Erwinia capable of promoting germination of seu juniperi seeds and application of Erwinia

Abstract

The invention discloses an Erwinia bilingiae strain STSG25-LZ24 capable of promoting germination of seu juniperi seeds and application of the Erwinia bilingiae strain STSG25-LZ24. The preservation number of the Erwinia bilingiae strain STSG25-LZ24 disclosed by the invention is CGMCC (China General Microbiological Culture Collection Center) No.33386, and a 16S rRNA (Ribose Nucleic Acid) gene of the Erwinia bilingiae strain STSG25-LZ24 comprises a sequence as shown in SEQ ID NO.1. The invention also discloses an application of the Erwinia The strain not only can obviously promote the germination of juniperus chinensis seeds, but also has a good application prospect in the aspects of juniperus chinensis seed breeding, seed coating manufacturing and synthetic flora construction.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms and biotechnology, and particularly relates to Erwinia capable of promoting germination of giant cedar seeds and application thereof. Background Art

[0002] Cupressus gigantean, also known as Yarlung Zangbo cypress, is a rare and unique evergreen tree, listed as a Class I endangered tree species in my country. Its population is mainly distributed in strips or sporadically in some river valleys along the Yarlung Zangbo River and its tributary, the Niyang River, in southeastern Tibet. With its excellent material and outstanding tolerance, Cupressus gigantean plays a key role in soil and water conservation and water conservation. As an ancient species, Cupressus gigantean is of great significance to the study of vegetation evolution and paleoclimate reconstruction, and is also of great significance to the protection of the Qinghai-Tibet Plateau ecosystem.

[0003] At present, the giant cypress population is facing severe challenges of natural regeneration, and the population is in decline as a whole. The primary problem is that the seed quality is poor, the natural germination rate is low, and the seedlings have weak adaptability to the environment. The harsh habitat conditions make the conversion rate from seeds to saplings extremely low. Secondly, the geographical distribution range of giant cypress is narrow, and the habitat is heterogeneous and fragmented, which poses a serious constraint on its growth and development. In addition, the climate in southeastern Tibet tends to be "warm and dry", droughts are frequent, and human factors such as the development and construction of the lower reaches of the Yajiang River have further aggravated the deterioration of the living environment of giant cypress. The germination of giant cypress seeds mainly depends on natural conditions, is inefficient, and is greatly affected by environmental factors. Therefore, it is crucial to develop an effective technology to promote the germination of giant cypress seeds, innovate the existing microbial agent production formula, and explore local efficient strains with stronger resistance and higher growth-promoting ability to make efficient microbial agents or construct synthetic flora to promote the renewal and recovery of giant cypress populations. Summary of the invention

[0004] Plant roots contain a large number of microorganisms, one of which can promote seed germination, plant growth and disease prevention, known as growth-promoting microorganisms. Growth-promoting microorganisms play a vital role in improving the soil environment, promoting plant nutrient absorption and enhancing plant resistance.

[0005] The growth-promoting bacteria of giant cypress can help to increase the growth rate and health of giant cypress, and help the plants resist stress such as drought and nutrient deficiency, thus significantly improving the environmental adaptability of giant cypress and promoting the renewal of the community. For example, bacteria with the function of dissolving phosphorus and potassium can significantly improve the environmental adaptability and ecosystem productivity of cypress plants, which is crucial to maintaining forest diversity, stability and resistance.

[0006] The present invention uses the root system of giant cypress as the separation material, and carries out the separation, screening and identification of its growth-promoting bacteria, with the purpose of providing a strain that promotes the germination of giant cypress seeds. The strain can promote the germination of giant cypress seeds, improve the seed germination rate and growth rate, and thus help promote the renewal of giant cypress communities.

[0007] The present invention is specifically implemented through the following technical solutions:

[0008] The present invention provides a strain of Erwinia billingiae, which has been deposited with a deposit number of CGMCC No. 33386, a depository of which is the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC), a deposit address of which is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, a taxonomic name of which is Erwinia billingiae, a deposit date of which is January 13, 2025, and a strain name of which is STSG25-LZ24.

[0009] The 16s rRNA gene of the Erwinia sp. CGMCC33386 comprises the sequence shown in SEQ ID NO.1.

[0010] Furthermore, the Erwinia sp. CGMCC33386 was isolated from endophytic bacteria in the roots of Tibetan cedar.

[0011] Further, the Erwinia sp. CGMCC33386 can promote plant seed germination. Preferably, the plant is a tree plant of the genus Cupressus in the family Cupressaceae. More preferably, the tree plant of the genus Cupressus in the family Cupressaceae is Cupressus giganteus.

[0012] The present invention provides an application of the Erwinia sp. CGMCC33386. Preferably, the application is the application of the strain in plant breeding and transplanting, the process is seed germination, artificial sowing, aerial sowing and seedling transplanting, and the plant is a tree plant of the genus Cupressaceae. Preferably, the tree plant of the genus Cupressaceae is Cupressus giganteus.

[0013] The present invention also provides the application of the Erwinia CGMCC33386. Specifically, the application includes the application in the preparation of a bacterial agent, seed coating, composition or cooperative flora for promoting cypress seed germination and seedling growth. In some embodiments, the bacterial agent can be obtained by conventional liquid or solid culture, including a bacterial cell culture, produced by conventional liquid fermentation, and then a surfactant such as a dispersant, a stabilizer, a wetting agent, a binder, a defoamer, a disintegrant, an antifreeze agent, etc. is added or mixed with an adsorption carrier in a certain proportion to prepare a seed coating, a dry powder, a pill, a wettable powder, a water-dispersible granule, a suspension, a suspoemulsion, an aqueous emulsion or a microemulsion.

[0014] The method for promoting seed germination by Erwinia CGMCC33386 described in the present invention also belongs to the protection scope of the present invention. The method is to perform spray treatment during the seed germination process; the spray is a preparation prepared from the bacterial suspension or fermentation liquid or metabolites of Erwinia CGMCC33386.

[0015] The present invention also provides the use of the Erwinia CGMCC33386 in promoting the germination of giant cedar seeds. At the same time, the use of Erwinia CGMCC33386 in constructing a synthetic bacterial community is also within the protection scope of the present invention.

[0016] The present invention also provides a bacterial agent, seed coating, composition or synthetic bacterial community comprising the Erwinia CGMCC33386. Further, the bacterial agent, seed coating, composition or synthetic bacterial community is the bacterial agent, seed coating, composition or synthetic bacterial community as described above.

[0017] The beneficial effects of the present invention are:

[0018] The Erwinia sp. CGMCC33386 provided by the invention can promote the germination and growth of giant cypress seeds, and improve the seed germination rate and growth rate. As a biological preparation material for Cupressaceae cypress plants, the strain has a good market prospect in terms of developing new biological control agents or growth regulators. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Showing the indoleacetic acid (IAA) production ability of different strains.

[0020] Figure 2 Showing the phosphate solubilization effect of different strains.

[0021] Figure 3 Showing the potassium-dissolving effects of different strains.

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

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

[0024] Figure 6 These are the colony growth characteristics of strain STSG25-LZ24. DETAILED DESCRIPTION

[0025] The technical scheme of the present invention is further described below in conjunction with the embodiments. The following description is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the technical content disclosed above to change or modify it into an equivalent embodiment with equivalent changes. Any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the content of the scheme of the present invention shall fall within the protection scope of the present invention.

[0026] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0027] Example 1 Isolation of strain STSG25-LZ24

[0028] 1.1 Strain source

[0029] The root samples of giant cypress in this example were collected from the Institute of Plateau Ecology in Linzhi City, Tibet Autonomous Region in June 2024. The samples were placed in sterile sealed plastic bags, labeled, brought back to the laboratory in time, and stored in a -20°C refrigerator.

[0030] 1.2 Isolation of endophytic bacteria from Cupressus giganteus

[0031] The roots of Giant Cupressus were disinfected with 1% sodium hypochlorite solution for 1 min and 75% alcohol for 1 min. After rinsing with sterile water for 5 min, the surface moisture of the roots was absorbed with sterile filter paper, and then the roots were added with sterile water for grinding. After 1 mL of the ground liquid was diluted twice, 150 μL of the diluted liquid was inoculated into LB medium and spread, and cultured inverted at 28°C for 3 days. Single colonies were picked and purified according to the apparent characteristics such as colony size, morphology and color. 60 cultivable strains were obtained through separation and purification.

[0032] Example 2 Qualitative analysis of indoleacetic acid (IAA) production capacity

[0033] Part of the strains isolated above were inoculated into King's medium and cultured to the logarithmic phase. They were shaken at 28°C and 180 r / min for 3 days. 50 μl was taken out and placed on a clean white porcelain plate. 50 μL of the developer was added and the plate was incubated at 28°C in the dark for 30 min. If a pink color appeared, it indicated that the strain was able to produce IAA. The stronger the pink color, the stronger the ability of the strain to produce IAA. Using King's medium without inoculation as control, the qualitative results of IAA production ability 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 Figure 1 , where CK is distilled water, CKM is the culture medium stock solution, Figure 1 It shows that STSG25-LZ24 (the original number in the figure was STSG-25, and the number was subsequently adjusted to STSG25-LZ24 to emphasize its origin) has a higher IAA production capacity than other strains.

[0034] Example 3 Inorganic phosphorus degradation ability

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

[0036] Table 1 Inorganic phosphorus degradation capacity of culturable bacteria in the roots of Cupressus giganteus 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

[0037] 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 , showing that the phosphate solubilizing ability of STSG25-LZ24 (the original number in the figure was STS-G25, and the number was subsequently adjusted to STSG25-LZ24 to emphasize its origin) is at the medium level of the experimental strains.

[0038] Example 4 Identification of Potassium-Resolving Ability

[0039] The isolated strains were inoculated on potassium-solubilizing medium and cultured at 28°C for 3 days. The transparent circle (H) and colony diameter (h) in the plate were measured, and the (H / h) ratio was calculated. The larger the ratio, the stronger the potassium-solubilizing ability. The potassium-solubilizing ability of different strains is shown in Table 2. As shown in Table 2, 25 of the 60 strains have potassium-solubilizing ability, accounting for 41.6% of the total isolated strains. Among them, the potassium-solubilizing ability of strain STSG25-LZ24 is 1.67.

[0040] Table 2 Potassium-solubilizing capacity of culturable bacteria in the roots of Cupressus giganteus 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

[0041] 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 3 In the qualitative test of potassium solubilization, the strain STSG25-LZ24 (the original number in the figure was STS-G25, and the number was subsequently adjusted to STSG25-LZ24 to emphasize its origin) had a medium potassium solubilization ability compared to other test strains.

[0042] Example 5 Identification of Siderophore Production Capacity

[0043] The isolated strains were inoculated into CAS medium and cultured at 28°C for 3 days. The medium was observed and determined to see whether a transparent circle was produced. The transparent circle (H) and colony diameter (h) in the plate were determined, and the ratio (H / h) was calculated. The larger the ratio, the stronger the ability to produce siderophores. The siderophore production capacity of different strains is shown in Table 3. As shown in Table 3, 8 of the 60 strains have the ability to produce siderophores, accounting for 55% of the total isolated strains. Among them, the siderophore production index of strain STSG25-LZ24 was 1.8, which was between the medium level of the above strains.

[0044] Table 3 Siderophore production capacity of culturable bacteria in the roots of Cupressus giganteus 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

[0045] 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 siderophore production effect of STSG26), STS-G37, STS-Y16, STS-G27, STS-G23, STS-Y8, STS-G25, STS-G26, STS-Y5) is shown in Figure 4 The strain STSG25-LZ24 (the original number in the figure was STS-G25, and the number was subsequently adjusted to STSG25-LZ24 to emphasize its origin) has a medium-level ability to produce siderophores compared to some other strains.

[0046] Example 6 Test on the ability to promote germination of giant cedar seeds

[0047] The growth-promoting strain STSG25-LZ24 was activated and inoculated into LB liquid medium, and cultured in a constant temperature shaker at 28°C and 180 r / min. After 72 hours of culture, it was used as seed liquid to inoculate LB liquid medium at a ratio of 1:1, and then cultured in a constant temperature shaker at 28°C and 160r / min for 120 hours to make the live bacterial concentration of the bacterial liquid reach 107 CFU / mL, and then taken out for use. 10 giant cypress seeds were spread on each sterile culture dish, and the bacterial suspension of strain STSG25-LZ24 was inoculated on the surface of giant cypress seeds by spraying to ensure that the surface of the seeds was evenly covered with bacterial liquid. The bacterial suspension was added once every 3 days, 5 ml each time, and irrigated 3 times in total. Distilled water (CK), blank culture medium (CKM) and some other strains (STS-Y16, YD5MG9-9, STS-G27) were used as control experiments according to the above treatment methods. After 10 days of cultivation, the germination of seeds was observed and recorded. The test results are shown in Figure 5 , proving that the germination of giant cedar seeds treated with the strain STSG25-LZ24 bacterial suspension was faster than that of the control group. Through the above implementation, multiple experiments have proved that the strain STSG25-LZ24 can significantly promote the germination rate and growth rate of giant cedar seeds, and has good application prospects.

[0048] Example 7 Molecular Biological Identification of STSG25-LZ24

[0049] The strain DNA was extracted using a bacterial genome extraction kit and diluted 50 times as a template for PCR reaction to amplify the strain 16S rRNA sequence. The amplification primers were 27F and 1492R. The amplified product was then recovered and sequenced. The measured sequence is shown in SEQ ID NO.1. The NCBI gene accession number is PQ849237.

[0050] Based on the above characteristics, strain STSG25-LZ24 was identified as Erwinia billingiae. Its colony characteristics are shown in Figure 6 The strain is white in the culture medium, the surface of the colony is smooth and sticky, and it is easy to pick up when wet. The strain has been deposited with the deposit number CGMCC No. 33386.

[0051] In summary, the present invention evaluates the IAA production, phosphate solubilization, potassium solubilization, iron carrier production and growth-promoting characteristics of bacteria in the roots of giant cedar, and selects strain STSG25-LZ24 based on various indicators to apply for a strain patent. Strain STSG25-LZ24 can promote the germination of giant cedar seeds, and has the ability to produce IAA, solubilize phosphate, solubilize potassium, and produce iron carriers, which can significantly promote the shortening of seed germination time.

Claims

1. A strain capable of promoting the germination of giant cedar seeds, characterized in that: It is Erwinia billingiae, and its deposit number is CGMCC No. 33386.

2. The strain capable of promoting germination of giant cedar seeds as claimed in claim 1, characterized in that: The 16sRNA thereof comprises the sequence shown as SEQ ID NO.

1.

3. The strain capable of promoting germination of giant cedar seeds according to claim 2, characterized in that: It is an endophyte of Cupressus giganteus.

4. Use of the strain capable of promoting the germination of giant cedar seeds as claimed in any one of claims 1 to 3 in the preparation of products for promoting the germination of giant cedar seeds and / or promoting the reproduction of giant cedar.

5. The use according to claim 4, characterized in that: The product comprises the strain capable of promoting the germination of giant cedar seeds as described in any one of claims 1 to 3.

6. The use according to claim 5, characterized in that The product is a bacterial agent, a seed coating, a composition or a synthetic bacterial community.

7. The use according to claim 6, characterized in that The bacterial agent is a dry powder, a pill, a wettable powder, a water-dispersible granule, a suspension, a suspoemulsion, an aqueous emulsion or a microemulsion.

8. A bacterial agent, seed coating, composition or synthetic bacterial community as claimed in any one of claims 5 to 7.

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