Isolation of endophytic bacteria
Twelve leaf samples were gathered from 3 date palm cultivars viz., Khalas, Nighal and Khinaizi (four samples from each location) at 3 different locations (GPS coordinates 23.232825,58.078146; 23.233959,58.082256; 23.233851,58.082413) in the village of Al-Fankh, Samael, Oman. Samples were collected in sterile paper bags and processed on the same day of collection. Leaves were ruptured into pieces and sterilized with 5% sodium hypochlorite for 3 min, followed by washing in distilled water for 3 times and dried by filter paper. The chopped leaves were then crushed in a sterile mortar and the sap was streaked on nutrient agar (NA) plate. The sterile water obtained from the last washing step was streaked on NA plate as a control. Followed by the incubation of the plates at 27 °C for 3 days. The selection of bacterial colonies was based on distinct morphological features (colour, size, shape) and pure cultures were obtained by streak-plate technique (Al-Hussini et al. 2019).
Fungal pathogens
The isolates of different pathogenic fungi viz., Fusarium solani 2114, Curvularia subpendrofi 2315, Tilletiopsis minor 2110, T. minor 2111, Alternaria sp. 2372, Nigrospora sp. 2394 and Thieloviopsis sp. 2107, used in this work, came from the previous collections of Al-Nadabi et al. (2020). These fungal pathogens were isolated from date palm leaves infected with leaf spot disease collected from different governorates of Oman. All of them were cultured on PDA medium at 25±2 °C.
In vitro testing for antagonistic activity
Twenty-four bacterial strains (13 strains from Nighal, 6 from Khinaizi and 5 from Khalas) were investigated in vitro for their antagonistic vitality against 7 foliar fungal pathogens of date palm by dual culture technique. A bacterial colony was streaked at one end of a 9-cm Petri dish containing NA medium. A 7-mm mycelial disc of a 7-day-old culture of the test fungal pathogens was placed on the other end and incubated at 27 °C until mycelial growth of the test fungus covers the entire Petri plate in the control. In the control plate, only a fungal disc was placed. At the end, the inhibition zone was measured by counting the distance between the 2 sides of the tested Petri dish (Al-Hussini et al. 2019).
Molecular characterizations
DNA extraction
The DNA of the bacterial strains was extracted using a foodproof StarPrep Two kit (BIOTECON Diagnostics, Germany). The quality and concentrations of DNA were measured by the NanoDrop 1000 Spectrophotometer (Thermo Scientific, USA) in accordance with the manufacturer’s direction.
PCR amplification and sequencing
The identification of endophytic bacterial isolates was achieved by PCR amplification of 16S rRNA genes with universal primers viz., 27F (5′AGAGTTTGATCMTGGCTCAG-3′), 1429R (5′-TACGGYTACCTTACGACTT-3′) and 534R (5′-ATTACCGCGGCTGCTGG-3′) (Al-Hussini et al. 2019). PCR was carried out with 25 μl of PCR reaction mixture containing PuRe Ready-to-go PCR beads (GE Healthcare), 22 μl of sterile distilled water, 1 μl of each primer and 1 μl of DNA. PCR conditions included of initial DNA denaturation at 95 °C for 2 min, followed by 35 cycles of 30 s at 95 °C, 30s at 54 °C, 1 min at 72 °C and a final extension at 72 °C for 10 min. Finally, PCR products were sent to Korea for purification and sequencing at Macrogen Inc.
Phylogenetic analysis
The software MEGA v.7.0.26 was used for generating the multiple sequence alignments. Maximum likelihood (ML) was analysed through RAxMLGUI v. 1.3 (Silvestro and Michalak 2012). The search of optimal ML tree was performed with 1000 separate runs from a random starting tree for each run using the default algorithm of the program. Based on suboptimal trees from each run, the final trees were selected by comparing likelihood scores under the GTR+GAMMA substitution model evolution by MrModeltest 2.2. The software MEGA v.7.0.26 was used for printing the generated tree, while Adobe Illustrator CS v.6 was employed for making the layout.
Morphological changes in the fungal pathogens due to antagonistic effects of endophytic bacterial strains
Scanning electron microscopy (SEM) was used to study the morphological changes of susceptible fungal species (F. solani and Alternaria sp.) due to antagonistic activity of bacterial endophytes. A small piece of fungal mycelia taken from the inhibition zone in the dual culture plate was transferred into a glass vial containing fixative buffer (2.0 g paraformaldehyde, 8.0 ml of 25% glutaraldehyde, 1 N NaOH, 1 N HCl, 10 ml of 0.1 ml sodium cacodylate, 2.0 g of sucrose, 1.0 ml of 1% CaCl2, 1.01% MgCl2 and 100 ml of distilled water) and incubated at 36 °C. On the next day, buffer was removed and sufficient quantity of the osmium tetroxide was added and mixed in a rotor for one hour. The previous step was repeated by using distilled water twice and kept in the rotor for 10 min, followed by washing with absolute ethanol four times for 10 min, 10 min, 20 min and 10 min. Then the samples were kept overnight and dried. Finally, samples were coated with platinum and observed using a JEOL scanning electron microscope model JSM-7800F (JEOL, MA, USA).
Evaluation of cell-free culture filtrates of endophytic bacteria for antifungal activity
The evaluation of antifungal activity of cell-free culture supernatants from bacterial strains B1, B7, B8 and B9 against F. solani and Alternaria sp. was achieved by poisoned food technique as described by Kumar et al. (2008). Antagonistic bacterial strains were cultured in nutrient broth for 48 h at 28 °C on an incubator shaker (150 rpm). Followed by centrifugation of bacterial culture for 10 min at 12,000 rpm at temperature of 4 °C and the supernatant was collected. After that, the bacterial culture supernatant was filtered by a 0.2 μm sterilized syringe filter. PDA plates amended with different concentrations (2.5, 5.0, 7.5 and 10%) of cell-free culture supernatants were prepared. Then, 6-mm diameter of a mycelial disc cut out from 7-day-old culture and placed on the centre of agar medium. Next, incubated at 25 °C and the mycelial growth was measured after 7 days. The percentage of inhibition of mycelial growth was calculated, using the following formula (Pandey et al. 1982)
$$ \mathrm{Percentage}\ \mathrm{of}\ \mathrm{mycelial}\ \mathrm{inhibition}=\frac{Dc- dt}{Dc}\times 100 $$
where Dc = average diameter of fungal colony in control and dt = average diameter of fungal colony in treatment.
Testing antifungal metabolites production by bacterial endophytes
The antifungal metabolites production by endophytic bacterial strains was evaluated using electrolyte leakage bioassay (Manhas and Kaur 2016). The supernatants of cell-free culture of the endophytic bacterial strains were prepared as described above. Then, 0.5 g of fungal mycelium (F. solani and Alternaria sp.) was added to 10 ml of cell-free bacterial culture supernatant in glass vials. Finally, the samples were placed at room temperature (25±2 °C) and then the conductivity of the mixture was assayed at 0 and 2 h by using the conductivity meter (Prapagdee et al. 2008).
Statistical analysis
Data were analysed using PSPPIRE (PSPP+GUI). Treatment means ± SD were compared using one-way ANOVA and Tukey test.