Bryophytes are not as widespread as other plants in higher plants, and their individual size and structure are relatively simple, so they are often neglected by people. In recent years, studies on the chemical constituents of bryophytes have shown that bryophytes produce some secondary metabolites, such as aromatic family, terpenoids, lipids and other active substances, which are potential bioactive products library (Wang and Luo, 2001; Zhu et al., 2002; Zhou, 2002, Journal of Guangxi University of Traditional Chinese Medicine, 5(4): 84-87), most of these secondary metabolites have a good inhibitory effect on pathogenic fungi and bacteria (Yu and Jiang, 2012, Guizhou Sciences, 30(2): 52-61), and are one of the important sources of natural antibiotics. Therefore, bryophytes have broad application prospects in antibacterial, anticancer, anti-corrosion, prevention and treatment of cardiovascular diseases, lowering blood lipids, anticoagulant and other diseases, and have been favored by researchers and become a hot research field of bryophytes (Wu and Xiao, 1998, Journal of Guizhou Normal University, 1998, 16(4): 25-28; Yi, 2001; Li et al., 2004; Chen et al., 2005, Journal of Ningde Normal University, 17(4): 353-355; Wang, 2006; Wang et al., 2007; Sha and Han, 2009; Liu, 2010; Zhang and Lin, 2011, Journal of Xinzhou Normal University, 27(5): 4-5; Zhou et al., 2011, Guizhou Sciences, 29(2): 40-43).

In this study, we studied the bacteriostasis of 7 species of Hypnobrya to find out the difference of bacteriostasis among different bryophytes, so as to provide basic data for the interspecific difference of bryophytes, resource protection, screening of medicinal bryophytes and further development and utilization of bryophytes.

1 Result Analysis

1.1 Analysis of bacteriostasis of alcohol extracts from single bryophytes

Firstly, the diameter of bryophytes' inhibitory zone was compared with that of control group anhydrous ethanol (Table 1). Those with a diameter greater than that of anhydrous ethanol were considered to be bacteriostatic, while those with a diameter less than or equal to that of control group were considered to be non-bacteriostatic. After the bryophytes with bacteriostatic activity were screened out, independent sample t test was performed on the diameters of the inhibition zones between the two groups. p<0.01 was considered as extremely significant difference, and p<0.05 was considered as significant difference.

The alcohol extract of Palamocladium euchloron and Trachypus bicolor was weaker than that of other 5 kinds of Bryophytes. Palamocladium euchloron alcohol extract only has bacteriostatic effect on Streptococcus pyogenes, Bacillus subtilis and Salmonella paratyphi-B. The alcohol extract of Trachypus bicolor had bacteriostatic effect on Pseudomonas aeruginosa, Streptococcus pyogenes and Salmonella typhimurium. However, from the perspective of antibacterial activity, the bacteriostasis of Trachypus bicolor alcohol extract to Pseudomonas aeruginosa and Streptococcus pyogenes was significantly different from that of the control group.

The alcohol extract of Hypnum calcicolum and Eurohypnum leptothallum was stronger than that of Palamocladium euchloron and Trachypus bicolor, but weaker than the rest of the three kinds of Bryophytes. Hypnum calcicolum has bacteriostatic effect on five kinds of bacteria (Staphylococcusepid ermidis, Pseudomonas aeruginosa, Vibrio parahemoly ticus, Salmonella typhimurium and Salmonella paratyphi-B) in 11 kinds of bacteria. The bacteriostasis of Pseudomonas aeruginosa and Salmonella paratyphi-B was significantly different from that of the control group. The Eurohypnum leptothallum has bacteriostatic effect on 6 kinds of bacteria (Staphylococcus epidermidis, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pyogenes, Salmonella typhimurium, Salmonella paratyphi-B) in 11 kinds of bacteria. The bacteriostatic effect on Streptococcus pyogenes and Salmonella paratyphi-B was significantly different from that of control group.

Entodon taiwanensis, Brachythecium moriense and H. fertile alcohol extract has broad antibacterial effect. Among the 11 kinds of bacteria, Entodon taiwanensis alcohol extract has bacteriostatic effect on 9 kinds of bacteria, except Staphylococcus aureus and Streptococcus pyogenes. And the Salmonella paratyphi-B has significant difference compared with control group. Among 11 kinds of bacteria, Entodon taiwanensis has bacteriostatic effect on 8 kinds of bacteria (Pseudomonas aeruginosa, Vibrio parahemolyticus, Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium, Salmonella paratyphi-B). The bacteriostasis of Pseudomonas aeruginosa, Salmonella typhimurium and Salmonella paratyphi-B was significantly different from that of the control group. The ethanol extract of H. fertile had bacteriostatic effect on 8 kinds of bacteria, except Streptococcus pyogenes, Escherichia coli and Bacillus subtilis. The bacteriostasis of Salmonella paratyphi-B was significantly different from that of control group.

1.2 Antibacterial activity analysis of different bryophytes in a single species of bacteria

In terms of a single bacterium, bacteriostasis of alcohol extracts of 7 species of Hypnobryales effect on the basis of the comparison. Independent sample t test was performed for the bacteriostatic effect of the Hypnobryales in pairs, p<0.05 means significant, p<0.01 means extremely significant. 7 species of Hypnobryales in three kinds of antibacterial activity show differences in the test bacteria. In Pseudomonas aeruginosa, the ethanol extract of Hypnum calcicolum. had the strongest bacteriostatic activity, and the bacteriostatic activity of Brachythecium moriense alcohol extract was stronger, and the bacteriostatic activity of Entodon taiwanensis alcohol extract was significantly different from the bacteriostatic activity of Brachythecium moriense alcohol extract. Among vibrio parahaemolyticus, the ethanol extract of Brachythecium moriense had the strongest antibacterial activity, which was significantly different from the ethanol extract of Hypnum calcicolum. Among Salmonella paratyphi-B, the ethanol extract of Hypnum calcicolum was the most antibacterial, followed by the ethanol extract of H. fertile Sendtn., and there were significant differences between the two kinds of Hypnobryales and Palamocladium euchloron. There was no difference in bacteriostasis of alcohol extracts of the other seven bryophytes (Figure 1).

1.3 Cluster analysis on antibacterial activity of 7 species of Hypnobryales

From clustering diagram can be seen, when T=2, 7 kinds of Hypnobryales plants were clustered into four categories. Hypnum calcicolum and H. fertile for a class, Entodon taiwanensis and Brachythecium moriense for a class, the Eurohypnum leptothallum and Trachypus bicolor for a class, suggests that the bryophytes clustered into one group have great similarity in the antibacterial activity of 11 species of bacteria, and that the properties of their secondary metabolites are also very similar. Palamocladium euchloron belong to one group, suggests its bacteriostatic activity against 11 species of bacteria was different from that of other Hypnobryales species. When T=7, 7 species of Hypnobryales were clustered into two categories, Hypnum calcicolum, H. fertile, Entodon taiwanensis and Brachythecium moriense for a class, Eurohypnum leptothallum, Trachypus bicolor and Palamocladium euchloron for a class, which indicates that several Hypnobryales plants have similarities in antibacterial activies, but also there are some differences (Figure 2).

2 Discussion

In terms of systematics, Palamocladium euchloron and Brachythecium moriense belong to the Brachytheciaceae, and the bacteriostasis of Brachythecium moriense is more extensive than that of the Palamocladium euchloron, indicating that the Brachythecium moriense may be slightly more evolved than the Palamocladium euchloron. Eurohypnum leptothallum, Hypnum calcicolum, and H. fertile belong to the Hypnaceae family, and H. fertile is more antibacterial than the other two boteboea species, indicating that the degree of evolution of boteboea polyboea may be slightly higher than the other two Hypnobryales species, which may be because bryophytes produced more complex secondary metabolites in the process of evolution to adapt to the environment. The alcohol extracts of Brachythecium moriense, Entodon taiwanensis and H. fertile all have extensive antibacterial activity, but they are not listed as medicinal bryophytes at present (Yi, 2000, Journal of Zibo University, 3(3): 77-79; Han and Zhao, 2008; Shi et al., 2016, Journal of Guizhou Normal University, 34(6): 39-44; Han, 2017, Biology Education, 33(12): 4-6).

The results also showed that Salmonella typhimurium and Salmonella paratyphi-B were most sensitive to the bacteriostasis of alcohol extracts of 6 species of Hypnobryales. Salmonella typhimurium was sensitive to the alcohol extracts of the other 6 bryophytes except for Palamocladium euchloron, and Salmonella paratyphi-B was sensitive to the alcohol extracts of the other 6 Hypnobryales except for Trachypus bicolor. This may be because Salmonella typhimurium and Salmonella paratyphi-B are sensitive to the major secondary metabolites produced by moths, such as flavonoids, triterpenes, sterols and long-chain insoluble fatty acids (Wang and Luo, 2001, Guizhou Sciences, 19(4): 93-100).

From the perspective of the antibacterial activities of the 7 bryophytes, not most of them showed significant differences compared with the control group, and only some of the alcohol extracts showed relatively strong antibacterial activities, which was similar to the study on the antibacterial activities of 67 bryophytes (Wang, 2006). This study made up for the blank of the research on the antibacterial activity of the Hypnobryales and provided the foundation for the further research on the antibacterial activity of the Hypnobryales. Antimicrobial studies are an important area for future research as there is growing global interest in the use of natural remedies. In addition, new sources of natural bacteriostatic agents need to be identified as the number of strains developing resistance to antibiotics increases.  Further research should be done on the antibacterial properties of bryophytes in order to screen out new antibacterial agents and protect these biological resources.

3 Materials and Methods

3.1 Test materials

11 tested bacteria were as follows: Staphylococcus epidermidis, Pseudomonas aeruginosa, Vibrio parahemolyticus, Staphylococcus aureus, Streptococcus pyogenes, Staphylococcus albus, Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium, Bacillus subtilis and Salmonella paratyphi-B all come from Shanghai Luwei Technology Co. Ltd. The test materials were 7 species of Hypnobryales (Table 2).

3.2 Preparation of bryophyte alcohol extract

The whole bryophyte was collected from the field, and other mixed bryophytes were removed. The remaining test samples were repeatedly washed with tap water, and then cleaned with deionized water for 3 times. After that, they were put into an oven and dried at a constant temperature of 50℃. Weighing 2.0 g moss sample powder, put it into triangle flask (with rubber plug), add 20 mL of anhydrous ethanol, shake it on shaking table for 20 h, centrifuge it at 1 500 r/min for 10 min, collect supernatant, extract residue with anhydrous ethanol for 2 times, the same extraction method for 3 times, combine 3 times extract into small beaker. Store in refrigerator at 4℃ for later use.

3.3 Preparation of culture medium

Liquid medium and solid medium were prepared according to the type of medium required for the experiment. The preparation of liquid medium was as follows: Sodium chloride 5.0 g peptone 10 g beef paste 3.0 g, add distilled water to 1 000 mL, the use of 0.5 mol/L NaOH pH modulation 7.2~7.4, specific preparation process according to the conventional method, and finally prepared a good liquid medium into the refrigerator 4℃ reserve.  Solid medium preparation: sodium chloride 5.0 g peptone 10 g beef paste 3.0 g AGAR powder 15 g, add distilled water to 1 000 mL, pH range 7.2~7.4, the specific preparation process according to the conventional method (Zhou et al., 1989, Microbiology Experiment Tutorial, pp.63-81).

3.4 Preparation of bacterial suspension

A little of the tested bacterial lichen was inoculated on the inclined surface of solid medium with inoculation ring, and incubated in a constant temperature incubator at 37℃ for 20 h for activation. After that, the activated strain was connected to a ring in 5 mL liquid medium and incubated at 37℃ for 6~8 h in a constant temperature oscillator, which was the bacterial suspension.

3.5 Determination of bacteriostasis by filter paper method

12 mm in diameter of sterilizing filter in the extract of 7 kinds of Hypnobryales soak overnight, the 0.5 mL from each bacteria suspension and made corresponding solid medium containing bacteria tablets, sterile forceps take contains moss plants extract filter on the tablet containing bacteria, every dish 4, each bacteria do repeat 3 times, using anhydrous ethanol soak filter as CK, After incubation at 37℃ for 24 h, the diameter of bacteriostatic zone was measured by cross crossing method with vernier calipers (Liu et al., 2015).

3.6 Experimental data processing

As all the antimicrobial active substances of 7 kinds of Hypnobryales were extracted with anhydrous ethanol, the mean antibacterial diameters of anhydrous ethanol were used as control group. System clustering method is used first to each sample as a category, has bacteriostasis down to 1, there was no bacteriostasis to 0, using the IBM SPSS Statistics 23 software for data collection and analysis, the Euclidean distance is used to calculate the distance between the samples, using the shortest distance method to calculate the distance between the groups, according to the system clustering analysis method for cluster analysis.

Authors’ Contributions

ZC and PT are the experimental designer and executor of this study. They have completed data analysis and written the first draft of the paper. LQ participated in specimen collection, data sorting and data analysis. ZC is in charge of the project, directing experimental design, data statistics, paper writing and revision. All authors read and approved the final manuscript.

Acknowledgments

This study was co-funded by the Natural Science Research Project of Guizhou Education Department (Qianjiaohe KY [2014] 274), the Joint Project of Guizhou Science and Technology Department (Qiankehe J LKB[2012]22), and the First-class Construction and Cultivation Discipline "Ecology" in Guizhou Province (Qianjiaoke Research and Development [2018]216).

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