*2.2. Isolation of Bacterial Endophytes*

On each plant, the first five leaves from a shoot tip were excised and washed under running tap water. Sterilization of leaf surfaces was done by soaking the tissues in a series of baths: sterile distilled water for 1 min, 70% ethanol for 1 min, 2.5% sodium hypochlorite for 4 min, 70% ethanol for 30 s, and a final series of rinsing thrice in sterile distilled water in three different containers. A 0.1 mL aliquot of the final rinse water was plated onto nutrient agar plates to confirm the success of surface sterilization.

The sterilized plant leaves were then cut into 5 mm segments, and twenty leaf segments per individual plant were placed in four petri dishes (9 cm; five segments/plate) containing luria broth (LB) media (tryptone 10 g·L<sup>−</sup>1; yeast extract 5 g·L<sup>−</sup>1; NaCl 10 g·L<sup>−</sup>1; agar 15 g·L<sup>−</sup>1; and 1 L dis. H2O, adjusted to pH 7) supplemented with nystatin (25 μg·mL<sup>−</sup>1) to suppress fungal growth, and incubated in the dark at 35 ± 2 ◦C. Another twenty segments of sterilized leaves per individual plant were together crushed in 10 mL sterile saline solution using a sterile digital homogenizer (PRO25D, Pro Scentific, 120 V, Willenbrock, Oxford, CT, USA), and 1 mL of the suspension was serially diluted until 10−<sup>3</sup> from which a 0.1 mL aliquot was spread onto each of the three Petri plates containing LB medium and incubated in the dark at 35 ± 2 ◦C [31]. The cultures were regularly observed for bacterial growth, for a period of 96 hours. Bacteria growing from the previous steps were streaked on fresh LB plates to obtain single colonies, which picked up and inoculated on LB slants and stored at 4 ◦C until further study.

#### *2.3. Molecular Identification of Bacterial Endophytes*

Bacterial identification was based on 16S rRNA gene sequence analysis. Genomic DNA of each isolate was extracted following the method of Miller et al. [32], with some modifications. Briefly, individual colonies from an agar plate were picked up either using a sterile toothpick or an inoculating loop and resuspended in 50 μL of sterile deionized water. The cell suspension was placed in a water bath at 97 ◦C and heated for 10 min, the cell lysate was centrifuged (15,000× *g*, 10 min), and the supernatant containing the DNA was recovered. DNA concentration was determined by measuring its absorbance at UV spectrum of 260 nm using a spectrophotometer (JENWAY 6350, 230 V/50 Hz, Staffordshire, UK). A partial 16S rDNA fragment was PCR amplified using the bacterial universal primers 27f (5 -AGAGTTTGATCCTGGCTCAG-3 ) and 1492r (5 -GGTTACCTTGTTACGACTT-3 ) [33]. The PCR reaction contained: 1 × PCR buffer, 0.5 mM MgCl2, 2.5 U Taq DNA polymerase (QIAGEN), 0.25 mM dNTP, 0.5 μM of each primer, and approximately 5 ng of bacterial genomic DNA. The PCR cycling conditions were 94 ◦C for 3 min, followed by 30 cycles of 94 ◦C for 0.5 min, 55 ◦C for 0.5 min, and 72 ◦C for 1 min, followed by a final extension performed at 72 ◦C for 10 min. The PCR products were forward and reverse sequenced using the Applied Biosystems 3730xl DNA Analyzer technology at the Genome Quebec Innovation Center (Montreal, QC, Canada). The sequences generated in this study were deposited in GenBank under accession numbers KY555785 to KY555797. The 16S rRNA sequences were then compared against the GenBank database using the NCBI BLAST nucleotide search. A multiple sequence alignment was constructed on approximately 1200 bp of 16S rRNA gene fragments using the ClustalX 1.8 software package (http://www.clustal.org/clustal2) and a phylogenetic tree was constructed using the neighbor-joining method in the MEGA v6.1 software (www.megasoftware.net), with confidence tested by bootstrap analysis (1000 repeats).

#### *2.4. Screening the Extracellular Enzymatic Activities of Bacterial Endophytes*

The production and activity of extracellular enzymes (amylase, cellulase, protease, pectinase, and xylanase) of isolated bacterial endophytes were assessed by growing the isolates in a mineral salt (MS) media (NaNO3 5 g·L<sup>−</sup>1; KH2PO4 1 g·L<sup>−</sup>1; K2HPO4 2 g·L<sup>−</sup>1; MgSO4.7H2O 0.5 g·L<sup>−</sup>1; KCl 0.1 g·L<sup>−</sup>1; CaCl2 0,01 g·L<sup>−</sup>1; FeSO4.7H2O 0.02 g·L<sup>−</sup>1; agar 15 g·L<sup>−</sup>1; and 1 L dis. H2O) complemented with various additives, depending on the enzyme being tested, as detailed below. Control treatments consisted of the same media without bacterial inoculation. After incubation for 24–48 h depending on the growth rates of the bacterial endophytes at 35 ± 2 ◦C, specific reagents were added (see paragraph below), and the size of the clear zone surrounding the bacterial colony was measured, indicating extracellular enzymatic activities. All assays were performed in triplicates.

Amylolytic and cellulase activity were assessed by growing the endophytic bacterial isolates on MS agar medium supplemented with 1% soluble starch and 1% cellulose or carboxy-methylcellulose (CMC) respectively. After incubation, the plates were flooded with 1% iodine. MS agar medium containing 1% gelatine was used to determine the bacterial proteolytic activity. After incubation, the degradation of gelatine was highlighted using acidic mercuric chloride as an indicator. Pectinolytic activity was determined by growing bacteria in MS medium containing 1% pectin. After the incubation period, the plates were flooded with 1% aqueous solution of hexadecyl trimethyl ammonium bromide. MS agar medium supplemented with 1% xylan from corncobs was used to measure bacterial xylanolytic activity. After the incubation period, the xylanase activity was assessed after flooding with absolute ethyl alcohol to indicate biodegradation [34].

#### *2.5. Antimicrobial Activity of Bacterial Endophytes*

To test the antimicrobial activity of the bacterial endophytes, the isolated strains were cultured in nutrient broth medium for 6 days at 35 ± 2 ◦C on a shaker (LABOAO, LH-2102C, Zhengzhou, China) at 180 rpm. Crude fermentation broth was blended thoroughly and centrifuged at 4000 rpm for 5 min. Liquid supernatant was extracted twice with an equal volume of ethyl acetate. The organic solvent

extract was then evaporated under reduced pressure using a rotary evaporator (RE-801, BM-500 water bath (4 L), glassware C set, Yamato scientific, Tokyo, Japan). The crude extracts were dissolved in dimethyl sulfoxide (DMSO) and used for antimicrobial screening using a well diffusion method [35]. Nutrient broth media without bacterial inoculation were extracted and dissolved in DMSO and were used as controls.

Microbial strains used for antimicrobial assays were: *Staphylococcus aureus* ATCC 6538 and *Bacillus subtilis* ATCC 6633 (Gram-positive bacteria), *Escherichia coli* ATCC 8739, *Pseudomonas aeruginosa* ATCC 9027 and *Salmonella typhimurium* ATCC 14028 (Gram-negative bacteria), and *Candida albicans* ATCC 10231 (yeast). Test organisms were inoculated in Petri dishes containing Muller–Hinton agar medium (Sigma-Aldrich) for bacteria or Sabouraud agar medium (Sigma-Aldrich) for yeast [19,36,37]. Three wells of 1 cm diameter were cut in the tested organism colony using a sterile cork borer and filled with 40 μL of endophytic bacterial extract. Negative control wells were filled with 40 μL of control extract. The plates were kept at 4 ◦C for 4 h to allow diffusion of antimicrobial compounds, and then incubated at 35 ± 2 ◦C for bacteria and 28 ± 2 ◦C for *C. albicans* for 24 h [38,39]. The inhibition zones around the wells were measured to assess the antimicrobial activity of bacterial extracts. All antimicrobial activity assays were performed in triplicates.

#### *2.6. Screening for In Vitro Plant Growth Promoting (PGP) Traits*

#### 2.6.1. Phosphate Solubilization

The bacterial endophytic isolates were screened for P-solubilization as follows. Pikovskaya medium (glucose 10 g·L<sup>−</sup>1; Ca3(PO4) 2·5 g·L<sup>−</sup>1; (NH4)2SO4 0.5 g·L<sup>−</sup>1; NaCl 0.2 g·L<sup>−</sup>1; MgSO4·7H2O 0.1 g·L<sup>−</sup>1; KCl 0.2 g·L<sup>−</sup>1; FeSO4·7H2O 0.002 g·L<sup>−</sup>1; yeast extract 0.5 g·L<sup>−</sup>1; MnSO4·2H2O 0.002 g·L<sup>−</sup>1; agar 15 g·L<sup>−</sup>1; and 1 L dis. H2O) was prepared and bromophenol blue was added as an indicator. The medium was inoculated with endophytic isolates and incubated for 48 h. The Pikovskaya medium without bacterial growth was used as a control. The formation of clear zones around the colony, due to the utilization of tricalcium phosphate, was measured to assess the ability of endophytes to solubilize phosphate [40].

#### 2.6.2. Ammonia Production

The ability of the isolated endophytic bacterial strains to produce NH3 was assessed after growing the bacterial strains in peptone water (peptone 10 g·L<sup>−</sup>1; NaCl 5 g·L<sup>−</sup>1; and 1 L dis. H2O) for 72 h at 35 ± 2 ◦C. Peptone water without bacterial inoculation was used as a control. The addition of 1 mL of Nessler's reagent in the peptone liquid medium was used to assess the ammonia production. A color change to faint yellow indicated the minimum ammonia production while deep yellow to brownish color indicated the maximum ammonia production [41].

#### *2.7. Quantitative Screening for Indole-3-acetic acid (IAA) Production*

The ability of bacterial endophytes to produce IAA was determined in nutrient broth at 35 ± 2 ◦C for 24 h. One milliliter of each bacterial suspension was added to 20 mL of nutrient broth medium containing 0, 1, 2, or 5 mg·mL−<sup>1</sup> tryptophan, and incubated for 14 days. Controls consisted of nutrient broth media containing 0, 1, 2, or 5 mg·mL−<sup>1</sup> tryptophan but without bacterial inoculation. Five milliliters of each culture were collected from the incubating broth after 14 days and centrifuged at 6000 rpm for 30 min. One milliliter of the supernatant was mixed with 1 drop of orthophosphoric acid and 2 mL of Salkowski's reagent (300 mL concentrated sulfuric acid, 500 mL distilled water, and 15 mL 0.5 M FeCl3). Development of a pink color indicated IAA production. The optical density at 530 nm was measured using a spectrophotometer (Jenway 6305 UV spectrophotometer, 230 V/50 Hz, Staffordshire, UK), and the amount of IAA produced was estimated using a standard curve for authentic IAA [42].

Five isolates were chosen based on their ability to produce IAA for further analysis. Equal amount of each inoculum was added to nutrient broth medium containing 5 mg mL−<sup>1</sup> tryptophan and incubated for 14 days at 35 ± 2 ◦C. IAA concentration was determined at 2 days intervals up to the 14th day after inoculation. Samples were centrifuged at 6000 rpm for 30 min and IAA production was determined as mentioned above. All the IAA production assays were performed in triplicates.

### *2.8. E*ff*ect of Bacterial Isolates on Zea mays L. Growth*

#### 2.8.1. Experimental Design

A pot experiment was conducted in a completely randomized design with five replicates of each treatment. Plants were inoculated with one of five individual bacterial isolates (*Brevibacillus* spp. Af.13, and Af.14, *Bacillus* spp. Fm.3, Fm.4, and Fm.6) or with a bacterial consortium formed of an equal amount of the five bacteria isolates. A control treatment consisted of uninoculated plants.

#### 2.8.2. Culture Conditions

A loamy soil was collected from an agricultural field in the El-Menoufia governorate. Physical and chemical characteristics of the soil are shown in Table 1. The soil was air-dried, sieved with a 2 mm sieve, mixed with quartz sand at a soil: sand ratio of 3:1 and autoclaved twice for one hour at 121 ◦C. The five most potent IAA producing bacterial isolates (as listed above) were inoculated in nutrient broth and incubated at 35 ± 2 ◦C for 24 h on a shaker (LABOAO, LH-2102C, Zhingzhou, China) at 180 rpm. Seeds of maize (*Zea mays*, Cultivar Giza 9) were surface sterilized by soaking in 2.5% sodium hypochlorite for 3 minutes and then washed 5 times in sterile distilled water. Six groups of pregerminated seeds were separately incubated in 50 mL aliquots of the culture medium inoculated each with one of the five bacterial strains or with the bacterial consortium, and incubated at room temperature for 4 h on a shaker at 180 rpm. After incubation, the soaked seeds were sown in 1 L plastic pots filled with 900 g of sterilized soil-sand mixture. Each pot received three germinated seeds. Plants were grown in a greenhouse with a temperature of 25–30 ◦C and were irrigated with tap water as required without fertilization.


**Table 1.** Physical and chemical characteristics of the soil used in the greenhouse experiment.

#### *2.9. Plant Tissue Analysis*

After 30 days, plants were harvested, shoot and root systems were separated, and roots were washed carefully with tap water to remove the attached soil particles. The dry weight of shoots and roots were measured after drying for 48 h at 60 ◦C. Phosphorus, nitrogen, and potassium contents were determined according to the methods described by AOAC international [43] and Rice et al. [44].

#### *2.10. Statistical Analysis*

Data were statistically analyzed using SPSS v17 (SPSS Inc., Chicago, IL, USA). When the normality and homogeneity of variance hypotheses were satisfied, one-way analysis of variance (ANOVA) was used to compare the bacterial isolates for extracellular enzymes production, antimicrobial activity, IAA and ammonia production, P-solubilization ability, and the effect of these endophytes on maize growth performance. A posteriori multiple comparisons were done using Tukey's range tests at *p* < 0.05. All results are the means of three to five independent replicates, as specified above.
