Caffeine-resistant burkholderia parakholderia and application of caffeine-resistant burkholderia parakholderia in tobacco growth promotion

Abstract

The invention relates to a caffeine-resistant burkholderia paracasei and application of the caffeine-resistant burkholderia paracasei in tobacco growth promotion, the caffeine-resistant burkholderia paracasei is named as a YIM B11874 strain, the preservation number of the caffeine-resistant burkholderia paracasei is CGMCC No.36352, and the preservation date is October 27, 2025. The bacterial strain has good nitrogen fixation and phosphorus solubilization capabilities, and pot experiments show that the bacterial strain has a good growth promotion effect on crops represented by tobacco, and has a positive construction effect on sustainable development of agriculture and environmental protection.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and relates to a caffeine-tolerant Burkholderia parapenetrans and application thereof in tobacco growth promotion. BACKGROUND

[0003] Nitrogen-fixing microorganisms can convert atmospheric nitrogen into plant-absorbable ammonia in soil, increase the nitrogen content in soil, provide nitrogen nutrients for plants, promote plant growth, and increase crop yield. In addition, the activity of nitrogen-fixing microorganisms helps maintain soil nitrogen balance and avoid soil acidification, which is of great significance to promoting the health and sustainability of the soil ecosystem.

[0004] Growth-promoting bacteria can decompose insoluble phosphorus and potassium in soil and secrete nutrients and plant hormones, thus playing an important role in promoting seed germination, extending root length, and increasing biomass, laying a good foundation for crop growth. Growth-promoting bacteria can also improve soil fertility and nutrient absorption efficiency of crops, reduce dependence on chemical fertilizers, reduce agricultural production costs, and reduce environmental pollution caused by excessive use of chemical fertilizers. In addition, the use of growth-promoting bacteria in crop cultivation can greatly reduce the use of pesticides and reduce the harm of pesticide residues to agricultural products and the environment.

[0005] At present, there are various types of growth-promoting microbial agents on the market, but the activity of microorganisms is affected by temperature, humidity, soil conditions, and other factors. In addition, the natural conditions of tobacco-growing areas in China are quite different. Therefore, it is crucial to screen growth-promoting bacteria suitable for Yunnan tobacco-growing areas and reconstruct rhizosphere beneficial microbial flora for large-scale application. SUMMARY

[0006] In view of the deficiencies in the prior art, the present application aims to provide a caffeine-tolerant Burkholderia parapenetrans and application thereof in tobacco growth promotion, in particular, a caffeine-tolerant Burkholderia parapenetrans with nitrogen-fixing and phosphorus-solubilizing effects and application thereof in tobacco growth promotion.

[0007] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0008] In a first aspect, the present application provides a caffeine-tolerant Burkholderia parapenetrans, which is named YIM B11874 strain, has a preservation number of CGMCC No.36352, and was preserved on October 27, 2025.

[0009] The application isolates and preserves a new Paraburkholderia caffeine-tolerant strain YIM B11874 from the soil of a Paraburkholderia bacteria pond in Wuding County, Chuxiong Yi Autonomous Prefecture, Yunnan Province, which has a fast breeding speed, is easy to cultivate, and has a simple preparation method and a short preparation period. The strain has good nitrogen fixation and phosphorus solubilization capabilities. Through a pot experiment, it is found that the strain has a good growth-promoting effect on crops represented by tobacco, which has great economic and social benefits for improving the production efficiency of tobacco, reducing the dependence on natural resources, and environmental protection, and has a positive role in the sustainable development of agriculture and the protection of the environment.

[0010] In a second aspect, the application provides a bacterial agent, wherein the strain in the bacterial agent comprises the YIM B11874 strain of the first aspect.

[0011] Preferably, the dosage form of the bacterial agent comprises a solution or a powder.

[0012] In the application, the specific dosage form of the bacterial agent is not particularly limited, and any commonly used bacterial agent type in the art can be applicable to the application, such as a common bacterial agent type, for example, a solid bacterial agent, a liquid bacterial agent, or a semi-solid (concentrated) bacterial agent.

[0013] Preferably, the dosage form of the bacterial agent is a solution, which can be prepared by the following method:

[0014] The YIM B11874 strain is inoculated into a culture medium for activation and fermentation culture in sequence to obtain a fermentation broth; or the fermentation broth is further centrifuged, resuspended with a solvent to obtain a bacterial suspension.

[0015] Preferably, the dosage form of the bacterial agent is a powder, which can be prepared by the following method:

[0016] The YIM B11874 strain is inoculated into a culture medium for activation and fermentation culture in sequence to obtain a fermentation broth; the fermentation broth is centrifuged, mixed with a protective agent, and then dried to obtain a bacterial powder.

[0017] Preferably, the viable bacterial count of the YIM B11874 strain in the bacterial agent is not less than 1×10 6 CFU / g or 1×10 6 CFU / mL, for example, 1×10 6 CFU / g (or CFU / mL), 1×10 7 CFU / g (or CFU / mL), 5×10 7 CFU / g (or CFU / mL), 1×10 8 CFU / g (or CFU / mL), 5×10 8 CFU / g (or CFU / mL), 1×10 9CFU / g (or CFU / mL) or the like, and other specific points in the numerical range can be selected, which are not listed here.

[0018] In a third aspect, the present application provides the use of the caffeine-resistant B. paraparkinsonii of the first aspect or the microbial agent of the second aspect in soil nitrogen fixation.

[0019] In a fourth aspect, the present application provides the use of the caffeine-resistant B. paraparkinsonii of the first aspect or the microbial agent of the second aspect in soil phosphorus solubilization.

[0020] The present application has found that the caffeine-resistant B. paraparkinsonii has good phosphorus solubilization and nitrogen fixation functions through the agricultural activity research on the caffeine-resistant B. paraparkinsonii, and the functions are rarely reported in the research on the caffeine-resistant B. paraparkinsonii.

[0021] In a fifth aspect, the present application provides the use of the caffeine-resistant B. paraparkinsonii of the first aspect or the microbial agent of the second aspect in promoting the growth of Solanaceae plants.

[0022] Preferably, the Solanaceae plants include tobacco.

[0023] In a sixth aspect, the present application provides a method for promoting the growth of Solanaceae plants, the method comprising: applying the caffeine-resistant B. paraparkinsonii of the first aspect or the microbial agent of the second aspect to the rhizosphere soil of the plants, wherein the application amount of the caffeine-resistant B. paraparkinsonii strain is not less than 1×10 7 CFU / strain / time.

[0024] Preferably, the Solanaceae plants include tobacco.

[0025] Compared with the prior art, the present application has the following beneficial effects:

[0026] The present application has isolated and preserved a new caffeine-resistant B. paraparkinsonii strain from the soil of a Ganba bacterial pond in Wuding County, Chuxiong Yi Autonomous Prefecture, Yunnan Province, and named it as YIM B11874 strain. The strain has a fast reproduction speed and is easy to cultivate. The preparation method of the microbial agent of the strain is simple and has a short preparation period. The strain has good nitrogen fixation and phosphorus solubilization capabilities. Through a pot experiment, it is found that the strain has a good growth promotion effect on crops represented by tobacco, which has a very huge economic benefit and social benefit for improving the production efficiency of tobacco, reducing the dependence on natural resources, and environmental protection, and has a positive construction role for the sustainable development of agriculture and the protection of the environment. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is an NJ phylogenetic tree diagram of the YIM B11874 strain;

[0028] Figure 2 is a genomic tree diagram of the YIM B11874 strain;

[0029] Figure 3 is a colony morphology diagram of the YIM B11874 strain on NA medium;

[0030] Figure 4 is an effect diagram of nitrogen fixation of the YIM B11874 strain;

[0031] Figure 5 is an effect diagram of phosphorus dissolution of the YIM B11874 strain;

[0032] Figure 6 is a comparison diagram of the height growth of tobacco seedlings in the control group and the experimental group;

[0033] Figure 7 is a comparison diagram of the maximum leaf length increase value of tobacco seedlings in the control group and the experimental group;

[0034] Figure 8 is a comparison diagram of the maximum leaf width increase value of tobacco seedlings in the control group and the experimental group;

[0035] Figure 9 is a comparison diagram of the ground diameter growth of tobacco seedlings in the control group and the experimental group;

[0036] Figure 10 is an appearance diagram of the whole tobacco plant in the control group and the experimental group;

[0037] The classification of the YIM B11874 strain involved in the present application is Paraburkholderia sp. resistant to caffeine, the preservation unit is China General Microbiological Culture Collection Center, the preservation date is October 27, 2025, the preservation number is CGMCC No. 36352, and the address is No. 3, Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0038] The technical solutions of the present application will be further described through specific embodiments. Those skilled in the art should understand that the embodiments are only to help understand the present application, and should not be regarded as specific limitations on the present application.

[0039] Example 1

[0040] Isolation, purification and identification of the YIM B11874 strain:

[0041] (1) Obtaining of the strain:

[0042] The soil of the mushroom pond with a depth of about 5 cm was collected from the dry-bamboo mushroom conservation area in Wuding County, Chuxiong Yi Autonomous Prefecture, Yunnan Province, China, and the fallen leaves and other litter within a diameter of 20 cm were removed. The soil was stored in a refrigerator at 4°C for standby use.

[0043] The culture medium used in the strain isolation and purification was obtained by mixing BG11 medium, photosynthetic bacterial culture medium and plate counting broth (PCB medium) in a weight ratio of 1:1:1 and adding 20 g / L agar, and sterilizing at 121°C for 20 min. The BG11 medium, photosynthetic bacterial culture medium and PCB medium were purchased from Haibo Biotechnology Co., Ltd. (product numbers HB8793, HB8882 and HB8756, respectively).

[0044] The soil sample was diluted and plated by using the dilution coating method. 2 g of soil sample was placed in a 300 mL conical flask containing 198 mL of sterile water, and an appropriate amount of glass beads was added. The flask was placed in a shaking incubator at 30°C and 140 r / min for 4-6 h. 200 μL of the solution was diluted to 10 -4 、10 -5 concentration and plated on the above-mentioned medium. After being cultured in a constant temperature incubator at 30°C for 3-5 d, single colonies with different morphological characteristics were picked and purified on a slope. A strain was obtained through the above-mentioned steps and was named YIM B11874.

[0045] (2) Molecular identification of the strain:

[0046] The total DNA of the strain YIM B11874 was obtained by using the Chelex-100 rapid extraction method as a template. The universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO: 1) and 1492R (5'-TACGACTTAACCCCAATCGC-3', SEQ ID NO: 2) were used. The reaction system and conditions in Table 1 were used for 16S rRNA gene amplification.

[0047] Table 1

[0048]

[0049] The amplification product was purified by 1% agarose gel electrophoresis (gel recovery and purification kit produced by Guangzhou Meiji Biological Technology Co., Ltd.) and then sent to Beijing Qikang Biological Technology Co., Ltd. for sequencing. The sequencing result is shown in SEQ ID NO: 3:

[0050]

[0051] The sequencing results were subjected to 16S rRNA gene alignment analysis, and the similarity of strain YIM B11874 to Paraburkholderia caffeinilytica was 99%. Then, the 16S rRNA sequence of the higher homologous model strain was selected as a reference for comparison, and subjected to multiple sequence alignment to calculate the similarity between the test strain and the reference strain; the Neighbor Joining analysis was used to exclude base deletion sites, and the phylogenetic tree between the test strain and the reference strain was constructed; the Bootstrap value was set to 1000, and the rest were default values.

[0052] Figure 1 The phylogenetic tree of strain YIM B11874 based on the Neighbor Joining method is shown, from which it can be seen that the homology of strain YIM B11874 to Paraburkholderia caffeinilytica CF1 is the highest.

[0053] Figure 2 The whole genome sequencing results of strain YIM B11874 are shown, from which it can be seen that the ANI value of strain YIM B11874 to its nearest relative strain Paraburkholderia caffeinilytica CF1 is 90%, and the dDDH value is 40%, which meets the division standard of subspecies level unit in the International Rules for the Nomenclature of Prokaryotes (ANI 85-95%; dDDH 40-70%); it is judged that strain YIM B11874 is a subspecies of Paraburkholderia caffeinilytica.

[0054] (3) Characteristics of the strain:

[0055] (3.1) Phenotypic characteristics: The colony morphology of the strain is round, semi-transparent white colony, the colony is flat, the edge is regular, and the wetness is opaque (see Figure 3 ), the strain can grow on NA and LB medium. The strain was identified as a gram-negative bacterium by gram staining experiment, and was rod-shaped; the oxidase and catalase were both positive.

[0056] (3.2) Genomic characteristics: The whole genome draft of strain YIM B11874 contains 16 contigs with a total length of 8,004,102 bp; after optimization, the strain is composed of 13 scaffolds with a total length of 8,004,253 bp, and the largest scaffold length is 1,570,725 bp; the GC content of all bases in each genome is 61.68%.

[0057] In addition, 7,082 protein coding sequences (CDSs) were annotated in the whole genome sketch of the strain YIM B11874, with a total length of 6,891,243 bp. The carbohydrate-active enzymes database annotated the families (GH15, GH3, GH54, GH39) related to starch hydrolysis and cellulose degradation, which have an impact on the environmental adaptation, fermentation performance, and the like of the strain. In the secondary metabolic product gene cluster of YIM B11874, there is one gene cluster with a similarity higher than 85% - ornibactin (86%); the ornibactin gene cluster can guide the synthesis of ornibactin, which can chelate Fe 3 + in a low-iron environment, thereby improving the efficiency of the strain in obtaining iron.

[0058] Example 2

[0059] Evaluation of the nitrogen fixation effect of the YIM B11874 strain:

[0060] (1) Preparation of Ashby's glucose medium:

[0061] Ashby's glucose medium (g / L): glucose 20.0; CaCO3 5.0; K2HPO4 0.2; MgSO4·7H2O 0.2; NaCl 0.2; K2SO4 0.1; agar 20.0; pH 7.4±0.2.

[0062] (2) Evaluation method:

[0063] The method of observing whether a transparent ring appears in the medium was adopted. The caffeine-resistant B. parapari chlorica YIM B11874 was inoculated on the Ashby's glucose plate by the three-point method and placed in a 30°C constant temperature incubator for 5 days of culture, and whether a transparent ring appeared around the colony was observed, and the ratio of the halo diameter (D) to the colony diameter (d) was recorded.

[0064] (3) Evaluation result:

[0065] Figure 4 The transparent ring of the caffeine-resistant B. parapari chlorica YIM B11874 on the Ashby's culture medium was shown, and the ratio of the transparent ring diameter to the colony diameter D / d was 2.1 / 0.3, which indicated that it had good nitrogen fixation ability.

[0066] Example 3

[0067] Evaluation of the phosphorus solubilizing effect of the YIM B11874 strain:

[0068] (1) Preparation of inorganic phosphorus bacterial culture medium:

[0069] Inorganic phosphorus bacterial culture medium (g / L): glucose 10.0; ammonium sulfate 0.5; sodium chloride 0.3; potassium chloride 0.3; magnesium sulfate 0.3; ferrous sulfate 0.03; manganese sulfate 0.03; calcium phosphate 5.0; agar 20.0; pH 7.4±0.2.

[0070] (2) Evaluation method:

[0071] The method of observing whether a transparent circle appears in the culture medium is adopted. The coffee-resistant Paraburkholderia YIM B11874 is inoculated on the inorganic phosphorus plate by three-point method and placed in a 30℃ constant temperature incubator for 5 days of culture, and whether a transparent circle appears around the colony is observed, and the ratio of the halo diameter (D) to the colony diameter (d) is recorded.

[0072] (3) Evaluation result:

[0073] Figure 5 The transparent circle of the coffee-resistant Paraburkholderia YIM B11874 on the inorganic phosphorus culture medium is shown, and the ratio of the transparent circle diameter to the colony diameter D / d is 1.1 / 0.2, which shows that it has the ability to dissolve phosphorus.

[0074] Example 4

[0075] Evaluation of the growth-promoting effect of YIM B11874 strain on tobacco:

[0076] (1) Preparation of bacterial agent

[0077] The YIM B11874 strain isolated in Example 1 is inoculated in 40 mL of NB culture medium, and cultured at 30℃ for 48 h in a 180 r / min shaking bed, until the viable bacterial count is about 1×10 9 CFU / mL.

[0078] (2) Preparation of soil and tobacco seedlings

[0079] The same weight of soil is added in each flowerpot, and 3 pots are randomly grouped according to each treatment, and 3 tobacco seedlings with similar growth vigor are planted in each pot.

[0080] (3) Design of experimental groups

[0081] CK group: sterilized NB liquid culture medium; T1 group: YIM B11874 bacterial liquid with a concentration of 1×10 9 CFU / mL; T2 group: YIM B11874 bacterial liquid with a concentration of 1×10 8 CFU / mL; T3 group: YIM B11874 bacterial liquid with a concentration of 1×10 7 CFU / mL.

[0082] (4) Verification of growth-promoting effect

[0083] The tobacco seedlings were treated with 40 mL / plant of the YIM B11874 inoculum described above on the 7th day after transplanting to the flower pots, and the same amount of NB medium cultured under the same conditions for 48 h was used to treat the tobacco seedlings as a control group. The tobacco plants were placed in a greenhouse and kept in adequate soil moisture during the period. The ground diameter, plant height, maximum leaf length, and maximum leaf width of the tobacco plants were measured every week for 4 weeks, and the tobacco plants were taken out for growth comparison at the 4th week.

[0084] (5) Measurement method of each plant trait

[0085] Ground diameter: The diameter of each tobacco plant was measured at 1 cm from the soil surface of the flower pot using a vernier caliper, and the average value ± standard deviation was taken for each group of data.

[0086] Plant height: The height of the top of each tobacco plant to the soil surface of the flower pot was measured using a ruler, and the average value ± standard deviation was taken for each group of data.

[0087] Maximum leaf length: A ruler was placed on the midrib of the tobacco leaf to ensure that the ruler was parallel to the midrib, and the longest distance from the top to the base of the leaf was the maximum leaf length. The average value ± standard deviation was taken for each group of data.

[0088] Maximum leaf width: A ruler was placed perpendicular to the midrib of the leaf, and the distance at the widest part of the leaf was the maximum leaf width. The average value ± standard deviation was taken for each group of data.

[0089] (6) Measurement results

[0090] The measurement results of the 3rd and 4th weeks are shown in Tables 2 and 3, respectively (Note: In Tables 2 and 3, different superscripts indicate significant differences (P<0.05), and the data in the tables are the average value ± standard deviation).

[0091] Table 2

[0092]

[0093] Table 3

[0094]

[0095] From Table 2 and Table 3, it can be seen that in the third week after the first root irrigation, the average of all traits of the treatment group is higher than that of the control group CK, but the growth promotion of the maximum leaf length and the maximum leaf width is not significant; the growth promotion effect of the T1 group is relatively good, and the average of the plant height and the ground diameter is significantly greater than that of the CK group, and is higher than that of the CK group. In the fourth week after the first root irrigation, the average of the plant height, the maximum leaf length, the maximum leaf width and the ground diameter of the T1 group is significantly greater than that of the CK group, and is 16%, 23%, 21% and 33% higher than that of the CK group, respectively; the maximum leaf length and the maximum leaf width of the T3 group are significantly greater than those of the CK group by 20% and 19%, respectively.

[0096] Figure 6-9 The measurement result comparison of the plant height growth amount ( Figure 6 ), the maximum leaf length increase value ( Figure 7 ), the maximum leaf width increase value ( Figure 8 ) and the ground diameter growth amount ( Figure 9 ) of the tobacco plants of the control group CK and the experimental groups T1, T2 and T3 at the end of growth and at the beginning are shown in Table 4. It can be seen that there is no significant difference in the plant height growth amount between different groups, but the plant height growth amount of each experimental group is higher than that of the CK group; the maximum leaf length increase value, the maximum leaf width increase value and the ground diameter growth amount of the T1 group are significantly greater than those of the CK group, and are 1.23 times, 1.23 times and 1.56 times higher than those of the CK group, respectively; in addition, the maximum leaf length increase value of the T2 group is also significantly 1.23 times higher than that of the CK group.

[0097] Figure 10 The comparison results of the final growth of the tobacco plants of the CK, T1, T2 and T3 groups from left to right are shown in Fig. 2, and it can be seen that the root length, plant size, leaf size and number, and root density of the experimental groups are obviously better than those of the CK group; in addition, the plant growth of the experimental groups, especially the T1 group, is the best.

[0098] From the above results, it can be seen that after the YIM B11874 strain is applied to the rhizosphere soil, it has a good growth promotion effect on the plant height, leaf length, leaf width, ground diameter and the like of the tobacco seedlings, especially the bacterial liquid with a concentration of 1x10 9 CFU / mL (T1 group) has a significant (P<0.05) growth promotion effect on the leaf length, leaf width and ground diameter of the tobacco, and has the best growth promotion effect on the tobacco seedlings; this has a certain positive influence on the yield and quality of the tobacco leaves; in addition, this is the first time to find that the B. caffoeatans has a growth promotion effect on the Solanaceae plants.

[0099] The applicant declares that the technical scheme of the present application is illustrated by the above-mentioned embodiments, but the present application is not limited to the above-mentioned embodiments, that is, the present application does not mean that it must rely on the above-mentioned embodiments to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific modes, etc. fall within the protection scope and disclosure scope of the present application.

[0100] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details in the above-mentioned embodiments, and various simple modifications can be made to the technical scheme of the present application within the technical concept of the present application, and these simple modifications all belong to the protection scope of the present application.

[0101] In addition, it should be noted that each specific technical feature described in the above-mentioned specific embodiments can be combined by any suitable method without contradiction, and in order to avoid unnecessary repetition, the present application will not further describe various possible combination methods.

Claims

1. A Paraburkholderia caffeine-resistente, characterized in that, The coffeeine-resistant Burkholderia paraputida is named YIM B11874 strain, with the preservation number of CGMCC No. 36352 and the preservation date of October 27, 2025.

2. An inoculant characterized in that, The strain in the microbial agent includes the YIM B11874 strain of claim 1.

3. The bacterial agent of claim 2, wherein The dosage form of the microbial agent includes a solution or a powder.

4. The bacterial agent of claim 2, wherein The viable cell number of YIM B11874 strain in the bacterial agent is not less than 1 x 10 6 CFU / g or 1 x 10 6 CFU / mL.

5. The coffeeine-resistant Burkholderia paraputida of claim 1 or the microbial agent of any one of claims 2-4 for use in soil nitrogen fixation.

6. The coffeeine-resistant Burkholderia paraputida of claim 1 or the microbial agent of any one of claims 2-4 for use in soil phosphorus solubilization.

7. The coffeeine-resistant Burkholderia paraputida of claim 1 or the microbial agent of any one of claims 2-4 for use in promoting the growth of Solanaceae plants.

8. Use according to claim 7, characterized in that, The Solanaceae plants include tobacco.

9. A method of promoting growth of a solanaceous plant, characterized by, The method comprises: applying the B. caffoeicola of claim 1 or the inoculant of any one of claims 2-4 to the rhizosphere soil of the plant, wherein the application amount of the B. caffoeicola strain is not less than 1 x 10 7 CFU / strain / time.

10. The method of claim 9, wherein, The Solanaceae plants include tobacco.

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