Samples Collection and Authentication
Healthy plant samples of Citrullus colocynthis (L.) Schrad were collected from Qasr-e Shirin (34° 3110 N and 45° 35 15 E), in the western part of Iran, and transported to the laboratory in sterile bags for surface sterilization. The samples were collected in October 2020 and May 2021 to cover seasonal variation. They were identified and authenticated by an expert, Dr. Nastaran Jalilian, at the Kermanshah Agricultural and Natural Resources Research and Education Centre, Kermanshah, Iran. and the voucher specimen (10080) was deposited in the Herbarium of RANK. The plants were dissected to stems, leaves, roots, and, fruits, surface-sterilized and cultured within 48 hours of sampling.
Surface sterilization
The surface sterilization of plant samples was performed according to Kaewkla and Franco (34). Briefly, the plant tissues were rinsed with running tap water to remove all contaminations and any physical debris. After natural air drying and rinsing with distilled water three times, the samples were then dissected to roots, stems, leaves, and fruits using a sterile saw/ knife, and tweezers. The segments were then chopped with scissors into small pieces of two-five cm lengths and undergone the surface sterilization with various surface sterilizing agents to prevent non-endophytic bacteria and fungi from growing on the surfaces of the samples.
Each sample was first immersed in 0.1 % sterile Tween 20 for five minutes. After being treated with 70% ethanol for five minutes, the samples were rinsed with a freshly made 6% sodium hypochlorite (NaOCl) solution. The samples were ten times rinsed with D.W water to remove the chemical residues. The samples were then immersed in sterile 10% (w/v) sodium bicarbonate (NaHCO3) for 10 minutes to retard the growth of endophytic fungi (35), and washed three times with sterile double distilled water. Upon sterilization, the samples were air-dried in a laminar air flow.
To ensure the efficacy of the surface sterilization, 100 μl of the last double distilled washed water was cultivated onto ISP-2 media and incubated at 28±2 °C for one week. The absence of microbial growth on the culture media after plating the last washing water demonstrated the effectiveness of surface sterilization (36).
Isolation of actinobacteria
The selection of an isolation medium is critical because it directly impacts on the diversity of endophytic bacteria that may be isolated from plant tissues and, consequently, on the outcome of the experiment (37). Four different isolation media (Table 1), namely Yeast extract-casein hydrolysate agar (YECD) (38), potato dextrose agar (PDA) (39), Tap water yeast extract (TWYE) (40), and humic acid-vitamin (HV) agar (41) were selected and supplemented with nystatin at final concentration (20 µg/mL) and nalidixic acid (10 µg/mL) to prevent and repress the growth of fungi and Gram-negative bacteria (42, 43). The surface-sterilized samples were then aseptically cut into 1-2 cm and placed directly on the culture media in triplicate and the plates were incubated at 28±2 °C, and the growth of EA from tissues was daily monitored for 16 weeks. After emerging, different colonies with putative actinobacteria features were introduced and sub-cultured onto international Streptomyces Project-2 (ISP2) (44). Pure cultures were obtained after two to three successive sub-culturing rounds and transferred to fresh isolation media. Given the possibility that each colony resembles a distinct strain (45). The pure isolated strains were then maintained on ISP2 cultures and used as master plates to establish stock cultures and the isolates stored in Tryptic Soybean Broth medium (TSB) containing 30% glycerol at -20°C for further analysis.
Table 1. Composition of culture media used to isolate endophytic actinobacteria
Name of culture medium
|
Components of the Culture Medium (1 Liter)
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References
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PDA
(Potato Dextrose Agar)
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39 gm
|
(46)
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TWYE
(Tap Water Yeast Extract Agar)
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Yeast extract 0.25 g, K2HPO4 0.50 g, Agar 18.00 g
|
(47)
|
YECD
(Yeast extract-casein hydrolysate agar)
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0.3 g of yeast extract. 0.3 g of D.glucose. 2 g of K2HPO4, and 18 g of Agar
|
(48)
|
Humic acid-vitamin B (HV) agar
|
Humic acid 1 g, Na2HPO4 0.25 g, KCl 0.85 g, MgSO4.7H2O 0.025 g, FeSO4.7H2O 0.05 g, CaCO3 0.01 g, Agar18 g, Vitamin B 100x, (added after media autoclaved) 1 ml
|
(41)
|
Identification of Actinomycetes
Actinobacteria-like colonies were identified after 8–16 weeks of incubation at 28°C from the original isolation plates. Well-separated Actinobacteria-like colonies were picked and sub-cultured on ISP2 and purified using ISP2 medium. They were cultivated at 28 °C for 10 days on ISP2 agar medium. The isolates were identified based on microscopic characteristic and morphological criteria, such as colony features, colour of aerial and substrate mycelia, the colour of culture, pigmentation, and sporulation, following the general guidelines of the International Streptomyces Project.
Typically, light microscopy was used to examine the basic morphology of hyphae and spores, while scanning electron microscope (SEM) (FEI, Quanta 450, America) was used to examine the microscopic structures of hyphae and spores on the surface (49). The isolates were cultured onto ISP2, the aerial mycelium and spores were obtained on a cover slip. Cover slides were cut into one cm pieces and sterilized. The Sterile coverslips were inserted into solidified ISP2 at an angle of 45°. The inoculum was spread along with the glass-agar medium interface and incubated at 28°C for 10 days. During incubation, the organisms grew over the surface of the glass pieces. The cover slip containing organisms was then coated with a film of gold about 150-200 A° thickness and observed under SEM at an accelerating voltage of 25.000–30.000 V for spore surface ornamentation (50). The spore chain morphology and ornamentation of the potential strains were analysed.
Culture and preparation of living cell mass (BIOMASS)
Isolated strains were first cultured on ISP2 and then incubated at 28 °C for five to seven days. Microscopic monitoring of stained and unstained (wet mount) bacteria was further applied for identification. The purity of actinomycete colonies, grown on ISP2 plates were inspected directly, using wet mount or Gram staining method under a light microscope (Nikon Eclipse E100). After ensuring their purity and the absence of contamination, loopful of colonies were cultured on Tryptic Soy Agar (TSA) and/or into Tryptic Soy Broth (TSB), at 28 °C for 7–10 days. The broth culture was incubated in a shaker incubator, (HYSC, Korea) at 160 rpm. Then, it was centrifugated with a Hettich 320R, Germany (4,000 rpm, 10 min, 4 °C) to separate the mycelia. The bacteria were then used to extract DNA or study their antimicrobial activities (51).
DNA extraction
With some modifications, genomic DNA was extracted according to Coombs et al (38). Initially, 70-100 mg of the colony were placed in a two ml sterile microtube and washed twice with 500µ of Tris-EDTA (10 mM Tris; 1 mM EDTA, pH 8.0) by vortex and centrifuging (5 min, 5000 RCF). The sample was then suspended in 500 µL TE buffer containing 0.2 mg.ml-1 of lysozyme, then vortexed/ spin down (3-5 sec.). Following a 60-minute incubation at 37°C, 10µ of 1% (w/v) proteinase K and 10 µ SDS 10% were added, and the mixture was incubated in a water bath at 55°C for 60 minutes. The tubes were re-incubated at 55oC for 10 minutes after adding 100 µL of 5 M NaCl and 65 µL of CTAB/NaCl (700 mM NaCl, 275 mM CTAB). The equal volume of phehol:chlorofornm:isoamyl alcohol (25:24:1) 600µ was added to the suspension in a fume hood before being left at room temperature for 30 minutes with intermittent shaking. To pellet the cell debris, the tubes were centrifuged at 12,000 rpm for 15 minutes using a microcentrifuge Hermle Z216M, Germany, and the supernatant was carefully transferred to a new 1.5ml tube. The sample was washed with 500 µL chloroform after incubation for 15 minutes at room temperature (inverse mix every 7-8 min). Following centrifugation at 12,000 RCF for 15 minutes, the aqueous phase was transferred to a new sterile 1.5 ml Eppendorf tube. 20 µl of RNase (10 mg/ml) was added and incubated at 37C for 60 minutes. Washing with chloroform was repeated. The aqueous phase was then conveyed to a new sterile 1.5 ml Eppendorf tube after centrifugation at 12,000 RCF for 15 minutes. The supernatant was treated with 1x volume of 3M sodium acetate and 3X volume of 100 percent cold ethanol and kept in a freezer at -20℃ overnight. The DNA was then pelleted by centrifugation at 16,000 RCF for 15 minutes and supernatant was carefully discarded. The pellet was washed twice with 70% ethanol. The pellet was dried by placing the tubes in the heating block at 55◦C for about 10 minutes with the lids open, or until the pellet was dry. Finally, the pellets were resuspended in 50µL of injection water and the quality and quantity of genomic DNA extracts were checked at A260nm/280 nm and A260nm/230 nm ratios by Nanodrop 2000 spectrophotometer (Thermo Scientific, USA), and on 1% agarose gel and stored at -20 degrees Celsius. Using agarose gel electrophoresis, the quality of the DNA samples was determined.
Polymerase Chain Reaction (PCR)
To demonstrate the microbial diversity of samples, the polymerase chain reaction (PCR) is most commonly employed technique in conjunction with universal primers (52). PCR amplification of the bacterial 16S rRNA gene was performed as described by Coombs and Franco (38) using Mastercycler® nexus, Eppendorf and a set of universal primers 27F 5’-AGAGTTTGATCMTGGCTCAG -3’, 1492R 5’-TACGGGTACCTTGTTACGACTT-3’ synthesized by Metabion, Germany. The PCR products were about 1,500 bp.
Briefly, the amplifications were carried out in a total volume of 25µL reaction mixture, including 12.5µ of master mix (Sinaclon Cat.No.MM2062), 0.3 μL of each primer (10 pmol/𝜇L), 1µL of DNA template (50-100 ng), and 10.9 µL of D.W. Then insert the microtubes to the PCR device. PCR conditions included an initial denaturation at 94°C for two min, followed by 30 cycles of denaturation at 94°C for one min, annealing at 55°C for one min, and extension at 72°C for two min. The final cycle was ended by extension at 72°C for 10 min and then cooled to 4°C. To continue, the device was given a temperature of 4°C at the end of the cycles to prevent the PCR product from reacting and degrading.
Electrophoresis was performed for 45 minutes at 70 volts, and the gel was then imaged under Gel Doc device (Vilber, France) to evaluate the PCR products. The PCR products was sent to Macrogen Inc. (Seoul, Korea) for Sanger sequencing.
Sequencing and phylogenetic analysis
Bidirectional Sanger sequencing was performed by Macrogen )South Korea( using the same sets of primers used for PCR amplification. MEGA X (Molecular Evolutionary Genetics Analysis) software was used to process the nucleotide sequence of the 16S rRNA gene. The 16S rDNA gene sequences were obtained using the BLASTn tool (Basic Local Alignment Search Tool) (http://www.ncbi.nlm.nih.gov/BLAST/Blast.cgi), and then compared to existing sequences of bacteria in the NCBI (National Center for Biotechnology Information) and EzBioCloud databases to estimate similarity percentages (53). The hits of subjects’ sequences deposited in international nucleotide databases (e.g., GenBank, EMBL, DDJD, etc.) that provide the best match with the query. Multiple DNA sequence alignment of selected 16S rDNA was performed using the ClustalW algorithm in the MEGA X software (54). The neighbour-joining method was used to create phylogenetic trees.
Isolates were classified according to their taxonomy using 16S rDNA analysis. The phylogenetic tree was created by MEGA X using the maximum-likelihood method , which was based on the Kimura 2-parameter model (55). The Kimura two-parameter model was used to calculate pairwise distances for the neighbour-joining procedure (Kimura 1980). Strength and reliability (topologies of the neighbor- joining) of the resultant tree was evaluated after a 1000 bootstrap -replicate analysis (56). Escherichia coli was used as an outgroup strain for tree constructions (57). The 16S rDNA gene sequences of the isolates were then deposited in GenBank and their accession numbers acquired. GenBank accession numbers for the resultant sequences are listed in Table 3. The phylogenetic tree was constructed using the neighbor-joining algorithm/method (based on 1000 bootstrap iterations) of nucleotides sequence of 16S rDNA gene.
Preparation and Screening of isolates for Antibacterial Activity
To test the antibacterial activity of endophytic isolated strains, the isolates were extracted using ethyl acetate and methanol, respectively. Briefly, the pure isolates were grown on TSA medium and Liquid TSB (7 days, 28±2 °C). The Solid culture (full TSA plate) containing the culture and the agar medium was then cut into small pieces with a sterile scalpel, and placed into 250 ml Erlenmeyer flasks containing 50 ml of ethyl acetate, and shaken for 24h (200 RPM) at room temperature. The ethyl acetate solution was filtered and the supernatant collected. Then, 50 ml of methanol was added into the flasks, allowing the device to operate for another 24 hours under the same conditions, the extract was separated. Both extracts were concentrated under reduced pressure using rotary evaporator (70 RPM, 38°C) to yield a dry extract. Then, the extracts were stored at -20°C until used for evaluation of antimicrobial activity. The TSB cultures were incubated onto a rotary shaker (HYSC, Korea) at 28°C with 180 rpm for 72 hours (58). The colonies were then harvested by centrifugation at 4,000 G for 10 min to pellet bacterial cells, and extracted by ethyl acetate and methanol, respectively using the same procedure as used for TSA.
Antimicrobial activity of fractions using diffusion method
The endophytic isolates were primarily examined for their antimicrobial activity against the test bacterial strains including Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa using agar well diffusion methods. Initially, 1-2 loops of test bacteria (E. coli ATCC 25922, Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 27853) were inoculated into TSB media at 37 °C for 24 hours. The growth of the cultures was then adjusted to half the McFarland (OD = 0.2) by measuring the optical density (OD) using a Spekol 1500, spectrophotometer at 600 nm (OD600 nm). The antibiotic agar medium No.1 (AAM- MERCK) was seeded with the test culture (1% v/v) and dispensed into petri dish plates, and 6 mm diameter wells were prepared using a sterile cork borer at regular intervals to make 10 wells.
The antibacterial activity of the crude extract was determined using the agar well diffusion method. Each well was loaded with 50 μL of extracts, allowing the wells to dry completely. The plates were then incubated at 37°C for 24 hours. Vancomycin (20 µg/ml) and Imipenem (15 µg/ml P. aeruginosa ATCC 27853 and 1.5 µg/ml E. coli ATCC 25922) were used as positive controls for Gram-positive and Gram-negative bacteria, respectively. Negative controls were wells containing the same volume of methanol or ethyl acetate (50 mL). Each test was done in duplicates and repeated trice. The width of the zone of inhibition around each well was measured and the average recorded in millimetres (mm).