|Year : 2022 | Volume
| Issue : 2 | Page : 236-240
Growth promotion effects of Bacillus subtilis on Bletilla striata seedlings
Di Liu1, Jian Chen1, Hui Yang1, Zubaida Yousaf2, Chao-Yang Liu1, Bi-Sheng Huang1
1 College of Pharmacy, Hubei University of Chinese Medicine, Wuhan 430065, China
2 Department of Botany, Lahore College for Women University, Lahore, Pakistan
|Date of Submission||20-Oct-2020|
|Date of Acceptance||10-Mar-2021|
|Date of Web Publication||03-Jun-2021|
College of Pharmacy, Hubei University of Chinese Medicine, Huangjiahu West Road #16, Hongshan, Wuhan, Hubei 430065
Source of Support: None, Conflict of Interest: None
Objective: The objective of the study is to study the growth promotion effects of Bacillus subtilis inoculants on the growth of Bletilla striata seedlings. Methods: Various concentrations (1/10×, 1/50×, 1/100×, and 1/500 × dilutions) of B. subtilis inoculants were applied on the B. striata seedlings. Plant height, base diameter, leaf length, leaf width, relative chlorophyll content, tuber diameter, average fibrous root length, number of fibrous roots, and fresh weight were measured after incubation for 50 days. Results: Treatments with different concentrations of B. subtilis significantly increased the number of fibrous roots of the B. striata seedlings and promoted root elongation, and a higher concentration (1/10 × dilution) is associated with more significant promotion effects. B. striata seedlings treated with different concentrations of B. subtilis all showed an increase of relative chlorophyll contents in leaves with the increasing inoculant concentration. Only B. subtilis inoculant at a high concentration (1/10 × dilution) significantly promoted the plant height and base diameter of B. striata seedlings. The application of B. subtilis did not significantly promote the tuber diameter and fresh weight of B. striata seedlings. Principal component analysis confirmed the maximum growth promotion by B. subtilis inoculant in 1/10 × dilution than the other treatments. Conclusions: The application of B. subtilis can increase the relative chlorophyll content, promote growth and development of roots, and increase plant height and base diameter of B. striata seedlings. Therefore, B. subtilis has significant growth promotion effects on B. striata seedlings. These findings provide theoretical basis for the ecological cultivation of B. striata.
Keywords: Chlorophyll content, fibrous roots, medicinal plant, principal component analysis, seedling growth promotion
|How to cite this article:|
Liu D, Chen J, Yang H, Yousaf Z, Liu CY, Huang BS. Growth promotion effects of Bacillus subtilis on Bletilla striata seedlings. World J Tradit Chin Med 2022;8:236-40
|How to cite this URL:|
Liu D, Chen J, Yang H, Yousaf Z, Liu CY, Huang BS. Growth promotion effects of Bacillus subtilis on Bletilla striata seedlings. World J Tradit Chin Med [serial online] 2022 [cited 2022 Dec 10];8:236-40. Available from: https://www.wjtcm.net/text.asp?2022/8/2/236/317484
| Introduction|| |
Bletilla striata (Thunb.) Reichb.f. is a perennial herb of the Orchidaceae, and the dry tubers of this plant are used in traditional Chinese medicine. B. striata can enhance the hemostasis in the lungs, liver, and stomach. It has the characteristics of astringent, detumescence, and promoting granulation and has been usually used to treat hemoptysis, traumatic bleeding, abscesses, and chapped skins. B. striata has the effect of improving the nutritional status of the skin and preventing the formation of wrinkles.,, Recent studies have shown that polysaccharides in B. striata possess antibacterial and antitumor effects., The polysaccharide gum in B. striata tubers is widely used in medicinal and daily chemical products, and thus, B. striata serves as an important raw material for modern pharmaceutical and chemical industries in China. In addition to the medicinal and economic values, B. striata also has high ornamental value.,,, The color of B. striata flowers is bright and saturated; the flower shape is dignified and elegant; and the leaves are stretched and beautiful. The artificial breeding of B. striata includes sexual reproduction and asexual reproduction. Due to the low seed germination rate, the ramet propagation method is mainly adopted in B. striata breeding, which is time-consuming, low efficiency, and material consuming. Moreover, long-term asexual reproduction would lead to reduced genetic diversity and germplasm degradation. Therefore, in recent years, an increasing number of studies on technologies of direct generation of B. striata seedlings from seeds has been conducted.,
Traditional plant disease control is mainly chemical control and long-term use of which can easily lead to pathogen resistance. In addition, chemical pesticides remaining in the fields will result to serious environmental pollutions. The requirements of ecological environment protection and sustainable development have made the research of new biological fertilizers a hotspot. Bioinoculants, which are made by artificially cultivating certain beneficial microorganisms, play important roles in improving soil nutrient structure and fertility, promoting plant growth, and enhancing plant resistance to diseases., The widely distributed Bacillus subtilis, which is mesophilic and aerobic, locates at crop rhizosphere and can promote plant growth. B. subtilis colonizes the soil and plant rhizosphere and exhibits the characteristics of pollution-free, environment-friendly, fast growing, and simple nutrition. It produces spores and a variety of antibiotics and enzymes and has strong broad-spectrum antibacterial activity and stress resistance. In addition, the mass production of B. subtilis is simple and low cost; the application of B. subtilis is convenient; and the B. subtilis merchandise has a long shelf life. Therefore, B. subtilis is a promising microorganism for biological control and growth promotion., Many studies have reported the important roles of B. subtilis as a bioinoculant in growth promotion and biological control on plants, such as field crops including wheat, rice, and corn; vegetables and fruits including cabbage, cucumber, pepper, and watermelon; as well as medicinal plants Atractylodes macrocephala (“Bai Zhu” in Chinese) and Siraitia grosvenori (Luo Han Kuo).
It is well known that orchid mycorrhizal fungi are essential for the seed germination and vegetative growth of orchids, and many studies have investigated the effects of mycorrhizal fungi on germination and the early growth stage of B. striata, especially the mycorrhizal fungi isolated from B. striata or orchid species.,,, However, little is known about the effect of B. subtilis on B. striata growth. In this study, we applied different concentrations of B. subtilis on B. striata seedlings and analyzed the growth and biomass of the aboveground and underground parts of B. striata to reveal the growth promotion effects of B. subtilis on B. striata seedlings. The optimal application dose range of B. subtilis was obtained. The findings from this study provide a theoretical basis for the ecological cultivation of B. striata.
| Methods|| |
The B. striata seedlings used in this study were provided by B. striata Breeding Base of the Yichang Zhongke Gastrodia Professional Cooperative, Yichang City, Hubei Province, China. These B. striata seedlings were obtained by direct germination of B. striata seeds raised in soil, 15 months old. The B. subtilis powder formulation used in the experiments is wettable powders (Shandong Lyulong Biological Co., Ltd., China), with an effective number of viable bacteria ≥2 × 1010/g.
Preparation of Bacillus subtilis suspension
Ten grams of B. subtilis powders were weighed by an analytical balance (METTLER TOLEDOD) and fully dissolved with 250 ml of sterile water to prepare B. subtilis stock suspension. Four aliquots of B. subtilis storage suspension were measured using a sterile pipette and diluted 10 times (1/10×), 50 times (1/50×), 100 times (1/100×), and 500 times (1/500×), respectively, to prepare B. subtilis working suspensions. These working suspensions were sealed and stored at room temperature away from light for later use.
Bacillus subtilis treatment of Bletilla striata seedlings
B. striata seedlings with uniform growing vigor were selected and randomly divided into five groups, with 45 seedlings in each group. The seedlings used in the study were not mycorrhized before their use in the experiment. The substrate (peat: coco coir: perlite = 1:1:1) for growing seedlings was sterilized before use. The seedlings were transplanted into pots (length: 50 cm, width: 20 cm, and height: 15 cm) marked with numbers 1, 2, 3, 4, and 5, respectively. Group 1 was set as the control group, and B. striata seedlings in this group were treated with sterile water; seedlings in Group 2, Group 3, Group 4, and Group 5 were treated with 1/500×, 1/100×, 1/50×, and 1/10 × B. subtilis suspensions, respectively. The seedlings were inoculated with B. subtilis through application at the root zone by root irrigation. The five groups of B. striata seedlings were cultured in the greenhouse (20°C ± 2°C, 1200 ~ 1800lx, under a 16 h/8 h day/night regimen and 50%~60% humidity at daytime and 80%~90% at night) of Medicinal Botanical Garden in Hubei University of Traditional Chinese Medicine, Wuhan, Hubei Province, China. For root irrigations, on the day of transplanting, the experimental groups were irrigated with 500 ml of the corresponding concentrations of B. subtilis suspension, and the control group was irrigated with the same amount of sterile water. The roots were irrigated with B. subtilis suspension again after 3 days and then every 7 days in later time, and a dosage of 500 ml bacteria suspension (for experimental groups) or sterile water (for control group) was applied each time. The frequency of plant irrigation with water was every 3 days. After culturing for 50 days, the growth indices of the B. striata seedlings were measured.
Determination of growth indices of Bletilla striata seedlings
The plant height, base diameter, leaf length, leaf width, relative chlorophyll content, tuber diameter, number of fibrous roots, average fibrous root length, and fresh weight were measured for each B. striata seedling. The leaf length and leaf width were determined by measuring the length and width of the longest leaf of each seedling. The tuber diameter and base diameter were measured using digital vernier calipers (LERBS). The fresh weight was determined by an analytical balance (METTLER TOLEDOD). The SPAD (Soil Plant Analysis Development) values of B. striata leaves were employed to reflect the relative chlorophyll content and determined by a SPAD-502 PLUS chlorophyll meter (Konica Minolta).
The data were analyzed using SPSS (version 26.0) software (IBM Corporation, Chicago, IL, USA). A one-way analysis of variance (ANOVA) was used to compare the differences in various growth indices among B. striata groups treated with B. subtilis at different concentrations. The Levene's test of variance homogeneity was used to determine whether the variance of a given growth index was homogeneous. For indices with homogeneous variances, the least significant difference multiple comparison was performed among groups. For indices with nonhomogeneous variances, the Welch and Brown–Forsythe tests as well as the Tamhane's T2 multiple comparison were used. Principal component analysis (PCA) was carried out to assess the influence of B. subtilis treatments at different concentrations. The violin diagrams were created using R (v3.5.3) software.
| Results|| |
Growth promotion effect of Bacillus subtilis on the aerial part of Bletilla striata seedlings
There were 35, 35, 36, 36, and 40 individuals survived in Group 1, 2, 3, 4, and 5, respectively, after culturing 50 days. When evaluating the effect of inoculation with B. subtilis on the aerial part of seedlings, a slightly increase in plant height and base diameter when inoculated with 1/10× B. subtilis suspension was observed, with average increases of 29.50% and 22.50% than control [Table 1] and [Figure S1]a, [Figure S1]b. Both leaf length and leaf width were not affected by any treatment compared to the control group [Figure S1]c, [Figure S1]d, so treatments of B. subtilis suspensions had no significant effect on the leaf size of B. striata seedlings [Table 1]. However, the significant enhancement of average relative chlorophyll content in B. striata seedling leaves was observed in the treatments of B. subtilis suspensions at higher concentration level (1/100×, 1/50×, and 1/10×), with the relative increases of 14.62%, 19.26%, and 28.20%, respectively [Table 1]. Moreover, the sample distribution frequencies over mean value in the treatments of B. subtilis suspensions at higher concentration level (1/50× and 1/10×) were obviously higher than control group. Moreover, compared with the inoculation with B. subtilis suspensions at relatively lower concentrations (1/100× and 1/50×), a higher concentration (1/10×) of B. subtilis suspension has more significant promotion effects on the relative chlorophyll content in the leaves of B. striata seedlings [Table 1] and [Figure S1]e.
|Table 1: Effect of Bacillus subtilis on the growth characteristics of Bletilla striata seedlings (x¯±s)|
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Growth promotion effect of Bacillus subtilis on the underground part of Bletilla striata seedlings
As for the effect on the underground part, B. subtilis treatments at all concentrations showed no significant effect on the tuber diameter of B. striata seedlings compared with the control group [Table 1] and [Figure S1]f. Otherwise, B. subtilis treatments exerted a significant influence on root growth in terms of average fibrous root length and number of fibrous root per seedling. The increase in average fibrous root length was 44.19%–124.65%, and fibrous root number was 35.81%–98.02% in various treatments than control. The highest concentration treatment (1/10×) produced the maximum average fibrous root length (10.64 cm) and fibrous root number (14 per seedling), corresponding to increases of 201.42% and 133.33% than the mean values of control [Table 1] and [Figure S1]g, [Figure S1]h.
Effect of Bacillus subtilis on the biomass of Bletilla striata seedlings
No obvious effect was observed on fresh weight by all the B. subtilis treatments, and means of fresh weight were not statistically different between treatments [Table 1] and [Figure S1]i.
Principal component analysis from treatments by Bacillus subtilis inoculation at different concentration
The PCA analysis of all the samples of the five different treatments by B. subtilis inoculation at different concentration showed that the three groups of treatments (Group 2, 3, and 4) almost clustered together and appeared with the control (Group 1). Group 5 slightly overlapped with Group 4 but was entirely separated from the control [Figure 1]. From the PCA analysis, it could be concluded that the treatment by B. subtilis inoculation at the highest concentration in this study significantly differs from the control and had the greatest influence on plant growth compared to other groups.
|Figure 1: Principal component analysis (PCA) of all the Bletilla striata seedlings treated by Bacillus subtilis inoculation at different concentration.|
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| Discussion|| |
The results of this study show that B. subtilis can significantly promote the root growth of B. striata seedlings. B. subtilis treatments at all concentrations can significantly increase the number of fibrous roots and promote the root elongation of B. striata seedlings. In particular, the promotion effects of B. subtilis treatment at higher concentrations are more significant. These results are consistent with the effect of B. subtilis on promoting root growth in other plants. Cai et al. found that B. subtilis can increase the synthesis of indole acetic acid (IAA) and other auxins and reduce plant hormones (e.g., abscisic acid) that inhibit plant growth, thereby stimulating root development and plant growth. Zhang et al. revealed that the number of roots and root elongation of sweet potato seedlings were increased significantly after treatment with B. subtilis mutant strains with increased 3-IAA and cytokinin production capacity. Therefore, the effect of B. subtilis on plant root development is probably achieved by regulating the hormones in plants.
In this study, the application of B. subtilis suspensions had no obvious effect on the leaf size of B. striata seedlings. However, B. striata seedlings treated with various concentrations of B. subtilis suspensions all showed different degrees of increases in the relative chlorophyll content in leaves, which increased with the increasing B. subtilis concentration. The relative chlorophyll content of plant leaves is an important indicator that can effectively reflect the photosynthesis of plants. It has been found from the studies on wheat and pepper that B. subtilis can significantly increase the chlorophyll content in the leaves.,, The application of B. subtilis inoculants can promote the synthesis of chlorophyll, thereby increasing the photosynthesis rate in the leaf tissues to provide more nutrients for plant growth.
Results of this study show that the high concentration of B. subtilis suspension (1/10 × dilution) can significantly promote the plant height and base diameter of B. striata seedlings. On the contrary, B. subtilis treatments at low concentrations have little or no promotion effects on the base diameter; although they can promote plant height to a certain extent, the differences are not significant (P > 0.05) compared to the control group. On the other hand, the application of different concentrations of B. subtilis suspensions did not show significant promotion effects on the tuber diameter and fresh weight of B. striata seedlings. Wang et al. studied the effects of B. subtilis on a series of physiological and biochemical indices of winter wheat without observing significant promotion effects of B. subtilis on plant height and root length and believed that this may be due to the varied functions among different B. subtilis strains.
Besides the significant growth promotion effect was observed in this study, the B. striata seedling mortality occurred in all the treatments, varying from 11.11% to 22.22%. It is tempting to speculate that this seedling mortality was related to less habitable environment in the study site for B. striata seedlings despite growing in greenhouse. The optimal habitat for B. striata is located at an altitude of 600 m–1000 m, whereas the average altitude of Wuhan (city where the study site located) is 23.3 m. On the other hand, the seedling mortality observed might be related to the variation of symbiotic effect in the symbiosis in different individual B. striata seedling due to the change of growth condition. There are studies shown that fungal colonization and nutrition transfer can be differentially regulated in the symbiosis, and the inherent differences among the fungi influence mycorrhizal symbioses might be more than previously suspected, implying the effect and mechanism of symbiosis are much more complicated than previously proposed. For those seedlings not survived, the regulation of symbiosis network might be influenced when growth condition altered.
The mechanisms of B. subtilis on promoting plant growth have been extensively studied. For example, it has been shown that B. subtilis can produce a variety of biologically active enzymes, which can regulate plant growth and chlorophyll accumulation in varying degrees; however, further studies remain to be conducted on the roles of these enzymes. Other studies have also shown that B. subtilis can promote plant growth by improving soil qualities such as soil texture, soil nutrients, and soil microbial environment., In an era of increasingly development of multiomics technologies, the research and utilization of B. subtilis are also intensified, which provide the possibility for better and in-depth utilization and development of B. subtilis.,
The study of the medicinal properties of Chinese medicinal materials poses higher requirements for the production. In the production of Chinese medicinal materials, guarantee not only the yield but also the quality requires reduced application of chemical fertilizers and pesticides, as well as the implementation of ecological cultivation. Meanwhile, secondary metabolites, which are usually the active chemical components and contribute to the quality of Chinese medicinal materials, require environmental stress., Hence, the balance between growth and secondary metabolites is essential in the cultivation of Chinese medicinal plant. “Simulative habitat cultivation” is proposed to have advantages in this balance and guaranteeing the quality of traditional Chinese medicinal materials. As a new type of bioinoculant, B. subtilis is pollution-free, environment-friendly and can improve the soil conditions, which is in line with the philosophy of “simulative habitat cultivation.” Therefore, B. subtilis has broad application prospects in the ecological cultivation of Chinese medicinal materials.
| Conclusion|| |
In this study, the Bacillus subtilis has significant growth promotion effects on Bletilla striata seedlings through increasing the relative chlorophyll content, promoting growth and development of roots, and increasing plant height and base diameter of B. striata seedlings. These findings are instructive for the ecological cultivation practice of B. striata.
We would like to thank TopEdit (www.topeditsci.com) for English language editing of this manuscript.
Financial support and sponsorship
This work was supported by the National Key R&D Program of China (2019YFC1711100).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 1]
[Table 1], [Table 1]