Bacillus pacificus strain and application thereof

Abstract

The application provides a bacillus pacificus G124. The application also provides an application of the bacillus pacificus G124 or a related product thereof in promoting plant drought resistance.

Description

Technical Field

[0001] This invention relates to a drought-resistant Pacific Bacillus strain and its applications, belonging to the field of agricultural biotechnology. Background Technology

[0002] Due to complex natural environments, plants face numerous adverse conditions during their growth. Drought is the most common environmental stress in agriculture, severely limiting crop growth and yield. Climate change and global warming are accelerating the recurrence of severe drought events, and it is projected that by 2050, more than 50% of the Earth's surface will suffer from water shortages, further exacerbating serious ecological and food security problems. Therefore, there is an urgent need to develop sustainable solutions to enhance plant resilience to drought.

[0003] Plant growth-promoting rhizobacteria (PGPRs) are bacteria that colonize the rhizosphere or roots of plants. They directly or indirectly improve plant growth, development, and stress resistance under adverse conditions by promoting nitrogen fixation, increasing nutrient absorption, improving soil properties, inhibiting plant pathogens, and enhancing plant drought tolerance. Increasing evidence reveals that utilizing the interaction between PGPRs and the host is an effective strategy for improving plant drought resistance. For example, PGPRs can improve the tolerance of various crops, including rice, maize, wheat, and barley, to drought stress, showing varying degrees of improvement in growth indicators, such as more developed root systems, increased aboveground biomass, improved photosynthesis, and reduced oxidative damage. The mechanisms by which PGPRs mediate plant drought resistance are complex, mainly including altering root structure, enhancing antioxidant capacity, secreting osmotic regulators (such as extracellular polysaccharides EPS), synthesizing plant hormones such as auxin (IAA), abscisic acid (ABA), and gibberellin (GA), and producing ACC deaminase. Therefore, the exploration of PGPR resources is of great significance for improving plant drought resistance.

[0004] Microorganisms that live in extremely arid habitats are often more tolerant of drought conditions and can serve as an important source of PGPR to improve plant drought resistance. Summary of the Invention

[0005] In this invention, a bacterium G124 with the potential to enhance plant drought resistance was isolated from arid soil in Haixi, Qinghai. Through morphological and molecular identification, growth-promoting characteristic analysis and pot inoculation experiments, the growth-promoting and drought-resistant functions of this strain were evaluated, laying the foundation for the subsequent development of plant drought-resistant bacterial agents.

[0006] Specifically, this invention provides Bacillus pacificus G124, with accession number CGMCC 30821.

[0007] The present invention also provides a microbial fermentation broth, the preparation method of which is as follows:

[0008] Bacillus pacificus G124 (accession number CGMCC 30821) was inoculated into TSB medium and cultured at 26–28°C and 180 rpm on a shaker until OD reached. 600 A value of 1.0 is sufficient.

[0009] The present invention also provides a bacterial suspension, which is prepared by the following method: take the above-mentioned bacterial fermentation broth, discard the culture medium, and resuspend it in sterile water to obtain the bacterial suspension.

[0010] The present invention also provides the application of one of the above-mentioned Bacillus paclitaxel, bacterial fermentation broth, and bacterial suspension in promoting plant drought resistance.

[0011] The present invention also provides the application of one of the above-mentioned Bacillus paclitaxel, bacterial fermentation broth, and bacterial suspension in the preparation of plant growth promoters.

[0012] The present invention also provides the application of one of the above-mentioned Bacillus paclitaxel, bacterial fermentation broth, and bacterial suspension in promoting the absorption of iron by plants.

[0013] Furthermore, the plants include Arabidopsis thaliana and alfalfa.

[0014] The present invention also provides a formulation for promoting plant drought resistance, wherein the formulation uses one of the above-mentioned Bacillus paclitaxel, bacterial fermentation broth, and bacterial suspension as an active ingredient.

[0015] The present invention also provides a method for increasing plant yield, wherein during the seedling stage, one of Bacillus paclitaxel, bacterial fermentation broth, or bacterial suspension is inoculated into the rhizosphere.

[0016] This invention has discovered that Bacillus pacificus G124 or its fermentation broth and suspension have a high iron-carrier production capacity, can produce a significant phosphorus-solubilizing zone, can mitigate or even reverse the adverse effects of drought stress on plant seedlings, and can increase the soluble sugar content and proline content of plants. Attached Figure Description

[0017] Figure 1 IAA Standard Curve

[0018] Figure 2 glucose standard curve

[0019] Figure 3 Standard curves for the determination of α-butanone and protein content

[0020] Figure 4Morphology of strain G124 on MS medium (A) and Gram staining microscopic observation (B)

[0021] Figure 5 Phylogenetic tree constructed based on 16S rDNA gene sequence

[0022] Figure 6 Effects of Bacillus pacificus G124 inoculation on the phenotypes of Arabidopsis thaliana (A and B) and alfalfa seedlings under drought stress (C and D)

[0023] Figure 7 Effects of Bacillus pacificus G124 on the growth of Arabidopsis thaliana and alfalfa seedlings under drought stress Figure 8 Effects of Bacillus pacificus G124 on the biochemical characteristics of alfalfa seedlings under drought stress Detailed Implementation

[0024] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the embodiments do not limit the present invention in any way. For those skilled in the art, any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered equivalent substitutions and are included within the protection scope of the present invention.

[0025] Example 1

[0026] 1. Research Materials

[0027] Strain G124 was isolated from arid soil in Haixi Prefecture, Qinghai Province, and is deposited at the China General Microbiological Culture Collection Center, accession number: CGMCC 30821.

[0028] The potted plants were Arabidopsis thaliana (Columbia, Col-0) and alfalfa. The soil for the Arabidopsis potted experiment was a 1:1 mixture of peat moss and vermiculite, while the soil for the alfalfa potted experiment was a 1:1:1 mixture of sand, peat moss, and vermiculite.

[0029] 2. Strain morphology and molecular identification

[0030] Morphological observation of strain G124 was performed according to Bergey's Manual of Bacterial Identification (8th edition) and the Manual of Systematic Identification of Common Bacteria. The activated strain was streaked onto MS solid medium. After colonies grew, their growth was observed and recorded, including color, shape, size, transparency, gloss, texture, colony edge characteristics, and raised shape. Gram staining was performed, followed by microscopic observation to preliminarily determine the bacterial properties. Total DNA was extracted from strain G124 using the SDS-CTAB method. Using the extracted total DNA as a template, PCR amplification was performed using universal bacterial primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-GGTTACCTTGTTACGACTT-3'). The purified PCR products were sent to Genewiz Biotechnology Co., Ltd. for sequencing. Subsequently, the assembled sequencing sequences were submitted to the online tool Identify (https: / / www.ezbiocloud.net / identify) in the EzBioCloud database for homology sequence comparison to identify type strains with high homology to the sequenced strain. The 16S rDNA sequences of similar strains were downloaded, and a Neighbor-joining (NJ) phylogenetic tree was constructed using MEGA 11.0 with a breakthrough value of 1000.

[0031] 3. Determination of the growth-promoting ability of the strain

[0032] 3.1 Determination of the strain's ability to secrete siderophores

[0033] Take the bacterial strain frozen at -80℃ with glycerol, inoculate it into 3mL of TSB liquid medium at a 1% inoculum volume to activate it, and culture it at 28℃ with shaking at 180rpm until the bacterial culture reaches OD. 600 The value was approximately 1.0. The strains were spot-caught on CAS detection plates, and each treatment was repeated three times. The cells were incubated at 28°C for 72 hours. Strains with a distinct orange-yellow halo around the colony were considered positive for siderogenetic activity. The diameter (D) of the transparent halo of a single colony and the diameter (d) of the colony were then measured using a colony analyzer. The ability to produce siderogenetic activity was evaluated by the D / d ratio.

[0034] 3.2 Determination of the phosphate-solubilizing ability of the strain

[0035] The phosphorus-solubilizing zone method was used. Activated strains were spot-inoculated on inorganic phosphorus medium in triplicate and incubated at 28°C for 72 h. Phosphorus-solubilizing zones were observed, and their diameter (D) and colony diameter (d) were measured. The phosphorus-solubilizing ability was evaluated by the D / d ratio.

[0036] 3.3 Determination of the strain's ability to secrete IAA

[0037] The isolated strain was inoculated with a solution containing tryptophan (100 mg·L⁻¹). -1In TSB liquid medium, after shaking culture for 48 h, centrifuge and collect 2 mL of the supernatant. Add 50 μL of 83% phosphoric acid and 4 mL of Salkowski reagent. The solution turns pink, indicating the production of IAA. Use uninoculated medium as a blank group and measure the absorbance of the reaction solution at 530 nm. Perform the same reaction with standard IAA solution, plot the standard curve and fit the linear equation (…). Figure 1 ), to the OD of the sample 530 Substitute the values ​​into the equation to calculate the IAA concentration in each bacterial culture.

[0038] 3.4 Determination of EPS production capacity of the strain

[0039] Crude polysaccharides were extracted, and a glucose standard curve was plotted using the phenol-sulfuric acid method. Figure 2 The absorbance was measured at 490 nm, and the measured absorbance was substituted into the standard curve fitting equation to obtain the polysaccharide concentration.

[0040] 3.5 Assay of ACC deaminase activity produced by the strain

[0041] The strain was cultured with shaking for 12 h, and the bacterial cells were collected by centrifugation and resuspended in nitrogen-free DF medium. Filtered and sterilized ACC solution (0.5 mol·L⁻¹) was then added. -1 The bacterial strain was cultured with shaking for 24 h to induce ACC deaminase activity. The bacterial pellet was collected and resuspended in Tris-HCl buffer (pH=7.6). The ACC deaminase activity was determined using 2,4-dinitrophenylhydrazine as a dye and α-butanone as the detection index. The protein content in the bacterial suspension was determined using the Bradford method, with bovine serum albumin as a standard sample as a control. Figure 3 ACC deaminase activity is measured by the amount of α-butanol produced per unit of protein content per unit time. One unit of enzyme activity (U) is defined as 1 μmol of α-butanol produced per minute. Specific activity (U·mg) is calculated by dividing the enzyme activity by the total protein content. -1 ).

[0042] 4. Preparation of bacterial suspension and pot inoculation experiment

[0043] Strain G124 was inoculated into TSB medium and cultured at 28°C and 180 rpm on a shaker until OD reached. 600The concentration was approximately 1.0. After centrifugation, the supernatant was removed, the culture medium was washed away, and the bacterial cells were resuspended in sterile ddH2O to obtain the bacterial suspension for inoculation. In the pot experiment, Arabidopsis thaliana and alfalfa were used as test plants. Arabidopsis thaliana seeds were sterilized and disinfected, then sown on 1 / 2 MS medium. After being placed at 4℃ for 12 hours, they were placed in an artificial climate chamber for germination. The culture conditions were 16 hours of light / 8 hours of darkness, and the temperature was 22-25℃. After 7 days of culture, Arabidopsis thaliana seedlings with uniform growth were selected and transplanted into flower pots. When they had 4-6 true leaves, they were inoculated with the bacterial strain. The treated alfalfa seeds were germinated in Petri dishes lined with moist filter paper. The culture conditions were the same as those for Arabidopsis thaliana. After 4 days, seedlings with uniform growth were selected and transplanted into flower pots. On day 20, they were inoculated with the bacterial strain.

[0044] The pot experiment was divided into three groups: normal watering group (CK), drought stress group (DR), and drought stress-inoculated group (G124). Inoculation was performed by root inoculation. 1 mL of bacterial suspension was poured around the rhizosphere of Arabidopsis thaliana and 2 mL of bacterial suspension was poured around the rhizosphere of alfalfa. Inoculation was carried out for three consecutive days. At the same time, the DR group was watered with the corresponding volume of distilled water in the same manner and at the same time. Each treatment was replicated three times.

[0045] 5. Determination of physiological and biochemical indicators of seedling growth

[0046] Leaves and root lengths (from the same tissue part) of plants in different treatment groups were photographed, and the images were imported into ImageJ to calculate leaf area and root length; plant height was measured with a measuring tape. Chlorophyll fluorescence indices of the same tissue part of plants in different treatment groups were measured using a chlorophyll fluorescence meter. Maximum photochemical efficiency (Fv / Fm) was measured after 30 minutes of dark adaptation before measurement; chlorophyll content, relative conductivity, and relative water content in leaves were measured according to relevant literature. For the determination of biochemical indicators of alfalfa, alfalfa seedling leaves were first collected, thoroughly ground in liquid nitrogen, and then the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), as well as the activities of hydrogen peroxide (H2O2) and superoxide anion (O2O2), were measured using a kit (purchased from Suzhou Keming Biotechnology Co., Ltd.) according to the instructions. - The content of malondialdehyde (MDA), soluble sugars, and proline.

[0047] 6. Data Processing

[0048] One-way ANOVA was performed on the data results using SPSS 26.0 software, and the significance of differences was analyzed. Then, Graphpad Prime 8.0 was used to plot the experimental results.

[0049] III. Research Results

[0050] 1. Morphological observation of G124 growth-promoting bacteria

[0051] When strain G124 grows on MS medium, it exhibits yellowish-white colonies with irregular edges, wrinkled surfaces, and a rough texture. Figure 4 A), consistent with the morphological characteristics of Bacillus. Furthermore, single colonies picked from the culture medium were Gram-stained and observed under a microscope. The results showed that the strain stained purple with Gram and was rod-shaped or club-shaped, indicating that this strain belongs to Bacillus (A). Figure 4 B).

[0052] 2. Molecular biological identification of strain G124

[0053] A 1440 bp sequence was obtained by amplifying and sequencing the 16S rDNA gene of strain G124. Homology searching was performed using the Identify function in the EzBioCloud database, showing a maximum similarity of 99.93% between this gene sequence and the 16S rDNA sequence of Bacillus pacificus EB422. A phylogenetic tree was constructed using MAGA 11.0. Figure 5 The strain G124 was found to belong to the genus Bacillus and clustered with Bacillus pacificus EB422. Therefore, the strain was named Bacillus pacificus G124.

[0054] 3. Identification of the growth-promoting characteristics of strain G124

[0055] Strain G124 produced an orange-yellow halo on CAS plates, with a mean D / d ratio of 1.895, indicating a high siderophore production capacity. G124 also produced a distinct phosphate-solubilizing zone on PKO solid medium, with a mean D / d ratio of 0.587, demonstrating strong phosphate solubilization ability. The IAA production capacity of strain G124 was determined to be 0.319 μg·mg. −1 In addition, strain G124 also has the ability to produce EPS, with an EPS concentration of 1.238 mg·mg⁻¹. −1 The ACC deaminase activity of strain G124 was 0.054 U·mg. −1 These indicators suggest that the bacterium has potential growth-promoting and drought-resistant properties (Table 1).

[0056] Table 1. Determination of the growth-promoting ability of the strains

[0057]

[0058] +: Stronger; ++: Very strong; —: Not obvious

[0059] 4. The alleviating effect of strain G124 on drought stress inhibition in plant seedlings.

[0060] To evaluate whether Bacillus pacificus G124 could alleviate drought stress in plants, Bacillus pacificus G124 cultures were inoculated into Arabidopsis thaliana and alfalfa. The results showed that, compared to the uninoculated group (DR group) under drought stress, Bacillus pacificus G124 significantly promoted the growth phenotype of seedlings in both plants. Figure 6 Furthermore, their root systems are more developed, and their phenotypes under drought stress are significantly reversed.

[0061] Further analysis revealed that, compared with uninoculated seedlings under drought stress, Arabidopsis and alfalfa seedlings inoculated with Bacillus pacificus G124 showed significantly increased plant heights of 15.7% and 33.4%, respectively, with no significant difference in plant height compared to the control group. Figure 7 A); the leaf area of ​​seedlings also increased by 219.2% and 64.6% respectively, but was slightly lower than that of the CK group seedlings ( Figure 7 B). Furthermore, under drought stress, the leaves of the inoculated seedlings were greener. Further analysis of chlorophyll content and photosynthetic capacity revealed that the chlorophyll content of the inoculated Arabidopsis and alfalfa seedlings was significantly higher than that of the drought-stressed non-inoculated groups, increasing by 46.2% and 16.2%, respectively. Moreover, the maximum photochemical efficiency (Fv / Fm) was also significantly higher in the inoculated groups than in the non-inoculated groups. Figure 7 The results (CD) indicate that inoculation with Bacillus pacificus G124 promoted the photosynthetic capacity of seedlings under drought stress. After drought stress, the relative water content of seedling leaves decreased sharply, and the relative conductivity increased significantly, suggesting damage to the cell integrity of seedling leaves. However, the relative water content of leaves in the Bacillus pacificus G124-inoculated groups of Arabidopsis and alfalfa recovered somewhat, and the relative conductivity decreased by 25.1% and 30.6% respectively compared to the uninoculated groups under drought stress, essentially returning to the CK group level (7E-F). This indicates that Bacillus pacificus G124 can alleviate the adverse effects of drought stress on the seedlings of both plants.

[0062] 5. Effects of PGPR strains on the biochemical characteristics of alfalfa seedlings under drought stress

[0063] To reveal the response of Bacillus pacificus G124-inoculated plants to drought stress, this study used alfalfa as the research object and analyzed the levels of peroxides, antioxidant enzyme activities, and osmotic regulation in alfalfa seedlings under different treatment groups. Figure 8The results showed that, compared to the control group, alfalfa seedlings subjected to drought stress had lower levels of peroxides such as H2O2 and O2 in their leaves. - The significantly increased MDA content indicates that drought treatment subjected alfalfa seedlings to severe oxidative stress. However, inoculation with Bacillus pacificus G124 significantly reduced the levels of these oxidants in the seedlings, decreasing by 22.8%, 50.8%, and 20.5%, respectively, compared to the drought-free group. Figure 8 The results (AC) indicate that inoculation with Bacillus pacificus G124 effectively alleviates oxidative damage caused by drought stress. Antioxidant enzyme systems are important defense mechanisms in plants against oxidative stress from adverse environments. Compared with the control group, drought induced a significant increase in the activities of SOD, POD, and CAT in alfalfa seedlings, and inoculation with Bacillus pacificus G124 further enhanced the activities of these antioxidant enzymes, increasing them by 62.4%, 158.7%, and 341.8%, respectively. Figure 8 (DF), which may be an important reason for the reduced oxide levels in the leaves of seedlings in the inoculated group.

[0064] Furthermore, soluble sugars and proline are important substances for osmotic regulation in plant cells. Compared with the control group, drought stress also induced the biosynthesis and accumulation of these two substances in seedlings. The soluble sugar and proline contents in seedlings inoculated with Bacillus pacificus G124 were further increased, with increases of 66.5% and 234.2% respectively compared with the control group. Figure 8 In summary, Bacillus pacificus G124 can improve the drought resistance of alfalfa by regulating the activity of plant antioxidant enzymes, oxide levels, and osmotic regulators, thereby mitigating drought stress-induced seedling damage.

Claims

1. Pacific Bacillus ( Bacillus pacificus G124, collection number: CGMCC 30821.

2. A microbial fermentation broth, characterized in that: The preparation method is as follows: Take the Pacific Bacillus G124 as described in claim 1, inoculate it into TSB medium, and culture it at 26-28°C and 180 rpm on a shaker until the OD600 value is 1.

0.

3. A bacterial suspension, characterized in that: The preparation method is as follows: Take the fermentation broth of the strain described in claim 2, discard the culture medium, and resuspend it in sterile water to obtain a bacterial suspension.

4. The application of any one of the following in promoting drought resistance in plants: the Bacillus paclitaxel of claim 1, the fermentation broth of claim 2, and the bacterial suspension of claim 3; wherein the plant is Arabidopsis thaliana or alfalfa.

5. The application of any one of the following in the preparation of a plant growth promoter: the Bacillus paclitaxel of claim 1, the fermentation broth of claim 2, or the bacterial suspension of claim 3; wherein the plant is Arabidopsis thaliana or alfalfa.

6. The application of any one of the following in promoting iron absorption by plants: the Bacillus paclitaxel of claim 1, the fermentation broth of claim 2, and the bacterial suspension of claim 3; wherein the plant is Arabidopsis thaliana or alfalfa.

7. A formulation for promoting drought resistance in plants, characterized in that, The formulation uses Bacillus paclitaxel as the active ingredient as described in claim 1; the plant is Arabidopsis thaliana and alfalfa.

8. A method for increasing plant yield, characterized in that, During the seedling stage, one of the following is inoculated into the rhizosphere: Bacillus paclitaxelus as described in claim 1, the fermentation broth of the strain as described in claim 2, or the bacterial suspension as described in claim 3; the plant is Arabidopsis thaliana or alfalfa.

Images