Escherichia capable of promoting seed germination of cupressus gigantea and application thereof

Abstract

The application discloses an Erwinia billingiae strain STSG25-LZ24 for promoting the germination of Thuja sutchuenensis seed and application thereof, the preservation number of the Erwinia billingiae strain STSG25-LZ24 disclosed by the application is CGMCC No.33386, and the 16S rRNA gene of the strain comprises a sequence as shown in SEQ ID NO.1. The strain can not only obviously promote the germination of Thuja sutchuenensis seed, but also has a good application prospect in the aspects of seed breeding, seed coating preparation and synthetic microbial population construction.

Description

Technical Field

[0001] This invention belongs to the field of microbiology and biotechnology, specifically relating to Erwinia bacterium that can promote the germination of giant cypress seeds and its applications. Background Technology

[0002] Giant cypress (Cupressus gigantean), also known as Yarlung Tsangpo cypress, is a rare and endemic evergreen tree, listed as a Class I endangered tree species in my country. Its populations are mainly distributed in strips or sporadically along parts of the valleys of the Yarlung Tsangpo River and its tributary, the Nyang River, in southeastern Tibet. With its excellent timber and remarkable resilience, the giant cypress plays a crucial role in soil and water conservation and water source protection. As an ancient species, the giant cypress is of great significance for research on vegetation evolution and paleoclimate reconstruction, and also plays an important role in the protection of the Qinghai-Tibet Plateau ecosystem.

[0003] The current population of *Juniperus chinensis* faces severe challenges to natural regeneration, and the overall population is in decline. The primary problem lies in poor seed quality and low natural germination rates. Coupled with the weak adaptability of seedlings to the environment, the harsh habitat conditions result in an extremely low conversion rate from seed to sapling. Secondly, the narrow geographical distribution of *Juniperus chinensis* and the heterogeneity and fragmentation of its habitats severely restrict its growth and development. Furthermore, the climate in southeastern Tibet is trending towards warmer and drier conditions, with frequent droughts. Human factors such as development and construction in the lower reaches of the Yarlung Tsangpo River further exacerbate the deterioration of the *Juniperus chinensis*'s living environment. Seed germination of *Juniperus chinensis* mainly depends on natural conditions, is inefficient, and is greatly affected by environmental factors. Therefore, developing an effective technology to promote seed germination of *Juniperus chinensis*, innovating existing microbial agent production formulas, discovering more resistant and higher-growth-promoting native high-efficiency strains to produce high-efficiency microbial agents, or constructing synthetic microbial communities are crucial for promoting the regeneration and recovery of *Juniperus chinensis* populations. Summary of the Invention

[0004] Plant roots contain a large number of microorganisms, one type of which can promote seed germination, plant growth, and disease control; these are known as growth-promoting microorganisms. Growth-promoting microorganisms play a crucial role in improving the soil environment, promoting nutrient absorption by plants, and enhancing plant resistance to adverse conditions.

[0005] Growth-promoting bacteria in giant cypress help improve its growth rate and health, and help the plant resist adverse stresses such as drought and nutrient deficiency, thereby significantly enhancing the environmental adaptability of giant cypress and promoting community regeneration. For example, bacteria with phosphorus-solubilizing and potassium-solubilizing functions have a significant effect on improving the environmental adaptability and ecosystem productivity of cypress species, and are crucial for maintaining forest diversity, stability and resilience.

[0006] This invention uses the root system of *Juniperus chinensis* as the isolation material to isolate, screen and identify its growth-promoting bacteria. The purpose is to provide a strain that promotes the germination of *Juniperus chinensis* seeds. This strain can promote the germination of *Juniperus chinensis* seeds, increase the seed germination rate and growth rate, thereby helping to promote the regeneration of *Juniperus chinensis* communities.

[0007] This invention is specifically achieved through the following technical solutions:

[0008] This invention provides a strain of Erwinia billingiae, which has been deposited with accession number CGMCC No. 33386. The depository institution is the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The taxonomic name is Erwinia billingiae, the deposit date is January 13, 2025, and the strain name is STSG25-LZ24.

[0009] The 16S rRNA gene of Erwinia CGMCC33386 contains the sequence shown in SEQ ID NO.1.

[0010] Furthermore, the Erwinia CGMCC33386 was isolated from endophytic bacteria of the roots of Cypress tibetica.

[0011] Furthermore, the Erwinia CGMCC33386 can promote plant seed germination. Preferably, the plant is a tree belonging to the genus *Cupressus* of the family Cupressaceae. More preferably, the tree belonging to the genus *Cupressus* of the family Cupressaceae is *Cupressus gigantea*.

[0012] This invention provides the application of the Erwinia CGMCC33386 strain. Preferably, the application is in the process of plant propagation and transplanting, including seed germination, artificial sowing, aerial sowing, and seedling transplanting, wherein the plant is a tree belonging to the genus *Cupressus* of the family Cupressaceae. Preferably, the tree is *Cupressus gigantea*.

[0013] This invention also provides applications of the aforementioned Erwinia CGMCC33386. Specifically, these applications include its use as a microbial agent, seed coating, composition, or cooperative microbial community for preparing cypress seed germination and seedling growth. In some embodiments, the microbial agent can be obtained through conventional liquid or solid culture to produce a bacterial cell culture, which is then produced through conventional liquid fermentation. One or more surfactants, such as dispersants, stabilizers, wetting agents, binders, defoamers, disintegrants, and antifreeze agents, or adsorbents, are then added in a certain proportion to prepare seed coatings, dry powders, pellets, wettable powders, water-dispersible granules, suspensions, emulsions, water-in-oil emulsions, or microemulsions.

[0014] The method for promoting seed germination with Erwinia CGMCC33386 described in this invention also falls within the scope of protection of this invention. This method involves spraying during the seed germination process; the spray is a preparation made from a bacterial suspension, fermentation broth, or metabolites of Erwinia CGMCC33386.

[0015] This invention also provides the application of Erwinia CGMCC33386 in promoting the germination of giant cypress seeds, and the application of Erwinia CGMCC33386 in constructing synthetic microbial communities is also within the scope of protection of this invention.

[0016] The present invention also provides microbial agents, seed coatings, compositions, or synthetic microbial communities comprising the aforementioned Erwinia CGMCC33386. Further, the microbial agents, seed coatings, compositions, or synthetic microbial communities are as described above.

[0017] The beneficial effects of this invention are as follows:

[0018] The Erwinia CGMCC33386 strain provided by this invention can promote the germination and growth of giant cypress seeds, increasing the seed germination rate and growth rate. As a biological agent material for plants of the Cupressaceae family and the genus *Cupressus*, this strain has excellent market prospects, whether for developing new biological control agents or growth regulators. Attached Figure Description

[0019] Figure 1 This demonstrates the ability of different strains to produce indoleacetic acid (IAA).

[0020] Figure 2 The phosphorus-solubilizing effect of different strains is shown.

[0021] Figure 3 The potassium-solubilizing effects of different strains are shown.

[0022] Figure 4 This demonstrates the siderophore production effects of different strains.

[0023] Figure 5 The results of an experiment to promote the germination ability of giant cypress seeds.

[0024] Figure 6 The colony growth characteristics of strain STSG25-LZ24. Detailed Implementation

[0025] The technical solution of the present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. Any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention fall within the protection scope of the present invention.

[0026] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0027] Example 1: Isolation of strain STSG25-LZ24

[0028] 1.1 Source of strains

[0029] The root samples of *Juniperus chinensis* in this embodiment were collected in June 2024 from the Plateau Ecological Research Institute of Linzhi City, Tibet Autonomous Region. The samples were placed in sterile sealed plastic bags, labeled, and promptly brought back to the laboratory and stored in a -20°C refrigerator.

[0030] 1.2 Isolation of endophytic bacteria from giant cypress

[0031] The roots of *Platycladus orientalis* were disinfected with 1% sodium hypochlorite solution for 1 minute and 75% alcohol for 1 minute. After rinsing with sterile water for 5 minutes, the surface moisture of the roots was absorbed with sterile filter paper. The roots were then ground with sterile water. 1 mL of the ground liquid was diluted twice and 150 μL of the diluted solution was inoculated into LB medium and spread. The medium was incubated upside down at 28°C for 3 days. Single colonies were picked and purified based on their size, morphology, color and other appearance characteristics. 60 culturable strains were obtained through isolation and purification.

[0032] Example 2 Qualitative analysis of ability to produce indoleacetic acid (IAA)

[0033] Some of the isolated strains were inoculated into King's medium and cultured to the logarithmic phase. They were then cultured at 28°C and 180 r / min for 3 days with shaking. 50 μl of the culture was placed in a clean white porcelain plate, and 50 μL of colorimetric reagent was added. The plate was then incubated at 28°C in the dark for 30 minutes. If a pink color appeared, it indicated that the strain could produce IAA. The stronger the pink color, the stronger the IAA production capacity of the strain. Using uninoculated King's medium as a control, the qualitative results of the IAA production capacity of different strains (YD5MG9-34, YD5MG9-13, YD5MG9-11, YD5MG9-60, YD5MG9043, YD5MG9-30, YD5MG9-15, YD5MG9-12, YD5MG9-42, YD5MG9-10, YD5MG9-47, YD5MG9-9, YD5MG9-56, YD5MG9-2, YD5MG9-6, YD5MG9-14, STS-G19, STS-Y18, STS-G33, STS-G25, STS-Y8, STS-Y16, STS-G26, STS-G27, STS-G37, STS-Y5) are shown in [the table below]. Figure 1 Where CK is distilled water and CKM is the stock solution of the culture medium. Figure 1 The results show that STSG25-LZ24 (originally designated STSG-25 in the figure, but later changed to STSG25-LZ24 to emphasize its origin) has a higher IAA production capacity than other strains.

[0034] Example 3 Inorganic phosphorus degradation capacity

[0035] Some of the isolated strains were inoculated onto PVK medium and cultured at 28°C for 3 days. The inorganic phosphorus solubility zone (H) and colony diameter (h) were measured, and the (H / h) ratio was calculated. The larger the ratio, the greater the phosphorus solubility index. The phosphorus solubility indexes of different strains are shown in Table 1, demonstrating that 18 out of 60 strains have phosphorus solubility, accounting for 30% of the isolated culturable strains. Among them, the solubility index of strain STSG25-LZ24 was 1.5, which is at a moderate level among the strains mentioned above.

[0036] Table 1. Inorganic phosphorus degradation capacity of culturable bacteria in the roots of *Juniperus chinensis*

[0037] Strain number Phosphate solubilization index (H / h) Strain number Phosphate solubilization index (H / h) YD5MG9-2 1.6 STS-G1 nd YD5MG9-3 nd STS-G2 nd YD5MG9-4 nd STS-G3 nd YD5MG9-5 1.5 STS-G4 nd YD5MG9-6 1.5 STS-G6 nd YD5MG9-7 nd STS-G12 nd YD5MG9-8 nd STS-G19 1.83 YD5MG9-9 1.75 STSG25-LZ24 1.5 YD5MG9-10 1.75 STS-G26 1.2 YD5MG9-12 1.6 STS-G27 nd YD5MG9-13 1.4 STS-G33 nd YD5MG9-14 1.6 STS-G37 nd YD5MG9-15 1.5 STS-G38 nd YD5MG9-16 nd STS-Y4 nd YD5MG9-18 nd STS-Y5 nd YD5MG9-19 nd STS-Y6 1.29 YD5MG9-20 nd STS-Y7 nd YD5MG9-21 1.25 STS-Y8 nd YD5MG9-22 nd STS-Y11 nd YD5MG9-23 nd STS-Y14 nd YD5MG9-24 nd STS-Y30 nd YD5MG9-26 1.75 STS-Y31 nd YD5MG9-29 1.6 STS-Y38 nd YD5MG9-30 1.25 STS-Y39 nd YD5MG9-34 nd STS-Y40 nd YD5MG9-38 nd STS-G32 nd YD5MG9-43 nd STS-G40 nd YD5MG9-47 1.2 STS-G20 nd YD5MG9-56 nd YD5MG9-42 nd YD5MG9-60 nd YD5MG9-11 nd

[0038] Different strains (YD5MG9-11, YD5MG9-42, YD5MG9-30, YD5MG9-60, YD5MG9-12, YD5MG9-43, YD5MG9-14, YD5MG9-2, YD5MG9-47, YD5MG9-13, YD5MG9-15, YD5MG9-6, YD The phosphorus dissolving effect of 5MG9-10, YD5MG9-9, YD5MG9-56, STS-G25, STS-G26, STS-Y16, STS-G37, STS-Y8, STS-G33, STS-G27, STS-G19, STS-G26, STS-Y8, STS-G19, STS-Y5) is shown in Figure 2 The results show that the phosphorus-solubilizing ability of STSG25-LZ24 (originally numbered STS-G25 in the figure, but later changed to STSG25-LZ24 to emphasize its origin) is at a medium level compared to the experimental strains.

[0039] Example 4: Identification of Potassium Solubility

[0040] The isolated strains were inoculated onto potassium-solubilizing medium and incubated at 28°C for 3 days. The clear zone (H) and colony diameter (h) in the plates were measured, and the (H / h) ratio was calculated. The higher the ratio, the stronger the potassium-solubilizing ability. The potassium-solubilizing abilities of different strains are shown in Table 2. As shown in Table 2, 25 out of 60 strains had potassium-solubilizing ability, accounting for 41.6% of the total isolated strains. Among them, strain STSG25-LZ24 had a potassium-solubilizing ability of 1.67.

[0041] Table 2 Potassium-solubilizing capacity of culturable bacteria in the roots of *Juniperus chinensis

[0042] Strain number Potassium solubilization index (H / h) Strain number Potassium solubilization index (H / h) YD5MG9-2 1.67 STS-G1 1.25 YD5MG9-3 nd STS-G2 1.66 YD5MG9-4 nd STS-G3 nd YD5MG9-5 1.5 STS-G4 nd YD5MG9-6 2.0 STS-G6 1.5 YD5MG9-7 nd STS-G12 nd YD5MG9-8 nd STS-G19 1.57 YD5MG9-9 1.5 STSG25-LZ24 1.67 YD5MG9-10 2.0 STS-G26 1.4 YD5MG9-12 2.28 STS-G27 1.29 YD5MG9-13 2.5 STS-G33 nd YD5MG9-14 2.25 STS-G37 nd YD5MG9-15 1.5 STS-G38 nd YD5MG9-16 nd STS-Y4 nd YD5MG9-18 nd STS-Y5 1.33 YD5MG9-19 nd STS-Y6 3.5 YD5MG9-20 nd STS-Y7 1.67 YD5MG9-21 2.33 STS-Y8 1.33 YD5MG9-22 nd STS-Y11 nd YD5MG9-23 nd STS-Y14 nd YD5MG9-24 nd STS-Y30 nd YD5MG9-26 2.0 STS-Y31 nd YD5MG9-29 1.67 STS-Y38 nd YD5MG9-30 1.67 STS-Y39 nd YD5MG9-34 1.0 STS-Y40 nd YD5MG9-38 nd STS-G32 nd YD5MG9-43 nd STS-G40 nd YD5MG9-47 2.25 STS-G20 nd YD5MG9-56 nd YD5MG9-42 nd YD5MG9-60 nd YD5MG9-11 2.33

[0043] Different strains (STS-G26, STS-G19, STS-Y8, STS-Y6, STS-G37, STS-G27, STS-Y5, STS-G19, STS-Y8, STS-G33, STS-G25, STS-G26, YD5MG9-15, YD5MG9-30, YD5MG9-1 2. The potassium-solubilizing effect of YD5MG9-2, YD5MG9-47, YD5MG9-14, YD5MG9-56, YD5MG9-9, YD5MG9-13, YD5MG9-43, YD5MG9-42, YD5MG9-11, YD5MG9-10, YD5MG9-6, YD5MG9-60) is shown in Figure 3In the qualitative test of potassium solubilization, strain STSG25-LZ24 (originally numbered STS-G25 in the figure, but later changed to STSG25-LZ24 to emphasize its origin) had a medium potassium solubilization ability compared to other tested strains.

[0044] Example 5: Identification of Iron Carrier Production Capacity

[0045] The isolated strains were inoculated onto CAS medium and incubated at 28°C for 3 days. The presence of a clear zone was observed and measured. The clear zone (H) and colony diameter (h) were measured on the plates, and the (H / h) ratio was calculated. A higher ratio indicates a stronger siderophore-producing ability. The siderophore-producing abilities of different strains are shown in Table 3. Table 3 shows that 8 out of 60 strains possessed siderophore-producing ability, accounting for 55% of the total isolated strains. Among them, strain STSG25-LZ24 had a siderophore-producing index of 1.8, which is at a moderate level compared to the other strains.

[0046] Table 3. Siderophore production capacity of culturable bacteria in the roots of *Juniperus chinensis*

[0047] Strain number Siderophore production index (H / h) Strain number Siderophore production index (H / h) YD5MG9-2 1.5 STS-G1 2.0 YD5MG9-3 nd STS-G2 1.5 YD5MG9-4 nd STS-G3 nd YD5MG9-5 1.5 STS-G4 nd YD5MG9-6 1.6 STS-G6 1.25 YD5MG9-7 nd STS-G12 nd YD5MG9-8 nd STS-G19 1.8 YD5MG9-9 1.2 STSG25-LZ24 1.8 YD5MG9-10 2.0 STS-G26 nd YD5MG9-12 1.5 STS-G27 nd YD5MG9-13 2.33 STS-G33 nd YD5MG9-14 1.5 STS-G37 nd YD5MG9-15 1.4 STS-G38 nd YD5MG9-16 nd STS-Y4 nd YD5MG9-18 1.75 STS-Y5 1.2 YD5MG9-19 1.5 STS-Y6 2.0 YD5MG9-20 1.5 STS-Y7 1.75 YD5MG9-21 1.25 STS-Y8 1.6 YD5MG9-22 1.5 STS-Y11 nd YD5MG9-23 1.67 STS-Y14 nd YD5MG9-24 nd STS-Y30 nd YD5MG9-26 1.75 STS-Y31 nd YD5MG9-29 1.5 STS-Y38 nd YD5MG9-30 1.75 STS-Y39 nd YD5MG9-34 1.75 STS-Y40 nd YD5MG9-38 1.67 STS-G32 nd YD5MG9-43 nd STS-G40 nd YD5MG9-47 1.2 STS-G20 nd YD5MG9-56 1.6 YD5MG9-42 1.4 YD5MG9-60 nd YD5MG9-11 1.75

[0048] Different strains (YD5MG9-9, YD5MG9-47, YD5MG9-2, YD5MG9-56) Figure 4 YD5MG956 in G9-42, YD5MG9-43, YD5MG9-6, YD5MG9-2, YD5MG9-10, YD5MG9-13, STS-Y8, STS-G19, STS-G26( Figure 4 The iron-producing carrier effects of STSG26), STS-G37, STS-Y16, STS-G27, STS-G23, STS-Y8, STS-G25, STS-G26, and STS-Y5 are shown in [reference needed]. Figure 4 The siderophore production capacity of strain STSG25-LZ24 (originally numbered STS-G25 in the figure, but later changed to STSG25-LZ24 to emphasize its origin) is at a medium level compared to other strains.

[0049] Example 6: Experiment on the ability to promote the germination of giant cypress seeds

[0050] The STSG25-LZ24 strain, which promotes growth, was activated and inoculated into LB liquid medium. It was cultured at 28°C and 180 r / min in a constant-temperature shaker for 72 h. After 72 h, it was used as a seed culture and inoculated into LB liquid medium at a 1:1 ratio. The culture was then incubated at 28°C and 160 r / min in a constant-temperature shaker for 120 h to achieve a viable bacterial concentration of 10⁷ CFU / mL. Ten *Juniperus chinensis* seeds were evenly spread on each sterile petri dish. The STSG25-LZ24 bacterial suspension was sprayed onto the surface of each seed, ensuring even coverage. The bacterial suspension was repeated every 3 days, 5 ml each time, for a total of 3 applications. Distilled water (CK), blank culture medium (CKM), and some other strains (STS-Y16, YD5MG9-9, STS-G27) were used as controls in the same treatment. After 10 days of cultivation, observe and record the seed germination status. The experimental results are shown below. Figure 5 This study demonstrated that the germination rate of *Juniperus chinensis* seeds treated with strain STSG25-LZ24 was faster than that of the control group. Through the above-described methods, multiple experiments have proven that strain STSG25-LZ24 can significantly promote the germination rate and growth speed of *Juniperus chinensis* seeds, showing promising application prospects.

[0051] Example 7 Molecular biological identification of STSG25-LZ24

[0052] DNA was extracted from the strain using a bacterial genome extraction kit and diluted 50-fold as a template for PCR amplification of the strain's 16S rRNA sequence. The amplification primers were 27F and 1492R. The amplification product was then recovered and sequenced. The sequence obtained is shown in SEQ ID NO.1, and the NCBI accession number is PQ849237.

[0053] Based on the above characteristics, strain STSG25-LZ24 was identified as Erwinia billingiae, and its colony characteristics are shown in [the table below]. Figure 6 The strain appears white on the culture medium, with smooth, viscous, moist colonies that are easily picked up. This strain has been deposited under the accession number CGMCC No. 33386.

[0054] In summary, this invention evaluated the IAA production, phosphorus solubilization, potassium solubilization, siderophore production, and growth-promoting characteristics of bacteria within the roots of *Juniperus chinensis*, and selected strain STSG25-LZ24 for patent application based on a comprehensive assessment of these indicators. Strain STSG25-LZ24 can promote the germination of *Juniperus chinensis* seeds and possesses the abilities to produce IAA, solubilize phosphorus, solubilize potassium, and produce siderophores, significantly shortening seed germination time.

Claims

1. Use of a strain in the manufacture of a product for promoting the germination of Thuja plicata seeds and / or for promoting the breeding of Thuja plicata, characterized in that, The strain is Erwinia billingiae, and its preservation number is CGMCC No. 33386.

2. Use according to claim 1, wherein The 16sRNA of the strain comprises a sequence as shown in SEQ ID NO.

1.

3. Use according to any one of claims 1-2, characterized in that, The product is a microbial agent, a seed coating, a composition or a synthetic microbial flora.

4. The use according to claim 3, wherein the compound is ###0002### The microbial agent is a dry powder, a pill, a wettable powder, a water dispersible granule, a suspension, a suspoemulsion, an aqueous emulsion or a microemulsion.

5. A microbial agent, a seed coating, a composition or a synthetic microbial flora, which is the microbial agent, the seed coating, the composition or the synthetic microbial flora as claimed in claim 3.

Images