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

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 (C₂H₄) using Hewlett-Packard 5890 gas chromatography Series 2 plus [26, 27]. The nitrogenase activity was assessed as a mol n of C₂H₄/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% Ca₃(PO₄)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: NaNO₃, 5; KH₂PO₄, 1; K₂HPO₄, 2; MgSO₄·7H₂O, 0.5; KCl, 0.1; CaCl₂, 0.01; FeSO₄·7H₂O, 0.02; agar, 15; distilled H₂O, 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% H₂O₂. 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; K₂HPO₄, 1.5; MgSO₄, 1.5; Agar, 15, 1.0 L distilled H₂O; pH, 7.2 ± 0.2) supplemented with chrome azurol S (CAS), Fe³⁺ 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 H₂O, followed by mixing with 10 ml of Fe³⁺ 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 H₂O). 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 FeCl₃). 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.

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.

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. Bacillus licheniformis T11r and Bacillus velezensis T13r showed the highest nitrogen fixation with amounts of 322.4±2.1 and 346.6±1.4 n-mole C₂H₄/ ml/24h, respectively. The lowest activity was recorded for Bacillus haynesii T9r with an amount of 82.133±1.4 n-mole C₂H₄/ ml/24h. Similarly, out of 250 endophytic bacterial strains isolated from stems, mature seeds, roots, and leaves of rice, only 21 bacterial strains were positive for the acetylene reduction test with varied nitrogenase activity ranging from 7.6 to 225.9 n-mol C₂H₄/h/mg/protein [45].

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 Ca₃(PO₄)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 Ca²⁺ 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.

Figure 2

Quantitative phosphate solubilizing assay in Pikovskaya’s broth medium supplemented with Ca₃(PO₄)₂. A is denotes the amount of liberated phosphorus (µg ml⁻¹) at interval times; B is denotes the pH values of Pikovskaya’s media at interval times. Values within the same time interval with different letters are significantly different (p ≤ 0.05), values are means ± SE (n = 3).

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 CO₂ and NH₄ [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 T12r, and Bacillus velezensis T13r were positive for amylase. Moreover, out of the 5 most potent endophytic bacterial isolates, four isolates designated as T9r, T11r, T12r, and T13r are cellulase and catalase producers. In general, the highest activity for enzymes production was recorded for endophytic bacterial isolate Bacillus velezensis T13r with clear zones of 19.0±1.53, 35.0±0.6, 35.7±1.5, and 24.0±0.7 mm for amylase, cellulase, protease, and chitinase, respectively. The bacterial isolate Citrobacter farmeri T10r showed the lowest protease and chitinase enzymes activity with a clear zone of 19.7±1.2 and 17.5±1.7 mm, respectively. In a similar study, out of 52 endophytic bacterial isolates, 45 isolates (86.5%), 44 isolates (84.6%), and 47 isolates (90.3%) were positive for cellulase, amylase, and protease, respectively [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 (plant-parasitic 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 Fe³⁺ 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 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 metal-sensitive 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].

Figure 3

Indole-3-acetic acid (IAA) production by the most endophytic bacterial strains isolated from T. vulgaris. A is denotes the qualitative IAA assay at different tryptophan concentrations (0, 1, 2, and 5 mg ml⁻¹), B is denoted the quantitative IAA assay in broth media amended with 5 mg ml⁻¹ tryptophan at various time courses (2 to 14 days). Values with different letters are significantly different (p ≤ 0.05), values are means ± SE (n = 3).

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 time-dependent, 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].

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. The obtained results revealed that the thymus plants inoculated with the mixture of endophytic bacterial strain B. licheniformins T11r and B. velezensis T13r mostly gave the highest oil volume in comparison to that individually inoculated in the presence and absence of chemical fertilizer ratios, which gave slightly lower oil content. Data of oil extraction showed that the treatment of T vulgaris with bacterial endophytes have plant growth-promoting properties are stimulate and improvement the oil contents as compared with those untreated. Data showed that the amount of oil/100 g dry weight (mixture of first and second harvest) of T. vulgaris treated with T11r, T13r and consortium (T11r+T13r) were (2.27, 2.12, 1.28, 0.51 ml), (2.31, 2.16, 1.29, 0.52 ml), and (2.39, 2.18, 1.31, 0.54 ml) in presence of 100, 75, 50, and 0% mineral fertilization respectively (Table 6). Whereas the amount of oil in the untreated plant was 2.13, 1.57, 1.13, 0.34 ml in presence of various concentrations of mineral fertilization (100, 75, 50, 0% respectively) (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].

Figure 4

Total Ion Chromatograms (TIC) of GC-MS analyses of oils extracted from T. vulgaris plants treated or not with bacterial endophytes (T11r, T13r, and their consortium) under different mineral fertilization doses. The description of symbols A1 to A16 was recorded in the material and method section.

Figure 5

Percentages of oil fractions based on GC-MS analysis in the presence and absence of mineral fertilization (0, 50, 75, and 100%) and bacterial inoculation. C is a control (presence of mineral fertilization and absence of endophytic bacteria; T11r is B. licheniformins; T13r is B. velezensis; BC is an endophytic bacterial consortium (T11r+T13r). The description of each peak number is listed in Table 7.

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 concentrations of mineral fertilization (0. 50, 75, and 100%), whereas the second group included all other bacterial inoculated plants under mineral fertilization range of 0–100%. However, cluster analysis discriminated against plants inoculated with B. licheniformins T11r than other groups (Fig. 6 and S3).

Figure 6

Two-way cluster analysis to group the different treatments into a number of clusters based on the observed values of the compounds and their percentages existing in the extracted oil from T. vulgaris plant treated or not with bacterial endophytes (T11r, T13r, and their consortium) under different mineral fertilization doses. The description of each peak number is listed in Table 7.

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 complex network of interactions between the plant and bacterial endophytes would help to enhance productivity and sustainability.