Endophytic bacterium for tissue culture seedling of flintley and application of endophytic bacterium

Abstract

The invention discloses endophytic bacteria for tissue culture seedlings of flintley and application of the endophytic bacteria, and relates to the technical field of microorganisms. The endophytic bacterium (Bacillus sterilis) OdE-III of the tissue culture seedling of the flintley is preserved in the China Center for Type Culture Collection on September 1, 2025, and the preservation number of the endophytic bacterium is CCTCC (China Center for Type Culture Collection) NO: M 20251913. The flintleaf flintleaf herb endophytic bacterium OdE-III provided by the invention is derived from a flintleaf flintleaf herb tissue culture seedling plant stem, can inhibit the growth of pathogenic bacteria in soil and improve the growth vigor of the flintleaf flintleaf herb tissue culture seedling after transplanting, breaks through the technical bottleneck that the high-quality flintleaf flintleaf herb is dead of illness and poor in growth after transplanting, and is beneficial to promoting the artificial planting of the flintleaf flintleaf herb.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to endophytic bacteria from a tissue culture seedling of *Ficus microcarpa* and its applications. Background Technology

[0002] flint flower ( Oreoseris delavayi (Franch.) is native to Yunnan, southern Sichuan, and other regions. It is a perennial herbaceous plant belonging to the Asteraceae family. It gets its name from the fact that the fibers on the back of its leaves were used as tinder for starting fires with flint. It is also known as fire grass or hook-budded large-headed grass. Gerbera delavayi Franch.). Currently, through taxonomic research, it has been reclassified from the genus *Gnaphalium* to the genus *Gnaphalium* (Flammulina). Oreoseris Currently, *Firewood* is mainly used in textiles and cut flower production. Because its roots contain antibacterial components such as coumarins, it can also be used as a traditional Chinese medicine to treat dysentery, stomachache, and indigestion. *Firewood* textiles are the only technique that directly uses the felt fibers from the back of plant leaves for hand-weaving, and it has been listed as a national intangible cultural heritage at various levels. *Firewood* textiles are also highly sought-after collectibles by many enthusiasts of ethnic culture both domestically and internationally; thus, the market potential for *Firewood* fiber is enormous.

[0003] However, wild *Firewood* resources are scarce, making artificial cultivation the most effective way for its protective development. During artificial cultivation, the supply of high-quality seedlings is a prerequisite for successful cultivation. Research has found that *Firewood* is a completely heteropollinated plant; offspring propagated from seeds exhibit significant phenotypic segregation, resulting in poor consistency and stability in fiber quality and yield, which is extremely detrimental to the industrial-scale cultivation of *Firewood*. Therefore, the most suitable method for producing high-quality *Firewood* seedlings is tissue culture rapid propagation. However, through seven years of practice, the applicant has found that transplanting tissue-cultured *Firewood* seedlings has become a technical bottleneck in seedling propagation. Specifically, after transplanting, tissue-cultured *Firewood* seedlings are prone to stem rot, leading to shortened stems and necrosis, which is the main reason for the low survival rate of transplanted tissue-cultured *Firewood* seedlings. Generally, the pathogens causing stem rot and root rot in potted plants are mainly *Phytophthora* species (…). Phytophthora Fungi, Pythium ( Pythium Fungi, Fusarium ( ) Fusarium Fungi (such as Fusarium oxysporum) F. oxysporum ), Rhizoctonia spp. ( Rhizoctonia Fungi, Agrobacterium ( AgrobacteriumThese pathogens are caused by bacteria, among other things. While these pathogens are also abundant in the soil of wild *Flammulina lucida*, wild *Flammulina lucida* rarely suffers from stem rot and root rot. Conversely, tissue-cultured *Flammulina lucida* seedlings are mostly sterile because the endophytic bacteria in the explants may have been killed during the initial sterilization process. When transplanting tissue-cultured *Flammulina lucida* seedlings, the lack of beneficial endophytic bacteria antagonizing the pathogens causing stem rot and root rot leads to widespread stem and root rot, ultimately resulting in a transplant survival rate of less than 50%.

[0004] In view of the above, it is necessary to study the endophytic bacteria in *Firestone* tissue culture seedlings and their applications to solve the aforementioned technical problems. Summary of the Invention

[0005] The purpose of this invention is to provide an endophytic bacterium for a tissue culture seedling of *Ficus microcarpa*. This endophytic bacterium, OdE-III, is derived from the stem of the *Ficus microcarpa* tissue culture seedling and can inhibit the growth of pathogens in the soil and improve the growth of *Ficus microcarpa* tissue culture seedlings after transplanting. This invention breaks through the technical bottleneck of disease and death and poor growth of high-quality *Ficus microcarpa* seedlings after transplanting, and is conducive to promoting the artificial cultivation of *Ficus microcarpa*.

[0006] To achieve the above objectives, the present invention provides an endophytic bacteria from a tissue culture seedling of *Ficus pumila*, wherein the endophytic bacteria from the tissue culture seedling of *Ficus pumila* (… Bacillus stercoris OdE-III was deposited on September 1, 2025 at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China; accession number: CCTCC NO: M 20251913.

[0007] Specifically, the application of the endophytic bacteria OdE-III in the tissue culture seedlings of *Firewood* in inhibiting the growth of pathogens in the soil and thus improving the growth of *Firewood* tissue culture seedlings after transplanting.

[0008] Specifically, the pathogen is Fusarium graminearum II, Fusarium equisetifolium, Fusarium graminearum I, or Fusarium oxysporum.

[0009] The present invention also provides a formulation comprising OdE-III, an endophytic bacterium from *Firestone* tissue culture seedlings.

[0010] Specifically, the preparation is an agent that inhibits the growth of pathogens in the soil, thereby improving the growth of *Firewood* tissue culture seedlings after transplanting.

[0011] To achieve the above objectives, the present invention employs the following method: (1) Isolation and purification of endophytic bacteria from *Ficus pumila* tissue culture seedlings: Fresh fleshy roots, shortened stems, and petioles of well-grown *Ficus pumila* tissue culture seedlings were taken. After disinfecting the outside of the tissue culture bottles with alcohol, they were placed in a clean bench after UV sterilization for 15 min. The fleshy roots, shortened stems, and petioles were soaked in 70% ethanol for 60 s. After soaking, they were rinsed three times with sterile water. The water was then absorbed with sterile filter paper. The tissues were cut into small pieces (5 mm × 5 mm) using a sterile scalpel and scissors and placed on a PDA culture plate. The culture medium was placed in a constant temperature incubator at 28℃ for incubation. After incubation, the colony morphology was observed. After 3–5 generations of purification, pure colonies were obtained, and the purified strain was obtained.

[0012] (2) Molecular identification: Genomic DNA was extracted from the purified strain using the CTAB method. PCR amplification of the genomic DNA was performed using universal primers for the 16S rRNA gene (27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3'). The reference PCR conditions were: 94℃ pre-denaturation for 3 min, followed by 34 cycles (94℃ 25 s, 60℃ 25 s, 72℃ 2 min), and a final extension at 72℃ for 5 min. PCR products were detected by 1% agarose gel electrophoresis and sent to a biotechnology company for sequencing. The obtained sequences were manually verified and then compared online using NCBI's BLAST database. Based on base similarity, the strain was molecularly identified, and its phylogenetic tree was constructed.

[0013] (3) Transplanting of tissue culture rooted seedlings: After the root length of the tissue culture seedlings of *Firestone* exceeds 3 cm, transplant the seedlings after hardening them for 4-5 days. The transplanting substrate is a mixture of humus and perlite in a mass ratio of 2:1.

[0014] (4) Re-inoculation of endophytic bacteria from *Firewood*: The bacterial strain in solid culture medium was inoculated into a conical flask containing liquid culture medium and cultured by shaking for 30 h to prepare a liquid bacterial agent. The concentration was adjusted to 10% using blank liquid culture medium. 6 -10 8 CFU·mL -1 (OD value between 0.4 and 0.6). The inoculum was inoculated using the root drenching method, while the control group used a blank liquid culture medium. 5 ml was inoculated. After transplanting, the *Firewood* plants were covered with plastic film for 5-7 days, and their growth and development were observed 15-20 days after transplanting.

[0015] (5) Screening of beneficial endophytic bacteria: The survival rate of transplanted seedlings and their growth compared with the control group were used to determine whether the transplanted seedlings could improve the growth and reproduction of *Firewood* tissue culture seedlings in the later stage of transplanting. In this way, beneficial endophytic bacteria that can improve the growth and reproduction of *Firewood* transplanted seedlings were screened.

[0016] (6) Antagonistic experiment against soil pathogenic fungi: After activating the pathogenic fungi, they were transferred to new PDA medium. Endophytic bacteria were inoculated in three parallel streaks, one strain each in the four cardinal directions (north, south, east, and west) of the pathogenic fungi, spaced 3 cm apart. The streaks were approximately 2-3 cm long. The streaks were repeated three times. After incubation at 28°C for 6-8 days, the presence of antagonistic activity was observed.

[0017] The antagonistic bacteria from the previous step were inoculated onto newly inoculated plates of the same pathogenic fungus. The same bacteria was inoculated on both sides, 3 cm apart, with streaks approximately 2-3 cm long, three times in parallel. The plates were incubated at 28°C for 10 days, and the inhibition rate was calculated. The control group consisted of pathogenic bacteria plates without endophytic bacteria.

[0018] Antibacterial rate (%) = (Fungal expansion radius of control group - Colony radius of endophytic bacteria culture) / Fungal expansion radius of control group.

[0019] (7) After the transplanted flint flowers survive, select the flint flowers with good growth and use the strains of flint flower fleshy roots, shortened stems and petioles in step (1) to re-isolate and culture them to verify whether the inoculation of endophytic fungi was successful.

[0020] The OdE-III endophytic bacteria for *Firewood* of this invention is derived from *Firewood* tissue culture seedlings. It can inhibit the growth of pathogens in the soil and improve the growth of *Firewood* tissue culture seedlings after transplanting, breaking through the technical bottleneck of high-quality *Firewood* seedling propagation and facilitating the artificial cultivation of *Firewood*. Furthermore, *Firewood* plants inoculated with beneficial endophytic bacteria exhibit good growth vigor in later stages of growth and development. Attached Figure Description

[0021] Figure 1 Purification and culture of endophytic bacteria for this invention; Figure 2 This is the microscopic morphology of *Femtobacter* in this invention; Figure 3 This is a phylogenetic tree of *Faecalibacterium* according to the present invention; Figure 4 The growth curve of *Faecalibacterium* in this invention is shown. Figure 5 This invention relates to rooting and culturing *Flintstoneia* tissue culture seedlings. Figure 6 This is a growth observation diagram of *Flammulina velutipes* tissue culture seedlings transplanted after inoculation with *Bacillus flavus* according to the present invention. Figure 7 This invention relates to the antagonism of *Femtobacter faecium* against soil pathogens. Detailed Implementation

[0022] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application. Example

[0023] 1.1 Using PDA medium, culturable endophytic bacteria were isolated from the shortened stems of *Sedum lineare* tissue culture seedlings. Colony morphology data during the culture process were recorded, and the results are shown below. Figure 1 As shown. By Figure 1 Ten endophytic bacteria strains were screened from the *Firewood* tissue culture seedlings, and their colonies were milky white. Colonies of OdE I-VI had irregular, diffuse edges, a frosted glass-like luster, and were opaque; colonies of OdE VII-X were round with regular edges, smooth surfaces, smaller diameters, and were semi-transparent. Simultaneously, PCR amplification and BLAST comparison of the 16S rRNA gene of the endophytic bacteria were performed to determine the taxonomic position of the isolated strains.

[0024] 1.2 Isolation and purification of endophytic bacteria from *Ficus pumila* tissue culture seedlings: Fresh fleshy roots, shortened stems, and petioles from well-rooted *Ficus pumila* tissue culture seedlings were collected. The tissue culture bottles were disinfected externally with alcohol and then placed in a clean bench after UV sterilization for 15 minutes. The fleshy roots, shortened stems, and petioles were soaked in 70% ethanol for 60 seconds, followed by rinsing three times with sterile water. The tissue was then blotted dry with sterile filter paper. The tissue was cut into 5 mm × 5 mm pieces using a sterile scalpel and scissors and placed on a PDA culture plate. The culture medium was incubated at 28℃. After 3–5 generations of purification, pure colonies were obtained. The purified bacterial strain was obtained, and the colony micromorphology was observed. The colony micromorphology is shown in the figure below. Figure 2 As shown. By Figure 2 As can be seen under the microscope, OdE-III appears as a short rod, indicating that it is a Gram-negative bacterium. The two ends of the bacterial body are straight or slightly rounded, and there is no obvious internal structure.

[0025] 1.3 Molecular identification of endophytic bacteria: Genomic DNA was extracted from the purified strain using the CTAB method. PCR amplification of the endophytic bacteria's genomic DNA was performed using universal primers for the 16S rRNA gene. 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-GGTTACCTTGTTACGACTT-3'); The PCR reaction conditions were as follows: pre-denaturation at 94℃ for 3 min, followed by 34 cycles (94℃ for 25 s, 60℃ for 25 s, 72℃ for 2 min), and extension at 72℃ for 5 min after each cycle. PCR products were detected by 1% agarose gel electrophoresis and sent to a sequencing company for sequencing. The sequence is shown in SEQ ID NO:1.

[0026] SEQ ID NO:1: After manual verification, the obtained SEQ ID NO:1 sequence was compared with the NCBI online database using BLAST. Based on sequence similarity, the strain was molecularly identified, and its phylogenetic tree was constructed. OdE-III was sequenced using 16S rRNA, identified by comparison with NCBI, and a phylogenetic tree was drawn, as shown below. Figure 3 As shown. Figure 3 The results showed that fecal bacteria ( Bacillus stercoris The OdE III strain showed the highest 16S rRNA sequence similarity (99.31%) to the D7XPN1 strain (NR 181952.1); OdE-III and Bacillus stercoris strain The D7XPN1 strain (NR181952.1) has a high degree of kinship.

[0027] Endophytic bacteria in the tissue culture seedlings of *Ficus pumila* ( Bacillus stercoris OdE-III was deposited on September 1, 2025 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China; accession number CCTCC NO: M20251913.

[0028] 1.4 After rooting and acclimation of *Flammulina velutipes* tissue culture seedlings, hardening-off transplanting was carried out. New strains of *F. velutipes* cultured in liquid were then inoculated. The growth after transplanting was compared with that of the control to determine whether the transplanting improved the growth of *F. velutipes* tissue culture seedlings. At the same time, antagonistic experiments with pathogenic fungi in the soil were combined to determine whether the new strains of *F. velutipes* were beneficial endophytic bacteria for *F. velutipes*.

[0029] The method for re-inoculating endophytic bacteria is as follows: promote the root growth and development of *Pterocarya stenoptera* tissue culture seedlings using conventional rooting culture medium, and wait until the root length of the *Pterocarya stenoptera* tissue culture seedlings exceeds 4 cm ( Figure 5 After 7-10 days of hardening off, the tissue culture seedlings are transplanted into a substrate of perlite and humus in a mass ratio of 2:1.

[0030] Growth curves of *Bacillus faecalis* OdE-III strain were plotted to determine the start times of its logarithmic growth and stationary phases. This provided a basis for selecting the culture time of the bacterial suspension with the highest activity in subsequent plant inoculation experiments. The growth curves are shown below. Figure 4 As shown. By Figure 4 It can be seen that *Faecalibacterium* OdE-III is in the logarithmic growth phase within 12-36 hours, and enters the stationary phase after 36 hours. Therefore, to ensure the activity of the strain, seed culture solution that has been cultured for 30 hours was used for inoculation in subsequent re-inoculation experiments of tissue culture seedlings.

[0031] Re-inoculation of endophytic bacteria from *Firewood*: Inoculate the bacterial strain from solid culture medium into a conical flask containing liquid culture medium, and culture by shaking for 30 hours to prepare a liquid inoculum. Adjust the concentration to 10% using blank liquid culture medium. 6 -10 8 CFU·mL -1 (OD value between 0.4 and 0.6). The inoculum was inoculated using the root drenching method, with a blank liquid culture medium used as the control group; 5 ml was inoculated. After transplanting, the *Flammulina velutipes* plants were covered with plastic film for 5-7 days, and their growth and development were observed 15 days after transplanting. The growth observation of *Flammulina velutipes* tissue culture seedlings re-inoculated with *Bacillus foetida* after transplanting is as follows: Figure 6 As shown, by inoculating tissue culture seedlings with OdE-III bacterial suspension (treatment) and blank culture medium (control), and observing 15 days after transplanting, it was found that OdE-III had a certain growth-promoting effect on the growth of tissue culture seedlings of *Firewood* after transplanting.

[0032] 1.5 Antagonistic Experiment of Endophytic Bacteria from *Firewood* Tissue Culture Seedlings against Soil-borne Pathogenic Fungi: After activating the pathogenic fungi, they were transferred to fresh PDA medium. Endophytic bacteria were streaked onto four sides of the pathogenic fungus, one strain each in the four cardinal directions (north, south, east, and west), spaced 3 cm apart, with streaks approximately 2-3 cm long, and repeated three times. After 7 days of incubation at 28℃, the presence of antagonistic activity was observed. The bacteria exhibiting antagonism in the previous step were inoculated on newly inoculated pathogenic fungi plates. The same bacteria was inoculated on both sides at a distance of 3 cm, with streaks of approximately 2-3 cm in length, and repeated three times. The plates were incubated at 28°C for 10 days, and the inhibition rate was calculated. The control group consisted of pathogenic bacteria plates without endophytic bacteria.

[0033] Antibacterial rate (%) = (Fungal expansion radius of control group - Colony radius of endophytic bacteria culture) / Fungal expansion radius of control group.

[0034] The antagonistic effects of *Faecalibacterium* on soil pathogens are shown in Table 1 and... Figure 7 As shown in Table 1 and Figure 7 It can be seen that *Femtobacter foetida* OdE-III exhibits antagonistic activity against *Fusarium graminearum* 1, *Fusarium graminearum* 2, *Fusarium equisetifolium*, and *Fusarium oxysporum* pathogens in the soil; among them, the antagonistic activity against *Fusarium graminearum* 2 and *Fusarium oxysporum* is more pronounced, with an inhibition rate of over 60%.

[0035]

[0036] The screening method for beneficial endophytic bacteria is as follows: After the *Firewood* plants have survived transplanting, select the *Firewood* plants with good growth and use step (1) to re-isolate and culture the strains from the fleshy roots, shortened stems and petioles of *Firewood* to verify whether the inoculation of endophytic bacteria was successful.

[0037] Through screening, the endophytic bacteria that can improve the survival rate of *Firewood* tissue culture seedlings after transplanting were identified as *Faecalibacterium* (Fragillus spp.). Bacillus stercoris The growth of *Flintstoneia* tissue culture seedlings inoculated with a single endophytic bacterium was significantly better than that of the control plants.

[0038] In summary, the OdE III endophytic bacteria of *Firewood* provided by this invention is derived from *Firewood* plant tissue culture seedlings. It can inhibit the growth of pathogens in the soil and improve the growth and development of *Firewood* tissue culture seedlings in the later stages of transplanting, which is beneficial for promoting the artificial cultivation of *Firewood*.

[0039] The specific embodiments of the invention have been described in detail above, but these are merely examples, and the invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications or substitutions to the invention are also within the scope of this invention. Therefore, all equivalent transformations, modifications, and improvements made without departing from the spirit and principles of this invention should be covered within the scope of this invention.

Claims

1. An endophytic bacteria of tissue culture seedling of Epipactis helleborine, characterized in that, The endophytic bacteria in the firestone flower tissue culture seedlings Bacillus stercoris OdE-III, deposited on September 1, 2025 in China Center for Type Culture Collection, with the preservation number of CCTCC NO: M 20251913.

2. The endophytic bacteria of the tissue culture seedling of Flos Carthami as claimed in claim 1, characterized in that, The application of the endophytic bacteria OdE-III in the firestone flower tissue culture seedlings to inhibit the growth of pathogenic bacteria in the soil and further improve the growth of the firestone flower tissue culture seedlings after transplanting.

3. The endophytic bacteria of the tissue culture seedling of Flos Carthami as claimed in claim 1, wherein, The pathogenic bacteria are Fusarium graminearum II, Fusarium equiseti, Fusarium graminearum I or Fusarium oxysporum.

4. A formulation characterized in that, The preparation comprises the endophytic bacteria OdE-III in the firestone flower tissue culture seedlings as claimed in any one of claims 1-3.

5. The formulation of claim 4, wherein, The preparation is a preparation for inhibiting the growth of pathogenic bacteria in the soil and further improving the growth of the firestone flower tissue culture seedlings after transplanting.

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