Bacteriostatic agent for nymphaea tetragona tissue culture as well as preparation method and application thereof

Abstract

The invention belongs to the field of plant tissue culture, and particularly relates to a bacteriostatic agent for tissue culture of snow white water lily as well as a preparation method and application of the bacteriostatic agent. The bacteriostatic agent comprises an azoxystrobin suspending agent, carbendazim and mancozeb wettable powder. The bacteriostatic agent consists of a plurality of effective bacteriostatic components, can effectively control the growth of endophytes of the snow-white water lily, reduces the fungal infection rate of the snow-white water lily, and has an inhibition effect on different endophytic fungi. In the tissue culture process, after the bacteriostatic agent is used, the contamination rate of the tissue in the culture process can be remarkably reduced, the normal operation of the tissue dedifferentiation process is effectively ensured, and the success rate of the tissue culture is improved.

Description

Technical Field

[0001] The present invention belongs to the field of plant tissue culture, and particularly relates to an antibacterial agent for tissue culture of Nymphaea candida, a preparation method thereof, and an application thereof. Background Art

[0002] Nymphaea candida Presl is the only cold-tolerant water lily in China, and is mainly distributed in the Bosten Lake in Xinjiang, China. The petals of Nymphaea candida contain flavonoids, saponins, phenolic substances, etc., which have pharmacological effects such as antibacterial and anti-inflammatory, antioxidant, etc., and have high medicinal value. It has been developed into a traditional Chinese medicine for relieving summer heat, relieving alcoholism, and calming the nerves. As a floating-leaved plant, Nymphaea candida can effectively reduce water pollutants and has the effect of purifying water quality. The growth of Nymphaea candida is easily affected by water-borne diseases and water quality changes, and its seeds will be predated by a large number of waterfowl, resulting in a very low natural reproduction rate. At present, Nymphaea candida has been included in the "Red List of Endangered Species of the International Union for Conservation of Nature" (IUCN) and the list of the second-level national key protected wild plants in China. To prevent the extinction of this rare species, it is urgent to carry out the protection of germplasm resources and seedling breeding of Nymphaea candida. The plant tissue culture technology is based on the theory of plant cell totipotency, and uses isolated organs, tissues or cells to induce callus, adventitious buds, adventitious roots under sterile and suitable conditions, and finally form complete plants. At present, the plant tissue culture technology has become an important tool for plant improvement and rapid propagation, and has been successfully applied not only to terrestrial plants, but also to a large number of aquatic plants. In addition, the plant tissue culture technology has been successfully used for the breeding of endangered species and is expected to become an effective means for protecting the germplasm resources of Nymphaea candida.

[0003] The success of plant tissue culture is closely related to the prevention and control of microbial contamination. Multiple factors such as incomplete sterilization of explants and the transfer process of tissue culture bottles will cause microbial contamination of the culture medium. The culture medium also contains many nutrients and vitamins, etc., which will promote the growth of microorganisms and cause the explants to fail to form callus smoothly. During the plant tissue culture process, the surface of the explants will be disinfected, which will kill the surface microorganisms. Endogenous microbial contamination of plants is the main reason limiting the success of plant tissue culture. By adding antibiotics during the plant tissue culture process, it has played a good role in preventing and controlling endogenous bacterial contamination.

[0004] Therefore, the plant tissue culture technology is usually limited by endogenous fungal contamination, which seriously affects the growth of explants during the plant tissue culture process. Due to the proliferation of endogenous fungi, many explants are damaged, resulting in a tissue culture failure rate as high as 40-60%, and finally resulting in slow growth or even death of the callus. A good antibacterial agent can not only improve the success rate of tissue culture, but also reduce the complexity of manual operations, simplify aseptic operations, and reduce the cost of tissue culture. Summary of the Invention

[0005] To solve the above problems, the present invention provides an antibacterial agent for tissue culture of Nymphaea candida, and the components include azoxystrobin, carbendazim and mancozeb.

[0006] Further, the dosage form of azoxystrobin is a suspension concentrate.

[0007] Further, the dosage form of mancozeb is a wettable powder.

[0008] Further, the concentration ratio of azoxystrobin, carbendazim and mancozeb is 1:10:10.

[0009] The present invention also provides a preparation method of the above antibacterial agent, including the following steps:

[0010] Dissolve 1 mg of azoxystrobin suspension concentrate and 10 mg of mancozeb wettable powder in 900 ml of distilled water sterilized at high temperature. After the mixed solution is subjected to the first ultrasonic treatment, the mixed solution is filtered through a 0.22 μm filter membrane, then 10 mg of carbendazim is added, and the second ultrasonic treatment and filter membrane filtration are carried out. Finally, the volume is fixed to 1000 ml to prepare a mother liquor of the antibacterial agent. When in use, it is added to the culture medium in a ratio of 1:9 so that the final concentration is 1 / 10 of the mother liquor.

[0011] Further, the conditions for the first ultrasonic treatment are: 50 °C, 50 KHz; the conditions for the second ultrasonic treatment are: room temperature, 50 KHz.

[0012] The present invention also provides the application of the above antibacterial agent in antibacterial of plant tissue culture.

[0013] Further, it is used for inhibiting the growth of endophytic bacteria of Nymphaea candida.

[0014] The present invention has the following beneficial effects:

[0015] In the current technology, carbendazim is widely used as an antibacterial agent for plant tissue culture, but azoxystrobin suspension concentrate and mancozeb wettable powder are rarely used in the tissue culture process. Experiments show that only using a single antibacterial agent only has an inhibitory effect on one or several types of fungi, while the combined antibacterial agent can achieve broad-spectrum fungal inhibition. At the same time, for example, the antibacterial agents for tissue culture such as Chinese Patent No. 201711167809.3 mostly target exogenous fungi and infected fungi, and rarely design antibacterial agents for endophytic fungi in plant tissues. Therefore, the present invention designs a combined antibacterial agent of multiple antibacterial drugs for the endophytic fungi of Nymphaea candida in order to achieve the inhibitory effect on endophytic fungi.

[0016] The present invention uses three chemical substances as raw materials and mixes them in proportion to form an antibacterial liquid. And through the fungal isolation technology, the endophytes of Nymphaea candida were isolated. This antibacterial agent is designed for endophytes, which can not only effectively inhibit the growth of endophytes, reduce the infection rate, but also improve the success rate of tissue culture, reduce the cost of tissue culture, simplify the steps of manual operation, and reduce the cost and failure rate. Brief Description of the Drawings

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0018] Figure 1 It is the community composition and abundance of endophytic fungi in Example 1. The community composition of endophytic fungi in the root, stem, and leaf tissues of Nymphaea candida and the relative abundances of each species in different tissues (A - B). In (A), different colors represent different groups (or samples), the numbers in the overlapping parts represent the number of species common to multiple groups (or samples), and the numbers in the non - overlapping parts represent the number of species unique to the corresponding group (or sample); in (B), the horizontal axis is the sample number, and the vertical axis is the relative abundance ratio of the species. The color corresponds to the name of each species at this taxonomic level, and the width of different color blocks represents the relative abundance ratio of different species.

[0019] Figure 2 It is the morphology, species, and evolutionary status of endophytic fungi in Example 1. (A - D) respectively represent the fungi R1, R2, R3, and R4 isolated from the tissue culture bottles of the roots of Nymphaea candida; (E - F) respectively represent the fungi L1 and L2 isolated from the tissue culture bottles of the leaves of Nymphaea candida; (G - H) respectively represent the fungi B1 and B2 isolated from the tissue culture bottles of the buds of Nymphaea candida; (I) represents the fungus S1 isolated from the tissue culture bottle of the stem of Nymphaea candida; (J) is the infection rate of each purified endophytic fungus; (K) is the evolutionary status of the identified endophytic fungi. The branching pattern was generated by the neighbor - joining method, and a bootstrap analysis with 1000 replicates was performed, with 0.5 or 1.0 nucleotide substitutions per site.

[0020] Figure 3Antibacterial effect of the antibacterial agent on endophytic fungi in Example 1. Antibacterial conditions of five antibacterial drugs at different concentrations against three endophytic fungi (A-C). (A-C) respectively represent the antibacterial rates of five antibacterial drugs at different concentrations against Alternariasp., Phytopythium helicoides, and Fusarium oxysporum. SD represents sodium dichloroisocyanurate, C represents carbendazim, CWP represents chlorothalonil wettable powder, PS represents azoxystrobin suspension concentrate, and MWP represents mancozeb wettable powder.

[0021] Figure 4 Bacterial contamination situation of tissue culture without adding antibacterial agent in Example 2. Morphology of different tissues of Nymphaea candida and phenotypic of fungal contamination in tissue culture flasks (A-J). (A) shows the tissue morphology and bacterial contamination rate of roots, leaves, stems, flowers, and buds during the tissue culture of Nymphaea candida; (B-J) respectively show the growth conditions of different fungi around the explants in the tissue culture flasks of Nymphaea candida.

[0022] Figure 5 Bacterial contamination situation of tissue culture with added antibacterial agent in Example 2. Bacterial contamination rate after adding antibacterial agent and its influence on the organs formed after the redifferentiation of root tissue and callus during the tissue culture of Nymphaea candida. (A) shows the bacterial contamination rate of each tissue after adding antibacterial agent, and (B-C) show the influence of adding antibacterial agent on the organs formed by root tissue and callus during the tissue culture of Nymphaea candida. Detailed implementation manners

[0023] Now, various exemplary implementation manners of the present invention will be described in detail. In the examples, the methods are all conventional methods unless otherwise specified, and the reagents are all conventional commercially available reagents or reagents prepared by conventional methods unless otherwise specified. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0024] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0026] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the description of the present invention, which will be apparent to those skilled in the art. Other embodiments obtained from the description of the present invention will be apparent to those skilled in the art. The description and examples of the present invention are merely exemplary.

[0027] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0028] Example 1 Exploration of the Inhibitory Effect of Bacteriostatic Agents on Endophytes in Plant Tissues

[0029] 1. Isolation, purification, DNA extraction, and amplicon identification of endophytic fungi from Nymphaea candida. (1) Refer to tissue culture for the collected Nymphaea candida. First, select 5 - 6 cm root, stem, and leaf tissue samples from the Nymphaea candida plant, rinse them with running tap water for 5 - 10 min to remove surface sludge. Subsequently, cut each tissue into small pieces about 1 cm in size, soak them in 75% ethanol for 10 s, rinse once with sterile water, then soak them in 0.1% mercuric chloride for 12 min, and rinse 5 times with sterile water to complete the surface sterilization treatment of the plant tissue. Add 50 ml of MS medium, 3.00 mg / L TDZ, and 0.20 mg / L NAA to a 240 ml culture flask, and adjust the pH of the mixed medium to 5.8 ± 0.1 with 1 mol / L HCl. Autoclave the container with the mixed medium (121 °C, 15 min). After the mixed medium solidifies, add 50 ml of sterile distilled water to prepare a double-layer medium. Directly inoculate the treated tissue samples into the culture container containing the double-layer medium for tissue culture. The culture process is carried out in a 28 °C dark constant temperature incubator. Each culture flask contains one explant. Observe once every 3 days until callus is successfully differentiated, count the contamination situation of different tissues during this process and take pictures.

[0030] (2) For the fungi that appear during the cultivation process, first, use an inoculation needle to cut a culture medium with a diameter of about 5 mm and containing hyphae of different phenotypes, and invert it in the center of a new PDA culture medium plate. Repeat the cultivation until the strain is completely purified. Subsequently, inoculate the purified strain onto a PDA slant medium by the streaking method. After the hyphae cover the slant, store it temporarily in a 4°C refrigerator.

[0031] (3) Grind the collected and purified endophytic fungal hyphae with sterile glass beads. Use a fungal DNA isolation kit (Beijing) to extract the genomic DNA of the strain, and use a 2×HieffTM PCR MasterMix conventional Polymerase Chain Reaction (PCR) premixed solution for ITS molecular identification. Use the universal primers NS1 (5'-GTAGTCATATGCTTGTCTC-3') / NS6

[0032] (5'-GCATCACAGACCTGTTATTGCCTC-3') to perform a PCR reaction to amplify the ITS sequence of the endophytic fungus of Nymphaea candida. The PCR reaction conditions are as follows: pre-cycle denaturation temperature of 95°C for 5 min, followed by 34 cycles, each cycle including denaturation, annealing, and extension at 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min. Finally, the extension temperature is 72°C for 10 min for extension. Sequencing is carried out by Bioengineering Co., Ltd. (Shanghai), and the obtained sequence is compared with other fungal sequences in the National Center for Biotechnology Information (NCBI) database by BLAST sequence homology comparison to obtain homologous sequences.

[0033] 2. Study on the inhibitory effects of different bacteriostatic agents on the endophytic fungus of Nymphaea candida.

[0034] Five antibacterial drugs, namely sodium dichloroisocyanurate, carbendazim, chlorothalonil wettable powder, azoxystrobin suspension concentrate, and mancozeb wettable powder, and three pathogenic bacteria, Alternaria sp., Phytopythium helicoides, and Fusarium venenatum, were selected for the experiment. This antibacterial test was divided into 21 test groups, including 1 control group (using a PDA plate without the agent as the control) and 20 treatment groups with five antibacterial drugs at concentrations of 0.01, 0.1, 1, and 10 μg / mL. First, the PDA medium was autoclaved (121 °C, 0.105 MPa, 15 min). When it cooled to 40 - 50 °C, different concentrations of antibacterial drugs were added according to the divided treatments in a laminar flow hood and the plates were poured. Subsequently, the three pathogenic bacteria, Alternaria sp., Phytopythium helicoides, and Fusarium venenatum, were inoculated onto the plates of each test group (n = 6). After culturing at 28 °C for 3 days, the colony morphology of each test group was observed, the colony diameter was measured by the cross method, and the growth inhibition rate of the antibacterial drugs on the hyphae of the three pathogenic bacteria in each treatment group was calculated according to the method of Sheikholeslami et al. (2020). The minimum concentration at which the hyphal growth of all the tested strains was inhibited was used as the minimum inhibitory concentration (MIC) of the tested agent against the tested strains.

[0035] The experimental results are as follows:

[0036] Figure 1 It was shown that there were no significant differences in the Alpha diversity of the endophytic fungi in the roots, stems, and leaves of Nymphaea candida. However, further analysis of the endophytic fungal community composition in different tissues of Nymphaea candida found that compared with other tissues, there were 3 unique endophytic fungal communities in the root tissue of Nymphaea candida, namely Amanita, Kohlmeyeriopsis, and Monoblepharella. Compared with the stem and leaf tissues, the proportions of Amphisphaeriaceae, Sclerotium, and Minutisphaera in the root tissue of Nymphaea candida were relatively high, being 38.03%, 6.83%, and 2.41% respectively. In the stem tissue, the proportions of Trichosporonaceae, Cladosporium, and norank - Erythrobasidiaseae were relatively high, being 6.43%, 3.00%, and 2.65% respectively. While in the leaf tissue, the proportions of Colletotrichum and Symmetrospora were relatively high, being 2.87% and 0.67% respectively.

[0037] Figure 2It was shown that a total of 9 endophytic fungi were obtained. The ITS sequencing results showed that the 9 fungi were Alternaria sp., Chaetomium globosum, Alternaria sp. SPS-04, Aspergillus fumigatus, Phytopythium helicoides, fungal sp., Trichoderma koningiopsis, Fusarium venenatum, and Trichoderma sp. 2F, respectively. Among them, Alternaria sp. and Alternaria sp. SPS-04 were both grayish-white, Fusarium venenatum was pinkish-white, fungal sp., Trichoderma koningiopsis, and Phytopythium helicoides were all white, Chaetomium globosum and Trichoderma sp. 2F were yellowish-white, and Aspergillus fumigatus was black. A total of 3 pathogenic bacteria were found among the 9 purified endophytic fungi: Alternaria sp., Phytopythium helicoides, and Fusarium venenatum, and their occurrence frequencies during the tissue culture of Nymphaea candida were 30.77%, 23.08%, and 23.08%, respectively.

[0038] Figure 3 It was shown that carbendazim had the best antibacterial effect on Alternaria sp., and the antibacterial rate was 100% when its concentration was 1 μg / mL. Mancozeb wettable powder had the best antibacterial effect on Phytopythium helicoides, and the antibacterial rate was 100% when its concentration was 1 μg / mL. Azoxystrobin suspension had the best antibacterial effect on Fusarium oxysporum, and the antibacterial rate was 100% when its concentration was 0.1 μg / mL.

[0039] Example 2 Exploration on the Effect of Bacteriostatic Agents in Reducing the Bacterial Infection Rate of Plant Tissue Culture

[0040] Add 50 ml of MS medium, 3.00 mg / L TDZ, and 0.20 mg / L NAA to the culture vessel. Before autoclaving, adjust the pH to 5.8 ± 0.1 with 1 mol / L HCl (121 °C, 0.105 MPa, 15 min). After sterilization, when the medium cools to 40 - 50 °C, add the antibacterial drug combination and cool to room temperature until it solidifies. Then add 50 ml of sterile distilled water to the culture vessel to prepare a double-layer medium. Finally, inoculate the disinfected explants onto the MS solid medium supplemented with the antibacterial drug combination. The subsequent experimental operations are the same as the tissue culture described in Example 1, and compare with the growth status of the explants in the tissue culture flask without the antibacterial drug combination to evaluate the effect of the antibacterial drug combination during tissue culture.

[0041] The experimental results are as follows:

[0042] Figure 4 It shows that when no antibacterial agent is added, endophytic fungal contamination occurs during the tissue culture of Nymphaea candida, and the highest infection rate of the root tissue is 33.33% ± 0.348; the infection rate of the leaf is the second highest, which is 27.77% ± 0.345; the infection rate of the flower is the lowest, which is 5.55% ± 0.159.

[0043] Figure 5 It shows that in the tissue culture of Nymphaea candida with the antibacterial agent combination of 0.1 μg / mL azoxystrobin suspension, 1 μg / mL mancozeb wettable powder, and 1 μg / mL carbendazim, the infection rate of the root tissue is 7.5% ± 0.033, a decrease of 25.83%, and the infection rates of the stem and leaf tissues drop to 0.

[0044] The above-described embodiments are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. An antibacterial agent for tissue culture of Nymphaea candida, characterized in that, The components include azoxystrobin, carbendazim and mancozeb.

2. The bacteriostatic agent according to claim 1, wherein The dosage form of azoxystrobin is a suspension.

3. The bacteriostatic agent according to claim 1, characterized in that, The dosage form of mancozeb is a wettable powder.

4. The bacteriostatic agent according to claim 1, wherein The concentration ratio of azoxystrobin, carbendazim and mancozeb is 1:10:

10.

5. The preparation method of the bacteriostatic agent according to claim 1, characterized in that, It includes the following steps: Dissolve 1 mg of azoxystrobin suspension and 10 mg of mancozeb wettable powder in 900 ml of high-temperature sterilized distilled water. After the mixed solution is subjected to the first ultrasonic treatment, the mixed solution is filtered through a 0.22 μm filter membrane, then add 10 mg of carbendazim, conduct the second ultrasonic treatment and filter membrane filtration, and finally make up the volume to 1000 ml to prepare a bacteriostatic agent stock solution. When in use, add it to the culture medium at a ratio of 1:9 so that the final concentration is 1 / 10 of the stock solution.

6. The preparation method according to claim 5, wherein, The conditions for the first ultrasonic treatment are: 50 °C, 50 KHz; the conditions for the second ultrasonic treatment are: room temperature, 50 KHz.

7. The application of the bacteriostatic agent according to claim 1 in inhibiting bacteria in plant tissue culture.

8. The application according to claim 7, characterized in that, It is used for inhibiting the growth of endophytes of Nymphaea candida.

Images