Isolation and identification of fungus
Amasya apples showing characteristic symptoms of brown rot were obtained from a tree nursery in Amasya, Turkey in August-October 2021. The causal agent of the disease was isolated from apple fruits as described by Kurt et al. (2020). The fruits were sterilized using NaOCl 1.5% for 6 min.. Rot tissue pieces were cut from the fruit samples using a knife and then divided into small parts (1.5 cm2). These parts were placed on the surface of a potato dextrose agar (PDA) (potato: 200 g, glucose: 20 g, agar: 20 g, sterile water to 1000 ml, pH 7) medium supplemented with 250 mg/l of streptomycin and then incubated at 30°C for 5 d. The pathogenic fungus A. alternata was identified using microscopic observations and molecular methods with ITS1 and ITS4 primers spanning the ITS1, 5.8-S rRNA, and ITS2 regions, 5-TCCGTAGGTGAACCTGCGG-3 and 5-TCCTCCGCTTATTGATATGC-3 (White et al. 1990).The nucleotide sequences were assembled using the CAP program available on the NCBI website (http://www.ncbi.nlm.njh.gov/blast). The nucleotide sequences were deposited in the Gen Bank database, and the access numbers were obtained.
Pathogenicity tests of fungus
For pathogenicity testing, using healthy Amasya apples, three lesions (3 mm in width and 3 mm deep) were created at the lateral sides of each fruit with a sterile perforator. 100 µl conidia suspensions of A. alternata (2×102, 2×104, 2×106, and 2×108 spores/ml) were inoculated in each lesion, and sterilized distilled water served as the control. The test was performed in five repetitions. Apples were placed in sterilized plastic boxes and then incubated at 27°C for 7 day. After the virulence of the fungus was defined, the growth of the brown rot lesions around the inoculated lesions was measured (in mm) and compared to the measurements of the control fruits (Mohamed and Saad 2009).
Isolation of endophytic bacteria
Different samples were collected from among healthy Amasya apples cultivated in the Amasya region of Turkey. The fruits were washed in running tap water and then surface-sterilized first in 70% ethanol for 5 min and then in 1% NaOCl for 20 min. Eventually, the fruits were fully washed thrice with sterilized distilled water. For sterility check, water collected from the final wash process was placed on a Nutrient Agar (NA) medium and incubated at 30°C for 24 h. The surface-sterilized apple fruits were dissected into 1-2-cm pieces, transferred to the NA medium and incubated at 30°C for 70 h for the development of colonies. Bacteria colonies were isolated based on phenotypic characteristics and purified using plate streaking techniques. Finally, each colony was stored at -20°C in 30% sterilized glycerol for testing antagonism (Sun et al. 2013).
Screening of antagonistic bacteria
The antagonistic properties of the endophytic bacteria against A. alternata were determined using in vitro dual culture confrontation assays on PDA plates following the method of Bektas and Kusek (2019) with some modifications. Briefly, 0.5-cm2 discs of fungal mycelia of A. alternata were placed at the center of each 90-mm disposable plastic Petri dish containing PDA. A single colony of each bacterium was inoculated in 10 ml NB and incubated at 27°C for 72 h by continuous shaking (120 rpm). Pellets obtained by centrifuging the bacterial cultures at 5000 rpm for 20 min were re-suspended in sterile water at a concentration of 108 CFU ml−. 10-µl endophytic bacterium suspensions were inoculated overnight around the fungal disc at a distance of approximately 25 mm from the fungus. For the control, a fungal disc was placed on PDA agar, but instead of endophytic bacteria, only 10 µl of sterile water was streaked. The experiments were conducted in triplicates. For comparison, the fungal growth inhibition effects of the endophytic bacteria against the pathogen were determined. All plates were incubated at 27°C for 10 day. After incubation, each antagonistic bacterium inhibition zone was measured, and the percent inhibition of radial/mycelial growth (PIRG) was calculated according to the following formula:
PIRG = (R1 − R2)/R1 x 100
Where R1 is the average diameter of the fungus on the control, and R2 is the average diameter of the fungus on the endophytic bacteria co-cultured plate.
Effects of antagonistic endophytic bacterium treatment on the control of black spot rot in apple fruits
The antifungal effect of the endophytic bacteria on black spot rot development on Amasya apple fruits was investigated based on the method reported by Ge et al. (2019) with minor changes. Healthy apple fruits (cv. Amasya) were harvested at commercial maturity from an orchard in Amasya and transported directly to the Suluova Vocational School’s microbiology laboratory. The surfaces of the apple fruits were disinfected with 1.0% (v/v) NaOCl for 3 min, thoroughly washed with distilled water and then dried at room temperature (25°C). Then, 100 µl of the spore suspension of A. alternata at a concentration of 2×108 ml− was injected into the uniformly sized lesions (7-mm deep, 3-mm-wide) of the fruit. The fruits were air-dried for 2 h, and the same volumes of bacterium solutions were injected into the same lesions (i) simultaneously, (ii) 24 h after A. alternata, and (iii) 24 h before A. alternata. The positive control apples were injected with A. alternata only, while the negative control apples were injected with distilled water only at the lesion sites. Each treatment included three replicates. Lastly, the fruits were incubated at 25°C in clean boxes by maintaining a humidity of 80–85%. After 10 day of incubation, the disease scores were measured. The disease severity index (DSI) was calculated according to the formula below:
(0 = no rot, 1 = rot area 0–20%, 2 = 20–40%, 3 = 40–60%, 4 = 60–80%, 5 = 80–100% with tissue rot), and the
Observations were converted to DSI values based on the method of Promwee et al.(2017) as follows: DSI(%) =[∑(SXA)/MxT]/100
S: Scale, A: Amount of fruit, M: Maximum level and T: Total number of fruits
The % of disease reduction (R) rate was calculated by using the Abbott formula: R(%) = (A − B)/Ax100
A = disease severity of positive control, B = disease severity of samples treated with endophytic bacteria against A. alternata
Genomic DNA extraction and 16S rDNA gene amplification of antagonistic bacteria
The genomic DNA of All bacteria was isolated using the enzymatic hydrolysis method (Y.-H. Liu et al. 2016). The bacteria grown in fresh cultures were sampled by approximately 1 ml in 1.5 ml Eppendorf tubes and centrifuged at high speed (14,000 rpm) to remove supernatants. Then, 50 mg of the bacterial samples was put into the pellet portions of the tube along with 480 ml of TE buffer and 20 µl of lysozyme solution (2 mg/ml). The bacterial suspension was incubated in a shaking hot water bath (37°C) for 2 h. The blend was then mixed with 50 ml of SDS solution (20%, w/v) and 5 µl of Proteinase K solution (20 mg/ml), and it was incubated for 1 h at 55°C in a water bath. Preparations were made for DNA extraction by treating the samples twice with a solution of phenol, chloroform, and isoamyl alcohol (25:24:1 v/v/v), followed by precipitation with 100 µl of sodium acetate (3 mol/l, pH 4.8–5.2) and 900 µl of absolute ethanol. After rinsing with 70% ethanol and allowing the samples to air dry, the resulting DNA precipitate was centrifuged at 5°C (12,000 rpm, 10 min. The extracted DNA was resuspended in 50 µl of sterile water and stored at -20°C for PCR reactions. Using the universal primers UB_16SF (5’-AGAGTTTGATCCTGGCTCAG-3’) and UB_16SR (5’-GTACGCTACCTTGTTACGAC-3’), bacterial endophyte isolates were identified by 16S rDNA gene partial sequencing (Mauti et al., 2013). For each reaction, amplification was carried out in 40-µl PCR tubes containing 1 µl MgCl2, 1 µl DNA, 4 µl Taq buffer, 0.5 µl Taq DNA Polymerase, 1 µl dNTPs, 2 µl of the primers, and 32 µl of nuclease-free dH2O. The PCR instrument was optimized to operate at the following temperatures: initial denaturation at 94°C for 5 minutes, denaturation at 94°C for 45 seconds, annealing at 55°C for 45 seconds, elongation at 72°C for 1 minute, and final elongation at 72°C for 5 minutes. There were 35 iterations of the denaturation, annealing, and elongation cycles. The separation of the amplification products on 1% (w/v) agarose gel in 1X TBE buffer was followed by ethidium bromide dye staining and UV visualization.
Phylogenetic analysis and DNA sequencing of isolated endophytic bacteria
Using the Sanger sequencing method of PCR products, DNA sequencing was performed bidirectionally with the help of the 3130xl Genetic Analyzer (Applied Biosystems, USA). At 84–100% similarity on the species level, 16S rDNA sequence data were subjected to Basic Local Alignment Search Tool (BLAST) analysis on the National Center for Biotechnology Information (NCBI) and selected as the nearest phylogenetic neighbor (Hentschel et al. 2001). These sequences were then aligned using Clustal X v.2.1 (Larkin et al. 2007). Phylogenetic dendrograms based on the 16S rDNA gene sequences were then generated using the MEGA 11 software with the Maximum Likelihood (ML) method and the Tamura-Nei model, with a p-distance matrix for nucleotides with the pair-wise gap deletion option selected and with 1,000 bootstrap repetitions (Tamura et al. 2021). The resulting phylogenetic trees were converted to the Newick format, and a more detailed visualization of the phylogenetic trees was performed using the Interactive Tree of Life (iTOL) (https://itol.embl.de/) server (Letunic and Bork 2021).
Production of extracellular enzymes
Chitinase activity: Chitinase enzyme antagonistic bacteria isolates were detected in the colloidal chitin agar medium (g/l: Na2HPO4, 6; KH2PO4, 3; NH4Cl, 1; NaCl, 0.5; yeast extract, 0.05; agar, 15, and colloidal chitin 1% (w/v), pH 7). Extracellular chitinase activities of the bacteria were observed after 7 d of incubation at 27°C, and the observation of a clear zone around the colony was considered positive for chitinase production (Souza et al. 2009).
Cellulase activity
Cellulase activities of the antagonistic bacteria were tested using the Bushnell and Hass carboxymethyl cellulose (CMC) medium. The antagonistic bacteria isolates were spot-inoculated and incubated at 27°◦C for 10 day. Then, the plates were flooded with 0.5% Congo red for 15 min, followed by 0.5-M NaCl wash for 10 min. The observation of a clear halo zone around the colony was considered positive for cellulase production (Singh et al. 2013).
Amylase activity: The amylase production capacity of the bacteria isolates was tested using starch agar medium ((l): raw starch, 20 g; NaNO3, 1 g; K2HPO4, 1 g; MgS04, 0.5g; FeS04, 0.01 g; agar ,15 g). The isolates were spot-inoculated and incubated at 27°C for 72 h. Then, the plates were flooded with iodine solution for 8 min. The observation of a clear zone around the colony was considered positive for amylase production (Priest, 1977).
Protease activity
The protease activity of the isolates was tested in skim milk agar medium. The bacterial isolates were spotted onto plates and incubated for 48 h at 30°C, and the observation of a clear zone around the colony was considered positive for protease production (Gardini et al. 2006).
Statistical analysis
The data were statistically analyzed using the SPSS 20.0 program. The mean values of the control and treatment samples were compared using Duncan’s multiple range test at the significance level of 5% (p < 0.05).