The microbial strain used in this study was isolated from diseased spots on tea leaves. Isolated bacteria were identified as P. syringae via molecular identification and routinely cultured in Luria–Bertani (LB) liquid medium at 30°C. The P. syringae phage LDT325 was isolated from soil sampled from the flower base in Dongchangfu District, Liaocheng City, Shandong Province, China. The tested tea variety was Longjing No. 43 (Camellia sinensis), purchased from Juchuang Agricultural Development Co., Ltd., Shaoxing City, Zhejiang Province, China.

Bacteriophages were isolated from the soil of the Liaocheng flower base in Shandong Province. Phage LDT325 was isolated and purified using P. syringae as the host. Initially, 5 g of soil was mixed with 5 mL of sterile water and vortexed to achieve a homogeneous mixture. The soil sample was centrifuged at 10,000 × g for 5 min. The supernatant was removed and passed through a microporous filter with a pore diameter of 0.22 μm to remove bacteria. The 15 mL filtrate was mixed with 3 mL of exponentially growing bacteria (2 × 10⁷ CFU/mL). The mixture was inoculated into 15 mL of LB liquid medium and then incubated at 30°C for 4 h, centrifuged at 10,000 × g for 5 min (centrifuge H1650, Xiangyi, China), and filtered through a 0.22-μm filter to obtain the supernatant. Phage separation was conducted using the double-layer agar (DLA) plate method. Then, 200 μL of log-phase bacteria (2 × 10⁷ CFU/mL) were incubated with 5 mL of soft agar [LB containing 0.4% (w/v) agar] and poured onto solid bottom agar [LB containing 1.5% (w/v) agar]. Approximately 5 μL of the filtration solution was applied on to the solidified LB agar plate and incubated for 12 h at 30°C. Agar petri dishes were assessed for the presence of see-through regions or formations at the inoculation points.

The purified bacteriophage was obtained using the dual-layer agar plate method. See-through plaques were collected and resuspended in a test tube containing 1.5 mL of SM buffer. After amplification of the bacteriophage at approximately 25°C, the obtained solution was centrifuged for 1 min at 12,000 × g. The supernatant was filtered through a 0.22-μm filter. The double-layer plate method was used to titrate the filtered supernatant again. Based on their size and transparency, different plaques were selected after incubation at 30°C for 12 h and were resuspended in 900 μL of SM buffer. The purification process was repeated five times until homogeneously distributed plaque-containing phage isolates were obtained. Phage particles were stored in precooled LB medium, mixed with 50% (v/v) glycerol, and refrigerated at −80°C (Xing et al., 2017; Chen et al., 2018).

Bacteriophage isolation was initiated by incubation with 5 mL of LB broth at 30°C, followed by the collection of the clarified supernatant. The plaque number was counted using the dual-layer agar plate method. The initial bacteriophage titration was computed using the following equation: initial bacteriophage titration = number of plaques × 5× dilution factor.

A high-titer phage lysis buffer was prepared via plate amplification. The isolated bacteriophage (2 × 10⁷ PFU/mL) was added to the bacterial solution (2 × 10⁷ CFU/mL) and cultured for 4 h. The solution was mixed with 5 mL of dissolved liquid LB agar [LB containing 0.4% (w/v) agar] and the mixture was poured onto a plate containing LB agar [LB containing 1.5% (w/v) agar]. The plate was incubated at 30°C for 12 h, and 10 mL of SM buffer was added to the culture plate and gently agitated for 4 h. Subsequently, the culture mixture was centrifuged at 10,000 × g for 4 min, and the resulting supernatant was filtered through a 0.22-μm membrane. The concentration of the bacteriophage liquid filtrate was determined using the double-agar plate method. A bacteriophage suspension was negatively stained with 2% phosphotungstic acid and viewed using electron microscopy (EM). Images were obtained at an acceleration voltage of 100 kV, and the phage head and tail dimensions were determined using ImageJ (Peng and Yuan, 2018).

To determine the optimal MOI, the P. syringae bacterial solution was diluted to 2 × 10⁷ CFU/mL. The bacteriophage suspension was mixed with the diluted P. syringae solution at ratios of 10, 1, 0.1, and 0.01. The mixtures were incubated for 3 h at 30°C under constant agitation at 180 rpm, centrifuged at 10,000 × g for 10 min; and filtered through a 0.22-μm filter. The concentration of the bacteriophage suspension was assessed using the dual-layer agar plate method. The experiment was performed in triplicate. The dilution with the highest number of phage particles was selected as the dilution with the most favorable MOI (Sui et al., 2021).

The growth curve was determined as described previously (Cao et al., 2022). Phage LDT325 was mixed with the bacterial solution during the logarithmic growth phase at an MOI of 0.01 (Sun et al., 2012). The mixture was incubated at 37°C for 5 min and centrifuged at 10,000 × g for 10 min. The supernatant was removed. The pellet was rinsed twice with LB broth, suspended in an equivalent amount of LB liquid medium, and incubated at 180 rpm under continuous agitation at 30°C. Sampling was performed at 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 140, 160, 180, and 200 min, and bacteriophage concentrations were determined using the dual-layer agar plate method. After calculating the number of plaque-forming units per milliliter (PFU/mL), the latent period and burst size were determined by dividing the mean PFU/mL during the latent period by that during the last three time points of the experiment (Sanmukh et al., 2023). The experiment was performed in triplicate.

Thermal stability was evaluated by incubating purified bacteriophages at different temperatures (40°C, 50°C, 60°C, 70°C, and 80°C). Bacteriophage concentration was measured using the dual-layer agar plate method at 20, 40, and 60 min after incubation (Tie et al., 2018; Shang et al., 2021; Sui et al., 2021). To assess pH sensitivity, purified phages (2 × 10⁷ PFU/mL) were exposed to SM buffer at pH (1–13) for 1 h at 37°C (Tie et al., 2018; Yang et al., 2020; Sui et al., 2021).

The phages (2 × 10⁷ PFU/mL) were incubated with 20% chloroform on a shaker for 1 h. The control group lacked chloroform. Bacteriophage concentrations were evaluated using the dual-layer agar plate method and incubated at 30°C for 12 h in triplicate. The experiment was performed three times (Gašić et al., 2022).

Further refinement was performed based on the method (Iriarte et al., 2007; Czajkowski et al., 2014) and the impact of ultraviolet (UV) radiation on phage strains was investigated. A UV lamp integrated within a laminar flow hood was used as the UV light source (365 nm, 75 μW/cm²). Phage suspensions were prepared at a density of 2 × 10⁷ PFU/mL. In total, 5 mL of phage particles were introduced into each petri dish. The suspensions were irradiated with UV light at a distance of 30 cm for 0, 1, 2, 3, 6, 9, 12, 15, 18, and 21 min. The samples were diluted serially. Subsequently, phage concentrations were determined using the dual-layer agar plate method, and the samples were incubated at 30°C for 12 h. Three replicates of the experiment were performed.

The ability of the LDT325 bacteriophage (2 × 10⁷ PFU/mL) to lyse target bacteria was assessed by introducing a bacteriophage (200 μL) sample into a suspension of P. syringae (200 μL). An equal volume of LB medium was added to a bacterial culture with no phage inoculum as a control. The mixture was cultured at 30°C under shaking conditions at 180 rpm. The phage bacteriolytic activity was assessed by monitoring the absorbance of the culture solution (OD600) at 6-h intervals for up to 48 h. The assay was performed in triplicate (Wang et al., 2016).

The susceptible tea cultivar Longjing No. 43 was used as the study material. Tea leaves were sterilized with 75% alcohol, washed multiple times with sterile water, and left to dry. The number of bacteria in leaf tissue was evaluated using the needle-prick method (Aftab et al., 2022). First, 40 μL of P. syringae solution (2 × 10⁷ CFU/mL) was applied to the top surface of each tea leaf (dispersed across the four needle puncture apertures) and air-dried. Then, 40 μL of phage suspension (2 × 10⁷ PFU/mL) was applied to the top surface of each tea leaf (dispersed across the four needle puncture apertures), and injured leaves were protected with sterile cotton. Untreated leaves were simultaneously infected with P. syringae. The treated leaves were stored in a biochemical incubator at 28°C for 4 days under a relative humidity (RH) of 90%. Leaf samples from each group were collected after infection. For each specimen, 0.5 g of leaf tissue was mixed with 1 mL sterile water to determine the P. syringae concentration (CFU/mL). The experiments were performed in triplicate.

The lesion length was determined as described previously (Wang et al., 2017). Before inoculation, the leaves were treated with 75% alcohol, washed multiple times with sterile water, and air-dried. The leaves were punctured four times using a 0.45-mm diameter needle. Then, 40 μL of P. syringae solution (2 × 10⁷ CFU/mL) was applied onto the upper surface of each leaf, targeting the four puncture wounds, and the leaf was air-dried. Subsequently, 40 μL of the phage suspension (2 × 10⁷ PFU/mL) was applied onto the upper surface of each tea leaf (targeting the four puncture wounds). The wounded leaves were protected with sterilized cotton, control leaves were inoculated with P. syringae, and treated leaves were stored in a biochemical incubator at 28°C for 72 h with a relative humidity of 90%. The size of the disease spot was assessed 6 h after inoculation. The experiment was performed in triplicate.

The leaves were fixed with glutaraldehyde for at least 24 h for tissue observation. The fixed tissues were first dehydrated in 75% ethanol for 2 h, followed by 85% ethanol for 2 h, 90% ethanol for 2 h, 95% ethanol for 1 h, and finally anhydrous ethanol for 2 h. The tissues were dehydrated twice with xylene for 45 min and infiltrated with paraffin. Paraffin-embedded tissues were sliced into 4-μm sections using a tissue slicer. The slices were baked at 65°C; deparaffinized twice with xylene; washed twice with anhydrous ethanol for 10 min per rinse; washed with 95, 90, 80, and 70% ethyl alcohol for 5 min; and stained with hematoxylin for 10 min and eosin for 1.5 min. The slices were desiccated via immersion twice in 95% ethanol for 5 min, twice in pure ethanol for 5 min, and twice in xylene for 5 min. The slices were air-dried and sealed with neutral balsam (Liu et al., 2020). Under a Nikon 80i microscope (Japan), the paraffin sections were observed.

Data were analyzed using GraphPad Prism 8.0.2, specifically employing one-way analysis of variance. At least three independent replicates were performed under identical conditions, and data were presented as the mean ± standard deviation. Statistical significance was assessed based on p-value, and p-values of <0.05 were considered to indicate statistical significance. The significance levels were denoted as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

In this study, 12 soil samples were obtained from the Liaocheng flower base in Liaocheng, Shandong Province. To confirm the presence of phages, the samples were assessed using the dual-layered plate method. The findings indicated that only one of the soil samples harbored P. syringae phage, and the lysis area was the same (Figure 1A). To examine the biological characteristics of LDT325, the bacteriophage was isolated using the double-layer agar plate method. The findings revealed plaques with varying dimensions and types on the double-layer agar plate (Figure 1B). After six rounds of refinement, LDT325 displayed plaques with consistent dimensions and types on double-layer agar plates (Figure 1C). Thus, a phage that lysed P. syringae was isolated and refined.

To categorize bacteriophage LDT325 into groups specific to morphotypes, its structure was examined using electron microscopy. Initially, LDT325 lysate was prepared via plate amplification, yielding a concentration of 1.45 × 10¹⁰ PFU/mL. The structure of bacteriophage LDT325 is shown in Figures 2A,B. Structurally, bacteriophage LDT325 contains an icosahedral head with a diameter of 53 ± 1 nm and elongated, nonretractable tails measuring 110 ± 1 nm. Based on morphological characteristics, bacteriophage LDT325 belongs to the family Siphoviridae and order Caudovirales, according to the recommendations of the International Committee on Taxonomy of Viruses (Fokine and Rossmann, 2014).

To determine the optimal MOI for bacteriophage LDT325, varying quantities of the phage were used to infect P. syringae. After incubation for 3 h, the phage titer was assessed. At an MOI of 0.01, the peak titer of phage LDT325 was 4.6 × 10⁷ PFU/mL (Figure 3A). Growth curve analysis of LDT325 indicated minimal alteration in the initial incubation period during the first 10 min, followed by a rapid growth period between 10 and 160 min, finally reaching a plateau after 160 min (Figure 3B). The latent period and burst size of the phage were 10 min and 17 PFU/cell, respectively. The stability of phage LDT325 in various environments was assessed based on its pH, thermal stability, and viability rate, which was determined by counting PFUs. In addition, pH significantly influenced adsorption, contagiousness, intracellular multiplication, and enhancement of the bacteriophage. Unfavorable pH values can disrupt lysozyme or other phage capsid proteins, ultimately hindering phage attachment to receptor sites on the host cell (Liu et al., 2021). Bacteriophage LDT325 was highly active at pH 3–11 but could not be detected at pH 1–2 and pH 13. Thus, phage LDT325 showed increased stability under alkaline conditions but displayed susceptibility to acidic or strong basic environments (Figure 3C). LDT325 maintained significant levels of infectivity after incubation in water at varying temperatures (Figure 3D). Phage stability declined gradually once the temperature exceeded 60°C, and phage activity steadily diminished with prolonged incubation under identical conditions. The phage was undetectable after incubation at 80°C for 1 h.

Chloroform treatment decreased the bacteriophage titer from 2.68 × 10⁷ to 1.55 × 10⁷ PFU/mL, with no significant difference. The bacteriophages were tolerant to chloroform (Figure 4A). Ultraviolet (UV) radiation kills viruses, mainly because ultraviolet light can directly destroy the nucleic acid molecules of viruses, causing DNA and RNA to break and damage, thereby preventing virus replication and transmission. The bacteriophage titer decreased from 1.78 × 10⁷ to 1.56 × 10² PFU/mL with increased exposure time to UV (Figure 4B). Thus, bacteriophage LDT325 was sensitive to UV light.

The potential application of bacteriophage LDT325 in inhibiting P. syringae was evaluated. To assess the duration of LDT325 bactericidal activity against P. syringae, cellular density (OD600) was monitored for 48 h (Figure 5A). The concentration of P. syringae devoid of bacteriophage increased by 0.34 after 48 h. After 48 h phage therapy, the bacterial load was markedly reduced in treated cultures compared to untreated cultures.

The in vivo biological control test results are shown in Figure 5B. The control and experimental groups exhibited notable differences in bacterial count at the same time point. After 4 days of treatment, bacterial CFU was 10⁶ in tea leaves untreated with phage. However, in tea leaves treated with phages, the bacterial CFU was 10⁵. Thus, LDT325 was effective against P. syringae.

Treatment effect analysis of the P. syringae phage was initially performed on the leaves of Longjing No. 43. Under needling conditions, tea leaves were inoculated with sterile water and the bacteriophage did not yield any symptoms on the tea leaves. However, the trend of lesion length on tea leaves inoculated with P. syringae significantly differed between the experimental groups. The wounds were brown, and their sizes exhibited substantial variability among the four groups. After 72 h inoculation, wound width in the untreated group was 5.2 to 5.4 mm. The wound width in the experimental group ranged from 2.7 to 2.9 mm, suggesting that LDT325 is highly effective against P. syringae (Figure 5C).

Ultrastructural and micropathological analyses of pathological sections of the sterile water group revealed clear cell walls and nuclei, with the nuclei appearing round and blue. The cytoplasm was pale red in color. The nucleus was scattered throughout the tissue (Figure 6A). These characteristics are specific to the internal morphological structures of normal leaves. Pathological sections of the control group inoculated with P. syringae showed nuclear rupture, indicating nuclear necrosis (Figure 6B). In the experimental groups, cell wall boundaries were clearer, and nuclei appeared round and blue, without shrinkage. The cytoplasm was pale red in color. The nucleus was scattered throughout the tissue (Figure 6C), suggesting that LDT325 is highly effective against P. syringae.