Plant growth-promoting properties of bacterial endophytes isolated from roots of Thymus vulgaris L. and investigate their role as biofertilizers to enhance the essential oil contents

: The main objective of the current study was to improve the essential oil contents of Thymus vulgaris L. using bio-inoculation with bacterial endophytes. Therefore, out of fourteen endophytic bacterial isolates obtained from roots of T. vulgaris , five isolates were selected based on the highest nitrogen-fixation and phosphate solubilization activity and identified as: Bacillus haynesii T9r, Citrobacter farmeri T10r, Bacillus licheniformis T11r, Bacillus velezensis T12r, and Bacillus velezensis T13r. These five strains have been recorded as ammonia, hydrogen cyanide (HCN), siderophores, and indole-3-acetic acid (IAA) producers. These strains have the efficacy to fix-nitrogen by reduction of acetylene with values of 82.133±1.4–346.6±1.4 n-mole-C 2 H 4 / ml/24 h. The IAA, gibberellic acid, abscisic acid, benzyl, kinten, and ziaten production were confirmed using HPLC. Two strains of T11r and T13r showed the highest plant growth-promoting properties and were selected for bio-inoculation of T. vulgaris individually or in a consortium with different mineral fertilization doses (0, 50, 75, and 100%) under field conditions. The highest growth performance was attained with the endophytic consortium (T11r+T13r) in the presence of 100% mineral fertilization. The GC-MS analysis of thyme oil contents showed the presence of 23 various compounds with varying percentages and the thymol fraction represented the highest percentages (39.1%) in the presence of the bacterial consortium.


Introduction
Thymus vulgaris L. or thyme (Family: Lamiaceae) is a flowering plant and is considered indigenous in Northern Africa, the Mediterranean, and various parts of Asia. Thyme plants are widely cultivated in Africa, for instance: Morocco, Egypt, Libya, Algeria, Cameroon, Tunisia, South Africa, and Nigeria [1]. The thyme plant is a common flavoring agent which has various importance in herbal medicine for the treatment of fever, cough, cold, diabetes, and chest infections. Also, it can be used to treat digestive disorders, calmative for throat infections due to its antibiotic, antiseptic, and antifungal characteristics [2]. Moreover, the thyme plant has been successfully displayed in the treatment of several intestinal infections including ascarids, hookworms, pathogenic Grampositive and Gram-negative bacteria, uni-and multi-cellular fungi [3]. The importance of thyme plants is mainly related to their essential oils, which have antiviral, antimicrobial, and antioxidant activities along with their carminative actions [4]. According to European Pharmacopoeia, the minimum essential oil contents in the thyme plant is 12 ml kg -1 , and it contains mainly six chemotypes, geraniol, γ-terpineol, linalool, thymol, carvacrol, and thujan-4-ol [5]. Therefore, these findings raise the great necessity to improve the development and growth of the thyme plants for their essential oil contents.
Recently, the endophytic microbes and their metabolites have been integrated into various biomedical and biotechnological applications [6,7]. Bacterial endophytes are considered the major endophytic group consisting of approximately 200 genera belonging to 16 phyla, and most of them associated with Proteobacteria, Actinobacteria, and Firmicutes [8]. Although the existence of the bacterial endophytes in plants is variable and in some cases, it is in transit, it has the efficacy to make various physiological and biochemical changes that regulate plant growth and performance under different environmental conditions [9]. Bacterial endophytes act as plant growth-promoting bacteria (PGPB) that can modulate plant growth through different mechanisms such as the production of phytohormones (indole-3-acetic acid, gibberellic acid, cytokinin, etc.), secretion of lytic enzymes, phosphate solubilization, nitrogen fixation, siderophores production, hydrogen cyanide (HCN), and ammonia production [10][11][12]. Moreover, bacterial endophytes can promote plant growth through the production of ACC deaminase enzymes that control the amount of ethylene in plants and increase the resistance of the plant to phytopathogens [9]. Interestingly, the presence of bacterial endophytes increases the resistance of the plant to various biotic and abiotic stresses [13]. Recently, various bacterial endophytes are used to regulate the growth of different plants such as rice, canola, tomato, mung bean, Vica faba, black pepper, Phaseolus vulgaris, and groundnut, to name of few [7,11,14].
The involvement of PGPB in the various biofertilizing systems started in the 1950s and now occupies approximately 5% of the fertilizer market, the majority of components of them are nitrogen-fixing bacteria [15]. The advantages of biofertilizers containing PGPB to agricultural sectors are improving soil fertility, providing plants with essential nutrition, enhancing the plant resistance against both biotic and abiotic stresses [15]. The main challenge of PGPB utilization for agronomic practice is the low efficiency of PGPB isolated from certain plant species to improve plant growth and performance in a wide range of other plant species [16]. Another complexity is the low stability of plant colonizing with PGPB under different environmental conditions along with the host plant specificity [17]. Therefore, the highest activity of PGPB can be achieved when these bacteria are applied to the same isolated plant and under the same conditions.
The main purpose of the current study is to improve the growth of T. vulgaris as oil-producing plants using bacterial endophytes. To accomplish this, bacterial endophytes were isolated from the healthy root of T. vulgaris collected from North Sinai. Five bacterial endophytes were selected based on their high potency for nitrogen fixation and phosphate solubilization. The plant growth-promoting properties of selected bacterial isolates include quantitative nitrogen fixation (using acetylene reduction), quantitative phosphate solubilization, ammonia production, hydrolytic enzyme activity, siderophores and HCN production, and synthesis of various phytohormones (IAA, gibberellic acid, abscisic acid, benzyl, kinten, and ziaten) were investigated. Finally, a field experiment was conducted to evaluate the effect of an individual or a consortium of the most two potent strains (based on higher plant growth-promoting properties) on the growth performance and essential oil contents of T. vulgaris in presence of different concentrations of mineral fertilization (0, 50, 75, and 100%).

Material and method Plant sample collection
The root of the medicinal plant Thymus vulgaris L. (family Lamiaceae) was collected from Balouza Research Station, North Sinai, Egypt (31°00'20.6"N 32°33'46.1"E). Four individual plants were collected per site. The collected plant samples were kept in sterile polyethylene bags and transported to the laboratory on the same day and subjected to isolation procedures within a short period of collection [18]. The plant identification was tentatively recorded in the field with help of a local Balouza Research Station agricultural engineer, and the botanical identification was carried out at the herbarium unit of Desert Research Center.

Isolation of endophytic bacterial strains
The collected roots samples of T. vulgaris were subjected to surface sterilization as follows: the plant samples were washed with tap water to exclude the soil debris and epiphytes adhered. After that, the plant samples were washed with sterilized distilled water for 60 sec. followed by 2.5% sodium hypochlorite for four min. and ethyl alcohol (70%, v/v) for 30 sec. Finally, the surface-sterilized plant samples were immersed in sterilized distilled water for 60 sec. To check the successful surface sterilization, approximately one ml of final distilled water was streaked onto Czapek Dox agar, nutrient agar, and starch nitrate agar for the growth of fungi, bacteria, and actinomycetes respectively. The success of surface sterilization was checked by the absence of microbial growth on the surface of the previous inoculated media after an appropriate incubation period [19].
The sterilized roots were cut into small fragments (5mm) under aseptic conditions. Approximately twenty fragments (20 fragments per sterilized root) were loaded on the surface of four nutrient agar Petri dishes (8 cm, 5 fragment/plate) supplemented with 25 μg ml -1 nystatin (as antifungal). The loaded plates are incubated at 30 ± 2°C and checked daily for bacterial growth. The appeared bacterial colonies from sterilized fragments were picked-up and re-streaked on the same media to confirm the purity. The purified colonies were inoculated into nutrient agar slants and kept at 4°C for further investigation. All obtained endophytic bacterial isolates were subjected to primary identification based on morphological, physiological, and biochemical tests according to standard keys [20,21].

Selection of the most potent endophytic bacterial stains
The selection of the most potent endophytic bacterial isolates was achieved based on their efficacy in qualitatively fixing nitrogen and solubilizing phosphate. The endophytic bacterial isolates were inoculated on nitrogen-deficient Jensen's agar medium (containing g L -1 : sucrose, 20; K 2 PO 4 , 1; MgSO 4 , 0.5; NaCl, 0.5; Fe 2 SO 4 , 0.1; Na 2 MoO 4 .2H 2 O, 0.005; CaCO 3 , 2; Agar, 15; 1.0 L distilled H 2 O) supplemented with bromothymol blue as an indicator. The positive nitrogen-fixing endophytic bacterial isolates were selected based on their efficacy to change media color to blue color [22].
The primary screening to investigate the efficacy of endophytic bacterial strains to phosphate solubilization was achieved using Pikovskaya agar media (containing g L -1 : glucose, 10; Ca 3 (PO 4 [23]. The experiment was achieved in triplicates.

Identification of the most potent endophytic bacterial isolates
The endophytic bacterial isolates that showed a positive result for nitrogen fixation and phosphate solubilization (T9r, T10r, T11r, T12r, and T13r) were subjected to molecular identification based on amplification and sequencing of the 16S rRNA gene. The genomic DNA was extracted according to the modified method [24]. Briefly, a separate bacterial colony was picked up by a sterile toothpick and suspended in 50 μl of sterilized deionized water. The cell suspension was put in a water bath for 10 min at 97°C, after that, the suspension was centrifuged for 10 min at 15000 rpm to recover the upper layer that contains the DNA. The intensity of DNA in the collected layer was calculated by measuring its absorbance at 260 nm by UV-spectrophotometer. A bacterial universal primers 27f (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492r (5'-GGTTACCTTGTTACGACTT-3') were used to PCR amplification of the 16S rDNA fragment. The PCR tube containing PCR buffer (1 x), MgCl 2 (0.5 mM), Tag DNA polymerase (2.5 U, QIAGEN Inc.), Deoxynucleoside triphosphate (dNTP, 0.25 mM), universal primer (0.5 μM), and bacterial DNA (5 ng/μl). The PCR cycling conditions were 94°C for three minutes, followed by 30 cycles at 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 60 seconds, and final extension at 72°C for ten minutes. The forward and reverse sequencing for PCR products was achieved using Applied Biosystems 3730xl DNA Analyzer technology at Sigma company, Cairo, Egypt.
The obtained sequences were analyzed using BLAST as compared with those deposited in the GenBank database. Multiple sequence alignment was performed using ClustalX 1.8 software package and a phylogenetic tree was constructed by the neighbor-joining method using MEGA (Version 6.1) software. The confidence level of each branch (1000 repeats) was tested by bootstrap analysis [25].

Characterization of bacterial endophytes as plant growth-promoting Quantitative nitrogen fixation using acetylene reduction assay
The efficacy of five selected endophytic bacterial strains to fixing nitrogen was investigated quantitatively through detecting the nitrogenase activity by the acetylene reduction method. In this method, each endophytic bacterial strain was inoculated in a test tube closed by a rubber stopper and containing 10 ml of nitrogen-free Jensen's semi-solid medium and incubated for 72 h at 33 ± 2°C. After that, approximately, one ml of acetylene gas was injected into the air of the headspace and incubated for 24 h at 33 ± 2°C. At the end of the incubation period, one ml of gas sample existing in the tube headspace was assayed for ethylene production (C 2 H 4 ) using Hewlett-Packard 5890 gas chromatography Series 2 plus [26,27]. The nitrogenase activity was assessed as a mol n of C 2 H 4 / ml/24 h. The experiment was performed in triplicate.

Quantitative phosphate solubilizing assay
The phosphate solubilizations were analyzed quantitatively by measuring pH values and P concentration at interval times (2 -10 days). Briefly, Pikovskaya's broth medium supplemented with 0.5% Ca 3 (PO 4 ) 2 was inoculated with endophytic bacterial strain and incubated at 33 ± 2°C for 10 days on a rotary shaker at 180 rpm. Five ml samples were taken daily and centrifuged at 10,000 rpm for 10 min. The phosphomolybdate method was used for the determination of available soluble phosphate in the culture supernatant using a spectrophotometer (Jenway 6305 UV) measuring at O.D. 700 nm. The concentration of P was calculated from the slope of the standard curve of P. Also, the pH of the broth medium was measured daily with a digital pH meter [13].

Ammonia production
The ability of endophytic bacterial strains for producing ammonia was assessed in peptone water broth. Each bacterial strain was inoculated into tubes containing 10 ml peptone water and incubated at 33 ± 2˚C for 48 h. At the end of the incubation period, approximately 0.5 ml of Nessler's reagent was added to the culture broth media. The development of yellow color was recorded as +, ++, +++ based on the color intensity [28].

Extracellular Enzymatic Activities
The production of extracellular enzymes (amylase, gelatinase, cellulase, and catalase) of the endophytic bacterial strains was investigated by inoculating the isolates in a mineral salt (MS) agar media (containing g L -1 : NaNO 3 , 5; KH 2 PO 4 , 1; K 2 HPO 4 , 2; MgSO 4 ·7H 2 O, 0.5; KCl, 0.1; CaCl 2 , 0.01; FeSO 4 ·7H 2 O, 0.02; agar, 15; distilled H 2 O, 1L) supplemented with specific additives, depending on the enzyme being tested. The MS agar media without bacterial inoculation was running with the experiment as a negative control. After incubation at 33 ± 2˚C for 24 h, specific reagents were added and the size of the clear zone (mm) surrounding the bacterial colony was measured, indicating extracellular enzymatic activities. All assays were performed in triplicates.
For amylolytic and cellulolytic activities, the bacterial strains were inoculated onto MS agar medium supplemented with 1% soluble starch and 1% carboxymethylcellulose (CMC) respectively. After an incubation period, the plates were flooded with 1% iodine.
For gelatinase activity, the MS agar medium containing 1% gelatin was used to determine the proteolytic activity of the bacteria. After an incubation period, gelatin degradation was checked using acidic mercuric chloride as an indicator [19].
For chitinase, the bacterial endophytes were spotted on MS agar media containing 1% of colloidal chitin formed according to Wen et al. [29]. The positive chitinase results were recorded as the diameter of the clear zone around bacterial growth.
For catalase activity, the pure bacterial colony from young culture (18-24h.) was put into a glass slide and mixed with a few drops of 3% H 2 O 2 . The appearance of oxygen bubbles indicates the presence of catalase activity [30].

Siderophore production
Secretion of siderophore was qualitatively analyzed using king's B agar medium (containing g L -1 : proteose peptone, 20; K 2 HPO 4 , 1.5; MgSO 4 , 1.5; Agar, 15, 1.0 L distilled H 2 O; pH, 7.2 ± 0.2) supplemented with chrome azurol S (CAS), Fe 3+ solution, and hexadecyltrimethylammonium bromide (HDTMA) as an indicator. The indicator preparation was achieved as follows: 60.5 mg of CAS was dissolved in 50 ml distilled H 2 O, followed by mixing with 10 ml of Fe 3+ solution (1 mmol L -1 dissolved in 10 mmol L -1 of HCl). Under the stirring condition, the previous mixture was dropped wisely added to the HDTMA solution (72.9 mg of HDTMA dissolved in 40 ml distilled H 2 O). The prepared indicator was added to King's B media with a ratio of 1:15. The fresh bacterial culture was spotted onto a king's B agar plate and incubated at 28°C for 72 h. The appearance of an orange halo around the endophytic bacterial growth indicates a positive siderophore production [14].

HCN production
The purified endophytic bacterial isolate was tested for HCN production through inoculation on king's B agar medium amended with glycine [31]. After that, a Whatman No. 1 filter paper was soaked in a solution of (2% sodium carbonate mixed with 0.05% picric acid solution) and put on the lid of inoculated king's B petri dish. The plates were sealed with parafilm and incubated at 28 ± 2°C for 48 h. The change in the color of the filter paper from deep yellow to reddish-brown color indicates a positive result for HCN production.

IAA production
The most potent endophytic bacterial isolates were screened for qualitative IAA production. Briefly, the bacterial isolates are inoculated into nutrient broth media amended with different concentrations of tryptophan (0, 1, 2, and 5 mg ml -1 ) and incubated at 33± 2˚C for 15 days. At the end of the incubation period, the inoculated culture was centrifuged at 3000 rpm for 30 min. After that, two ml of the obtained supernatant was mixed with 2 drops of orthophosphoric acid and 4 ml of Salkowski's reagent (300 ml concentrated Sulfuric acid: 500 ml distilled water: 15 ml 0.5 M FeCl 3 ). The development of pink color indicates successful IAA production. The intensity of the formed color was measured at 530 nm using a T60 UV-visible spectrophotometer [32]. The amount of IAA was estimated via a standard IAA graph to detect the best tryptophan concentration for IAA production.
For quantitative IAA assay, the endophytic bacterial isolates were inoculated into nutrient broth media supplemented with 5 mg ml -1 of tryptophan (the best concentration of tryptophan for IAA production based on the qualitative assay) and incubated at 33 ± 2°C for 14 days. Approximately two ml of inoculated culture was withdrawn after the 2 nd day up to the 14 th day with 2 days interval and centrifuged at 3000 rpm for 30 minutes. One ml of the supernatant was mixed with 1 drop of orthophosphoric acid and 2 ml of Salkowski's reagent. The intensity of development of pink color was measured at 530 nm using a T60 UV-visible spectrophotometer. The amount of IAA produced was estimated by the standard IAA graph to detect the best days for IAA production.

Field experiment
The seeds of Thymus vulgaris (local species) were purchased from the farm of the Faculty of Pharmacy, Cairo University, Cairo, Egypt. The bacterial isolates T11r and T13r were selected as the most potent based on previous plant growth-promoting activities to investigate their efficacy on the growth trail and oil content of T. vulgaris under filed experiment conditions. The experiment was carried out at Alqantra Sharq Research Station, North Sinai, Egypt (GPS: 31°00'21.6" N and 32°33'48.1" E) using a completely randomized design that contains four blocks, each block containing four plots based on different treatments. The texture and chemical constituents of soil used in the field experiment are recorded in table 1. Before the experiment, the soil was well-milled and thoroughly mixed with organic fertilizer (20 m 3 /feddan). Moreover, the mineral fertilization (MF) is designated as calcium super-phosphate (containing 15.5% P 2 O 5 ), calcium ammonium nitrate (containing 33.3% N), and Potassium sulfate (containing 48% K 2 O) were added twice, immediately after planting and after 65 days. The mineral fertilization doses were added as follows: 100%, 75%, 50%, and 0.0% with amounts (150, 112.5, 75 and 0 kg/ feddan) for calcium ammonium nitrate, (300, 225, 150 and 0 kg/feddan) for calcium ammonium nitrate, and (100, 75, 50 and 0 kg/feddan) for Potassium sulphate according to standard of Egyptian Agriculture Ministry. The addition of plant growth-promoting bacteria as biofertilizers (BF) was applied as follows: immerse plant seeds in bacterial culture for one hour before planting, the bacterial cell was suspended in saline solution and added to soil beside the plant root at 65 and 140 days of planting. The following treatment was conducted: A1: seeds planting on soil with 100% MF without BF; A2: seeds planting on soil with 75% MF without BF; A3: seeds planting on soil with 50% MF without BF; A4: seeds planting on soil with 0.0% MF without BF; A5: seeds treated with BF T11r and planting on soil with 100% MF; A6: seeds treated with BF T11r and planting on soil with 75% MF; A7: seeds treated with BF T11r and planting on soil with 50% MF; A8: seeds treated with BF T11r and planting on soil with 0.0% MF; A9: seeds treated with BF T13r and planting on soil with 100% MF; A10: seeds treated with BF T13r and planting on soil with 75% MF; A11: seeds treated with BF T13r and planting on soil with 50% MF; A12: seeds treated with BF T13r and planting on soil with 0.0% MF; A13: seeds treated with consortium BF (T11r+T13r) and planting on soil with 100% MF; A14: seeds treated with consortium BF (T11r+T13r) and planting on soil with 75% MF; A15: seeds treated with consortium BF (T11r+T13r) and planting on soil with 50% MF; A16: seeds treated with consortium BF (T11r+T13r) and planting on soil with 0.0% MF. All treatments were achieved in triplicates. The plant was harvested two times, at 65 days and 140 days.

Growth and Vegetative Parameters Measurement
The plant height after two harvests of 65 and 140 days was measured by cm. The shoots of 65 and 140-day-old thymus plants were separated from the plants and measured to detect the fresh weight. The collected shoots have dried in an oven at 70°C until constant weight, then, the dry weights of the shoots were measured.

Oil extraction
The extraction of oil was achieved for 100 g of T. vulgaris dry weight (collected from first and second harvest) using the oil distillation method according to British Pharmacopoeia [34]. Briefly, 100 g of the dry weight of the plant was added to a conical flask (1.0 L) and mixed with a sufficient water amount (4 -5 times of dry weight). A proper oil trap connected to a condenser was attached to the conical flask followed by the addition of water to fill the trap. After that, the flask undergoes heating using an electric hot plate. The distillation process was continued for approximately 2.0-2.5 hours till separate the essential oil from water and no further increase in the content of oil was observed. The amount of the obtained essential oil was calculated in proportion to the plant dry weight used.

Oil Fraction by Gas chromatography-mass spectrometry analysis (GC-MS)
The GC-MS system (Agilent Technologies) was equipped with gas chromatography (7890B) and a mass spectrometer detector (5977A) at Central Laboratories Network, National Research Centre, Cairo, Egypt. Samples were diluted with hexane (1:19, v/v). The GC was equipped with an HP-5MS column (30 m x 0.25 mm internal diameter and 0.25 μm film thickness). Analyses were carried out using helium as the carrier gas at a flow rate of 1.0 ml/min at a split ratio of 1:10, injection volume of 1 μl, and the following temperature program: 40°C for 1 min; rising at 4°C /min to 150°C and held for 6 min; rising at 4°C/min to 210°C and held for 1 min. The injector and detector were held at 280°C and 220°C, respectively. Mass spectra were obtained by electron ionization (EI) at 70 eV; using a spectral range of m/z 50-550 and solvent delay 5 min. Identification of different constituents was determined by comparing the spectrum fragmentation pattern with those stored in Wiley and NIST Mass Spectral Library data.

Statistical analysis
Split plot design was designed as a layout for the experiment with three replicates for each treatment. Data were analyzed statistically by a statistical program Sigma Plot v14. Analysis of variance (ANOVA) was used as a tool for calculating the mean difference comparison between the treatments and afterward by Tukey's test (honestly significant difference) at p ≤ 0.05. Hierarchical two-way cluster analysis was conducted on the data matrix of oil content percentages using the centered method of the standardized data.

Results and discussion
Isolation of bacterial endophytes that inhabit the roots of Thymus vulgaris L.  [35]. These 117 isolates were belonging to Firmicutes, proteobacteria, and Actinobacteria. In the recent study, the endophytic bacterial community isolated from the medicinal plant Pulicaria incisa were belonging to proteobacteria (α, β, and γ) and Firmicutes with percentages of 33.3% and 66.6%, respectively [32].

Selection of the most potent bacterial isolates
Phosphorus and nitrogen are the main nutrients for enhancement of the plant growth. In most cases, these elements are not available for plants [36]. Endophytic bacteria along with nitrogen-fixing and phosphate solubilizing bacteria facilitate nutrient requirement are widely used as biofertilizers [37]. Therefore, in the current study, the selection of the most potent bacterial isolates to be used as a biofertilizer was achieved based on higher nitrogen fixation and phosphate solubilization. Data represented in Table 2 showed that T3r and T8r to T13r had the ability of nitrogen-fixing with varying degrees according to the appearance of blue color on nitrogendeficient Jensen's agar medium. The highest color intensity was recorded for isolates T9r, T10r, T11r, T12r, andT13r. The obtained data are compatible with those recorded by Abdelshafy and co-authors who reported that approximately 84% of the bacterial endophytes isolated from the tissues of T. vulgaris had the ability for fixing nitrogen when grown on Ashby's media [35]. Moreover, the bacterial isolates T9r, T11r, T12r, and T13r showed varied efficacy of phosphate solubilization designated as diameters of clear zone formed around bacterial growth on Pikovskaya agar media. Endophytic bacterial isolates T11r and T13r exhibited the highest phosphate solubilizing activity with clear zones of 7.5±0.3 mm and 8.9±0.2 mm, respectively. Similarly, among 13 endophytic bacterial strains isolated from medicinal plants Fagonia mollis and Achillea fragrantissima collected from Saint Katherine Protectorate, South Sinai, Egypt, only 9 strains exhibit phosphate solubilizing activity on Pikovskaya agar media with a diameter of clear zone ranging from 7.6 ± 0.3 mm to 9.6 ± 0.3 mm [38]. Based on the obtained data, the endophytic bacterial strains T9r, T10r, T11r, T12r, and T13r were selected for identification and further investigation on their efficacy as plant growthpromoting rhizobacteria.

Identification of the most potent endophytic bacterial isolates
Five bacterial isolates were selected based on their efficacy for nitrogen fixation and phosphate solubilization and were identified using PCR amplification and sequencing of the 16S rRNA gene. The sequence analysis of strain T9r showed a similarity of 99.7% with the sequence of Bacillus haynesii, whereas the sequence of strains T10r and T11r showed 99.3% and 98.7% similarity percentages with Citrobacter farmeri and Bacillus licheniformis, respectively. Strains T12r and T13r showed 99.9% similarity percentages with the sequence of Bacillus velezensis ( Table 3, Fig. 1). Moreover, the outgroup of Gram-positive and Gram-negative endophytic bacterial isolates was represented in Figures S1 and S2 [32,35,38,40].
Recently, endophytic Bacillus haynesii was isolated from Ocimum tenuiflorum and exhibits high antagonistic actions against different phytopathogenic fungi [41]. Also, Bacillus velezensis is identified as a novel endophytic Bacillus strain with high biological and plant growthpromoting activity as reported previously [42]. To the best of our knowledge, the obtained strains in this study were recorded for the first time as endophytic bacterial strains isolated from T. vulgaris.

In-vitro characterization of bacterial endophytes as plant growth-promoting rhizobacteria
Quantitative nitrogen fixation using acetylene reduction assay Nitrogen element (N) is considered the main component of enzymes, proteins, nucleic acids, and chlorophyll. Therefore, it is a fundamental element for adequate plant growth and hence increases the yield through its contribution within several routes affecting plant physiological and biochemical functions [43]. For example, certain bacterial endophytes have the ability to provide plants with their required nitrogen through the process of nitrogen fixation. From these bacteria, one group are identified as diazotrophic bacterial endophytes, they are considered the critical source for the input of nitrogen into the agricultural implements, and candidates as a promising alternative source for chemical fertilizers [44]. In the current study, besides qualitative nitrogen fixation (Table 2), the quantitative nitrogen fixation efficacy of endophytic bacterial strains through reduction of acetylene was investigated. Data in Table 3 revealed that all selected bacterial endophytes have the ability to produce nitrogenase through the reduction of acetylene with a varied degree.

Quantitative phosphate solubilizing assay
Approximately, 95 to 99% of phosphorus levels in soils are unavailable for plant uptake because they present either as immobilized or precipitated, or insoluble forms. This leads to an increase in applying chemical phosphorus fertilization and harmfully affects the environment and soils [46]. Bacterial endophytes can increase the availability of phosphorus for plant uptake by various mechanisms, for instance, chelation, producing organic acids, exopolysaccharide secretion, ion exchange, and synthesis of different phosphate solubilizing enzymes such as phytase, phosphatase, and C-P lyase [9]. In the current study, the most potent endophytic bacteria showed varied phosphate solubilizing activity through detecting the amount of soluble phosphate in Pikovskaya's broth medium supplemented with Ca 3 (PO 4 ) 2 that was measured by the phosphomolybdate method. Analysis of variance showed that the highest P amount was liberated after nine days with values of 123.9 ± 2.8, 149.5 ± 0.9, 149.5 ± 1.01, and 159.5 ± 0.9 μg ml -1 for bacterial endophytes Bacillus haynesii T9r, Bacillus licheniformis T11r, Bacillus velezensis T12r, and Bacillus velezensis T13r, respectively ( Fig. 2A). The lowest P secretion was recorded for bacterial isolate Citrobacter farmeri T10r and these data were compatible with preliminary screening ( Table 2). The pH value of Pikovskaya's inoculated broth media was decreased with time increase as compared with control. In the present study, the medium pH was decreased from 6.6 on the first day to reach 4.5 on the ninth day as compared with control (pH=7.1 on the first day and reach 6.8 on the ninth day) (Fig 2B). The reduction in pH values was acidified in the medium and hence the Ca 2+ is replaced by H + and liberates the phosphate ions [47]. Various organic acids such as malic, gluconic, citric, oxalic, salicylic, fumaric, and tartaric acid are the main ones involved in phosphate solubilization [9]. Among selected endophytic bacterial strains, the strains Bacillus licheniformis T11r and Bacillus velezensis T13r possess the highest activity for phosphate solubilization.

Ammonia production
Bacterial endophytes can enhance plant growth through the production of ammonia, which significantly improves the shoot and root growth; moreover, ammonia enhances the growth of plants by the formation of different biomolecules containing nitrogen [48]. In the current study, all bacterial endophytes differed in their efficacy to produce ammonia through a color change of inoculated peptone water broth media after adding Nessler's reagent. Data showed the highest color change was recorded for the bacterial isolate of T13r followed by T11r and T12r (Table 3). Among 52 bacterial endophytes isolated from roots, stems, and leaves of Clerodendrum colebrookianum, 51 isolates (98%) exhibited ammonia production [28]. Endophytic bacteria possibly produce ammonia from different routes such as the hydrolysis of amino acids containing nitrogen or through deamination of by-products resulting from the breakdown of various macromolecules, or from the breakdown of urea to produce CO 2 and NH 4 [32,49].

Extracellular Enzymatic Activities
Data represented in Table 4 showed that all selected bacterial isolates were positive for protease and chitinase, whereas Bacillus licheniformis T11r, Bacillus velezensis   [28]. The ability of bacterial endophytes to penetrate the cells of their host can be related to their efficacy to secrete various extracellular lytic enzymes. Moreover, these lytic enzymes protect plant hosts from the attack by phytopathogenic fungi [50]. It has been reported that the endophytic bacterial strains characterized by catalase production are more resistant to different mechanical, environmental, and chemical stresses [30]. This phenomenon can be attributed to the catalase enzyme which is responsible for the scavenging of free radicals raised due to exposure to biotic and abiotic stresses. Also, the chitinase enzyme has a critical role in the hydrolysis of chitin which is the main component of the fungal cell wall, and hence protects the plant from infection by phytopathogenic fungi [7].
Microbes with amylase, cellulase, and protease enzymes activity have different advantages including organic matter degradation, plant growth promotion, and plant protection from diseases caused by soil-borne pathogens [51]. Amylase enzymes are considered the major industrial enzyme group represented by 25-30% of the total market enzyme [52]. Bacterial cellulase enzymes are not only important for depolymerization of cellulose, which is the major component in the plant cell but also assists and facilitate bacterial penetration to plant host [53,54]. Also, protease enzymes secreted by bacterial endophytes have primary importance to suppress phytopathogens injury and protect the plant from nematodes infection. The protease enzyme secreted by endophytic bacterial strain Bacillus cereus BCM2 showed high activity against Meloidogyne incognita (plantparasitic nematodes) through the degradation of chemical components of nematodes cuticle and their eggshells [55]. The finding that Bacillus spp. are considered the highest endophytic strains producing various lytic enzymes has been previously reported [13,28], and these data are in harmony with the current study. Based on enzymatic activity, bacterial endophytes can provide the plant with their requirement of nutrients and protect their host from phytopathogen infection by secretion of different lytic enzymes [28].

Siderophores
Siderophores are defined as secondary metabolites with a low molecular weight that has the ability to iron-chelating from complex substance. Siderophores are synthesized by different endophytic bacterial species and promote plant growth through the availability of Fe 3+ for iron-deficient plants [56]. In the current study, four bacterial strains of the selected most potent isolates can synthesize siderophores by forming an orange halo around the bacterial growth on king's B agar medium (Table 4). Similarly, out of five endophytic bacterial strains isolated from Cassia tora L. root, only three isolates produced siderophores [57]. Brígido and co-author reported that although the few -, +, ++ denotes negative, moderate, and high production bacterial endophytes isolated from chickpea root can be produced siderophores, most of them are isolated from a plant collected from soil contaminated with manganese [58]. Besides the critical role of siderophores in the high scavenging of insoluble iron, siderophores secreted by various bacterial species can bind with other non-iron metals, and hence reduce the toxicity of these metals in the environment [59]. Accordingly, bio-inoculation of metalsensitive plants with siderophore-producer bacterial endophytes could possibly acclimate plants within metal contaminated soils.

HCN production
Various plant-associated microbes can produce volatile substances as secondary metabolites known as hydrogen cyanide (HCN). This volatile substance has a crucial role in suppressive the growth of plant pathogenic microbes [60]. In the current study, all selected bacterial endophytes have the capacity to change the color of filter paper from deep yellow to reddish-brown with varying degrees. The highest bacterial strains producing HCN were Bacillus licheniformis T11r and Bacillus velezensis T13r, followed by Bacillus haynesii T9r (Table 4). As shown, the highest producing strain was Bacillus spp. which is compatible with a previous study [28]. In addition to the capacity of HCN to suppress phytopathogen, it is also used to control nematodes infection [61]. The endophytic bacterial strains that produce HCN along with siderophores are considered a good candidate in biocontrol investigation as previously mentioned [62].

IAA production
The main feature of plant growth-promoting microbes is their potency in the synthesis of various phytohormones such as indole-3-acetic acid (IAA). Various processes involved in plant growth such as cell division, stem elongation, geotropisms, root development and hence nutrient uptake, biomass production, apical dominance, phototropism, and cell differentiation are regulated by secretion of IAA [63,64]. In the current study, the efficacy of isolated endophytic bacterial strains to produce IAA was assessed after 15 days of incubation in the presence and absence of tryptophan (0, 1, 2, and 5 mg ml -1 ) as the precursor for IAA. Data analysis showed that the ability of various endophytic bacterial strains for the synthesis of IAA in absence of tryptophane was non-significant compared with control. At tryptophane of 1 and 2 mg ml -1 , the bacterial strains T9r, T10r, T11r, and T13r produced IAA with values of (16.9 ± 1.1, 14.1 ± 0.3, 28.7 ± 1.9, and 48.3 ± 1.5 mg ml -1 ) and (20.3 ± 0.2, 16.4 ± 0.7, 43.2 ± 2.9, and 50.4 ± 1.9 mg ml -1 ), respectively. These values are significant (p ≤ 0.001) different as compared with control (2.8 ± 0.4 and 3.8 ± 0.5 mg ml -1 ) and those produced by endophytic strain T12r (2.1 ± 0.3 and 4.1 ± 0.1 mg ml -1 ) (Fig. 3A). The concentration of IAA was increased by increasing tryptophan concentration. At 5 mg ml -1 tryptophan, the concentration of IAA was increased to reach 29.6 ± 1.3, 18.1 ± 0.6, 74.2 ± 1.8, 16.2 ± 0.8, and 81.8 ± 1.9 mg ml -1 for strains T9r, T10r, T11r, T12r, and T13r, respectively (Fig. 3A). It was reported that 48 out of 52 endophytic bacterial strains isolated from the roots of medicinal plant Clerodendrum colebrookianum showed efficacy to produce IAA in nutrient broth media supplemented with 2 mg ml -1 tryptophan [28]. The authors reported that the highest production was recorded for isolates Bacillus sp. BPSAC3 and Bacillus sp. BPSAC6 with values of 78.4 and 80.6 mg ml -1 . Besides the importance of IAA for plant growth, the secretion of this phytohormone displays a crucial key in the relationship between plant and endophytic microbes [65]. Based on the qualitative screening, the best concentration of tryptophan used for quantitative screening at interval times was 5 mg ml -1 . Data represented in Figure (3B) showed that the productivity was timedependent, increased by increasing incubation times. The highest IAA production was still recorded for bacterial strains T11r and T13r with amounts of 75.6 ± 3.7 and 80.8 ± 3.9 mg ml -1 after 14 days, respectively. The high IAA production can be attributed to the enzymes responsible for the conversion of tryptophan [66]. Previous studies that harmony with the current investigation showed that the production of IAA by bacterial and fungal endophytic strains is dependent on the concentration of tryptophan in liquid media and incubation times [13,19,67].

Detection of phytohormones using HPLC.
The secreted acidic (GA 3 , IAA, and ABA) and alkaline (Benzyl, Kinten, and Ziaten) plant hormones were quantified for the selected bacterial endophytes (T9r, T10r, T11r, T12r, and T13r) using HPLC (Table 5). For gibberellic acid (GA 3 ), the bacterial isolates Bacillus licheniformis T11r and Bacillus velezensis T13r exhibited the highest productivity with values of 7.08±0.15 and 6.597±0.69 mg/100 ml, respectively. Whereas the lowest production was recorded for bacterial isolate Bacillus haynesii T9r with a value of 2.31±0.05 mg/100 ml. On the other hand, the highest production of IAA was recorded for endophytic bacterial isolates T11r and T13 followed by T10r, and these data are compatible with a qualitative and quantitative assay using Salkowski's reagent. In a similar study, among four endophytic bacterial strains isolated from Zingiber officinale and identified as Bacillus sp. ZoB1, Pseudomonas sp. ZoB2, Stenotrophomonas sp. ZoB3, and Staphylococcus sp. ZoB4, only one strain ZoB2 has the efficacy to produce IAA and confirmed their productivity by HPLC [68]. Abscisic acid (ABA) has a critical role in ameliorating the various stresses and enhancing plant adaptation [69]. Moreover, under normal conditions, ABA at adequate concentration has an important role in the development and growth of the plant [70]. In the current study, all selected bacterial isolates have the potency to secrete ABA at low concertation ranging between 0.02±0.004 to 0.03±0.003 mg/100 ml. The HPLC analysis showed the activity of selected endophytic bacteria to secrete other phytohormones including benzyl adenine, kinten, and ziaten. The isolates T10r, T11r, and T13r still possess high activity in phytohormones production. Compatible with our study, the HPLC analysis for ethyl acetate extract of Bacillus sp. BPSAC6 isolated from roots of medicinal plant Clerodendrum colebrookianum exhibit varied activity for production of IAA, kinten, and benzyl adenine with values of 31.2, 12.3, and 3,24 μg ml -1 , respectively [28]. Based on plant growth-promoting traits of selected five endophytes bacterial strains, two strains designated as T11r and T13r were selected to investigate their activity on the growth and oil content of T. vulgaris under field conditions.

Effect of bacterial inoculation and mineral fertilization treatment on T. vulgaris growth
The improvement of medicinal and agronomical plants for integrating into various life sectors is being an important challenge to cope with the overpopulation growth [71,72]. Bacterial endophytes can enhance plant growth by various direct and indirect mechanisms, such as the production of phytohormones, phosphate solubilization, nitrogen fixation, ammonia production, secretion of lytic enzymes, production of siderophores and HCN, and protect the plant against phytopathogens [9,35]. In the current study, all selected bacterial endophytes exhibited various plant growth-promoting traits with varying degrees. The two bacterial endophytic strains Bacillus licheniformis T11r and Bacillus velezensis T13r possess the highest activity as plant growth-promoting rhizobacteria. Therefore, it can be used as biofertilizers to enhance T. vulgaris growth performance and the oil content and quality. The efficacy of the two most potent bacterial strains T11r and T13r and their consortium as bio-inoculants or biofertilizers (in presence and absence of different concentrations of mineral fertilization) on the growth performance of T. vulgaris was investigated at two stages, after 65 days and 140 days under field conditions. The height of T vulgaris in the absence of bacterial endophytes (control) was (10.0±0.8 cm and 12.7±0.9 cm) after 65 and 140 days in the absence of mineral fertilization (A4), this height was significantly increased to (28.7±0.8 cm and 39.7±0.9 cm) after adding 100% mineral fertilization (A1). On the other hand, the fresh weight and dry weight of the control (A4) was (13.0±0.8 g and 21.3±0.9 g) and (3.6±0.2 g and 6.1±0.3 g) after 65 and 140 days, respectively, and these weights were increased after adding 100% mineral fertilization (A1) to (31.7±0.8 g and 57.3±0.9 g) and (9.6±0.2 g and 16.2±0.3 g), respectively. Hence, the addition of mineral fertilization positively increased the growth parameters of T. vulgaris. These positive impacts were increased in the presence of bacterial endophytes (Bacillus licheniformis T11r and Bacillus velezensis T13r) individually or in a consortium (Table 6). For instance, the maximum plant heights (42.3±0.8 cm and 55.9±0.9 cm) were recorded for seeds treated with Bacillus licheniformis T11r (A5) in the presence of 100% mineral fertilization followed by those treated by the bacterial consortium in presence of 100% mineral fertilization (38.9±0.8 cm and 55.7 ±0.9 cm). On the other hand, the maximum fresh and dry weights of shoots were reported for seeds treated by the bacterial consortium (A13) followed by seeds treated by Bacillus velezensis T13r (A9) and those treated by Bacillus licheniformis T11r (A5) ( Table 6). Based on the obtained results, it can be concluded that the presence of bacterial endophytes promotes plant growth in the presence of mineral fertilization as compared with control. The potency of two selected endophytic strains to improve plant growth can be attributed to their plant growth-promoting properties and hence increase the availability of nutrients required for plant growth as reported previously [73]. Ullah and co-authors reported that the enhancement of plant morphological and physiological properties (root and shoot length, number of root tips responsible for nutrient uptake, plant biomass, and chlorophyll contents) due to microbial endophytes treatment are mainly because of their efficacy in producing phytohormones and phosphate solubilization [74]. Recently, the endophytic bacterial strains Bacillus cereus strain PI-8 and Bacillus subtilis strain PI-10 isolated from Pulicaria incisa leaves enhanced the morphological characteristics (plant heights, fresh shoots, and roots weight, and dry shoots and roots weight) of Zea mays compared with control (un-inoculated plants) [32]. Similarly, out of three bacterial endophytes, one strain identified as Bacillus sp. strain BPSAC6 exhibited high efficacy to improve the root length, height of shoots, and weight of tomato plants after 45 and 60 days after planting as compared with control (un-inoculated seeds) [28].

Effect of bacterial inoculation and mineral fertilization treatment on oil content and fractions using GC-MS analysis
The oil content of T. vulgaris due to different treatments was analyzed using GC-MS.  (Table 6). Hence, the bacterial endophytes have the efficacy to improve oil contents even though the absence of mineral fertilization. The obtained results are in harmony with those recorded that the dry weight, fresh weight, and content of oils in T. vulgaris were the highest in soil fortified with phosphate solubilizing bacteria plus manure, Nitroxin biofertilizer, and phosphate at full doses [75]. The effect of biofertilizations, mineral fertilizations, and/or organic fertilization on the oil contents was previously reported [76,77]. The GC is considered the main technique used for the separation of volatile and semi-volatile compounds in herbs and identifying each one. The attachment of MS with GC apparatus enables the fragmentation of each separated compound and compared these fragments with available database and identified it without using markers [78,79]. In the current study, the GC-MS analysis was achieved for sixteen different treatments of T. vulgaris to investigate the effects of bacterial endophytes in the presence and absence of mineral fertilization on thyme contents (Fig.  4). GC-MS data analysis showed that the thyme herb oil samples containing twenty-three (23) different compounds were identified based on retention times and authentic substances. The identification of these 23 compounds and their concentrations in each treatment were represented in Table 7 and Figure 5. The major components of thyme oil were α-Thujene, Sabinene, β-Myrcene, α-Terpinene, o-Cymene, γ-Terpinene, Terpinen-4-ol, α-Terpineol, Caryophyllene, and Thymol (Table 7) with varied contents according to treatments. Most of these fractions, especially Thymol, have medicinal and biotechnological importance [80,81]. The obtained data are compatible with that recorded by Al-Asmari and co-authors [82], who showed that the GC-MS analysis of T. vulgaris oil contains 24 various compounds and is identified based on their retention time. Moreover, the major compounds recorded in the current study are matched with those recorded in various published studies [82,83].
Our data recorded that the thymol compounds are the highest value (39.1%) when the plants were treated with a full dose of mineral fertilizers with the consortium of endophytic bacteria (B. licheniformins T11r and B. velezensis T13r), whereas the lowest value (29.85%) was recorded in plants which un-treated with chemical or bacterial fertilizations. In all cases, uninoculated thyme plants gave the lowest thymol compound value. Two-way cluster analysis showed that the different treatments were clustered in two groups. The first group included the control untreated plant in presence of various    (Fig. 6 and S3). It is clear that cooperation between both bacterial endophytes and mineral fertilizers activated biosynthesis reactions of thyme plants resulting from the highest value of the main component. This result agrees with Sharaf EL-Din et al, [84], who reported that the use of biofertilization can improve the biosynthesis of different constituents in thyme plants. Moreover, the application of plant growth-promoting bacteria increases the morphological characteristics and oil contents of Ocimum basilicum L. as compared with un-treated plants [85]. Based on obtained data, can be concluded that the plant growth-promoting bacterial endophytes as biofertilizers could serve as a potential treatment to increase the growth, yield, and oil fractions of the thymol plant. Further, the activity of thymol contents to incorporate into various biomedical applications will be assessed in-vitro and in-vivo. The ecological roles of these bacterial endophyte need to be studied in the future because they might be important in supporting plant survival in different regions. The benefits derived from the plant-endophytic bacteria interaction should be considered at genetic levels below the species. Overall, the bacterial endophytes of T. vulgaris might depend on several environmental factors that may structure their communities, more research is required to identify the functional and ecological significance of these bacterial endophytes. Some of the species identified have been described potential characteristics to promote plant growth. Therefore, a better understanding of this  Table 7.
complex network of interactions between the plant and bacterial endophytes would help to enhance productivity and sustainability.

Conclusion
In the current study, seven bacterial endophytes were isolated from the collected roots of T. vulgaris and showed varied qualitative activities for phosphate solubilization and nitrogen fixation. Five endophytic strains identified as Bacillus haynesii T9r, Citrobacter farmeri T10r, Bacillus licheniformis T11r, Bacillus velezensis T12r, and Bacillus velezensis T13r were selected as the most potent strains. These strains showed varied plant growth-promoting properties including acetylene reduction (nitrogenase), phosphate solubilization (in ranging of 123.9±2.8 -159.5±0.9 μg ml -1 ), various hydrolytic enzyme activity, siderophores and HCN production, and the synthesis of different phytohormones. The strains Bacillus licheniformis T11r and Bacillus velezensis T13r were further selected based on the highest plant growth-promoting activity to be used as bioinoculant for T. vulgaris under different mineral fertilization doses (0, 50, 75, and 100%). Data analysis showed that the growth characteristics (plant heights, fresh and dry weight of shoots) of inoculated thyme plants with the bacterial consortium (T11r + T13r) in presence of 100% mineral fertilization were high as compared with single endophytic strains and control. Moreover, the GC-MS analysis for thyme oil revealed that the presence of 23 various compounds, and the maximum percentages were recorded for the bacterial consortium in presence of 100% mineral fertilization. This study provides an efficient method to improve the oil contents of the thyme plant using plant growth-promoting bacterial endophytes. However, the biomedical activity of various oil fractions obtained in the current study has been proposed for further investigation. This study needs  Table 7. more investigation to show the efficacy of the obtained plant growth-promoting bacterial endophytes on the oil contents and growth performance of others agronomical plants under different environmental conditions. Also, the association between bacterial endophytes and plants needs more study.