Foliar Application of Bio-Stimulants Enhancing the Production and the Toxicity of Origanum majorana Essential Oils Against Four Rice Seed-Borne Fungi

In the present study, the enhancement of the production of Origanum majorana essential oils (EOs) was studied by treating plants with ascorbic acid (AA) and tryptophan (Trp) at concentrations of 100, 200 and 300 mg/L and Moringa oleifera leaf extract (MLE) at 2.5%, 5% and 10% as foliar applications during the seasons 2018–2019. The toxicities of the EOs were assayed against four seed-borne fungi (Bipolaris orzyae, Curvularia lunata, Fusarium verticilliodies and F. graminearum) isolated from rice grains (Oryzae sativa). Vegetative growth parameters and EO production were enhanced by the application of AA, Trp and MLE in both seasons. Analysis of the EOs by Gas chromatography–mass spectrometry (GC-MS) showed that the main chemical constituents were terpineol (cis-β-(1-terpinenol)), terpinen-4-ol, 4-thujanol (sabinene hydrate), α-terpineol, cymene and sabinene. The highest fungal mycelial growth inhibition (FMGI) percentages against F. verticilliodies were 94.57% and 92.63% as MLE at 5% and 10%, respectively, was applied to plants and 85.60% and 82.19% against F. graminearum as Trp was applied to plants at 300 and 200 mg/L, respectively. EOs from the treated plant with MLE (10%) observed the highest FMGI (84.46%) against B. oryzae, and EOs from plants treated with AA as foliar application at 300 and 200 mg/L showed the highest FMGI values of 81.11% and 81.85%, respectively, against the growth of C. lunata. Application of EOs extracted from plants treated with Trp, AA and MLE at 300 mg/L, 300 mg/L and 10%, respectively, or untreated plants to rice seeds inhibited or decreased the fungal infection percentage from 82.5% (naturally infected grains) to 1.75%, 10.5%, 17.5% and 18.5%, respectively. In conclusion, the extracted EOs affected by the foliar application of O. majorana plants with Trp, AA, and MLE could be useful as a biofungicide against rice seed-borne fungi.


Introduction
Origanum majorana L. (marjoram) belongs to the family Lamiaceae, is a perennial aromatic herb native to Cyprus in the Mediterranean region and is widely distributed in Egypt. It is one of the most of O. majorana plants and, secondly, to investigate the antifungal activity of the essential oils against major seed-born rice fungi.

Effects of Ascorbic Acid, Tryptophan and Moringa Leaf Extract on Vegetative Growth
Results in Table 1 show the significant effects of different concentrations of ascorbic acid (AA), tryptophan (Trp) and moringa leaf extract (MLE) on the vegetative parameters of O. majorana plants (plant height (cm), plant diameter (cm), branch number/plant, leaf fresh weight (g), leaf dry weight (g) and leaf area (cm 2 )) compared with control plants in both seasons (2018-2019). The highest value of growth was obtained by the plants treated with AA at a concentration of 300 mg/L. These results agree with Hashem [2] who found that foliar spraying of marjoram plants with AA at concentrations of 150 and 300 mg/L significantly increased all vegetative parameters compared to control plants. Another study showed that application of MLE at 75 mL/L positively increased the vegetative growth of Pelargonium graveolens L'Hér. [39]. Furthermore, at high rates MLE significantly increased the fresh and dry weights of tomato leaves [40]. Foliar application of AA increased the plant growth yield of peas (Pisum sativum L.,) roselle (Hibiscus sabdariffa L.) and fennel (Foeniculum vulgare Mill.) plants [41][42][43]. Application of AA at different concentrations showed significant increases in all growth parameters and fresh and dry weights of Ocimum basilicum L. plant [44].
The simulative effect of MLE on herb growth due to its beneficial effects and supplying the growing plants with the required micro and macronutrient elements plays an important role in metabolic processes such as photosynthesis and carbohydrate synthesis [39]. The simulative effects of AA and Trp on herb growth resulted from their important roles in controlling several physiological processes and synthesis of plant hormones [32]. Furthermore, Trp plays a direct role in regulating plant development and pathogen defense responses [45], which promote the growth of plants. Through eliciting the synthesis of endogenous phytohormones such as auxin and gibberellin, bio-stimulant applications are responsible for activating the transduction signal pathway, which subsequently leads to a higher yield [46,47].

Effects of Ascorbic Acid, Tryptophan and Moringa Extract on the Chemical Parameters of O. majorana
Oil Percentage (%) There was a significant effect of the different treatments on the essential oil (EO) percentage of O. majorana, where the highest percentage was obtained by plants treated with MLE (10%) in both seasons (2018-2019), with values of 1.06% and 0.86%, respectively, compared to control treatments (0.66% and 0.65%; Table 2). These results are in line with those obtained by Abd-el-Kader and Hamad [42], where they noticed that Roselle plants treated with 300 mg/L AA had improved vegetative parameters, yield production and chemical constituents of Roselle sepals. Sakr et al. [39] indicated that foliar applications of MLE significantly improved the EO percentage and yield per plant and feddan of geranium plants. Moreover, application of AA increased the EO yield of O. basilicum plant [43].

Total Chlorophyll and Carbohydrate Content
There were significant effects of the studied treatments on total chlorophyll and total carbohydrate contents of O. majorana plants compared with the control plants in both seasons ( Table 2). The best results were obtained by plants treated with the highest concentration of MLE (10%). These results agreed with Sakr et al. [39] who reported that foliar application to geranium plants with MLE at 6 g/L increased the values of chlorophyll a, chlorophyll b and carotenoids. This increase may be due to moringa leaves having a high content of different macro elements such as Mg, a constituent of chlorophyll that would be responsible for inducing chlorophyll (a and b) in leaves with increasing carbohydrate content [48].
Chemical Composition of the Essential Oils Table 3 lists the chemical compounds of O. majorana leaf EOs as analyzed by Gas chromatography-mass spectrometry (GC-MS). These compounds were affected by different foliar applications applied to the plants compared with the control. Terpineol ranged from 38.06% (AA at 300 mg/L) to 26.76% (AA at 100 mg/L); terpinen-4-ol ranged between 38.35% and 27.65%, where the highest value was obtained by Trp at 300 mg/L; and the percentage of 4-thujanol (sabinene hydrate) ranged from 46.47% (MLE 2.5%) to 9.13%. The cymene percentage ranged from 17.75% to 4.5%, where the maximum relative percentage was obtained by Trp at 300 mg/L. α-Terpineol was found in the range of 12.58% to 6.24%, where the highest percentage was obtained by MLE at 10%, and sabinene ranged from 8.66% to 2.76%. In addition, the oxygenated constituents of EOs ranged from 62.88% to 89.17% and non-oxygenated constituents of EOs ranged from 9.62% to 26.65%. These ranges show strong positive correlation coefficients among the treatments in terms of affecting the oxygenated and non-oxygenated percentages as shown in Table 4.   The results of the current investigation agree with other observations [2,49]. There were two different chemotypes of O. majorana L. The oil of the first chemotype was rich in monoterpene alcohols including terpinen-4-ol (10%-48%), cis-sabinene hydrate (0.2%-33.0%), trans-sabinene hydrate and α-terpineol. Terpinen-4-ol is the substance responsible for the odor of these oils. The second chemotype included the phenolics thymol (0.01%-21.1%) and carvacrol (0.4%-30.0%) as major constituents [50,51]. In addition, these results are in line with Elansary [52] and Gharib et al. [53], who found that the main EO constituents of marjoram growing in Northwest Egypt were cis-sabinene hydrate, α-terpinene, 4-terpineol and sabinene. These effects on EO production may be due to the foliar application of AA, which is an essential co-factor for α-ketoglutarate-dependent dioxygenases (e.g., prolyl hydroxylases), which play an important role in the formation of covalent adducts with electrophilic secondary metabolites in plants [54]. Furthermore, it is responsible for several metabolic enzymes involved in the fundamental developmental process of plants [55]. Additionally, aromatic amino acids-phenylalanine, tyrosine and Trp-are central molecules in plant metabolism. Besides their function in building proteins, they serve as precursors for a variety of plant hormones, such as auxin and salicylate, as well as for a very wide range of aromatic secondary metabolites [56,57].

Isolation and Identification of the Fungal Isolates
The identifed Curvularia lunata fungal isolate from rice grains of Giza 177 cultivar showed grain discoloration. Three fungal isolates, Fusarium verticilliodies (F.v.101), F. graminearum (F.g.101) and Bipolaris orzyae (B.o.177), were previously identified in the internal transcribed spacer (ITS) region [58] and were used in the present work. The cultivar name and fungal isolates are presented in Table 4. Isolate C.l.177 was identified as Curvularia sp. based on its colonial morphology, morphological characteristics, growth pattern, conidiophores and conidia.

Molecular Identification through the Internal Transcribed Spacer (ITS) Region
The cultural characteristics of fungi isolates from Egypt were in agreement with those in previous studies [59,60]; thus, their identification was confirmed. However, the molecular characteristics were more precise and provided information on genetically pathogenic fungi. Accurate identification of pathogenic fungi is crucial in disease management of economically important crops, especially rice.
A partial DNA sequence in the ITS region of the C.l.177 isolate was analyzed using the BLAST tool at the National Center of Biotechnology and Information site (http://www.ncbi.nlm.nih.gov). The partial DNA nucleotide sequence and analysis via BLAST and Genbank data showed the fungal isolate C.l.177 actually belonging to C. lunata. The homology of the C. lunata isolate to the Genbank strain reached 99%. The Genbank accession numbers of this isolate and other referenced isolates are shown in Table 5.  Table 6 shows that the EOs from the plants treated with AA and Trp at the concentrations of 100, 200 and 300 mg/L, and MLE at 2.5%, 5%, and 10%, compared with the control plants, were bioassayed against the growth of F. graminearum, F. verticilliodies, B. oryzae and C. lunata. The maximum fungal mycelial growth inhibition (FMGI) percentages of 94.57% and 92.63% against F. verticilliodies were found by MLE at 5% and 10%, followed by Trp (300 mg/L) and AA (100 mg/L) with FMGI values of 71.31% and 70.93%, respectively. In the case of F. graminearum, the best EO treatment that inhibited mycelial growth was Trp at the concentrations of 300 mg/L (  Table 7 shows that the minimum inhibitory concentrations calculated for the essential oil treatments ranged between 250 and 400 mg/L, which were lower than those from Mancozeb as a chemically positive control of all fungal isolates (range from 20 to 40 mg/L).  Previous work showed that oil extracted with n-hexane at a concentration of 3% from Pinus halepensis showed an FMGI value of 80% against the growth of B. oryzae, while the EO from Schinus terebinthifolius observed the values of 74.44% and 71.66% at 3% and 2%, respectively [35]. Moreover, Cymbopogon martinii, O. vulgare and Cinnamomum zeylanicum EOs showed significant antifungal activities against F. graminearum and F. culmorum in wheat grains with FMGI percentages of 90.99% and 68.13%, respectively [61]. The methanol extract of O. majorana showed antifungal activity against some plant pathogenic fungi including F. solani, Aspergillus niger and A. parasiticus [62]. Moreover, (-)-terpinen-4-ol caused the mycelial growth inhibition of fungi Aspergillus, Penicillium and Fusarium species [63][64][65]. In addition, previous studies demonstrated that some monoterpenes preformed their antifungal action at the membrane level or membrane-embedded enzymes [66].

Efficacy of Seed Treatment with Essential Oils Against Seed-Borne Fungi
From the previous antifungal activity bioassay results, the concentrations of the EOs from O. majorana plants treated with AA (300 mg/L), Trp (300 mg/L) and MLE at a concentration of 10% were used to evaluate their activity against seed-borne fungi as a seed treatment, and they were compared with the EO obtained from untreated plants and seeds without EOs. It is clear from the results of Table 8 and Figure 1 that the treatments with the EOs showed different levels of fungal inhibition compared with untreated grains. The percentage of infection in non-treated grains (naturally infected) was 82.5%, while the percentages of infection were suppressed as the rice seeds were treated with EOs extracted from O. majorana plants treated with Trp (300 mg/L), AA (300 mg/L) and MLE (10%), or from untreated plants, with values of 1.75%, 10.5%, 17.5% and 18.5%, respectively, compared to the control (82.5%).  The pathogens associated with discolored grains evidenced the transmission of pathogens through grains, which causes seedling and inflorescence abnormalities and rotting of grains in the soil [37,[67][68][69]. Therefore, the present results showed that the EOs inhibited the fungi associated with rice seeds.
Finally, this study found that treatment of rice grains with EOs of O. majorana before sowing the rice in nurseries inhibited grain rot as well as enhanced grain germination and, thus, crop yield. EOs play important roles as antifungal agents and prevent the formation of fungal biofilms, the production of mycotoxins and the mechanisms of cellular communication [70].

Preparation of Aqueous Extracts of Moringa Oleifera, Ascorbic Acid and Tryptophan
Healthy leaves of Moringa oleifera Lam. were collected from the nursery of the Department of Floriculture, Ornamental Horticulture and Landscape Gardening, Faculty of Agriculture, Alexandria University, Egypt. The leaves were shade-dried and ground to a fine powder using an electric grinder. About 100 g of powdered leaves was soaked in 1000 mL of distilled water at room temperature for 24 h, then filtered through cotton plugs and then with filter paper (Whatman No. 1). The obtained moringa leaf extract (MLE) was prepared at the concentrations of 2.5%, 5% and 10%. Ascorbic acid (AA) and Tryptophan (Trp) were prepared at the concentrations of 100, 200 and 300 mg/L.

Field Study
The field study was carried out during two successive seasons of 2018 and 2019 at the Nursery of Floriculture, Department of Ornamental Horticulture and Landscape Gardening, Faculty of Agriculture, Alexandria University, Egypt. Sweet marjoram (Origanum majorana L.) seeds were sown in March and transplanted after one month in a mixture of sand and clay (1:1 v/v) for two seasons, 2018 and 2019, where four plants for each treatment were chosen. The soil mixture analysis was done according to Jackson [71] and illustrated in Table 9. The plants were sprayed with MLE at different concentrations (2.5%, 5% and 10%) [72] as well as (AA) and (Trp) with concentrations of 100, 200 and 300 mg/L every 30 days starting from the 1st of May until the 1st of July in both seasons (the plants were sprayed three times: first of May, first of June and first of July) as shown in Table 10. The control plants were sprayed with tap water. The plants were harvested on the 1st of August in both seasons. In both seasons, all plants were fertilized with nitrogen-phosphorus-potassium (N-P-K) (Milagro Amino leaf 20-20-20) at a rate of 2 g/pot. Fertilization was repeated every 30 days throughout the growing season.

Vegetative Growth and Chemical Parameters
Vegetative growth parameters including plant height (cm), plant diameter (cm), number of branches per plant, leaf fresh weight (g), leaf dry weight (g) and leaf area (cm 2 ) [73] as well as the chemical parameters were measured. Chlorophyll content of fresh leaves was determined as SPAD unites at the end of the season using Minolta (chlorophyll meter) SPAD 502. Carbohydrate content of the leaves was determined according to Dubois et al. [74].

Extraction of the Essential Oils
Essential oils (EOs) were extracted by a water distillation method using a Clevenger-type apparatus [75]. About 50 g from each O. majorana leaf from different treatments was put in a 2-L flask and hydrodistillated for 3 h. The collected essential oils were put in brown bottles and kept dry at 4 • C in the refrigerator. The amount of essential oils obtained from the treated plants, the oil percentage (%), was calculated according to the following formula: where A is the quantity of the EO from plants (mL), and B is the weight of the plant sample (g).

GC/MS Analysis of the Essential Oils
The obtained EOs from O. majorana leaves were analyzed for their chemical compositions using a Trace GC 1300-TSQ Quantum mass spectrometer (Thermo Scientific, Austin, TX, USA) with a direct capillary column TG-5MS (30 m × 0.25 mm × 0.25 µm film thickness). The column oven temperature was initially held at 60 • C and then increased by 5 • C/min to 250 • C, held for 2 min, and then increased to 300 • C at 5 • C/min. The injector and MS transfer line temperatures were maintained at 270 • C. Helium was used as the carrier gas at a constant flow rate of 1 mL/min. The solvent delay was 3 min, and diluted samples of 1 µL were injected automatically using an Autosampler AS3000 (Thermo Scientific, Austin, TX, USA) coupled with GC in the split mode. EI mass spectra were collected at 70 eV ionization voltages over the range of m/z 40-650 in full scan mode. The ion source and transfer line were set at 200 • C and 280 • C, respectively. The components were identified by comparison of their retention times and mass spectra with those of the WILEY 09 and NIST 14 mass spectral database. GC/MS with Xcalibur 3.0 data system has threshold values to confirm that all the MS of the compounds were attached to the library. Therefore, measuring their standard index (SI) and reverse standard index (RSI), values ≥ 650 are acceptable to confirm the compounds [35,[75][76][77][78].

In Vitro Study for Fungal Isolation
The experiments were carried out at the Department of seed pathology, Plant Pathology Research Institute, Agriculture Research Center (ARC), Egypt. Rice variety Sakha 101 was taken from the Rice Research and Training Center (RRTC), Sakha, Kafr El-Sheikh. Randomly, 100 grains were picked from the Giza 177 cultivar, which was obtained according to the International Rules of Seed Testing Association [79]. These grains were disinfected, placed in Petri dishes on potato dextrose agar media (PDA) and incubated at 25 • C in the dark for 4-5 days. Isolation and purification techniques were done following the methods of Mothlagh and Kavian [80]. Three fungal isolates, Fusarium verticilliodies (F.v.101), F. graminearum (F.g.101) and Bipolaris orzyae (B.o.177), which were previously isolated and identified from Sakha 101 and Giza 177 rice cultivars, were used in the present study [58].

Culture Characteristics and Microscopic Examination
Basic culture characteristics, such as color and growth pattern, were studied on the PDA medium. Fungal growth was stained with lactin-blue phenol and examined under microscopic light for fungal diagnostic properties. Innate isolation was defined on the basis of the description by Sivanesan [59].

Molecular Identification through the Internal Transcribed Spacer (ITS) Region
Total genomic DNA was extracted from the fungal growth according to Edel et al. [81]. The DNA pellet was dissolved in 100 µL of TE buffer and stored at 4 • C until use. The internal transcribed spacer (ITS) region was amplified using two universal primers: ITS1 (5 -TCCGTAGGTGAACCTGCGG-3 ) and ITS4 (5 -TCCTCCGCTTATTGATATGC-3 ). PCR amplification was carried out in a total volume of 25 µL containing 2 µL of template DNA, 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 1.5 mM MgCl 2 , 0.2 mM of each dNTP, 10 pmol of each primer and 0.5 unit Dynazyme TM II DNA Polymerase. PCR amplification was performed as one cycle at 95 • C for 1 min followed by 35 cycles each with 30 s at 94 • C for denaturation, 2 min at 55 • C for annealing and 1 min at 72 • C for elongation. Reactions were then incubated at 72 • C for 10 min for the final extension. PCR products were separated on a 1.5% agarose gel in 1X TAE buffer at 120 Volt, stained with EtBr and visualized under UV light [82].

Sequencing of the Amplified ITS Region
The amplified ITS1-5.8s and ITS2 regions were sent for sequencing. The products were sequenced by the use of a Big Dye terminator cycle sequencing kit and resolved on an ABI PRISM model 310 automated DNA sequencer from Sigma Company. Identification was confirmed by applying a BLAST search on the National Center for Biotechnology information (NCBI) site (http://www.ncbi.nlm.nih.gov) using the obtained sequences of the amplified regions. The ITS1-5.8s and ITS2 region sequences have been deposited in the Genbank database.

Antifungal Activity and Minimum Inhibitory Concentration (MIC) Assays of Essential Oils (First Investigation)
The EOs were prepared at a concentration of 2000 mg/L by diluting the oil in 10% dimethyl sulfoxide (DMSO) with the addition of a few drops of Tween-80 [83]. The antifungal activity of the EOs from O. majorana leaves was assessed against four grain-borne fungi (F. verticilliodies, F. graminearum, B. orzyae and C. lunata) by radial growth. The isolates were cultivated on PDA medium. Then, a single 6-mm culture disk was taken from actively growing cultures using a cork-borer and placed in the middle of the Petri dishes. Sterilized filter discs of 17 mm diameter loaded with 50 µL of 2000 mg/L of each oil were placed around the fungal disk and compared with referenced fungicide drug Mancozeb at 200 mg/L. The plates were incubated at 28 • C for 5 days, and three replications were used for each fungal pathogen [84,85]. Fungal mycelial growth inhibition (%) of the tested fungi was calculated with the following formula: where G0 and Gt are the average diameters (mm) of fungal colonies under the control and experimental treatments, respectively. The minimum inhibitory concentrations (MICs) of the essential oils prepared at concentrations of 200-2000 mg/L and Mancozeb (reference chemical fungicide) prepared at concentrations of 10-200 mg/L were assessed using the broth dilution method according to CLSI [86].

Efficacy of Seed Treatment with Selected Essential Oils Against Seed-Borne Fungi (Second Investigation)
The effectiveness of essential oils against seed-borne fungi was evaluated on 400 rice grains by treating them with oils from the most effective concentrated treatment for the plants based on in vitro testing, along with untreated control (sterile water), and then placing them on PDA Petri dishes followed by incubation for 5 days at 28 • C [87,88]. The percentage rice grain infection was calculated by the following formula [89]: Rice grain in f ection (%) = No. o f seed containing a particular f ungus Total seeds × 100

Statistical Analysis
The experiment was conducted in a complete randomized block design (RCBD) containing 10 treatments with three replicates. The collected data of vegetative growth and chemical parameters from both seasons were subjected to one-way analysis of variance (ANOVA) using the SAS program, SAS Institute [90]. Data on mycelial growth inhibition (%) as affected by essential oils from plants treated with AA and tryptophan at concentrations of 100, 200 and 300 mg/L and M. oleifera leaf extract (2.5%, 5% and 10%) were subjected to one-way ANOVA. The means of the individual factors and their interactions were compared by the least significant difference (L.S.D) test at 5% level of probability according to Snedecor and Cochran [91].

Conclusions
This study found that different foliar applications of ascorbic acid, tryptophan and moringa leaf extract on O. majorana plants significantly affected vegetative growth and chemical constituents compared with untreated plants. Essential oils had potential antifungal activity against the growth of F. verticilliodies, F. graminearum, B. oryzae and C. lunata. Furthermore, the most effective essential oils were obtained from the O. majorana plants treated with ascorbic acid and tryptophan at 300 mg/L, whereas moringa leaf extract is suggested to be used at 10% to inhibit the growth of these fungi when applied to seed rice grains before sowing. Even though this is a high amount, the raw material of moringa is readily available as it is a fast-growing tree. This study suggests using these essential oils as a biofungicide against seed-born fungi, especially in treatments before sowing.