**3. Results**

Thirteen endophytic bacterial strains were isolated from the leaves of the two medicinal plants (Table 2). Nine strains were isolated from *F. mollis* plants and identified as *Bacillus* spp. (eight strains), and *Paenibacillus* sp. (one strain), while four bacterial strains were isolated from *A. fragrantissima* plants and identified as *Paenibacillus* sp. (one strain) and *Brevibacillus* sp. (three strains). The 16S rRNA gene sequences of strains Fm.2 to Fm.9 showed 96–99% sequence similarity with the sequences of *Bacillus amyloliquefaciens*, *Bacillus thuringiensis*, and *Bacillus cereus* (Figure 2 and Table 2). The 16S rRNA gene sequences of strains Fm.1 and Af.12 showed 99% of sequence similarity with *Paenibacillus barengoltzii*. Isolates Af.13 to Af.15 showed between 93 and 99% of 16S rRNA sequence similarity with *Brevibacillus agri*.

All bacterial endophytes isolated from *F. mollis* were positive for amylase, pectinase, carboxymethyl cellulase, cellulose, xylanase, and gelatinase, while those isolated from *A. fragrantissima* showed activities for only one to four enzymes (Table 3). The highest activities of cellulase and carboxymethyl cellulase were observed with *Bacillus* sp. Fm.5 (22.0 ± 1.1 and 21.3 ± 1.2 mm, respectively), while *Bacillus* sp. Fm.2 showed the highest activities of pectinase, xylanase, and gelatinase enzymes with clear zone 17.6 ± 0.6, 19.6 ± 0.3, and 22.0 ± 0.5 mm respectively. The highest gelatinase activity (22.3 ± 1.4 mm) was measured for *Bacillus* sp. Fm.3.

The antimicrobial activity of the bacterial endophytes against selected pathogenic bacterial and yeast strains are given in Table 4. The crude extract of *Brevibacillus* sp. Af.13 suppressed the growth of five tested pathogenic microorganisms, while *Bacillus* sp. Fm.8 inhibited the growth of *P. aeruginosa*, *S. typhi*, and *E. coli*. Endophytic strains *Bacillus* sp. Fm.2 and *Brevibacillus* sp. Af.13 were the only endophytes whose crude extracts showed an inhibitory effect against the pathogenic yeast *C. albicans* ATCC 10231 with clear zone 15 and 18 mm respectively. While the filtrates extracted from all strains showed some inhibition of *P. aeruginosa* ATCC 9027, the highest growth inhibition was noted from strains Fm.6, Fm.7, Fm.8, Fm.9, and Af.14 with inhibition zones ranging between 15 to 30 mm.


**Table 2.** The 16S rRNA sequence identification of endophytic bacterial strains from two different medicinal plants.

**Figure 2.** Phylogenetic analysis of 16S rRNA sequences of bacterial strains with reference sequences from NCBI. Fm.1–Fm.9 refers to 16S rRNA sequences of bacteria isolated from *Fagonia mollis* plants, whereas Af.13–Af.15 are the sequences from isolates from *Achillea fragrantissima*. Identity of the bacterial isolates is available in Table 2. The analysis was performed inMEGA 6 using the neighbor-joining method.



<sup>1</sup> C: controls without bacterial inoculation. Identity of the bacterial isolates is available in Table 2. <sup>2</sup> Different letters between lines denote that mean values are significantly different (*p* ≤ 0.05) by Tukey's test, means ± Standard Error (SE) (*n* = 3). <sup>3</sup> Carboxymethyl cellulase.


**Table 4.** Antimicrobial activities of bacterial endophytes.

<sup>1</sup> C: controls without bacterial inoculation. Identity of the bacterial isolates is available in Table 2.

All endophytes identified as *Brevibacillus* sp. produced the highest amount of ammonia compared to *Bacillus* spp. strains (Table 5). Moreover, nine endophytes (Fm.2 to Fm.9 and Af.12) displayed significant ability to solubilize inorganic phosphate with clear zone on the Pikovskaya medium ranging from 7.6 ± 0.3 to 9.6 ± 0.3 mm. Results showed that all the isolated strains were IAA producers, with or without tryptophan (Figure 3). However, increasing tryptophan concentration from 1 to 5 mg·mL−<sup>1</sup> resulted in increased bacterial ability to produce IAA from 10 to 60 μg·mL<sup>−</sup>1. Strains of *Brevibacillus* spp. Af.14, Af.13, *Bacillus* sp. Fm.6, *Bacillus* sp. Fm.4, and *Bacillus* sp. Fm.3 produced the highest amount of IAA, and were selected for further analysis to measure the production of IAA at 2 day intervals in a time course over 14 days. The results indicated that the maximum IAA production with tryptophan was 5 mg mL−<sup>1</sup> after 10 days. The results revealed that *Brevibacillus* sp. Af.14 produced the highest amount of IAA 59.7 μg·mL−<sup>1</sup> (*p* ≤ 0.05; Figure 4).



<sup>1</sup> C: controls without bacterial inoculation. Identity of the bacterial isolates is available in Table 2. <sup>2</sup> -, +, and ++ denote no, low, and high ammonia production, respectively. <sup>3</sup> Different letters between columns denote that mean values are significantly different (*p* ≤ 0.05) by Tukey's test, means ± SE (*n* = 3).

**Figure 3.** Quantitative production of IAA by endophytic bacterial strains with and without tryptophan. C, controls without bacterial inoculation. Identity of the bacterial isolates are available in Table 2. Data are statistically different at *p* ≤ 0.05 by Tukey's test, (*n* = 3); error bars are means ± SE. Bars with the same letter for each endophytic isolate did not differ significantly, different letters on bars denote that mean values are significantly different at significant level of (P ≤ 0.05), error bars are means ± SE.

In the greenhouse experiment, all maize plants inoculated with bacterial endophytes yielded significantly higher dry shoot weights (F6,28 = 11.09 and 10.33 respectively; *p* ≤ 0.001) compared to the uninoculated control plants (Table 6). Plants inoculated with bacterial endophytes produced dry root weight higher than those recorded in control plants, but the differences were not significant (F6,28 = 1.51; *p* = 0.21).

**Figure 4.** IAA production by the most potent bacterial strains in the presence of 5 mg mL−<sup>1</sup> tryptophan and over 14 days. C, controls without any bacterial inoculation. Identity of the bacterial isolates is available in Table 2. At each time point, bars with the same letter did not differ significantly at a significant level of (*p* ≤ 0.05) by Tukey's test, (*n* = 3).

**Table 6.** Effect of bacterial inoculations on the growth properties of maize plants.


<sup>1</sup> C: controls without bacterial inoculation. Identity of the bacterial isolates is available in Table 2. Mix, bacterial consortium consists of Fm.3, Fm.4, Fm.6, Af.13, and Af.14. <sup>2</sup> Different letters between columns denote that mean values are significantly different (*p* ≤ 0.05) by Tukey's test, means ± SE (*n* = 5).

Inoculation of maize plants with *Bacillus* spp. Fm.3, Fm.4, and Fm.6 significantly (F6,14 = 49.07; *p* ≤ 0.001) increased P shoot contents (1.05 ± 0.07, 0.72 ± 0.03, and 0.76 ± 0.02 mg respectively) as compared to the un-inoculated control plants (0.42 ± 0.01 mg), while *Brevibacillus* spp. Af.13, Af.14, and the bacterial consortium formed by a mixture of the five isolates did not affect shoot P content compared to control plants (Table 6). Analysis showed that bacterial inoculation significantly increased N shoot contents compared to the control plants (F6,14 = 35.76, *p* ≤ 0.001; Table 6). Plants inoculated with *Bacillus* spp. Fm.3, and Fm.6, *Brevibacillus* spp. Af.13, and Af.14 had significantly higher (*p* ≤ 0.05) N contents (range of 3.9 ± 0.19 to 7.2 ± 0.43 mg) than those treated with other strains or uninoculated. The bacterial strains *Bacillus* spp. Fm.3 and Fm.6 significantly (F6,14 = 4.15; *p* = 0.013) increased K shoot contents (11.35 ± 0.92 and 11.11 ± 0.31 mg) in comparison with uninoculated control plants (8.70 ± 0.03 mg).

#### **4. Discussion**

In this study, 13 putative bacterial endophytic strains were isolated from two medicinal plants growing under the adverse conditions of the Sinai desert. Nine bacterial endophytes were isolated from *F. mollis* and identified as different species of *Bacillus*, and *Paenibacillus*, and four bacterial endophytes were isolated from *A. fragrantissima* and identified as *Paenibacillus* spp. and *Brevibacillus* spp. (Table 2). The plant growth promoting (PGP) activities of these bacterial strains were characterized, including extracellular enzyme production, antimicrobial action, IAA and ammonia production, and P-solubilization. In the same regard, Eida et al. [45] reported isolation of endosphere and rhizosphere bacterial groups associated with four native Saudi desert plants and proved their plant growth promotion potential including phosphate solubilization and IAA production. Based on PGP characteristics, five endophytic bacterial strains were selected to evaluate their effects on plant growth and development. Results showed that the selected endophytes have key PGP properties, and significantly increased dry weight of tissues, and P concentrations in shoots of maize plants compared with uninoculated controls. Corresponding with our results, Marag and Suman [42] isolated six bacterial endophytes including *Bacillus cereus* from two cultivars of maize, and the pot experiment indicates the efficacy of the isolates in improving biomass parameters of inoculated maize plants, in addition to compensating for approximately 25% of the NPK fertilizer input.

The bacterial endophytes exhibited different enzymatic activities involving cellulase, pectinase, xylanase, amylase, and gelatinase production [46,47]. Cellulolytic and pectinolytic activities are known to enable microorganisms to penetrate plant tissues and establish a symbiotic relationship with their host plants. The *Bacillus* spp. strain isolated in this study showed high hydrolytic activity for cellulose and pectin, as well as proteolytic activity. Similarly, different endophytic strains of *Bacillus* were shown to be strong producers of cellulase and pectinase [48]. The extracellular hydrolytic enzymes produced by endophytes contribute indirectly to plant growth promotion and protection against pathogens [49,50]. The endophytes can be described as bioproducers for amylases and xylanases based on their amylolytic and xylanolytic activities. Similarly, bacterial endophytes isolated from mangrove plants had activities associated with amylases [50]. The diverse enzymatic activities of the isolated endophytes showed their capability to catalyze different biochemical reactions and their potential for agricultural and industrial applications. Likewise, Castro et al (2014) isolated endophytic *Bacillus* from two Brazilian mangrove species, the isolates displayed extracellular amylase, esterase, lipase, protease, and endoglucanase activities and thus can be used in industrial applications [50]. Moreover, theses enzymes could enable endophytes to penetrate plant tissues and build a symbiotic relationship with their host plant, besides protecting the host from pathogens by hydrolysis of the pathogen cell wall [10].

Antimicrobial activities of the isolated endophytes were evaluated based on the suppression of microbial growth caused by the crude extracts. The estimation of antimicrobial activity of crude extracts is the initial step required for the discovery of new antimicrobial compounds. Selection of bacterial isolates as inoculants based on their PGP traits, and on their inhibitory effect against different pathogens, has received attention and has been suggested as an approach to enhance plant growth and protect plants against diseases [51]. In the current study, the isolated endophytes showed a significant antagonistic effect against different pathogenic microorganisms. Endophytic bacteria can indirectly assist plant growth through the production of substances, which inhibit plant pathogens [52,53]. Endophytes isolated from other medicinal plants have also produced novel bioactive compounds [49,54]. Hassan [10] isolated six bacterial endophytes including *Bacillus cereus* and *Bacillus subtilis* from the native desert medicinal plant *Teucrium polium* L., the isolates manifested variable broad-spectrum activity against *Pseudomonas aeruginosa*, *Salmonella typhimurium*, *Escherichia coli*, *Staphylococcus aureus*, *Bacillus subtilis*, and *Candida albicans*. Accordingly, suggesting their application as biocontrol agents [10].

Sun et al. [55] showed that 10 endophytic bacterial strains of *Bacillus* and *Streptomyces* isolated from *Polygonum cuspidatum* exhibited antagonistic effects against different plant pathogens. *Bacillus licheniformis* and *Bacillus pumilus* endophytic isolates from *Platycodon grandiflorum* roots also exhibited a significant antifungal action against *Phytophthora capsici*, *Fusarium oxysporum*, *Rhizoctonia solanic*, and *Pythium ultimum*. The endophytic bacterial strain *Paenibacillus* sp. IIRAC-30 was

isolated from cassava and suppressed the growth of *Rhizoctonia solani* [56]. *Bacillus amyloliquefaciens* was isolated from the Chinese medicinal plant *Scutellaria baicalensis* Georgi. A crude extract of this strain exhibited antagonistic effects against some plant pathogens, food-borne pathogenic and spoilage microorganisms [53].

The PGP properties of bacteria have been investigated to select bacteria with high potential to be used as biofertilizers. These tests are critical in light of the fact that they identify bacteria with higher benefits for plants before testing them in field traits [57]. Ammonia and IAA production, as well P-solubilization, are among various mechanisms exhibited by bacteria that enhance plant growth [58]. Here, most endophytic bacterial isolates were able to produce different amounts of ammonia. It is often found that ammonia-producing bacteria can supply ammonia as a nitrogen source for plant growth [59]. Bacterial endophytes can enhance plant growth through the production of ammonia through the hydrolysis of urea into ammonia and carbon dioxide [60]. With regard to P-solubilization, most of the isolated endophytes showed variable capacity to solubilize phosphate. Rodrigues et al. [61] found that about 47% of bacterial endophytes isolated from sugarcane have low P-solubilizing indices. At soil with low phosphate supply, inoculation of P-solubilizing endophytic bacteria leading to increase of pant growth performance.

Indole-3-acetic acid (IAA) is a phytohormone that can be produced by plants and various microorganisms. This hormone not only enhances plant growth but also contributes in the interaction between plants and microorganisms [62]. In this study, all endophytic bacterial strains had the ability to produce IAA in the absence and presence of tryptophan, the precursor for IAA production. Although most microorganisms utilize tryptophan in IAA synthesis [63,64], the advantage of bacterial endophytes is that they can produce IAA without tryptophan supplementation. Rodrigues et al. [61] showed that 57% of bacterial endophytes secreted high IAA concentration of 21.05–139.21 μg mL−<sup>1</sup> in 72 h in the presence of 5 mM tryptophan. Endophytic bacterial strains were shown to produce higher IAA concentrations than rhizospheric strains, suggesting a closer link, and potential symbiosis, between endophytes and their hosts [12]. Thus, in the current study, a higher capacity to produce IAA was used to select five bacterial strains to determine their effect on maize growth performance. Bokhari et al. [65] isolated *Bacillus circulans* PK3-138 from plants grown in Pakistan desert, reported the potency of this isolate for IAA production. Similarly, four bacterial endophytes (*Sphingomonas* sp., *Bacillus* sp., *Pantoea* sp., and *Enterobacterc* sp.) isolated from the roots of elephant grass showed valuable PGP traits including IAA production at a range of 10.50–759.19 mg/L, and ammonia production capacity. So, these inoculants could be used for increasing crop yield in a sustainable mode [58].

We found that inoculated plants produced more biomass than uninoculated plants. Plant–microbe interactions are well known to influence nutrient transfer between microorganisms and plants [66]. Therefore, it is possible that plant biomass production varied with different microbial taxa assemblages in the roots due to their various abilities to supply nutrients to their host. The results showed that the shoot P concentration was significantly increased in plants inoculated with Fm.3, Fm.4, and Fm.6 compared to the uninoculated plants. P-solubilizing bacteria help plants to access insoluble forms of phosphate, such as apatite, through excretion of protons and organic acids, mainly gluconic acid, rendering phosphate available to plants for uptake [11,67]. These bacteria can also produce enzymes that mineralize organic phosphorus, which also render it available for plants [67]. The capacity of microorganisms to absorb immobile nutrients such as P from soils and transfer it to their host plants is one of the main effects of microbial symbiosis; however, microbial capacity for nutrient transfer varies with different microorganisms [68]. Basically, plant roots can be colonized simultaneously by multiple microorganisms, which can positively or negatively benefit the host plant [69,70].

Importantly, not only did the isolated endophytic *Bacillus* and *Brevibacillus* species display the highest level of IAA and ammonia production, but they also had various plant growth-promoting traits. Bacteria that were isolated and characterized in the present study are potential candidates for plant bioinoculation in agricultural practices, in particular those that inhibited pathogens and harbored the highest levels of IAA production and of nutrient uptake.

#### **5. Conclusions**

Very few isolates were obtained herein to claim that, bacterial endophytes inhabiting the two studied medicinal plants, *F mollis* and *A. fragrantissima*, mainly belong to *Bacillus* and *Brevibacillus* spp. Bacterial endophytes characterization including extracellular enzymatic activity, antimicrobial actions, P-solubilization activity, ammonia, and IAA production were performed in terms of their plant growth-promoting abilities in-vitro and in plants. Five bacterial strains identified as *Brevibacillus agri* Af.13, and Af.14, *Bacillus* sp. Fm3, *Bacillus* sp. Fm.4, and *Bacillus* sp. Fm.6 were selected and inoculated into maize plant to increase their growth performance under normal conditions. These endophytic bacterial isolates significantly promote plant growth and increase P and N shoot contents of maize plant. However, in order to demonstrate the beneficial role of these bacterial endophytes in plant growth promotion of their host plants, particularly under real field conditions, further investigation of their mechanisms of colonization and competition against other soil microbial communities will be required.

**Author Contributions:** Conceptualization, A.F., A.M.E., E.E.-D.E., S.E.-D.H., K.A.A.A., Y.M.H., K.A.A. and M.D.F.A.; methodology, A.F., A.M.E., E.E.-D.E., S.E.-D.H., K.A.A.A., Y.M.H., K.A.A. and M.D.F.A.; software, A.F., A.M.E., F.A.-O., M.H., M.S.-A., E.E.-D.E., S.E.-D.H., K.A.A.A., Y.M.H., K.A.A. and M.D.F.A.; formal analysis, A.F., A.M.E., E.E.-D.E., S.E.-D.H., F.A.-O., M.H., M.S.-A., K.A.A.A., Y.M.H., K.A.A., A.M.E. and N.K.; investigation, A.F., A.M.E., E.E.-D.E., S.E.-D.H., F.A.-O., M.H., M.S.-A., K.A.A.A., and N.K.; resources, A.F., A.M.E., E.E.-D.E., S.E.-D.H., K.A.A.A., Y.M.H.; data curation, A.F., A.M.E., E.E.-D.E., S.E.-D.H., F.A.-O., M.H., M.S.-A., K.A.A.A., Y.M.H. and K.A.A.; writing—original draft preparation, A.F., A.M.E., E.E.-D.E., F.A.-O., M.H., M.S.-A., S.E.-D.H. and K.A.A.A.; writing—review and editing, A.F., A.M.E., E.E.-D.E., S.E.-D.H., K.A.A.A., Y.M.H., K.A.A., A.M.E., M.A. and N.K.; visualization, A.F., A.M.E., E.E.-D.E., S.E.-D.H., K.A.A.A., Y.M.H. and K.A.A.; funding acquisition, M.D.F.A., K.A.A., Y.M. and K.A.A.A. All authors have equal contribution. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Deanship of Scientific Research at Princess Nourah bint Abdulrahman University through the Fast-track Research Funding Program.

**Acknowledgments:** The authors extend their appreciation to the Deanship of Scientific Research at Princess Nourah bint Abdulrahman University for funding this research through the Fast-track Research Funding Program.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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