Comparative study of the antibacterial effects of wound secretions of different cultivars of Chinese fir

Overview of the test site

The experimental site is situated in the Yangkou State-owned Forest Farm in Shunchang County, Fujian Province (26°50′N, 117°53′E). The location is categorized as grade II, featuring mountain red soil and an elevation of 260 m. With a mid-subtropical maritime monsoon climate, it has an average annual temperature of 18.5 °C, an average annual sunlight duration of 1740 h, an average annual frost-free period of 230 d, and an average annual precipitation of 1,880 mm. In 2019, various experimental Chinese fir forest strains were cultivated. Chinese fir clonal container plantation Yang 061 and Yang 020 were grown in the Forest Farm of Fujian Province for afforestation purposes. Hubei Chuizhisha developed a clonal container plantation that matured into Chinese firs. Yangkou 3 is a cultivated at the Forest Farm of Fujian Province, owned by Yangkou State, using seeds from a Chinese fir seed orchard dating back to the third generation. Red-heart Chinese fir were cultivated at the Jiangxi Province’s Chenshan Forest Farm in Anfu County.

Experimental design

Artificial afforestation of different Chinese fir cultivars was performed in February 2019 using a randomized block design. Each experimental plot, ranging from the foot of the mountain to the top of the mountain strip afforestation, covered an area of more than 500 m², with Schima Superba as the isolation zone. The planting density for each experimental plot was 3,000 plants hm⁻². A total of three blocks and 15 experimental plots were constructed. The growth of different cultivars in 3a Chinese fir plantations is shown in Table 1.

Fifteen typical standard trees were selected in October 2021 for artificial mechanical damage wound simulation treatment in each cultivar of Chinese fir in a three-year-old experimental forest. Sharp knives were used to cut ellipses with a long half-axis of three cm and a short half-axis of one cm in the four directions of the bark in the east, west, south, north and south. The angle between the long half-axis of the ellipse and the horizontal line was 45^∘, thus simulating the mechanical damage of the Chinese fir bark. Each strain of Chinese fir simulated mechanical damage treatment of 15 trees. After the bark was cut, 2 ml centrifuge tubes were placed at the lower part of the bark ellipse to collect wound secretions, and 4 centrifuge tubes were collected for each average tree of Chinese fir (Fig. 1). Each strain of Chinese fir collected 15 average wood exudates for 1 h, and the collection time was between 8:00 and 10:00 every morning. The samples were placed in dry ice at 4 °C and returned to the laboratory for further analysis.

Based on the management status (Li et al., 2021; Tian et al., 2022) regarding pests and diseases of Chinese fir plantations, Bacillus subtilis, Salmonella paratyphi B, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus were selected as the test species. Test bacteria were purchased from Beijing Lvyuan Bird Biotechnology Co., Ltd. The antibacterial effects of the collected exudates of Chinese firs were to test against different bacterial species.

Test treatment implementation

The antibacterial effects of exudates were ascertained using the filter paper diffusion method. A total of 3 g of beef extract, 5 g of sodium chloride, and 10 g of agar powder (20 g) were weighed using an electronic balance and placed in a 1,000-mL beaker. Ultrapure water was then added to the beaker, and the mixture was heated to slight boiling until the solids completely dissolved. The pH was then adjusted to 7.0, the mixture was sterilized for 30 min at 121 °C, and the mixture was refrigerated at 4 °C.

The preparation procedure for the liquid medium without the agar powder was the same as that for the solid medium. Test tubes were subpackaged after processing. The bacterial which grow in the sterile water stock solution was obtained by removing the dry powder test bacteria from the refrigerator at 4 °C, adding them to a test tube filled with sterile water, and shaking the mixture in a constant temperature (37 °C) shaker on the sterile ultra-clean bench. An appropriate amount of bacterial stock solution was inoculated onto the plate and activated in a constant temperature incubator at 37 °C for 24 h. The activated bacterial plate was inoculated with an inoculation rod, and the strain was transferred to a test tube containing sterile water. The bacterial concentration was diluted to 10⁵ to 10⁸ CFU/mL using a McBurney turbidity tube and stored in a refrigerator at 4 °C for later use. According to the above conditions, select the appropriate concentration of bacterial solution, if the bacteria can grow throughout the whole dish and the distribution is uniform. The sensitivity determination of the test antibacterial ring is shown in Table 2 (Wang & Zhang, 2023).

Determination of antibacterial effect

One hundred microliters of the bacterial suspension were placed on a plate and evenly coated with a coating rod. sterile water soaked by exudates (6 mm in diameter) was attached to a solid plate, and sterile water infiltration filter paper was used as the blank control (CK). The prepared plates were placed in a constant temperature incubator at 37 °C for 24 h. The plate was removed, and the diameter of the inhibition zone was measured using a Vernier caliper. Each experiment was performed in triplicates.

Determination of MIC

The MIC was determined following a previously established method by Ning et al. (2022). Briefly, sterile water was used as the diluent, and six concentration gradients of wound secretion were prepared at 8, 4, 2, 1, 0.5, and 0.25 mg/mL. A total of 100 µL of bacterial suspension was added to 9 mL of a liquid medium containing exudates and thoroughly mixed. After incubation in a constant temperature incubator for 24 h, the plate was inoculated and placed in a constant temperature incubator for an additional 24 h to observe colony growth on each plate. Each concentration gradient was repeated three times, with sterile water as the control, and the presence of growth inhibition was used as to determine the antibacterial activities of the wound secretions.

Data processing and analysis

SPSS software (version 24.0) was used for statistical analysis. Single factor T-test was used in RStudio to compare the effects of exudates of five strains of Chinese fir on different strains (α = 0.05). Excel 2010 software was used to create the charts.

Antibacterial effects of wound secretions from different Chinese fir cultivars on Bacillus subtilis

The antibacterial test results for the secretion of the five Chinese fir cultivars against B. subtilis are shown in Fig. 2. The exudates of the cultivars exhibited significant antibacterial effects against B. subtilis (P < 0.05). The average diameter of the inhibition zone for Yangkou 3, Yang 020, Red-heart Chinese fir, Yang 061, and Chuizhisha was 2.70, 2.48, 3.07, 2.56, and 1.90 times that for CK, respectively. The bacterial cells displayed moderate sensitivity toward Taxodium distichum but high sensitivity toward the other four secretions. The antibacterial effect of the exudates of the five Chinese fir cultivars was in the following order: Red-heart Chinese fir > Yangkou 3 > Yang 061 > Yang 020 > Chuizhisha.

The MIC test results for the different secretions against B. subtilis are shown in Table 3. The MIC of Red-heart Chinese fir, Yangkou 3 and Yang 061, Yang 020, and Chuizhisha was 0.5, 1, 2, and 4 mg/mL, respectively. The MICs of exudates of Chuizhisha, Yang 020, and Yang 061 on B. subtilis were greater than those of Yangkou 3 and Red-heart Chinese fir. The MIC of exudates of Yangkou 3rd generation on B. subtilis was greater than that of Red-heart Chinese fir, and the exudates of Red-heart Chinese fir exhibited the strongest inhibitory effect on B. subtilis.

Antibacterial effect of wound secretions from different Chinese fir cultivars on Salmonella paratyphi B

The antibacterial test results for the secretions of different Chinese fir cultivars against S. paratyphi B are shown in Fig. 3. All exudates exhibited significant antibacterial effects against S. paratyphi B (P < 0.05). The average diameter of the inhibition zone for Yangkou 3, Yang 020, Red-heart Chinese fir, Yang 061, and Chuizhisha was 2.21, 1.72, 2.00, 2.00, and 1.83 times that for the CK, respectively. The exudates exhibited moderate antibacterialactivity on S. paratyphi B, and their antibacterial effects were in the following order: Yangkou 3 > Red-heart Chinese fir > Yang 061 > Chuizhisha > Yang 020.

The MIC test results for the secretions of different Chinese fir cultivars against S. paratyphi B are shown in Table 4. The MICs of Yangkou 3, Red-heart Chinese fir, and Yang 061 were 2 mg/mL, and those of Yang 020 and Chuizhisha were 4 mg/mL. The MICs of the exudatesof Chinese fir Yang 020 and Chinese fir on S. paratyphi B were greater than those of Yangkou 3, Red-heart Chinese fir, and Yang 061, with Yangkou 3 exhibiting the strongest antibacterial effect on S. paratyphi B.

Antibacterial effect of wound secretions from different Chinese fir cultivars on Pseudomonas aeruginosa

The antibacterial test results of the secretions of different Chinese fir cultivars against P. aeruginosa are shown in Fig. 4. All exudates exhibited significant antibacterial effects against P. aeruginosa (P < 0.05). The average diameter of the inhibition zone for Yangkou 3, Yang 020, Red-heart Chinese fir, Yang 061, and Chuizhisha was 1.70, 1.35, 1.93, 1.83, and 1.51 times that for the CK, respectively. The inhibition effect was moderately sensitive, with an average diameter of the inhibition zone of 9.07 mm, indicating a relatively lower level of sensitivity. The antibacterial effect of the secretions was in the following order: Red-heart Chinese fir >Yang 061 > Yangkou 3 >  Chuizhisha > Yang 020.

The MIC test results for the secretions of the different cultivars of Chinese fir against P. aeruginosa are shown in Table 5. The MIC of Yangkou 3, Hongxinshan, and Yang 061 was 4 mg/mL, whereas that of Yang 020 and Chuizhisha was 8 mg/mL The MICs of the secretions of Yang 020 and Chuizhisha on P. aeruginosa were greater than those of Yangkou 3, Red-heart Chinese fir, and Yang 061.

Antibacterial effect of wound secretions from different Chinese fir cultivars on Escherichia coli

The antibacterial test results for the secretions of the different Chinese fir cultivars against E. coli are presented in Fig. 5. All exudates exhibited antibacterial effects against E. coli. The average diameter of the inhibition zone for Yangkou 3, Yang 020, Red-heart Chinese fir, Yang 061, and Chuizhisha was 2.68, 2.87, 2.79, 2.92, and 2.91 times that for the CK, respectively. The secretions of the five Chinese fir cultivars exhibited a high antibacterial effect on E. coli. The antibacterial effects of the secretions on E. coli were in the following order: Yang 061 > Chuizhisha > Yang 020 > Red-heart Chinese fir > Yangkou 3.

The MIC test results for the different secretions of the Chinese fir cultivars against E. coli are shown in Table 6. The MICs of Yang 020, Red-heart Chinese fir, Yang 061, and Chuizhisha were 0.5 mg/mL, and that of Yangkou 3 was 1 mg/mL. The MIC of exudates of Yangkou 3 on Escherichia coli was greater than that of Yang 020, Red-heart Chinese fir, Yang 061, and Chuizhisha. This result indicates that Yangkou 3 had the weakest antibacterialeffect on E. coli.

Antibacterial effect of wound secretions from different cultivars of Chinese fir against Staphylococcus aureus

The antibacterial test results for the secretions of the different Chinese fir cultivars against S. aureus are shown in Fig. 6. All secretions exhibited antibacterial effects against S. aureus. The average diameter of the inhibition zone for Yangkou 3, Yang 020, Red-heart Chinese fir, Yang 061, and Chuizhisha was 2.49, 2.55, 2.41, 2.44, and 2.53 times that for the CK, respectively. Notably, S. aureus exhibited moderate sensitivity towards Yangkou 3, Red-heart Chinese fir, and Yang 061, but high sensitivity towards Yang 020 and Chuizhisha. The antibacterial effect of the secretions on S. aureus was in the following order: Yang 061 > Chuizhisha > Yang 020 > Red-heart Chinese fir > Yangkou 3.

Table 7 shows the MIC test results for the secretions of the different Chinese fir cultivars against S. aureus. The MIC of Yang 0 and that of Yangkou 3, Red-heart Chinese fir, Yang 061, and Chuizhisha was 2 mg/mL. The MIC of Yang 020 exudates against S. aureus was lower than that of the secretions of the four other Chinese fir varieties.