Tea tree phyllobacillus sp. BFI-L8 and application thereof

Abstract

The invention relates to the technical field of agricultural microorganisms, in particular to a tea tree phyllobacillus sp. BFI-L8 and application of the tea tree phyllobacillus sp. BFI-L8. The tea tree phyllobacillus sp. BFI-L8 is bacillus pumilus, and the preservation number of the tea tree phyllobacillus sp. BFI-L8 is CGMCC (China General Microbiological Culture Collection Center) No. 29511. The strain has a nitrogen fixation effect and can produce IAA, siderophore, protease and cellulase, the produced VOCs can significantly promote the growth of arabidopsis thaliana, and the growth of tea tree pathogenicity sphaerotrichum fungi is inhibited on a flat plate.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbial technology, specifically to a tea leaf bacillus BFI-L8 and its applications. Background Technology

[0002] Currently, chemical fertilizers and pesticides are widely used in agricultural production, playing a crucial role in achieving high and stable crop yields. However, long-term application leads to the accumulation of fertilizer / pesticide residues in the soil and crops, causing problems such as soil nutrient imbalance, environmental pollution, and microecological imbalance. Therefore, the development of green and efficient fertilizers and pesticides has become a hot topic of concern.

[0003] With continuous breakthroughs in bioscience and technology, microbial agents have shown significant potential as a green alternative to chemical fertilizers and pesticides due to their low cost, green efficiency, and contribution to sustainable agricultural development. Current research indicates that strains with growth-promoting and disease-preventing functions are mainly isolated from plants or the environment itself. Introducing them as microbial agents into plant growth offers advantages such as being green, efficient, and long-lasting. Bacillus is a widely distributed Gram-positive bacterium in nature that produces spores and can survive in many adverse environments. Many strains of Bacillus possess special functions and have application value in agriculture, industry, and synthetic biology.

[0004] Microorganisms exhibit varying colonization abilities in different habitats, and microorganisms isolated from different ecological niches tend to colonize their original niches. Current development of growth-promoting and disease-preventing functional strains primarily focuses on rhizosphere microorganisms, with less development of phyllosphere microorganisms. Phyllosphere bacteria isolated from plants are more likely to colonize leaves, promoting plant growth and development and enhancing disease resistance in an eco-friendly manner. Phyllosphere accessibility is crucial for the successful introduction of beneficial microorganisms throughout the plant life cycle. Isolating microorganisms from the phyllosphere of foliage plants and applying them appropriately can reduce fertilizer application, improve fertilizer utilization, partially replace pesticide application, and mitigate the environmental impacts of chemical fertilizers and pesticides.

[0005] In recent years, with the continuous development of bioscience and technology, reports on the use of microbial inoculants / fertilizers isolated from plants have increased significantly. However, most of these microorganisms are isolated from the root system or rhizosphere, and are still rarely found in the foliage. This means that the efficacy of inoculants is easily limited to specific habitats, making it difficult to achieve a synergistic effect of promoting foliar growth and preventing diseases in the root and soil.

[0006] Therefore, there is an urgent need to develop plant foliar separation-promoting and biocontrol bacteria to reduce the use of chemical fertilizers and pesticides, thereby reducing environmental pollution. Summary of the Invention

[0007] This invention screened a tea foliar Bacillus strain BFI-L8 that promotes growth and prevents disease. It has nitrogen-fixing activity, produces IAA, siderophores, proteases, and cellulases. The VOCs produced can significantly promote the growth of Arabidopsis thaliana and inhibit the growth of pathogenic fungi of the genus Globosa on agar plates. It provides a high-quality resource for the research of Bacillus resources and the development and application of adaptive growth-promoting and disease-preventing strains.

[0008] The tea leaf bacillus BFI-L8 described in this invention is Bacillus pumilus, which was deposited on January 4, 2024, 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), and classified as Bacillus pumilus, with accession number CGMCC No. 29511.

[0009] The 16S rRNA gene (16S rDNA) sequence of Bacillus thuringiensis BFI-L8 described in this invention is shown in SEQ ID NO:1; in its transcribed RNA sequence, all Ts are replaced with Us.

[0010] The present invention also provides a culture of the tea leaf bacillus BFI-L8.

[0011] The present invention also provides a bacterial agent containing the tea leaf bacillus BFI-L8.

[0012] The present invention also provides secondary metabolites of the tea leaf bacillus BFI-L8.

[0013] The Bacillus thuringiensis BFI-L8 bacterial agent of tea leaves described in this invention can be a liquid bacterial agent or a solid bacterial agent.

[0014] In this invention, the culture of Bacillus thuringiensis BFI-L8, the bacterial agent containing Bacillus thuringiensis BFI-L8, and the secondary metabolites of Bacillus thuringiensis BFI-L8 can be prepared using conventional techniques.

[0015] The present invention also provides the application of the culture of the tea tree foliar Bacillus BFI-L8, the bacterial agent containing the tea tree foliar Bacillus BFI-L8, or the secondary metabolites of the tea tree foliar Bacillus BFI-L8 in promoting nitrogen fixation, producing IAA, producing siderophores, producing proteases and cellulases, promoting Arabidopsis thaliana growth, and inhibiting the growth of tea tree pathogenic fungi of the genus Globosa.

[0016] The tea leaf bacillus BFI-L8 described in this invention can grow on R2A, LB, and PDA media, with a culture temperature generally between 25-37℃.

[0017] This invention uses leaf niche isolation culture strains. The screened Bacillus BFI-L8 has a variety of growth-promoting characteristics, has a significant antibacterial effect on pathogenic tea plant fungi of the genus Globosa, and the VOCs produced have a significant growth-promoting effect, which is conducive to the synergistic effect of leaf growth promotion and root-soil disease prevention.

[0018] The Bacillus spp. BFI-L8 of this invention has growth-promoting and disease-preventing functions. It possesses growth-promoting characteristics such as nitrogen fixation, IAA production, siderophore production, protease production, and cellulase production. On agar plates, it has a significant inhibitory effect on pathogenic fungi of the genus *Bacillus* in tea trees, and the VOCs produced have a significant growth-promoting effect on Arabidopsis thaliana. Attached Figure Description

[0019] Figure 1 Microscopic images of Bacillus BFI-L8 colonies after Gram staining.

[0020] Figure 2 The image shows the results of the nitrogen fixation function identification of Bacillus BFI-L8.

[0021] Figure 3 The image shows the results of functional identification of IAA production by Bacillus BFI-L8.

[0022] Figure 4 The image shows the results of the identification of siderophore production function of Bacillus BFI-L8.

[0023] Figure 5 The image shows the results of functional identification of protease produced by Bacillus BFI-L8.

[0024] Figure 6 The image shows the results of functional identification of cellulase produced by Bacillus BFI-L8.

[0025] Figure 7 The graph shows the effect of Bacillus thaliana BFI-L8 on VOCs-promoting growth in Arabidopsis thaliana.

[0026] Figure 8 This image shows the antibacterial effect of Bacillus spp. BFI-L8 against pathogenic fungi of the genus *Gastrodia* in tea plants. Detailed Implementation

[0027] 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.

[0028] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0029] The materials and reagents used in the following examples are all commercially available.

[0030] 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.

[0031] In the following examples, all experiments were repeated three times, and the average value of the results was taken.

[0032] 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.

[0033] LB broth agar medium: tryptone 10.0 g / L, yeast extract 5.0 g / L, sodium chloride 10 g / L, agar 15 g / L.

[0034] LB broth medium: tryptone 10.0 g / L, yeast extract 5.0 g / L, sodium chloride 10 g / L.

[0035] 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.

[0036] NFA solid culture medium was purchased from Ruichu Biotechnology Co., Ltd.

[0037] 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.

[0038] Example 1: Isolation and Identification of Strains

[0039] I. Isolation of Bacillus

[0040] Bacillus pumilus BFI-L8 was isolated from the leaf margins of tea trees. Tea leaf samples were collected in May 2022 from Furong District, Changsha City, Hunan Province, China.

[0041] 1. Isolation and culture of leaf microorganisms in tea

[0042] Healthy tea shoots with one bud and two leaves were picked from the tea garden. The surface was washed with distilled water, sterilized in a laminar flow hood, and then ground with liquid nitrogen. The samples were then suspended in PBS buffer (0.4g sample: 2ml PBS buffer). The suspensions were serially diluted and spread on four types of culture media: R2A, LB, PDA, and NFA. Each treatment was repeated three times. The plates were incubated at 25℃, 30℃, and 37℃ for 5-10 days to obtain visible colonies. Single colonies were picked for subsequent isolation and purification.

[0043] 2. Identification and strain comparison of isolated bacteria

[0044] Take the purified bacterial culture and extract template DNA based on the Gram staining results. For detailed operation methods, please refer to the instructions of the BIOMIGA Bacterial Genomic DNA Extraction Kit (GD2411).

[0045] Based on the identification method of 16S rRNA from endophytic bacteria in tea plants in relevant literature, primers were used:

[0046] 16S-PA(5'-AGAGTTTGATCCTGGCTCAG-3');

[0047] 16S-PB(5'-AAGGAGGTGATCCAGCCGCA-3');

[0048] 16S rRNA amplification was performed on the bacterial strains, and the sequencing results were aligned using BLAST at NCBI to obtain the aligned bacterial species for each strain. The 16S rRNA gene sequence of the bacterium numbered BFI-L8 is shown in SEQ ID No. 1, as follows:

[0049]

[0050]

[0051] II. Identification of Bacillus BFI-L8

[0052] The strain BFI-L8 isolated above was identified by biological characteristics and morphological observation. The morphology, size, concavity, and edge of the colonies were observed, and Gram staining was performed. The method was in accordance with the "Handbook for Systematic Identification of Common Bacteria". For specific methods of physiological and biochemical characteristic testing, please refer to the manual.

[0053] Microscopic morphology of Bacillus BFI-L8 colonies after Gram staining is shown in Figure 1. Figure 1 .

[0054] The results showed that strain BFI-L8 had straight rod-shaped cells arranged in pairs or chains, with an average cell size of 0.5–1 μm × 1.5–10 μm, and was Gram-positive. When cultured on LB broth agar at the optimal temperature for 1–2 days, the colonies dried, with a rough, translucent surface, neat edges, and a milky-white color. Bacillus BFI-L8 was positive for lysozyme resistance, mannitol acid production, glucose utilization, VP reaction, motility test, citrate reaction, and 3% hydrogen peroxide test, but negative for starch reaction, nitrate reduction reaction, and gelatin test. The 16S rRNA of strain BFI-L8 showed 100% similarity to that of other Bacillus strains.

[0055] After identification, strain BFI-L8 was determined to be Bacillus pumilus.

[0056] The strain BFI-L8 was deposited on January 4, 2024, 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), and classified as Bacillus pumilus, with accession number CGMCCNo.29511.

[0057] Bacillus pumilus (CGMCC No. 29511), activated and cultured for 1 day, was inoculated at a ratio of 1:100 into Erlenmeyer flasks containing 100 mL of LB broth. The culture was incubated at 37°C and 200 rpm for 24 hours as a seed culture, and then diluted to a final volume of 10. 8 The following tests were performed using CFU / mL.

[0058] Example 2: Identification of nitrogen fixation function in Bacillus pumilus BFI-L8

[0059] Nitrogen fixation function of Bacillus BFI-L8 (CGMCC NO.29511) was identified by the ability of the strain to fix nitrogen, which was defined as the ability of the strain to grow normally after three consecutive passages in Ashby nitrogen-free medium.

[0060] The culture medium formula is as follows: mannitol 2g, CaCl2·2H2O 0.2g, K2HPO4 0.2g, MgSO4·7H2O 0.2g, MoO3 0.001g, FeCl3 0.005g, and Agar 15.0g. Accurately weigh the above reagents and dissolve them in 1000mL of distilled water. Adjust the pH to 7.2±0.2 (25℃), and autoclave for 25 minutes.

[0061] Results of testing BFI-L8 on Ashby medium showed that the strain possessed nitrogen-fixing activity. (See attached data.) Figure 2 .

[0062] Example 3: Identification of phosphorus-solubilizing function of Bacillus pumilus BFI-L8 (organic and inorganic phosphorus)

[0063] The clear zone method was used. Bacillus BFI-L8 (CGMCC NO:29511) was inoculated on phosphorus-solubilizing (organic phosphorus / inorganic phosphorus) medium and incubated in a constant temperature incubator at 28℃ for 3 days. When a clear zone appeared at the edge of the bacterial colony on the medium, the bacterial strain had the corresponding phosphorus-solubilizing ability. Each group of experiments was repeated 3 times.

[0064] Organic phosphorus broth (1L): glucose 10.0g, ammonium sulfate 0.5g, sodium chloride 0.3g, magnesium sulfate 0.3g, manganese sulfate 0.03g, potassium sulfate 0.3g, ferrous sulfate 0.03g, calcium phosphate 5.0g, lecithin 0.2g, agar 15.0g, pH 7.0-7.5 (25℃).

[0065] Inorganic phosphorus-free medium (1L): glucose 10.0g, ammonium sulfate 0.5g, sodium chloride 0.3g, magnesium sulfate 0.3g, manganese sulfate 0.03g, potassium sulfate 0.3g, ferrous sulfate 0.03g, calcium phosphate 5.0g, agar 15.0g, pH 7.0-7.5 (25℃).

[0066] Bacillus BFI-L8 does not exhibit a clear zone in either inorganic or organic phosphorus media, indicating that this strain lacks phosphorus-solubilizing capabilities.

[0067] Example 4: Functional identification of IAA production by Bacillus pumilus BFI-L8

[0068] Bacillus BFI-L8 (CGMCC NO:29511) was inoculated into LB broth and cultured at 37°C and 200 rpm for 3 days. After centrifugation at 12000 rpm for 10 min, 1 mL of fermentation supernatant was taken and mixed with 2 mL of Salkowski's colorimetric reagent (2 mL of 0.5 mol / L FeCl3·6H2O and 98 mL of 35% perchloric acid). The mixture was incubated in the dark at room temperature for 30 min, and the color was observed. A red color indicated that the strain produced IAA. An equal volume of LB broth served as a negative control, and the IAA standard solution served as a positive control.

[0069] The fermentation broth of Bacillus BFI-L8 (CGMCC NO:29511) showed a pink color, indicating that the strain produces IAA. (See attached image). Figure 3 .

[0070] Example 5: Identification of Siderophore Production Function in Bacillus pumilus BFI-L8

[0071] The siderophore production function of Bacillus BFI-L8 was identified using CAS detection medium. The formation of a yellow transparent zone indicated that the strain possessed the ability to produce siderophores. CAS detection medium (1L): Chromium azuril 60.5mg, cetyltrimethylammonium bromide 72.9mg, ferric chloride hexahydrate 2.635mg, sodium dihydrogen phosphate dihydrate 295.25mg, disodium hydrogen phosphate dodecahydrate 1213.5mg, ammonium chloride 125mg, potassium dihydrogen phosphate 37.5mg, sodium chloride 62.5mg, agar 9g, pH 6.8±0.1 (25℃).

[0072] CAS detection of the culture medium results showed that Bacillus BFI-L8 (CGMCC NO:29511) produced siderophores, as shown in the following figures. Figure 4 .

[0073] Example 6: Identification of the protease production function of Bacillus pumilus BFI-L8

[0074] The protease production function of Bacillus BFI-L8 was identified by the formation of a hydrolysis zone in the protease production screening medium. The protease production screening medium was prepared as follows: 8g casein, 1g disodium hydrogen phosphate, 0.36g dipotassium hydrogen phosphate, 20g agar, 5g sodium chloride, added to 1000mL distilled water, heated and stirred until the pH was 7.4±0.2.

[0075] The protease production screening test medium for Bacillus BFI-L8 showed that the strain had the ability to produce protease. (See attached results). Figure 5 .

[0076] Example 7: Functional identification of cellulase production by Bacillus pumilus BFI-L8

[0077] Cellulase production function of Bacillus BFI-L8 was identified by the formation of a hydrolysis zone on the cellulase screening solid medium. The cellulase screening medium was prepared as follows: 0.5 g KCl, 0.5 g MgSO4, 1.0 g K2HPO4, 3.0 g NaNO3, 5.0 g CMC-Na, and 20.0 g agar were dissolved in 1000 mL distilled water, with a pH of 7.4 ± 0.2.

[0078] The initial screening test for cellulase production in the culture medium of Bacillus BFI-L8 showed that the strain had the ability to produce cellulase. (See attached results.) Figure 6 .

[0079] Example 8: The effect of Bacillus pumilus BFI-L8 on VOCs growth in Arabidopsis thaliana.

[0080] One half of the bipartite dish was prepared with LB broth agar, and the other half with MS solid medium. 10 μL of bacterial culture was added to the LB broth agar, and Arabidopsis thaliana with uniform growth was inoculated into the MS solid medium after 4 days. After co-culturing for 14 days, growth was photographed and fresh weight was measured. The growth-promoting effect was as follows: Figure 7 As shown.

[0081] 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%

[0082] Table 1. The effect of VOCs produced by Bacillus BFI-L8 on the fresh weight growth rate of Arabidopsis thaliana.

[0083] CK(g) Processing (g) growth rate Bacillus BFI-L8 0.020±0.002 0.039±0.003 96.61％

[0084] Example 9: Inhibitory effect of Bacillus pumilus BFI-L8 against pathogenic *Plasmodium* fungi in tea plants.

[0085] The pathogenic *Phyllostachys* fungi used in this embodiment were screened from healthy tea leaves, and the re-inoculation experiment showed that they were pathogenic. Using the PDA plate five-point confrontation method, mycelial cakes (5 mm in diameter) containing BFI-L8 bacterial suspension were inoculated at the four corners of a 9 cm diameter PDA plate containing 10 mL of medium, approximately 2.5 cm from the center. A *Phyllostachys* fungal cake (5 mm in diameter) was inoculated at the center of the plate, and this was repeated three times. The plates were incubated at 28°C in the dark. After the control plate reached full colony size, the fungal colony radius was measured, and the inhibition rate was calculated using the following formula:

[0086] Antibacterial rate (%) = (Growth diameter of control group - Growth diameter of treatment group) / Growth diameter of control group × 100%

[0087] See results Figure 8 Bacillus BFI-L8 showed an average inhibition rate of 63.20% against fungi of the genus *Bacillus*, indicating that Bacillus BFI-L8 has a significant antibacterial effect against *Bacillus*.

[0088] 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. Bacillus teapilus BFI-L8, which is Bacillus pumilus, and its preservation number is CGMCC No. 29511.

2. The Bacillus teapilus BFI-L8 according to claim 1, and its 16S rRNA gene sequence is shown as SEQ ID NO:

1.

3. A culture of the Bacillus teapilus BFI-L8 according to claim 1 or 2.

4. A microbial agent comprising the Bacillus teapilus BFI-L8 according to claim 1 or 2.

5. A secondary metabolite of the Bacillus teapilus BFI-L8 according to claim 1 or 2.

6. Use of the culture of the Bacillus teapilus BFI-L8 according to claim 1 or 2, the microbial agent comprising the Bacillus teapilus BFI-L8 according to claim 1 or 2, or the secondary metabolite of the Bacillus teapilus BFI-L8 according to claim 1 or 2 in promoting nitrogen fixation, IAA production, siderophore production, protease and cellulase production, promoting Arabidopsis thaliana growth, or inhibiting the growth of Sphaerulina fungi pathogenic to tea trees.

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