Separation and identification method of helichrysum psammophila rhizosphere growth-promoting bacteria and application thereof

Abstract

The invention discloses a method for separating and identifying helichrysum psammophila rhizosphere growth-promoting bacteria and application thereof, and relates to the technical field of biologica.According to the technical key points, the helichrysum psammophila rhizosphere growth-promoting bacteria are screened through an abutilon ammophila culture medium, an organic phosphorus dissolving culture medium, an inorganic phosphorus dissolving culture medium, a CAS detection culture medium and a Salkowski colorimetric method, and the helichrysum psammophila rhizosphere growth-promoting bacteria are obtained; the growth promoting potential of the strain in the aspects of phosphorus solubilization, siderophore production, nitrogen fixation and the like is analyzed, a phylogenetic tree is constructed by adopting 16S rRNA sequence homology analysis to identify the strain, and finally, the growth promoting effect of the strain is verified by adopting an in-dish germination test of helichrysum psammophila and a potted helichrysum psammophila experiment. The bacillus sorola S31 provided by the invention can be used as a candidate growth-promoting rhizobacteria, and valuable strain resources are provided for the development of microbial agents.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a method for isolating and identifying rhizosphere growth promoting bacteria of Helichrysum arenarium (L.) Moench. and its application. BACKGROUND

[0002] Helichrysum arenarium (L.) Moench. is a perennial herb with high medicinal value, mainly distributed in Xinjiang and other regions. With the increasing demand for Helichrysum arenarium (L.) Moench. in the market, exploring environmentally friendly microbial inoculants and bio-organic fertilizers to improve plant growth has become a trend. Plant growth promoting rhizobacteria (PGPR) is a group of beneficial soil bacteria that can colonize the surface of plant roots and promote plant growth. They can improve the agricultural ecosystem by promoting plant nutrient uptake, inhibiting the growth of pathogenic bacteria, and regulating plant root community structure.

[0003] Currently, research on PGPR has involved a variety of crops, including Camellia oleifera, corn, and Schisandra chinensis, but research on Helichrysum arenarium (L.) Moench. rhizosphere growth promoting bacteria is relatively limited. Existing technologies include screening Helichrysum arenarium (L.) Moench. rhizosphere growth promoting bacteria through Ashby medium, organic phosphorus-degrading medium, inorganic phosphorus-degrading medium, and CAS detection medium, as well as Salkowski colorimetry. These technologies provide methods for screening and identifying growth promoting strains with high efficiency, but the specificity and multifunctionality of Helichrysum arenarium (L.) Moench. rhizosphere growth promoting bacteria are not deep enough.

[0004] Although existing research has involved rhizosphere growth promoting bacteria of other crops, research on Helichrysum arenarium (L.) Moench. rhizosphere growth promoting bacteria is relatively limited, and there is a lack of targeted strain screening and application research. Therefore, it is necessary to further explore the diversity and functionality of Helichrysum arenarium (L.) Moench. rhizosphere growth promoting bacteria and their application in practical agricultural production to improve the yield and quality of Helichrysum arenarium (L.) Moench. SUMMARY

[0005] The purpose of the present application is to solve the above problems and provide a method for isolating and identifying Helichrysum arenarium (L.) Moench. rhizosphere growth promoting bacteria and its application to address the problem that existing methods for screening and applying Helichrysum arenarium (L.) Moench. rhizosphere growth promoting bacteria are not systematic enough.

[0006] In summary, the technical solution of the present application is as follows: a method for isolating and identifying Helichrysum arenarium (L.) Moench. rhizosphere growth promoting bacteria, comprising the following steps:

[0007] S1: collecting Helichrysum arenarium (L.) Moench. rhizosphere soil samples;

[0008] S2: screening the rhizosphere growth promoting bacteria of H. sonorensis by using Ashby medium, organic phosphorus hydrolysis medium, inorganic phosphorus hydrolysis medium and CAS detection medium and Salkowski colorimetry;

[0009] S3: identifying the screened strains by constructing a phylogenetic tree through 16S rRNA sequence homology analysis.

[0010] Further, the growth promoting bacteria screening further comprises evaluating the growth promoting potential of the strains in terms of phosphorus dissolution, iron carrier production and nitrogen fixation.

[0011] Further, the growth promoting bacteria identification further comprises determining the IAA production of the strains.

[0012] Another object of the present application is to provide a method for promoting the growth of H. sonorensis by using the growth promoting bacteria isolated and identified by the above method, comprising the following steps:

[0013] SS1: inoculating the isolated and identified growth promoting bacteria to the seeds or plants of H. sonorensis;

[0014] SS2: verifying the growth promoting effect of the strains through H. sonorensis seed germination test and pot experiment.

[0015] Another object of the present application is to provide a microbial inoculant for H. sonorensis, wherein the inoculant comprises the growth promoting bacteria isolated and identified by the above method.

[0016] Another object of the present application is to provide a method for improving the biomass of H. sonorensis by using the above microbial inoculant, comprising the following steps:

[0017] a. applying the microbial inoculant to H. sonorensis;

[0018] b. measuring the growth indexes of fresh weight, stem length, stem diameter, aboveground fresh weight, underground fresh weight, nodules and dry weight of H. sonorensis.

[0019] Another object of the present application is to provide a rhizosphere growth promoting bacteria Bacillus sonorensis S31 for H. sonorensis, wherein the strain is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 33843, and the preservation date is March 17, 2025; the 16S rRNA gene sequence of the Bacillus sonorensis S31 is shown in Seq ID No. 1.

[0020] Further, the strain has the abilities of phosphorus dissolution, iron carrier production and IAA production, and can significantly improve the biomass of H. sonorensis.

[0021] Compared with the prior art, the beneficial effects of the scheme are: the present application realizes efficient screening of rhizosphere growth promoting bacteria of Hesperaloe funiflora by comprehensively using the technical means of Ashby medium, organic phosphorus dissolving medium, inorganic phosphorus dissolving medium and CAS detection medium and Salkowski colorimetric method, which can comprehensively evaluate the growth promoting potential of the strains in terms of phosphorus dissolution, iron carrier production and nitrogen fixation, and further identify the growth promoting ability of the strains by determining the IAA yield, so as to ensure that the screened strains have practical agricultural application value. The systematization and comprehensiveness of this method are lacking in the prior art, which can more accurately identify and utilize microbial resources with growth promoting effect.

[0022] The present application can significantly improve the germination rate, germination potential, bud length and biomass of Hesperaloe funiflora by applying the growth promoting bacteria screened and identified to the growth process of Hesperaloe funiflora. Especially the application of Bacillus sonorensis S31 not only shows excellent growth promoting effect due to its ability of phosphorus dissolution, iron carrier production and IAA production, but also has a particularly significant effect on improving the biomass of Hesperaloe funiflora, which has important practical significance for improving the yield and quality of Hesperaloe funiflora. In addition, the present application also provides an environmentally friendly microbial agent, which can not only promote the growth of Hesperaloe funiflora, but also help to improve the plant rhizosphere microbial community structure and improve the utilization efficiency of soil nutrients, and has a positive effect on the improvement of agricultural ecological environment. Considering the medicinal value and market demand of Hesperaloe funiflora, the implementation of the present application provides an important way for the sustainable development of Hesperaloe funiflora planting, which has significant economic and ecological value. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is a schematic diagram of the Hesperaloe funiflora soil sample collection site in the embodiment of the present application;

[0024] Figure 2 is a schematic diagram of the IAA production of the strain in the embodiment of the present application;

[0025] Figure 3 is a schematic diagram of the phylogenetic tree constructed based on 16S rDNA in the embodiment of the present application;

[0026] Figure 4 is a schematic diagram of the influence of the strain on the germination of Hesperaloe funiflora seeds in the embodiment of the present application;

[0027] wherein, Figure A is the influence of the strain on the germination rate of Hesperaloe funiflora seeds; Figure B is the influence of the strain on the germination potential of Hesperaloe funiflora seeds; Figure C is the influence of the strain on the bud length of Hesperaloe funiflora seeds germinated for 5 days;

[0028] Figure 5 is a schematic diagram of the influence of the growth promoting bacteria on the seedling length of the germinated seeds in the embodiment of the present application;

[0029] Figure 6 Figure is a schematic diagram of the influence of the plant growth promoting bacteria on the pot culture of the psammophyte Helipticum benghalense in the embodiments of the present application;

[0030] Figure 7 Figure is a schematic diagram of the influence of the strains on the biomass of the psammophyte Helipticum benghalense in the embodiments of the present application;

[0031] Among them, Figure A is the influence of the strains on the root length of the psammophyte Helipticum benghalense; Figure B is the influence of the strains on the stem length of the psammophyte Helipticum benghalense; Figure C is the influence of the strains on the stem thickness of the psammophyte Helipticum benghalense; Figure D is the influence of the strains on the fresh weight of the psammophyte Helipticum benghalense; Figure E is the influence of the strains on the aboveground fresh weight of the psammophyte Helipticum benghalense; Figure F is the influence of the strains on the underground fresh weight of the psammophyte Helipticum benghalense; Figure G is the influence of the strains on the nodal number of the psammophyte Helipticum benghalense; Figure H is the influence of the strains on the dry weight of the psammophyte Helipticum benghalense; Figure I is the influence of the strains on the aboveground dry weight of the psammophyte Helipticum benghalense; and Figure J is the influence of the strains on the underground dry weight of the psammophyte Helipticum benghalense. DETAILED DESCRIPTION

[0032] In order to enable those skilled in the art to better understand the present application, the technical solutions of the present application will be further described in detail below in combination with the embodiments of the present application and the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor should belong to the scope of protection of the present application.

[0033] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict. The present application will be described in detail in combination with the embodiments.

[0034] Experimental materials and culture medium:

[0035] Sample collection: The present application uses soil samples as the rhizosphere soil of the psammophyte Helipticum benghalense. The rhizosphere soil samples were collected from Bu'erjin County (48°18N, 86°78E) in Altay Prefecture of Xinjiang Uygur Autonomous Region, China at an altitude of 737.4 m in June 2023, and the psammophyte Helipticum benghalense soil samples were collected from Habahai County, as shown in the accompanying Figure 1

[0036] Culture medium: NA culture medium, inorganic phosphorus culture medium, Mengjina culture medium, Ashby nitrogen-free culture medium, CAS detection culture medium, and Jin's liquid culture medium.

[0037] Embodiment 1: Isolation and identification method of plant growth promoting bacteria in the rhizosphere of the psammophyte Helipticum benghalense

[0038] 1. Isolation and screening of strains

[0039] ​The rhizosphere soil was mixed thoroughly, and 10 g of the sample was taken into a conical flask containing 90 ml of sterile water, and incubated at 28°C for 4 h. 1 ml of the supernatant was taken and added to a test tube containing 9 ml of sterile water, and the concentration in the test tube was 10 –1 , and sequentially gradient diluted to 10 –2 , 10 –3 , 10 –4 , 10 –5 , 10 –6 ; 0.2 ml of the solution with a concentration of 10 -3 , 10 –4 , 10 -5 was taken and uniformly coated on a plate, and the coating was repeated for 3 times. The coated medium was placed in a 28°C constant temperature incubator and incubated for 24 to 48 h, and different morphological colonies were picked and purified.

[0040] 2.2 Screening of the strain with nitrogen fixation function

[0041] 2.1 Screening of the strain with phosphorus dissolving function

[0042] The obtained strains were point-inoculated on inorganic phosphorus and organic phosphorus medium, and each pure culture strain was inoculated on 3 culture media, and incubated at 28°C for 7 d, and the phosphorus dissolving ring was observed. The strains with obvious phosphorus dissolving ring were quantitatively determined by molybdenum-antimony anti-colorimetric method.

[0043] After the rhizosphere soil bacteria were screened by morphology, 43 strains of bacteria were obtained. The bacteria were inoculated on the selective medium, and the strain dissolving capacity was shown in Table 1. The strains S29 and S16 can dissolve inorganic phosphorus, and S31 can dissolve organic phosphorus and inorganic phosphorus, and there are obvious phosphorus dissolving rings on the selective medium.

[0044] Table 1: Phosphorus dissolving capacity of the strains

[0045]

[0046] 2.2 Screening of the strain with nitrogen fixation function

[0047] The obtained strains were point-inoculated on the Ashby medium, and each pure culture strain was inoculated on 3 culture media, and incubated at 28°C for 7 d, and the nitrogen fixation ring was observed. The strains with obvious nitrogen fixation ring were further tested, and the nitrogen fixation capacity of the strains was shown in Table 2. Two strains can fix nitrogen, and they are S7 and S10, and there are transparent rings on the Ashby medium. The dissolving index of S7 is the largest, and reaches 1.23, and the nitrogen fixation effect is the best.

[0048] Table 2: Nitrogen fixation capacity of the strains

[0049] Strains Colony diameter (d) / cm Lytic zone diameter (D) / cm Lytic index (D / d) S7 0.3 0.33 1.1 S10 0.43 0.53 1.23

[0050] 2.3 Screening of the strain with iron carrier production function

[0051] The obtained strains were inoculated on CAS detection medium, 3 media for each pure culture strain, and cultured at 28°C for 7d to observe the nitrogen fixation ring. Strains with significant orange chelation ring were screened for further experiments. The iron carrier production ability of the strains is shown in Table 3, wherein 10 strains can produce iron carrier, which are S2-1, S3, S7, S10, S13, S14, S16, S18, S29 and S31, and have orange chelation ring on CAS medium. The strain S16 has the strongest iron carrier production ability, and the dissolution index reaches 2.96.

[0052] Table 3 Iron carrier production ability of strains

[0053]

[0054]

[0055] 2.4 Screening of potassium-dissolving ability of strains

[0056] The obtained strains were inoculated on potassium-dissolving medium, 3 media for each pure culture strain, and cultured at 28°C for 7d to observe the nitrogen fixation ring. No potassium-dissolving strain was screened in the present application.

[0057] 2.5 Screening of IAA production ability of strains

[0058] The screened strains were inoculated in beef extract peptone liquid medium with a final concentration of 0.5g / L L-tryptophan, and cultured at 28°C, 200r / min for 3d. After centrifugation at 6000r / min, 4°C for 10min, the supernatant was taken. The content of auxin in the supernatant was determined by Salkowski colorimetry, and the color reaction was observed on a white porcelain plate. The observation results are shown in the accompanying Figure 2 Table 4. IAA production ability of strains

[0059] 3 Compatibility of strains

[0060] The growth-promoting bacteria strain was inoculated into beef extract peptone liquid medium and cultured at 28℃ and 200 rpm for 24 h. 100 μL of bacterial suspension A was spread onto the surface of beef extract peptone solid medium. After slight drying, the spread medium was perforated, and 100 μL of growth-promoting bacteria strain B was injected into each well. The culture was then incubated for 24 h, and the mutual inhibition between the strains was observed. If a clear colony growth zone appeared around strain B, it indicated an inhibitory effect between the two bacteria, indicated by "+"; if no growth traces appeared around strain B, it indicated no growth inhibition between the two bacteria, indicated by "-". The compatibility of the 10 isolated growth-promoting bacteria strains was analyzed. The results are shown in Table 4 below. Strains S10, S13, S14, and S16 showed good compatibility, while strain S31 was compatible with all other strains.

[0061] Table 4. Strain compatibility

[0062] Strain name S2-1 S3 S7 S10 S13 S14 S16 S18 S29 S3 - S7 - - S10 - - + S13 + + + + S14 + - - - - S16 + + + + - + S18 + + + + - + - S29 + + - + - - - - S31 - - - - - - - - -

[0063] 4. Constructing a phylogenetic tree

[0064] DNA was extracted from the selected growth-promoting bacteria, and 16S rDNA was amplified. The amplified products were detected by electrophoresis and sent to Shanghai Sangon Biotech for sequencing. After sequencing, the results were compared and analyzed on the NCBI website (www.ncbi.nlm.nih.gov), and a phylogenetic tree was constructed using MEGA11. Universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTT ACCTTGTTACGACTT-3′) were used to amplify the selected strains by bacterial PCR. The PCR reaction system (30 μL) consisted of: 15 μL Taq enzyme, 1 μL each of forward and reverse primers (10 μmol / L), 2 μL DNA template, and 11 μL ddH2O. PCR conditions were: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 45 s, 50℃ annealing for 30 s, 72℃ extension for 1 min, 35 cycles; and a final extension at 72℃ for 1 min. The PCR products were sequenced by Shanghai Sangon Biotech Co., Ltd., and then the sequences were compared with bacterial sequences in the NCBI database for homology analysis. Finally, a phylogenetic tree of the strains was constructed using the neighbor-joining method and MEGAX software, as shown in the attached figure. Figure 3 As shown.

[0065] Genomic DNA was extracted from 10 growth-promoting bacteria, and their 16S rDNA sequences were determined and compared with those sequences on the NCBI website. A phylogenetic tree was then constructed using MEGA11. Sequencing results showed that 9 of the growth-promoting bacteria matched the aligned sequences more than 99% of the original sequence. They belonged to the genera *Bacillus* (Firmicutes), *Alcaligenes* (Proteobacteria), and *Kocuria* (Actinobacteriota), respectively. Among them, 5 strains belonged to *Bacillus*, 3 to *Alcaligenes*, and 1 to *Kocuria*. Within the genus *Bacillus*, strain S2-1 was preliminarily identified as *Priestia megaterium*, strain S3 as *Bacillus mobilis*, strain S10 as *Bacillus bataviensis*, strain S14 as *Bacillus mycoides*, and strain S29 as *Bacillus paramobilis*; strain S31 was preliminarily identified as *Bacillus sonorensis*. Within the genus *Alcaligenes*, strains S18, S13, and S16 were preliminarily identified as *Alcaligenes faecalis*. Within the genus *Kocuria*, strain S7 was preliminarily identified as *Kocuria rosea*.

[0066] This invention isolated 10 growth-promoting bacteria from the rhizosphere of *Helichrysum fusiforme*, belonging to 3 genera. Among them, S31 exhibited strong phosphorus-solubilizing and siderophore-producing abilities, with an IAA yield as high as 122.57 μg mL⁻¹. 16S rRNA gene sequence analysis identified S31 as *Bacillus sonorensis*, S2-1 as *Priestia megaterium*, S3 as *Bacillus mobilis*, S10 as *Bacillus bataviensis*, S14 as *Bacillus mycoides*, S29 as *Bacillus paramobilis*, S18, S13, and S16 as *Alcaligenes faecalis*, and S7 as *Kocuria rosea*. All 10 growth-promoting bacteria had the ability to produce siderophores. S7 and S10 had the ability to fix nitrogen, S31, S29 and S16 had the ability to solubilize phosphorus, and S7, S10, S13, S14 and S31 had the ability to produce IAA.

[0067] Example 2: A rhizosphere growth-promoting bacterium of *Helichrysum lataniae*, *Bacillus desertica* S31

[0068] A rhizosphere growth-promoting bacterium of *Helichrysum fusiforme*, *Bacillus sonorensis* S31, has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCCNo. 33843, deposited on March 17, 2025, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing (Institute of Microbiology, Chinese Academy of Sciences), postal code 100101. The 16S rRNA gene sequence of *Bacillus sonorensis* S31 is shown in Seq ID No. 1.

[0069] Example 3: Effects of rhizosphere growth-promoting bacteria on seed germination of *Helichrysum praecox*

[0070] The tested bacterial strain was inoculated into NB medium and cultured at 150 r / min and 28℃ for 24 h to adjust the OD of the bacterial culture. 600 =0.05. Select plump, uniformly sized *Helichrysum praecox* seeds from the same batch. Surface disinfect with 70% alcohol for approximately 5 seconds at room temperature, rinse 3-4 times with sterile water, and evenly sprinkle 20 seeds per dish onto sterile filter paper moistened with sterile water. Control group 1 received 10 mL of sterile water, control group 2 received 10 mL of NB medium, and treatment groups received 10 mL of bacterial solution. Each treatment was replicated three times. Germination was carried out in the dark at 25°C, with sterile water added regularly to maintain moisture. Germination rate and germination potential were measured daily. On day 5, root length was measured. A small amount of water was poured onto a flat surface to maintain a shallow water layer. The roots were placed on the surface, and the roots were straightened with tweezers and the length of each root was measured with a ruler. This was repeated three times. The effect of the bacterial strain on the germination of *Helichrysum praecox* seeds is shown in the attached figure. Figure 4 As shown.

[0071] Germination rate = (Number of germinated seeds / Total number of experimental seeds) × 100%;

[0072] Germination potential = Number of seeds that germinated during peak germination period / Total number of seeds in the experiment × 100%.

[0073] This invention uses a growth-promoting bacteria fermentation broth to treat *Helichrysum lataniae* seeds for 5 days. The statistical results are shown in Table 5 below. The third day is the peak germination period. Seed germination potential was calculated. The growth of *Helichrysum lataniae* seedlings is shown in the attached figure. Figure 5 As shown, the seed germination rate changed under the influence of growth-promoting bacteria soaking.

[0074] Table 5. Effects of growth-promoting bacteria on seed germination of *Helichrysum lataniae*.

[0075]

[0076] In-vessel germination experiments showed that, compared with the control, the germination rate of strain S2-1 increased by 4.01%, and the germination rate of strain S31 increased by 6%. Soaking seeds with growth-promoting bacteria did not significantly change germination potential, but treatment with strains S3 and S7 increased germination potential by 12.5%, with strain S31 showing the most significant increase at 37.5%. Strains S2-1 and S31 had the most significant promoting effect on seed germination, with seedling length increasing by 87.87% and 104.70%, respectively.

[0077] Example 4: Effects of rhizosphere growth-promoting bacteria on potted *Helichrysum praecox*

[0078] Two superior growth-promoting bacteria were screened from Example 3 for further pot experiments. Seeds of *Impatiens scoparia* from the same batch, with plump and uniform size, were sown in seedling trays filled with peat moss and vermiculite. After 5 days, seedlings with consistent growth were transplanted. The tested strains were inoculated into NB medium and cultured at 150 rpm and 28°C for 24 hours, adjusting the OD of the bacterial solution. 600 =0.05. Root drenching treatment was performed, while the control group was treated with plain water. Root drenching was done every 7 days. After 120 days, growth indicators of *Helichrysum praecox* were measured. The effect of growth-promoting bacteria on potted *Helichrysum praecox* is shown in the attached figure. Figure 6 As shown. Growth indicators for *Helichrysum praecox* include root length, stem length, stem diameter, fresh weight, dry weight, and above-ground and below-ground fresh weight. Root length, stem length, and stem diameter were measured using a measuring tape and vernier calipers. After washing and drying the plant roots, the fresh weight of the plant, as well as the fresh weight of the above-ground parts and roots, were measured separately. Then, the plants were placed in an oven at 70℃ and dried to constant weight. After cooling, their dry weights were measured separately.

[0079] This invention selected two bacterial strains, S2-1 and S31, with good growth-promoting effects through germination experiments for pot experiments. The statistical results of the growth indicators of *Helichrysum praecox* are shown in Table 6 below. The growth of *Helichrysum praecox* is as follows: Figure 7 As shown in the figure. The results indicated that the biomass indicators of the plants changed to varying degrees after treatment with the growth-promoting bacteria. After inoculation with strain S2-1, the root length increased by 16.78%, fresh weight by 237.42%, stem length by 25.29%, stem diameter by 13.68%, aboveground fresh weight by 131.78%, underground fresh weight by 101.89%, nodules by 283.25%, and dry weight by 70.67%, with aboveground dry weight increasing by 24.66% and underground dry weight by 38.71%.

[0080] Table 6. Effects of growth-promoting bacteria on the seedling stage of *Helichrysum lataniae*.

[0081]

[0082]

[0083] Pot experiment results showed that inoculation with strain S31 significantly increased the biomass of *Helichrysum fusiforme*, with fresh weight increasing by 237.42%, stem length by 54.71%, stem diameter by 28.22%, above-ground fresh weight by 255.08%, underground fresh weight by 252.83%, nodules by 266.75%, dry weight by 228.00%, above-ground dry weight by 229.09%, and underground dry weight by 247.37%.

[0084] The above specific embodiments are merely explanations of the present invention and are not intended to limit the present invention. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A method for isolating and identifying rhizosphere growth-promoting bacteria of *Helichrysum fusiforme*, characterized by: The method includes the following steps: S1: Collect rhizosphere soil samples from *Helichrysum praecox*. S2: Rhizosphere growth-promoting bacteria of Helichrysum praecox were screened using Assumption medium, organic phosphorus-solubilizing medium, inorganic phosphorus-solubilizing medium, CAS detection medium, and Salkowski colorimetric method. S3: The selected strains were identified by constructing a phylogenetic tree through 16S rRNA sequence homology analysis.

2. The method as described in claim 1, characterized in that: The screening of growth-promoting bacteria also includes evaluating the growth-promoting potential of strains in phosphorus solubilization, siderophore production, and nitrogen fixation.

3. The method as described in claim 1, characterized in that: The identification of growth-promoting bacteria also includes measuring the IAA yield of the strain.

4. A method for promoting the growth of *Helichrysum fusiforme* using growth-promoting bacteria isolated and identified by any one of claims 1 to 3, characterized in that: Includes the following steps: SS1; Inoculate the isolated and identified growth-promoting bacteria onto seeds or plants of *Helichrysum fusiforme*. SS2; The growth-promoting effect of the strain was verified through seed germination experiments and pot experiments of *Helichrysum praecox*.

5. A microbial inoculant for *Impatiens balsamina*, characterized in that: The microbial agent comprises growth-promoting bacteria isolated and identified by the method of any one of claims 1 to 3.

6. A method for increasing the biomass of *Helichrysum lataniae* using the microbial inoculant of claim 5, characterized in that: Includes the following steps: a. Apply microbial inoculants to *Helichrysum fusiforme*; b. Measure the growth indicators of *Helichrysum fusiforme*, including fresh weight, stem length, stem diameter, above-ground fresh weight, underground fresh weight, nodules, and dry weight.

7. A rhizosphere growth-promoting bacterium of *Helichrysum lataniae*, *Bacillus desertica* S31, characterized by: The strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33843 and deposit date of March 17, 2025; the 16S rRNA gene sequence of the Sonora desert Bacillus S31 is shown in Seq ID No.

1.

8. The strain according to claim 7, characterized in that: The strain possesses the ability to solubilize phosphorus, produce siderophores and produce IAA, and can significantly increase the biomass of *Helichrysum fusiforme*.

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