot. The plant surface was sterilized with 0.1% sodium hypochlorite, and small infected pieces of leaves or roots were cut and placed in Petri dishes with potato dextrose agar medium for 2 weeks at 25 • C. The fungi were identified using microscopic morphological characteristics. The inoculum of each fungus was prepared by introducing a sterile needle into sterilized and filtered flasks of Richard's liquid medium, containing 5 g KH 2 PO 4 , 10 g KNO 3 , 2.5 g MgSO 4 , 0.02 g FeCl 3 , 50 g sucrose, and 1000 mL distilled water. They were then incubated for 2 weeks at 25 ± 1 • C, and the mycelia mat was isolated by filtering with filter paper No. 1, washing with distilled water, mixing 10 g of mycelium in 100 mL distilled water, blending the mix at 10,000 rpm in a Waring blender, and inoculating a suspension of 1 g fungus suspension to the young plants. Untreated plants with fungi were used as a control. Factorial experimental design was used. Three blocks were used: Each block contained 24 treatments (3 fungal treatments, including control × 2 soil mixes × 4 ALA doses). Each treatment was represented by 6 replicates, and the total number of plants was 144 plants per block. All chemicals were HPLC grade (Sigma-Aldrich, Cairo, Egypt).

Morphological Measurements
After 8 weeks, branch length (cm) was determined, number of branches per plant was counted, and the fresh (FWs, g) and dry weights (DWs, g) were obtained. The dry weight was determined by preliminary drying at room temperature (20 • C), then dried in the oven at 30 • C until reaching a constant weight, following Reference [32].

GC-MS of Essential Oil and Preparation of Leaf Extracts
Leaf essential oil was extracted via hydrodistillation of dried leaves (500 g) using a Clevenger-type apparatus. The oil was dried, filtered, and kept at 4 • C under dark conditions [33]. A gas chromatograph mass spectrometer (Thermo Scientific, Waltham, MA, USA) equipped with a TG-1MS column was used for the analyses. The temperature was ramped from 45 • C to 165 • C at 4 • C min −1 for 2 min, followed by a gradual increase reaching 280 • C for 15 min. Helium was used as a carrier gas, and the same temperature was used for gas chromatography with flame-ionization detection (GC-FID). The retention times and indices of n-alkanes (C 10 -C 36 ) were used for the identification of detected compounds along with computer matching in the National Institute of Standards and Technology (NIST) mass spectra ver. 2.0 and WILEY libraries. Literature references were also used for accurate identification and comparison [12,34]. Leaf extracts were obtained as described previously in [35] and then maintained at −80 • C for further analysis.

Antioxidant Activities
The total antioxidant activities were determined for leaves using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and β-carotene-linoleic acid assays [33]. Briefly, for DPPH, 50 µL (1 mg·mL −1 ) of the leaf methanolic extract was mixed with DPPH solution (5 mL, 0.004%) and incubated for 30 min in the dark, and then the absorbance was determined at 517 nm in a spectrophotometer. For the β-carotene-linoleic assay, β-carotene was incubated for 48 h with leaf methanolic extract (50 µL), and the absorbance was then determined at 470 nm. The results were expressed as IC 50 in µg mL −1 . Superoxide dismutase (SOD) and catalase (CAT) activities were quantified in leaf tissues [12,36]. Experiments were repeated twice in triplicate.

Chlorophyll, Soluble Sugars, and Proline Compositions
Total chlorophyll was determined in leaves, following Reference [37]. The soluble sugar in the leaves was determined (% of DW), following Reference [38], and proline was determined in the leaves, following Reference [39].

Gas Exchange and Water Measures
Net photosynthetic rate (A), transpiration rate (E), and stomatal conductance (g s ) were determined using portable gas exchange equipment (ADC LCi, Bioscientific Ltd., Hoddesdon, UK) in the morning (10:00) in mature leaves at the end of the experiment. The leaf water potential and relative water content (RWC) of mature leaves were determined at noon (12:00), following References [4,40].

Gene Expression
To assess the expression of stress-related genes, quantitative real-time PCR (qRT-PCR) was conducted for FeSOD, Cu/ZnSOD, MnSOD, DREB2, and ERF3 in three plants of each treatment subjected to ALA and fungal infection. The RNA was isolated from young leaves using the RNeasy Plant Mini Kit (Qiagen, Berlin, Germany). To obtain the cDNA, a Reverse Transcription Kit (Qiagen, Berlin, Germany) was used, and qRT-PCR was performed in triplicate using QuantiTect SYBR Green PCR (Qiagen, Berlin, Germany). PCR primers and conditions followed Reference [12]. The Actin housekeeping gene [41] was used as a control, and the 2 −∆∆Ct method was used to determine the relative expression levels.

Statistical Analyses
Factorial experimental design was used. Three blocks were used: Each block contained 24 treatments (3 fungal treatments, including control × 2 soil mixes × 4 ALA doses). Each treatment was represented by 6 replicates, and the total number of plants was 144 plants per block. The least significant differences (LSD) were determined for all means (SPSS, PASW Ver. 22, Chicago, IL, USA).

Conclusions
A novel finding discovered in this study confirmed that ALA could alleviate the biotic stress of rosemary plants caused by A. dauci/R. solani fungal infections. The biotic stress produced shorter plants with a lower number of leaves and caused reductions in the fresh and dry weights. ALA-treated plants produced the longest and highest number of branches, higher fresh and dry weights, increases in major essential oil components (1,8-cineole, linalool, camphor, and borneol), higher antioxidant activities in DPPH and β-carotene-bleaching assays, upregulated SOD and CAT antioxidant enzyme activities, increased total phenolics, chlorophyll, soluble sugars, and proline, increased gas exchange parameters, leaf water potential, and RWC, and upregulated expression of DREB2 and ERF3 (stress-related genes) and FeSOD, Cu/ZnSOD, and MnSOD (antioxidant genes). Several mechanisms were involved, including stress tolerance, antioxidative, and transcription regulation mechanisms. Furthermore, we found that ALA performance was increased in higher-quality soils with higher nutrient content.