Tea tree phyllostachys paratacidophilus BFI-L14 and application thereof
By using the leaf foliar acidophilus BFI-L14 and its BVCs, the problems of drug residues and drug resistance caused by chemical control of anthracnose in tea trees have been solved, achieving the effect of green control and promoting tea tree growth.
Patent Information
- Application Number
- CN202511587282.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-03
- Publication Date
- 2026-02-24
AI Technical Summary
Current technologies for controlling anthracnose in tea trees mainly rely on chemical methods, leading to problems such as drug residues and pathogen resistance, and lacking green and integrated control measures.
The leaflet of tea plant BFI-L14 and its released volatile compounds (BVCs) were used to inhibit the growth of Camellia anthracnose and promote the growth of tea plants through gas/water phase diffusion. The inoculant and secondary metabolites were used to control anthracnose in tea plants and promote the growth of Arabidopsis thaliana.
BVCs significantly inhibited the growth of *Anthracnose spp.*, reduced the lesion area by 71.61%, and increased the fresh weight of *Arabidopsis thaliana* by 132.20%. They worked without physical contact and left no residue.
Smart Images
Figure CN121555348A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of agricultural microbial technology, specifically to a tea leaflet acidophilus strain BFI-L14 and its applications. Background Technology
[0002] Tea trees are perennial woody evergreen plants with high economic value. Due to their preference for warm and humid conditions and limitations in cultivation techniques, tea trees are prone to fungal diseases, with anthracnose, dominated by *Anthracnose spp.*, being the most prevalent. This disease significantly reduces tea yield and quality by interfering with the physiological metabolism of tea trees, causing enormous economic losses to the global tea industry. Currently, the control of anthracnose in tea trees still relies mainly on chemical control. While some effectiveness has been achieved, long-term application has led to increasingly prominent problems such as pesticide residues, environmental pollution, and the development of drug resistance in pathogens. Therefore, how to achieve green and integrated control of anthracnose in tea trees has become a hot topic of concern.
[0003] Plant microbial communities are mainly composed of bacterial and fungal communities with well-defined taxonomic structures on above-ground and underground organs, with bacteria considered the most abundant group in the microecology. Tea leaves grown in natural habitats are rich in a vast and diverse bacterial community. This community can synthesize and release bacterial volatile compounds (BVCs) with different physicochemical properties and biological activities. These BVCs can diffuse through the gas / aqueous phase and act as long-distance signaling molecules or chemical defense substances on target organisms, producing effects such as inhibiting plant pathogens, promoting plant growth, and inducing systemic resistance. These BVCs have a wide range of action, are highly volatile and easily degradable, and can function without physical contact, possessing enormous development potential.
[0004] Paracidovorax is a Gram-negative genus of bacteria, a newly separated genus from the Acidovorax family. It has found numerous applications in environmental pollution control. However, research on Paracidovorax in the phyllosphere of plants remains relatively limited, and the function of this genus in the phyllosphere environment of tea trees has not been fully explored. Therefore, investigating the functions of its BVCs (biologically modified vitamins) in disease prevention and growth promotion in tea trees has significant practical application value. Summary of the Invention
[0005] This invention provides a tea leaflet bacterium, Paracidovorax oryzae BFI-L14, and its applications. The tea leaflet bacterium provided by this invention is Paracidovorax oryzae BFI-L14, whose released BVCs significantly inhibited the growth of *Anthracis chinensis* in a two-plate confrontation, with the viable count adjusted to 10-1. 9The strain exhibited a CFU / mL inhibition rate of 92.31% against *Anthracnose spp.* on a two-part plate, and reduced the lesion area of detached leaves by 71.61% after fumigation. Furthermore, the BVCs released by this strain promoted the growth of *Arabidopsis thaliana*, increasing its fresh weight by 132.20%. These results provide valuable resources for the research of acidophilus bacteria and the development and application of disease-preventing and growth-promoting strains.
[0006] This invention relates to the leaf foliage acidophilus BFI-L14, which is *Paracidovorax oryzae*, and was deposited on September 12, 2025, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), classified as *Paracidovorax oryzae*, with accession number CGMCCNo. 35900.
[0007] The 16S rRNA gene (16S rDNA) sequence of the tea leaf acidophilus BFI-L14 described in this invention is shown in SEQ ID NO:1; in its transcribed RNA sequence, all Ts are replaced with Us.
[0008] The present invention also provides a culture of the leaf foliar acidophilus BFI-L14 of the tea plant.
[0009] The present invention also provides a microbial agent containing the tea leaflet acidophilus BFI-L14.
[0010] The present invention also provides secondary metabolites of the tea leaflet acidophilus BFI-L14.
[0011] The leaflet of tea plant BFI-L14 described in this invention can be a liquid or solid inoculant.
[0012] In this invention, the culture of *Acidic acid bacteria BFI-L14* from the leaf folium of *Tea folium*, the inoculum containing *Acidic acid bacteria BFI-L14* from the leaf folium of *Tea folium*, and the secondary metabolites of *Acidic acid bacteria BFI-L14* from the leaf folium of *Tea folium* can be prepared using conventional techniques.
[0013] The present invention also provides the application of the culture of the tea foliar acidophilus BFI-L14, the inoculum containing the tea foliar acidophilus BFI-L14, or the secondary metabolites of the tea foliar acidophilus BFI-L14 in the prevention and control of anthracnose in tea or the promotion of Arabidopsis thaliana growth.
[0014] The secondary metabolites of the tea leaf acidophilus BFI-L14 described in this invention include volatile compounds (BVCs).
[0015] BVCs have good penetrability and can diffuse efficiently in air and soil pores. They have a wide range of effects, are volatile, easily degraded, and leave no residue. They can inhibit plant pathogens, promote plant growth, and induce systemic resistance without physical contact.
[0016] The tea leaflet acidophilus BFI-L14 described in this invention can grow on R2A, LB, PDA and NFA media, and the culture temperature is generally 25-37℃.
[0017] The BVCs released by the isolated acid bacteria BFI-L14 of this invention have a significant inhibitory effect on Camellia anthracnose, significantly reduce the severity of leaf disease, and also have the characteristic of promoting Arabidopsis thaliana growth.
[0018] The BVCs produced by *Paracidovorax oryzae* BFI-L14 provided by this invention exhibit significant advantages in the control of anthracnose in tea plants and in promoting plant growth. On two-petal dishes, the released BVCs significantly inhibited the growth of *Anthracnose fungi* spp. in *Camellia sinensis*, with the viable cell count adjusted to 10-1. 9 The inhibition rate reached 92.31% after CFU / mL, and the same number of viable bacteria reduced the lesion area by 71.61% in detached leaf experiments. Furthermore, the BVCs produced by this strain had a significant growth-promoting effect on Arabidopsis thaliana, increasing fresh weight by 132.20%. Attached Figure Description
[0019] Figure 1 This is a schematic diagram of the colony and microscopic morphology of BFI-L14 of Acidic Bacteria parasiticus after Gram staining.
[0020] Figure 2 Phylogenetic tree diagram of acid bacteria BFI-L14.
[0021] Figure 3 Figure showing the preliminary identification results of the ability of BVCs of acidophilus BFI-L14 to inhibit the growth of anthracnose bacillus camelliae.
[0022] Figure 4 Figure showing the optimal working concentration of BFI-L14 for inhibiting Bacillus anthracis.
[0023] Figure 5 The graph shows the control effect of BVCs produced by Acidic bacteria BFI-L14 on anthracnose in tea trees.
[0024] Figure 6 This image shows the BVCs-promoting function of the acid bacteria BFI-L14. Detailed Implementation
[0025] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.
[0026] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.
[0027] The materials and reagents used in the following examples are all commercially available.
[0028] In the following examples, the first position of each nucleotide sequence in the sequence listing is the 5′ terminal nucleotide of the corresponding DNA, and the last position is the 3′ terminal nucleotide of the corresponding DNA.
[0029] In the following examples, all experiments were repeated three times, and the average value of the results was taken.
[0030] R2A agar medium: yeast extract 0.5 g / L, peptone 0.5 g / L, casein hydrolysate 0.5 g / L, glucose 0.5 g / L, soluble starch 0.5 g / L, dipotassium hydrogen phosphate 0.3 g / L, anhydrous magnesium sulfate 0.024 g / L, sodium pyruvate 0.3 g / L, agar 15 g / L, pH 7.2 ± 0.2.
[0031] LB broth agar medium: tryptone 10.0 g / L, yeast extract 5.0 g / L, sodium chloride 10 g / L, agar 15 g / L.
[0032] LB broth medium: tryptone 10.0 g / L, yeast extract 5.0 g / L, sodium chloride 10 g / L.
[0033] Potato glucose agar (PDA) medium: 200.0 g / L potato, 20.0 g / L glucose, 15.0 g / L agar, pH 5.6 ± 0.2.
[0034] NFA solid culture medium was purchased from Ruichu Biotechnology Co., Ltd.
[0035] MS solid medium: MS medium basal salt (Phyto Technology, M524) 4.4 g / L, sucrose 30 g / L, agar 7 g / L, pH 5.8±0.2.
[0036] Example 1: Isolation of Paracidovorax oryzae BFI-L14
[0037] Paracidovorax oryzae BFI-L14 was isolated from the leaf margins of tea leaves. Tea leaf samples were collected in May 2022 from Furong District, Changsha City, Hunan Province, China.
[0038] Isolation and culture of leaf microorganisms in tea: Tea tree branches were cut and placed in sterile No. 9 side-sealed bags, sealed with water, and transported back to the laboratory at low temperature. The side-sealed bags containing the samples were placed in a laminar flow hood. Healthy one-bud-two-leaf shoots were placed in sterilized 500mL Erlenmeyer flasks. 5mL of sterile PBS buffer (pH 7.2–7.6) was added per gram of sample. After sealing with sealing film, the samples were ultrasonically washed for 2 minutes, then transferred to a shaker at 30℃, 200rpm, for 30 minutes. After the shaker was used, the washed one-bud-two-leaf samples were removed from the laminar flow hood. The remaining solution was serially diluted and used as an inoculum, spread on four types of media: R2A, LB, PDA, and NFA. Each treatment was repeated three times. The plates were then incubated at 25℃, 30℃, and 37℃ for 5–10 days to obtain visible colonies. Single colonies were picked for further isolation and purification. The resulting target strain was named BFI-L14.
[0039] Example 2: Morphological observation and strain identification of the acid bacteria BFI-L14
[0040] The strain BFI-L14 isolated in Example 1 was identified by morphological observation, including the morphology, size, texture, and edge of the colonies. Gram staining was performed according to the "Handbook of Systematic Identification of Common Bacteria": specific methods for physiological and biochemical characteristic tests are as described above. The results showed that strain BFI-L14 had short rod-shaped cells arranged in pairs, with an average cell size of 0.5–1 μm × 1–2 μm, and was Gram-negative. When cultured on LB solid culture base at 37°C for 1–2 days, the colonies dried, had a rough, transparent surface, neat edges, and were white (see [link to LB solid culture base]). Figure 1 ).
[0041] Bacterial cells cultured on LB agar plates were scraped into sterile centrifuge tubes, and genomic DNA was extracted using alkaline lysis. 16S rRNA amplification was performed on the strain using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3'). The 16S rRNA sequence of bacterium BFI-L14 is shown in SEQ ID No. 1. Sequencing results were compared on NCBI and EzBioCloud websites, followed by alignment analysis using MEGA11 software. Finally, a phylogenetic tree was constructed using neighbor-joining and phylogenetic analysis was performed. Comparison revealed that strain BFI-L14 belongs to the genus *Paracidovorax*. In EzBioCloud, BFI-L14 showed the highest sequence similarity (99.79%) to *Paracidovorax oryzae* (formerly *Acidovorax oryzae*), a member of the genus *Paracidovorax*. Furthermore, the two strains clustered on the same branch in the phylogenetic tree, indicating their closest phylogenetic relationship (see...). Figure 2 Therefore, strain BFI-L14 belongs to the genus *Acidobacterium*.
[0042] SEQ ID No. 1:
[0043]
[0044] The strain BFI-L14 was deposited on September 12, 2025, at the China General Microbiological Culture Collection Center (CGMCC, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), and classified as Paracidovorax oryzae, with accession number CGMCCNo.35900.
[0045] Example 3: Inhibitory effect of BVCs released by acidophilus bacteria BFI-L14 on anthracnose fungus of tea tree
[0046] The anthracnose bacterium used in this invention was screened from healthy tea tree leaves and is a highly infectious strain.
[0047] Strains were screened using bipartite plates (9 cm in diameter with a partition in the middle). LB solid medium was poured onto one side, and PDA solid medium onto the other, resulting in LB-PDA. Strain BFI-L14, stored at -80℃, was revived. The revived bacterial colony was added to 1 mL of liquid LB and incubated for 2 days before use. Anthracnose mycelia were inoculated onto PDA solid plates and incubated for 5 days before use. 100 μL of BFI-L14 bacterial suspension was inoculated onto the LB side of the LB-PDA plate, with an uninoculated plate serving as a control. Anthracnose mycelia (approximately 7 mm in diameter) were inoculated at the center of the other side. The plate edges were sealed with sealing film. At least three biological replicates were performed for each treatment. The plates were inverted and incubated at 28℃ until the fungus covered the entire width of the plate. The diameter of Anthracnose mycelia was measured using the cross-sectional method, and the inhibition rate was calculated using the following formula: Inhibition rate (%) = ((Control colony diameter - Treated colony diameter) / (Control colony diameter - Mycelia diameter)) × 100%.
[0048] The results are as follows Figure 3 As shown, strain BFI-L14 exhibits a significant inhibitory effect on *A. anthracnose* of tea trees without contact with the pathogenic fungus, effectively inhibiting the growth of the pathogenic mycelium with an inhibition rate of 90.79%. In vitro antibacterial experiments demonstrate that the BVCs released by strain BFI-L14 have a good antibacterial effect against *A. anthracnose* of tea trees.
[0049] Example 4: Effect of bacterial suspension inoculation concentration on the antibacterial effect of *Acidic bacteria ferruginea* BFI-L14
[0050] A suitable amount of strain BFI-L14 culture was picked from the cryopreservation tube of the preserved strain, streaked onto LB agar, and activated at 37°C for 24 h. Single colonies of BFI-L14 were picked and inoculated into Erlenmeyer flasks containing LB broth, and cultured at 37°C and 200 rpm for 24 h to obtain the seed culture. The seed culture was adjusted to approximately 10... 5 10 6 107 10 8 10 9 Different concentrations of BFI-L14 bacterial suspensions were obtained using CFU / mL. 100 μL of bacterial suspension was inoculated onto the LB side of an LB-PDA plate, and one day later, anthrax mycelium was inoculated into the center of the other side. The plate edges were sealed with sealing film. Three biological replicates were performed for each treatment. The plates were incubated upside down at 28°C until the fungus covered the entire width of the plate. The diameter of *Anthrax cambogia* was measured using the cross-sectional method, and the inhibition rate was calculated using the following formula: Inhibition rate (%) = ((Control colony diameter - Treated colony diameter) / (Control colony diameter - Mycelium diameter)) × 100%.
[0051] As shown in Table 1 and Figure 4 As shown, the antibacterial effect of BFI-L14 is positively correlated with the number of viable bacteria in the bacterial suspension, at 10... 9 CFU / mL showed a significant inhibitory effect, with an inhibition rate of 92.31%.
[0052] Table 1. Inhibitory effects of different inoculation concentrations on *Anthracis chinensis*.
[0053]
[0054] Example 5: Control of anthracnose in tea trees by BVCs produced by *Acidic bacteria BFI-L14*
[0055] Pour LB solid culture medium onto one side of the bipartite dish, leaving the other side untreated. Adjust the seed culture from Example 4 to 10 using 1xPBS. 9 CFU / mL, 100 μL was spread on LB solid medium, and after 1 day, treated tea leaves were placed in it to form the treatment group. Healthy tea leaves of similar size were selected, washed with running water, and then surface-sterilized with sodium hypochlorite. After washing with sterile water, the leaves were needled, and *Anthracnose cambogia* mycelial cakes were placed at the needle-piercing sites. The control group was coated with an equal volume of sterile water one day in advance, while the blank group received no other treatment. After incubation at 28℃ under high humidity for 2 days, the diseased area was calculated, and the inhibition rate was calculated according to the following formula.
[0056] Inhibition rate (%) = (Affected area in control group - Affected area in treatment group) / Affected area in control group × 100%
[0057] The results are as follows Figure 5 As shown, the area of anthrax lesions decreased by 71.61%.
[0058] Example 6: Effect of Acidobacterium glutamicum BFI-L14 on the growth promotion of BVCs in Arabidopsis thaliana.
[0059] One half of the distillation dish was used to prepare LB solid medium, and the other half was used to prepare MS solid medium. In Example 4, the seed culture was adjusted to 10. 8After CFU / mL, 10 μL of LB solid medium was added, and Arabidopsis thaliana with uniform growth was inoculated into MS solid medium. After 4 days of co-culture, growth was photographed and fresh weight was measured. The growth-promoting effect is shown in Table 2. Figure 6 As shown.
[0060] Fresh weight growth rate (%) = [(Weight of Arabidopsis thaliana in the treatment group - Weight of Arabidopsis thaliana in the control group) / Weight of Arabidopsis thaliana in the control group] × 100%
[0061] Table 2 shows the effect of BVCs produced by BFI-L4 on the fresh weight growth rate of Arabidopsis thaliana.
[0062]
[0063] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.
Claims
1. Leafy paracidal acid bacteria BFI-L14 of tea leaves, which is paracidal acid bacteria (Paracidovorax oryzae), with the accession number: CGMCC No.35900.
2. The 16S rRNA gene sequence of the tea leaf acid bacteria BFI-L14 according to claim 1 is shown in SEQ ID NO:
1.
3. The culture of the tea leaflet acidophilus BFI-L14 as described in claim 1 or 2.
4. An inoculum containing the leaf phytoseptica BFI-L14 of tea as described in claim 1 or 2.
5. The secondary metabolite of the tea leaflet acidophilus BFI-L14 as described in claim 1 or 2.
6. The application of the culture of *Acidithiobacillus foliata* BFI-L14 of tea leaves as described in claim 1 or 2, the inoculum containing *Acidithiobacillus foliata* BFI-L14 of tea leaves as described in claim 1 or 2, or the secondary metabolites of *Acidithiobacillus foliata* BFI-L14 of tea leaves as described in claim 1 or 2, in the prevention and control of anthracnose in tea trees or in the promotion of *Arabidopsis thaliana* growth.