Drought-resistant growth-promoting bacterium, fungicide, application and plant drought-resistant growth-promoting method

Abstract

The invention relates to the field of microorganisms, in particular to a drought-resistant growth-promoting bacterium, a fungicide, application and a plant drought-resistant growth-promoting method. The strain is Rhodobacter sp. K28, is preserved in the China Center for Type Culture Collection on January 29, 2024, and has the preservation number of CCTCC (China Center for Type Culture Collection) NO: M 2024252. The strain can enhance the growth of arabidopsis thaliana under drought stress and promote the increase of the amount of dry matter of arabidopsis thaliana. The method is used for improving the drought resistance of arabidopsis thaliana and promoting the growth of arabidopsis thaliana.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to a drought-resistant growth-promoting bacterium, a bacterial agent and an application thereof, and a method for resisting drought and promoting growth of plants. Background Art

[0002] Drought is currently the biggest threat to global agricultural production. Drought stress increases soil heterogeneity, limits nutrient transfer, increases soil oxygen, reduces microbial biomass and ultimately causes crop yield reduction. Rhizosphere microorganisms are considered to be the second gene bank of plants. In terms of helping plants resist drought, on the one hand, they can use their own structures and functions, such as Gram-positive bacteria producing dormant structures such as spores, and some strains that can produce biofilms and osmotic regulating substances, which can adjust their osmotic pressure to adapt to drought environments in the absence of water. On the other hand, they can help plants enhance antioxidant enzyme activity and improve water use efficiency through their own characteristics to adapt to drought, such as Streptomyces and arbuscular mycorrhizal fungi.

[0003] Arabidopsis thaliana is used as a model plant for research because of its short life cycle, complete genome sequencing, rich genetic resources and mutant library, mature genetic transformation technology, and simple and relatively simple growth conditions. Therefore, Arabidopsis rhizosphere microorganisms are an important way to study the ability to improve plant drought stress resistance. The drought-resistant growth-promoting bacteria in the existing technology include Bacillus velezensis, Aspergillus oryzae, Pseudomonas nitroreducens, Bacillus cereus, Pantoea agglomerans, etc. More types of drought-resistant growth-promoting bacteria are beneficial to improving the ability of plants to resist drought stress, so more types of drought-resistant growth-promoting bacteria need to be provided. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a drought-resistant and growth-promoting bacterium, a bacterial agent, and an application and a method for resisting drought and promoting growth of plants.

[0005] A drought-resistant growth-promoting bacterium, wherein the drought-resistant growth-promoting bacterium is Rhodobacter roehei ( Rhodanobacter sp.) K28, deposited in China Center for Type Culture Collection, the deposit date is January 29, 2024, and the deposit number is CCTCC NO: M 2024252.

[0006] A bacterial agent comprises the drought-resistant and growth-promoting bacteria.

[0007] Preferably, TSB medium is also included.

[0008] The application of the drought-resistant and growth-promoting bacteria or the bacterial agent in plant drought resistance and growth promotion.

[0009] Preferably, the drought-resistant growth-promoting bacteria are diluted with sterile water, and the concentration of the drought-resistant growth-promoting bacteria is greater than or equal to 10 6 CFU / mL.

[0010] Preferably, the plant is Arabidopsis thaliana.

[0011] A method for resisting drought and promoting growth of plants, comprising applying the drought-resistant and growth-promoting bacteria or the bacterial agent to the seeds of the plants, or applying the drought-resistant and growth-promoting bacteria or the bacterial agent to the planting matrix of the plants.

[0012] Preferably, the drought-resistant growth-promoting bacteria are diluted with sterile water, and the concentration of the drought-resistant growth-promoting bacteria is greater than or equal to 10 6 CFU / mL.

[0013] Preferably, the plant is Arabidopsis thaliana, soybean or rice.

[0014] Preferably, the growth promotion refers to an increase in biomass, an increase in the number of leaves and / or an increase in the leaf area.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a drought-resistant growth-promoting bacterium, wherein the drought-resistant growth-promoting bacterium is Rhodobacter roehei ( Rhodanobacter sp.) K28, which has the ability to resist drought stress and has a growth-promoting effect under drought conditions.

[0016] The present invention is the Rhodanobacter sp.) K28 was inoculated into Arabidopsis thaliana and found that Rhodanobacter sp.) K28, the biomass, leaf number and leaf area of ​​Arabidopsis thaliana increased, and the biomass was dry weight. Therefore, the present invention provides a method for plant drought resistance and growth promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 For Rhodeobacter Rhodanobacter Observation of colony morphology of sp. K28 cultured on 50% TSB plates.

[0018] Figure 2 For Rhodeobacter Rhodanobacter sp.) Phylogenetic tree of K28.

[0019] Figure 3 For Rhodeobacter Rhodanobacter Fermentation diagram of K28 in 10% TSB medium.

[0020] Figure 4 For Rhodeobacter Rhodanobacter sp.) K28 on drought resistance and growth promotion of Arabidopsis thaliana.

[0021] Figure 5 For inoculation with Rhodeobacter Rhodanobacter sp.) K28 and Arabidopsis thaliana not inoculated with R. Rhodanobacter sp.) K28 Arabidopsis thaliana growth comparison. DETAILED DESCRIPTION

[0022] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

[0023] In the study of drought stress induction on Arabidopsis thaliana, the present invention firstly takes non-drought induced Arabidopsis rhizosphere soil and uses high-throughput bacterial library construction technology to construct an Arabidopsis rhizosphere bacterial library. Then, the drought-induced and non-induced Arabidopsis rhizosphere bacterial communities are subjected to bioinformatics analysis and back-inoculation verification, and a strain of Rhodobacter roxburghii ( Rhodanobacter sp.) K28 drought-resistant bacteria promoted biomass and trait indicators in Arabidopsis thaliana.

[0024] Example 1 The bacteria in this example are Rhodeobacter spp. Rhodanobacter sp.) K28, deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, the deposit date is January 29, 2024, and the deposit number is CCTCC NO: M2024252.

[0025] In this example, Rhodeobacter Rhodanobacter sp.) The method for obtaining K28 is: Constructing a bacterial library: Select Arabidopsis thaliana that has grown normally for five weeks, take fresh rhizosphere soil, and -3 , 10 -4 , 10 -5 The cells were diluted with 10% TSB and then placed in a 96-well cell culture plate for incubation at room temperature (25°C) for 2 weeks. -5 The subsequent bacterial library was constructed using 96-well cell culture plates.

[0026] First, take 10 µL of the bacterial solution in the 96-well cell culture plate, add 16.6 µL of alkaline lysis buffer (25 mM NaOH and 0.2 mM Na2-EDTA (pH = 12)) and lyse at 95°C for 30 min, add 16.6 µL of neutralization buffer (40 mM Tris-HCl (pH = 7.5)) and mix well to obtain the DNA of the bacterial solution, and store it at -20°C.

[0027] The first round of PCR amplification was performed on the DNA of the preserved bacterial solution: bacterial universal amplification primers 799F and 1193R were selected, the nucleotide sequence of 799F was: AACMGGATTAGATACCCKG, recorded as SEQ ID NO.1, and the nucleotide sequence of 1193R was: ACGTCATCCCCACCTTCC, recorded as SEQ ID NO.2, wherein, in the nucleotide sequence of 779F, M was base A or base C, and K was base G or base T. In the present invention, M was base A, and K was base G.

[0028] The amplification system was: 3 µL template, 3 µL 10× Buffer, 2.4 µL dNTPs (2.5 mM each), 0.3 µL 799F (10 µM), 0.3 µL 1193R (10 µM), 0.15 µL HS taq (5U / µL). The amplification program was: 95°C 5 min pre-denaturation; 95°C 10 s, 55°C 15 s, 72°C 1 min for 25 cycles; finally 72°C 5 min; stored at 4°C.

[0029] In the second round of PCR, the template is the product of the first round of PCR, which is diluted 40 times. The primers used are 799F and 1193R primers with labels (Barcodes) and adapters required for sequencing. The specific sequences are as follows: the author is Jingying Zhang et al., Nature Protocols olume 16, pages 988-1012 (2021), and the name is High-throughput cultivation and identification of bacteria from the plant root microbiota. The amplification system and amplification procedure are the same as the first round of PCR. After the two rounds of PCR amplification, the obtained DNA samples are subjected to agarose gel electrophoresis, and then gel recovery is performed according to the instructions of the kit. Finally, the concentration of the gel recovery samples is detected by Nanodrop, and qualified samples are sequenced on the Illumina machine.

[0030] Bioinformatics analysis and identification of isolated and cultured bacteria: According to Professor Bai Yang's method of "High-throughput isolation, cultivation and identification of plant root bacteria", data analysis was performed on the network server http: / / bailab.genetics.ac.cn / culturome.

[0031] Isolation and purification: Isolate and purify the bacteria identified by bioinformatics analysis. Select the identified bacterial wells from the 96-well cell culture plate, draw 10 µL of the bacterial solution and streak it on a 50% TSB plate, with three groups of parallels per well. Cultivate the strain at 28°C for 3 days and purify it again, repeat twice until pure colonies are obtained. The selected pure strains are cultured in 10% TSB liquid medium, 28°C, 180 rpm for 3 days for 16S sequencing confirmation.

[0032] TSB liquid culture medium formula: trypticase 17.0 g / L, sodium chloride 5.0 g / L, soybean papain hydrolysate 3.0 g / L, dipotassium hydrogen phosphate 2.5 g / L, glucose 2.5 g / L, ultrapure water added to 1L.

[0033] TSB plate formula: trypticase 17.0 g / L, sodium chloride 5.0 g / L, soybean papain hydrolysate 3.0 g / L, potassium dihydrogen phosphate 2.5 g / L, glucose 2.5 g / L, agar 1.5%.

[0034] Example 2 1. Identification of strain K28 The strain K28 was identified physiologically and biochemically according to the Manual of Common Bacterial Systematic Identification.

[0035] Strain K28 is a Gram-positive bacterium. The colony color is yellow and slightly transparent. The colony surface is smooth and moist, with neat edges. It can grow in TSB medium and grows faster. The morphology of strain K28 on a 50% TSB plate is shown in the figure below. Figure 1 shown.

[0036] 2. Molecular identification of strain K28 DNA of strain K28 was extracted using alkaline lysis solution and buffer, and the V5-V7 region of 16SrRNA gene was selected for detection. The primers for detection were 799F sequence shown in SEQ ID NO.1 and 1193R sequence shown in SEQ ID NO.2. The reaction system was: 3 μL template, 3 μL 10×Buffer, 2.4 μL dNTPs (2.5 mM each), 0.3 μL 799F (10 μM), 0.3 μL 1193R (10 μM), 0.15 μL HS taq (5U / μL). The amplification program was: pre-denaturation at 95℃ for 5 min; 25 cycles of 95℃ for 10 s, 55℃ for 15 s, and 72℃ for 1 min; finally 72℃ for 5 min; and stored at 4℃.

[0037] After PCR amplification, the product was tested by 1.5% agarose gel electrophoresis, and there were obvious characteristic bands. The PCR amplification product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the obtained sequence was uploaded to NCBI. The homology was compared with the sequence in the gene library by Blast analysis, and the phylogenetic tree was constructed by Neihbor Joinhing in MEGA5.1 software, as shown in Figure 2 shown.

[0038] strain K28 and Rhodeobacter roebuckii ( Rhodanobacter sp.) has the highest homology, reaching 99%. Combined with the identification results of bacterial morphology, growth conditions, etc., it is determined that the strain K28 of the present invention is Rhodeobacter ( Rhodanobacter sp.) and submitted the strain K28 sequence to the GenBank database, obtaining the gene accession number CP157948.1.

[0039]

[0040] Example 3 Rhodeobacter Rhodanobacter sp.)K28 bacterial solution preparation and irrigation application Rhodeobacter Rhodanobacter sp.) K28 can grow well in 10% TSB liquid medium. It reproduces rapidly in the medium. Figure 3 As shown. Rhodanobacter sp.) K28 can grow well in 10% TSB liquid medium.

[0041] Pretreatment of bacterial solution: Pick a single colony of the isolated and purified strain K28 and inoculate it into 50 mL of 10% TSB liquid culture medium. Cultivate it at 28°C and 180 rpm for 48 h, and measure the OD600 of the strain (the minimum value of 0.2 is used to judge the growth status of the strain).

[0042] Transfer the cultured bacterial solution into a sterile 50 mL centrifuge tube, centrifuge at 5000 rpm, add sterile water to reselect the bacterial solution and centrifuge again, repeat the washing twice to remove the culture medium. Then add sterile water to adjust the bacterial solution OD to about 0.5. Then add sterile water at a volume ratio of 1:100 (bacterial solution: sterile water) for dilution. The diluted bacterial solution is then diluted at a ratio of 1mL:1g (each gram of soil contains about 10 6 CFU) was added to sterilized Arabidopsis planting soil (peat soil: vermiculite: perlite volume ratio of 1:1:1), the planting soil with bacterial solution was stirred evenly and left for 8 hours to make it homogenous. Finally, it was placed in a small planting pot (60±5g / pot).

[0043] After two weeks of Arabidopsis seedling cultivation (2 days of vernalization at 4°C after seed disinfection; 12 days of seedling cultivation at 22°C, 16 h light, 8 h dark, relative humidity 65%), the seedlings were transplanted into the homogenized planting soil with bacterial solution added. Twelve seedlings of the same treatment were placed in the same bottom pot and 1 L of Huabao No. 2 nutrient solution was added. The seedling pot was covered with plastic wrap. After one day, a hole was pierced in the plastic wrap with a bamboo stick. After two days, a corner of the plastic wrap was lifted. After three days, the plastic wrap was lifted and continued to be cultivated in the indoor greenhouse (25°C, 16 h light, 8 h dark) for 10 days, that is, normal watering (sterile water) was applied during the whole process.

[0044] Example 4 Applying Rhodeobacter Rhodanobacter sp.) K28 on the growth-promoting and drought-resistance effects of Arabidopsis thaliana The Arabidopsis thaliana was treated by the K28 bacterial solution watering method in Example 3, and sterilized water was used as a control (blank control). Calculated from the beginning of transplanting, each bottom pot was watered to saturation at the end of the 10th day, and the drought treatment group (blank drought) stopped watering, and continued to be cultivated in the indoor greenhouse (25°C, 16 h light, 8 h dark). At the same time, the control was watered normally until about 14 days (any group of drought treatment showed wilting or withering of leaves). The blank control was watered with Schwannella ( Rhodanobacter sp.) K28, and obtain K28 control, and pour Schroederella sp. ( Rhodanobacter sp.) K28, obtained K28 drought, observed Rhodobacterium ( Rhodanobacter sp.) K28 on growth promotion and drought resistance of Arabidopsis thaliana.

[0045] Weighing of dry weight: insert scissors into the root of the Arabidopsis plant, cut the root system and pull it out, take out the above-ground part of the plant, trim the root system and remove the residual soil and other debris on the plant, then put the plant in an oven at 68°C to dry for 48 hours. When it can be crushed into powder by hand, weigh the dry weight.

[0046] Rhodeobacter Rhodanobacter The growth promotion and drought resistance of sp. K28 Figure 4 , Figure 5 shown. Figure 4 , Figure 5 The results showed that Rhodopsin ( Rhodanobacter sp.) K28 can assist Arabidopsis in drought-resistant growth, which is manifested by increased dry weight, increased leaf number, and increased leaf area.

[0047] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0048] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0049] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A drought-resistant growth-promoting bacterium, characterized in that: The drought-resistant growth-promoting bacteria are Rhodopsin ( Rhodanobacter sp.) K28, deposited in China Center for Type Culture Collection, the deposit date is January 29, 2024, and the deposit number is CCTCC NO: M2024252.

2. A bacterial agent, characterized in that Including the drought-resistant growth-promoting bacteria described in claim 1.

3. The bacterial agent according to claim 2, characterized in that TSB medium is also included.

4. Use of the drought-resistant and growth-promoting bacteria according to claim 1 or the bacterial agent according to claim 2 in plant drought resistance and growth promotion.

5. The use according to claim 4, characterized in that: The drought-resistant growth-promoting bacteria are diluted with sterile water, and the concentration of the drought-resistant growth-promoting bacteria is greater than or equal to 10 6 CFU / mL.

6. The use according to claim 4, characterized in that: The plant is Arabidopsis thaliana.

7. A method for resisting drought and promoting growth of plants, characterized in that: The drought-resistant and growth-promoting bacteria according to claim 1 or the bacterial agent according to claim 2 is applied to the seeds of the plant, or applied to the planting matrix of the plant.

8. The method according to claim 7, characterized in that The drought-resistant growth-promoting bacteria are diluted with sterile water, and the concentration of the drought-resistant growth-promoting bacteria is greater than or equal to 10 6 CFU / mL.

9. The method according to claim 7, characterized in that: The plant is Arabidopsis thaliana, soybean or rice.

10. The method according to claim 7, characterized in that The growth promotion refers to an increase in biomass, an increase in the number of leaves and / or an increase in the leaf area.

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