CN118745400A

CN118745400A - A drought-resistant Velez bacillus and its application

Info

Publication number: CN118745400A
Application number: CN202410886016.0A
Authority: CN
Inventors: 殷恒霞; 马晓兰; 张本印; 向信
Original assignee: Qinghai University
Current assignee: Qinghai University
Priority date: 2024-07-03
Filing date: 2024-07-03
Publication date: 2024-10-08

Abstract

The invention provides bacillus beijerinus G138. The invention also provides application of bacillus bailii G138 or related products thereof in promoting drought resistance of plants.

Description

Drought-resistant bacillus beliae and application thereof

Technical Field

The invention relates to a drought-resistant bacillus belicus strain and application thereof, belonging to the technical field of agricultural biology.

Background

The increase in population and grain demand brings great pressure to global agriculture, however extreme weather disasters are frequent worldwide, where extensive continuous drought has become a major threat to sustainable development of agriculture. When drought stress is applied, plant physiological and morphological characteristics can be obviously changed, such as growth inhibition, root system structure change, serious oxidative stress, osmotic pressure change and the like. Plants develop a variety of adaptation mechanisms at morphological, physiological, biochemical and molecular levels during long-term evolution.

In addition, drought resistance of plants is also closely related to plant rhizosphere microbial community structure. Rhizosphere microorganisms not only promote plant growth but also induce systemic resistance in plants. Beneficial bacteria that live in the soil or attach to plant root systems to promote plant growth and its uptake and utilization of mineral nutrients and to inhibit pests are known as rhizosphere growth promoting bacteria (plant growthpromotingbacteria, PGPB).

In recent years, PGPB has been increasingly studied for its role in drought stress, and its mediated drought-resistance mechanisms in plants mainly include 4 pathways: synthesizing plant growth regulating factors such as indole-3-acetic acid (indole-3-ACETIC ACID, IAA) and the like; producing 1-aminocyclopropane-1-carboxylic Acid (ACC) deaminase, reducing root ethylene content; enhancing the activity of plant antioxidant enzyme and eliminating the harm of active oxygen; bacterial extracellular polysaccharide (exopolysaccharides, EPS) is produced, enhancing water retention.

Currently, PGPB is used on many crops such as rice, wheat, corn, tomato, pepper, etc. to enhance environmental tolerance of plants. Therefore, the development of PGPB resources has important significance for crop yield and grain safety guarantee in the global warming background.

Disclosure of Invention

The research separates bacillus with potential growth promoting and plant stress resisting effects from Qinghai-Shaoxing drought soil, analyzes the taxonomic status of the bacillus and enhances the drought resistance of plants through 16S rRNA gene sequence comparison, phylogenetic tree construction and potting inoculation experiments, and provides theoretical support and strain resources for developing PGPB drought resistant biofertilizer.

Specifically, the invention provides bacillus belicus Bacillus velezensis G138,138 with the deposit number: CGMCC30822.

The invention also provides a strain fermentation broth, which is prepared by the following steps:

taking a preservation number: bacillus belicus Bacillus velezensis G138,138 of CGMCC30822 is inoculated in TSB culture medium and cultured at 26-30 deg.C and 180 rpm.

The invention also provides a bacterial suspension, and the preparation method comprises the following steps: taking the strain fermentation liquor, discarding the culture medium, and re-suspending with sterile water to obtain a bacterial suspension.

The invention also provides application of one of the bacillus belicus, the strain fermentation liquor and the strain suspension in promoting drought resistance of plants.

The invention also provides application of one of the bacillus beijerinus, the strain fermentation liquor and the strain suspension in preparation of the plant growth promoting agent.

The invention also provides application of one of the bacillus belicus, the strain fermentation liquor and the strain suspension in promoting plants to absorb iron elements.

Further, the plant includes arabidopsis thaliana, alfalfa.

The invention also provides a preparation for promoting drought resistance of plants, which takes one of bacillus belicus, strain fermentation liquor and strain suspension as an active ingredient.

The invention also provides a method for improving the plant yield, and one of bacillus beijerinus, strain fermentation liquor and strain suspension is inoculated in the rhizosphere in the plant seedling stage.

The bacillus belicus Bacillus velezensis G or the strain fermentation liquid and the strain suspension thereof are found to improve the antioxidant enzyme activity of plants, reduce the peroxide level and promote the accumulation of osmotic adjusting substances so as to reduce the drought stress damage of the plants.

Drawings

FIG. 1 Strain G138 phylogenetic tree based on 16S rRNA Gene

FIG. 2 shows the siderophore and phosphorus-dissolving properties of strain G138

FIG. 3 influence of Strain G138 on Arabidopsis and alfalfa growth phenotype (A), plant height (B) and aboveground biomass (C) under drought stress

FIG. 4 influence of Strain G138 on physiological parameters of Arabidopsis thaliana and alfalfa under drought stress

FIG. 5 influence of Strain G138 on the biochemical characteristics of Arabidopsis and alfalfa under drought stress

Detailed Description

The invention will be further illustrated by the following detailed description in conjunction with the drawings and examples, which are not intended to limit the invention in any way. It will be apparent to those skilled in the art that various changes, modifications, substitutions, combinations, and simplifications can be made without departing from the spirit and principles of the invention and these are intended to be equivalent arrangements.

Example 1

(1) Bacterial strain origin and preservation

The strain G138 used in the research is separated from Qinghai sea and western drought soil and is stored in China center for type culture collection with the preservation number of CGMCC30822.

(2) Identification of strains

Culturing the activated strain on LB solid plate by streaking, standing at 28 ℃, picking up purified single colony, inoculating in TSB liquid culture medium, culturing at 28 ℃ 180 r.min ^-1 to logarithmic phase, sucking 1mL bacterial liquid to 1.5mL centrifuge tube, centrifuging to collect bacterial cells, extracting total DNA of the strain G138 by SDS-CTAB method, taking the bacterial general primers 27F (5 '-AGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') as templates, carrying out PCR amplification on the full-length sequence of 16S rRNA gene, and sending the PCR product to Jin Weizhi Biotech Co, sequencing, uploading the sequence to GenBank database, and recording the number PP809070. The measured 16S rDNA gene sequence is subjected to homologous sequence alignment in Ezbiocloud database (https:// www.ezbiocloud.net/identification), then homologous sequences of similar strains are downloaded, and a phylogenetic tree based on a Neighbor-Joining method is constructed by using MEGA 11.0 software, and the self-expanding value is 1000 times.

(3) Determination of growth-promoting Properties of Strain G138

Determination of iron production carrier capacity: strain G138 was inoculated into CAS medium and after 3 days of incubation at 28 ℃ was observed for the production of orange halos, which if produced indicated that the strain had ferric carrier production and the capacity size was characterized by the ratio of halo to colony diameter. Determination of phosphate-solubilizing ability: the strain is inoculated on an inorganic phosphorus culture medium, and after the strain is cultured for 3 days at the temperature of 28 ℃, the strain is observed, if obvious phosphorus dissolving rings or transparent rings appear on the culture medium, the strain has phosphorus dissolving capability, if the strain does not appear, the strain does not have phosphorus dissolving capability, and the capability is represented by the ratio of the transparent rings to the colony diameter. IAA content measurement: strain G138 was inoculated into a liquid medium containing tryptophan (100 mg·l ^-1), shake-cultured for 48 hours, centrifuged to obtain 2mL of supernatant, and 50 μl of 83% by volume orthophosphoric acid and 4mL Salkowski reagent were added, and the solution turned pink, indicating IAA production. The IAA standard solution is subjected to the same reaction, a standard curve is drawn, a linear equation is fitted, and the OD ₅₃₀ value of the sample is substituted into the fitting equation to calculate the IAA concentration in the bacterial liquid. EPS content determination: first, an equal volume of deionized water was added to the culture medium for 2 days of TSB liquid culture, and the supernatant was collected by centrifugation. And adding 95% ethanol with the volume of 2 times to the supernatant, uniformly mixing, and centrifugally collecting the precipitate to obtain the crude polysaccharide. Drawing a glucose standard curve by using a phenol-sulfuric acid method, sampling 1.0mL, adding 1.0mL of distilled water, then adding 1.0mL of 6% phenol, rapidly adding 5.0mL of concentrated sulfuric acid, fully and uniformly mixing, standing for 30 minutes, measuring absorbance at 490nm, and calculating polysaccharide concentration according to the standard curve. ACC deaminase activity assay: strain G138 was shake-cultured in TSB liquid medium for 12 hours, collected by centrifugation, resuspended in DF medium without nitrogen source, added 45 μl of filter sterilized ACC solution (0.5M), continued to shake-culture for 24 hours to induce ACC deaminase activity, then collected by centrifugation to precipitate and resuspended in Tris-HCl buffer (ph=7.6), this step was repeated 3 times to thoroughly remove DF medium, resuspended in 600 μl Tris-HCl buffer (ph=8.5), added 30 μl toluene, The cells were broken by shaking for 30 seconds. 200 mu L of broken bacterial liquid is sucked, 20 mu LACC solution is added, the mixture is uniformly mixed, water bath is carried out for 15 minutes at 37 ℃, 1mL of 0.56M HCl is added, 1mL of supernatant fluid is centrifugally taken, 800 mu LHCl and 300 mu L of 2, 4-dinitrophenylhydrazine are added, water bath is carried out for 30 minutes at 30 ℃, and finally 2mL of 2M NaOH is added for color development, the light absorption value is measured at 540nm, and distilled water is used as a blank control. According to the same color reaction, a standard curve of the alpha-butanoic acid is drawn, the OD ₅₄₀ value of a sample is substituted into a standard equation to calculate the content of the alpha-butanoic acid, and the quantity (mu M) of the alpha-butanoic acid produced by the ACC under the catalysis of ACC deaminase in unit time, namely the unit enzyme activity U is calculated. Firstly, drawing standard curves of bovine serum albumin with different concentrations and absorbance by using a Coomassie brilliant blue G-250 method, and then calculating the protein content in the bacterial suspension. ACC deaminase activity is expressed as the ratio of unit enzyme activity U to total protein mass (U.mg ^-1). Biofilm assay: quantifying in vitro biofilm formation by adopting a microtiter plate measurement method, firstly, standing and incubating 100 mu L of bacterial liquid in a 96-well plate at 37 ℃ for 36 hours, sucking out the bacterial liquid, washing 3 times by using a sterile PBS buffer solution, fixing methanol for 15 minutes, sucking out the bacterial liquid, air-drying, dyeing for 5 minutes by using crystal violet, washing redundant dye by using flowing water, drying, adding glacial acetic acid with the concentration of 33%, dissolving the crystal violet in a 37 ℃ oven, finally measuring the absorbance at the wavelength of 590nm by using an enzyme-labeling instrument, taking the absorbance of a culture liquid without bacteria as a negative control, taking the absorbance of the culture liquid without bacteria as a limit value (Dc) which is 2 times as a strong biofilm when the OD ₅₉₀ is more than 2Dc, Dc is less than OD ₅₉₀ and less than or equal to 2Dc is a weak biofilm, and OD ₅₉₀ is less than or equal to Dc and no biofilm is formed.

(4) Preparation of G138 bacterial suspension

Strain G138 was streaked on LB medium, cultured at 28 ℃ for 24 hours, single colonies were picked up in TSB medium, cultured at 28 ℃ at 180rpm for 24 hours, centrifuged at 5000rpm for 10min to collect cells, and then resuspended in sterile deionized water, and the bacterial suspension OD ₆₀₀ was adjusted to 1.0 for inoculation experiments.

(5) Potting and inoculation test

The plants for pot culture and inoculation test are Arabidopsis thaliana and alfalfa, and for Arabidopsis thaliana cultivation, arabidopsis thaliana seeds are first surface sterilized with 1% sodium hypochlorite for 5 minutes, then sterilized with 75% ethanol for 45 seconds, and then rinsed with sterilized deionized water 5 times. Subsequently, the seeds were sown in 1/2MS medium (ph=5.8) containing 1.5% sucrose and 1% agar, 70 seeds were sown in each petri dish (10×10 cm), vernalized at 4 ℃ for 24 hours, and germinated in 16h light (light intensity 8000 LUX)/8 h dark incubator for 7 days. Then selecting seedlings with consistent growth vigor, transplanting the seedlings into square flowerpots (the flowerpots are 8 multiplied by 7cm in size, the soil is peat soil 1:1 vermiculite), watering five seedlings in each pot every three days, continuously culturing for 7 days, and then carrying out drought treatment. For alfalfa cultivation, selecting full alfalfa seeds, sterilizing with 2% sodium hypochlorite for 5 minutes, sterilizing with 75% ethanol for 45 seconds, rinsing with sterilized water for 5 times, placing in a sterile culture dish with wet filter paper, germinating for 4 days, picking seedlings with consistent growth vigor, transplanting into a round flowerpot (diameter 10cm, height 9cm; soil matrix is peat soil, vermiculite and sandy soil mixture of 1:1), watering five seedlings every basin every three days, and drought treatment after 20 days of growth. The potting experiments were set to 3 groups, namely, normal watering group (CK), drought treatment group (DR), drought treatment+g138 inoculation group (G138). The CK group is poured with water once every three days, and 30mL of water is used each time; DR group was not watered for 14 days from drought treatment; group G138 was watered with 1mL of the bacterial suspension around the plant rhizosphere three consecutive days after the start of drought treatment, and, in addition, 1mL of distilled water was watered around the plant rhizosphere of group DR at the same time. The plants were photographed and observed for phenotype after 14 days of drought stress treatment and relevant physiological and biochemical indicators were determined.

(6) Physiological and biochemical index determination

After 14 days of drought stress, the plant heights and root lengths of the plants of the different treatment groups are measured by using a tape measure, and the overground biomass of the seedlings is weighed by a balance. 0.2g of the same-height expanded leaf samples were taken, de-enzymed in an oven at 105℃for 15 minutes and dried to constant weight at 80℃and weighed, the relative moisture content calculated as shown below, i.e., relative moisture (%) = (FW-DW)/(TW-DW). Times.100, where FW, TW and DW represent fresh weight, saturated fresh weight and dry weight, respectively. Leaf maximum photochemical efficiency (Fv/Fm) was determined for the same tissue site of different treatment groups of plants using a chlorophyll fluorometer (FluorPen FP110,110); in addition, 0.1g of fresh leaves was thoroughly ground with calcium carbonate and 95% alcohol in a dark treatment, the 95% alcohol was fixed to 25mL after filtration, absorbance values at wavelengths of 665nm and 649nm were measured and chlorophyll content was calculated. The conductivities of fresh and boiled blades were measured using a conductivity meter EC215 (Hanna) and then the relative conductivities were calculated from the conductivity ratio of the two. Fresh She Yang 0.1.1 g of alfalfa seedlings were collected, first ground using liquid nitrogen, and then assayed for superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) activities, and for the content of soluble sugars, proline, malondialdehyde (MDA), hydrogen peroxide (H ₂O₂₎, superoxide anion (O ₂ ^-) using a biochemical kit (purchased from su-state Ming biotechnology limited) according to the instructions, respectively, with 3 biological replicates per test.

Results of the study

(1) Molecular characterization of Strain G138

The 16S rRNA gene amplification and sequencing result of the strain G138 shows that the length of the 16S rRNA gene sequence is 1434bp. By carrying out homologous sequence alignment in EzBioCloud database, the maximum similarity of the strain G138 is found to be Bacillus bailii CR-502 (Bacillus velezensis CR-502) respectively, and the similarity is 99.50% respectively. The taxonomic position of the strain G138 was further determined by phylogenetic tree analysis based on the 16S rRNA gene construction, which revealed that the strain G138 was clustered with the largest similar strain (FIG. 1), and thus, the strain G138 was named Bacillus belicus G138 (B.velezensis G138).

(2) Growth-promoting Property analysis of Strain G138

Strain G138 was able to produce a pronounced orange-yellow halo on CAS plates and the D/D ratio was 1.895, indicating that strain G138 had a stronger siderophore production capacity. In addition, the strain G138 can generate obvious phosphate solubilizing ring on PKO solid culture medium, and the D/D ratio is 2.186, which shows that the strain has stronger phosphate solubilizing capability (figure 2). As a result of IAA content measurement, it was found that the strain G138 also had an IAA-producing ability and a secretion amount of 6.712. Mu.g.mL ^-1. For EPS producing ability, the concentration of EPS produced by strain G138 was 1.790 mg.mL ^-1, indicating that the strain has the ability to produce EPS. The results of ACC deaminase activity test show that the strain G138 has strong ACC deaminase activity and is 0.013 U.mg ^-1. In addition, biofilm formation test found that OD ₅₉₀ of strain G138 was 1.185, approximately 4 times that of Dc (0.301), indicating that strain G138 had stronger biofilm formation ability. These properties indicate that bacillus bailii G138 has potential growth promoting and drought resisting properties.

(3) Influence of Strain G138 on physiological characteristics of Arabidopsis thaliana and alfalfa under drought stress

To further evaluate whether strain G138 is capable of drought tolerance in plants, this study observed the phenotypic and physiological characteristic responses of inoculated G138 arabidopsis and alfalfa to drought stress. As a result, it was found that the growth of Arabidopsis thaliana and alfalfa was significantly inhibited after 14 days of drought stress (FIG. 3A), and that the plant height and the on-ground fresh weight biomass of Arabidopsis thaliana and alfalfa were significantly lower than those of the normally watered group (FIGS. 3B and C). Interestingly, after G138 inoculation, the phenotypes of arabidopsis and alfalfa under drought stress were largely reversed relative to the drought stress group, both plant seedlings had significantly increased plant height and ground fresh weight biomass, and arabidopsis stem length and alfalfa ground fresh weight reached the CK group level (fig. 3A-C). The stem length of G138 inoculated arabidopsis and alfalfa was increased by 24.3% and 39.8%, respectively, and the above-ground fresh biomass was increased by 59.3% and 54.2%, respectively, relative to the drought stress group (fig. 3A-C).

In addition, root development of both arabidopsis and alfalfa seedlings under drought stress was inhibited and root length was significantly reduced relative to CK group (fig. 4A). Under drought stress, the root length of the seedling of the Arabidopsis inoculated G138 group is obviously higher than that of the seedling of the DR group, and the seedling is improved by 39.3 percent. Although the alfalfa vaccinated seedlings were all significantly lower in root length than the control, but significantly higher than the DR group, an increase of 46.9% (fig. 4B). The same leaf area of plants in different treatment groups was measured to find that drought stress resulted in a significant reduction in leaf area of both plants, but after inoculation of both bacillus strains, leaf area was restored to normal group levels, and strain G138 increased leaf area of arabidopsis and alfalfa by 197.3% and 41.4% respectively under drought stress (fig. 4C). The measurement results of the phenotype and physiological parameters show that the strains G128 and G138 can obviously improve the drought resistance of plants. Further analysis of the relative moisture content, relative conductivity and photosynthesis related physiological parameters showed that the relative moisture content of arabidopsis and alfalfa seedling leaves under drought stress was significantly reduced relative to CK group, while the inoculation strain G138 greatly increased the relative moisture content of plant leaves under drought stress, strain G138 increased the relative moisture content of arabidopsis and alfalfa seedlings by 57.1% and 28.3%, respectively, but lower than the relative moisture content of seedlings of the control group (fig. 4D).

Relative conductivity of arabidopsis and alfalfa seedling leaves was significantly improved compared to CK group, while relative conductivity of two inoculant inoculated group seedlings was significantly reduced under drought stress, strain G138 reduced relative conductivity of arabidopsis and alfalfa seedlings by 21.2% and 30.2%, respectively, without significant difference from the relative conductivity of normal group seedling leaves (fig. 4E). Furthermore, the chlorophyll content of arabidopsis seedlings was significantly reduced under drought stress compared to CK group, while the chlorophyll content of G138 inoculated group seedlings was greatly increased, but below normal group level, interestingly without significant differences in maximum photochemical efficiency (Fv/Fm) between the different treatment groups (fig. 4F and G). Compared with the CK group, the alfalfa has insignificant chlorophyll content change of plant seedlings after drought stress, but compared with the drought stress group, the chlorophyll content of seedlings of the G138 inoculated group is significantly improved compared with the control group, and Fv/Fm is significantly higher than that of the drought stress group (figures 4F and G), which shows that the strain G138 promotes photosynthesis of plants under drought stress.

(4) Effect of Strain G138 on alfalfa Biochemical Properties under drought stress

To further investigate the change in response to drought stress in seedlings inoculated with strain G138 at the biochemical level, alfalfa biochemical indicators from different treatment groups were selected for further analysis. As a result, it was found that, during drought stress, alfalfa seedling H ₂O₂、O₂ ^- and MDA content were significantly increased relative to the control group (FIGS. 5A-C), and it was presumed that drought stress caused severe oxidative stress to both plants. Compared with the DR group, the peroxide content in alfalfa seedlings inoculated by the strain G138 is obviously reduced, the H ₂O₂、O₂ ^- and MDA contents of the seedlings under drought stress are respectively reduced by 44.1%,51.4% and 21.9% by the strain G138, and the peroxide contents are almost reduced to the normal group level (fig. 5A-C), so that the strain G138 can relieve oxidative damage caused by plant drought.

In terms of antioxidant enzyme activity, drought stress significantly increased antioxidant enzyme SOD, POD and CAT activities of alfalfa seedlings by 24.1%,51.9% and 103.8%, respectively (fig. 5D-F), relative to CK group, while the addition of microbial inoculum G138 further increased antioxidant enzyme activity of seedlings under drought stress, strain G138 increased antioxidant enzyme activities of these three in alfalfa seedlings by 28.9%,36.2% and 28.4%, respectively (fig. 5D-F), possibly related to their ability to clear peroxides, relative to drought stress group. In terms of accumulation of osmoregulation substances, drought stress caused accumulation of osmoregulation substances soluble sugars and prolines relative to the CK group (fig. 5G and H), whereas inoculation of strain G138 slightly increased the content of soluble sugars and prolines in seedlings relative to the DR group, but without significant differences (fig. 5G and H). In summary, strain G138 alleviates drought stress injury in plants by increasing antioxidant enzyme activity, decreasing peroxide levels, and promoting accumulation of osmoregulation substances in alfalfa seedlings.

Claims

1. Bacillus bailii Bacillus velezensis G138,138 accession number: CGMCC30822.

2. A strain fermentation broth, characterized in that: the preparation method comprises the following steps:

3. A bacterial suspension characterized by: the preparation method comprises the following steps: taking the strain fermentation broth of claim 2, discarding the culture medium, and re-suspending with sterile water to obtain a strain suspension.

4. Use of one of bacillus belicus according to claim 1, a strain fermentation broth according to claim 2, and a strain suspension according to claim 3 for promoting drought resistance of plants.

5. Use of one of bacillus belicus according to claim 1, a strain fermentation broth according to claim 2, and a strain suspension according to claim 3 for the preparation of a composition for promoting plant growth.

6. Use of one of bacillus belicus according to claim 1, a strain fermentation broth according to claim 2, and a strain suspension according to claim 3 for promoting the absorption of elemental iron by plants.

7. The use according to claim 4 or 5, wherein the plant comprises arabidopsis thaliana, medicago sativa.

8. A preparation for promoting drought resistance of plants, which is characterized in that the preparation takes one of bacillus belicus according to claim 1, strain fermentation broth according to claim 2 and strain suspension according to claim 3 as an active ingredient.

9. A method for improving plant yield, characterized in that in the seedling stage of the plant, one of bacillus belicus according to claim 1, the strain fermentation broth according to claim 2 and the strain suspension according to claim 3 is inoculated in the rhizosphere.