Burkholderia parakholderia capable of promoting growth of tobacco seedlings under low-temperature and drought conditions as well as screening method and application of burkholderia parakholderia

Abstract

The invention relates to burkholderia parakholderia capable of promoting growth of tobacco seedlings under low-temperature and drought conditions as well as a screening method and application of the burkholderia parakholderia. The Burkholderia paragallinarum is the Burkholderia paragallinarum sp, and the preservation number of the Burkholderia paragallinarum sp is CCTCC (China Center for Type Culture Collection) No: M 20242310, and the Burkholderia paragallinarum sp is the Burkholderia paragallinarum sp. The invention also provides an application of the burkholderia paragallinarum in preparation of a microbial agent. The invention provides a microbial agent, and the microbial agent contains the burkholderia paragallinarum. The invention also provides an application of the burkholderia parasuis in a product for promoting plant growth under a low-temperature or drought condition. The invention further provides a screening method of the burkholderia paragallinarum. The method solves the problem that the growth of the tobacco in the high-altitude (altitude gt; 1200 meters) tobacco region is hindered when meeting low-temperature and drought weather in the seedling stage.

Description

Technical Field

[0001] The invention relates to the technical field of plant microorganisms, and in particular to a Burkholderia parasitica capable of promoting the growth of tobacco seedlings under low temperature and drought conditions, a screening method and an application thereof. Background Art

[0002] As an important economic crop in China, tobacco has high requirements for temperature for its growth. In the high-altitude tobacco-growing areas in the southwest (>1200 meters above sea level), due to the low temperature in early spring, tobacco seedlings are often severely affected by cold damage, which leads to slow plant growth, reduced biomass, and extended growth period, which ultimately affects the yield and quality of flue-cured tobacco. Although certain results have been achieved in alleviating cold damage through breeding and agricultural measures, these methods have the problems of long cycles and high costs, and their applicability is also limited in high-altitude tobacco-growing areas with changeable environments. In addition, spring droughts frequently occur in the southwest before and after the transplanting of tobacco seedlings. Spring drought is also one of the main stress factors restricting tobacco production in the southwest, and the Yunyan 87 variety with the largest planting area in the southwest is sensitive to drought.

[0003] Rhizosphere growth-promoting bacteria can establish a mutually beneficial symbiotic relationship with plants, promote plant growth, enhance stress resistance and disease resistance by releasing volatile or soluble substances, and have the advantages of low cost and environmental friendliness, so they have attracted much attention from researchers. Studies have shown that specific rhizosphere growth-promoting bacteria play an important role in improving crop stress resistance and promoting growth by secreting volatile metabolites or inducing plants to produce stress resistance responses. However, most of the current research is concentrated in warm climate areas, and there are few studies on strains that can significantly promote growth in low temperature environments, especially reports on cold-resistant growth-promoting bacteria for tobacco crops. In addition, many studies are limited to laboratory or greenhouse environments and lack verification of actual field experiments, which limits the widespread application of microbial growth-promoting bacteria in actual production.

[0004] Therefore, there is an urgent need to seek a strain with cold-resistant and growth-promoting properties, in order to provide a new type of microbial resource for tobacco production in high-altitude tobacco areas and promote the growth of tobacco seedlings under low temperature and drought conditions. Summary of the invention

[0005] The purpose of the present invention is to provide a Burkholderia parasitica that can promote the growth of tobacco seedlings under low temperature and drought conditions, a screening method and an application, so as to solve the problem that tobacco seedlings in high-altitude (altitude>1200 meters) tobacco areas encounter low temperatures (<15°C) during the seedling stage and are prone to spring drought during the transplanting period, resulting in growth stunted.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] A paraburkholderia that can promote the growth of tobacco seedlings under low temperature and drought conditions. The paraburkholderia is Paraburkholderia sp. PS-1, the preservation time is October 23, 2024, the preservation address is China Type Culture Collection (CCTCC) of Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the preservation number is CCTCC No: M 20242310.

[0008] The above-mentioned Burkholderia PS-1 was isolated from the rhizosphere soil of tobacco at high altitudes, and experiments have shown that the strain PS-1 exhibits a significant growth-promoting effect on tobacco seedlings under low temperature or drought conditions, not only significantly increasing the biomass of tobacco seedlings, but also improving the root morphology and aboveground growth, including plant height, leaf length and width, etc., which is especially suitable for tobacco seedling breeding and tobacco cultivation in high-altitude and low-temperature environments. It effectively solves the problem of growth stagnation in low temperature and drought weather in tobacco seedlings in high-altitude (>1200 meters above sea level) tobacco areas. Compared with chemical measures, the use of microbial growth-promoting bacteria resources is more environmentally friendly, achieves sustainable development and is low in cost. At the same time, the culture medium required for strain fermentation is low in cost, simple and easy to prepare, which helps promote the development of green agriculture.

[0009] Among them, Burkholderia parasitica PS-1 is a Burkholderia parasitica that efficiently promotes the growth of tobacco seedlings through volatile substances under low temperature conditions. At the same time, experiments have shown that peptone substances in the culture medium will cause the strain to synthesize excessive dimethyl disulfide and dimethyl trisulfide, inhibiting plant growth, while rich glucose and sucrose are conducive to the PS-1 strain to produce growth-promoting volatiles, providing an important theoretical basis for the large-scale use of the strain.

[0010] Preferably, the optimum pH range for the growth of the Burkholderia parakholderia is 5-6, and the optimum temperature range for the growth is 4-37°C.

[0011] Preferably, the optimum temperature for the growth of Burkholderia parakholderia is 30°C.

[0012] The present invention also provides a use of the Burkholderia parakholderia described in the present invention in preparing a microbial agent.

[0013] The present invention also provides a microbial agent, wherein the microbial agent contains the Burkholderia parakaloids.

[0014] Preferably, the bacterial agent is a strain fermentation broth.

[0015] Preferably, the microbial agent further comprises an auxiliary material, and the auxiliary material comprises a carbon source.

[0016] Among them, the carbon source is one or more of starch, cellulose and lignin. These auxiliary materials can enhance the stability and activity of the bacterial agent and improve its adaptability in different environments.

[0017] Experimental studies have shown that when Burkholderia PS-1 uses carbohydrates such as glucose and sucrose as carbon sources, it will produce the volatile substance 2-ethylhexanoate methyl ester to promote the growth of tobacco seedlings, while when it grows on a medium rich in peptone, it can produce high concentrations of volatile substances dimethyl disulfide and dimethyl trisulfide to inhibit the growth of tobacco seedlings. Therefore, during the application process, it should be avoided that it grows in a matrix rich in protein and sulfur-containing organic matter.

[0018] The invention also provides an application of the Burkholderia parakholderia in products promoting plant growth.

[0019] Preferably, the Burkholderia parakholderia can produce the volatile substance methyl 2-ethylhexanoate and / or secrete siderophore to promote plant growth.

[0020] Among them, methyl 2-ethylhexanoate, as a volatile substance, indirectly promotes plant growth and development by affecting the microenvironment around plants, such as the structure of soil microbial communities. Siderophores are low-molecular-weight compounds secreted by bacteria that can efficiently chelate iron ions. Under conditions where iron is limited, they can help plants absorb more iron from the soil, thereby alleviating the iron deficiency symptoms of plants and improving plant growth performance and yield. This makes Burkholderia parasitica a potential plant growth-promoting bacterium with broad application prospects in agricultural production and plant protection.

[0021] Preferably, the plant is a tobacco seedling.

[0022] The present invention also provides a method for screening the paraburkholderia, comprising the following steps:

[0023] Tobacco rhizosphere soil at an altitude of more than 1200 meters was collected as soil samples, and the soil samples were added to sterile water, shaken on a shaker, and diluted to obtain a soil suspension;

[0024] The soil suspension is coated on LB medium and cultured at low temperature until a single colony grows. A single colony is picked according to the colony morphology and streaked on the LB medium for separation and purification until all single colonies have uniform morphology to obtain low-temperature resistant bacteria. The low-temperature resistant bacteria are inoculated on PDA solid medium for culture to obtain an agar block with bacteria;

[0025] Planting tobacco seeds in MS culture medium, culturing and growing seedlings to obtain tobacco seedlings;

[0026] The tobacco seedlings are transplanted into a new MS culture medium, and the agar block with the bacteria is placed under the roots of the tobacco seedlings for low temperature culture, thereby obtaining the Burkholderia parasitica PS-1 that can promote the growth of tobacco seedlings under low temperature and drought conditions.

[0027] Preferably, the temperature at which the low-temperature culture is performed until a single colony grows is lower than 10°C.

[0028] Preferably, the temperature for culturing the low-temperature resistant bacteria on the PDA solid culture medium is 28° C. and the time is 24 hours.

[0029] Preferably, the seedling cultivation specifically includes: under the conditions of normal temperature or 25°C, a light intensity of 8000lx, a humidity of 70%, a light-dark cycle of 12h:12h germination culture, and seedling cultivation for 8 days.

[0030] Preferably, placing the agar block with bacteria below the roots of tobacco seedlings for low-temperature cultivation specifically includes cultivating under the conditions of temperature below 15°C, light intensity of 8000lx, humidity of 70%, and light-dark cycle ratio of 12h:12h.

[0031] Preferably, the LB medium has a formula of: peptone, yeast powder, sodium chloride, solid medium with agar and deionized water; the LB medium is sterilized by high pressure steam at 121° C. for 25 min before use, and then poured into a plate for later use.

[0032] Preferably, the ratio of peptone, yeast powder, sodium chloride, solid culture medium added agar and deionized water is 10g peptone, 5g yeast powder, 5g sodium chloride, 15g solid culture medium added agar and 1000mL deionized water.

[0033] Preferably, the formula of the MS medium is: potassium nitrate, ammonium nitrate, calcium chloride, potassium iodide, zinc sulfate, cobalt chloride, ferrous sulfate, glycine, nicotinic acid, potassium dihydrogen phosphate, magnesium sulfate, boric acid, manganese sulfate, sodium molybdate, copper sulfate, inositol, thiamine hydrochloride, pyridoxine hydrochloride, disodium ethylenediaminetetraacetic acid, sucrose, agar and deionized water.

[0034] Preferably, the MS culture medium is sterilized by high pressure steam at 116° C. for 30 min before use, and then poured into a plate for later use.

[0035] Preferably, the ratio of potassium nitrate, ammonium nitrate, calcium chloride, potassium iodide, zinc sulfate, cobalt chloride, ferrous sulfate, glycine, nicotinic acid, potassium dihydrogen phosphate, magnesium sulfate, boric acid, manganese sulfate, sodium molybdate, copper sulfate, inositol, thiamine hydrochloride, pyridoxine hydrochloride, disodium ethylenediaminetetraacetic acid, sucrose, agar and deionized water is 1.9 g potassium nitrate, 1.65 g ammonium nitrate, 0.44 g calcium chloride, 0.83 mg potassium iodide, 8.6 mg zinc sulfate, 0.0 25mg, ferrous sulfate 27.8mg, glycine 2mg, niacin 0.5mg, potassium dihydrogen phosphate 0.17g, magnesium sulfate 0.37g, boric acid 6.2mg, manganese sulfate 22.3mg, sodium molybdate 0.25mg, copper sulfate 0.025mg, inositol 0.1g, thiamine hydrochloride 0.1mg, pyridoxine hydrochloride 0.5mg, disodium ethylenediaminetetraacetic acid 0.0373g, sucrose 30g, agar 8g and deionized water 1000mL.

[0036] Preferably, the preparation method of the PDA culture medium is: boil the peeled potatoes with water until they are soft but not mushy, then filter with double-layer gauze, add glucose to the filtrate and stir to dissolve, add agar powder, set the pH to natural, sterilize with high pressure steam at 121° C. for 25 min, and pour the plate for later use.

[0037] Preferably, the ratio of potato, water, glucose and agar powder is 200 g potato, 1000 ml water, 20 g glucose and 15 g agar powder.

[0038] Preferably, the screening method for Burkholderia parakholderia comprises the following steps:

[0039] S1. Collect rhizosphere soil of tobacco at an altitude of more than 1200 meters as soil samples. After mixing, weigh 10g of soil and add it to 90mL of sterile water. Oscillate it on a shaker at a speed of 120rpm for 20min, then dilute it 10 times with sterile water. Take the dilution factor of 10. -3 , 10 -4 , 10 -5 The soil suspension is coated on LB medium, and low-temperature culture is carried out at a temperature of 10°C until a single colony grows; according to the different morphologies of the colonies, single colonies are picked and streaked on the LB medium for separation and purification, and all single colonies are considered pure bacteria when they have uniform morphologies; because the culture temperature of the mycelial culture is 10°C, the Burkholderia PS-1 described in this application has the characteristics of rapid growth under low temperature conditions and an optimal growth temperature above 20°C, and all of them are low-temperature resistant bacteria. The strains are preserved with glycerol at a final concentration of 20% for later use.

[0040] S2. Soak tobacco seeds in 0.1% CuSO4 for 20 minutes to disinfect, rinse with sterile water for 3 to 5 times, soak the seeds at 40°C for 30 minutes, and soak them in cold water for 24 hours to obtain sterile tobacco seeds; plant the sterile tobacco seeds on MS culture medium, germinate and culture them at room temperature or 25°C, with a light intensity of 8000lx, a humidity of 70%, and a light-dark cycle of 12h:12h. After 8 days of seedling cultivation, select tobacco seedlings with relatively consistent germination and transplant them to new MS culture medium, with 5 tobacco seedlings per plate as a group, and set 3 replicates;

[0041] S3. Purify the strain preserved in S1, pick out a single colony of the purified strain and inoculate it on PDA solid culture medium, and culture it at a temperature of 28°C for 24 hours; then take the agar block with the bacteria and place it in the MS culture medium with transplanted tobacco seedlings, the agar block is placed under the roots of the tobacco seedlings, and a PDA culture medium agar block of the same size is placed under the roots of the tobacco seedlings as a control; the inoculated tobacco seedlings are cultured for a certain period of time under low temperature conditions of 15°C, a light intensity of 8000lx, a humidity of 70%, and a light-dark cycle ratio of 12h:12h; according to the growth conditions of the roots and aboveground parts of the tobacco seedlings, the strain with the best growth-promoting effect is selected, that is, according to the growth of the tobacco seedlings compared with the non-inoculated control, a strain of Burkholderia parakholderia PS-1 that has a significant promoting effect on the growth of tobacco seedlings is selected, and then at least 3 rounds of rescreening are performed to confirm that Burkholderia parakholderia PS-1 has stable growth-promoting properties.

[0042] The beneficial effects of the present invention are as follows:

[0043] The Burkholderia PS-1 of the present invention is isolated from rhizosphere soil of tobacco at high altitude, and the strain PS-1 has been proved to have a significant growth-promoting effect on tobacco seedlings under low temperature or drought conditions, which not only significantly increases the biomass of tobacco seedlings, but also improves the root morphology and aboveground growth, including plant height, leaf length and width, etc., and is particularly suitable for tobacco seedling breeding and tobacco cultivation in high altitude, low temperature and drought environments. Compared with chemical measures, the use of microbial growth-promoting bacteria resources is more environmentally friendly, achieves sustainable development and is low in cost. At the same time, the culture medium required for strain fermentation is low in cost, simple and easy to prepare, which helps to promote the development of green agriculture and has a wide range of promotion and practical value in the field of plant microbial technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The colony characteristics of strain PS-1 on LB plate;

[0045] Figure 2 This is the morphology of strain PS-1 under an optical microscope;

[0046] Figure 3This is the phylogenetic tree of strain PS-1 constructed based on the 16S rDNA gene sequence;

[0047] Figure 4 This is a graph showing the effect of different pH values ​​on the growth of Burkholderia parakholderia PS-1 isolated in Example 1;

[0048] Figure 5 This is a graph showing the effect of different temperatures on the growth of Burkholderia parakholderia PS-1 isolated in Example 1;

[0049] Figure 6 This is a comparison chart of the effect of strain PS-1 on tobacco seedlings under 15°C.

[0050] Figure 7 This is a comparison of the fresh weight of aboveground / underground parts of tobacco seedlings treated with CK and strain PS-1 on MS medium at 15°C;

[0051] Figure 8 This is a comparison of the fresh weight of aboveground / underground parts of tobacco seedlings treated with CK and strain PS-1 on MS medium at 25°C;

[0052] Fig. 9 This is a comparison of the siderophore production results of strain PS-1 at 15°C and 25°C;

[0053] Fig.10 This is a comparison chart of the results of using two-plate plates to confirm that strain PS-1 promotes tobacco seedling growth through volatile substances;

[0054] Fig.11 The PS-1 strain was grown on MS plates supplemented with 5% PEG-6000 to simulate drought. DETAILED DESCRIPTION

[0055] The following will describe the embodiments of the present invention with reference to the accompanying drawings and preferred embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be understood that the preferred embodiments are only for illustrating the present invention, not for limiting the scope of protection of the present invention.

[0056] Example 1

[0057] A method for screening Burkholderia parakholderia comprises the following steps:

[0058] S1. Collect tobacco rhizosphere soil at an altitude of more than 1200 meters as soil samples. After mixing, weigh 10g of soil and add it to 90mL of sterile water. Oscillate on a shaker at a speed of 120rpm for 20min, then dilute it 10 times with sterile water. -3 , 10 -4 , 10 -5 The soil suspension was coated on LB medium and cultured at 10°C until a single colony grew. According to the different morphology of the colonies, single colonies were picked and streaked on the LB medium for separation and purification. All single colonies with uniform morphology were considered as pure bacteria of low-temperature-resistant bacteria. The low-temperature-resistant bacteria were preserved with glycerol at a final concentration of 20% for later use.

[0059] S2. Soak tobacco seeds in a 0.1% CuSO4 solution for 20 minutes to disinfect, rinse with sterile water for 3 to 5 times, soak the seeds at 40°C for 30 minutes, and soak them in cold water for 24 hours to obtain sterile tobacco seeds; the sterile tobacco seeds are planted on MS culture medium, and germinated and cultured at a temperature of 25°C, a light intensity of 8000lx, a humidity of 70%, and a light-dark cycle of 12h:12h. After 8 days of seedling cultivation, select the tobacco seedlings with consistent germination observed by naked eye and transplant them to new MS culture medium, with 5 tobacco seedlings per plate as a group, and 3 replicates are set;

[0060] S3, purify the low-temperature resistant bacteria preserved in S1, pick out a single colony of the purified strain in advance and inoculate it on PDA solid culture medium, and culture it at a temperature of 28°C for 24 hours; then take the agar block with bacteria and place it in the MS culture medium with transplanted tobacco seedlings, and place the agar block under the roots of the tobacco seedlings, and take the PDA culture medium agar block of the same size without bacteria and place it under the roots of the tobacco seedlings as a control; the tobacco seedlings inoculated with the bacteria are used as the experimental group, and the tobacco seedlings not inoculated with the bacteria are used as the control group. The cells were cultured for 25 to 30 days at a temperature of 15°C, a light intensity of 8000lx, a humidity of 70%, and a light-dark cycle ratio of 12h:12h. The strains with the best growth-promoting effect were selected according to the growth conditions of the roots and above-ground parts of the tobacco seedlings. That is, based on the growth of the tobacco seedlings compared with the non-inoculated control, a low-temperature-resistant rhizosphere growth-promoting bacterium PS-1 was selected that had a significant promoting effect on the growth of tobacco seedlings. Subsequently, at least 3 rounds of rescreening confirmed that the low-temperature-resistant rhizosphere growth-promoting bacterium PS-1 had stable growth-promoting activity.

[0061] The formula of the LB medium is as follows: 10 g of peptone, 5 g of yeast powder, 5 g of sodium chloride, 15 g of agar added to the solid medium, and 1000 mL of deionized water. The LB medium needs to be sterilized by high-pressure steam at 121°C for 25 min before use and poured into a plate for later use.

[0062] The formula of the above-mentioned MS medium is: 1.9g potassium nitrate, 1.65g ammonium nitrate, 0.44g calcium chloride, 0.83mg potassium iodide, 8.6mg zinc sulfate, 0.025mg cobalt chloride, 27.8mg ferrous sulfate, 2mg glycine, 0.5mg nicotinic acid, 0.17g potassium dihydrogen phosphate, 0.37g magnesium sulfate, 6.2mg boric acid, 22.3mg manganese sulfate, 0.25mg sodium molybdate, 0.025mg copper sulfate, 0.1g inositol, 0.1mg thiamine hydrochloride, 0.5mg pyridoxine hydrochloride, 0.0373g disodium ethylenediaminetetraacetic acid, 30g sucrose, 8g agar and 1000mL deionized water. Before use, the MS medium needs to be sterilized by high-pressure steam at 116°C for 30min and poured into plates for use.

[0063] The preparation method of the above-mentioned PDA culture medium is as follows: 200g of peeled and washed potatoes are cut into small pieces, 1000ml of water is added and boiled until soft but not mushy, then filtered with double-layer gauze, 20g of glucose is added to the filtrate and stirred to dissolve, 15g of agar powder is added, the pH is natural, and then high-pressure steam sterilization is carried out at a temperature of 121°C for 25min, and the plate is poured for use.

[0064] 16S rDNA sequencing of the obtained low-temperature-tolerant rhizospheric growth-promoting bacteria PS-1 showed that it was Burkholderia paraspora.

[0065] The Paraburkholderia sp. PS-1 was deposited in the China Type Culture Collection (CCTCC) of Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province on October 23, 2024, with the deposit number: CCTCCNo:M20242310.

[0066] Example 2

[0067] Identification of strains

[0068] 1) Morphological observation of strain PS-1

[0069] The colony characteristics of the low-temperature-resistant rhizosphere growth-promoting bacteria PS-1 isolated in Example 1 on LB solid medium are as follows: Figure 1 The morphology of the low-temperature-resistant rhizosphere growth-promoting bacteria PS-1 isolated in Example 1 under an optical microscope is as follows Figure 2 shown.

[0070] from Figure 1 It can be seen from the observation that the low-temperature-resistant rhizosphere growth-promoting bacteria PS-1 isolated in Example 1 forms white, round protrusions and neatly edged colonies on the LB solid culture medium.

[0071] from Figure 2It can be seen from the observation that the cold-resistant rhizosphere growth-promoting bacteria PS-1 isolated in Example 1 is short rod-shaped under optical microscope and Gram-negative.

[0072] 2) Molecular identification of strain PS-1

[0073] The cold-resistant rhizosphere growth-promoting bacteria PS-1 strain isolated in Example 1 was inoculated into liquid LB medium, the bacteria were collected by centrifugation, and the bacterial DNA was extracted as a template. The bacterial universal primer 27F (SEQ ID No. 1: 5′

[0074] -AGAGTTTGATCMTGGCTCAG-3′) and 1492R (SEQ ID No. 2: 5′

[0075] -GGTTACCTTGTTACGACTT-3′) for PCR amplification of 16S rRNA gene fragments. After confirming the size of the PCR product by agarose gel electrophoresis, it was sent to Sangon Biotech (Shanghai) Co., Ltd. for gene sequence determination. The obtained sequence was searched and compared with multiple sequences with high similarity in GenBank using BLASTn, and the 16S rRNA gene fragments of bacteria of similar species were downloaded. The sequences were aligned in the software MEGA 11.0, and the Neighbor-Joining algorithm was selected to construct a phylogenetic tree, such as Figure 3 As shown, the strain was initially identified as Paraburkholderia sp.PS-1.

[0076] The amplification conditions of PCR were as follows: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 1 min, annealing at 55°C for 0.5 min, extension at 72°C for 1 min, 30 cycles; and final extension at 72°C for 10 min.

[0077] The specific sequence of 16S rDNA is SEQ ID No.3:

[0078] GGAGTGGGGGGCATGCTTACCATGCAGTCGAACGGCAGCACGGGGGCAACCCTGGTG

[0079] GCGAGTGGCGAACGGGTGAGTAATACATCGGAACGTGTCCTGGAGTGGGGGATAGCC

[0080] CGGCGAAAGCCGGATTAATACCGCATACGATCTAGGGATGAAAGCGGGGGACCGAAA

[0081] GGCCTCGCGCTCAAGGGGCGGCCGATGGCAGATTAGCTAGTTGGTGGGGTAAAGGCCT

[0082] ACCAAGGCGACGATCTGTAGCTGGTCTGAGAGGACGACCAGCCACACTGGGACTGAG

[0083] ACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGGGCAA

[0084] CCCTGATCCAGCAATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTTG

[0085] TCCGGAAAGAAAACCTCCGTCCTAATACGGTGGGGGGATGACGGTACCGGAAGAATA

[0086] AGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTAATC

[0087] GGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTCGCTAAGACCGATGTGAAATCCCC

[0088] GGGCTTAACCTGGGAACTGCATTGGTGACTGGCGGGCTAGAGTATGGCAGAGGGGGG

[0089] TAGAATTCCACGTGTAGCAGTGAAATGCGTAGAGATGTGGAGGAATACCGATGGCGAA

[0090] GGCAGCCCCCTGGGCCAATACTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGG

[0091] ATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAACTAGTTGTCGGGTCTTCATT

[0092] GACTTGGTAACGAAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGTCGCAAGA

[0093] TTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGATTAATT

[0094] CGATGCAACGCGAAAAACCTTACCTACCCTTGACATGTACGGAATCCTGCTGAGAGTGGGGAGTGCCCGAAAGGGAGCCG.

[0095] Example 3

[0096] Effect of different pH values ​​on the growth of Burkholderia PS-1 isolated in Example 1

[0097] The specific operation is as follows: the Burkholderia PS-1 isolated in Example 1 was inoculated at an inoculum of 1% into a culture medium (200 g of peeled potatoes, 20 g of brown sugar and 1000 ml of water) with different initial pH values ​​(pH values ​​include 4, 5, 6, 7, 8 and 9), wherein the initial pH value of the culture medium was adjusted by sodium hydroxide and hydrochloric acid, and the culture was shaken for 18 h at a temperature of 28° C. and a rotation speed of 150 rpm to obtain a bacterial solution, and the OD of the bacterial solution was measured by a spectrophotometer. 600 Value, the result is Figure 4 shown.

[0098] from Figure 4 From the analysis, we can see that the optimum pH of PS-1 is 5-6, but it can grow in both acidic and alkaline environments. From the perspective of pH adaptability, it is suitable for field application.

[0099] Example 4

[0100] Effect of different temperatures on the growth of Burkholderia PS-1 isolated in Example 1

[0101] The specific operation is as follows: the Burkholderia PS-1 isolated in Example 1 was inoculated into a culture medium (200 g of peeled potatoes, 20 g of brown sugar and 1000 ml of water), and cultured under shaking conditions of 15°C, 20°C, 30°C and 37°C and a rotation speed of 150 rpm for 18 h to obtain a bacterial solution, and the OD600 value of the bacterial solution was measured by a spectrophotometer. The results are as follows: Figure 5 shown.

[0102] from Figure 5 The analysis showed that PS-1 can grow at temperatures between 15 and 37°C, with the optimum growth temperature being 30°C, indicating that PS-1 is also suitable for tobacco seedling breeding and field application from a temperature perspective.

[0103] Example 5

[0104] A method of using the Burkholderia parakholderia PS-1 isolated in Example 1 to promote tobacco seedling growth comprises the following steps:

[0105] S1. The Burkholderia parakholderiae PS-1 isolated in Example 1 was inoculated into a culture medium (200 g of peeled potatoes, 20 g of brown sugar and 1000 ml of water), and cultured under shaking conditions of 28° C. and 150 rpm for 18 h to obtain a bacterial solution;

[0106] S2. Take 1 μL of the bacterial solution obtained in S1 and inoculate it near the roots of each tobacco seedling growing in MS medium, which is used as the experimental group (PS-1), and the blank group (CK) is used as the control. The culture is carried out at a temperature of 15°C for 20 days to observe the growth-promoting effect of the strain on tobacco seedlings. The results are as follows: Figure 6 As shown, the cultivated tobacco seedlings were processed to obtain the fresh weight of the above-ground part and the underground part. Figure 7 shown.

[0107] from Figure 6 The comparative analysis showed that Burkholderia PS-1 significantly promoted the growth and root development of tobacco seedlings.

[0108] from Figure 7 The comparative analysis showed that under the condition of temperature of 15℃, Burkholderia PS-1 significantly increased the fresh weight of the aboveground and underground parts of tobacco seedlings.

[0109] Example 6

[0110] A method of using the Burkholderia parakholderia PS-1 isolated in Example 1 for growing tobacco seedlings comprises the following steps:

[0111] S1. The Burkholderia parakholderiae PS-1 isolated in Example 1 was inoculated into a culture medium (200 g of peeled potatoes, 20 g of brown sugar and 1000 ml of water), and cultured under shaking conditions of 28° C. and 150 rpm for 18 h to obtain a bacterial solution;

[0112] S2. Take 1 μL of the bacterial solution obtained in S1 and inoculate it near the roots of each tobacco seedling grown in MS medium, which is used as the experimental group (PS-1), and the blank group (CK) is used as the control. The tobacco seedlings obtained by culture are treated at a temperature of 25°C for 20 days, and the fresh weight of the above-ground part and the underground part is obtained as shown in the following table. Figure 8 shown.

[0113] from Figure 8The comparative analysis showed that under the condition of 25℃, Burkholderia PS-1 significantly increased the aboveground fresh weight of tobacco seedlings and slightly promoted the underground fresh weight. It can be seen that PS-1 has a good promoting effect on the growth of tobacco seedlings under non-low temperature conditions and has a wide range of application temperatures.

[0114] Example 7

[0115] Tests for siderophore production

[0116] The specific operation steps are as follows: the Burkholderia PS-1 strain isolated in Example 1 is inoculated on a CAS blue detection medium plate, the screened strain is tested for siderophore secretion, and cultured at low temperature 15°C and room temperature 28°C for 3 days, respectively, and whether the siderophore is produced is determined based on the halo around the colony.

[0117] CAS culture medium: ① Prepare CAS staining solution first: dissolve 0.0605g CAS (chrome azure) in 50mL deionized water, add 10mL 1mmol / LFeCl3 solution (containing 12mmol / LHCl), this is solution A; dissolve 0.0729g HDTMA (hexadecyltrimethylammonium bromide) in 40mL deionized water to obtain solution B; sterilize solutions A and B separately (121℃, 20min), store them separately, mix solution A and solution B at a ratio of 6:4 (v / v) when used, slowly add solution A to solution B, stir evenly to obtain CAS staining solution, and add it to the CAS detection plate at a ratio of 5% when used. ② Prepare 0.1 mol / L phosphate buffer (pH = 6.8): each 100 mL contains Na2HPO4 0.9627 g, NaH2PO4 0.4542 g, KH2PO4 0.075 g, NH4Cl 0.250 g, and NaCl 0.125 g. When using, dilute it 10 times and then add it to the CAS detection plate at a ratio of 5%. ③ Finally, prepare the CAS detection plate: add 15g of Agar to 1000mL of deionized water, sterilize at 121℃ for 20min, then add 30mL of 10% casamino acids (sterilized at 115℃ for 15min, stored separately), 10mL of 20% sucrose (sterilized at 115℃ for 15min, stored separately), 1mL of 1mmol / LCaCl2 (sterilized at 121℃ for 20min, stored separately), 20mL of 1mmol / LMgSO4·7H2O (sterilized at 121℃ for 20min, stored separately), then add the diluted phosphate buffer and the mixed CAS dye solution at about 60℃, pour the plate to obtain the CAS detection plate, and the results are as follows. Fig. 9 shown.

[0118] from Fig. 9It can be seen that PS-1 produced a light yellow halo around the colony when cultured at 15℃ and 25℃, and the halo at 15℃ was larger. It can be seen that the bacteria produced iron carriers under these two temperature conditions and was not restricted by low temperature.

[0119] Example 8

[0120] Using bisection plates to confirm that PS-1 promotes tobacco seedling growth through volatile substances

[0121] The specific operation steps are as follows: add 10 mL of MS solid culture medium containing 3% sucrose to the two chambers of the two-part culture dish, plant 10 sterilized tobacco seeds on one side, take the WXZ-9 agar block grown on PDA, and inoculate it on the other side of the MS culture medium. Use the PDA culture medium agar block without bacteria as the control. Do 5 replicates for each treatment, seal the mouth of the culture dish with parafilm, place it horizontally in a 15℃ light incubator and culture it for 25 days, and observe the growth difference between the control group and the inoculated group. The results are as follows Fig.10 shown.

[0122] from Fig.10 It can be seen that the tobacco seedlings inoculated with PS-1 on one side grew significantly better than those in the control group, with wider leaves and more developed root systems. The two sides of the two-part culture dish are only connected near the lid, and the culture medium side is completely separated. In this case, PS-1 also has significant growth-promoting properties, indicating that the bacteria mainly promotes tobacco seedling growth through volatile substances.

[0123] Example 9

[0124] Experiments were conducted in actual field environments in high-altitude tobacco-growing areas (altitude > 1200m) in Pengshui County, Chongqing, to verify the growth-promoting effect of strain PS-1.

[0125] The specific operations are:

[0126] Bacterial culture: The Burkholderia parakholderiae PS-1 isolated in Example 1 was inoculated into a culture medium (200 g of peeled potatoes, 20 g of brown sugar and 1000 ml of water), and cultured under shaking conditions of 28° C. and 150 rpm for 18 h to obtain a bacterial solution;

[0127] Strain application: When the tobacco seedlings were transplanted in the field, the cultured bacterial solution was added to the rooting water applied during transplanting at an amount of 5% (v / v). Other transplanting work was performed according to routine operations. The experimental group (PS-1) was added with bacterial solution, and the control group (CK) was added with an equal amount of sterile culture medium. After the tobacco leaves were transplanted and grown to the cluster stage and the dome stage, relevant tests were performed on the tobacco leaves. The results are shown in Tables 1 to 6.

[0128] Table 1 Effects of strains on the appearance and growth of K326 tobacco leaves in Pengshui base

[0129]

[0130] Table 2 Effects of strains on the appearance and growth of Yunyan 87 tobacco leaves at Pengshui base

[0131]

[0132] Table 3 Effects of strains on the dry and fresh weights of different organs of K326 at the Pengshui base during the cluster period

[0133]

[0134] Table 4 Effects of different low-temperature resistant bacteria on the dry and fresh weights of different organs of Yunyan 87 at the Pengshui base

[0135]

[0136]

[0137] Table 5 Effect of strains on economic benefits of K326 in Pengshui base

[0138]

[0139] Table 6 Effect of strains on economic benefits of Yunyan 87 in Pengshui base

[0140]

[0141] The field test results in Tables 1 to 6 show that the root-inoculated paraburkholderia PS-1 solution significantly promoted tobacco growth. Compared with the control group, paraburkholderia PS-1 increased tobacco plant height, stem girth, maximum leaf length, maximum leaf width, and fresh weight and dry weight of leaves, stems and roots. After a single application (the cost of use is about 10 yuan / mu), the yield per mu increased by 400-700 yuan. This proves that paraburkholderia PS-1 can effectively promote tobacco growth under chilling conditions in high-altitude tobacco areas, indicating the feasibility of the actual application of the strain.

[0142] Example 10

[0143] PS-1 promotes the growth of tobacco seedlings under drought conditions

[0144] 5% polyethylene glycol PEG-6000 was added to the above MS medium to simulate drought environment, and the tobacco seedlings that had germinated for 8 days were transplanted on the MS medium with PEG added. A hole was punched to take out the PDA medium with PS-1 growing on it and placed under the roots of the tobacco seedlings. A control group without inoculation and PEG addition and a control group without inoculation and PEG addition were set up. The cells were placed in a 25℃ light incubator for 27 days, and the growth differences between the control group and the inoculated group were observed. Fig.11 shown.

[0145] from Fig.11It can be seen that adding 5% PEG-6000 to simulate drought caused the growth of tobacco seedlings to be hindered, and the root length and leaf width were significantly smaller than those of the control without PEG-6000. However, adding PS-1 can completely relieve the drought stress caused by adding PEG-6000, and the growth of tobacco seedlings returned to normal, and the growth was significantly better than the blank control without PEG.

[0146] In summary, the Burkholderia PS-1 of the present invention is isolated from the rhizosphere soil of tobacco at high altitudes, and the strain PS-1 has been proved to have a significant growth-promoting effect on tobacco seedlings under low temperature conditions, which not only significantly increases the biomass of tobacco seedlings, but also improves the root morphology and aboveground growth, including plant height, leaf length and width, etc., and is particularly suitable for tobacco seedling breeding and tobacco cultivation in high altitude, low temperature or arid environments. Compared with chemical measures, the use of microbial growth-promoting bacteria resources is more environmentally friendly, achieves sustainable development and is low in cost. At the same time, the culture medium required for strain fermentation is low in cost, simple and easy to prepare, which helps to promote the development of green agriculture and has a wide range of promotion and practical value in the field of plant microbial technology.

[0147] The above embodiments are only preferred implementations of the present invention, and the protection scope is not limited thereto. Equivalent substitutions or changes made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention.

Claims

1. A Burkholderia parasitica that can promote the growth of tobacco seedlings under low temperature and drought conditions, characterized in that: The Paraburkholderia is Paraburkholderia sp. PS-1, the preservation time is October 23, 2024, the preservation address is China Type Culture Collection (CCTCC) of Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the preservation number is CCTCC No: M 20242310.

2. The Burkholderia parakholderia according to claim 1, characterized in that The pH value range for the growth of the Burkholderia parakholderia is 5-6, and the temperature range for the growth is 4-37°C.

3. Use of the Burkholderia parakholderiae as claimed in claim 1 in the preparation of microbial inoculants.

4. A microbial agent, characterized in that: The bacterial agent contains the Burkholderia parakholderia as claimed in claim 1.

5. Use of the Burkholderia parakholderiae as claimed in claim 1 in a product for promoting plant growth.

6. The use according to claim 5, characterized in that: The paraburkholderia can produce the volatile substance 2-ethylhexanoic acid methyl ester and / or secrete iron carrier to promote plant growth.

7. The use according to claim 5, characterized in that: The plant is a tobacco seedling.

8. A method for screening Burkholderia parakholderia according to claim 1, characterized in that: The following steps are involved: Tobacco rhizosphere soil at an altitude of more than 1200 meters was collected as soil samples, and the soil samples were added to sterile water, shaken on a shaker, and diluted to obtain a soil suspension; The soil suspension is coated on LB medium and cultured at low temperature until a single colony grows. A single colony is picked according to the colony morphology and streaked on the LB medium for separation and purification until all single colonies have uniform morphology to obtain low-temperature resistant bacteria. The low-temperature resistant bacteria are inoculated on PDA solid medium for culture to obtain an agar block with bacteria; Planting tobacco seeds in MS culture medium, culturing and growing seedlings to obtain tobacco seedlings; The tobacco seedlings are transplanted into a new MS culture medium, and the agar block with the bacteria is placed under the roots of the tobacco seedlings for low temperature culture, thereby obtaining the Burkholderia parasitica that can promote the growth of tobacco seedlings under low temperature and drought conditions.

9. The method for screening Burkholderia parakholderia according to claim 8, characterized in that: The temperature of the low-temperature culture until a single colony grows is lower than 10°C; and / or, inoculating the low-temperature resistant bacteria on PDA solid medium and culturing at 28° C. for 24 h; And / or, the culturing and seedling raising specifically includes: germination and culturing at room temperature or 25°C, with a light intensity of 8000 lx, a humidity of 70%, a light-dark cycle of 12h:12h, and seedling raising for 8 days; And / or, placing the agar block with bacteria under the roots of tobacco seedlings for low-temperature cultivation specifically includes cultivating under the conditions of temperature below 15°C, light intensity of 8000lx, humidity of 70%, and light-dark cycle ratio of 12h:12h.

10. The method for screening Burkholderia parakholderia according to claim 8, characterized in that: The formula of the LB medium is: peptone, yeast powder, sodium chloride, solid medium with agar and deionized water; The LB medium was sterilized by high pressure steam at 121° C. for 25 min before use; And / or, the formula of the MS medium is: potassium nitrate, ammonium nitrate, calcium chloride, potassium iodide, zinc sulfate, cobalt chloride, ferrous sulfate, glycine, nicotinic acid, potassium dihydrogen phosphate, magnesium sulfate, boric acid, manganese sulfate, sodium molybdate, copper sulfate, inositol, thiamine hydrochloride, pyridoxine hydrochloride, disodium ethylenediaminetetraacetic acid, sucrose, agar and deionized water; The MS medium was sterilized by high pressure steam at 116° C. for 30 min before use; And / or, the preparation method of the PDA culture medium is: adding water to peeled potatoes and boiling until they are soft but not mushy, then filtering with double-layer gauze, adding glucose to the filtrate and stirring to dissolve, adding agar powder, and sterilizing with high-pressure steam at a temperature of 121° C. for 25 minutes to obtain the culture medium.

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