A grass-roots Burkholderia parahaemolyticus and its bacterial agent and application

Abstract

The present invention provides a grass-roots paraburkholderia, a bacterial agent and an application thereof. The grass-roots paraburkholderia ( Paraburkholderia graminis ) is deposited as CCTCC NO: M20242399. The grass-roots paraburkholderia provided by the present invention has the functions of nitrogen fixation, phosphorus solubilization, siderophore production and indoleacetic acid production. The grass-roots paraburkholderia of the present invention and the bacterial agent using the same as the effective active ingredient can promote the germination of plant seeds and the growth of crops.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a Burkholderia paraherbivora and a bacterial agent and application thereof. Background Art

[0002] PGPB is a kind of beneficial microorganism that can promote plant growth. It can directly or indirectly improve crop yield, quality and soil fertility through various mechanisms. Among them, the direct mechanism includes the production of plant hormones to regulate plant growth and improve plant nutrient supply (fixing nitrogen in the atmosphere, dissolving insoluble phosphorus in the soil and decomposing potassium-containing minerals, etc.); the indirect mechanism refers to biological control, such as inducing plant systemic resistance, fighting plant pathogens by synthesizing antibiotics and chitinase, and competing for rhizosphere niches or effective iron in the soil.

[0003] However, with the development of biotechnology, the development of microbial resources that promote plant growth currently relies more on artificial means such as artificial mutagenesis screening and genetic engineering modification. The strains obtained are prone to problems of difficulty in colonization or the effects are far from the laboratory detection level in actual production. In order to meet the needs of agricultural production, it is urgent to develop new beneficial strains with better and more stable growth-promoting effects, which are easier to colonize and can be applied to actual production. Summary of the invention

[0004] In order to overcome the problems existing in the background technology, the present invention provides a grass-roots paraburkholderia and its bacterial agent and application. The grass-roots paraburkholderia and its bacterial agent have good nitrogen fixation, phosphate solubilization, siderophore production and indoleacetic acid production capabilities, and can effectively promote seed germination and crop growth.

[0005] To achieve the above object, the present invention provides a first aspect of a Burkholderia paraherbivora strain ( Paraburkholderia graminis ), the deposit number of the strain is: CCTCC NO: M20242399.

[0006] The second aspect of the present invention provides the use of the above-mentioned Burkholderia parafollicularis in nitrogen fixation, phosphate solubilization, siderophore production or indoleacetic acid production.

[0007] The third aspect of the present invention provides a bacterial agent having the function of promoting plant growth, wherein the active ingredient in the bacterial agent includes the above-mentioned Burkholderia paraherbivora.

[0008] The fourth aspect of the present invention provides a method for promoting plant seed germination, wherein the plant seeds are immersed in a bacterial solution containing the above-mentioned bacterial agent and / or the above-mentioned Burkholderia serrata, wherein the effective viable bacteria count of Burkholderia serrata in the bacterial solution is (1-10)×10 5 CFU / mL.

[0009] A fifth aspect of the present invention provides a method for promoting plant growth, the method comprising applying the above-mentioned Burkholderia pararhizogenes or the above-mentioned bacterial agent to the rhizosphere soil of the plant.

[0010] Through the above technical solution, the present invention can at least achieve the following beneficial effects:

[0011] 1. The grass root Burkholderia provided by the present invention has excellent nitrogen fixation and phosphorus solubilization effects. It can not only solubilize inorganic phosphorus, but also organic phosphorus. In addition, the strain can also produce siderophores and indoleacetic acid, thereby effectively improving soil quality and increasing the content of nutrients that can be used by plants in the soil, thereby having a good effect of promoting plant growth.

[0012] 2. The grass-roots Burkholderia provided by the present invention is a strain isolated from the soil of tobacco-wheat rotation farmland. Compared with the strains screened by laboratory mutagenesis, it has better adaptability to the planting environment and can better colonize in the soil, thereby exerting a more stable growth-promoting effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a colony morphology of strain YNK PB0001 on NA medium in Example 1;

[0014] Figure 2 This is the phylogenetic tree of strain YNK PB0001 in Example 1 drawn using the Neighbor-Joining method;

[0015] Figure 3 The following are the results of strain YNK PB0001 in Example 2: (A) nitrogen fixation effect; (B) organic phosphorus decomposition effect; (C) inorganic phosphorus decomposition effect; (D) siderophore production effect;

[0016] Figure 4 This is a qualitative result diagram of the IAA production capacity of strain YNK PB0001 in Example 2;

[0017] Figure 5 The figure is a graph showing the results of the germination promotion experiment of strain YNK PB0001 in Example 3, in which CK1 represents the sterile water control group, CK2 represents the NB blank medium, and T1-T4 represent experimental groups 1-4;

[0018] Figure 6 The figure is a graph showing the growth promotion experiment results of strain YNK PB0001 in Example 4, in which CK1 represents the sterile water control group, CK2 represents the NB blank medium, and T1-T3 represent experimental groups 1-3;

[0019] Biological Deposit

[0020] The grass root periphyton Burkholderia YNK PB0001 provided by the present invention is classified and named Paraburkholderia graminis YNK PB0001 was deposited in the China Center for Type Culture Collection (CCTCC) on October 31, 2024, and the deposit address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, and its deposit number is CCTCC NO: M20242399. DETAILED DESCRIPTION

[0021] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0022] During the research, the inventors of the present invention isolated a grass rhizosphere Burkholderia paraspora from the long-term flue-cured tobacco-barley rotation field soil. It was found through testing that the strain has good nitrogen fixation, phosphorus solubilization, siderophore production and indoleacetic acid production performance. After further research, the inventors also found that the strain can effectively promote seed germination, and the fermentation liquid of the strain is applied to the rhizosphere soil of plants, which can effectively promote plant growth, improve agricultural productivity, and promote the green and sustainable development of ecological agriculture.

[0023] Based on the above findings, the present invention provides a Burkholderia parafollicularis strain ( Paraburkholderia graminis ), the deposit number of the strain is CCTCC NO: M20242399.

[0024] The second aspect of the present invention provides the use of the grass root periphery Burkholderia described in the first aspect in nitrogen fixation, phosphate solubilization, siderophore production and indoleacetic acid production.

[0025] In the present invention, "nitrogen fixation" and "phosphorus solubilization" refer to the increase of the N and P content in the soil available for plant use through the action of the grass root periphery Burkholderia provided by the present invention. For example, the inorganic phosphorus components (such as Ca 3 (PO 4 ) 2 、FePO 4 The invention can convert the organic phosphorus components (such as phosphatidylcholine, inositol hexaphosphate, etc.) into a form that can be used by plants, thereby promoting the absorption and utilization of phosphorus in the soil by plants. "Sterophore production" means that the strain provided by the invention can secrete iron carriers under iron deficiency conditions to help microorganisms absorb Fe from the environment. 3+ , increase Fe in the environment 3+Move and promote crops to absorb Fe 3+ absorption.

[0026] The third aspect of the present invention provides a bacterial agent having the function of promoting plant growth, wherein the active ingredient in the bacterial agent includes the Burkholderia pararhizogenes described in the first aspect.

[0027] According to a preferred embodiment of the present invention, the bacterial agent is a liquid bacterial agent.

[0028] Preferably, the content of the Burkholderia paragracilis in the liquid bacterial agent is not less than 1×10 6 CFU / mL. Preferably 1×10 7 -1×10 10 CFU / mL. More preferably 5×10 8 -5×10 9 CFU / mL.

[0029] The fourth aspect of the present invention provides the use of the Burkholderia paraherbivora described in the first aspect, or the bacterial agent described in the third aspect, in promoting plant seed germination and plant growth.

[0030] A fifth aspect of the present invention provides a method for promoting plant seed germination and plant growth.

[0031] In the present invention, the method for promoting plant seed germination is: soaking the plant seeds in the bacterial solution of the Burkholderia paragracilis described in the first aspect and / or the bacterial agent described in the third aspect, wherein the effective viable bacteria count of the Burkholderia paragracilis in the bacterial solution is 1×10 5 -1×10 6 CFU / mL.

[0032] In the present invention, the method for promoting plant growth is: applying the Burkholderia pararhizosphere described in the first aspect or the bacterial agent described in the third aspect to the rhizosphere soil of the plant.

[0033] According to a preferred embodiment of the present invention, the application amount of the grass root paraburkholderia is not less than 1×10 8 CFU / strain / time, preferably 1×10 9 -1×10 12 CFU / strain / time.

[0034] According to a preferred embodiment of the present invention, the dosage of the bacterial agent is such that the application amount of Burkholderia paragracilis is not less than 1×10 8 CFU / strain / time, preferably 1×10 9 -1×10 12CFU / strain / time. Preferably, the grass root periphyton Burkholderia or bacterial agent is applied 1-3 times per crop.

[0035] Preferably, the plant is selected from the Solanaceae family, genus Solanum, most preferably tomato.

[0036] The present invention will be described in detail below by way of examples. It should be understood that the following examples are only used to further explain and illustrate the content of the present invention by way of example, and are not intended to limit the present invention.

[0037] In the following examples, unless otherwise specified, the reagents and materials used were all commercial products purchased from regular chemical / biological reagent or material suppliers, and the reagents were all analytical grade.

[0038] In the following examples, unless otherwise specified, the operating temperature is room temperature (25±5° C.).

[0039] Example 1

[0040] This example is used to illustrate the acquisition, identification and preservation of Burkholderia spp. CCTCC NO: M20242399.

[0041] (I) Strain isolation and purification

[0042] Nutrient agar medium (NA medium) was used in the process of strain isolation and purification. The preparation method was as follows: 10 g of peptone, 3 g of beef powder, and 5 g of sodium chloride were weighed and added to 1000 mL of water, the pH was adjusted to 7.3 ± 0.1, and then 15 g of agar was added and sterilized at 121 °C under high pressure for 15 min.

[0043] A bacterial strain named YNK PB0001 was isolated and purified from soil samples collected from long-term flue-cured tobacco-barley rotation fields by using the dilution spread plate method and the plate streak method.

[0044] (II) Strain identification

[0045] 1. Identification of bacterial morphological, physiological and biochemical characteristics

[0046] Physiological and biochemical tests were performed on strain YNK PB0001 according to the Bergey's Manual of Bacterial Identification and the Manual of Identification of Common Bacterial Systems, and the colony morphology characteristics were described.

[0047] Colony & Cell Morphology: Figure 1 The colony morphology of strain YNK PB0001 on NA medium is shown. It can be seen from the figure that the colony of the strain is nearly circular, with neat edges, white, and a convex middle, translucent, moist and shiny. Observation under an optical microscope shows that the strain is a rod-shaped bacterium with flagella.

[0048] Physiological and biochemical characteristics: Gram staining of strain YNK PB0001 is negative, and the pH tolerance range is 6-8. Catalase, oxidase, urease and arginine dihydrogenase activities are all positive; it can reduce nitrate to nitrite, but does not denitrify. It can use the following substances as the sole carbon source: glycerol, D- and L-arabinose, ribose, galactose, D-glucose, D-fructose, D-mannose, inositol, mannitol, sorbitol, D-arabitol, gluconic acid and 2-keto-gluconic acid; it cannot use L-sorbose, methyl α-D-xyloside, methyl α-D-mannoside, methyl α-D-glucoside, inulin, melleose, starch, glycogen or D-furanose as the sole carbon source. At the same time, the strain does not acidify glucose, hydrolyze suberin, or produce indole or gelatinase. It can grow on L-xylose, lactose, rhamnose, trehalose, D-xylulose, L-arabitol, xylitol, and beet sugar, but not on sorbitol or D-tagose as the sole carbon source.

[0049] 2. Molecular identification

[0050] The total DNA of strain YNK PB0001 was extracted by Chelex extraction method as a template, 27F (5'-AGAGTTTGATCCTGGCTCAG-3') was used as the upstream primer, 1492R (5'-TACGGCTACCTTGTTACGACTT-3') was used as the downstream primer, and the reaction system and conditions in Table 1 were used for 16S rRNA amplification.

[0051] Table 1 PCR system and conditions

[0052]

[0053] The amplified product was purified and recovered by 1% agarose gel electrophoresis (using the gel recovery and purification kit produced by Guangzhou Meiji Biotechnology Co., Ltd.), and then sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results are shown in SEQ ID NO.1. The sequencing results were compared and analyzed in the EZBiocloud database (https: / / www.ezbiocloud.net / ). The phylogenetic analysis method was used, and the 16S rRNA sequence of the model strain with high homology was selected as the reference object. The Clustal X 1.8 software was used for multiple sequence alignment to calculate the similarity between the test strain and the reference strain sequence. The base deletion sites were excluded during the phylogenetic analysis, and the neighbor-joining analysis was used to construct the phylogenetic tree between the test strain and the reference strain using MEGA 7.0. Among them, the Bootstrap value was set to 1000, and the rest were the default values.

[0054] Strain YNK PB0001 16S rRNA sequence (SEQ ID NO.1):

[0055]

[0056] Figure 2 The phylogenetic tree of strain YNK PB0001 is shown, from which it can be seen that YNK PB0001 is closely related to Burkholderia spp. Paraburkholderia graminis )C4D1M has the highest homology, with a homology rate of 99.30%.

[0057] 3. Identification results

[0058] Combined with the molecular detection results of strain YNK PB0001 and the results of bacterial morphological and physiological and biochemical characteristics, the strain was identified as Paraburkholderia praecox ( Paraburkholderia graminis ).

[0059] 3. Strain preservation

[0060] The above obtained Paraburkholderia graminis YNK PB0001 was deposited on October 31, 2024 in the China Center for Type Culture Collection, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University, with the deposit number CCTCCNO: M20242399.

[0061] Example 2

[0062] This example is used to illustrate the nitrogen fixation, phosphate solubilization, siderophore production and indoleacetic acid production effects of Paraburkholderia turcica CCTCC NO: M20242399.

[0063] 1. Nitrogen fixation effect test

[0064] Preparation method of Asbhy nitrogen-free medium: weigh 10g glucose, 0.2g dipotassium hydrogen phosphate, 0.2g sodium chloride, 0.2g magnesium sulfate monohydrate, 0.2g potassium sulfate, 5g calcium carbonate, add to 1000mL water to dissolve, add 18±2g agar, and sterilize at 121℃ for 20min.

[0065] The strain YNK PB0001 obtained in Example 1 was inoculated on the Asbe nitrogen-free culture plate using the four-point plate inoculation method, and the inoculation was repeated on three plates. After the inoculation was completed, the plate was placed in a 30°C constant temperature incubator for 7 days, and the bacterial growth and the formation of transparent circles around the colonies were observed every day during the incubation period.

[0066] Figure 3(A) shows the nitrogen fixation effect of strain YNK PB0001. As can be seen from the figure, strain YNK PB0001 can produce a transparent circle when cultured on Aspergillus nitrogen-free medium, indicating that the strain has the ability to fix nitrogen. The size of the transparent circle was measured by the cross method, and the ratio of the transparent circle diameter (D) to the colony diameter (d) was calculated to be D / d = 4.49 ± 0.25, indicating that strain YNK PB0001 has a strong nitrogen fixation ability.

[0067] (II) Phosphorus dissolution effect test

[0068] 1. Organophosphorus

[0069] Preparation method of organophosphorus solid culture medium: weigh 10g 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, 0.2g lecithin, 1g calcium carbonate, add to 1000mL water to dissolve, adjust pH to 7.3±0.2, add 18±2g agar, and sterilize at 121℃ for 20min.

[0070] The strain YNK PB0001 obtained in Example 1 was inoculated on an organophosphate culture plate using the four-point plate inoculation method, and the inoculation was repeated for three plates. After the inoculation was completed, the plate was placed in a 30°C constant temperature incubator for 7 days, and the bacterial growth and the formation of transparent circles around the colonies were observed every day during the incubation period.

[0071] Figure 3 (B) shows the effect of strain YNK PB0001 on degrading organophosphates. As can be seen from the figure, strain YNKPB0001 can produce transparent circles when cultured on organophosphate culture plates. The size of the transparent circle was measured by the cross method, and the ratio of the transparent circle diameter (D) to the colony diameter (d) was calculated to be D / d = 2.95 ± 0.11. This shows that the strain has a good ability to degrade organophosphates.

[0072] 2. Inorganic phosphorus

[0073] Preparation method of inorganic phosphorus solid culture medium: weigh 10g glucose, 0.5g ammonium sulfate, 0.3g sodium chloride, 0.3g potassium chloride, 0.3g magnesium sulfate heptahydrate, 0.03g ferrous sulfate heptahydrate, 0.03g manganese sulfate tetrahydrate, 5g tricalcium phosphate, add to 1000mL water to dissolve, adjust pH to 7.3±0.2, add 18±2g agar, and sterilize at 121℃ for 20min.

[0074] The strain YNK PB0001 obtained in Example 1 was inoculated on an inorganic phosphorus culture plate using the four-point plate inoculation method, and the inoculation was repeated for 3 plates. After the inoculation was completed, the plate was placed in a 30°C constant temperature incubator for 7 days, and the bacterial growth and the formation of transparent circles around the colonies were observed every day during the incubation period.

[0075] Figure 3 (C) shows the inorganic phosphorus decomposition effect of strain YNK PB0001. As can be seen from the figure, strain YNKPB0001 can produce a transparent circle when cultured on an inorganic phosphorus culture plate, indicating that the strain has the ability to decompose inorganic phosphorus. The size of the transparent circle was measured using the cross method, and the ratio of the transparent circle diameter (D) to the colony diameter (d) was calculated to be D / d = 1.29 ± 0.05.

[0076] 3. Test of the effect of producing siderophore

[0077] 1. Qualitative testing

[0078] Preparation method of CAS solid test medium: weigh 60.5 mg of chrome azuro blue S (CAS), 72.9 mg of hexadecyl trimethyl ammonium bromide (HDTMA), 2.645 mg of ferric chloride hexahydrate, 295.25 mg of sodium dihydrogen phosphate dihydrate, 1213.5 mg of disodium hydrogen phosphate dodecahydrate, 125 mg of ammonium chloride, 37.5 mg of potassium dihydrogen phosphate, and 62.5 mg of sodium chloride, add to 1000 mL of water to dissolve, adjust the pH to 6.8 ± 0.1, and add 18 ± 2 g of agar. Autoclave at 121°C for 20 min.

[0079] The strain YNK PB0001 obtained in Example 1 was inoculated on a CAS solid culture plate using the four-point plate inoculation method, and the inoculation was repeated for 3 plates. After the inoculation was completed, the plate was placed in a 30°C constant temperature incubator for 7 days, and the bacterial growth and the generation of orange halo around the colonies were observed every day during the incubation period.

[0080] Figure 3 (D) shows the siderophore production effect of strain YNK PB0001. As can be seen from the figure, strain YNKPB0001 can produce an orange halo when cultured on a CAS solid culture plate, indicating that the strain has the ability to produce siderophores. The size of the transparent circle was measured using the cross method, and the ratio of the transparent circle diameter (D) to the colony diameter (d) was calculated to be D / d = 1.69 ± 0.17.

[0081] 2. Quantitative detection

[0082] Preparation of CAS color developer: Add 60.5 mg of Chrome Azurol S (CAS) to 50 mL of deionized water, then mix with 10 mL of Fe 3+Solution (1 mM FeCl 3 6H 2 O ,10 mM HCl) mixed.

[0083] The strain YNK PB0001 was inoculated in NB liquid culture medium and cultured on a constant temperature shaker at 30°C at 150rpm for 48h. After the culture was completed, about 2mL of the culture solution was aspirated and filtered with a 0.22um sterile filter membrane, and an equal volume of CAS detection solution was added. After standing for 1h, the OD630 of the inoculated bacterial solution was measured using a full-wavelength microplate reader (denoted as "As"). The OD630 of the liquid culture medium without inoculation was measured in the same way as the reference value (denoted as "Ar"). The concentration of the iron carrier was expressed as the iron carrier activity unit (siderophore unit, SU), SU = [(Ar-As) / Ar] × 100%, and the determination was repeated 3 times, and the average value was taken for comparison.

[0084] According to the test results, it was calculated that the iron carrier concentration produced by strain YNK PB0001 in NB liquid culture medium for 48 hours was 15.47%.

[0085] (IV) Test on the effect of producing indoleacetic acid

[0086] 1. Qualitative testing

[0087] KB medium: peptone 20g, glycerol 15ml, K 2 HPO 4 1.5g, MgS0 4 7H 2 O1.5g, tryptophan 0.1g, add to 1000mL water to dissolve, adjust pH to 7.2±0.2. Sackowcki's color developer: slowly add 150mL of concentrated sulfuric acid to 250mL of deionized water, stirring while adding, and after the solution cools, add 7.5mL of 0.5mol / L FeCl 3 6H 2 O solution.

[0088] A single colony of strain YNK PB0001 obtained in Example 1 was picked and inoculated into KB medium, and cultured at 30°C and 180 rpm / min for 24 h. 1 mL of fermentation broth was aseptically pipetted into a centrifuge tube, quickly mixed with 4 mL of Sackowcki's color developer, and allowed to stand at room temperature in the dark for 40 min to develop color. The color change was observed and recorded. If pink appeared, it was positive, indicating that the strain could secrete IAA.

[0089] Figure 4 The color development of strain YNK PB0001 is shown in the figure. Its pink color indicates the production of IAA.

[0090] 2. Quantitative detection

[0091] After the strain was cultured in NB medium for 1 day, the seed solution was prepared and inoculated into KB liquid medium containing L-tryptophan at a 1% inoculum. After being cultured in a 30°C constant temperature shaker at 180 r / min, 4 ml of the supernatant was aspirated every day and mixed with 4 ml of Sackowcki's colorimetric reagent. After standing in the dark for 40 minutes, the OD value was measured at a wavelength of 535 nm. The absorbance value obtained was substituted into the standard curve for calculation to obtain the indoleacetic acid content produced by the strain. It was determined that the indoleacetic acid content produced by the strain YNK PB0001 was the highest at 23.36±0.26μg / mL on the third day.

[0092] Example 3

[0093] This example is used to illustrate the germination-promoting effect of Burkholderia spp. CCTCC NO: M20242399 on tomato seeds.

[0094] Experimental group settings: CK1: sterile water; CK2: NB blank culture medium; T1: 1.0×10 8 CFU / mL of the fermentation supernatant of the strain (obtained by inoculating the strain YNK PB0001 obtained in Example 1 into NB medium for 24 h of fermentation and centrifuging at 12000 rpm for 5 min); T2: 1.0×10 7 CFU / mL of the fermentation supernatant of the strain; T3: 1.0×10 6 CFU / mL of the strain fermentation supernatant; T4: 1.0×10 5 CFU / mL of the fermentation supernatant of the strain.

[0095] Specific implementation method: Tomato seeds were selected as experimental objects. The seeds were soaked in 70% alcohol for 10 minutes and then washed with sterile water for 3 times to complete disinfection. Take the seeds that have settled under the water, soak them in water at room temperature for 12 hours, and then take the seeds of each group and place them in a 9.00cm transparent culture dish with 2-3 layers of sterilized filter paper. Each treatment has 15 seeds per dish, and repeat 3 times. Then take the liquid treatment of each group to moisten the filter paper, place it in a 26℃ artificial climate box, and culture it under the alternating light and dark time of 16h and 8h. After that, 2.00mL of sterile water should be added regularly every day to keep the filter paper moist. After 7 days, record the germination situation and measure the root length and the whole plant length.

[0096] Table 2 Seed germination data results

[0097]

[0098] *The data in the table with different letters indicate significant differences

[0099] Results of the germination promotion experiment: Figure 5 The germination of seeds in each experimental group on the 7th day after treatment is shown. The seeds of control group 2 did not germinate on the 7th day after treatment with NB blank medium; the seeds of experimental group 1 were treated with 1.0×10 8 CFU / mL of the fermentation supernatant of the strain was used to wet the filter paper. After 7 days of treatment, the seeds germinated but no cotyledons were seen. 5 CFU / mL of the fermentation supernatant of the strain wetted the filter paper for 7 days, and the seeds germinated significantly. The root length of this group was 6.37±0.25cm, and the whole plant length was 9.18±0.23cm, which was significantly higher than that of other groups. The second was experimental group 3 using 1.0×10 6 CFU / mL of the supernatant of the fermentation of the strain was used to wet the filter paper for 7 days. The root length of this group was 5.28±0.18cm and the whole plant length was 8.25±0.33cm. The specific results are shown in Table 2. 5 -1.0×10 6 The fermentation supernatant of the CFU / mL strain has a better effect on promoting seed germination. The experiment shows that the application of Burkholderia praecox YNK PB0001 has a significant promoting effect on the germination of tomato seeds.

[0100] Example 4

[0101] This example is used to illustrate the growth-promoting effect of Burkholderia spp. CCTCC NO: M20242399 on tomato plants.

[0102] Preparation of bacterial agent: The strain YNK PB0001 obtained in Example 1 was inoculated into NB liquid culture medium and cultured at 30°C with shaking at 180 rpm for 72 h. The resulting fermentation broth was the bacterial agent of strain YNK PB0001 (with a viable cell count of about 1×10 10 CFU / mL).

[0103] Plant planting: Add the same weight of soil to each pot, randomly group 3 pots per treatment, and plant 6 tomato seedlings of similar growth in each pot.

[0104] Experimental group settings: CK1: sterile water; CK2: sterile NB liquid culture medium; T1: concentration of about 1×10 10 CFU / mL of strain YNK PB0001; T2: concentration is about 1×10 9 CFU / mL of strain YNK PB0001; T3: concentration is about 1×10 8 CFU / mL of strain YNK PB0001 bacterial liquid.

[0105] Verification of growth promotion effect: On the 8th day after the tomato seedlings were transplanted into the pots, the tomato seedlings were treated with 200 mL / plant of the above-mentioned bacterial agent for root irrigation. At the same time, the tomato seedlings were treated with the same amount of sterile water and NB medium cultured under the same culture conditions for 72 hours as the control group (CK1 and CK2). The tomato seedlings after root irrigation were placed in a greenhouse to grow naturally, and the soil was kept moist during the period. After 28 days of root irrigation, the aboveground fresh weight, underground (root) fresh weight, stem diameter, root length and plant height of the tomato plants were measured.

[0106] The specific measurement method is as follows:

[0107] Aboveground / belowground fresh weight: Cut the part of the tomato plant above the root base and weigh the aboveground / belowground fresh weight on an analytical balance, retaining two decimal places.

[0108] Stem Diameter: Use a caliper to measure the diameter of the thickest stem part of the plant.

[0109] Root length: Straighten the roots and measure the root length using a ruler.

[0110] Plant height: Straighten the plant and use a ruler to measure the length of the above-ground part from the highest point of the leaves.

[0111] Table 3 shows the growth index measurement results of tomato seedlings in the control group and the experimental group.

[0112] Table 3 Growth status of tomato seedlings

[0113]

[0114] *The data in the table with different letters indicate significant differences

[0115] From the data in the above table, it can be seen that after 28 days of root irrigation treatment with strain YNK PB0001, the plant height, stem diameter, aboveground fresh weight, root length, and underground fresh weight of tomato plants were significantly improved compared with the control group (CK1) with water and the control group (CK2) with NB medium. Among them, the aboveground fresh weight of the T1 treatment group was 21.72% and 77.57% higher than the control groups CK1 and CK2, respectively, the underground fresh weight of the T1 treatment group was 38.44% and 80.82% higher than the control groups CK1 and CK2, respectively, the stem diameter of the T1 treatment group was 18.56% and 37.18% higher than the control groups CK1 and CK2, respectively, the root length of the T1 treatment group was 12.55% and 42.54% higher than the control groups CK1 and CK2, respectively, and the plant height of the T1 treatment group was 10.33% and 29.06% higher than the control groups CK1 and CK2, respectively. It can be seen that whether or not to apply bacterial solution for treatment has a significant effect on plant growth, proving that the application of strain YNK PB0001 has a significant promoting effect on the growth of tomatoes.

[0116] Figure 6 The figure shows a comparison of tomato plants in the experimental group and the control group after 28 days of root irrigation. It can be seen from the figure that after root irrigation with strain YNK PB0001, the tomato plants as a whole grew faster than those treated with only water and NB liquid medium, and the sizes of roots, stems and leaves were larger, and the tomato root system was more developed.

[0117] The above results show that strain YNK PB0001 can effectively promote the growth of tomato seedlings.

[0118] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A Burkholderia paraphyletica strain ( Paraburkholderia graminis ), characterized in that The deposit number of the grass-roots Burkholderia is: CCTCC NO: M20242399.

2. A bacterial agent, characterized in that The active ingredient of the bacterial agent includes the Burkholderia paraherbivora described in claim 1.

3. The bacterial agent according to claim 2, wherein The bacterial agent is a liquid bacterial agent; in the liquid bacterial agent, the content of the grass root periphyton Burkholderia is not less than 1×10 6 CFU / mL.

4. A method for promoting plant seed germination, characterized in that: The plant seeds are soaked in the bacterial agent as claimed in claim 2 or 3.

5. A method for promoting plant growth, characterized in that: The method comprises applying the Burkholderia pararhizogenes of claim 1 or the bacterial agent of claim 2 or 3 to the rhizosphere soil of the plant.

6. The method according to claim 5, wherein: The application amount of the grass root paraburkholderia is not less than 1×10 8 CFU / strain / time; or the dosage of the bacterial agent is such that the application amount of Burkholderia paragracilis is not less than 1×10 8 CFU / strain / time.

7. The method according to claim 5 or 6, wherein: The application frequency of the grass root paraburkholderia or bacterial agent is 1-3 times per crop; the crop is tomato.

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