Agrobacterium cavarae RZ10 and its applications

Abstract

Agrobacterium cavarae RZ10 and its applications belong to the field of microbial technology. On the one hand, the present invention provides an Agrobacterium cavarae RZ10 with a deposit number of CCTCC No: M 20221414. On the other hand, the present invention provides the uses of the Agrobacterium cavarae RZ10. The strain provided by the present invention has inhibitory effects on Magnaporthe oryzae and Fusarium moniliforme of rice; can secrete indole acetic acid; and has characteristics such as promoting plant growth, alkali tolerance, and drought tolerance, providing a stable, efficient, and intergenerational microbial resource for promoting green and sustainable agricultural development.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to Agrobacterium cavarae RZ10 and its application. Background Art

[0002] Rice is the world's major crop and the staple food for nearly half of the world's population, and its yield plays a central role in maintaining the global food supply. Worldwide, rice is mainly affected by three diseases: rice blast, sheath blight, and bakanae disease. Among them, rice blast is the most serious of the three diseases. Its occurrence can lead to a 15% - 20% reduction in rice yield, and in severe cases, it can cause local fields to have no harvest. Rice blast is a fungal disease caused by the rice blast fungus (Magnaporthe oryzae). Its transmission principle is mainly through the spread of spores by air currents. When the number of spores on rice plants reaches a certain level, the rice plants will be infected with rice blast. It mainly damages the leaves, stems, and panicles of rice. According to the different damage periods and parts, rice blast can be divided into seedling blast, leaf blast, node blast, neck blast, grain blast, and collar blast.

[0003] Currently, the control measures for rice blast mainly include chemical pesticides and resistance breeding. However, the long-term use of chemical pesticides seriously pollutes the environment and harms human health. Resistance breeding relying on a single resistance gene of rice also has limitations in application. The high mutation rate of the rice blast fungus can enable it to produce resistant physiological races in a relatively short time, thus threatening resistant rice varieties. Therefore, finding more environmentally friendly and effective methods for controlling rice blast has attracted global attention. In recent years, plant endophytes have been favored by researchers due to their advantages such as low toxicity, no environmental pollution, and no ability to make the rice blast fungus produce resistance. It is a sustainable and environmentally friendly biological control measure.

[0004] Biological control refers to a method of effectively controlling plant diseases by using microorganisms in the environment or their metabolites. Its essence is to utilize the mutual relationship between biological species to inhibit the growth of one or a class of organisms with another or another class of organisms. There are many types of biocontrol bacteria. Currently, the biocontrol bacteria widely applied in production mainly include fungi, bacteria, actinomycetes, and viruses, etc. Fungi mainly include Trichoderma and yeast, bacteria mainly include Bacillus and Pseudomonas, and actinomycetes mainly include Streptomyces. For example, Hongying Shan et al. isolated a methylotrophic Bacillus subtilis from the soil of a virgin forest that could significantly inhibit the mycelial growth and spore germination of the rice blast fungus. The greenhouse experiment results showed that it also had a good control effect on rice blast.

[0005] As microbial flora living inside plants, endophytes have formed a harmonious and friendly symbiotic relationship with host plants during the long-term evolution process. The host plant can provide living space and nutrients for endophytes, and endophytes can promote plant growth and improve plant disease resistance. Therefore, this new type of biological control factor, endophytes, has gradually become a hot topic in biological control. Currently, there are few studies on screening rice endophytes to control rice blast. Therefore, isolating and screening endophytes with inhibitory effects on Magnaporthe oryzae from rice seeds is of great significance for finding new resources and strategies for the biological control of rice blast. Summary of the Invention

[0006] Aiming at the problems existing in the prior art, the purpose of the present invention is to design and provide a technical solution for Agrobacterium cavarae RZ10 and its application.

[0007] The present invention is specifically realized through the following technical solutions:

[0008] The present invention provides strain RZ10, which is isolated from the seeds of Yangdao 6. This strain is preserved in the China Center for Type Culture Collection, with the preservation number CCTCC No: M 20221414, the preservation date being September 13, 2022, and the preservation address being Wuhan University, Hubei Province, Postcode: 430072. Its systematic classification is Agrobacterium cavarae.

[0009] The present invention provides a microbial inoculant containing the above-mentioned Agrobacterium cavarae RZ10.

[0010] Furthermore, the application of this inoculant as an agent for controlling Magnaporthe oryzae in rice is provided.

[0011] Furthermore, the application of this inoculant as an agent for controlling Fusarium moniliforme in rice is provided.

[0012] Furthermore, the application of this inoculant as an IAA-producing inoculant is provided.

[0013] Furthermore, the application of this inoculant as a stress-resistant inoculant is provided, and the stress resistance includes acid and alkali tolerance and drought tolerance.

[0014] Furthermore, the application of this inoculant as an inoculant for promoting plant growth is provided.

[0015] Furthermore, the application of this inoculant as an ecological organic fertilizer is provided.

[0016] The strain provided by the present invention is isolated from the seeds of the rice variety Yangdao 6 with high nitrogen utilization efficiency and belongs to endophytic bacteria in seeds. Endophytic bacteria in seeds live inside seeds for a long time, and their unique living environment endows endophytic microorganisms with specific biological functions, such as: direct or indirect growth promotion, strong stress resistance, and can be transmitted between consecutive plant generations through vertical transmission, etc. The strain of the present invention has an inhibitory effect on Magnaporthe oryzae and Fusarium moniliforme; it can secrete indole acetic acid, has characteristics such as promoting plant growth, alkali tolerance, and drought tolerance, is a biosafe strain, can effectively reduce the use of pesticides, and is of great significance for promoting green and sustainable agriculture. Description of the Drawings

[0017] Figure 1 Phylogenetic tree of RZ10 and type strains of different species in the same genus;

[0018] Figure 2 Inhibitory effect of RZ10 strain on Magnaporthe oryzae Guy11;

[0019] Figure 3 Inhibitory effect of RZ10 strain on Fusarium moniliforme EM-48;

[0020] Figure 4 Qualitative test results of indole acetic acid production by RZ10 strain;

[0021] Figure 5 Acid and alkali tolerance test results of RZ10 strain;

[0022] Figure 6 Drought tolerance test results of RZ10 strain. Detailed Description of the Invention

[0023] The present invention will be further described in detail below with specific examples, which are explanations of the present invention rather than limitations.

[0024] Example 1: Isolation and Identification of Agrobacterium cavarae RZ10

[0025] (1) Culture Medium

[0026] LB medium (1L): Tryptone 10g, Yeast Extract 5g, NaCl 10g, Agar 15g.

[0027] TSA medium (1L): Trypticase Peptone 5g, NaCl 5g, Soybean Peptone 5g, Agar 15g.

[0028] PDA medium (1LA): Potato 200g, Glucose 20g, Agar 15g.

[0029] (2) Pathogenic Bacteria

[0030] The tested pathogenic fungi, Magnaporthe oryzae Guy11 and Fusarium fujikuroi EM-48, were kindly provided by the laboratory of Huang Shiwen at the China National Rice Research Institute.

[0031] (3) Isolation and purification of strains

[0032] Pick 0.5 g of uniform and intact rice seeds and wash them twice with sterile deionized water; then wash them with 75% ethanol for 2 minutes in sequence; the seed surface disinfectant (a mixture of 20% sodium hypochlorite, 3% sodium chloride, 0.1% sodium carbonate, and 0.15% sodium hydroxide) was shaken on a shaker (150 rpm) for 12 minutes; wash the seeds 7 times with physiological saline to clean the residual disinfectant on the seed surface. The surface-sterilized seeds were ground in a mortar containing 2 mL of sterile 10 mM MgCl2 solution, and 100 μL of the solution was taken for serial dilution to 10 -3 10 -4 10 -5 and then spread on LB medium plates. Each dilution was repeated 2 times and incubated at 28 °C in an incubator for 3 days. After the colonies grew, pick colonies with different morphologies and streak them for purification on fresh LB medium plates. Then pick single colonies onto solid LB slant medium for subsequent confrontation tests. At the same time, pick single colonies and culture them in LB liquid medium for 1 - 2 days. Mix the bacterial solution with an equal volume of 40% sterilized glycerol and store it in a -80 °C refrigerator for long-term preservation. Take 100 μL of the last washing solution and spread it on TSA medium for culturing to detect the thoroughness of seed surface disinfection.

[0033] (3) 16S rDNA sequence amplification and identification of strain taxonomic status

[0034] The DNA of the isolated and purified strain was extracted with reference to the instruction manual of the FastDNA Spin Kit kit from Tiangen Biotech (Beijing) Co., Ltd. Using the DNA as a template, PCR amplification was performed with primers for the 16S rDNA sequence. The forward primer was: 5’-AGAGTTTGATCATGGCTCAG-3’ (27F), and the reverse primer was: 3’-CGCTTACCTTGTTACGACTT-5’ (1492R). The PCR amplification conditions were as follows: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 1 min; annealing at 53 °C for 1 min; extension at 72 °C for 90 s; final extension at 72 °C for 5 min, for a total of 35 cycles. The PCR product was separated by 1% agarose gel electrophoresis to obtain a band of about 1.5 kb. After recovering and purifying this band, it was sent to Tsingke Biotechnology Co., Ltd. for sequencing. The obtained sequence was 1379 bp (as shown in SEQ ID NO.1). The sequencing results were submitted to the EzBioCloud website for homology analysis. The results showed that the 16S rDNA sequence of this strain was most similar to the 16S rDNA sequence of Agrobacterium cavarae RZME10, with a homology of 99.20%. And the phylogenetic tree was constructed using MEGA7.0 software ( Figure 1 ). The strain RZ10 had the closest evolutionary distance to Agrobacterium cavarae RZME10, reflecting the closest genetic relationship between them. Therefore, the strain RZ10 was determined to be Agrobacterium cavarae.

[0035] Subsequently, this strain was deposited in the China Center for Type Culture Collection, with the deposit number CCTCC No: M20221414, the deposit date of September 13, 2022, and the deposit address of Wuhan University, Hubei Province, postal code: 430072. Its systematic classification is Agrobacterium cavarae.

[0036] Example 2: Inhibitory effect of Agrobacterium cavarae RZ10 on Magnaporthe oryzae

[0037] (1) Activation of Magnaporthe oryzae and Agrobacterium cavarae

[0038] RZ10 was picked from a -80 °C glycerol tube and streaked onto an LB culture dish to activate the strain. After the strain grew into single colonies, single colonies were picked and inoculated into an LB liquid medium, and cultured with shaking at 28 °C. The test pathogenic fungus Magnaporthe oryzae Guy11 was activated on a PDA solid medium, and then punched into discs along the hyphal edge with a puncher for later use.

[0039] (2) Confrontation culture

[0040] Take the well-activated Magnaporthe oryzae fungal cake, place it in the center of a PDA plate with the mycelium side facing downwards. Then, respectively pipette 3 μL of the RZ10 bacterial solution cultured to the logarithmic growth phase and spot it at an equal distance (1.5 cm) to the left and right of the Magnaporthe oryzae. Inoculate 3 plates as replicates. At the same time, use the plate inoculated only with Magnaporthe oryzae without inoculating RZ10 as a control. Conduct confrontation culture at 28 °C and observe and calculate the relative inhibition rate on the 5th and 7th days respectively.

[0041] Relative inhibition rate (%) = (radius of the control colony - radius of the treated colony) / radius of the control colony

[0042] The antibacterial situation is shown in Figure 2 , on the 5th day, the growth radii of Magnaporthe oryzae antagonized by the RZ10 strain were 0.8 cm, 0.7 cm, and 0.6 cm respectively, and the growth radii of the control Magnaporthe oryzae were 1.7 cm, 1.6 cm, and 1.5 cm respectively. After taking the average value of the three replicates and calculating according to the above relative inhibition rate (%) formula, the relative inhibition of the RZ10 strain on Magnaporthe oryzae was 56.4%.

[0043] Example 3: Inhibitory effect of Agrobacterium cavarae RZ10 on Fusarium moniliforme

[0044] (1) Activation of Fusarium moniliforme and Agrobacterium cavarae

[0045] Pick RZ10 from the -80 °C glycerol tube and streak it onto an LB culture dish to activate the strain. After the strain grows into single colonies, pick the single colonies and inoculate them into an LB liquid medium, and culture them with shaking at 28 °C. The test pathogenic fungus Fusarium moniliforme (Fusarium fujikuroi EM-48) is activated on a PDA solid medium, and then a fungal cake is punched out along the mycelium edge with a puncher for later use.

[0046] (2) Confrontation culture

[0047] Take the well-activated Fusarium moniliforme fungal cake, place it in the center of a PDA plate with the mycelium side facing downwards. Then, respectively pipette 3 μL of the RZ10 bacterial solution cultured to the logarithmic growth phase and spot it at an equal distance (1.5 cm) to the left and right of the Fusarium moniliforme. Inoculate 3 plates as replicates. At the same time, use the plate inoculated only with Fusarium moniliforme without inoculating RZ10 as a control. Conduct confrontation culture at 28 °C and observe and calculate the relative inhibition rate on the 5th and 7th days respectively.

[0048] Relative inhibition rate (%) = (radius of the control colony - radius of the treated colony) / radius of the control colony. The antibacterial situation is shown in Figure 3, on the fifth day, the growth radii of the bakanae disease bacteria antagonized by the RZ10 strain were 0.8 cm, 0.6 cm, and 0.9 cm respectively, and the growth radii of the control bakanae disease bacteria were 1.8 cm, 2.1 cm, and 2.5 cm respectively. After taking the average value of three repetitions, the relative inhibition of the RZ10 strain against the bakanae disease bacteria of rice was calculated to be 63.7% according to the above relative inhibition rate (%) formula.

[0049] Example 4: Growth promotion effect of Agrobacterium cavarae RZ10

[0050] (1) Qualitative analysis of the plant hormone indole-3-acetic acid (IAA)

[0051] KingB medium: 0.5 g of tryptophan, 20 g of peptone, 1.15 g of K2HPO4, 1.5 g of MgSO4·7H2O, 1.5% glycerol, sterilized at 112 °C for 20 min, and tryptophan was filter-sterilized.

[0052] Salkowski colorimetric reagent: Dissolve 4.5 g of FeCl3 in 300 mL of distilled water, then slowly add 587.4 mL of 98% H2SO4, and make up the volume to 1 L after cooling. The detection range of IAA is 5 - 200 mg / L.

[0053] Inoculate the activated RZ10 strain into KingB liquid medium and culture for 4 days. Pipette 2 mL of the bacterial liquid into a 5 mL centrifuge tube, then add 2 mL of Salkowski colorimetric reagent, mix well, and react in the dark for 30 min. The strain whose liquid turns red or pink can produce IAA. Use 2 mL of KingB medium plus 2 mL of Salkowski reagent as the negative control, and use Bacillus as the positive control. The results showed that the RZ10 strain could produce the plant hormone indole-3-acetic acid ( Figure 4 ).

[0054] (2) Quantitative detection of the plant hormone indole-3-acetic acid (IAA)

[0055] Drawing of the indole-3-acetic acid standard curve: Weigh 5 mg of indole-3-acetic acid finished product, dissolve it with a small amount of ethanol, and then make up the volume to 50 mL with distilled water to prepare a stock solution of 100 μg / mL. Then use the stock solution to prepare a series of concentration standard solutions of 0, 0.5, 1.0, 5.0, 10, 15, 20, 25 μg / mL. Pipette 2 mL of each standard solution into 8 test tubes respectively, then add an equal volume of Salkowski colorimetric reagent, mix well and react in the dark for 30 min. The blank is a mixture of 2 mL of distilled water and 2 mL of Salkowski reagent. Measure the absorbance of the reaction solution at 530 nm and draw the standard curve of indole-3-acetic acid (y = 0.0285352x + 0.0116077, r 2 = 0.9972).

[0056] Inoculate the activated RZ10 strain into 5 tubes of King B liquid medium and culture for 4 days. First, use spectrophotometry to measure the bacterial concentration of the bacterial solution at 600 nm. Then, centrifuge the bacterial suspension at 10,000 rpm for 10 min, take 2 mL of the supernatant, add an equal volume of Salkowski colorimetric solution, let it stand in the dark for 30 min, and measure its OD 530 absorbance value. Calculate the content of IAA in the bacterial solution per unit volume by referring to the standard curve. The results show that the average IAA concentration produced by the RZ10 strain per OD 600 is 19.541 μg / mL.

[0057] Example 5: Determination of stress resistance and safety of Agrobacterium cavarae RZ10

[0058] (1) Detection of acid and alkali resistance

[0059] Use LB medium and adjust the pH to 3, 4, 5, 6, 7, 8, 9, 10, and 11 respectively. Inoculate the RZ10 strain into the above media with different treatments, with 3 replicates for each treatment. Culture at 28 °C, observe, and record the acid and alkali resistance of the strain. The results show that Agrobacterium cavarae RZ10 grows normal single colonies after 24 h of culture at 28 °C under the conditions of pH 7, 8, and 9. Under the conditions of pH 10 and 11, the growth rate slows down, and it takes 2 days to grow single colonies ( Figure 5 ).

[0060] (2) Detection of drought tolerance

[0061] For the drought tolerance medium, use polyethylene glycol (PEG 6000) to adjust the water potential and identify drought-tolerant strains under artificially simulated drought conditions. A total of 4 treatments are set, and the PEG 6000 contents are: 0 (CK), 10% (mild drought), 20% (moderate drought), and 30% (severe drought). The corresponding water potentials are: 0, -0.185, -0.559, and -1.122 MPa. Inoculate the RZ10 strain into the above media with different treatments, with 3 replicates for each treatment. Culture at 28 °C, observe, and record the drought tolerance of the strain. The results are as Figure 6 shown. After 48 h of culture, the strain RZ10 can grow under the drought conditions of 10% (mild drought) and 20% PEG 6000 (moderate drought).

[0062] (3) Safety detection

[0063] Blood agar medium: 18 g of peptone, 1 g of yeast powder, 5 g of NaCl, 15 g - 20 g of agar, 1000 mL of deionized water, pH 6.8 - 7.2. After autoclaving at 121 °C for 30 min, wait for the medium to cool to 50 °C, add 5% (5 ml / 100 ml) defibrinated sheep blood, mix well, and pour into plates.

[0064] The RZ10 strain was inoculated into blood agar medium and cultured at 28 °C for 7 days to observe the presence of a hemolytic zone. The results showed that no hemolytic zone appeared, indicating that this strain is a safe strain and can be used as a microbial inoculant.

[0065] Example 6: Promoting effect of Agrobacterium cavarae RZ10 on rice seed germination

[0066] (1) Fermentation of RZ10 inoculant

[0067] The RZ10 strain was activated on LB medium. A single colony was picked and transferred to LB liquid medium for shaking culture. When the bacterial solution reached the logarithmic growth phase, the bacterial concentration was adjusted to 1×10 10-11 CFU / mL as the mother liquor. Before use, the mother liquor of the inoculant was diluted three-fold with sterile normal saline to obtain the bacterial solution, and then trace element solution (1 μL of trace element solution was added to every 3 mL of bacterial solution) was added dropwise to the bacterial solution and mixed evenly to obtain the liquid inoculant. Among them, the contents of each component of the trace element solution were as follows: H3BO3 2.86 g / L; MnSO4 1.81 g / L; CuSO4·5H2O 0.80 g / L; ZnSO4 0.22 g / L; H2MoO4 0.02 g / L. After the above components were dissolved in appropriate water in sequence, deionized water was added to make up to 1 L.

[0068] (2) Rice germination experiment

[0069] Plump and uniform rice seeds were picked, added with the above inoculant, and placed in an incubator for dark seed soaking for 2 days (30 °C, 70% humidity). The control was treated with sterile normal saline, and the bacterial solution and sterile normal saline were replaced every day to avoid contamination. Then the seeds were transferred to a petri dish containing moist filter paper for germination. 50 seeds were placed in each petri dish, and each treatment was repeated 5 times. The petri dishes were placed in the dark at 28 °C for culture. After 3 days and 7 days of culture, the lengths of the buds and roots, germination rate, and germination index of the seeds in each treatment were measured respectively.

[0070] Germination rate (%) = total number of germinated seeds / total number of tested seeds × 100%

[0071] Germination index = Σ (number of germinated seeds within T time / corresponding germination days T)

[0072] The results are shown in Table 1. After 3 days of inoculation with the RZ10 strain, compared with the control, the average germination rate of rice seeds treated with the inoculated strain increased by 6.41%, the average root length increased by 10.29%, and the average bud length increased by 24.83%; after 7 days of germination, compared with the control, the average root length of rice inoculated with the RZ10 strain increased by 9.10%, the average bud length increased by 11.05%; the germination index increased by 3.40%.

[0073] Table 1. Promoting effect of RZ10 strain on rice seed germination

[0074]

Claims

1. An Agrobacterium cavarae ( Agrobacterium cavarae ) RZ10, characterized in that It was deposited at the China Center for Type Culture Collection on September 13, 2022, with the deposit number CCTCC No: M 20221414.

2. A microbial inoculum containing Agrobacterium cavarae RZ10 as described in claim 1.

3. Use of the inoculum as described in claim 2 as an agent for controlling Magnaporthe oryzae in rice.

4. Use of the inoculum as described in claim 2 as an agent for controlling Fusarium fujikuroi in rice.

5. Use of the inoculum as described in claim 2 in the production of the plant hormone indole-3-acetic acid.

6. Use of the inoculum as described in claim 2 as an inoculum for improving the stress resistance of rice, where the stress resistance is acid and alkali tolerance and drought tolerance.

7. Use of the inoculum as described in claim 2 as an inoculum for promoting rice seed germination.

8. Use of the inoculum as described in claim 2 as a rice ecological organic fertilizer for controlling Magnaporthe oryzae and Fusarium fujikuroi in rice.

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