Nigella (Nigella sativum L), is an annual medicinal plant belongs to family Raunculaceae, growing in countries, bordering the Mediterranean region (Including Egypt) western Asia countries, including India, Pakistan, Saudi Arabia and Eastern Europe  . In traditional remedy, N. sativa (NS) seeds commonly used as a spice and carminative . In addition, several properties such as liver tonics, diuretic, digestive, anti-diarrheal, appetite stimulant, analgesics. NS seed capsule is effective on decreasing the severity of physical symptom of premenstrual syndrome . Also used as anti-cancer drug and protective agent against gamma radiation induced adverse effects in cell line .
N. sativa Seed contains more than 30% fixed oil and 0.45% essential oil . Thymoquinone (TQ) is an abundant component of black seed essential oil extract . As antidote or a protective agent against natural or chemical toxicities . There are many reports on the biological activities including, antioxidant , analgestic, anti-inflammatory  , antitussive , anti-hypertensive , anti-diabetic , anti-bacterial  and anticancer  . In addition, the protective effects of N. sativa and its main constituents in different tissues including brain   , heart , liver , Kideny , lung  , have been established against some toxic agents . This is also revealed that most of therapeutic properties of N. sativa are due to the presence of TQ which is major bioactive components of essential oil , along with high value component such as linoleic acid, dithymoquinone   . It is now well documented that most of therapeutic benefits of NS are mainly attributed to TQ, which is almost one of the key constituents of its volatile oil  .
Seeds and seed oil of NS as to explore their potential applications for the development of innovative functional food, nutriceutical and pharmaceutical as well as to help scientific basis of the widespread traditional uses of this multipurpose spice . NS fatty oil includes one important fatty acid of linoleic acid which high industrial importance and recognized an essential biochemical component for human diet . In particular, polyunsaturated fatty acids are very important for maintaining biological function in mammalians, hence regarded essential fatty acids . Aside, it clearly recognized that humans and many animals can abstained monounsaturated fatty acids from sugar (existing in the booly) while due to lake of desaturase enzyme, human body is unable to convert oleic acid into linoleic omega 6 and lenolenic (omega 3) . Therefore, humans forced to append these polyunsaturated fatty acids . Polyunsaturated in human are converted again inavaliable molecules such as antioxidants which protect to body  . Unsaturated fatty acids are naturally important wherease Oleic acid is the most popular mona-unsaturated fatty acids capable of decreasing cholesterol, linoleic (omega 6) and lenaleinic (omega 3) are polyunsaturated fatty acids which are effective in reducing the risk of blood pressure and vascular diseases . NS seed and oil, potential sourcer of high-value components for development of functional foods and untriceuticals/pharaceuticals .
Elicitation; improve quale-quantitative bioactive secondary metabolites (BMS) which improve the health pronating qualities  - . It also modified growth and development, can have benifical effect on morphological, physiological, biochemical characteristics that increase biomass yield production and quality   . Elicitation had distinct role in regulation of plant and pathogen attack, that cause hug loss in yield production under agrochemical. Traditional agriculture  which often implicated to induce systemic resistance ISR) by regulating the expression genes involved for production and accumulation SMs phytoalexins (PHS) specific toxins characterized brood spectrum bio-anti-pesticide and microbicides making them improving against microbial diseases and pests infestation    . Exceedingly elicitation plays a major role in adaptation of plants to the changing environmental, overcoming biotic/abiotic stresses   .
Allied on the best of our knowledge, no findings have been reported on multi-repeating elicitation technology for improvement sustainable agriculture medicinal and aromatic plants. Therefore, the present study aimed to evaluate multi-repeating elicitation aside biofertilizers in respect to ameliorate N. sativa—yield production and quality.
2. Material and Methods
2.1. Elicitor Application
Foliar spray with two abiotic elicitor; (SA) salicylic acid (E1) 50 mg/L, (NPSe) nano-Selenium oxide (E2) 50 mg/L and 2 biotic elicitors; (YS) yeast, Saccharomyces servisiae, (E3) 150 PPm, (CH) chitosan (E4) 25% with 500 ML acid; with tween 80, 0.1% w/v. Meanwhile Zero elicitor control (EO) plants were sprayed only with Tween 80 solution.
2.2. Fertigation Management
Four fertilizers were applied; NPK 20:20:20 chemical fertilizer, 20 g/m2 as traditional (F1) control and three biofertilizers, 20 g/m2 dry moringa (F2) (Moringa olifera L.) dray leaves extract (MOLE), 20 g/m2 neem (F3) (Azodiracha indica A. Juss) dray leaves extract (NMLE), 20 g/m2 Humic acid (HA) as (F4).
2.3. Execute Field Experiment
Two subsequent field experiment trial, 2018 and 2019, on orchard farm at Agriculture Research center were designed as factorial split plot besed on randomized complete block with three replications. Four elicitors as main plot and 3 biofertilizer (F2 - F4) and NPK (F1) as sub-main plot. N. sativa seeds were sowing 10 October at both two seasons 2018 and 2019 in plots 1.5 × 2 m. size in rows 40, 20 intra and enter space to form 12.5 plant/m2. Resultant plants aged 60, 120, 180 were foliarly sprayed with elicitors (EO, E1 - E4). Irrigation and fertigation managements through drip surface irrigation system. Harvesting during full flowering stage in two subsequent seasons at 10 June 2018 and 2019.
2.4. Biometric Growth Traits
Five randomly selected plants were recorded for plant height, cm (PH, cm), number of primary branches (NPBP) number of secondary branches (NSBP), number of capsule (NCP) number of seeds per capsule (NSC), seed yield, g (SYP, g). Means of these traits were subjected to ANOVA statistical analyses.
2.5. Quali-Quantitative Yield Traits
2.5.1. Seed Yield
Seed yield per plots were recorded that were converted to seed yield, g/m2.
2.5.2. Fixed Seed Oil
Fifty gram of powdered seed sample/plot subjected to soxhlet apparatus with 250 ml. of petroleum ether for 4 h. . The extract was concentrated under reduced temperature and pressure and fatty oil (fixed oil) percentage was computed using the following formula:
Fixed seed oil yield, g/m2 (SFOY, g/m2) was estimated by multiplying (SFO%) with SY, g/m2 for each plot. Concerning SFO quality, fatty acid composition were determined as % by GC analysis using a thermoquest gas chromatography through a flame ionized detector.
2.5.3. Seed Essential Oil
Fifty gram seed/plot were subjected hydro-distillation for 3 h. to optain EO content. EO was dried using anhydrous sodium sulfate and kept in amber glass seed with Teflon septra at 4˚C intel analysis. Essential oil yield content (EOY, g/kg) and EO yield kg/m2 were calculated by the following equations.
EO composition was analyzed by GC/MS using on Agilent Technologies 7890 gas chromatograph coupled to quadruple El-mass analysis and Agilent 9575C mass selective detector.
3. Statistical Analysis
The data sets were firstly tested for normality by the Anderson and Darling normality test using a statistical analysis system (SAS) (SAS 2003). The pooled mean values of 2 years for all the traits were subjected to statistical analysis of variance was done for all traits. A least significant difference (LSD, 1%) test was used for mean comparison of treatment.
4. Results and Discussion
4.1. Growth Traits
Statistical analysis of variance revealed that malti-repeating elicitation with abiotic elicitors, salicylic acid, SA. (E1), nano-particale selinum, NPSe. (E2) and biotic elicitors; yeast, SY. (E3), chitosan, CH (E3) coincide. biofertilizers; moringa leaves extract, MOE (F2), neem leaves extract, NME (F3), humic acid, HA (F4) actuated significant promotion on growth traits actuated over that of NPKEo control (Table 1). At such trait, biotic elicitor; [CH (F4) > YS (F3)] > abiotic elicitor, [NPSe (E2) > SA (E1) inseparate with biofertilizers, [MOE (F2) > NME (F3) > HA (F2)] > NPK chemical fertilizer (F1). Extensive investigations were in line concerning biotic/abiotic elicitation premating growth trits under chemical fertilizer system    . Whereas, studies have been not available concurring biofertilizers, system with biotic/abiotic elicitation specially for N. sativa. However, solitary biofertilizer acted promotion growth traits has been reported   .
Table 1. Mean growth traits values for 2 years (2018) and (2019) for N-sativa in response to 5 elicitors (E1-4) under 4-fertilizers (F1-4).
E0-4; control, salicylic acid (SA), nano-selenium oxide (SeN), yeast (Y), chitosan (CHT), E0 control respectively. F1-4; NPK, Moring a dry leaf extract (MOLE), Neem dry leaf extract (NMLE), humic acid (HA), respectively. Values between parenthesis (were percent of control).
4.2. Quantitative Yield Traits
F1-4 coincide E1-4 performed significant augmentation for DSY, g/m2 SFY, g/m2 and SE0Y, g/m2 as% over control (F1E0) as shown (Table 2) and represented Figures 1-3. Aside, it follows that [F2 (MO) concur E4 (CH) > E3 (YS) > E2 (NPSe) > E1 (SA)] exceeded [F3 (NM) concur E4 > E3 > E2 > E1] exceeded [F3 (NM) concur F4 > F3 > F2 > E1] exceeded [F2 (HA) concur E4 > E3 > E1] exceeded [F1 (NPK) concur E4 > E3 > F2 > E1. These results were attributed to that significant promoting for growth traits that have been declared in Table 1. In despite, that there is no pest and microbial discusses incidence in the field experiment (except for NPKEo control) in both two seasons without using any agrochemical pesticide and miocrobiocide (except for npkwo control) which in consequence to biotic/abiotic elicitors under investigation-since, these elicitor trigger plants to induce systemic resistance (ISR)by regulating the expression of genes involved for production secondary metabolites (SMs) phytoalexins (PAs) which non-specific toxins against microbial deceases and pest infestation    that cause huge loss in yield production  under chemical fertilizer. Also abiotic/biotic elicitation evoked enhancing SMs production and quality 
Table 2. Mean seed yield traits values for 2 years (2017-2018) and (2018-2019) for N-sativa in response to 5 elicitors (F1-4) under 4-fertilizers (E1-4).
E0-4; control, salicylic acid (SA), nano-selenium oxide (SeN), yeast (Y), chitosan (CHT), E0 control respectively. F1-4; NPK, Moringa dry leaf extract (MOLE), Neem dry leaf extract (NMLE), humic acid (HA), respectively. Values between parenthesis (were percent of control).
Figure 1. Dray seed yield, g/m2 impacted with 4-elicitors (E1-4) under 4-fertilizers (F1-4).
Figure 2. Fixed oil yield, g/m2 impacted with 4-elicitors (E1-4) under 4-fertilizers (F1-4).
Figure 3. Essential oil yield, g/m2 impacted with 5-elicitors (E1-4) under 4-fertilizers (F1-4).
     that overcoming biotic and abiotic stresses. Exceedingly chitosan (E4) proved bio-fungicide, bio-nemiotoside, bio-viroside  . Also, SA elicitor enhanced growth and development and increased significantly  and improvement EO contents in organic agriculture system  MO effect on growth and yield of crops and thus can be promoting as a possible sublenent or substitute to inorganic fertilizers  these investigations confirmed our results shown in Table 1 and Table 2.
4.3. Qualitative Yield Traits
4.3.1. Seed Fixed Oil (SFO)
The major compounds SFO (Table 3) were comprised; lenoleinic (40.15%), carvon (36.21%), dihydroxylenolenic (21.12%) and oleic (12.75%) for NPK (F1E0) control. At such biofertilizer; MO (F2), NM (F3), HA (F4) concur with
Table 3. Proportions of N. sativa fixed oil constituents impacted by four elicitors (E1-4) and three fertilizers (F1-4) (an average over of the two years experiment).
E0-4; control, salicylic acid (SA), nano-selenium oxide (SeN), yeast (Y), chitosan (CHT), EO control respectively. F1-4; NPK, Moringa dry leaf extract (MOLE), Ne. em dry leaf extract (NMLE), humic acid (HA), respectively. Values between parenthesis (were percent of control).
bioelicitors. CH (E4), YS (E3) and abiotic elicitors, NPSe (E2), SA (E1) performed significant increment in the four major components for SFO over that of traditional chemical fertilizer [NPK (F1)] coincide with non-elicitor control (E0). Aside, it follows that, [MO (F2) coincide CH (E4) > YS (E3) > NPSe (E2) > SA (E1) > NPK E0] excel [NM (F3) coincide E4 > E3 > E2 > E1 > NPK (F1E0)] excel [HA (F4) coincide E4 > E3 > E2 > E1 > NPK (F1) E0] excel [NPK F1 coincide E4 > E3 > E2 > E1 > NPK (F1) E0] Based on that biofertilizer concur bioelicitors excel significantly biofirtilizer coincide abiotic elicitor which were exceeded significantly over that traditional chemical fertilizer NPK (F1) under Zero-elicitor (E0) control, improving SFO quality consequently promoting health benefits.
4.3.2. Seed Essential Oil (SEO)
The major SEO contents (Table 4) were comprising; lamonine (39.45%), Thymoquione, TQ (30.51%), Penine, (10.55%) for control, NPKE0 (Table 1). Biotic/abiotic elicitors inseparable biofertilizer and traditional chemical NPK fertilizer performed significant positive impacts for the four major components of
Table 4. Proportions of N. sativa essential oil constituents impacted by five elicitors (E1-4) and four fertilizers (F1-4) (an average over of the two years experiment).
E0-4; control , salicylic acid (SA), nano-selenium oxide (SeN), yeast (Y), chitosan (CHT), E0 control respectively. F1-4; NPK, Moring a dry leaf extract (MOLE), Neem dry leaf extract (NMLE), humic acid (HA), respectively. Values between parenthesis (were percent of control).
SEO (Table 1). Aside, [MO (F2) coincur; CH (E4) > YS (E3) > NPSe (E2) > SA (E1) > NM (F3) concur; CH (E4) > YS (E3) > NPSe (E2) > SA(E1)] > HA (F1) concur, CH (E4) > YS (E3) > NPse (E2 > SA (E1)] > NPK (F1) concur CH (E4) > YS (E3 ) > NPSe (F2) > SA (E1)] therefore biotic elicitors; CH (E4),YS (F3) exceed significantly abiotic elicitors; NPSe (E2), SA(E1), concur biofertilizer; MO (F2), NM (F3), HA (F2), which excel significantly NPK(F1)E0 to improve SEO quality and ameliorated health promoting benefits Extensing study for elicitation revealed upraise EOs production and quality undertrditional chemical fertilizers  . Also solitary biofertilizer application have been shown the positive impacts of EOs quality and quantity   the different EOs compositions and different mutal ratio of compounds contained in EOs may excert significant on their biological efficacy and their components demonstrated beneficial impacts on human health, antimicrobial, antifungal, antiviral and food preservation  . Also, the resultant increment in the main major component of SFO and SEO was more or less in line with what has been reported recently  .
Based on overall obtained, it could be considered multi-repeating biotic/abiotic elicitation correlative biofertilizers as a reliable technological strategy to boast up significantly N. sativa, dray seed, seed fixed oil and seed essential oil yield production. Alongside, ameliorating significantly bioactive compound contents for both seed fixed, essential oils. Consequently, highlight to achieve sustainable development for N. sativa under organic and traditional inorganic system.
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