Laser Light Treatment Improves the Mineral Composition, Essential Oil Production and Antimicrobial Activity of Mycorrhizal Treated Pelargonium graveolens

Pelargonium graveolens, rose-scented geranium, is commonly used in the perfume industry. P. graveolens is enriched with essential oils, phenolics, flavonoids, which account for its tremendous biological activities. Laser light treatment and arbuscular mycorrhizal fungi (AMF) inoculation can further enhance the phytochemical content in a significant manner. In this study, we aimed to explore the synergistic impact of these two factors on P. graveolens. For this, we used four groups of surface-sterilized seeds: (1) control group1 (non-irradiated; non-colonized group); (2) control group2 (mycorrhizal colonized group); (3) helium-neon (He-Ne) laser-irradiated group; (4) mycorrhizal colonization coupled with He-Ne laser-irradiation group. Treated seeds were growing in artificial soil inculcated with Rhizophagus irregularis MUCL 41833, in a climate-controlled chamber. After 6 weeks, P. graveolens plants were checked for their phytochemical content and antibacterial potential. Laser light application improved the mycorrhizal colonization in P. graveolens plants which subsequently increased biomass accumulation, minerals uptake, and biological value of P. graveolens. The increase in the biological value was evident by the increase in the essential oils production. The concomitant application of laser light and mycorrhizal colonization also boosted the antimicrobial activity of P. graveolens. These results suggest that AMF co-treatment with laser light could be used as a promising approach to enhance the metabolic content and yield of P. graveolens for industrial and pharmaceutical use.


Introduction
The worldwide interest in the use of medicinal plants as a therapeutic agent has been arising due to their ease of availability and administration, cost-effectiveness, and perhaps fewer side effects than synthetic drugs. Medicinal plants are a rich source of active ingredients known as primary and secondary metabolites. Many ethnobotanical surveys of laser light application on the mycorrhizal treated P. graveolens in terms of its mineral composition, essential oil production, and antimicrobial activity.

Laser Improved Mycorrhizal Colonization in P. graveolens Plants
The application of laser light in the current study has significantly improved the mycorrhiza colonization in P. graveolens as represented in Table 1. Six weeks after AM fungal inoculation, no signs of AMF were detected in the roots obtained from non-inoculated plants. The rate of root colonization in the laser-treated group was increased by about 29% compared to that in the control. Moreover, the hyphal length of mycorrhiza has been increased by about 48% in the laser-treated group in comparison to the control group. Additionally, the number of arbuscules has been increased in the laser-treated group by about 30% in comparison to the control group. Table 1. Effect of irradiation and mycorrhizal inoculation on growth parameters.

Improved Colonization in Plant-Initiated from Laser Light Treated Seeds Increased Biomass Accumulation and Biological Value of P. graveolens
The bio stimulatory effects of laser light application in mycorrhiza-colonized P. graveolens were investigated. As illustrated in Figure 1, the plant biomass (g fresh weight/plant) was significantly increased (p < 0.05) in the laser-treated mycorrhiza-colonized group (2.53 ± 0.1 g FW/plant) of P. graveolens compared to the laser non-treated group (2.01 ± 0.1 g FW/plant) and control plant group (p < 0.01). Interestingly, the laser light irradiation also displayed improved biomass in the control group (1.73 ± 0.1 g FW/plant) when compared to their non-irradiated normal control counterparts (1.29 ± 0.1 g FW/plant).   Primary and secondary metabolic contents were highest in the laser and mycorrhizal cotreated groups (Table 2). This coupled treatment almost doubled the total phenolics and flavonoids content (21.5 ± 1 and 1.2 ± 0.1 mg/g DW, respectively) when compared to the untreated control plants (11.07 ± 1.2 and 0.53 ± 0.1 mg/g DW, respectively). Moreover, total protein, total sugar, and total alkaloid contents were also significantly higher (p < 0.05) in the laser and mycorrhizal cotreated group than the rest of the treatment groups. Similarly, treated samples produced a significantly higher amount of ash (p < 0.05). However, crude fiber level was somewhat similar in all samples (Figure 2a,b). Overall, different metabolites ranked in the order of laser and mycorrhizal cotreated group > laser-treated control > only mycorrhizal treated group > untreated control group with increasing nutrient content. Values are presented as mean ± SD whereas, different superscripts (a-c) show statistical significance among the data at p < 0.05. Figure 1. Impact of laser light and mycorrhizal co-treatment on plant biomass. Values are presented as mean ± SD whereas, * = p < 0.05, ** = p < 0.01. Values are presented as mean ± SD whereas, different superscripts (a-c) show statistical significance among the data at p < 0.05. Values are presented as mean ± SD whereas, * = p < 0.05, ** = p < 0.01, ns = non-significant. Table 3 shows the impact of mycorrhizal and laser light treatment on the mineral content of P. graveolens. Laser light combined with mycorrhizal treatment significantly (p Values are presented as mean ± SD whereas, * = p < 0.05, ** = p < 0.01, ns = non-significant. Table 3 shows the impact of mycorrhizal and laser light treatment on the mineral content of P. graveolens. Laser light combined with mycorrhizal treatment significantly (p < 0.05) induced the accumulation of different minerals in P. graveolens. Meanwhile, less significant changes were observed in laser light treated plants compared to their untreated controls. Just like the nutrient content, laser light combined with mycorrhizal treatments almost doubled the levels of potassium (K), phosphorus (P), calcium (Ca), magnesium (Mg), and iron (Fe). Similarly, laser light and mycorrhiza alone also improved the mineral uptake when compared to untreated control plants. However, Zinc (Zn) remained unchanged in treated and untreated plants. Values are presented as mean ± SD whereas, different superscripts (a-c) show statistical significance among the data at p < 0.05.

Co-Application of Mycorrhiza and Laser Light Treatment Induced the Production of Essential Oils and Their Precursors
Here, 28 essential oils and 3 important precursors (phenylalanine, cinnamic acid, and shikimic acid) were found in all samples. Among identified essential oils, geraniol and beta-citronellol were most predominant in all plants. Whereas shikimic acid was the most abundant essential oil precursor (72.9 ± 2.5-90.4 ± 1.4%) in treated and untreated samples. Generally, mycorrhizal-laser light cotreatment showed the most significant (p < 0.05) impact on the oil yield and its content. However, laser light and mycorrhizal alone treatments showed somewhat similar oil content when compared to untreated controls. Table 4 shows that the percentage of α-pinene, β-phellandrene, citronellyl formate, geraniol, geraniol formate, linalool, Cis-β-ocimene, I-Menthone, nerol, beta-bourbonene, beta-cubebene, zcitral, trans-caryophyllene, isoledene, ledene, neryl acetate, geranyl tiglate, and mintsulfide was higher in laser-treated group than the control. Surprisingly, all the treatments reduced myrcene content (~0.19%) when compared to untreated control plants (0.33%). Overall, laser light treatment displayed a more pronounced impact on the essential oil content in P. graveolens. Table 4. Essential oil content in control and treated P. graveolens.

Parameters
No Irradiation Laser Irradiation

Essential oil precursors
Values are presented as mean ± SD whereas, different superscripts (a-c) show statistical significance among the data at p < 0.05.

Laser Light Boosted the Antibacterial Properties of Mycorrhiza Inoculated P. graveolens Plant
Both gram-positive and negative bacterial strains were used to evaluate the antibacterial activity of P. graveolens through the disc diffusion method. Among all the tested bacterial strains, P. graveolens showed maximum growth inhibition of E. coli (gram-negative) followed by Streptococcus salivarius (gram-positive). Yet again, the laser-mycorrhizal coupled treatment group had the largest zones of inhibition (23.54 ± 1.1-35.31 ± 1.2 mm) against all bacterial strains (Table 5). In general, different plant samples followed the pattern of laser light and mycorrhizal group > mycorrhiza treated group > laser light control group > and untreated control group for antibacterial activity. Values are presented as mean ± SD whereas, different superscripts (a-c) show statistical significance among the data at p < 0.05.

Discussion
In the present study, we investigated the synergistic effect of laser light and mycorrhizal treatment on the phytochemical content of P. graveolens. For this, the P. graveolens sterilized seeds were co-cultivated with Rhizophagus irregularis and/or He-Ne laser light in a controlled environment. These treated P. graveolens samples were compared with untreated control P. graveolens for their biomass, mineral content, nutritional value, and antibacterial activity.
Here, laser light treatment significantly improved the mycorrhizal root colonization, hyphal length, and the number of arbuscular roots when compared with untreated control roots. Laser light induction along with mycorrhizal treatment also improved biomass production. Previously, Alam et al. (2011) [26] also reported that mycorrhizal treatment increased the growth and biomass in rose-scented geranium. The synergistic effects of laser light application could be understood in the light of the postulation that the energy from laser light can stimulate the intensity of the transmembrane electrochemical proton gradient in mitochondria and cell proliferation and produce morphological changes in cells and organisms [27]. It was found that some fungi that have been grown in in vitro culture, have shown an increased rate of cellular bioenergetic processes after exposure to laser light of a specific wavelength [28]. Moreover, plant macromolecules absorb laser light that triggers photosynthetic activity, resulting in increased growth, fresh weight, and biomass [29]. Our recent studies have augmented this postulation whereas laser light application was able to improve the nutritional value, antioxidant capacity, and anti-inflammatory activity of flavonoid-rich buckwheat sprouts and lemongrass sprouts [19].
Increased biomass production could be correlated with the enhanced primary and secondary metabolic content in treated P. graveolens plants. We observed enhanced amounts of protein, sugar (primary metabolites), phenolics, flavonoids, and alkaloids (secondary metabolites) in the laser-mycorrhizal treated plants. The laser-light-induced enhanced photosynthesis can mediate the biosynthetic pathways resulting in an increased sugar content [30]. The surplus photosynthetic sugar can generate the cellular energy required for the biosynthesis of primary and secondary metabolites [31] and/or used by AMP for their energy metabolism and growth [32]. Inoculation of olive trees and chamomile with R. irregularis significantly improved the accumulation of phenolic and flavonoids content in fungal infected plants [33,34]. Phenolics and flavonoids are the most abundant antioxidants involved in plant defense mechanisms. Several reports suggest the AMP induces the accumulation of Phenylalanine ammonia-lyase (PAL) in the infected roots. PAL is the key enzyme regulating the phenolic biosynthetic pathway. This accounts for higher levels of phenolics in AMP-associated plants [35]. In return, the phenolics and flavonoids exert a positive impact on hyphal growth and arbuscules development during symbiosis [36].
Minerals are essential elements to regulate the proper growth and development of plants. Under field conditions, the unavailability of adequate levels of these minerals can impede plant growth [37]. The previous meta-analysis confirms that AMF increases the soil enzyme activity that releases soil nutrients required for plant growth [38]. Moreover, AMF also inhibits the plant nutrient leaching into the soil thus mitigating the salt/drought stress and maintaining the ion balance [39]. In this study, an enhanced mineral uptake was observed in mycorrhiza and laser-treated plants than untreated control plants. Shao et al. (2018) also reported significantly higher leaf N, P, K, Ca, Mg, Zn, and Mn contents in AMFinoculated tea plants than in non-AMF-inoculated ones [40]. Similarly, AMP inoculation alleviated the drought stress in P. graveolens and improved its metabolic content [14]. Many researchers have reported the enhanced uptake of N, P, and K by AMF-inoculated plants under saline conditions [41,42]. The higher mineral uptake by AMP-inoculated plants, likely results from the extra-radical mycorrhizal hyphae that extend beyond the roots and exploit nutrient-filled soil [43]. Besides increasing the absorption area of the roots, AMF also releases important chemical compounds such as glomalin, a glycoprotein secreted by hyphae and spores of AMF. Glomalin in the soil helps the uptake of nutrients such as Fe and P that are difficult to dissolve [44].
On the other hand, several studies suggest that nutrient absorption and utilization by plants could be modulated according to the light source and the period of duration to this source. Several studies have been uncovered the relationship between light perception and nutrient uptake [30,45]. In particular, laser light application has been proven to improve the nutritional value of many plant species [46,47]. As a result, the combination of mycorrhizal colonization and laser application in the current study has shown a significant synergistic effect on the minerals uptake by P. graveolens The combination of plant essential oils with synthetic drugs offers a promising approach for producing new antimicrobial drugs [48]. Pelargonium genus produces the top 20 essential oils in the world, well known for their antimicrobial, anti-inflammatory, and spasmolytic properties [49,50]. In the present study, GC-MS revealed the presence of 28 essential oils in varying levels. Among the detected essential oils, geraniol and beta-citronellol were most abundant in all samples. Previous literature shows that P. graveolens essential oil is an enriched source of citronellol and geraniol. It is commonly known as rose geranium oil and is extensively used in perfumes and skincare products [51]. Geranium oils have gained worldwide acceptance in the food industry since they have been recognized as safe products and approved by the American Food and Drug Administration (FDA) for food use [50].
Earlier, laser light improved the levels of essential oils and their precursors in anise [52] which supports our results. Moreover, Balkhyour et al. (2021) [53] reported an induced accumulation of essential oils, phenylalanine, and cinnamic acid in He-Ne laser-treated ajwain seedlings, which is also in agreement with our findings. AMF colonization differentially improved the essential oils of oregano, mint, dill, and coriander [54,55].
These improved essential oils could account for significant antibacterial activities of P. graveolens in this study. Several authors have been able to demonstrate the broadspectrum antibacterial activity of essential oils and plant extracts including rosemary, oregano, basil, tea tree, and myrtle [56][57][58][59] [5,60,61]. Generally, gram-negative bacteria show high resistance against plant essential oils due to the presence of a hydrophilic outer membrane containing a hydrophilic polysaccharide chain, which acts as a barrier to a hydrophobic essential oil [62]. Contrarily, we observed that E. coli (gram-negative) was most susceptible against the laser light and mycorrhizal treated plants followed by S. salivarius (gram-negative). Previously, parsley and lovage essential oils demonstrated similar activity against gram-positive and negative bacteria [63]. This can be attributed to the antibacterial potential of geraniol and beta-citronellol (alcohol) in P. graveolens plants and part to the metabolic byproducts produced by AMF which impart antioxidant, antiviral, and antibacterial effects [64]. Previous reports have illustrated the antimicrobial activity of narrow-wavelength light, which eliminates a range of common pathogens by inducing oxidative stress that ultimately can kill microbial pathogens [65].
Consequently, the observed antibacterial activities show that laser light and mycorrhizal co-treated P. graveolens plants can be ideal candidates against drug-resistant bacterial strains. However, further studies are needed to explore the efficiency of suitable concentration and safety of essential oils as a potential therapeutic agent.

AMF Symbiont
A pure commercial inoculum of R. irregularis (Błaszk., Wubet, Renker & Buscot) C. Walker & A. Schüßler (MUCL 41833) was obtained from Glomeromycota in vitro collection (GINCO) in a pot (25 × 15 cm). Considering the bibliography, R. irregularis is the most popular species, it is easy to propagate in pot culture in association with different host plants. It is also well known for its growth-promoting effect on plants. AMF spores were grown for 6 months on the roots of Sorghum sudanenses Pers. The mycorrhizal inoculum was added as 5 g of trapped soil per plant or pot (~50 spores/g soil). The inoculum was inserted before sowing, at a depth of 6 cm below the surface, thus producing mycorrhizal treatment. The controlled plots received equal amounts of autoclaved inoculum to supply equivalent nutrients aside from mycorrhizal spores.

Experimental Setup
A homogenous lot of seeds were surface sterilized in 5% (v/v) of sodium hypochlorite for 20 min. Seeds of P. graveolens were soaked for 2 h in distilled H 2 O before laser illumination using a laser equipment system (DMC Equipment Ltd. Firenze, Italy). Four groups of seeds were used, each group contained 100 seeds: (1) control group1 (non-irradiated; non-colonized group); (2) control group2 (mycorrhizal colonized group); (3) helium-neon (He-Ne) laser-irradiated group; (4) mycorrhizal colonization coupled with He-Ne laserirradiation group. P. graveolens seeds were irradiated by He-Ne laser (632 nm, 5 mW) with a beam diameter of 1 mm at 500 mJ energy for 5 min from the side of the embryonic area. These conditions were selected following a preliminary experiment for detecting the most optimum laser treatment conditions based on the increase in the growth of P. graveolens species to laser in terms of fresh weight. The laser was applied perpendicularly at the distance of 12 cm between the laser source and the seeds.

AMF Growth Analysis
The roots for each treatment were washed, cut into segments, and cleaned with KOH solution. Then, the roots (30 root fragments total) were stained by trypan blue in lactoglycerol and were examined with a stereomicroscope [66]. The colonization was calculated by using the gridline intersect method and arbuscule abundance in the root system were calculated by the number of arbuscules cm −1 root [67]. The hyphal length was determined in rhizosphere and hydrosphere soils by the method of Andrade et al. (1997) [68].

Determination of the Nutritional Value
To give insights into the food functional value of P. graveolens samples, the levels of amino acids, organic acids, fatty acids, minerals, and vitamins were evaluated as described below.

Proximate Composition Analysis
Carbohydrates were extracted by boiling a known weight P. graveolens plant tissue in 1 N HCl for 2 h. The extract was cooled and centrifuged at 6000× g for 15 min. The supernatant was neutralized with 1 N NaOH and made up to a known volume with distilled water. The concentrations were evaluated following Nelson's method [70] from each sample. Protein concentration was extracted from 0.5 gFW in 1 M NaOH (5 mL) at 37 • C for 60 min and the soluble material was analyzed for total protein. The concentrations were measured following the Folin-Lowry methods, the absorbance of standards was measured at 660 nm against blank. Total lipids were assessed based on Folch modified by Bligh and Dyer (1959) [71], where the plant samples were homogenized in a mixture of chloroform/ methanol (2:1, v/v). Then, centrifuged (15 min, 3000× g). The pellets were re-dissolved in a mixture of toluene/ethanol (4/1, v/v). Afterward, the extract was mixed with a saline solution. The extracted lipids were concentrated, and the total lipid content was calculated. Crude fibers were extracted and measured according to AOAC (1990) [72]. Samples were gelatinized with a heat-stable alpha-amylase (pH 6, 100 • C, 30 min), and then enzymatically digested with protease (pH 7.5, 60 • C, 30 min) and amyloglucosidase (pH 6, 0 • C, 30 min) to remove undesirable components. Precipitation of Fibers was done with ethanol, then the residue was weighed.
4.6. Determination of Essential Oils P. graveolens plants were dried at room temperature and were used for essential oils extraction. The dried plants were exposed to steam distillation for 4 h using a Clevenger instrument. The levels of essential oils in plants were measured by GC/MS as described by Hassanpour et al. (2014) [73]. A gas chromatograph HP-5 (Crosslinked 5% PH ME Siloxane, 15 m × 0.53 mm × 1.5 µm film) column was used for separation at a helium flow rate 2 mL min −1 , injector temperature 220 • C and detector temperature 240 • C using temperature program 60 • C, 40 • C min −1 up to 220 • C, 2 min at 220 • C. Portions of 2 µL of the essential oil (dissolved in n-hexane) were injected into the used analytical column. Identification of oil components was achieved based on their retention indices and by comparison of their mass spectral fragmentation patterns (NIST) database. The concentrations of essential oil, as a percentage (%), were calculated.

Antibacterial Activity
The antibacterial activity of the tested plants was evaluated against B. subtilis, S. salivarius, E. coli, Pseudomonas aeruginosa, Sarcina lutea, and S. aureus by the application of the Disc diffusion method [74], using 100 µL of suspension containing 10 8 CFU/mL of bacteria spread on Muller Hinton agar. The antibacterial activity was indicated based on the inhibition zone, determined by the Vernier caliper.

Statistical Analysis
Statistical analysis was performed using the SPSS statistical package (SPSS Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) was applied to all data. Tukey's Test (p < 0.05) was carried out as the post-hoc test for mean separations. Each experiment was replicated at least three times (n = 3).

Conclusions
Both the application of laser light and mycorrhizal colonization have several beneficial effects on the biological value of many plants. However, laser stimulation coupled with mycorrhizal inoculation significantly improved the plant biomass and its nutrient content in P. graveolens. The treated samples also showed improved essential oils and primary and secondary metabolites when compared to untreated control plants. Moreover, laser light and mycorrhizal co-treatment boosted the antibacterial activities of P. graveolens against Gram-positive and negative bacteria. Thus, this study supports the co-application of laser light and AMF as an advantageous approach for increasing the nutritional and health-promoting effects of P. graveolens.

Data Availability Statement:
The data that support the findings of this study are available on request from the corresponding author.