Front. Plant Sci.Frontiers in Plant ScienceFront. Plant Sci.1664-462XFrontiers Media S.A.10.3389/fpls.2024.1474401Plant ScienceOriginal ResearchAntibacterial activity of the biogenic volatile organic compounds from three species of bambooDuanYifan1LvBingyang2ZhangChunlong2ShiLisha3LiJingting1LiuYanjun4ChenQibing12*1College of Art, Sichuan Tourism University, Chengdu, China2College of Landscape Architecture, Sichuan Agricultural University, Chengdu, China3College of Fine Art and Calligraphy, Sichuan Normal University, Chengdu, China4School of Business, Sichuan University Jinjiang College, Meishan, China
Edited by: Eduardo Jacob-Lopes, Federal University of Santa Maria, Brazil
Reviewed by: Mohiuddin Kabir, East West University, Bangladesh
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Plant biogenic volatile organic compounds (BVOCs) possess ecological functions in antimicrobial benefits and air purification. The objectives of the study were to determine the differences in antimicrobial capacity of bamboo forests at different sampling sites. Three common bamboo species—Phyllostachys edulis, Bambusa emeiensis, and Phyllostachys violascens—were selected to determinate the antimicrobial activity of bamboo forests as well as under ex vivo conditions. Natural sedimentation method was used to determine the microbe counts in bamboo forests, and the microbe counts in grassland in the same area was measured as control treatment. The results showed that except for the P. violascens in Ya’an, the airborne microbial content of the sampling sites in bamboo forests was significantly lower relative to that of grassland in the same area, and inhibition rate reached 74.14% in the P. violascens forest in Dujiangyan. P. edulis forest and P. violascens forest in Ya'an had significantly lower inhibition rates than the other sampling sites, and there was no significant difference in the inhibition rates among the rest of the bamboo forest. The bacterial inhibition rate of bamboo leaves under ex vivo conditions varied with bamboo species and bacterial strains, with higher antibacterial activity against Gram-negative bacteria overall. Escherichia coli was sensitive to B. emeiensis leaves, while Staphylococcus aureus and Bacillus subtilis were sensitive to P. violascens leaves. Moreover, Candida albicans, S. cremoris, and Shigella Castellani were sensitive to P. edulis leaves. An analysis of the BVOCs composition from P. edulis collected in Changning by SPME-GC/MS revealed that the relative content of ocimene was obviously higher than other components. This study showed that P. edulis BVOCs have strong inhibitory ability to the tested microorganisms, and its main constituent, ocimene, has health benefit. P. edulis has the potential to become a forest recreation bamboo species.
Bamboo forests have strong antimicrobial capacity against microorganisms relative to grasslands.
Bamboo growth status influences the BVOC inhibition capacity significantly.
Stronger antibacterial activity of BVOCs from bamboo leaves against Gram-negative than Gram-positive bacteria.
Overall, Phyllostachys edulis had a stronger and wider range antibacterial effect among the three bamboo species.
Terpenes are the main components in Phyllostachys edulis BVOCs.
Introduction
Plant biogenic volatile organic compounds (BVOCs) are low-boiling point and complex mixtures of organic compounds synthesized and released by plants (Schimitberger et al., 2018). These compounds are predominately terpenes, phenols, aldehydes, alcohols, among others (Penuelas and Llusia, 2003). The release of plant BVOCs is influenced by both plant intrinsic and environmental factors. Temperature and light are the main environmental factors affecting BVOC release, with the highest release rates generally observed during summer (Wolfertz et al., 2003; Motonori et al., 2018). BVOCs contribute to ecological benefits because of its significant inhibitory and bactericidal effects on specific plant pathogens and human pathogens (Sharifi et al., 2018; Božik et al., 2017). Terpenoids are the main antibacterial active components in BVOCs, and monoterpene 3-carene could inhibit the growth of Pseudomonas aeruginosa, damage the normal cell morphology, leading to bacterial cell death by the destruction of cell membrane (Liu et al., 2019). Sesquiterpenes caryophyllene showed strong bacteriostatic effects against Escherichia coli, Bacillus subtilis, Micrococcus tetragenus, B. thermosphacta (Shu et al., 2020). Besides, aldehydes also have significant antibacterial effects, and studies have shown that cinnamaldehyde has antibacterial effects on pathogenic bacteria such as Escherichia coli and Staphylococcus aureus (Sanla-Ead et al., 2012; Labib et al., 2017). Benzaldehyde have been reported to cause morphological and ultrastructural changes in the cells of Rhodobacter sphaeroides, resulting in a bacteriostatic effect (Tahir et al., 2017). Additionally, BOVCs has direct benefits for human health. Inhaling BVOCs like limonene and pinene can result in antioxidant and anti-inflammatory effects on the airways, and the pharmacological activity of some terpenes absorbed through inhalation may be also beneficial to promote brain functions by decreasing mental fatigue, inducing relaxation, and improving cognitive performance and mood (Joung et al., 2015; Antonelli et al., 2020). However, the presence of some harmful components such as paraxylene, dichloromethane, and trichloromethane has also been detected in some plant BVOCs (Ronald, 1997; Li et al., 2022).
Bamboo is an abundant forest resources with wide distribution, large biomass, rapid growth and metabolism, and long growing periods (Lima et al., 2012; Buziquia et al., 2019; Xu et al., 2020). More than 1500 species of bamboo inhabit 22 million hectares worldwide with China accounting for 20% of the total area of bamboo globally (Zhou et al., 2017). Bamboo leaves are rich in a variety of bioactive components, including flavonoids, polysaccharides, amino acids, phenolic acids, volatile oils, etc (Shen et al., 2022; Wang et al., 2015), and have attracted much attention for their pharmacological activities such as antioxidant and antimicrobial (Liao et al., 2012; Wang et al., 2021; Zhang et al., 2005). Several classes of antimicrobial compounds have been described as responsible for antibacterial potential in bamboos, including benzoquinone (Nishina and Uchibori, 1991), chitin-binding peptides (Fujimura et al, 2005), fatty acids, such as linoleic acid (Soumya et al., 2014), and phytosterols (Tanaka et al., 2013). Studies have shown that essential oils extracted from bamboo possess a variety of active constituents with significant antibacterial effects on environmentally common fungi like yeast and bacteria including Gram-positive (Staphylococcus aureus, Staphylococcus epidermidis and Bacillus subtilis) and Gram-negative (Shigella flexneri, Proteus vulgaris and Escherichia coli) bacterial strains (Das et al., 2020; Jin et al., 2011; Shen et al., 2014). Therefore, bamboo forests not only have high economic and ecological values, but are also potentially suitable sites for air purification and healing (Zhou et al., 2005). The ability of plant BVOCs to inhibit bacterial growth depends on their composition as well as their release rate (Aguiar et al., 2014; Ben Hsouna et al., 2014; Krumal et al., 2015). It has been reported that essential oils containing mainly aldehydes or phenols, such as cinnamaldehyde, citral, carvacrol, eugenol, or thymol were characterized by the highest antibacterial activity, followed by essential oils containing terpene alcohols (Dorman and Deans, 2000; Davidson and Taylor, 2013).The composition of BVOCs released by different bamboo species differ (Motonori et al., 2018; Crespo et al., 2013), so there may be differences in the inhibitory capacity. Current studies on the bacteriostatic activity of BVOCs in bamboo focus mostly on the bacteriostatic effect of extracted essential oils. Although essential oils do contain BVOCs, not all BVOCs spontaneously released by plants are present in essential oils, and some molecules included in essential oils are artefacts of distillation (Senatore, 2000; Najar et al., 2020). Therefore, the bacteriostatic effect of essential oils do not fully reflect the bacteriostatic effect of spontaneously released BVOCs from living plants.Phyllostachys edulis and Bambusa emeiensis are the main native bamboo species in China and widely distributed. Their leaves have been reported to contain antibacterial active substances (Motonori et al., 2018; Yuan et al., 2020; Shen et al., 2024). Phyllostachys violascens is commonly introduced and cultivated in China but the antimicrobial activity has not been reported. In order to compare the differences in the inhibitory effects of BVOCs on environmental microorganisms between different bamboo species and different bamboo forest types during natural growth, the present study selected Phyllostachys edulis and Bambusa emeiensis and Phyllostachys violascens as research subjects. By comparing the inhibitory effect of BVOCs spontaneously released on environmental microorganisms in bamboo forests and bamboo leaves in ex vivo conditions, we investigated the differences in the inhibitory activity of different bamboo species and determination of BVOCs composition of the bamboo species with the best inhibitory effect.This study provided a scientific basis for planting configurations in recreational bamboo forests.
Materials and methodsGeneral overview of the study area
Sichuan Province in China has a warm climate, abundant rainfall, and a long growing season, making most of the area suitable for bamboo growth. The experimental sample plots were selected from natural bamboo landscape forests and planted bamboo ecological gardens located in Sichuan, between longitude 103°03’–103°50’E and latitude 28°28’–30°07’N, with the bamboo forest area exceeding 10 ha. There were a total of seven sample plots, and the location information is shown in Table 1, involving three bamboo species, P. edulis, B. emeiensis, and P. violascens (Table 1).
Distribution and geographic location of the sample plots of the three bamboo species for the bacterial suppression experiment.
Bamboo Species
Location
East Longitude; North Latitude
Area (ha)
Phyllostachys edulis
Yucheng District, Ya’an City
103°4′24″E; 30°10′5″N
20
Changning County, Yibin City
105°0′23″E; 28°28′23″N
12000
Bambusa emeiensis
Yucheng District, Ya’an City
103°2′5″E; 29°54′19″N
15
Muchuan County, Leshan City
103°57′57″E; 28°52′35″N
5000
Phyllostachys violascens
Puyang Town, Dujiangyan Irrigation Project City
103°38′60″E; 31°2’22″N
30
Yucheng District, Ya’an City
103°3′17″E; 30°7′60″N
15
Pujiang County, Chengdu City
103°19′19″E; 30°15′24″N
20
Determination of the antibacterial inhibition rates in bamboo forest environments
The natural sedimentation method was used to determine the microbial counts in bamboo forest environments and control grassland. The antibacterial inhibition rate in bamboo forest environments was calculated based on the microbial count in the bamboo forest and control grassland environments (Formulas 1 and 2). The sampling time was from 9:00 AM to 11:00 AM from July to August to minimize the effect of differences in light and temperature on the experimental results. Sampling periods with clear and windless weather were selected, and sampling points were set up in areas with minimal human interference in bamboo forests and adjacent grasslands. A total of five sampling points for each plot were randomly selected, with three replicates per sampling point. Petri dishes containing beef extract peptone medium (BPY medium) were placed horizontally on a support plate 1.5 m above the ground, and the medium was exposed to air for 15 min by opening the lid of the petri dish, then the lid of the dish was closed and sealed with a sealing film. After labeling, the plates were returned to the laboratory and incubated at a constant temperature of 37°C for 24 h, and the petri dish colonies were counted. The number of airborne microorganisms was calculated using the Omelensky formula:
E=1000 N /(A/100×t×10/5)=50000 N/At
where E is the number of bacteria per unit volume of air (CFU/m3), A is the area in the petri dish (cm2), t is the sampling time (min), and N is the average number of colonies at each sampling point after incubation (pcs).
The formula for calculating the antibacterial inhibition rate:
Antibacterial inhibition rate=(N2−N1)/N2×100%
where N2 is the number of microorganisms in the air of the control grassland, and N1 is the number of microorganisms in the bamboo forest environment.
Determination of the antibacterial effect of volatile organic compounds from bamboo leavesActivation culture of test strains
The bacteriostatic activity of BVOCs from bamboo leaves was determined using the method described by Krumal et al. (2015). Common pathogenic microorganism in environment, which can cause various diseases were selected including Gram-negative Escherichia coli, Shigella Castellani, Gram-positive Staphylococcus cremoris, Staphylococcus aureus, Bacillus subtilis, and fungi Candida albicans. All strains were purchased from Boulder Vanguard Ltd. Bacterial strains were inoculated with BPY medium using the scribing method and activated for 24 h at 37°C in an incubator. Candida albicans was inoculated on potato glucose agar (PDA) medium and activated for 48 h in an incubator at 28°C.
Preparation of test strain suspensions
The activated bacterial strains were gently scraped off with an inoculating ring, which was immersed into a triangular flask of BPY liquid medium. After the strain was placed freely into the liquid medium, the mixture was sealed and shaken gently in a 37°C shaker constant temperature shock culture. The absorbance was measured at 600 nm, and OD600 = 0.3 was used as the standard bacterial solution. The activated C. albicans were inoculated into a triangular flask of potato dextrose broth using the same method. After sealing, they were placed in a table concentrator at 28°C for constant temperature shaking and cultivation. Their absorbance was measured at a wavelength of 560 nm, and OD560 = 0.3 was used as the standard bacterial liquid culture. The standard bacterial solution was prepared as a bacterial suspension containing about 105 CFU·mL−1 using the dilution method, which serves as the test bacterial suspension.
Collection and treatment of plant leaves
After determining the environmental bacterial inhibition rate of the bamboo forests, bamboo leaves were collected and returned to the laboratory for ex vivo bacterial inhibition testing. Five sample plots were randomly selected in each bamboo forest distant from anthropogenic disturbances, and branches and leaves with good and uniform growth conditions were collected with high pruning shears, stored in a constant temperature box at 4°C, and transported to the laboratory on the same day. After returning the samples to the laboratory, mature and fresh leaves of an appropriate size (without insect spots) were cut, removed from the petiole, and rinsed three times with sterile distilled water to remove surface impurities from the bamboo leaves. After rinsing the leaves, an absorbent paper was used to absorb surface moisture from the leaves. Then, degreased cotton dipped in 75% alcohol was used to remove surface residues from the leaves. Subsequently, the leaves were rinsed with sterile distilled water three times, the surface moisture was removed using absorbent paper, and the leaves were used to measure the antibacterial inhibition rate.
Determination of the antibacterial activity in the presence of volatile substances from bamboo leaves
A volume of 0.2 mL of the prepared suspension was used to test each bacteria (E. coli, Shigella Castellani, S. cremoris, S. aureus, B. subtilis) by placing it onto a BPY medium plate and applying it evenly using a triangular spreader. Using the same method, 0.2 mL of yeast suspension was evenly applied onto the PDA medium. After the culture medium absorbed the bacterial solution, the culture dish was inverted, and 0.2 g or 0.4 g of whole or broken leaves from the test plant were added to the lid of the dish, respectively. A culture dish without added leaves served as the control and each group was tested in triplicate (the leaves did not contact the culture medium). The bacterial group was cultured in a 37°C incubator for 24 hours, while the yeast group was cultured in a 28°C incubator for 48 hours. The growth and colony size of bacteria on the culture plate medium were observed, and the number of colonies was calculated and compared with the control to calculate the antibacterial inhibition rate:
Antibacterial inhibition rate=N2−N1N2×100%
where N1 is the number of colonies in the treatment medium, and N2 is the number of colonies in the control medium.
Determination of BVOCs from bamboo leaves
Bamboo leaves from the sampling site with the strongest inhibition effect were selected to determine the BVOC composition using headspace solid-phase microextraction combined with gas chromatography-mass spectrometry (SPME-GC-MS). Bamboo leaves were collected and processed as described in 1.3.3. A mass of 1.00 g of sample was weighed and placed into a 20 mL headspace injection vial.
The extraction conditions were set at a constant temperature of 50°C, adsorption time of 40 min, equilibration time of 20 min, and resolution time of 5 min. The chromatographic column used was a DA-5MS (30 m × 0.25 mm (ID) × 0.25 µm).
Warming program: 40°C initial temperature, held for 3 min, heated to 120°C at a rate of 6°C·min−1, held for 3 min, heated to 250°C at a rate of 6°C·min−1, and then heated to 270°C at a rate of 10°C·min−1 and held for 5 min.
MS operating conditions: the temperature of the transmission line was 280°C; the temperature of the electron impact (EI) ion source was 280°C; the electron bombardment energy was 70 eV; the full scan range was 45~500 m/z. The results were analyzed using TurboMass Ver 5.4.2 version software and searched by the NIST 2.0 mass spectrometry database, and the volatile compositions were determined by referring to the relevant literature.
Data analysis
Data were analyzed using SPSS 19.0 (SPSS Inc., Chicago, IL, USA) for Windows. Two-way ANOVA and Student’s t-test were used to compare the means of different treatments for each data set at the significance level of p< 0.05 and at an extreme significance level of p< 0.01.
ResultsAntibacterial results of the bamboo forest environments
The results of this study showed that bamboo forests have a significant inhibitory effect on environmental microorganisms. Except for the P. violascens forest in Ya’an, the airborne microbial counts in the bamboo forests were significantly decreased compared with the urban control environment (p< 0.05, Figure 1). There were differences in the inhibition rates of each bamboo forest. The P. violascens forest in Dujiangyan had the highest inhibition rate of 74.14%, which was 2.27 times higher than the P. violascens forest in Ya’an, which had the lowest inhibition rate. Figure 2
Microbial population of bamboo forest environments and adjacent grassland environments * represents a significant difference in microbial quantity between the bamboo forest environment and urban environment (p < 0.05), and ** represents an extremely significant difference in microbial quantity between the bamboo forest environment and urban environment (p < 0.01).
Comparison of bacterial inhibition rates at different bamboo forest sampling sites. Different lowercase letters represent three different bamboo species with significant differences in bacteriostasis rates (p< 0.05).
Inhibitory effect of three species of bamboo leaves on gram-negative bacteria in <italic>ex vivo</italic> conditions
The results of the bacteriostatic test results on bamboo leaves against two Gram-negative bacteria showed that the three bamboo species had an inhibitory effect on both bacteria, and the difference in bamboo species had a small effect on this inhibitory effect. Only in the 0.4 g treatment group was the inhibition of E. coli by B. emeiensis leaves significantly higher than that of P. edulis and P. violascens, and the other treatments did not differ significantly. The bacterial inhibition effect of fragmented leaves from the same bamboo species was significantly higher than that of whole leaves, and a greater leaf mass induced a stronger bacterial inhibition effect (Table 2). There were significant differences in the tolerance of the two Gram-negative bacteria to bamboo leaf BVOCs, with Shigella Castellani being significantly less tolerant than E coli to P. edulis but more tolerant than E. coli to B. emeiensis and P. violascens.
Inhibition of two Gram-negative bacteria by different treatments of bamboo leaves from three bamboo species.
Bamboo species
Bacteriostasis rate (%)
Escherichia coli
Shigella Castellani
0.2 g
0.4 g
0.2 g
0.4 g
whole leaves
fragmented leaves
whole leaves
fragmented leaves
whole leaves
fragmented leaves
whole leaves
fragmented leaves
Phyllostachys edulis
12.56aB
15.59aB
25.18bB
32.69bB
17.24aA
24.81aA
28.47aA
35.51aA
Bambusa emeiensis
11.29aA
22.78aA
30.96aA
41.93aA
11.73aA
16.87aB
24.38aB
33.7aB
Phyllostachys violascens
13.49aA
17.51aA
24.82bA
32.42bA
10.4aB
17.19aB
22.21aB
27.68aB
Different lowercase letters represent significant differences in the bacteriostatic effect of different bamboo species under the same treatment conditions (p< 0.05), and different uppercase letters represent significant differences in the inhibitory effect of the same bamboo species under different treatment conditions on the two Gram-negative bacteria (p< 0.05).
For the same bamboo species, the bacteriostatic effect of bamboo leaves varied depending on the sampling site. The inhibition rate of P. edulis 0.2 g collected from Changning against E. coli was 2.28 times higher than that collected from Ya’an; however, the inhibition rate against Shigella Castellani was only 44.28% of that from Ya’an. The inhibition rate of P. violascens for Shigella Castellani was significantly higher in bamboo leaves from Dujiangyan than from Ya’an and 19.08 times higher in the 0.2 g whole-leaf treatment. However, the difference in inhibition rate between P. violascens leaves from Dujiangyan and Ya’an against E. coli was not significant (Tables 3 , 4). This suggests that in addition to differences in bamboo species, the growth status of the plant may also affect the composition and release rate of BVOCs.
Inhibition of Escherichia coli by three species of bamboo leaves.
Bamboo species
Location
Bacteriostasis rate (%)
0.2 g whole leaves
0.2 g fragmented leaves
0.4 g whole leaves
0.4 g fragmented leaves
Phyllostachys edulis
Ya’an
7.51cC
9.56eC
25.60aB
33.79bA
Changning
17.36aD
21.49cbC
24.38aB
31.00bA
Bambusa emeiensis
Ya’an
11.33bcC
27.73aB
28.91aB
44.92aA
Muchuan
10.26bcD
17.95cdC
33.33aB
38.46abA
Phyllostachys violascens
Dujiangyan
14.5abD
16.03deC
25.57aB
36.64abA
Ya’an
11.42bcD
14.19deC
23.18aB
26.64cA
Pujiang
14.1abD
23.20bC
25.71aB
37.3abA
Different lowercase letters represent significant differences in the bacteriostatic effect of different bamboo leaves under the same treatment conditions in different regions (p< 0.05), while different uppercase letters represent significant differences in the bacteriostatic effect between different treatments in the same region (p< 0.05). The table below displays equivalent information for different bacteria species.
Inhibition of Shigella Castellani by three species of bamboo leaves.
Bamboo species
Location
Bacteriostasis rate (%)
0.2 g whole leaves
0.2 g fragmented leaves
0.4 g whole leaves
0.4 g fragmented leaves
Phyllostachys edulis
Ya’an
23.53aD
27.81aC
32.62bB
36.90bA
Changning
10.42bC
22.01bB
23.55cdB
33.58bA
Bambusa emeiensis
Ya’an
11.11bD
16.46bC
20.58dB
36.21bA
Muchuan
12.38bD
17.33bC
28.22bcB
31.19bA
Phyllostachys violascens
Dujiangyan
20.42aC
29.93aB
45.07aA
45.07aA
Ya’an
1.07cD
2.86cC
3.57eB
6.07cA
Pujiang
9.64bC
17.77bB
18.78dB
31.98bA
Different lowercase letters represent significant differences in the bacteriostatic effect of different bamboo leaves under the same treatment conditions in different regions (p < 0.05), while different uppercase letters represent significant differences in the bacteriostatic effect between different treatments in the same region (p < 0.05).
Inhibitory effect of bamboo leaves from three species on Gram-positive bacteria in <italic>ex vivo</italic> conditions
Differences in bacteriostatic effects on Gram-positive bacteria were found among the bamboo species. Interestingly, the differences in bacteriostatic effects of bamboo leaves subjected to fragmentation treatment varied, and it is possible that the fragmentation treatment of bamboo leaves from different bamboo species caused differing effects on their BVOC release. Specifically, among the S. cremoris inhibition treatments, B. emeiensis had the best inhibition effect using the whole-leaf treatment with an inhibition rate of 28.94%, which was significantly higher than P. edulis and P. violascens. However, the inhibition effect of P. edulis was significantly higher than B. emeiensis when using the leaf fragmentation treatment.
In the S. aureus inhibition treatment, the inhibition rate of P. violascens was significantly higher than the other two bamboo species, and no significant difference in the inhibition of B. subtilis between the three bamboo species was observed (Table 5).
Inhibition of Gram-positive bacteria by different treatments of bamboo leaves from three bamboo species.
Bamboo species
Bacteriostasis rate (%)
Staphylococcus cremoris
Staphylococcus aureus
Bacillus subtilis
0.2 gwhole leaves
0.2 gFragmented leaves
0.4 gwhole leaves
0.4 gFragmented leaves
0.2 gwhole leaves
0.2 gFragmented leaves
0.4 gwhole leaves
0.4 gFragmented leaves
0.2 gwhole leaves
0.2 gFragmented leaves
0.4 gwhole leaves
0.4 gFragmented leaves
Phyllostachys edulis
5.80aB
11.04aC
19.79bB
41.35aA
7.89bA
16.99abA
21.72abA
28.27aB
7.14aA
12.01aB
13.91aC
23.95aC
Bambusa emeiensis
7.03aB
16.40aB
28.94aA
35.42bA
7.82bB
11.04bC
14.51bA
24.84aC
12.69aA
19.49aA
24.42aB
31.34aB
Phyllostachys violascens
8.55aC
15.19aB
19.56bB
23.53cC
16.70aA
23.18aA
25.81aA
34.10aB
10.87aB
22.10aA
18.68aB
37.31Aa
Different lowercase letters represent significant differences in the bacteriostatic effect of different bamboo species under the same treatment conditions (p< 0.05), while different uppercase letters represent significant differences in the inhibitory effect of the same bamboo species under different treatment conditions on the three Gram-positive bacteria (p< 0.05).
Similar to the inhibition results of Gram-negative bacteria, the inhibitory effect of bamboo leaves on Gram-positive bacteria increased significantly with an increase in the degree of fragmentation and mass, with the highest inhibition rate in the 0.4 g fragmented leaves treatment group. The inhibition rate of the P. edulis 0.4 g fragmented leaves treatment against S. cremoris was up to 12.33 times that of the 0.2 g whole-leaf treatment.
Differences in sampling sites also affected the inhibitory effect of Gram-positive bacteria (Tables 6–8). Significant differences in the inhibition of P. edulis among different sampling sites were only observed in the 0.4 g of leaves treatment against S. cremoris. B. emeiensis from different locations showed significant differences against B. subtilis and Shigella Castellani, with 0.2 g of fragmented leaves and 0.4 g of whole leaves. Leaves from the three P. violascens sampling sites showed significant differences in all treatments except for 0.2 g of fragmented leaves against S. cremoris.
Inhibition of Staphylococcus cremoris by three species of bamboo leaves.
Bamboo species
Location
Bacteriostasis rate (%)
0.2 gwhole leaves
0.2 g fragmented leaves
0.4 gwhole leaves
0.4 g fragmented leaves
Phyllostachys edulis
Ya’an
7.95bcC
11.93aB
12.50cB
38.07bA
Changning
3.63cdD
10.08aC
27.42aB
44.76aA
Bambusa emeiensis
Ya’an
7.27bcD
18.18aC
28.18aB
34.09bA
Muchuan
6.72bcD
14.18aC
29.48aB
36.57bA
Phyllostachys violascens
Dujiangyan
9.79bC
17.02aB
19.15bB
26.38cA
Ya’an
13.79aD
18.28aC
25.52aB
27.59cA
Pujiang
2.02dD
9.76aC
14.14bcB
16.84dA
Different lowercase letters represent significant differences in the bacteriostatic effect of different bamboo leaves under the same treatment conditions in different regions (p < 0.05), while different uppercase letters represent significant differences in the bacteriostatic effect between different treatments in the same region (p < 0.05).
Inhibition of Staphylococcus aureus by three species of bamboo leaves.
Bamboo species
Location
Bacteriostasis rate (%)
0.2 gwhole leaves
0.2 g fragmented leaves
0.4 gwhole leaves
0.4 g fragmented leaves
Phyllostachys edulis
Ya’an
6.72cdD
17.23bcC
22.27bB
31.09bA
Changning
9.21bcD
16.83bcC
21.59bB
25.71bA
Bambusa emeiensis
Ya’an
7.49cD
13.86cC
17.23bcB
25.09bA
Muchuan
3.68cdD
7.89dC
11.58dB
24.21bA
Phyllostachys violascens
Dujiangyan
13.03bC
22.18bB
22.89bB
26.06bA
Ya’an
1.51dD
11.56cdC
15.08bcB
29.15bA
Pujiang
26.91aD
34.94aC
39.76aB
47.79aA
Different lowercase letters represent significant differences in the bacteriostatic effect of different bamboo leaves under the same treatment conditions in different regions (p < 0.05), while different uppercase letters represent significant differences in the bacteriostatic effect between different treatments in the same region (p < 0.05).
Inhibition of Bacillus subtilis by three species of bamboo leaves.
Bamboo species
Location
Bacteriostasis rate (%)
0.2 gwhole leaves
0.2 g fragmented leaves
0.4 gwhole leaves
0.4 g fragmented leaves
Phyllostachys edulis
Ya’an
7.22bcD
11.79bC
14.83bB
22.05cdA
Changning
6.81bcC
11.91bB
12.77bB
25.53cA
Bambusa emeiensis
Ya’an
9.39bcD
12.27bC
16.61bB
19.13cdA
Muchuan
16.49aD
26.80aC
31.96aB
43.30bA
Phyllostachys violascens
Dujiangyan
12.57abD
28.96aB
14.75bC
54.64aA
Ya’an
2.54cC
10.87bB
11.59bB
13.77dA
Pujiang
18.60aD
27.91aC
30.23aB
44.19bA
Different lowercase letters represent significant differences in the bacteriostatic effect of different bamboo leaves under the same treatment conditions in different regions (p < 0.05), while different uppercase letters represent significant differences in the bacteriostatic effect between different treatments in the same region (p < 0.05).
Inhibitory effect of bamboo leaves from three species on yeast in <italic>ex vivo</italic> conditions
Under the same treatment conditions, P. edulis inhibited yeast significantly more than the other two bamboo species (Table 9). The inhibition rate of P. edulis was 2.05 and 3.52 times higher than that of B. emeiensis and P. violascens under the treatment of 0.4 g of fragmented leaves, respectively. The difference between B. emeiensis and P. violascens in terms of the inhibition of yeasts was not significant.
Inhibition of Candida albicans by bamboo leaves from three bamboo species.
Bamboo species
Bacteriostasis rate (%)
Candida albicans
0.2 gwhole leaves
0.2 gfragmented leaves
0.4 gwhole leaves
0.4 gfragmentedleaves
Phyllostachys edulis
22.28aC
33.58aB
35.61aB
45.05aA
Bambusa emeiensis
6.89bD
11.73bC
16.81bB
21.99bA
Phyllostachys violascens
9.46bA
10.4bA
12.28bA
12.81bA
Different lowercase letters represent significant differences in the bacteriostatic effect of different bamboo leaves under the same treatment conditions in different regions (p < 0.05), while different uppercase letters represent significant differences in the bacteriostatic effect between different treatments in the same region (p < 0.05).
Differences in sampling sites for the same bamboo species had a significant effect on the inhibitory effect on yeast (Table 10). The inhibition rate of P. edulis leaves collected in Ya’an was significantly higher than in Changning, and the inhibition rate of B. emeiensis leaves collected in Ya’an was also significantly higher than in Muchuan.
Inhibitory effect of bamboo leaves from different sampling sites on Candida albicans.
Bamboo species
Location
Bacteriostasis rate (%)
0.2 gwhole leaves
0.2 gfragmented leaves
0.4 gwhole leaves
0.4 gfragmented leaves
Phyllostachys edulis
Ya’an
26.22aC
43.71aB
45.10aB
52.45aA
Changning
18.06bC
23.61bB
26.39bB
37.50bA
Bambusa emeiensis
Ya’an
12.04bcD
18.52bcC
26.39bB
29.63cA
Muchuan
1.09dD
4.92dC
7.10dB
14.75dA
Phyllostachys violascens
Dujiangyan
9.56cA
10.29cA
12.50cA
12.87dA
Different lowercase letters represent significant differences in the bacteriostatic effect of different bamboo leaves under the same treatment conditions in different regions (p < 0.05), while different uppercase letters represent significant differences in the bacteriostatic effect between different treatments in the same region (p < 0.05).
BVOCs composition in <italic>P. edulis</italic> leaves from Changning
P. edulis collected in Changning showed a relatively high antibacterial rate against six tested microorganisms and the highest proportion of terpenes was found among the BVOCs in its leaves. Terpenes comprised 63% of all measured BVOCs; ocimene was the most abundant BVOC component in P. edulis from Changning. Figure 3
Total ion chromatoGram of BVOCs in the leaves of Phyllostachys edulis collected in Changning by using headspace SPME-GC/MS.
DiscussionAntibacterial effects of bamboo forests are influenced by the growing conditions
The type and rate of BVOCs plants released is related to the plant’s ability to inhibit bacteria (Farmer, 2001; Sartorelli et al., 2007). Some BVOCs have strong antimicrobial effects, such as terpenes and aldehydes (Dorman and Deans, 2000). Environmental factors can also directly affect the release of plant BVOCs (Tyagi and Malik, 2011; Eller et al., 2016; Effah et al., 2020), and among the various environmental factors light and temperature are the key factors affecting BVOCs release (Staudt and Lhoutellier, 2011; Lindwall et al., 2015; Yu et al., 2021). In order to make the temperature and light intensity as similar as possible, we all chose to take samples in sunny and windless weather from July to August in Sichuan Province. The results showed that, except for the artificially planted nursery (P. violascens forest in Ya’an), the number of airborne microorganisms in bamboo forests was significantly lower compared with those in grasslands in the same area (control treatment), and significant differences were observed in antimicrobial capacity among different sampling sites for the same bamboo species. The varieties and composition of BVOCs are chemically diverse by the plant species and circumstances in which the plants grow (Iijima, 2014). Therefore, the antimicrobial capacity of the same bamboo species varies in different sampling sites, which is similar to the antimicrobial results of essential oil of bamboo leaves (Jin et al., 2011). The P. violascens in Ya’an were planted in nurseries, and the age of the bamboo was only eight years, so the growing environment and growth were not as good as other naturally growing bamboo forests, and the antimicrobial capacity was significantly lower than others. Furthermore, the differences in the inhibition rates of the natural bamboo forests were not significant.
BVOCs spontaneously released from bamboo leaves are more effective in inhibiting Gram-negative bacteria than Gram-positive bacteria
In the present study, the inhibitory ability of spontaneously released BVOCs from bamboo leaves against Gram-negative bacteria was generally stronger than that of Gram-positive bacteria, and was particularly significant in the 0.2 g treatment group. This is similar to the results of the bacterial inhibition of the essential oil of bamboo leaf (Tao et al., 2019). BVOCs are hydrophobic, which allows them to penetrate the lipids of bacterial cell membranes, disrupting the structure, and making it more permeable (Sikkema et al., 1994). This inhibits bacterial growth and even leads to death (Denyer and Hugo, 1991). The cell walls of Gram-negative bacteria are thinner than those of Gram-positive bacteria, and are more sensitive to BVOCs.
Antimicrobial activity of bamboo BVOCs showed strain specificity
P. edulis, B. emeiensis, and P. violascens in this study belong to the Bambusoideae (Poaceae) subfamily, but the antibacterial capacity differed significantly. It has been suggested that species differences are the main reason for the difference in BVOCs release (Llusia et al., 2002; Iijima, 2014), thus affecting antimicrobial capacity. Interestingly, the strength of inhibition of bamboo leaf BVOCs was strain-dependent. For example, the BVOCs released from B. emeiensis leaves were weakly inhibitory to bacteria compared to other bamboo species, but strongly inhibitory to yeasts, significantly higher than from bamboo leaves collected in the other bamboo forests. Also, P. edulis leaves collected in Ya’an inhibited S. cremoris significantly less than those collected in Changning, however, they inhibited C. albicans significantly more than those of P. edulis from Changning. Similar results were observed when bamboo leaves were cut up. This result is similar to the antimicrobial results of essential oils of different bamboo species (Jin et al., 2011). Studies on the antibacterial activity of BVOCs mainly focused on BVOCs essential oils, and it was concluded that essential oils with aldehydes or phenols as their main components had the strongest antimicrobial activity, followed by terpene alcohols (Anselmo-Moreira et al., 2021; Jin et al., 2011; Dhifi et al., 2016). However, the specific composition of BVOCs with strong inhibitory ability to specific microorganisms needs to be further investigated.
Growth condition is an important factor influencing the release of BVOCs, and differences in environmental conditions such as light, temperature, humidity and soil conditions in the sampling sites resulted in different plant growth and composition of BVOCs (Jin et al., 2011). P. edulis in Changning is a natural bamboo forest with a large area and a good natural ecological environment, which results in vigorous growth of P. edulis and strong antibacterial activity in ex vivo conditions. In addition, although all the leaves collected in this experiment were mature leaves, the bamboo forests had undergone different growing times, so the seedling ages of the collected bamboo also differed, resulted in differing releases of BVOCs from the same bamboo species (Bai et al., 2016; Motonori et al., 2018; Tang et al., 2023). The overall difference in the inhibitory ability of P. edulis and B. emeiensis collected in Ya’an was not significant, and it was hypothesized that the growth status of the bamboo was the main influencing factor on the inhibitory ability.
With Ocimene is the main BVOCs component, <italic>P. edulis</italic> is an excellent bamboo species for forest recreation
Bamboo’s volatile organic compounds possess health benefits (Guo et al., 2015). In this study, the main component of P. edulis BVOCs with the strongest overall inhibitory capacity was terpenes.Terpenoids are common constituents of BVOCs in plants and are especially released as signal substances for defense mechanisms when plants are injured (Kroes et al., 2017; Vivaldo et al., 2017). Studies have shown that most terpenoids have strong antibacterial properties (Senatore, 2000). Besides, terpenes have shown health benefits. For example, monoterpenes increases α-waves and decreases β-waves in brain, inducing a state of relative sedation (Linck et al., 2010; Kasuya et al., 2015). The BVOCs composition of P. edulis is safe and can be used not only for forest healing but also has the potential to be applied as a biologically active antimicrobial agent.
It has been shown that alcohols and acids are the main components of the essential oil of P. edulis (Jin et al., 2011; Tao et al., 2019), while terpenoids is the main component of BVOCs in this study. It indicated that BVOCs spontaneously released from bamboo leaves are highly reactive chemical species, which differ from the composition of essential oils. For forest recreation, volatile gases are the main active substances with healing effect. More attention should be paid to the analysis of volatile constituents in the study of forest recreation.
Conclusion
BVOCs from bamboo plants exhibit strong antibacterial properties that vary depending on the environmental conditions of the bamboo forest and the growth status of the plants. Generally speaking,the inhibitory ability of BVOCs from bamboo leaves against Gram-negative bacteria was generally stronger than that of Gram-positive bacteria. Antimicrobial activity of bamboo BVOCs showed strain specificity and B. emeiensis leaves collected in Ya’an show strong antibacterial ability against yeast, and P. violascens show strong antibacterial ability against Staphylococcus aureus and Bacillus subtilis. The ex vivo leaves of P. edulis collected in Changning possess stronger antibacterial activity against yeast, S. cremoris, and Shigella Castellani than the other sampled bamboo leaves. Additionally, the BVOCs of P. edulis leaves primarily comprise beneficial terpenes, making it a good choice for recreational bamboo forests.CRediT authorship contribution statement
Data availability statement
The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.
The author(s) declare financial support was received for the research, authorship, and/or publication of this article. the Dual Project of Discipline Construction (project no. T202101), the Sichuan Province Science and Technology Support Program (project no. 3227140499), and the National Natural Science Foundation of China (project no. 32271944), and the Innovation Training Program for College Students (project no. 202411552204).
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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