A strain with growth-promoting and salt-tolerant function derived from mangrove ecosystem and its application

Abstract

The present invention discloses a strain with growth-promoting and salt-tolerant function derived from a mangrove ecosystem and its application. The strain was deposited in the Guangdong Provincial Microbial Culture Collection Center on July 13, 2022, with a taxonomic name of Pantoeastewartii and a deposit number of GDMCC No: 62620; the strain can fix nitrogen, dissolve potassium, dissolve phosphorus, promote plant growth, and improve the relief of plant salt stress; compared with the reported strains, it can produce volatile organic compounds with growth-promoting functions under salt treatment, which can be used to prepare plant growth promoters, microbial fertilizers, soil conditioners, etc.; it has good application prospects.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of bacteria derived from the mangrove ecosystem that has growth-promoting and salt-tolerant functions and its applications. Background Technology

[0002] Salt stress during plant growth and development can lead to ion imbalance, nutrient deficiency, disruption of carbon and nitrogen absorption pathways, reduced photosynthetic rate, and the production of reactive oxygen species (ROS), causing osmotic and oxygen stress problems that hinder crop growth and development and reduce yield. Plant growth-promoting bacteria (PGPs) are important strains for developing microbial fertilizers and pesticides, and can be used to improve plant salt stress tolerance. They can increase yield by improving nutrient absorption through nitrogen fixation and phosphorus solubilization, and also positively influence plants through mechanisms related to stress tolerance, such as altering protein expression, extracellular polysaccharide (EPS) synthesis, osmotic accumulation, increasing plant hormone levels, and increasing the production of antioxidant enzymes (such as ascorbate peroxidase (APX), catalase (CAT), and glutathione reductase (GR)) under stress conditions.

[0003] Mangroves, as unique ecosystems subjected to periodic seawater inundation, possess a long-term high-salt environment, fostering unique microbial resources, including salt-tolerant growth-promoting bacteria that can play a positive role under salt stress. However, the application of mangrove microbial resources in promoting growth is currently limited. Therefore, it is necessary to develop microbial resources in mangrove ecosystems that can alleviate salt stress. Summary of the Invention

[0004] The purpose of this invention is to provide a strain that can alleviate salt stress and promote plant growth.

[0005] The technical solution adopted in this invention is:

[0006] In a first aspect, the present invention provides a Pantoea stewartii strain, which was deposited on July 13, 2022, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with the taxonomic name Pantoea stewartii and accession number GDMCC No: 62620.

[0007] In a second aspect, the present invention provides a bacterial agent comprising at least one of the bacterial solution, bacterial cells, and volatile organic compounds of the strain described in the first aspect of the present invention.

[0008] In some embodiments of the present invention, the volatile organic compound comprises: 10-15% 3-hydroxy-2-butanone, 40-50% 3-methyl-butanol, and 10-15% phenylethanol.

[0009] A third aspect of the present invention provides the application of the strains described in the first aspect of the present invention or the bacterial agents described in the second aspect of the present invention in potassium solubilization and / or nitrogen fixation and / or phosphorus solubilization and / or promotion of plant growth and / or improvement of plant tolerance to salt stress environments.

[0010] In some embodiments of the present invention, the salt concentration is 5% to 10%.

[0011] In some embodiments of the present invention, the plants include: rice, water spinach, and bok choy.

[0012] A fourth aspect of the present invention provides the use of the strains described in the first aspect of the present invention or the bacterial agents described in the second aspect of the present invention in the preparation of products that solubilize potassium and / or fix nitrogen and / or solubilize phosphorus and / or promote plant growth and / or improve plant tolerance to salt stress environments.

[0013] In some embodiments of the present invention, the product is a plant growth promoter, a plant salt-tolerant growth promoter, a microbial fertilizer, or a soil conditioner.

[0014] In some embodiments of the present invention, the plants include: rice, water spinach, and bok choy.

[0015] In a fifth aspect, the present invention provides a product comprising the strain described in the first aspect of the present invention or the microbial agent described in the second aspect of the present invention.

[0016] In some embodiments of the present invention, the product functions as potassium solubilization and / or nitrogen fixation and / or phosphorus solubilization and / or promoting plant growth and / or improving plant tolerance to salt stress environments.

[0017] In some embodiments of the present invention, the product is a plant growth promoter, a plant salt-tolerant growth promoter, a microbial fertilizer, or a soil conditioner.

[0018] In some embodiments of the present invention, the plants include: rice, water spinach, and bok choy.

[0019] A sixth aspect of the present invention provides a method for potassium solubilization and / or nitrogen fixation and / or phosphorus solubilization and / or promoting plant growth and / or improving plant tolerance to salt stress environments, by adding the product described in the fifth aspect of the present invention to the plant growth environment.

[0020] In some embodiments of the present invention, the plants include rice, water spinach, and bok choy.

[0021] The beneficial effects of this invention are:

[0022] This invention provides a novel plant strain, *Pantoeastewartii* DYX, which promotes plant growth and alleviates salt stress. This strain was deposited at the Guangdong Provincial Microbial Culture Collection Center on July 13, 2022, with the taxonomic name *Pantoea stewartii* and accession number GDMCC No. 62620. The strain described in this invention can fix nitrogen, solubilize potassium and phosphorus, promote plant growth, and improve the ability to alleviate salt stress in plants. Compared with previously reported strains, it can produce volatile organic compounds with growth-promoting functions under salt treatment, which can be used to prepare plant growth promoters, microbial fertilizers, soil conditioners, etc., and has excellent application prospects. Attached Figure Description

[0023] Figure 1 These are the morphological characteristics of strain DYX.

[0024] Figure 2 This is the phylogenetic tree of the 16S rDNA of strain DYX.

[0025] Figure 3 This shows the growth of strain DYX in organic phosphorus medium.

[0026] Figure 4 This shows the growth of strain DYX in inorganic phosphorus medium.

[0027] Figure 5 This shows the growth of strain DYX in silicate medium.

[0028] Figure 6 This shows the growth of strain DYX in nitrogen-free medium.

[0029] Figure 7 The results show the effect of adding strain DYX on the total length of rice seedlings.

[0030] Figure 8 These are the GC / MS results of volatile compounds from strain DYX. Detailed Implementation

[0031] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0032] Example 1: Enrichment and Isolation of Strain DYX

[0033] 1) Samples of hypocotyls of the mangrove plant *Kandelia candel* were collected in Shenzhen Bay, Futian District, Shenzhen, Guangdong Province (22.5231°N, 113.9947°E).

[0034] 2) Prepare R2A solid medium with a NaCl content of 5%. The components of R2A solid medium are: 0.25 g / L tryptone, 0.5 g / L acid-hydrolyzed casein, 0.5 g / L yeast extract, 0.5 g / L soluble starch, 0.3 g / L dipotassium hydrogen phosphate, 0.1 g / L magnesium sulfate, 0.3 g / L sodium pyruvate, 0.25 g / L peptone, 0.5 g / L glucose, 15.0 g / L agar, and pH 7.2 ± 0.2 (25℃).

[0035] Under aseptic conditions, a suitable amount of Kandelia candel hypocotyl was chopped with scissors, transferred to a mortar, and 7 mL of sterile ultrapure water, along with a small amount of sterile quartz sand and calcium carbonate. The mixture was ground thoroughly for 10 minutes. 1 mL of the grinding liquid was then pipetted into a 10 mL centrifuge tube containing 9 mL of sterile water and mixed thoroughly by pipetting. This yielded 10 mL of the mixture. -1 Diluent; pipette 1 mL of 10 -1 The diluent was transferred to a 10 mL centrifuge tube containing 9 mL of sterile water and mixed by pipetting to obtain a 10 mL centrifuge tube. -2 Diluent; take another 10 -2 Transfer 1 mL of the diluent to a centrifuge tube containing 9 mL of sterile water, mix thoroughly by pipetting, and you will have 10 mL of the solution. -3 Diluent. Use a pipette to draw 10... -1 10 -2 10 -3 Three concentrations of diluted bacterial suspension, 100 μL each, were inoculated onto plates of different dilutions. The culture medium was placed in a 30°C incubator. After colonies grew on the plates, characteristic colonies were picked and streaked onto fresh medium for pure culture. Each strain was repeated three times until a single colony was obtained.

[0036] The obtained strain is Pantoea stewartii subsp. indologenes DYX, which was deposited on July 13, 2022, at the Guangdong Provincial Microbial Culture Collection Center, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Its taxonomic name is Pantoea stewartii, and its accession number is GDMCC No. 62620. The viability of the sample was also confirmed on the same day.

[0037] Example 2: Morphological characteristics of strain DYX

[0038] 1) Cell morphology characteristics

[0039] The strain DYX is Gram-negative, non-capsulated, non-spore-forming, and rod-shaped.

[0040] 2) Colony morphology characteristics

[0041] The results of strain DYX after 48 h of aerobic R2A agar plate culture are as follows: Figure 1 As shown. The colonies are yellow, with a smooth, opaque surface, neat edges, and a diameter of approximately 1-2 mm.

[0042] Physiological and biochemical characteristics of strain DYX

[0043] The physiological and biochemical characteristics of the strains were tested according to the standard procedure described in the ninth edition of Bergey's Manual of Bacterial Identification. The biochemical characteristics are shown in Table 1.

[0044] Table 1 Phenotypic characteristics of strain DYX

[0045]

[0046] + indicates a positive result; - indicates a negative result.

[0047] 3) Molecular taxonomic position of strain DYX

[0048] Total DNA was extracted from strain DYX and amplified by PCR using universal 16S rDNA primers 27F (5'-AGAGTTT-G-ATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The PCR products were then amplified into 16S rDNA and sequenced.

[0049] The 16S rDNA sequence of the strain was submitted to the GenBank database, obtaining accession number ON203057. Homology was analyzed by comparing it with known sequences. Base composition and GC content were analyzed using MEGA software, and genetic distance was calculated using the Kimura2 parameter. Based on sampling analysis with 1000 replicates, a phylogenetic tree was constructed using the NJ nearest neighbor method. Figure 2 As shown. According to phylogenetic theory, strains can only be considered as a single strain if their similarity is greater than 97%. Strain DYX has a similarity of 99.6% to Pantoea stewartii subsp. indologenes; therefore, strain DYX is classified as Pantoea stewartii subsp. indologenes.

[0050] Example 3: Detection of the growth-promoting effect of the strain

[0051] 1) Evaluation test of growth promotion indicators of strain DYX

[0052] Organophosphorus culture medium formula: glucose 10.0 g / L, ammonium sulfate 0.5 g / L, yeast extract 0.5 g / L, sodium chloride 0.3 g / L, potassium chloride 0.3 g / L, magnesium sulfate 0.3 g / L, ferrous sulfate 0.03 g / L, manganese sulfate 0.03 g / L, lecithin 0.2 g / L, calcium carbonate 1.0 g / L, agar 15.0 g / L. pH 7.0-7.5, 25℃.

[0053] Inorganic phosphorus culture medium formula: glucose 10.0 g / L, ammonium sulfate 0.5 g / L, sodium chloride 0.3 g / L, magnesium sulfate 0.3 g / L, manganese sulfate 0.03 g / L, potassium sulfate 0.3 g / L, ferrous sulfate 0.03 g / L, calcium phosphate 5.0 g / L, agar 15.0 g / L. pH 7.0-7.5, 25℃.

[0054] Nitrogen-free culture medium formula: mannitol 10.0 g / L, potassium dihydrogen phosphate 0.2 g / L, magnesium sulfate heptahydrate 0.2 g / L, sodium chloride 0.2 g / L, calcium sulfate dihydrate 0.2 g / L, calcium carbonate 5.0 g / L, agar 20.0 g / L. pH 7.0-7.2, 25℃.

[0055] Silicate culture medium formula: sucrose 5.0 g / L, magnesium sulfate 0.5 g / L, calcium sulfate 0.1 g / L, disodium hydrogen phosphate 2.0 g / L, ferric chloride 0.005 g / L, glass powder 1.0 g / L, agar 15.0 g / L. pH 7.0 ± 0.2, 25℃.

[0056] After preparing the above three culture media, autoclave them at 121℃ for 20 minutes and then cool them for later use.

[0057] Bacterial colonies from the preserved strain DYX were picked from the slant and inoculated into R2A liquid medium. The cells were activated at 30°C and 180 rpm / min on a shaker for 18 hours to allow the bacteria to reach the exponential growth phase. After centrifugation at 8000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in sterile water. 7 μL of the bacterial suspension was pipetted into a fixed area of ​​the medium, with three replicates on each medium. After inoculation, the culture was incubated at 30°C. It was observed that strain DYX grew rapidly on organophosphate medium (…). Figure 3 ) and inorganic phosphorus culture medium ( Figure 4 The presence of a transparent zone on both silicate medium and inorganic phosphorus medium indicates that strain DYX has the ability to dissolve both organic and inorganic phosphorus; strain DYX can produce a transparent zone on both silicate medium and inorganic phosphorus medium. Figure 5 ) and nitrogen-free culture medium ( Figure 6 The fact that it can produce and reproduce normally on the surface indicates that it has the ability to fix nitrogen and dissolve potassium.

[0058] 2) Evaluation test of salt reduction rate of strain DYX

[0059] Prepare a liquid culture medium with a NaCl concentration of 5% and measure the salinity using a salinity meter. Then, inoculate the prepared bacterial solution into the medium at a 5% inoculum and incubate for 48 hours (180 rpm / min, 30℃). Afterward, take an appropriate amount of fermentation broth sample and measure the salinity again using a salinity meter. Calculate the salt reduction efficiency of the strain according to the following formula.

[0060] Salt reduction efficiency of the strain = [(salinity of liquid culture medium - salinity of fermentation broth) / salinity of liquid culture medium] × 100%.

[0061] The results showed that strain DYX achieved a salt reduction rate of 3.7% under the treatment conditions.

[0062] Regarding the strain's highest salt tolerance value, current testing indicates it is between 5% and 10%.

[0063] 3) Effects of strain DYX addition on rice seed growth under salt stress

[0064] This study investigated the effects of salt-tolerant bacteria addition on rice seed germination and seedling growth under salt stress, and verified its growth-promoting function. The specific methods are as follows:

[0065] Seed selection and surface disinfection: Select rice seeds of uniform size and plumpness. Soak them in 10% NaClO solution for 10 minutes, then rinse them 5-8 times with sterile deionized water. Soak them in 75% alcohol for 5 minutes, and finally rinse them 5-7 times with sterile deionized water. After disinfection, soak the seeds in sterile deionized water at room temperature for 24 hours for later use.

[0066] Preparation of bacterial suspension and co-culture plates: The bacterial strain was inoculated into liquid culture medium and incubated with shaking for 24 h (30℃, 180 rpm). After incubation, the culture was centrifuged for 5 min (8000 rpm, 25℃), the precipitated bacterial cells were collected, washed twice, resuspended in 60 mmol / L sterile NaCl solution, and the OD600 was adjusted to 0.3. The suspension was then diluted 30 times to obtain the bacterial suspension. The same operation was performed, but the bacterial cells were resuspended in double-distilled water and inoculated onto solid culture medium by drop method (6 drops per plate, 7 μL per drop). After incubation at 30℃ for three days, the co-culture plates were prepared.

[0067] Seed processing settings:

[0068] (1) T1, sterile water; add 5 mL of sterile water to the culture dish containing the seeds, and change the treatment solution every 2 days during the culture period to ensure that the stress environment remains unchanged.

[0069] (2) T2, use A bacterial suspension with 60 mmol / L NaCl solution; add 5 mL of the prepared salt-containing bacterial suspension to the culture dish containing the seeds, and change the treatment solution every 2 days during the culture period to ensure that the stress environment remains unchanged.

[0070] (3) T3, co-culture of seeds and microorganisms; add 10 mL of 60 mmol / L NaCl solution to the culture dish containing the seeds, invert the co-culture plate and the culture dish containing the seeds together, and seal it with Parafilm sealing film.

[0071] (4) T4, 60 mmol / L NaCl solution; Add 5 mL of 60 mmol / L NaCl solution to the culture dish containing the seeds. Change the treatment solution every 2 days during the culture period to ensure that the stress environment remains unchanged.

[0072] Seed culture: Each treatment was performed in triplicate, with 30 seeds per replicate placed in a single 9cm petri dish. Two layers of sterilized filter paper were placed on the dish to retain moisture, and the dish was covered to maintain humidity. After evenly distributing the seeds, the dishes were placed in a 25°C incubator. Germination was defined as the sprout reaching half the seed length. The number of germinating seeds was recorded daily during the culture period, and the germination status was observed and recorded. Root and sprout lengths of the seedlings were also measured. The seed vigor index was calculated using the following formula.

[0073] Vitality Index VI = ∑(Gt / Dt) × Average seedling length (root length + shoot length)

[0074] In the formula: Gt is the number of germinations on day t; Dt is the number of germination days.

[0075] The processing numbers are shown in Table 2:

[0076] Table 2 Processing Number

[0077]

[0078] (1) Effect of strain DYX addition on rice seed vigor index under salt stress

[0079] Compared with traditional germination rate measurement methods, seed vigor reflects the germination speed and uniformity of seeds under actual conditions, as well as the robust growth potential of seedlings. Table 3 shows that 60 mmol / L NaCl stress significantly reduces seed vigor (P<0.05), and the seed vigor index without stress is 1.81 times that under salt stress. Both the bacterial suspension of strain DYX and the addition of VOCs can improve seed vigor under salt stress, with VOCs showing a significant effect, increasing the seed vigor index by 1.69 times under salt stress.

[0080] Table 3 Effects of bacterial strain addition on rice seed germination under salt stress

[0081]

[0082] (2) Effect of strain DYX addition on rice seedling length under salt stress

[0083] Figure 7 In the figure, identical letters (a and b) indicate no significant difference, while different letters indicate significant difference (P<0.05). As shown in the figure, 60 mmol / L NaCl stress inhibited rice seedling growth, and the total seedling length (the sum of root and shoot length) was significantly shorter than under salt stress. The addition of DYX bacterial suspension and DYX VOCs significantly promoted the growth of seedling roots and shoots, with data essentially the same as under salt stress, showing no significant difference. This indicates that these treatments can alleviate the toxic effects of salt stress on rice seedlings and promote seedling growth.

[0084] Example 4: Effects of strain DYX addition on seed growth of other crops under salt stress

[0085] Two types of vegetable seeds, water spinach and bok choy, were selected as the research subjects.

[0086] Select seeds of uniform size and plumpness for surface disinfection. Water spinach and bok choy seeds were first disinfected with 75% ethanol for 3 minutes, then rinsed 3-5 times with sterile water. The disinfected seeds were then soaked in sterile deionized water at room temperature for 8 hours. Each treatment group consisted of 30 seeds as a replicate, with three repetitions.

[0087] The treatment codes for water spinach seeds and bok choy seeds are shown in Table 4:

[0088] Table 4 Treatment numbers for the two types of seeds

[0089]

[0090]

[0091] Table 5 shows that for water spinach seeds, the seed vigor index after adding VOCs from DYX bacteria was 1.19 times that under salt stress; for pak choi seeds, the seed vigor index after adding A bacteria suspension was 1.37 times that under salt stress. This indicates that these treatments can alleviate the toxic effects of salt stress on the corresponding vegetable seeds and promote seed germination and growth.

[0092] Table 5. Effects of bacterial strain addition on seed germination under salt stress.

[0093]

[0094] Example 5: Detection of VOCs produced by strain DYX

[0095] Dispense 5 mL portions of sterilized, undiluted R2A medium into 20 mL headspace vials. Tilt the vials at 30° to allow them to cool and solidify. Take a bacterial plate and use an inoculation loop to pick up colonies and inoculate them onto the slant of the vial, streaking as completely as possible to ensure the bacteria will cover the entire slant. Cover with a sealing film and incubate at 30°C for two days. Remove the sealing film and seal the vial with a hollow screw cap containing a PTFE septum for one day. Simultaneously, set up a blank control with only medium but no inoculation in the headspace vials and extract the gas phase components using solid-phase microextraction (SPME) fiber.

[0096] Immediately after extraction, the samples were analyzed using an Agilent Technologies 7890A-5975C gas chromatograph-mass spectrometer (Agilent Technologies, USA). Chromatographic conditions: injection port temperature 250℃, injection time 3 min; splitless mode, carrier gas 99.999% high-purity helium, column flow rate 1 mL / min. Column oven temperature program: initial temperature 50℃, hold for 2 min, increase to 180℃ at 8℃ / min, then increase to 240℃ at 10℃ / min, hold for 6 min. Mass spectrometry conditions: ionization mode EI, 70 eV; ion source temperature 230℃, quadrupole temperature 150℃, transfer line temperature 250℃; full scan mode, scan range 35–450 amu. The obtained volatile organic compound mass spectrometry results are as follows: Figure 8 As shown, comparison and identification in the NIST / EPA / NIH database revealed that the VOCs produced by strain DYX include three substances: 3-hydroxy-2-butanone (10%-15%), 3-methyl-butanol (40%-50%), and phenethyl alcohol (10%-15%). Among these, 3-hydroxy-2-butanone, also known as acetoin, has been reported to promote plant growth and resist stress, and can interconvert with 2,3-butanediol, which can resist salt stress. The substances 3-methyl-butanol and phenethyl alcohol may also have potential growth-promoting effects.

[0097] The above detailed embodiments have provided a comprehensive description of the present invention. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

Claims

1. A strain of Pantoea stutzeri, whose taxonomic name is Pantoea stewartii , deposited in Guangdong Provincial Microbiological Culture Collection Center on July 13, 2022, with the deposit number: GDMCC No: 62620.

2. A bacterial agent, characterized in that The bacterial agent comprises the bacterial cells of the strain according to claim 1.

3. Use of the strain according to claim 1 or the bacterial agent according to claim 2 in potassium solution, nitrogen fixation, phosphorus dissolution, plant growth promotion or improving plant tolerance to salt stress environment.

4. Use of the strain according to claim 1 or the bacterial agent according to claim 2 in preparing products for dissolving potassium, fixing nitrogen, dissolving phosphorus, promoting plant growth or improving plant tolerance to salt stress environments.

5. The use according to claim 4, characterized in that: The product is a plant growth promoter, a microbial fertilizer or a soil conditioner.

6. The use according to claim 5, characterized in that: The plant growth promoting agent includes a plant salt-tolerant growth promoting agent.

7. A product, characterized in that The product comprises the strain according to claim 1 or the bacterial agent according to claim 2.

8. A method for dissolving potassium, fixing nitrogen, dissolving phosphorus, promoting plant growth or improving plant tolerance to salt stress environment, characterized in that: Add the product of claim 7 to the plant growth environment.

9. The use according to any one of claims 3 to 6 or the method according to claim 8, characterized in that: The plants include: rice, water spinach and Chinese cabbage.

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