Brevibacterium pekinensis, microbial agent and application of brevibacterium pekinensis

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to brevibacterium pekinensis, a microbial agent and application of the brevibacterium pekinensis, the microbial agent and the application of the brevibacterium pekinensis, the brevibacterium pekinensis is brevibacterium pekinensis B474, the brevibacterium pekinensis is preserved in the China Center for Type Culture Collection, the preservation date is November 18, 2024, the preservation number is CCTCC NO: M 20242584, the brevibacterium pekinensis has high siderophore production activity, the activity unit of siderophore in 72 h is 49.5%, and the activity unit of siderophore in 72 h is 9.5%. A superior level (+ + +) in the standard siderophore bacteria; the strain can generate IAA (indoleacetic acid) auxin, can secrete 8.05 mu g / mL of IAA within 48 hours under the induction of L-tryptophan (500 mg / L), and can significantly improve the soybean emergence rate, significantly promote the growth of soybean seedlings and significantly relieve the inhibition effect of salt stress on the growth of radicles and buds of oilseed rape under salt stress.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a ginseng Brevibacterium, a microbial inoculant and application thereof. BACKGROUND

[0002] Soybean and rapeseed, as field crops with certain salt tolerance, show obvious advantages in saline-alkali agricultural production. Soybean is an important oil and protein source crop in China, and also has moderate salt tolerance, which makes it have the potential to adapt to saline-alkali habitats. Its symbiotic nitrogen fixation with rhizobia helps to improve soil nitrogen content, improve soil fertility, reduce dependence on chemical fertilizers, and achieve the combination of land use and land cultivation. Rapeseed, as the main wintering crop in many regions of China, is not only an important edible oil and biodiesel raw material, but also shows certain salt tolerance. Its root system is developed, which can effectively utilize deep soil water and nutrients, and enhance the survival ability under salt-alkali stress. In addition, rapeseed has beautiful landscape during flowering period, which has the potential for leisure and sightseeing, and helps to expand the multi-value of saline-alkali land. Therefore, promoting the large-scale planting of soybean and rapeseed in saline-alkali land is expected to realize the advantages of improving, utilizing and generating ecological and economic benefits of saline-alkali land, and provide an effective path for the sustainable development of saline-alkali land agriculture.

[0003] However, high concentrations of salt ions in saline-alkali soil can cause stress on crop growth in various ways. First, it causes osmotic stress, hindering normal water absorption by plants and causing physiological drought. Second, it leads to ion imbalance, with excessive sodium ions (Na + ) and chloride ions (Cl - ) interfering with the absorption of essential mineral elements, causing ion toxicity. In addition, salt stress also induces the accumulation of a large amount of active oxygen, causing oxidative damage and disrupting cell structure and function. Soil salinization has become one of the key environmental stress factors restricting the sustainable development of global agriculture.

[0004] Seed germination and seedling stage are critical periods for plant growth, and are also the most sensitive stages to salt-alkali stress. Under salt-alkali conditions, seed germination is hindered, emergence is slow and irregular; seedling stage is prone to dead seedlings, stunted seedlings, and uneven seedling distribution, and the overall growth of plants is weak, which seriously affects the yield formation in the later stage. In view of the above problems in the planting of soybean and rapeseed in saline-alkali land, it is currently necessary to explore microorganisms with multiple plant growth activities and effective salt stress relief to enhance the salt tolerance of crops and improve the agricultural production potential of saline-alkali land.

[0005] In recent years, utilizing plant growth-promoting bacteria (PGPB) to enhance crop salt tolerance has become an increasingly important strategy for mitigating the harmful effects of salt stress on crops due to its environmental friendliness and low cost. Rhizosphere microorganisms, often referred to as the plant's second genome, play a crucial and irreplaceable role in assisting plants to resist adversity such as salt stress. PGPB can promote plant growth under salt stress through various mechanisms: synthesizing plant hormones (such as IAA) to promote root development; secreting ACC deaminase to reduce ethylene accumulation under stress conditions, thus weakening the inhibitory effect of ethylene on crop growth; improving soil nutrient status through nitrogen fixation, phosphorus solubilization, and potassium solubilization; secreting siderophores to enhance iron availability, thereby alleviating the deficiency of available iron in saline-alkali soils and indirectly enhancing the salt tolerance of crops; some PGPBs can also produce extracellular polysaccharides, forming a protective matrix in the rhizosphere to effectively encapsulate or isolate sodium. + This inhibits the migration of bacteria into the plant, thereby reducing ion toxicity. Currently, the most studied plant growth-promoting bacteria are mainly distributed in the genus *Pseudomonas* (…). Pseudomonas ), Bacillus (Bacillus spp.), etc. Regarding bacteria of the genus *Bacillus*, Chinese patent CN202510680913.0 discloses a strain of *Bacillus* resistant to phenol (…). Brachybacterium phenoliresistens YSY80, this bacterium can improve the salt and alkali tolerance of peppers and cabbage, promote their growth in saline-alkali land, and reduce the content of heavy metal ions in soil contaminated with heavy metals; Chinese patent CN202510601847.3 discloses a *Bacillus huanglongensis* resistant to root rot (…). Brachybacterium hlantirrotis Application of this technology in the biological control of root rot in medicinal plants (such as Atractylodes macrocephala, Panax notoginseng, and ginseng); Chinese patent CN202510334594.8 discloses a short food bacterium with salt and alkali tolerance. Brachybacterium alimentarium The application of [unclear text - possibly related to a specific application or technology] in inhibiting beet root rot; Chinese patent CN202510334593.3 discloses a compound microbial agent suitable for salt-tolerant and growth-promoting corn, which contains *Microbacterium ginseng*. [The text then abruptly shifts to a discussion of *Microbacterium ginseng* and its application in inhibiting beet root rot; and finally, a patent for *Microbacterium ginseng*.] Brachybacterium ginsengisoli Current research is still focused on taxonomic identification; no studies have been reported on its application in saline-alkali land agriculture. Furthermore, no research has yet been conducted on *Gynostemma pentaphyllum* (Gynostemma pentaphyllum). Brachybacterium ginsengisoli Reports on promoting salt tolerance in soybeans and rapeseed. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a *Gynostemma pentaphyllum*, a microbial agent, and its applications.

[0007] A type of ginseng-derived short bacillus ( Brachybacterium ginsengisoli) B474, the Bacteroidetes ginseng soil short rod is B474 of Bacteroidetes ginseng soil short rod, is preserved in China typical culture preservation center, and the preservation date is November 18, 2024, and the preservation number is CCTCC NO: M 20242584.

[0008] The Bacteroidetes ginseng soil short rod B474 of the application has higher siderophore activity, and the 72h siderophore activity unit is 49.5%, the strain can produce IAA (indole acetic acid) auxin, under the induction of L-tryptophan, 48h IAA secretion reaches 8.05 μg / mL, under salt stress, can significantly improve the soybean emergence rate, significantly promote the growth of soybean seedlings, and can significantly alleviate the inhibition of salt stress on the growth of rape radicle and bud.

[0009] A microbial agent, the agent takes the B474 of Bacteroidetes ginseng soil short rod as active ingredient.

[0010] Preferably, the microbial agent is the suspension or fermentation broth of the B474 of Bacteroidetes ginseng soil short rod.

[0011] The B474 of Bacteroidetes ginseng soil short rod is applied to improve the salt tolerance of crops and / or promote the growth of crops.

[0012] Preferably, the improvement of the salt tolerance of crops refers to promoting the germination of crop seeds, promoting the emergence of crops or promoting the growth of crops in the seedling stage under salt stress.

[0013] Preferably, the step of improving the salt tolerance of crops is: inoculating the B474 of Bacteroidetes ginseng soil short rod into culture medium, culturing for 16h-18h, preparing bacterial suspension, centrifuging to collect bacterial bodies, adjusting OD 600 ≥1, to obtain a microbial agent, and using the microbial agent to treat crops to improve the salt tolerance of crops.

[0014] Preferably, the treatment includes seed soaking of the microbial agent on crop seeds and / or root irrigation of the microbial agent on planted crops.

[0015] Preferably, promoting the growth of crops refers to promoting the increase of plant height, increasing the aboveground biomass, increasing the root biomass, or increasing the functional leaf area.

[0016] Preferably, the crops are soybeans or rape.

[0017] Compared with the prior art, the beneficial effects of the application are: The B474 of Bacteroidetes ginseng soil short rod provided by the application has the advantages of high siderophore activity, high IAA secretion, and the like. Brachybacterium ginsengisoliB474 exhibits high siderophore activity, with a siderophore activity unit of 49.5% after 72 hours, reaching the upper-middle level (+++) among siderophore-producing bacteria. This strain can produce IAA (indoleacetic acid) auxin, and under the induction of L-tryptophan (500 mg / L), it secretes 8.05 μg / mL of IAA after 48 hours.

[0018] Experiments showed that under 1.5‰ (w / w) NaCl salt stress, inoculation with strain B474 significantly improved soybean emergence rate, increasing it by 88.9%. Soybean seeds were immersed in OD... 600 A 1.0 g B474 bacterial suspension was used for seed inoculation, and OD was applied to the soil before soybean sowing. 600 A 1.0 B474 bacterial solution was applied to the soybean roots again via root drenching 7-10 days after soybean sowing. 600 A B474 bacterial suspension with a pH of 1.0 significantly promoted the growth of soybean seedlings under salt stress. Under NaCl stress, 25 days after soybean sowing, inoculation with B474 increased the aboveground fresh weight by 48.0%, root fresh weight by 30.6%, soybean plant height by 35.3%, and the area of ​​the top three leaves (i.e., functional leaves) by 26.6% compared to the uninoculated plants.

[0019] Meanwhile, rapeseed germination experiments showed that under 150 mM NaCl stress, OD levels were lower than those of 1% and 10% bacterial inoculum. 600 Inoculating a 1.0% solution of B474 bacteria into a NaCl solution can significantly alleviate the inhibitory effect of salt stress on the growth of rapeseed radicles and shoots, increasing radicle length by 1.10 times and 1.03 times, and shoot length by 0.85 times and 0.66 times, respectively. Attached Figure Description

[0020] Figure 1 The ginseng short bacillus provided for this invention ( Brachybacterium ginsengisoli Colony morphology of B474 on LB solid medium.

[0021] Figure 2 Ginseng Microbractis ( Brachybacterium ginsengisoli B474 is a phylogenetic tree based on the 16S rRNA gene sequence.

[0022] Figure 3 Ginseng Microbractis ( Brachybacterium ginsengisoli The growth of B474 on CAS detection plates.

[0023] Figure 4 The ginseng short bacillus under 1.5‰ NaCl stress ( Brachybacterium ginsengisoli (Image showing the effect of B474 on promoting soybean emergence (Day 5: one replicate of each treatment was photographed; Day 9: all four replicates of each treatment were included).

[0024] Figure 5 Soybean emergence rate under the treatments of original soil (CK), original soil added with 1.5‰ NaCl (NaCl), and original soil added with 1.5‰ NaCl and inoculated with B474 (NaCl+B474).

[0025] Figure 6 Bacillus pumilus under 1.5‰ NaCl stress Brachybacterium ginsengisoli Figure of effect of B474 on promoting growth of soybean seedlings.

[0026] Figure 7 Soybean plant height under the treatments of original soil (CK), original soil added with 1.5‰ NaCl (NaCl), and original soil added with 1.5‰ NaCl and inoculated with B474 (NaCl+B474), wherein (A) is 11 days after treatment, and (B) is 25 days after treatment.

[0027] Figure 8 Soybean aboveground fresh weight (A), root fresh weight (B), and trifoliate leaf area (C) under the treatments of original soil (CK), original soil added with 1.5‰ NaCl (NaCl), and original soil added with 1.5‰ NaCl and inoculated with B474 (NaCl+B474).

[0028] Figure 9 Colony morphology of B474 on LB solid medium (A) and figure of bacterial growth on LB solid medium coated with 10-4 rhizosphere soil suspension after 72 hours of culture (B) (one representative dilution (10-4) of one repetition was selected for photographing).

[0029] Figure 10 Figure of effect of B474 on promoting seed germination of oilseed rape under salt stress, with three repetitions in each group. DETAILED DESCRIPTION

[0030] The specific embodiments of the present application are described in detail below, but it should be understood that the scope of protection of the present application is not limited by the specific embodiments. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present application. The experimental methods described in the embodiments of the present application are all conventional methods unless otherwise specified.

[0031] Unless otherwise specified, the reagents used in the embodiments of the present application are all ordinary commercially available products.

[0032] LB medium: 10g tryptone, 5g yeast extract, 10g sodium chloride (NaCl), 1000mL distilled water, pH 7.0, to prepare LB liquid medium. Then add 15-20g agar powder to make LB solid medium. Sterilize at 121℃ for 20min.

[0033] Salkowski colorimetric solution: Mix 1 part of 0.5 mol / L FeCl3 and 49 parts of 35% HClO4, and store away from light.

[0034] CAS solid medium: 60.5 mg of Chromium Azurite S (CAS) was dissolved in 50 mL of deionized water and mixed with 10 mL of 1 mM FeCl3 (prepared with 10 mmol / L HCl) to obtain component A. 72.9 mg of HDTMA was dissolved in 40 mL of deionized water to obtain component B. Component A was slowly added to component B, sterilized at 115 °C for 30 min, and then mixed with 900 mL of sterilized LB solid medium. The mixture was then poured into CAS plates for later use.

[0035] CAS detection solution: Dissolve 60.5 mg of Chromium Azurite S (CAS) in 50 mL of deionized water, and mix with 10 mL of 1 mM FeCl3 (prepared with 10 mmol / L HCl) to obtain component A. Dissolve 72.9 mg of HDTMA in 40 mL of deionized water to obtain component B. Slowly add component A to component B, sterilize at 115 °C for 30 min, and set aside for later use.

[0036] This invention is the first to isolate a plant growth-promoting microbacterium ginseng from the rhizosphere soil of Artemisia annua in the saline-alkali land of coastal Jiangsu. Brachybacterium ginsengisoli B474. This strain possesses dual growth-promoting properties, including siderophore production and IAA secretion, which can significantly alleviate the inhibitory effects of salt stress on crop germination, emergence, and growth. This *Panax ginseng* strain (B474) was used to... Brachybacterium ginsengisoli Microbial germination promoters and growth promoters prepared from B474 can significantly improve soybean emergence rate, increase functional leaf area, promote seedling growth, and alleviate the inhibitory effect of salt stress on soybean emergence and seedling growth under salt stress, thereby enhancing soybean's tolerance to salt stress; they can also enhance rapeseed seed germination vitality, specifically by promoting the growth of radicle and shoot under salt stress. Therefore, as a new microbial resource, *Gnaphalium affine* (B474) is a promising candidate for development. Brachybacteriumginsengisoli B474 and germination and growth promoters containing B474 have broad application prospects in promoting seed germination, emergence, and growth of dryland crops such as soybeans and rapeseed in saline-alkali land. This not only provides high-quality resources and technical support for developing microbial agents or microbial fertilizers suitable for saline-alkali land agriculture, but also provides important materials and technical support for the efficient utilization of saline-alkali land and the realization of good ecological and economic benefits.

[0037] Example 1 Isolation and identification of strain B474 The rhizosphere soil of Suaeda salsa was collected from a severe saline-alkali soil in Jiangsu coastal area in June 2022. 10.0 g of soil was weighed into a triangular flask containing 90 mL of sterile water and shaken at 180 rpm for 30 min to obtain a 10 -1 dilution soil suspension. Then 1 mL of the suspension was taken and added to a test tube containing 9 mL of sterile water, mixed well to obtain a 10 -2 dilution soil suspension. This method was continuously performed for 4 times of gradient dilution, and 10 -3 , 10 -4 , 10 -5 , and 10 -6 dilution soil suspensions were obtained in turn. 100 μL of the 10 -4 , 10 -5 , and 10 -6 dilution soil suspensions were taken respectively, and each 100 μL was added dropwise to LB solid medium containing 2% NaCl, and the suspension was uniformly coated with a coating rod. The plates were incubated at 28°C for 3-4 days, and then single colonies with obvious morphological differences were picked, numbered, purified, and cultured.

[0038] The purified strain was inoculated into LB liquid medium containing 100 mg / L L-tryptophan and incubated at 28°C, 180 rpm for 48 h to obtain a bacterial solution. 100 μL of the bacterial solution was mixed with an equal volume of Salkowski colorimetric solution in a 96-well plate, and a blank control (LB medium instead of bacterial solution mixed with colorimetric solution at a ratio of 1:1) was set. After the 96-well plate was placed in the dark for 30 minutes, it was observed: if the strain produces IAA, the mixed solution will appear pink, and the deeper the pink color, the stronger the IAA production ability.

[0039] The strain capable of producing IAA was further subjected to qualitative and quantitative analysis of siderophore production ability. Based on the IAA production ability and siderophore production ability of the strain, a strain capable of secreting IAA and siderophore was screened. The IAA production of the strain after 48 h of culture was 8.05 μg / mL, and the siderophore production ability after 72 h was strong (+++), and the siderophore activity unit was 49.5%.

[0040] The 16S rRNA gene of the strain was amplified by colony PCR method using bacterial universal primers 27F (5' AGAGTTTGATCMTGGCTCAG-3', denoted as SEQ ID NO. 1) and 1492R (5' TACGGYTACCTTGTTACGACTT-3', denoted as SEQ ID NO. 2), wherein M represents base A or C, and Y represents base C or T. The obtained PCR product was sent to a sequencing company for sequencing to obtain the corresponding 16S rRNA gene sequence (as shown in SEQ ID NO. 3). The sequence was submitted to the EzBioCloud database (https: / / www.ezbiocloud.net / ) for homology comparison. The results showed that the 16S rRNA gene sequence of the strain had a sequence similarity of 99.00% with that of DCY80 (Type). A phylogenetic tree (as shown in FIG. 1) was further drawn, and the strain was most closely related to DCY80 in the evolutionary relationship. The strain was identified as Bacteroides sp. (Bacteroides sp.). Brachybacterium ginsengisoli DCY80 (Type). The strain was named B474. The strain has been preserved in the China Center for Type Culture Collection (CCTCC). The preservation number is CCTCC NO: M20242584. Figure 2 Brachybacterium ginsengisoli Brachybacterium ginsengisoli

[0041] ​​​

[0042] Example 2 Determination of growth-promoting characteristics of strain B474 The purified strain was subjected to IAA production and siderophore production capacity determination.

[0043] Preparation of seed culture for strain B474: Strain B474 was inoculated into LB liquid medium and cultured with shaking at 28℃ and 180 rpm for 16-18 h. After centrifugation, the bacterial cells were resuspended in sterile water and the OD was adjusted. 600 A value of 1 indicates the seed solution is obtained.

[0044] IAA production capacity assay: 250 μL of B474 seed culture was added to 5 mL of LB liquid medium containing L-tryptophan (500 mg / L, approximately 2.5 mmol / L), repeated three times. After incubation at 28℃ and 180 rpm for 48 hours with shaking, the culture was centrifuged, and 100 μL of the supernatant was added to a 96-well plate containing 100 μL of Salkowski colorimetric solution. Uninoculated medium was used as a blank control. After standing in the dark for 30 minutes, the absorbance at 530 nm was measured. The IAA concentration in the bacterial suspension was calculated according to the IAA standard curve. The results (Table 1) showed that after 48 hours of culture, strain B474 secreted 8.05 μg / mL of IAA.

[0045] Qualitative detection of the strain's siderophore secretion ability: 2 μL of B474 seed culture was aspirated into the center of a CAS plate, repeated three times. The plates were placed in a 28℃ incubator and incubated statically for 4 days. The presence and size of a yellow halo around the colony were observed. The siderophore secretion ability was assessed by the ratio of the halo diameter (D) to the colony diameter (d); a higher ratio indicated stronger ability. Results are as follows: Figure 3 As shown, B474 has a strong ability to secrete siderophores, and its halo diameter D / colony diameter d value is 1.16.

[0046] Quantitative determination of siderophores secreted by the strain: 300 μL OD 600B474 seed liquid with OD600 of 1 was inoculated into 3 mL LB medium and incubated at 28°C, 180 rpm for 72 h. After incubation, the supernatant was mixed with CAS detection solution at equal volume, and the mixture was placed for 1 h before measuring the absorbance of the sample at 680 nm (denoted as As). The absorbance of the mixture of blank LB medium and CAS detection solution at 680 nm was used as the reference (denoted as Ar). According to the classification of the ability of bacteria to produce siderophores in the literature Manjanatha MG, Loynachan TE, Atherly AG. Tn5 mutagenesis of Chinese Rhizobium fredii for siderophore overproduction. Soil Biology and Biochemistry, 1992, 24(2): 151-155, As / Ar was from 1.0-0 with an interval of 0.2, and each decrease of 0.2 increased a +. The As / Ar value of strain B474 was 0.495, which was in the interval of 0.4-0.6, and the siderophore secretion ability was rated as +++, reaching the medium level. Therefore, B474 belongs to the medium siderophore-producing bacteria.

[0047] Table 1: Secretion of IAA and siderophores by strain B474 Example 3 Strain B474 promotes soybean emergence under salt stress Test treatment setting: 0-20 cm farmland soil was collected from Xintian test station in Yancheng, Jiangsu Province, and the soluble salt content of the soil was 0.5‰. 150 g (dry soil) of soil was weighed into a culture cup. The following three treatments were set: adding 1.5‰ NaCl (1.5 g NaCl / kg soil) treatment, denoted as NaCl; adding 1.5‰ NaCl and inoculating strain B474, denoted as NaCl+B474; and the control treatment without NaCl and inoculation, denoted as CK.

[0048] Soybean seed surface disinfection: After the soybean seeds were disinfected with 75% alcohol for 1 min, they were washed once with sterile water, disinfected with 2.5% sodium hypochlorite for 2 min, and washed with sterile water for 3 times. The last eluted water was used to coat the plate, and no microbial growth was observed on the plate after 2-5 days of culture, indicating that the surface disinfection was complete.

[0049] Bacterial suspension preparation and inoculation: Strain B474 was inoculated into LB liquid medium and incubated at 28°C, 180 rpm for 16-18 h to obtain a bacterial suspension. The bacterial cells were collected by centrifugation, resuspended with sterile water, and adjusted to OD600 of 1 (the number of viable bacteria was about 10 600 CFU / mL). 9 ​

[0050] B474 was inoculated by soil inoculation and seed inoculation. The inoculation concentration of B474 in soil was 10 9 CFU / g soil; and the surface-sterilized soybean seeds were immersed in OD 600 1 bacterial suspension, and the bacterial cells were uniformly attached to the surface of the seeds by sufficient stirring.

[0051] Soybean planting and cultivation: 5 soybean seeds were planted in each culture cup with a planting depth of about 1 cm. The culture cup was placed in an intelligent greenhouse for cultivation, with a day temperature of 25-30°C and a night temperature of 18-24°C. Water was supplemented by weighing every day to maintain the soil water content at 60% of the saturated water holding capacity (SWHC). After the soybean grew to a single leaf, the germination rate was counted. Then, the seedlings were thinned, and 2 seedlings with consistent growth were retained in each cup. To enhance the colonization effect of the strain, the B474 bacterial suspension was inoculated again by root irrigation after thinning. The soybean germination was observed every day during the cultivation. The soybean germination after 5 days and 9 days of cultivation is shown in Figure 4 , and the soybean germination rate is shown in Figure 5 . NaCl treatment significantly inhibited the germination of soybean, and inoculation of the B474 strain completely eliminated the inhibition of NaCl stress on the germination of soybean. Specifically (as shown in Figure 5 ), the germination rate of the control treatment (CK) was 85%, which decreased to 45% under NaCl treatment, and the germination rate of soybean recovered to 85% after inoculation of the B474 strain, which was equivalent to the control treatment without NaCl stress.

[0052] Example 4 Strain B474 promotes soybean growth at the seedling stage under salt stress The test treatment setting, preparation of B474 bacterial suspension and inoculation method, and soybean planting and cultivation conditions were the same as in Example 3. After 25 days of cultivation, the growth of soybean in each treatment group is shown in Figure 6 . Under NaCl stress conditions, the growth of soybean after inoculation of the B474 strain was significantly better than that without inoculation. The height of the aboveground part of soybean in each treatment group was measured and counted to obtain Figure 7 ; the aboveground biomass, root biomass, and trifoliate leaf area of soybean were measured and counted to obtain Figure 8 .

[0053] The results showed that NaCl treatment significantly (P<0.05) reduced the height of soybean, the fresh weight of the aboveground part, the fresh weight of the root system, and the area of the trifoliate leaf, indicating that salt significantly inhibited the growth of soybean. Inoculation of the B474 strain effectively alleviated the inhibitory effect of NaCl stress on the growth of soybean, and in some indicators, the inoculation of the B474 strain was even better than the control treatment without stress. Specifically, after 11 days of cultivation, NaCl treatment reduced the height of soybean from 9.66 cm in the control treatment to 4.36 cm, and inoculation of the B474 strain restored the height to 8.69 cm, which was equivalent to the control level.Figure 7 A). After 25 days of culture, the soybean plant height was 23.5 cm under no NaCl stress; the soybean plant height decreased to 20.1 cm under NaCl treatment; after inoculation of B474, the soybean plant height was 27.7 cm, which was 18.0% higher than that of the no NaCl control treatment Figure 7 B). After 25 days of culture, under NaCl stress, inoculation of the B474 strain restored the fresh weight of the aboveground part and the area of the third leaf from the top to the level comparable to that of the no NaCl control treatment Figure 8 A, 8C), and restored the root fresh weight to 60.4% of that of the no NaCl control treatment Figure 8 B). Therefore, the B474 strain can significantly alleviate the inhibition of NaCl stress on the growth of soybean seedlings.

[0054] Example 5 Stable colonization of the rhizosphere of soybean by the B474 strain under salt stress The test treatment setting, preparation of bacterial suspension and inoculation, soybean seeding and culture conditions were the same as in Example 3. After 25 days of culture of soybean, the rhizosphere soil of soybean was collected. For the rhizosphere soil of the NaCl+B474 treatment, gradient dilution method was used to prepare 10 -4 , 10 -5 , 10 -6 concentration gradients of soil suspension. 100 μL of each dilution suspension was added to LB solid medium plates, which were uniformly coated with a sterile coating rod, and incubated at 28°C for 3 days, and the growth of the target strain B474 was observed.

[0055] According to the colony morphological characteristics of B474 Figure 9 A), the growth of bacteria on the LB solid medium coated with soil suspension after 3 days of culture was observed Figure 9 B), a toothpick was used to pick single colonies with colony morphological characteristics similar to those of the B474 strain on the LB solid medium coated with soil suspension, and the 16S rRNA gene of the colonies was amplified by PCR using bacterial universal primers 27F (5' AGAGTTTGATCMTGGCTCAG-3') and 1492R (5' TACGGYTACCTTGTTACGACTT-3'). After detection and verification of the integrity and fragment length of the PCR products by agarose gel electrophoresis, the qualified products were sent to a sequencing company for sequencing, and the sequences obtained by sequencing were uploaded to the EzBioCloud database (https: / / www.ezbiocloud.net / ) for homology comparison. The results showed that the 16S rRNA gene sequence of the colonies on the detection plate was 99.9% identical to that of the type strain B. subtilis ATCC 6051 (GenBank accession number: NR_025549.1), and the sequence of the colonies on the detection plate was 99.9% identical to that of the type strain B. subtilis ATCC 6051 (GenBank accession number: NR_025549.1). Brachybacterium ginsengisoliThe sequence similarity of DCY80 (A. ginsengsoil) reached 98.27%-99.00% (Table 2), and its taxonomic status was consistent with the inoculated strain B474, confirming that the single colony on the detection plate also belonged to Brachybacterium ginsengisoli (A. ginsengsoil).

[0056] According to the colony morphological characteristics of B474 and the 16S rRNA gene identification results of the colony, the single colonies with the same colony morphology as B474 on the LB solid medium coated with soil suspension were counted. The results showed that, by the time of soybean harvest, strain B474 could still colonize in the rhizosphere soil of soybean, and the colonization number reached 2.52±0.34×10 7 CFU / g dry soil.

[0057] Table 2: Detection of B474 colonization in rhizosphere soil (RS) after 25 days of soybean culture under salt stress Example 6 Strain B474 promotes rape seed germination under salt stress Preparation of culture dishes: 15 culture dishes with a diameter of 9 cm were prepared, and two layers of sterilized filter paper were laid in each culture dish.

[0058] Preparation of bacterial solution: the B474 strain was inoculated into LB liquid medium and cultured at 28°C, 180 rpm for 16-18 h, and then the bacterial cells were collected by centrifugation, resuspended with 150 mM NaCl solution, and the OD 600 value of the bacterial suspension was detected by ultraviolet spectrophotometer, and the OD 600 value was adjusted to 1.0 with 150 mM NaCl solution for standby.

[0059] Rape seed disinfection: select rape seeds with intact seed coat and uniform size, and perform the following disinfection treatment in sequence: immerse the seeds in 75% ethanol for 30 seconds, rinse once with sterile water; then immerse the seeds in 15% H2O2 for 30 seconds, and rinse 3 times with sterile water.

[0060] Test setup: 3 treatment groups and 1 control group were set up, with 3 replicates in each group. The salt concentration of the treatment groups was uniformly 150 mM NaCl (labeled as 150NaCl), and the inoculation amount of the B474 strain was set at 1% (v / v), 10% (v / v), and 50% (v / v) three levels; the control group was replaced with sterile water (labeled as H2O). 10 mL of corresponding treatment solution was added to each culture dish with two layers of filter paper, and then 15 sterilized rape seeds were sown in the culture dish. The culture dishes were placed in a 25°C incubator, and the radicle length and bud length were measured after 5 days.

[0061] As shown in Table 3, compared with the control of sterile water, the radicle length and bud length of rape under 150 mM NaCl treatment were significantly reduced, which indicated that NaCl stress significantly inhibited the growth of radicle and bud. Inoculation of 1% and 10% B474 bacterial solution could significantly (P<0.05) alleviate the inhibition of NaCl on the growth of radicle and bud, and the effect of 1% inoculation concentration was the best. Specifically, the length of radicle was significantly reduced from 342.9 mm of the control treatment to 8.1 mm under NaCl stress, and the radicle length increased to 17.1 mm and 16.5 mm after inoculation of 1% and 10% B474 respectively, which was 1.10 times and 1.03 times of that of NaCl treatment; the length of bud was significantly reduced from 164.3 mm of the control treatment to 21.3 mm under NaCl stress, with a reduction of 87.0%, and the length of bud increased to 39.4 mm and 35.5 mm after inoculation of 1% and 10% B474 respectively, which was 85% and 66% of that of NaCl treatment. In addition, although inoculation of 50% B474 could also significantly alleviate the reduction of radicle length of rape under NaCl stress, the alleviating effect was weaker than that of 1% and 10% inoculation treatment. The germination of rape after inoculation of different amounts of B474 bacterial solution under 150 mM NaCl stress is shown in Figure 10 .

[0062] Table 3: Radicle length and bud length of rape under different treatments Note: The same column indicates that the same index is significantly different (P<0.05) between different small letters.

[0063] It should be noted that when the present application claims involve numerical ranges, it should be understood that each numerical range has two endpoints and any number between the two endpoints can be selected. In order to prevent repetition, the present application describes preferred embodiments.

[0064] Although preferred embodiments of the present application have been described, those skilled in the art can make further changes and modifications to these embodiments once they know the basic inventive concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications falling within the scope of the present application.

[0065] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application also intends to include these modifications and variations.

Claims

1. A type of ginseng-derived short bacillus ( Brachybacterium ginsengisoli B474, characterized in that, The *Gnaphalium affine* B474 is deposited at the China Center for Type Culture Collection (CCTCC) on November 18, 2024, with accession number CCTCC NO: M 20242584.

2. A microbial inoculant, characterized in that, The bacterial agent uses *Gnaphalium affine* B474 as the active ingredient as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The microbial agent is a suspension or fermentation broth of *Gnaphalium affine* B474.

4. The application of the *Panax ginseng* B474 as described in claim 1 or the microbial agent as described in any one of claims 2 to 3 in improving crop salt tolerance and / or promoting crop growth.

5. The application according to claim 4, characterized in that, Improving crop salt tolerance refers to promoting crop seed germination, crop emergence, or crop seedling growth under salt stress.

6. The application according to claim 4, characterized in that, The steps to improve crop salt tolerance are as follows: inoculate the *Gynostemma pentaphyllum* B474 into the culture medium, culture for 16-18 hours, prepare a bacterial suspension, collect the bacterial cells by centrifugation, and adjust the OD value. 600 ≥1, to obtain a microbial inoculant, and to treat crops with the microbial inoculant to improve crop salt tolerance.

7. The application according to claim 6, characterized in that, The treatment includes soaking crop seeds in the microbial agent and / or irrigating the roots of the planted crop with the microbial agent.

8. The application according to claim 7, characterized in that, Promoting crop growth refers to increasing crop height, aboveground biomass, root biomass, or functional leaf area.

9. The application according to claim 4 or claim 7, characterized in that, The crop in question is either soybean or rapeseed.

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