Bordetella glathei rs-36 and uses thereof

Abstract

The application discloses a Burkholderia glumae RS-36 and application thereof, and belongs to the technical field of microorganisms. Burkholderia gladioli The application provides a Burkholderia glumae (B. glumae) RS-36 with a preservation number of CCTCC No: M 20221584, and application of the B. glumae RS-36. Burkholderia gladioli The B. glumae RS-36 provided by the application has inhibiting effects on Magnaporthe oryzae, Ustilaginoidea virens, Exserohilum turcicum and Pyricularia grisea, can secrete indole acetic acid for promoting plant growth, can keep good activity in an acidic environment with a pH of 5 and a drought environment, has strong stress resistance, and has important significance for promoting green and sustainable development agriculture.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to Burkholderia gladioli RS-36 and its applications. Background Technology

[0002] Rice is one of my country's important food crops. However, the continuous occurrence of various rice diseases has led to a reduction in rice yield. Conservative estimates suggest that diseases cause at least 1-5% losses in grain production annually. Among these, rice blast, rice false smut, and bakanae disease are the most common and serious fungal diseases. Rice blast is the most serious disease of rice and a major challenge for rice production worldwide. It occurs in both northern and southern rice-growing areas of my country, causing severe damage. It generally causes 10-20% yield loss, and in severe cases, yield reduction can reach 40-50%, or even total crop failure. The rice blast fungus can infect almost all above-ground parts of the rice plant, causing seedling blast, leaf blast, node blast, neck blast, branch blast, and grain blast. Bakanae disease, also known as excessive growth disease, is caused by Fusarium moniliforme (…). Fusarium moniliforme Bakanae disease, a global rice disease caused by infection, is primarily spread through seed-borne transmission, and secondarily through soil and infected plants. Field symptoms in infected rice plants mainly include: abnormal elongation of stems or internodes, adventitious root growth at stem nodes, elongated and thin plants, pale leaves, increased leaf angles, reduced tillering, smaller panicles, decreased grain filling rate, and even death. In recent years, rice false smut has become increasingly severe, replacing bacterial blight as one of the three major new rice diseases. Rice false smut mainly infects some spikelets in the rice panicle, forming false grains that can steal photosynthetic products, affecting the grain filling of other normal spikelets, leading to a decrease in thousand-grain weight and quality.

[0003] Currently, the main control measures for rice fungal diseases include screening for resistant varieties and seed soaking with chemical agents, with chemical pesticides remaining the preferred choice. The active ingredients in chemical pesticides include 20 types such as cyazofamid, prochloraz, and carbendazim, with approximately 85% of the active ingredients being prochloraz, carbendazim, or fludioxonil. Although chemical pesticides have significant short-term control effects on rice bakanae disease, long-term use can easily lead to drug resistance in pathogens, posing a threat to the environment and human and animal health. Therefore, finding more environmentally friendly and effective methods for controlling rice fungal diseases is particularly important. In recent years, screening for microorganisms with antagonistic effects against pathogens has become the primary biological control measure due to its advantages such as low toxicity, sustainability, and lack of resistance to bakanae disease pathogens.

[0004] Biological control refers to the effective control of plant diseases using microorganisms in the environment or their metabolic products. Its essence lies in utilizing the interrelationships between biological species, with one type of organism inhibiting the growth of another. Screening for strains with high biological activity and a broad antibacterial spectrum is of great significance. Currently, numerous studies have shown that biocontrol bacteria have significant control effects against rice bakanae disease pathogens. For example, Li Yuyang et al. screened *Bacillus polymyxa* SH15 from the rice rhizosphere, which can affect the mycelial growth of *bakanae disease pathogens*. Tian Jieping applied a diluted fermentation solution of *Bacillus amyloliquefaciens* TF28 to treat seeds, achieving a field control efficacy of over 87% against bakanae disease.

[0005] Endophytic bacteria, as microbial communities living within plants, have developed a harmonious and friendly symbiotic relationship with their host plants over a long period of evolution. The host plant provides endophytic bacteria with living space and nutrients, while the endophytic bacteria promote plant growth and enhance disease resistance. Compared to rhizosphere microorganisms, plant endophytic bacteria are easier to colonize after inoculation and exhibit more durable and stable preventative effects. Therefore, isolating and screening endophytic bacteria from rice seeds that inhibit rice fungal pathogens is of great significance for finding new resources and strategies for the biological control of rice diseases. Summary of the Invention

[0006] To address the problems existing in the prior art, the purpose of this invention is to design and provide Burkholderia gladioli RS-36, which has a broad-spectrum antagonistic effect against rice fungal diseases (rice blast fungus, rice false smut fungus, bakanae fungus, and panicle rot fungus) and its applications.

[0007] This invention is specifically achieved through the following technical solutions:

[0008] The strain RS-36 provided by this invention was isolated from the seeds of Yangdao 6 (Yangdao No. 6). This strain is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC No.: M 20221584, deposited on October 17, 2022, at Wuhan University, Hubei Province, 430072, China. Its systematic classification is *Burkholderia gladioli* (…). Burkholderia gladioli ).

[0009] This invention provides a bacterium containing the above-mentioned Gladiolus burkerii ( Burkholderia gladioli RS-36 microbial inoculant.

[0010] Furthermore, the application of this inoculant as a fungicide for controlling rice blast disease is provided.

[0011] Furthermore, the application of this inoculant as a fungicide for controlling rice false smut is provided.

[0012] Furthermore, the application of this inoculant as a fungicide for controlling rice bakanae disease is provided.

[0013] Furthermore, the application of this inoculant as a fungicide for controlling rice panicle rot is provided.

[0014] Furthermore, the application of this microbial agent as an IAA-producing agent is provided.

[0015] Furthermore, the application of this microbial agent as a stress-resistant agent is provided, the stress including acid resistance and drought resistance.

[0016] Furthermore, the application of this microbial agent as an ecological organic fertilizer is provided.

[0017] The Gladiolus burkerii RS-36 described in this invention has been identified as having multiple functions, mainly including: (1) antagonism against rice blast fungus, rice false smut fungus, bakanae disease fungus and panicle rot fungus, with inhibition rates of 80.9%, 74.3%, 58.3% and 70.3% respectively; (2) growth promotion function: secretes IAA, with a synthesis amount of 13.56 ug / mL, which promotes rice seed germination; (3) stress resistance: can tolerate mild and moderate drought and acidity (can grow normally under pH 5 conditions).

[0018] This invention provides a method for inhibiting the effects of Burkholderia gladioli RS-36 inoculum on rice blast fungus, rice false smut fungus, bakanae disease fungus, and panicle rot fungus. The inhibition rates of RS-36 against these pathogens reach 80.9%, 74.3%, 58.3%, and 70.3%, respectively. Furthermore, the RS-36 strain secretes indoleacetic acid, which promotes plant growth, and maintains good activity in both acidic environments (pH 5) and drought conditions, demonstrating strong stress resistance. Therefore, using the RS-36 strain of this invention as a microbial inoculum to control common fungal diseases of rice (rice blast, rice false smut, bakanae disease, and panicle rot) can effectively reduce pesticide use and is of great significance for promoting green and sustainable agricultural development. Attached Figure Description

[0019] Figure 1 Phylogenetic tree of RS-36 and different species of type strains of the same genus;

[0020] Figure 2 The inhibitory effect of RS-36 strain on Guy11, the rice blast fungus;

[0021] Figure 3 The inhibitory effect of strain RS-36 on rice false smut;

[0022] Figure 4 The inhibitory effect of strain RS-36 on rice bakanae disease pathogen;

[0023] Figure 5 The inhibitory effect of strain RS-36 on rice panicle rot pathogen;

[0024] Figure 6 Qualitative test results for indoleacetic acid production by strain RS-36;

[0025] Figure 7 The results of the acid and alkali resistance test for strain RS-36;

[0026] Figure 8 The results show the drought resistance test results for strain RS-36. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to specific examples, so as to better understand the technical solution.

[0028] Example 1: Isolation of Burkholderia gladioli RS-36

[0029] (1) Culture medium

[0030] LB medium (1L): 10g tryptone, 5g yeast extract, 10g NaCl, 15g agar.

[0031] TSA medium (1L): 5g tryptone, 5g NaCl, 5g soybean peptone, 15g agar.

[0032] PDA medium (1LA): 200g potato, 20g glucose, 15g agar.

[0033] (2) Pathogens

[0034] The tested pathogenic fungus, rice blast fungus ( Magnaporthe oryzae Guy11), rice seedling blight pathogen ( Fusarium fujikuroi EM-48), rice false smut ( Ustilaginoidea virens JZ11-28), rice panicle rot fungus ( Curvularia lunata WB05 was a gift from the Huang Shiwen Laboratory of the China National Rice Research Institute.

[0035] (3) Isolation and purification of strains

[0036] Select 0.5g of uniform and intact rice seeds and wash them twice with sterile deionized water. Then wash them sequentially with 75% ethanol for 2 minutes each. Place the seeds in a seed surface disinfectant solution (a mixture of 20% sodium hypochlorite, 3% sodium chloride, 0.1% sodium carbonate, and 0.15% sodium hydroxide) on a shaker (150 rpm) for 12 minutes. Wash the seeds 7 times with physiological saline to remove any remaining disinfectant. After surface disinfection, grind the seeds in a mortar with 2mL of sterile 10mM MgCl2 solution. Dilute the solution serially to 10⁻¹⁰ using 100μL of the diluted solution. -3 10 -410 -5 The culture was then spread onto LB agar plates, with each gradient repeated twice, and incubated upside down at 28°C for 3 days. After colonies grew, colonies of different morphologies were picked and purified by streak plating in three zones on fresh LB agar plates. Single colonies were then picked and spread onto solid LB slant agar for subsequent confrontation experiments. Simultaneously, single colonies were picked and cultured in LB liquid agar with shaking for 1-2 days. The bacterial suspension was then mixed with an equal volume of 40% sterile glycerol and stored at -80°C for long-term preservation. 100 μL of the final washing solution was spread onto TSA agar and incubated to assess the thoroughness of seed surface sterilization.

[0037] Example 2: 16S rDNA sequence amplification and strain taxonomic identification

[0038] DNA was extracted from the strain obtained in Example 1 according to the instructions of the FastDNA Spin Kit from Tiangen Company. Using the DNA as a template, PCR amplification was performed using primers with 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: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 1 min; 53℃ annealing for 1 min; 72℃ extension for 90 s; and 72℃ final extension for 5 min, for a total of 35 cycles. The PCR product was separated by 1% agarose gel electrophoresis, yielding a band of approximately 1.5 kb. This band was recovered, purified, and sent to Qingke Company for sequencing. The obtained sequence was 1401 bp (as shown in SEQ ID NO.1). The sequencing results were submitted to EzBioCloud for homology analysis. The results showed that the 16S rDNA sequence of this strain was similar to that of Burkholderia gladioli (…). Burkholderia gladioli The 16S rDNA sequence of NBRC 13700 is most similar, with a homology of 99.57%. A phylogenetic tree was constructed using MEGA 7.0 software. Figure 1 Strain RS-36 and Burkholderia gladioli ( Burkholderia gladioli NBRC 13700 is the closest in evolutionary distance, reflecting their closest phylogenetic relationship. Therefore, strain RS-36 is identified as Burkholderia gladioli.

[0039] Therefore, this strain was deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC No: M20221584, deposited on October 17, 2022, at Wuhan University, Hubei Province, 430072, China. Its systematic classification is *Burkholderia gladioli* (…). Burkholderia gladioli ).

[0040] Example 3: Inhibitory effect of Burkholderia gladioli RS-36 on rice pathogenic fungi

[0041] (1) Activation of rice pathogenic fungi and Burkholderia gladioli

[0042] RS-36 strain was streaked from a -80℃ glycerol tube into an LB agar dish to activate the strain. Once a single colony grew, it was picked and inoculated into LB liquid medium, then incubated with shaking at 28℃. Tested pathogenic fungus: *Pseudomonas aeruginosa* (rice blast fungus). M. oryzae Guy11), rice seedling blight pathogen ( F. fujikuroi EM-48), rice false smut ( U. virens JZ11-28), rice panicle rot fungus ( C. lunata After WB05 is activated on PDA solid medium, holes are punched along the edge of the hyphae to form a cake for later use.

[0043] (2) Confrontation Cultivation

[0044] Take a cake of activated rice pathogenic fungus and place it in the center of a PDA plate with the mycelial side facing down. Then, take 3 μL of RS-36 bacterial suspension cultured to the logarithmic growth phase and spot it at a distance (1.5 cm) from the pathogenic fungus on both sides. Inoculate 3 plates as replicates. Meanwhile, use plates inoculated only with the pathogenic fungus without RS-36 as controls. Incubate at 28℃ for confrontation. Observe and calculate the relative inhibition rate after 7 days.

[0045] Relative inhibition rate (%) = (Control colony radius - Treatment colony radius) / Control colony radius

[0046] (3) Results of relative inhibition rate

[0047] The inhibitory effect of Gladiolus burkerii RS-36 on rice blast fungus is shown in [reference needed]. Figure 2 On the seventh day, the growth radii of rice blast fungus antagonized by RS-36 strain were measured to be 0.3 cm, 0.5 cm, and 0.4 cm, respectively, while the growth radii of the control rice blast fungus were 2.3 cm, 2.6 cm, and 2.7 cm, respectively. After taking the average value of three replicates, the relative inhibition rate (%) of RS-36 strain against rice blast fungus was calculated to be 80.9%.

[0048] The inhibitory effect of Gladiolus burkerii RS-36 on rice false smut is shown in [reference needed]. Figure 3On the seventh day, the growth radii of rice false smut antagonized by strain RS-36 were measured to be 0.4 cm, 0.3 cm, and 0.5 cm, respectively, while the growth radii of the control rice false smut were 1.6 cm, 1.7 cm, and 1.5 cm, respectively. After taking the average value of three replicates, the relative inhibition rate (%) of strain RS-36 against rice false smut was calculated to be 74.3%.

[0049] The inhibitory effect of Gladiolus burkerii RS-36 on rice bakanae disease pathogens is shown in [reference needed]. Figure 4 On the seventh day, the growth radii of rice bakanae disease antagonized by RS-36 strain were measured to be 0.8 cm, 1.0 cm, and 0.8 cm, respectively, while the growth radii of the control rice bakanae disease were 1.8 cm, 2.1 cm, and 2.5 cm, respectively. After taking the average value of three replicates, the relative inhibition rate (%) of RS-36 strain against rice bakanae disease was calculated to be 58.3%.

[0050] The inhibitory effect of Gladiolus burkerii RS-36 on rice panicle rot pathogens is shown in [reference needed]. Figure 5 On the seventh day, the growth radii of rice panicle rot pathogen antagonized by strain RS-36 were measured to be 0.9 cm, 0.7 cm, and 0.8 cm, respectively, while the growth radii of the control rice panicle rot pathogen were 2.5 cm, 2.9 cm, and 2.9 cm, respectively. After taking the average value of three replicates, the relative inhibition rate (%) of strain RS-36 against rice panicle rot pathogen was calculated to be 70.3%.

[0051] Example 4: Growth-promoting effect of Burkholderia gladioli RS-36

[0052] (1) Qualitative analysis of the plant hormone indoleacetic acid (IAA)

[0053] King B medium: tryptophan 0.5g, peptone 20g, K2HPO4 1.15g, MgSO4 4. 7H2O 1.5g, glycerol 1.5%, sterilized at 112℃ for 20min, sterilized by tryptophan filtration.

[0054] Salkowski colorimetric reagent: Dissolve 4.5g FeCl3 in 300mL distilled water, then slowly add 587.4mL 98%H2SO4. After cooling, bring the volume to 1L. The IAA range is 5-200mg / L.

[0055] The activated RS-36 strain was inoculated into KingB liquid medium and cultured for 4 days. 2 mL of the bacterial culture was transferred to a 5 mL centrifuge tube, and 2 mL of Salkowski reagent was added. The mixture was then incubated in the dark for 30 minutes. The strain whose liquid turned red or pink was the one capable of producing IAA. 2 mL of KingB medium with 2 mL of Salkowski reagent was used as a negative control, and Bacillus was used as a positive control. The results showed that the RS-36 strain could produce the plant hormone indoleacetic acid (IAA). Figure 6 ).

[0056] (2) Quantitative detection of the plant hormone indoleacetic acid (IAA)

[0057] Construction of the indoleacetic acid standard curve: Weigh 5 mg of indoleacetic acid product, dissolve it in a small amount of ethanol, and then dilute to 50 mL with distilled water to prepare a 100 μg / mL stock solution. Prepare a series of standard solutions with concentrations of 0, 0.5, 1.0, 5.0, 10, 15, 20, and 25 μg / mL using the stock solution. Add 2 mL of each standard solution to 8 test tubes, then add an equal volume of Salkowski reagent, mix well, and react in the dark for 30 min. For the blank, mix 2 mL of distilled water and 2 mL of Salkowski reagent. Measure the absorbance of the reaction solution at 530 nm and plot the indoleacetic acid standard curve (y = 0.0413099x + 6.38504e). -004 , r 2 =0.997.4).

[0058] The activated RS-36 strain was inoculated into 5 tubes of KingB liquid medium and cultured for 4 days. First, the bacterial concentration was determined spectrophotometrically at 600 nm. Then, the bacterial suspension was centrifuged at 10,000 rpm for 10 min, and 2 mL of the supernatant was added to an equal volume of Salkowski colorimetric solution. After standing in the dark for 30 min, its OD was measured. 530 Absorbance. The IAA content per unit volume of bacterial culture was calculated using a standard curve. Results showed that strain RS-36 had an average absorbance value per OD0.05. 600 The IAA concentration produced was 13.56 ug / mL.

[0059] Example 5: Determination of the stress resistance and safety of Burkholderia gladioli RS-36

[0060] (1) Acid and alkali resistance test

[0061] Using LB medium, the pH was adjusted to 3, 4, 5, 6, 7, 8, 9, 10, and 11. The RS-36 strain was inoculated into the media with different treatments, with each treatment replicated three times. The strains were incubated at 28°C, and their acid and alkali tolerance was observed and recorded. The results showed that *Burkholderia gladioli* RS-36 grew single colonies normally after 24 hours of incubation at 28°C under pH conditions of 5, 6, and 7, but did not grow under other pH conditions. Figure 7 This indicates that the strain is acid-resistant.

[0062] (2) Drought resistance test

[0063] The drought-resistant culture medium was adjusted with polyethylene glycol (PEG 6000) to artificially simulate drought conditions for the identification of drought-resistant strains. Four treatments were set up with PEG 6000 contents of 0 (CK), 10% (mild drought), 20% (moderate drought), and 30% (severe drought), corresponding to water potentials of 0, -0.185, -0.559, and -1.122 MPa, respectively. RS-36 strains were inoculated into the media of the different treatments, with each treatment replicated three times. The strains were incubated at 28°C, and their drought resistance was observed and recorded. Results are as follows: Figure 7 As shown, the strain can tolerate 10% and 20% PEG 6000 (mild and moderate drought).

[0064] (3) Safety testing

[0065] Blood agar medium: 18g peptone, 1g yeast extract, 5g NaCl, 15g-20g agar, 1000mL deionized water, pH 6.8-7.2. After sterilizing at 121℃ for 30min, add 5% (5ml / 100ml) defibrinated sheep blood to the medium after it cools to 50℃, mix well and pour into plates.

[0066] The RS-36 strain was inoculated into blood agar medium and cultured at 28°C for 7 days. The presence or absence of hemolytic zones was observed. The results showed that the absence of hemolytic zones indicated that the strain lacked hemolytic activity and was a safe strain suitable for use as a microbial inoculant.

[0067] Example 6: The promoting effect of Gladiolus Burkholderia RS-36 on rice seed germination

[0068] (1) Fermentation with RS-36 microbial agent

[0069] The RS-36 strain was activated on LB medium. Single colonies were picked and transferred to LB liquid medium for shaking. Once the bacterial culture reached the logarithmic growth phase, the bacterial concentration was adjusted to 1×10⁻⁶ with physiological saline. 10-11The stock solution was prepared using CFU / mL. Before use, the stock solution was diluted three times its volume with sterile physiological saline to obtain the bacterial solution. Then, trace element solution was added dropwise to the bacterial solution (1 μL of trace element solution was added for every 3 mL of bacterial solution) and mixed well to obtain the liquid bacterial agent. 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 dissolving all the above components in an appropriate amount of water, the volume was made up to 1 L with deionized water.

[0070] (2) Rice germination experiment

[0071] Select plump and uniform rice seeds, add the above-mentioned bacterial agent, and soak them in the dark in an incubator for 2 days (30℃, 70% humidity). Use sterile physiological saline as a control. Change the bacterial solution and sterile physiological saline daily to avoid contamination. Then transfer the seeds to petri dishes containing moistened filter paper for germination. Place 50 seeds in each petri dish, and repeat each treatment 5 times. Incubate the petri dishes in the dark at 28℃. After 3 and 7 days of incubation, measure the length of seed buds and roots, germination rate, and germination index for each treatment.

[0072] Germination rate (%) = (Total number of germinated seeds / Total number of seeds in the experiment) × 100%

[0073] Germination Index = Σ (Number of germinated buds within time T / Corresponding number of germination days T)

[0074] The results are shown in Table 1. Three days after inoculation with RS-36 strain, compared with the control, the sprout length of rice seeds treated with the inoculated strain increased by 11.76%; the average germination rate also increased, but the increase was not significant. Seven days after germination, compared with the control, the average germination rate of rice seeds treated with the inoculated strain increased by 4.38%, the average sprout length increased by 5.65%, and the germination index increased by 1.92%.

[0075] Table 1. The promoting effect of strain RS-36 on rice seed germination

[0076]

Claims

1. Burkholderia gladioli ( Burkholderia gladioli RS-36, accession number CCTCC No: M20221584.

2. Containing *Burkholderia gladioli* as described in claim 1 ( Burkholderia gladioli RS-36 microbial inoculant.

3. The application of the inoculant as described in claim 2 as an inoculant for controlling rice blast disease.

4. The application of the microbial agent as described in claim 2 as a fungicide for controlling rice false smut.

5. The application of the microbial agent as described in claim 2 as a fungicide for controlling rice seedling blight.

6. The application of the microbial agent as described in claim 2 as a microbial agent for controlling rice panicle rot.

7. The application of the microbial agent as described in claim 2 as an IAA-producing microbial agent.

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