Burkholderia and application thereof in tobacco growth promotion

Abstract

The invention discloses burkholderia and application thereof in tobacco growth promotion, and belongs to the technical field of microorganisms. The Burkholderia sp. XCD4 is preserved in the China Center for Type Culture Collection (CCTCC), the preservation number of the Burkholderia sp. XCD4 is CCTCC NO: M 20241837, and the preservation time of the Burkholderia sp. XCD4 is August 23, 2024. The Burkholderia cepacia XCD4 is obtained through separation for the first time, the Burkholderia cepacia XCD4 has the good capacity of dissolving phosphorus and potassium, secreting IAA, producing siderophores, producing ACC deaminase and producing gibberellin, seed germination is promoted by remarkably improving the germination rate, germination potential, seedling length and root length of seeds, plant growth can be remarkably promoted, and the yield of plants is increased. Meanwhile, enrichment of phosphorus, potassium and other elements in tobacco planting plough layer soil can be reduced, and the utilization efficiency of the fertilizer is improved, so that the application prospect is good.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a Burkholderia strain and application thereof in tobacco growth promotion. Background Art

[0002] Flue-cured tobacco is an important cash crop in my country, with its total planting area, total output and total sales ranking among the highest in the world. Hubei Province has a long history of tobacco cultivation and is one of the main tobacco producing areas in China. In recent years, the long-term application of chemical fertilizers in Hubei tobacco-growing areas has led to an imbalance in the structure of soil microbial communities, which in turn has caused the enrichment of nutrients such as nitrogen, phosphorus and potassium in the soil of tobacco-growing areas, resulting in low fertilizer utilization and reduced tobacco yield and quality.

[0003] Growth-promoting bacteria can decompose insoluble elements such as phosphorus and potassium in the soil, secrete plant hormones, produce siderophores and ACC deaminase, and therefore play an important role in promoting plant growth. Specifically, the phosphate-dissolving microorganisms in the growth-promoting bacteria can convert insoluble phosphorus into soluble phosphorus through acidification, chelation and exchange reactions and release it into the soil for plant absorption and utilization. The organic and inorganic acids produced by the potassium-dissolving bacteria in the growth-promoting bacteria can directly promote the weathering of potassium-containing minerals by lowering the pH of the surrounding environment, resulting in the slow release of exchangeable potassium and increasing the environmental available potassium content, thereby promoting plant growth. The microorganisms in the growth-promoting bacteria can promote plant growth by directly secreting plant hormones (IAA). The microorganisms in the growth-promoting bacteria can produce siderophores to form iron chelates, so as to effectively absorb the low-solubility iron in the environment to promote plant growth. The microorganisms in the growth-promoting bacteria produce ACC deaminase, which decomposes ACC in the synthetic ethylene into α-ketobutyric acid and NH3, reducing the ethylene level of the plant, alleviating the stress on the plant, and promoting plant growth.

[0004] Gibberellic acid (GAs) is a tetracyclic diterpenoid compound, 136 species of which have been discovered so far, of which GA3 is the most active. As one of the five major plant hormones, gibberellins are a natural plant growth regulator that can end seed dormancy early, induce seed germination, promote stem growth, induce flowering, etc., and play a key role in controlling plant height.

[0005] At present, there are many types of growth-promoting microbial agents on the market, with different strains. However, since the activity of microorganisms is affected by factors such as temperature, humidity, and soil conditions, and China's tobacco-growing areas are widely distributed and the natural conditions vary greatly, therefore, screening growth-promoting bacteria suitable for Hubei tobacco-growing areas and reconstructing the beneficial rhizosphere microbial flora are the keys to their large-scale promotion and application.

[0006] However, there are few reports that Burkholderia has the functions of solubilizing phosphate and potassium, secreting IAA, producing siderophores, producing ACC deaminase, producing gibberellins, promoting seed germination, and promoting plant growth. Summary of the invention

[0007] The purpose of the present invention is to provide a strain of Burkholderia and its application in tobacco growth promotion. The Burkholderia XCD4 provided by the present invention has good ability to solubilize phosphorus and potassium, secrete IAA, produce siderophores, produce ACC deaminase, produce gibberellins, promote seed germination and promote plant growth, and therefore has good application prospects in improving fertilizer utilization and / or promoting plant growth.

[0008] In a first aspect, the present invention provides a strain of Burkholderia ( Burkholderia cepacia )XCD4, Burkholderia ( Burkholderia cepacia )The deposit number of XCD4 is CCTCC NO:M 20241837.

[0009] The strain XCD4 was isolated from the rhizosphere soil of flue-cured tobacco in the typical tobacco area of ​​Shima Village, Jiaoyuan Town, Xuanen County, Enshi Tujia and Miao Autonomous Prefecture, Hubei Province. The strain XCD4 was cultured on LB solid medium plates at 28°C for 1-2 days and was round, milky white, about 1-2 mm in diameter, with a smooth surface, a viscous texture, a translucent colony, clear edges, and a distinct odor.

[0010] Furthermore, the strain XCD4 was sequenced by 16S rDNA, and its 16S rDNA sequence was compared by BLAST multiple sequence alignment in NCBI (https: / / www.ncbi.nlm.nih.gov / ), and a phylogenetic tree was constructed. The results showed that the strain XCD4 had a high homology with Burkholderia, and Burkholderia cepacia JCM 13198 belonged to the same branch; combined with the morphological characteristics and molecular biological identification results, strain XCD4 was named Burkholderia ( Burkholderia cepacia )XCD4.

[0011] In a second aspect, the present invention provides a biological bacterial agent, including the above-mentioned Burkholderia ( Burkholderia cepacia )XCD4.

[0012] In some embodiments, the bioagent is a liquid agent.

[0013] In some embodiments, the liquid inoculum contains Burkholderia ( Burkholderia cepaci )XCD4 content ≥1×10 7 cfu / mL.

[0014] It is understandable that the biological bacterial agent can be selected according to the actual use needs to select the conventional dosage form in the prior art, for example, it can also be a solid bacterial agent, and the solid bacterial agent can be prepared by conventional methods in the art.

[0015] In a third aspect, the present invention provides a method for preparing the above-mentioned biological bacterial agent, comprising the following steps: Burkholderia cepaci )XCD4 is inoculated into a liquid culture medium for cultivation, and the obtained culture is separated and resuspended in a solvent to obtain a biological bacterial agent.

[0016] The preparation method of the biological bacterial agent provided by the invention is simple, environmentally friendly and has good biological safety.

[0017] It is understood that the liquid culture medium can be a common culture medium in the art, as long as it can meet the requirements of the Burkholderia ( Burkholderia cepaci ) The normal growth of XCD4. The culture conditions can be selected from conventional conditions. In the present invention, the liquid culture medium is preferably LB culture medium.

[0018] In some embodiments, the separation comprises: centrifugation at 5000-7000 r / min for 3-10 min.

[0019] In some embodiments, the solvent comprises at least one of PBS buffer and sterile water.

[0020] In a fourth aspect, the present invention provides the Burkholderia ( Burkholderia cepaci ) Use of XCD4, any of the above biological agents or the biological agent obtained by the above preparation method in at least one of the following: A1) solubilizing phosphate; A2) solubilizing potassium; A3) secreting IAA; A4) producing siderophore; A5) producing ACC deaminase; A6) producing gibberellin.

[0021] In a fifth aspect, the present invention provides the Burkholderia ( Burkholderia cepaci ) Use of XCD4, any of the above biological agents or the biological agents obtained by the above preparation methods in promoting seed germination.

[0022] In some embodiments, the seeds include at least one of rapeseed seeds, Chinese cabbage seeds, rice seeds, and flue-cured tobacco seeds.

[0023] It is understandable that the seeds may be conventional plant seeds in the prior art. In the present invention, the seeds preferably include at least one of rapeseed seeds, Chinese cabbage seeds, rice seeds, and flue-cured tobacco seeds.

[0024] In a sixth aspect, the present invention provides the Burkholderia ( Burkholderia cepaci ) Use of XCD4, any of the above biological agents or the biological agents obtained by the above preparation methods in improving fertilizer utilization and / or promoting plant growth.

[0025] In some embodiments, the plant comprises tobacco.

[0026] It can be understood that the plant can be any conventional plant in the prior art. In the present invention, the plant preferably includes tobacco.

[0027] The beneficial effects of the present invention are as follows: Different from the prior art, the present invention is the first to isolate a strain of Burkholderia ( Burkholderia cepaci )XCD4, the strain XCD4 has good ability to solubilize phosphorus and potassium, secrete IAA, produce siderophores, produce ACC deaminase, and produce gibberellins. It promotes seed germination by significantly improving the germination rate, germination potential, seedling length and root length of seeds, and can significantly promote plant growth. At the same time, it can reduce the enrichment of elements such as phosphorus and potassium in the tobacco planting topsoil and improve the fertilizer utilization efficiency. Therefore, it has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a colony morphology diagram of strain XCD4 on an LB plate in Example 1 of the present invention; Figure 2 The phosphate dissolution circle diagram formed after the strain XCD4 in Example 1 of the present invention is cultured on an inorganic phosphorus solid medium; Figure 3 It is a potassium-dissolving circle diagram formed after the strain XCD4 in Example 1 of the present invention is cultured on the M1997 potassium-dissolving bacteria solid culture medium; Figure 4 This is an orange halo image formed after the strain XCD4 in Example 1 of the present invention is cultured on a CAS detection solid medium; Figure 5 The phylogenetic tree diagram constructed in Example 1 of the present invention; Figure 6 This is a color reaction diagram after adding Salkowski reagent colorimetric solution to the supernatant of strain XCD4 in Example 5 of the present invention, wrapping it with tin foil and reacting it at room temperature in the dark for 30 minutes; Figure 7 This is a graph showing the results of ACC deaminase activity produced by strain XCD48 in Example 6 of the present invention; Figure 8 This is a graph showing the gibberellin content produced by strain XCD48 in Example 7 of the present invention; Fig. 9 This is a trend diagram of the strain XCD48 promoting seed germination in Example 8 of the present invention; Fig.10 This is a graph showing the fresh weight and dry weight of the aboveground and underground parts of flue-cured tobacco after growth promotion by strain XCD48 in Example 9 of the present invention.

[0029] Biological Deposit The strain provided by the present invention is deposited in China Center for Type Culture Collection (CCTCC), deposit address: Wuhan University, Wuhan, China, deposit number: CCTCC NO:M 20241837, deposit time: August 23, 2024, classification name: Burkholderia ( Burkholderia cepaci )XCD4. DETAILED DESCRIPTION

[0030] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] The experimental methods without specific conditions in the examples are usually carried out according to conventional experimental methods in the field of molecular biology, including but not limited to the experimental methods described in Molecular Cloning: A Laboratory Manual by MR Green, Molecular Biology by Robert F. Weaver, etc., or according to the experimental methods recommended by the kit and instrument manufacturers. The reagents and biological materials used in the examples can be obtained from commercial channels unless otherwise specified.

[0032] In the present invention, the culture medium used is as follows: The formula of LB liquid culture medium is: 10.0 g tryptone, 5.0 g yeast extract powder, 10.0 g sodium chloride, and 1000 mL distilled water.

[0033] The formula of inorganic phosphorus solid culture medium is: 10.0g glucose, 0.5g ammonium sulfate, 0.5g yeast extract powder, 0.3g sodium chloride, 0.3g potassium chloride, 0.3g magnesium sulfate, 0.03g ferrous sulfate, 0.03g manganese sulfate, 5.0g tricalcium phosphate, 15g agar, and 1000mL distilled water.

[0034] The formula of M1997 potassium-soluble bacterial solid culture medium is: 0.5g magnesium sulfate, 0.1g calcium carbonate, 2.0g potassium aluminum silicate, 5.0g glucose, 0.005g ferric chloride, 2.0g calcium carbonate, 15g agar, and 1000mL distilled water.

[0035] The formula of silicate solid culture medium is: 5.0g glucose, 0.5g magnesium sulfate, 0.1g calcium carbonate, 2.0g disodium bicarbonate, 0.005g ferric chloride, 1.0g glass powder, 15g agar, and 1000mL distilled water.

[0036] The formula of LB liquid culture medium containing 0.5 g / L L-tryptophan is: 0.5 g L-tryptophan, 10.0 g tryptone, 5.0 g yeast extract powder, 10.0 g sodium chloride, and 1000 mL distilled water.

[0037] The formula of CAS detection medium is: chrome azuro blue S (CAS) 60.5 mg, hexadecyltrimethylammonium bromide (HDTMA) 72.9 mg, ferric chloride hexahydrate 2.645 mg, sodium dihydrogen phosphate dihydrate 295.25 mg, disodium hydrogen phosphate dodecahydrate 1213.5 mg, ammonium chloride 125 mg, potassium dihydrogen phosphate 37.5 mg, sodium chloride 62.5 mg, agar 9000 mg, adjust pH to 6.8±0.1, and distilled water 1000 mL.

[0038] The formula of MKB iron-limited culture medium is: ① 50g of casein amino acids, 15mL of glycerol (propylene glycol) plus 785mL of deionized water; ② 2.5g of potassium dihydrogen phosphate dissolved in 100mL of deionized water; ③ 2.5g of magnesium sulfate heptahydrate dissolved in 100mL of deionized water; after separate preparation, sterilize them separately, adjust the pH to 7.2, and mix ①, ②, and ③ together before use.

[0039] The formula of DF culture medium without nitrogen source is: 4 g potassium dihydrogen phosphate, 6 g disodium hydrogen phosphate, 0.2 g magnesium sulfate heptahydrate, 2 g glucose, 2 g gluconic acid, 2 g citric acid, 0.1 mL of trace element mother solution ①, 0.1 mL of trace element mother solution ②, and 1000 mL of distilled water; among them, the components of trace element mother solution ① are as follows: 10 mg boric acid, 11.2 mg magnesium sulfate, 124.6 mg zinc sulfate, 78.2 mg copper sulfate and 10 mg molybdenum trioxide per 100 mL, stored at 4°C; the components of trace element mother solution ② are as follows: 100 mg ferrous sulfate heptahydrate per 10 mL, stored at 4°C.

[0040] In the present invention, the preparation method of CAS detection solution is as follows: First, prepare the following solution: 1mM ferric chloride stock solution: weigh 0.2703g ferric chloride hexahydrate and dissolve in 1L 10mM hydrochloric acid; CAS stock solution: weigh 0.2421 g CAS (chrome azurol) and dissolve it in 200 mL deionized water; HTDMA (hexadecyltrimethylammonium bromide) solution: weigh 0.0219 g HTDMA and dissolve it in 50 mL water; Piperazine buffer: Weigh 4.3079 g of anhydrous piperazine and dissolve it in 30 mL of water. Adjust the pH to 5.6 with hydrochloric acid. Then, take 1.5 mL of 1 mM ferric chloride stock solution, add 7.5 mL of CAS stock solution to it and mix well, continue to add 50 mL of HTDMA solution while stirring, then continue to add 30 mL of piperazine buffer, and finally add deionized water to finally prepare 100 mL of CAS detection solution.

[0041] In the present invention, the components of the Salkowski reagent colorimetric solution are: 1 mL of solution A and 50 mL of solution B are mixed and used immediately after preparation; wherein, the preparation of solution A and solution B is as follows: Solution A (0.5M FeCl3): weigh 4.055g FeCl3 and add water to make up to 50mL; Solution B (35% HClO4): Measure 50 mL of HClO4 and add water to make up to 100 mL.

[0042] 100 mg / L IAA standard stock solution: Weigh 10 mg of IAA, dissolve it in a small amount of ethanol, and then dilute to 100 mL with deionized water to obtain an IAA standard stock solution with a concentration of 100 mg / L.

[0043] Example 1 Screening and identification of strain XCD4 1.1 Sample source The rhizosphere soil of flue-cured tobacco in a typical tobacco-growing area of ​​Shima Village, Jiaoyuan Town, Xuanen County, Enshi Tujia and Miao Autonomous Prefecture, Hubei Province was selected, and random sampling was carried out using the five-point method. The whole tobacco plant was pulled out, and the loose soil at the root was shaken off. The remaining part was scraped into a numbered ziplock bag with a brush. The soil samples were mixed and transported to the laboratory with dry ice and stored at -80℃.

[0044] 1.2 Screening of strain XCD4 Prepare a sterilized 150 mL Erlenmeyer flask, weigh 1.00 g of tobacco rhizosphere soil, add 99 mL of sterile water, and shake at 28 °C, 180 r / min for 30 min to obtain 10 -2 Dilution gradient soil suspension. Use a pipette to absorb the supernatant of the soil bacterial suspension after standing, and dilute it according to the gradient to make a dilution gradient of 10 -3 , 10 -4 , 10 -5Dilution. Take 100 μL of gradient dilution and use the alcohol burner flame to burn the sterilized glass coating rod, evenly spread it on the inorganic phosphorus solid culture medium, do three replicates for each gradient, and invert and culture in a 28℃ constant temperature incubator for 5 days. After 5 days, pick the colonies with obvious phosphorus decomposition rings, streak and purify them on the inorganic phosphorus solid culture medium, and invert and culture in a 28℃ constant temperature incubator for 3 days. Repeat the streak purification steps until a single colony is obtained. Inoculate the purified single colony into a mixture of 1mL glycerol + 1mL LB liquid culture medium and store it in a -80℃ refrigerator for later use.

[0045] The strains obtained above were inoculated in M1997 potassium-dissolving bacteria solid culture medium and silicate solid culture medium, and inverted in a constant temperature incubator at 28°C for 3 days. The growth of the strains and the production of transparent circles were observed to preliminarily determine the potassium-dissolving ability of the strains, and 12 strains with both phosphorus and potassium dissolving abilities were obtained. The 12 strains were inoculated in CAS detection medium, inverted at 28°C for 3 days, and a strain with a purple-red halo was obtained, which was named XCD4.

[0046] The colony morphology of the strain XCD4 after being cultured on LB solid medium at 28°C for 1-2 days is shown in the figure below. Figure 1 shown.

[0047] from Figure 1 It can be seen that strain XCD4 is round, milky white, about 1-2 mm in diameter, smooth in surface, viscous in texture, translucent in colony, with clear edges and a distinct odor.

[0048] The phosphate solubilization effect of strain XCD4 after being cultured on inorganic phosphorus solid medium at 28°C for 5 days is as follows Figure 2 shown.

[0049] from Figure 2 It can be seen that strain XCD4 forms a phosphate-solubilizing circle after being cultured on inorganic phosphorus solid culture medium. The results show that strain XCD4 has good phosphate-solubilizing ability.

[0050] The potassium-dissolving effect of strain XCD4 after culturing on M1997 potassium-dissolving bacteria solid medium at 28°C for 3 days is as follows Figure 3 shown.

[0051] from Figure 3 It can be seen that strain XCD4 forms a potassium-solubilizing circle after being cultured on M1997 potassium-solubilizing bacteria solid culture medium. The results show that strain XCD4 has a good potassium-solubilizing ability.

[0052] The siderophore production of strain XCD4 after culturing at 28°C for 3 days on CAS assay medium is shown in Figure 4 shown.

[0053] from Figure 4 It can be seen that strain XCD4 formed an orange halo after being cultured on the CAS detection medium. The results showed that strain XCD4 had a good ability to produce siderophores.

[0054] 1.3 Biological identification of strain XCD4 The strain XCD4 obtained by screening was sent to Guangdong Meige Gene Technology Co., Ltd. for sequencing. After obtaining the genomic DNA of the target strain, the corresponding primers (such as 27F / 1492R primers for 16S rDNA fragments) were used for amplification. The amplified product was sequenced using the ABI3730 sequencing platform.

[0055] The sequence (SEQ ID NO: 1) was aligned by BLAST in NCBI (https: / / www.ncbi.nlm.nih.gov / ), and a phylogenetic tree was constructed. The results are shown in Figure 5 shown.

[0056] from Figure 5 It can be seen that the strain XCD4 has a high homology with Burkholderia, and Burkholderia cepacia JCM 13198 belonged to the same branch; combined with the morphological characteristics and molecular biological identification results, strain XCD4 was named Burkholderia ( Burkholderia cepacia )XCD4.

[0057] Example 2 Quantitative determination of phosphate solubilization ability of strain XCD4 The strain XCD4 was inoculated into LB liquid culture medium, and cultured in a shaker at 28°C and 180 r / min for 24 h. Then, 1 mL of the bacterial solution was added to 100 mL of inorganic phosphorus liquid culture medium (agar is not required in the inorganic phosphorus solid culture medium). Each treatment was repeated 3 times, and a treatment without inoculation of strain XCD4 was set as a control group. The culture was shaken at 28°C and 180 r / min for 5 days. Samples were taken on the 5th day, and the solution was determined by the molybdenum antimony colorimetric method after centrifugation at 10,000 r / min for 5 min.

[0058] Among them, the phosphorus standard solution is used as the standard sample to obtain the standard curve, and its equation is: y=0.6994x-0.0005 (R 2 =0.9994).

[0059] The phosphate solubilizing capacity of strain XCD4 was calculated to be 98.30 mg / L using the above standard curve.

[0060] Example 3 Quantitative determination of potassium-solubilizing ability of strain XCD4 The XCD4 strain was inoculated into LB liquid culture medium, and cultured in a shaking incubator at 28°C and 180 r / min for 24 h. Then, 1 mL of the bacterial solution was added into 100 mL of M1997 potassium-soluble bacterial liquid culture medium (agar is not required in the M1997 potassium-soluble bacterial solid culture medium), with 3 replicates for each treatment. At the same time, a treatment without inoculation of strain XCD4 was set as a control group. After cultured in a shaking incubator at 28°C and 180 r / min for 5 days, samples were taken, and the potassium-soluble amount was determined by flame photometry after centrifugation at 10,000 r / min for 5 min.

[0061] Among them, potassium standard solution is used as the standard sample to obtain the standard curve, and its equation is: y=2.9276x+0.3707 (R 2 =0.9996).

[0062] The potassium-solubilizing capacity of strain XCD4 was calculated to be 10.23 mg / L using the above standard curve.

[0063] Example 4 Quantitative determination of the ability of strain XCD4 to produce siderophore The strain XCD4 was activated and inoculated into the MKB iron-limited medium at 1% of the amount. Each strain was replicated 3 times and cultured in a shaker at 28°C and 180rpm for 48h. After 48h, centrifuge for 15min at a speed of 3500r / min, take 3mL of the supernatant and add it to the CAS detection solution and mix thoroughly. After 1h, the absorbance value (As) at a wavelength of 630nm was measured by a spectrophotometer, and double distilled water was used as a control to adjust the zero. Take another 3mL of CAS detection solution and mix it thoroughly with 3mL of uninoculated MKB liquid culture medium supernatant, and measure it in the same way. The absorbance value is the reference value (Ar), and the As / Ar value is measured.

[0064] Among them, bacteria producing iron carriers can react with the reaction solution to show orange or purple red. If they do not produce, the color will not change and they will remain blue. Sterile water was used as a control in the experiment. Since the absorbance value (Ar) of the test solution is large, the smaller the As / Ar value, the stronger the ability of the strain to produce iron carriers. The general reference standard is: As / Ar values ​​are as follows: 0-0.25, 0.25-0.5, 0.5-0.75, 0.75-1.0, >1.0, which correspond to ++++, +++, ++, +, - respectively.

[0065] The results of the siderophore production ability of strain XCD4 are shown in Table 1 below.

[0066] Table 1 Results of the ability of strain XCD4 to produce siderophores

[0067] As can be seen from Table 1, strain XCD4 has a good ability to produce siderophores.

[0068] Example 5 Quantitative determination of IAA production capacity of strain XCD4 Accurately pipette a quantitative amount of IAA standard stock solution into a 100mL volumetric flask, dilute to volume with deionized water, and obtain a series of IAA standard solutions with standard concentrations of 0, 20, 40, 60, 80, 100, and 200 mg / L, respectively. Store in a dark place after preparation. Take 3mL of each concentration standard solution, add an equal volume of Salkowski reagent colorimetric solution, and treat in a dark place for 30 minutes. Use a spectrophotometer to detect the absorbance at 530nm. Use the IAA standard concentration as the horizontal coordinate and the absorbance as the vertical coordinate to draw the IAA standard curve. The standard curve equation is: y=0.0087x+0.0052(R 2 =0.997).

[0069] The strain XCD4 was activated and inoculated into LB medium supplemented with 0.5 g / L tryptophan at 1% concentration. After culturing at 28°C and 180 rpm for 3 days, ultracentrifuged at 10,000 rpm for 10 minutes. After centrifugation, 1 mL of supernatant was pipetted into a 24-well plate, and an equal volume of Salkowski reagent colorimetric solution was added. The plate was wrapped with tin foil and reacted at room temperature in the dark for 30 minutes. The color reaction was observed. At the same time, 3 mL of supernatant was added to an equal volume of Salkowski reagent colorimetric solution in a 10 mL centrifuge tube, and reacted at room temperature in the dark for 30 minutes ( Figure 6 After the reaction, the absorbance at 530 nm was measured using a spectrophotometer.

[0070] The IAA production capacity of strain XCD4 was calculated to be 78.31 μg / mL using the above IAA standard curve.

[0071] Example 6 Quantitative determination of the ability of strain XCD4 to produce ACC deaminase The strain XCD4 was inoculated into a flask containing 20 mL of LB liquid medium, and cultured at 28°C and 200 rpm for 12 h under shaking conditions; centrifuged at 4°C and 8000 rpm for 10 min; the bacteria were resuspended in 7.5 mL of DF medium without nitrogen source, and 45 μL of filtered sterilized 0.5 mol / L ACC solution was added, and cultured at 28°C and 200 rpm for 24 h under shaking conditions to induce the deaminase activity of bacterial ACC; centrifuged at 4°C and 8000 rpm for 10 min, the supernatant was removed, and the bacterial precipitate was collected; resuspended in 5 mL of 0.1 mol / L Tris-HCl buffer (pH = 7.6), and the precipitate was collected after further centrifugation at 4°C and 8000 rpm for 10 min. This step was repeated 3 times to completely remove the DF medium; 1 mL of 0.1 mol / L Tris-HCl buffer (pH = 7.6) was added to the bacteria, and centrifuged at 16000 rpm for 5 min; then the bacteria were resuspended in 600 μL 0.1mol / L Tris-HCl buffer (pH=8.5), add 30μL toluene, and oscillate on a vortex shaker for 30s to break the bacteria; after standing at 4℃ for 1h, centrifuge at 12000rpm for 10min. Transfer the supernatant into two 2mL centrifuge tubes, taking 200μL of each. One portion is stored in a 4℃ refrigerator for protein detection, and the other portion is immediately used for ACC deaminase activity detection.

[0072] ACC deaminase activity assay: Drawing of the standard curve: Use 0.1mol / L Tris-HCl buffer (pH=8.5) to prepare α-ketobutyric acid into gradient solutions of 0.02, 0.04, 0.06, 0.08, and 0.1μmol / L, and add 300μL of 2,4-dinitrophenylhydrazine respectively. After mixing evenly, water bath at 30℃ for 30min. Finally, add 2mL 2mol / L NaOH for color development. After stabilization, measure its absorbance at 540nm, and use Tris-HCl (pH=8.5) solution as blank control. The standard curve is made according to the concentration of α-ketobutyric acid solution and its corresponding absorbance value. The equation is: y=0.1739x-0.0057(R 2 =0.9905).

[0073] Pipette 200 μL of broken bacterial liquid, add 20 μL of 0.5mol / L ACC solution, mix well and incubate in a water bath at 37°C for 15 min, then add 1 mL of 0.56mol / L HCl, centrifuge at 25°C and 16000 rpm for 5 min, take 1 mL of the supernatant, add 800 μL of 0.56mol / L HCl, mix well and add 300 μL of 2,4-dinitrophenylhydrazine, incubate in a water bath at 30°C for 30 min, finally add 2 mL of 2mol / L NaOH for color development, measure the absorbance at 540 nm, and use distilled water as the blank control.

[0074] The OD of the sample 540 Substitute the above equation to calculate the content of α-ketobutyric acid, and then calculate the amount (μmol) of ACC deaminase-catalyzed ACC to produce α-ketobutyric acid per unit time, that is, the unit enzyme activity U.

[0075] Protein detection: Preparation of standard curve: Preparation of 0~100μg / mL standard curve: Take 6 clean 10mL stoppered test tubes and take samples according to Table 2. After plugging, invert the solution in each test tube vertically to mix, let it stand for 2 minutes, and then use a 1cm optical diameter colorimetric cup at a wavelength of 595nm to compare the color, and record the optical density OD of each tube. 595nm , and make a standard curve, the equation of which is: y=0.188x-0.1241 (R 2 =0.9905).

[0076] Table 2 Standard curve sampling

[0077] Take another 2 10mL stoppered test tubes, draw 1mL of the extract (do one repeat), put it into the stoppered graduated test tube, add 5mL of Coomassie Brilliant Blue G-250 protein reagent, mix thoroughly, let it stand for 2 minutes, and then use a 1cm optical path colorimetric cup to compare the color at 595nm, and record the optical density OD 595nm The protein content in the sample extract to be tested is calculated through the standard curve, and test tube No. 1 of the standard curve is used as the blank.

[0078] The specific activity (U / mg) was obtained by dividing the unit enzyme activity U by the total protein mass, which represents the ACC deaminase activity of strain XCD4.

[0079] The ACC deaminase activity of strain XCD4 was calculated to be 9.90 U / mg ( Figure 7 ).

[0080] Example 7 Quantitative determination of the ability of strain XCD4 to produce gibberellins Gibberellic acid was dissolved in 70% ethanol to prepare a 100 mg / L gibberellin standard solution. The solution was diluted according to a certain concentration gradient. 0.5 mL of gibberellin solution of each concentration was mixed with 4.5 mL of concentrated sulfuric acid (mass fraction 98%). The solution was placed in an ice bath for 10 min, then placed in a 28°C warm water bath for 1 h, and then placed at room temperature for 5 min. The OD 412 The standard curve is drawn based on the absorbance value at , and the standard curve equation is: y = 0.0282x, R 2 =0.9995.

[0081] The strain XCD4 was inoculated into LB liquid medium, cultured at 28°C and 180r / min for 24h to prepare seed solution. After the seed solution was transferred to LB liquid medium at a 1% inoculation rate for 2h, the bacterial suspension was centrifuged at 10000r / min for 10min, the supernatant was taken and continued to be cultured at 28°C and 180r / min for 24h, 48h and 72h, 0.5mL of bacterial solution was taken and mixed with 4.5mL of concentrated sulfuric acid (mass fraction 98%), placed in an ice bath for 10min, and then placed in a 28°C warm water bath for 1h, taken out and placed at room temperature for 5min, and the OD was measured. 412 The absorbance value was measured at 100 nm, and each group was repeated 3 times.

[0082] The gibberellin production capacity of strain XCD4 was calculated by the above standard curve to be 8.78 mg / L ( Figure 8 ).

[0083] In summary, the amount of phosphorus solubilized by strain XCD4 after being cultured at 28℃ for 5 days in inorganic phosphorus liquid medium was 98.30 mg / L; the amount of potassium solubilized after being cultured at 28℃ for 5 days in M1997 potassium-soluble bacteria liquid medium was 10.23 mg / L; after being cultured at 28℃ for 2 days in MKB iron-limited medium, its As / Ar=0.691, and iron carriers can be produced; the amount of IAA produced after being cultured at 28℃ for 3 days in LB medium containing 0.5 g / L tryptophan was as high as 78.31 μg / mL; the activity of ACC deaminase produced after being cultured at 28℃ for 2 days in DF medium without nitrogen source was 9.90 U / mg; the amount of gibberellin produced after being cultured at 28℃ for 72 hours in LB liquid medium was 8.78 mg / L.

[0084] Example 8 Experiment on promoting seed germination by strain XCD4 Strain preparation: Activate the XCD4 strain, wait for colonies to grow, inoculate the selected colonies into 10 mL LB liquid medium, shake at 28°C, 180 r / min, for 16-24 h, and count the number of bacteria by hemocytometer to ≥1×10 7The XCD4 bacterial solution was centrifuged at 6000r / min for 5min, the supernatant was removed and resuspended in 100mL sterile water to prepare the XCD4 bacterial agent.

[0085] The experimental Chinese cabbage seeds used were the original Shanghai Qing, the rapeseed seeds used were Zhongshuang No. 9, the rice seeds used were Hanyou No. 73, and the tobacco seeds used were Yunyan No. 87.

[0086] After the above seeds were quickly disinfected with 75% ethanol, they were soaked in 1% sodium hypochlorite for 15 minutes and then rinsed with sterile water for 3-5 times for later use. The disinfected seeds were soaked in XCD4 bacterial agent for 30 minutes. The control (CK) was uninoculated seeds soaked in sterile water. Filter paper was laid in a sterile culture dish, moistened with an appropriate amount of sterile water, and the treated seeds were evenly spread on the filter paper, with 100 seeds per dish (50 rice seeds per dish). Each treatment was repeated 3 times, and cultured at room temperature for 144 hours (tobacco cultured for 240 hours). The seed germination was observed every 12 hours, and the seedling length, root length, germination rate, and germination potential were measured on the 7th day (tobacco on the 10th day). The results are shown in Tables 3 and 4. Fig. 9 shown.

[0087] Table 3 Germination index

[0088] From Table 3 and Fig. 9 It can be seen that the XCD4 treatment significantly increased the germination potential of Chinese cabbage, rapeseed, rice and flue-cured tobacco. Compared with the CK group, Chinese cabbage increased by 31.34%, rapeseed increased by 3.34%, rice increased by 13.34%, and flue-cured tobacco increased by 22.17%. This shows that the XCD4 treatment has a general promoting effect on the early germination of crop seeds.

[0089] Treatment with XCD4 inoculant also increased the germination rate of crops. The germination rates of Chinese cabbage and rapeseed increased by 7.67% and 12.00% respectively, reaching 96.67% and 92.67%; the germination rates of rice and flue-cured tobacco also increased slightly, but to a smaller extent, at 4.00% and 12.50% respectively.

[0090] In terms of seedling length, XCD4 treatment significantly promoted the seedling length of Chinese cabbage, rapeseed and rice. Compared with the CK group, the seedling length of these three crops increased by 7.13mm, 8.27mm and 12.46mm respectively. Although the growth of flue-cured tobacco seedling length was not significant, it also increased by 0.73mm. This shows that XCD4 treatment can effectively promote the growth of most crops in the seedling stage.

[0091] At the same time, XCD4 treatment significantly promoted the root length of Chinese cabbage and rapeseed, increasing by 4.55mm and 4.39mm respectively. However, it had no significant effect on the root length of rice and flue-cured tobacco, which may reflect the different responses of different crops to XCD4 treatment.

[0092] In summary, the XCD4 inoculant treatment promoted the germination and early growth of Chinese cabbage, rapeseed, rice and flue-cured tobacco to varying degrees, especially the germination potential and seedling length.

[0093] Example 9 Experiment on the Growth-Promoting Effect of Strain XCD4 on Flue-cured Tobacco The polyethylene plastic flower pots used in the test had a diameter of 23.5 cm and a height of 24.9 cm. Each pot contained 5 kg of mixed soil with a mass ratio of soil to vermiculite of 4:1. Fertilization treatment: Before transplanting the tobacco seedlings, 0.2 g / kg N, 0.1 g / kg P2O5, and 0.1 g / kg K2O were applied as base fertilizers, and urea, diammonium phosphate, and potassium sulfate were used as fertilizer sources, respectively. Yunyan No. 87 was used as the test object. The tobacco seedlings were treated with the bacterial agent prepared in Example 8 for root irrigation, and the roots were irrigated with an equal amount of sterile water as a control. The dosage per pot was 100 mL, and the treatment was performed once every 7 days. Deionized water was used for irrigation during the test, wherein: N-CK group: yellow-brown soil from Huazhong Agricultural University; F-CK group: yellow-brown soil from Huazhong Agricultural University + fertilizer; N-XCD4 group: yellow-brown soil from Huazhong Agricultural University + XCD4 bacterial agent; F-XCD4 group: yellow-brown soil from Huazhong Agricultural University + fertilizer + XCD4 bacterial agent; Each treatment group was harvested on the 30th day of plant growth. The plant height, stem diameter, maximum leaf length, maximum leaf width, aboveground fresh weight, aboveground dry weight, underground fresh weight, underground dry weight, and leaf SPAD value were measured on the harvest day. The results are shown in Table 4 and Fig.10 shown.

[0094] Table 4 Effects of XCD4 bacterial agent on flue-cured tobacco growth

[0095] As can be seen from Table 4, the mixed application of fertilizer and XCD4 can significantly increase the plant height, stem diameter, maximum leaf length, maximum leaf width, and SPAD value of flue-cured tobacco. The plant height increased by 57.02% compared with the fertilized control, the stem diameter increased by 38.96% compared with the fertilized control, the maximum leaf length increased by 38.57% compared with the fertilized control, the maximum leaf width increased by 17.39% compared with the fertilized control, and the SPAD value increased by 8.39% compared with the fertilized control. The single application of XCD4 also had a significant effect on the agronomic traits of flue-cured tobacco, with the plant height increasing by 97.26% compared with the unfertilized control, the stem diameter increasing by 27.66% compared with the unfertilized control, the maximum leaf length increasing by 21.67% compared with the unfertilized control, the maximum leaf width increasing by 11.89% compared with the unfertilized control, and the SPAD value increasing by 3.99% compared with the unfertilized control.

[0096] from Fig.10 It can be seen that the mixed application of XCD4 bacteria and fertilizer has a significant effect on the biomass of flue-cured tobacco. The root irrigation treatment strain first attaches to the root system of the tobacco plant, which then promotes the growth of the plant, thereby increasing the biomass of the plant. The mixed application of fertilizer and XCD4 bacteria has a significant effect on the aboveground fresh weight and underground fresh weight of the tobacco plant. The aboveground fresh weight increased by 36.69% compared with the fertilized control, the aboveground dry weight increased by 52.13% compared with the fertilized control, the underground fresh weight increased by 40.10% compared with the fertilized control, and the underground dry weight increased by 100.79% compared with the fertilized control. The single application of XCD4 bacteria also has a significant effect on the biomass of flue-cured tobacco. The aboveground fresh weight increased by 63.56% compared with the unfertilized control, the aboveground dry weight increased by 43.84% compared with the unfertilized control, the underground fresh weight increased by 74.77% compared with the unfertilized control, and the underground dry weight increased by 24.39% compared with the unfertilized control.

[0097] In summary, the Burkholderia provided by the present invention ( Burkholderia cepacia ) XCD4 has good ability to solubilize phosphate and potassium, secrete IAA, produce siderophore, produce ACC deaminase, and produce gibberellin. It promotes seed germination by significantly improving seed germination rate, germination potential, seedling length and root length, and can significantly promote plant growth.

[0098] It should be noted that the above embodiments all belong to the same inventive concept, and the description of each embodiment has its own focus. For details not described in individual embodiments, reference may be made to the description in other embodiments.

[0099] The above-mentioned embodiments only express the implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

[0100] 16S rDNA sequence of strain XCD4 (SEQ ID NO: 1)

Claims

1. A Burkholderia strain XCD4, characterized in that The deposit number of the Burkholderia XCD4 is CCTCC NO:M 20241837.

2. A biological bacterial agent, characterized in that: Comprising the Burkholderia XCD4 of claim 1.

3. The biological agent according to claim 2, characterized in that: The biological bacterial agent is a liquid bacterial agent.

4. The biological agent according to claim 3, characterized in that: The content of Burkholderia XCD4 in the liquid bacterial agent is ≥1×10 7 cfu / mL.

5. A method for preparing a biological agent as claimed in any one of claims 2 to 4, characterized in that: The method comprises the following steps: inoculating the Burkholderia XCD4 described in claim 1 into a liquid culture medium for culturing, separating the obtained culture and resuspending it with a solvent to obtain a biological bacterial agent.

6. Use of the Burkholderia XCD4 according to claim 1, the biological agent according to any one of claims 2 to 4, or the biological agent prepared by the method according to claim 5 in at least one of the following: A1) Solubilizes phosphate; A2) Solubilizes potassium; A3) Secretes IAA; A4) Produces siderophore; A5) Produces ACC deaminase; A6) Produces gibberellins.

7. Use of the Burkholderia XCD4 according to claim 1, the biological agent according to any one of claims 2 to 4, or the biological agent obtained by the preparation method according to claim 5 in promoting seed germination.

8. The use according to claim 7, characterized in that: The seeds include at least one of rapeseed seeds, Chinese cabbage seeds, rice seeds, and flue-cured tobacco seeds.

9. Use of the Burkholderia XCD4 according to claim 1, the biological agent according to any one of claims 2 to 4, or the biological agent obtained by the preparation method according to claim 5 in improving fertilizer utilization and / or promoting plant growth.

10. The use according to claim 9, characterized in that: The plants include tobacco.

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