CN114934001B

CN114934001B - Endophytic antagonistic bacteria for tobacco seeds and application thereof

Info

Publication number: CN114934001B
Application number: CN202210769278.XA
Authority: CN
Inventors: 丁伟; 龚杰; 余佳敏; 江其朋; 余祥文; 董晏伶; 陈芷莹; 林正全; 李峰
Original assignee: Southwest University; China National Tobacco Corp Sichuan Branch
Current assignee: Southwest University; China National Tobacco Corp Sichuan Branch
Priority date: 2022-07-01
Filing date: 2022-07-01
Publication date: 2023-05-26
Anticipated expiration: 2042-07-01
Also published as: CN114934001A

Abstract

The invention relates to a tobacco seed endophytic antagonistic bacterium DW15, which has the preservation number: cctccc NO: m2022820, classification designated Pseudomonas paraxanthum Pseudomonas parafulva, was deposited with the China center for type culture Collection. The strain is yellow and round, and has wet and smooth surface and micro-bulge. The endophyte DW15 screened by the invention has strong antibacterial effect on Ralstonia solanacearum, is a biocontrol bacterium with good effect on tobacco bacterial wilt, can generate more protease, and has stronger phosphorus-dissolving and nitrogen-fixing capabilities. Meanwhile, the plant growth regulator has good growth promoting effect on tobacco plants, can obviously improve the biomass of tobacco seedlings, can be used singly or in combination, has wide application prospect on biological control of tobacco bacterial wilt, and has important significance on protecting ecological environment.

Description

Endophytic antagonistic bacteria for tobacco seeds and application thereof

Technical Field

The invention belongs to the technical field of agricultural microbial control, and relates to a tobacco seed endophytic antagonistic bacterium and application thereof.

Background

Bacterial wilt is a destructive soil-borne disease caused by ralstonia solanacearum (Ralstonia solanacearum), and pathogenic bacteria of the bacterial wilt are extremely destructive and can infect more than 200 plants, so that huge economic losses are caused, such as 0-91% of tomato yield reduction, 33-90% of potato yield reduction, 10-30% of tobacco yield reduction, 80-100% of banana yield reduction, 20% of peanut yield reduction and the like. Tobacco bacterial wilt (Tobacco bacterial wilt) is a soil-borne disease which is seriously harmful, difficult to prevent and control in the current tobacco production. Tobacco bacterial wilt is widely harmful in southwest tobacco areas of China, has a tendency of moving to cold areas at high altitude, and is a plant disease to be solved in actual production. In China, due to factors such as improper continuous cropping, fertilization and management modes of tobacco and the like for a long time, continuous cropping obstacle of the tobacco is serious, tobacco bacterial wilt is exploded in a large area, and the production quality of tobacco leaves in each large tobacco area is seriously reduced.

Tobacco bacterial wilt control faces a great challenge. For the prevention and treatment of tobacco bacterial wilt, measures such as breeding resistant varieties, biological prevention and treatment, chemical prevention and treatment and the like are mainly adopted at present. Because chemical control has the problems of harm to human health, ecological balance damage and the like, and biological control has the advantages of safety to people and livestock, environmental friendliness and the like, the biological control on the soil-borne disease tobacco bacterial wilt is more and more paid attention in recent years, and the research on the biological control bacteria for controlling the tobacco bacterial wilt is more extensive.

At present, the research on the prevention and treatment of the tobacco bacterial wilt mainly focuses on the prevention and treatment effect and mechanism of induced resistance compounds on the tobacco bacterial wilt, the interaction between the bacterial wilt and rhizosphere microorganisms and the like, and the related research on the interaction relationship between plant endophytes and the tobacco bacterial wilt is less. The co-evolution of plants and endophytes, according to the action of endophytes, divides them into three classes: firstly, neutral endophytic microorganisms which have no obvious harmful or beneficial effects on plants; secondly, beneficial endophyte can play a beneficial role of protecting plants from pathogen or herbivorous organisms, promoting plant growth and the like; thirdly, the potential pathogenic endophytic microorganisms generate substances which are unfavorable for plant growth. Finding beneficial endophytic microorganisms to tobacco plants is a major problem to be solved.

Disclosure of Invention

In view of the above, the present invention has an object to provide a tobacco seed endophytic antagonistic bacterium having various properties such as a growth promoting effect, and to provide various applications thereof in agricultural industrialization.

In order to achieve the above purpose, the present invention provides the following technical solutions:

1. the tobacco seed endophytic antagonistic bacterium is characterized by being DW15 and is named by classification: pseudomonas paraflavum Pseudomonas parafulva, deposited in China center for type culture Collection, address: martial arts, date of preservation: 2022, 6 and 7, deposit number: cctccc NO: m2022820.

Further, the bacterial strain of the endophytic antagonistic bacteria of the tobacco seeds is yellow and round, and the surface is moist and smooth and is micro-raised.

Further, the tobacco seed endophytic antagonistic bacteria strain is capable of producing proteases.

2. The use of the tobacco seed endophytic antagonistic bacteria according to any one of the above as a biological agent for inhibiting and/or controlling bacterial wilt.

Further, the application of the tobacco seed endophytic antagonistic bacteria as a biological bacterial agent in inhibiting and/or preventing bacterial wilt is provided, wherein the use concentration of the tobacco seed endophytic antagonistic bacteria in the biological bacterial agent is 1 multiplied by 10 ⁶ cfu/mL-1×10 ¹² cfu/mL。

Further, the tobacco seed endophytic antagonistic bacteria serving as a biological bacterial agent is applied to inhibiting and/or preventing bacterial wilt, and the preparation method of the biological bacterial agent comprises the following steps: activating the endophytic antagonistic strain DW15 strain of tobacco seeds, selecting single colony, inoculating to LB liquid culture medium, culturing at 30deg.C and 180r/min to logarithmic phase, diluting with sterile water to 1×10 ⁶ cfu/mL-1×10 ¹² cfu/mL root irrigation treatment is carried out on plants.

Preferably, dilution to 1X 10 with sterile water is performed ⁸ cfu/mL root irrigation treatment is carried out on plants.

3. The use of any of the above described having a tobacco seed endophytic antagonistic bacterium as a biological agent for promoting the growth of tobacco plants.

Further, the tobacco seed endophytic antagonistic bacteria is used as a biological bacterial agent in the application of promoting the growth of tobacco plants, and the use concentration of the tobacco seed endophytic antagonistic bacteria in the biological bacterial agent is 1 multiplied by 10 ⁶ cfu/mL-1×10 ¹² cfu/mL。

Further, the tobacco seed endophytic antagonistic bacteria are used as biological bacteria for promoting the growth of tobacco plants, and the preparation method of the biological bacteria comprises the following steps: activating endophytic antagonistic bacteria strain of tobacco seed, selecting single colony, inoculating to LB liquid culture medium, culturing at 30deg.C and 180r/min to logarithmic phase, diluting with sterile water to 1×10 ⁶ cfu/mL-1×10 ¹² cfu/mL root irrigation treatment is carried out on plants.

4. A biological agent comprising a tobacco seed endophytic antagonistic bacterium, wherein the tobacco seed endophytic antagonistic bacterium is DW15, is classified and named as pseudomonas paraxanthomonas Pseudomonas parafulva, and is preserved in China center for type culture collection, and the preservation number is as follows: cctccc NO: m2022820.

The invention has the beneficial effects that: the invention has the beneficial effects that: on the basis of earlier-stage work in a laboratory, the disease resistance phenotype of different tobacco varieties to bacterial wilt is screened through two methods of indoor root irrigation inoculation and field disease nursery; comparing tobacco seed endophytic bacterial community characteristics of different resistance phenotypes by using an Illumina high throughput sequencing technology system; further separating, purifying and screening out endogenous antagonistic bacteria of the resistant tobacco variety seeds by a plurality of culture mediums, and verifying the prevention and control effect of the endogenous antagonistic bacteria on tobacco bacterial wilt and the growth of tobacco seedlings by the endogenous antagonistic bacteria. The invention screens 5 tobacco seed endophytic antagonistic bacteria from middle layer of 328 strains of seed endophytic bacteria which are screened and purified in the earlier stage, has a certain antagonism to Ralstonia solanacearum and has different growth promotion effects. The best biocontrol effect is that the strain G3-KB-15 (DW 15) and the strain 6036-R2A-26 (DW 26) are subjected to dip dyeing by the ralstonia solanacearum CQPS-1 with strong pathogenicity, the average control effect of the strain G3-KB-15 after the inoculation treatment is more than 60 percent in 7-15 days, the average control effect of the strain G3-KB-15 after the inoculation treatment is still up to 52.17 percent in 19 days (at the end of the onset), and the strain G3-KB-15 shows good antagonism and has better effect of inhibiting or preventing bacterial wilt of tobacco.

The invention can produce more protease by screening endophyte G3-KB-15, the average transparent circle of the produced protease can reach 4.14cm, and the cell wall of pathogenic bacteria is destroyed, so that the pathogenic bacteria are killed or inhibited, and the invention has better biocontrol function in cooperation. The strain has a certain biological film forming capability, which indicates that the strain has good colonization capability. The strain also has strong phosphate-dissolving capability and nitrogen-fixing capability, can dissociate indissolvable phosphate in soil, is converted into a compound which can be absorbed and utilized by plants, promotes plant growth through the actions of phosphate dissolving, nitrogen fixing and the like, and can also achieve the aim of indirectly preventing and treating diseases.

The strain G3-KB-15 has good antagonism of Ralstonia solanacearum and growth promotion effect on tobacco plants, and the seed soaking treatment of the bacterial suspension can obviously promote the germination of tobacco seeds and improve the relative germination vigor and the relative germination rate of the seeds. The fermentation liquor can be used for continuously root irrigation, so that the biomass of tobacco seedlings such as overground fresh weight, underground fresh weight, overground dry weight, underground dry weight, maximum root length, SPAD and the like can be improved, the growth of tobacco in seedling stage can be effectively promoted, and the effect of early growth and rapid initiation of tobacco can be achieved. The strain G3-KB-15 also has the function of affecting the functional diversity of microorganisms in rhizosphere soil of tobacco plants, can solve the problems of nutrient supply and microecological unbalance of tobacco planting soil caused by the increase of continuous cropping years of tobacco, and avoids slow growth and development, quality and yield reduction of tobacco. Therefore, the endophytic antagonistic bacteria screened by the method has wide application prospect and has a certain guiding significance for relieving continuous cropping obstacle in actual production and application.

Preservation information

Strain name: DW15, classification naming: pseudomonas paraflavum Pseudomonas parafulva, deposited in China center for type culture Collection, address: martial arts, date of preservation: 2022, 6 and 7, deposit number: cctccc NO: m2022820.

Drawings

In order to make the objects, technical solutions and advantageous effects of the present invention more clear, the present invention provides the following drawings for description:

FIG. 1 shows the incidence of bacterial wilt in the room for different tobacco varieties.

FIG. 2 shows the in-house bacterial wilt index of different tobacco varieties.

FIG. 3 shows the field incidence and index of tobacco bacterial wilt of different tobacco varieties.

Fig. 4 is a field representation of tobacco bacterial wilt of different tobacco varieties.

FIG. 5 shows the area under the disease progression curve for different tobacco varieties based on disease index.

FIG. 6 is a partially screened and purified resistant tobacco variety seed endophytic bacteria.

FIG. 7 is a graph of single colonies of different endophytic antagonistic bacteria screened.

FIG. 8 shows the biofilm formation capacity of different endophytic antagonistic bacteria screened.

FIG. 9 is a transparent circle formed by extracellular enzyme secretion by different endophytic antagonistic bacteria screened.

FIG. 10 shows growth of different endophytic antagonistic bacteria screened on Ashby medium.

FIG. 11 shows the effect of different endophytic antagonistic bacterial treatments on tobacco seedling biomass.

FIG. 12 shows the effect of root irrigation treatment of different endophytic antagonistic bacteria fermentation broths on bacterial wilt disease index.

FIG. 13 shows the control effect of root irrigation treatment on bacterial wilt by screening different endophytic antagonistic bacteria fermentation broths.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The experimental methods for which specific conditions are not specified in the examples are generally conducted under conventional conditions or under conditions recommended by the manufacturer.

Example 1

1. Materials and methods:

based on the early-stage multi-variety resistance screening evaluation, 6 tobacco varieties are further screened out for indoor root irrigation inoculation and comprehensive resistance evaluation of bacterial wilt in field disease nursery, and a foundation is laid for characteristic analysis of endophytic bacterial communities of subsequent anti-infection tobacco variety seeds. In the embodiment, four-leaf first-heart tobacco seedlings of different tobacco varieties are taken as research objects, and bacterial wilt is used for root irrigation inoculation, so that the disease resistance phenotype of the different tobacco varieties to bacterial wilt is evaluated.

Test tobacco seeds: the tobacco variety to be tested was planted in Peng Shuixian Runxi tea tree plateau test district (altitude 1360m, 29 ° 7'43″ north latitude, 107 ° 56'38 ") in Chongqing city 5 months in 2020. Before flowering, selecting 15-20 standard plants with typical characters of each variety, and bagging and reserving seeds after flower thinning and fruit thinning; harvesting when the pericarp turns brown, air drying, threshing, and sealing in a refrigerator at-20deg.C. The original information of each variety is shown in table 1.

TABLE 1 original material information of test varieties

The pathogenic bacteria to be tested are strong pathogenic ralstonia solanacearum CQPS-1 (Ralstonia solanacearum) (called ralstonia solanacearum for short) separated from pathogenic smoke strains of Talcum village (altitude 1210m, north latitude 29 DEG 8 '12', east longitude 107 DEG 56 '31') in Chongqing city Peng Shui county in the research laboratory.

BG medium: 10g/L of bactopeptone, 1g/L of casein amino acid, 1g/L of yeast extract, 15g/L of agar powder and 5g/L of glucose. After sterilization, cooling to about 55 ℃, adding 1% TTC (2, 3, 5-triphenyltetrazolium chloride) aqueous solution to a final concentration of 0.005%,

liquid medium B: 10g/L of bactopeptone, 1g/L of yeast extract and 1g/L of tyrosine peptone.

All the medium components were mixed and sterilized by autoclaving (sterilization conditions: 121 ℃ C., 20min, the same applies hereinafter).

Tobacco cultivation: floating seedling raising is carried out by using the collected tobacco variety seeds, and the seedling raising conditions are as follows: the temperature is 25 ℃, the relative humidity is 60 percent, the time ratio of illumination to darkness is 14/10h, and the culture is carried out until four leaves are reserved at one heart.

Preparation of bacterial wilt inoculation liquid: streaking and activating bacterial wilt on BG plate, culturing at 30deg.C for 48 hr, selecting single colony, transferring into B liquid culture medium, shake culturing (30deg.C, 180 r/min) to logarithmic phase (OD) _600nm =0.8-1.0). Then dilute to OD _600nm ＝0.1(10 ⁸ CFU/mL).

Inoculating bacterial suspension by root irrigation: root-filling inoculation of each tobacco seedling 10 ⁸ CFU/mL of 10mL of bacterial suspension was repeated 4 times for each treatment, 8 seedlings each. After inoculation of pathogenic bacteria, the cells are placed in a greenhouse with a temperature of 30 ℃ and a relative humidity of 75% and a light-to-dark time ratio of 14/10h for cultivation.

Investigation of tobacco bacterial wilt disease: the disease condition of different varieties is systematically investigated at the beginning of the disease period, once every other day, until the end of the disease period, by referring to the indoor investigation grading standard of Zheng Jifa et al, and the disease rate and disease index are calculated.

The indoor resistance evaluation criteria of different tobacco varieties to bacterial wilt are as follows: the disease index is 0-20: high Resistance (HR); the disease index is 21-40: disease resistance (R); the disease index is 41-60: a neutralizing antibody (MR); the disease index is 61-80: low Resistance (LR); the disease index is 81-90: a disease (S); the disease index is 91-100: high Sense (HS).

Data analysis: summary data was collated with Excel 2016, analyzed by variance using IBM SPSS Statistics 23 and checked for significance (P < 0.05), plotted using Origin 2017 and GraphPad Prism 9.

2. Results and analysis:

bacterial wilt disease onset conditions of different tobacco varieties: the inoculation concentration is 10 ⁸ cfu/mL, the end of onset (23 d after inoculation) is shown in FIG. 1, and the index of disease throughout the investigation period is shown in FIG. 2. As the inoculation concentration is relatively high, and the tested pathogenic bacteria are strong pathogenic strains, the occurrence of different varieties of tobacco is serious. Each tobacco variety began to develop disease 5 days after inoculation. Over time, the disease index of each variety gradually increased, but throughout the disease investigation period, the disease index of varieties HD, K326 and YY87 was greater than that of varieties 6036, YY97 and G3. 23d after inoculation is the end stage of the disease, the average disease index of varieties HD, K326 and YY87 are 95.83, 95.83 and 91.67 respectively, which are significantly higher than the average disease index of varieties 6036, YY97 and G3 (66.44, 64.58 and 60.42 respectively). The disease index analysis of the whole investigation period is integrated, and the resistance of each tobacco variety to bacterial wilt is sequentially from high to low: G3G 3>YY97>6036>YY87>HD≥K326。

Bacterial wilt resistance grade of different tobacco varieties: the results of grading the disease index at the end of the onset (23 d after inoculation) are shown in Table 2. As the pathogenic bacteria are strong pathogenic strains, the inoculation concentration is 10 ⁸ At CFU/mL, the disease index of each variety is higher, G3 shows Medium Resistance (MR), 6036 and YY97 show Low Resistance (LR), and the disease indexes of K326, YY87 and HD are obviously higher than those of other varieties, and the disease indexes of the varieties show High Sense (HS).

TABLE 2 bacterial wilt resistance ranking of different tobacco varieties

Example 2

Evaluation of field resistance of different tobacco varieties to bacterial wilt: tobacco seeds are subjected to unified field transplanting and later management according to local related technical standards by adopting floating seedling culture as in the embodiment 1. A flat land block (altitude 1210m, north latitude 29 DEG 8 '12', east longitude 107 DEG 56 '31') with serious tobacco bacterial wilt and continuous cropping throughout the year of the mountain area of Peng Shuixian Runxi village is experimentally selected. 3 cells are repeated for different varieties, 18 cells are added, the design of the groups is random, and 40 tobacco plants are planted in each cell.

Field investigation of tobacco bacterial wilt:

the investigation and grading standard of the tobacco bacterial wilt are carried out by referring to national standard GB/T23222-2008 'tobacco plant diseases and insect pests grading and investigation method'. Investigation is started at the initial stage of bacterial wilt, and investigation is carried out every 5-7d until the later harvest period. The area under the curve of morbidity, disease index and disease progression (Area Under the Disease Progress Curve, AUDPC) was calculated:

(X _i index t of disease at the ith investigation _i Date at the ith investigation

The evaluation standard of the field resistance of different varieties refers to GB/T23224-2008 'disease resistance identification of tobacco varieties', and specifically comprises the following steps: the disease index is 0: high resistance or immunity (I); the disease index is 0.1-20: disease resistance (R); the disease index is 20.1-40: a neutralizing antibody (MR); the disease index is 40.1-60: a sense of well (MS); the disease index is 60.1-80: a disease (S); the disease index is 80.1-100: high Sense (HS).

Bacterial wilt disease onset conditions of different tobacco varieties: when bacterial wilt happens sporadically, the system surveys the occurrence of bacterial wilt of different tobacco varieties, the morbidity and the disease index are respectively shown in figure 3, wherein a: incidence, b: index of disease. The mid-onset (2021.07.28) and mid-late onset (2021.08.07) field performance is shown in fig. 4, where a: mid-period of onset; b: middle and late onset of disease. Over time, the incidence and index of tobacco bacterial wilt gradually increase. The incidence rate and the disease index of varieties HD, YY87 and K326 in the middle and late stages of the disease are greatly increased under the influence of high-temperature and high-humidity climate. The morbidity and the disease index of different varieties are expressed as follows by integrating the morbidity condition of the whole growth period: HD > YY87> K326> G3>6036> YY97.

Bacterial wilt resistance grade of different tobacco varieties

The area under the disease progression curve (AUDPC) results based on the disease index are shown in fig. 5. The results show that the area under the course progression curve (AUDPC) is sequentially from large to small: HD. YY87, K326, G3, 6036, YY97, wherein varieties HD, YY87, K326 are significantly higher than varieties G3, 6036, YY97, there are significant differences between varieties HD, YY87, K326, there are no significant differences between varieties G3, 6036, YY97, and overall, the resistance of different tobacco varieties to bacterial wilt appears as follows: YY97>6036> g3> k326> YY87> hd. The results of grading the disease index of tobacco in different stages of fertility and onset are shown in Table 3. The topping period (7 months and 16 days) is in the middle stage of disease, the disease index of HD is the largest, and other varieties show disease resistance. The middle and later harvest period (8 months and 16 days) is in the disease peak period, and the disease index of the HD is the largest and is manifested as the disease; the disease index of YY87 is several times, and is manifested as moderate sensation; the disease index of K326 is central and is expressed as moderate resistance; other varieties are disease-resistant. The later harvest period (22 days of 8 months) is at the end of disease, and the disease index of the HD is the largest and obviously higher than other varieties, and the HD is manifested as a disease; the disease index of YY87 is several times, and is manifested as moderate sensation; the disease indexes of K326 and G3 show moderate resistance, while other varieties show disease resistance.

TABLE 3 bacterial wilt resistance ranking of different tobacco varieties

Note that: index of disease: mean ± standard error, letter differences indicate significant differences at the 0.05 level for each treatment.

Example 3

Structural characterization of endophytic bacteria of different resistant tobacco varieties: tobacco seeds were prepared as in example 1, sterilized seeds were transferred to a sterile mortar, and ground with liquid nitrogen for 3-5min until the seeds were ground. 0.5g of ground seed was weighed each time repeatedly using FastDNA ^TM SPIN kit, DNA was extracted according to the protocol.

Establishing a PCR sequence library and performing Illumina high-throughput sequencing:

first, the integrity of the DNA is checked, and the concentration and purity of the DNA are checked. After quality inspection, the V5-V7 variable region of the 16S rDNA of the endophyte is subjected to PCR amplification.

The PCR reaction system was 20. Mu.L: 4. Mu.L of 5 XFastpfu Buffer, 2. Mu.L of 2.5mM dNTPs, 0.8. Mu.L of Forward Primer (5. Mu.M), 0.8. Mu.L of Reverse Primer (5. Mu.M), 0.4. Mu. L FastPfu Polymerase, 0.2. Mu.L of BSA,10ng of Template DNA, make up ddH ₂ O to 20. Mu.l.

The amplification procedure was two rounds of nested PCR: the first round of amplification uses primers 799F (AACMGGATTAGATACCCKG, 5 '-3') and 1392R (ACGGGCGGTGTGTRC, 5 '-3'), PCR amplification was performed on the V5-V8 variable region of 16S rDNA of endophytic bacteria using the extracted DNA as a template, pre-denatured at 95℃for 3min,27 cycles (denaturation at 95℃for 30S, annealing at 55℃for 30S, extension at 72℃for 45S), and extension at 72℃for 10min; the second round of PCR amplification was performed on the 16S rDNA V5-V7 variable region using primers 799F (AACMGGATTAGATACCCKG, 5 '-3') and 1193R (ACGTCATCCCCACCTTCC, 5 '-3') using the first round of amplification product as a DNA template for 13 cycles, with the remaining conditions being identical to the first round of amplification. After the amplification products are qualified in quality inspection, the method entrusts the Shanghai Meijia biological medicine technology Co.Ltd to carry out Illumina MiSeq sequencing.

According to the embodiment, high-throughput sequencing analysis is carried out on the screened tobacco variety seed endophytes with different bacterial wilt resistance phenotypes, 405651 optimized sequence fragments are obtained, and a sufficient data basis is provided for analysis of endophyte community composition and diversity and excavation of resistant variety seed endophyte biocontrol bacteria. High throughput sequencing analysis results: (1) The Beta diversity analysis results show that the resistance and the tobacco seed endophytic bacterial community structure have great difference. (2) The relative abundance of the firmicutes of resistant varieties G3, YY97 and 6036 is significantly higher than that of the susceptible varieties, 2.34, 5.58 and 8.46 times that of susceptible variety YY87, respectively, 1.54, 3.68 and 5.58 times that of susceptible variety HD; the actinomycota relative abundance of resistant varieties YY97 and 6036 is significantly higher than that of susceptible varieties, 2.77 times and 2.40 times that of susceptible variety YY87, and 2.59 times and 2.24 times that of susceptible variety HD, respectively. (3) Endophytic bacterial populations of different tobacco variety seeds are more similar in composition at the genus level, but have a greater difference in relative abundance. Analyzing the bacteria with significant difference of relative abundance among resistant varieties, wherein the unclassified_f __ Enterobacteriaceae of the resistant variety G3 has the highest relative abundance and is significantly higher than that of the resistant variety HD; the relative abundance of Pseudomonas (Pseudomonas) of the resistant varieties G3 and YY97 is higher than that of the susceptible variety YY87; paenibacillus (Paenibacillus) relative abundance of resistant varieties G3, 6036 and YY97 is significantly higher than that of the susceptible variety; the Bacillus (Bacillus) relative abundance of resistant variety 6036 is highest, significantly higher than that of the susceptible variety.

Example 4

Based on the evaluation of the resistance of different tobacco varieties and the structural characteristic system analysis of the endophyte bacteria of the seeds, the method further separates, purifies and screens the endophyte culturable bacteria of the resistant varieties G3, YY97 and 6036, and examines the relevant characteristics of the endophyte antagonistic bacteria of the seeds so as to obtain the endophyte biocontrol bacteria resource of the seeds.

Tobacco seeds were prepared as in example 1, test medium (after all medium components were mixed, autoclaved):

LB medium: 10g/L tryptone, 5g/L, naCl g/L yeast extract.

BG medium: as before.

NA medium: 5g/L peptone, 1g/L yeast extract, 3g/L beef extract, 10g/L glucose and 15g/L agar.

(OXOID Co., UK)

TSA medium: 15g/L of tryptone, 5g/L of soybean peptone, 5g/L of NaCl and 15g/L of agar. (Haibo biotechnology Co., ltd.)

KB medium: peptone 20g/L, K ₂ SO ₄ 10g/L，MgCl ₂ 1.4g/L, 10g/L glycerol and 15g/L agar.

R2A medium: 0.5g/L bactopeptone, 0.5g/L soluble starch, 0.5g/L yeast extract, 0.5g/L glucose, 0.5g/L Casein amino acid, KH ₂ PO ₄ 0.3g/L, 0.3g/L sodium pyruvate, mgSO ₄ 0.024g/L, 15g/L of agar.

1. Isolation and purification of endophytic bacteria

2g of seeds of different tobacco varieties with the surfaces disinfected are weighed by adopting a dilution flat plate coating method, fully ground in a mortar, and added with 8mL of sterile water for uniform mixing. Standing for 5min, sucking 100 μl of supernatant, adding 900 μl of sterile water, diluting, and recording as 10 ^-2 In this way, the mixture is diluted to 10 in a gradient ^-3 、10 ^-4 And 10 ^-5 . Respectively taking 100 mu L of each dilution gradient of the dilution liquid on NA, TSA, R2A and KB plates, uniformly coating the dilution liquid by using glass beads, setting 3 repeats for each concentration and each type of plate, culturing the plates at 30 ℃ for 2-5d in an inverted manner, continuously observing the growth condition of the plates, selecting different representative strains according to phenotype differences of colony morphology, color, transparency and the like, naming the different representative strains (naming rules as variety-culture medium-serial number), picking single colonies on the corresponding plates, continuing to score and purify the plates for 3-4 times until the colonies on the plates are consistent and sterile, and treating the purification to be completed.

And (3) carrying out streak purification according to phenotype differences on different culture mediums, and finally obtaining 101 strains of bacteria of a variety YY97, 115 strains of bacteria of a variety 6036 and 112 strains of bacteria of a variety G3, and obtaining 328 strains of endophytic bacteria of seeds of all resistant varieties. FIG. 6 is a partially resistant tobacco variety seed endophyte. The purified strain was scraped with a sterile cotton swab to obtain a single colony, and placed in a 1.5mL centrifuge tube containing 25% glycerol, and stored at-80℃under ultra-low temperature.

2. Antagonistic bacterial screening

(1) Plate antibacterial activity screening

And (5) performing primary screening on the antagonistic strains of the bacteria stored after purification by adopting a spraying method. First, single colonies were picked up and inoculated in the center of NA plate, and each strain was repeated 3 times. Culturing at 30deg.C overnight with inverted OD using sterile watering can _600nm ＝0.1(10 ⁸ CFU/mL) bacterial wilt liquid is evenly sprayed on an NA flat plate, and after inversion culture for 24 hours at 30 ℃, the diameter of a bacteriostasis ring is measured by adopting a crisscross method. And selecting a strain with a larger inhibition zone for subculture, repeatedly detecting the inhibition stability of the strain, and finally determining the potential antagonistic bacteria with good and stable plate inhibition effect. By evaluating the plate antibacterial activity against endophytic bacteria of the resistant variety seeds, bacteria with good and stable inhibition zone effect are finally selected as shown in table 4. There are many potential antagonistic bacteria in the resistant variety seeds, wherein the variety YY97 is 3 strains, the variety G3 is 7 strains, and the variety 6036 is 5 strains.

(2) Potted plant effect evaluation and screening

And (5) taking the strain with good plate antibacterial activity obtained by screening as a material, and evaluating the potting effect. Picking single colony of potential antagonistic bacteria, transferring to LB culture medium, shake culturing, picking single colony of ralstonia solanacearum, transferring to B liquid culture medium, shake culturing to OD _600nm =0.8-1.0, diluted with sterile water to OD _600nm ＝0.1(10 ⁸ CFU/mL).

First, a root irrigation treatment (10) is performed on a fermentation broth of a potential antagonistic bacterium ⁸ CFU/mL, 10 mL/strain), 3d bacterial wilt-root (10) ⁸ CFU/mL, 10 mL/plant), and placed in a greenhouse at 30℃and 75% relative humidity with a light to dark time ratio of 14/10h, 2 replicates per treatment, 8 seedlings per replicate. The disease condition of tobacco bacterial wilt was investigated from the initial stage of the disease, once every two days, and the disease investigation method was the same as that of the tobacco bacterial wilt of example 1.

The potted activity evaluation is carried out on the screened 15 potential endophytic antagonistic bacteria, and the result shows that the strain with the large diameter of the flat bacteriostasis zone has an indefinite greenhouse biocontrol effect, thereby indicating that the biocontrol effect and the size of the flat bacteriostasis zone are not necessarily in positive correlation. Through repeated potting experiments, 5 endophytic antagonistic bacteria (YY 97-KB-20, G3-NA-3, G3-KB-15, 6036-KB-29 and 6036-R2A-26) with better and stable effect are finally selected for subsequent experiments.

TABLE 4 evaluation of antibacterial effect of potential antagonistic bacteria

4. Colony morphology observation of endogenous antagonistic bacteria

And (3) streaking and activating the screened endogenous antagonistic bacteria on a corresponding plate, culturing for 36 hours at 30 ℃, and observing the appearance and morphological characteristics of single colonies.

Individual colonies of different endophytic antagonistic bacteria are shown in figure 7. The strain YY97-KB-20 is white and round, and the surface is moist and smooth; the strain G3-NA-3 is white and round, the surface is moist and smooth, and the surface is slightly raised; the strain G3-KB-15 is yellow and round, the surface is moist and smooth, and the surface is slightly raised; bacterial strain 6036-KB-29 is milky white, irregular and opaque in edge, and uneven in colony surface and provided with folds and ridges; strain 6036-R2A-26 is white and round and opaque, and elliptical spores are arranged in the enlarged cysts.

5. Identification of endogenous antagonistic bacterial molecules

(1) Bacterial genomic DNA extraction

Single colonies are picked and transferred into LB liquid medium, cultured for 12-14h at 30 ℃ and 180r/min, and the thalli are collected by centrifugation, and DNA extraction is carried out by using a bacterial DNA extraction kit.

(2) PCR amplification

The extracted total DNA is used as a template, and bacterial 16S rDNA amplification universal primers 27F and 1429R, gyrA gene amplification primers gyrA-F and gyrA-R, gyrB gene amplification primers gyrB-F and gyrB-R are used for amplification. Table 5 shows the sequencing primer sequences for each bacterium.

PCR reaction procedure: pre-denaturation at 95℃for 5min,34 cycles (denaturation at 95℃for 30s, annealing at 54℃for 30s, extension at 72℃for 1 min), extension at 72℃for 10min,4 ℃. The PCR reaction system was 25. Mu.L: template DNA 1.0. Mu.L; 1.0. Mu.L of each of the upstream and downstream primers; 2 XTaq Master Mix 12.5. Mu.L; ddH ₂ O 9.5μL。

TABLE 5 bacterial sequencing primer sequences

Sequencing PCR products:

and (3) after the PCR product is detected to be qualified by 1% agarose gel electrophoresis, sequencing is carried out by the large gene Limited company. Sequencing results were compared for BLAST homology via NCBI (National Center for Biotechnology Information) database and phylogenetic tree was constructed using Neighbor-Joining (NJ) using MEGA X software, and tested using Bootstrap (Bootstrap) with 1000 Bootstrap datasets.

The combination of 16S rRNA and gyrA gene or gyrB gene sequence analysis can carry out more accurate molecular identification on target bacteria. Target gene sequences obtained by different endophytic antagonistic bacteria are subjected to BLAST comparison in a GenBank database, and a phylogenetic tree is constructed. The results show that YY97-KB-20 strain is defensive pseudomonas (Pseudomonas protegens), G3-NA-3 strain is enterobacter albolsis (Enterobacter asburiae), G3-KB-15 strain is P.paraxanthum (Pseudomonas parafulva), 6036-KB-29 strain is geobacillus (Bacillus altitudinis), and 6036-R2A-26 strain is Paenibacillus odorifer.

Strain G3-KB-15 was designated DW15, classified under the following designations: pseudomonas paraflavum Pseudomonas parafulva, deposited in China center for type culture Collection, address: martial arts, date of preservation: 2022, 6 and 7, deposit number: cctccc NO: m2022820.

Strain 6036-R2A-26 was designated DW26, classified under the following names: paenibacillus odorifer, deposited in China center for type culture Collection, address: martial arts, date of preservation: 2022, 6 and 7, deposit number: cctccc NO: m2022821.

Example 5

The characteristics of the endophyte resistance-related characteristics (biofilm formation ability, extracellular enzyme secretion ability, phosphate solubilizing ability and nitrogen fixation ability) selected in example 4 were studied.

Test medium (after all medium components are mixed, autoclaved):

enzyme culture medium: 1% casein was added to LB solid medium.

CMC medium: sodium carboxymethylcellulose 20g/L, KNO ₃ 1g/L，MgSO ₄ ·7H ₂ O 0.5g/L，NaCl 0.5g/L，K ₂ HPO ₄ 0.5g/L，FeSO ₄ ·7H ₂ O0.01 g/L and agar 15g/L.

Starch agar medium: 2g/L of soluble starch, 5g/L of beef extract, 10g/L, naCl g/L of bactopeptone and 20g/L of agar.

Gram iodine solution: iodine 1.0g, potassium iodide 2.0g, distilled water 300mL.

NBRIP medium: glucose 10g/L, KCl 0.2g/L, ca ₃ (PO ₄ )25g/L，(NH ₄ ) ₂ SO ₄ 0.1g/L，MgSO ₄ ·7H ₂ O 0.25g/L，MgCl·6H ₂ O5 g/L, agar 20g/L.

Ashby medium: KH (KH) ₂ PO ₄ 0.2g/L，MgSO ₄ ·7H ₂ O 0.2g/L，NaCl 0.2g/L，CaCO ₃ 5g/L, mannitol 10.0g/L, caSO ₄ ·2H ₂ O0.1 g/L, agar 18g/L.

1. Evaluation of the Forming Capacity of endogenous antagonistic bacteria biofilm

The properties of the biofilm that can bind to the dye crystal violet were used to determine the biofilm of different bacteria using the crystal violet staining method. Transferring single colony of different antagonistic bacteria into LB liquid culture medium, culturing to logarithmic phase, centrifuging, washing with sterile water to obtain bacterial precipitate with residual culture medium removed, and re-suspending and diluting with new LB liquid culture medium (diluting to OD _600nm =0.1, again diluted 10 times) and sucked200. Mu.L was added to 96-well plates, each treatment was repeated 6 times, and the plates were incubated at 30℃for 48 hours under sealed conditions. After the completion of the culture, the culture was removed, 200. Mu.L of sterile water was added for 2 times, then 150. Mu.L of crystal violet solution (0.1%) was added for staining for 30 minutes, then each well was again washed with 200. Mu.L of sterile water for 2 times, dried at room temperature for 30 minutes, then 200. Mu.L of 95% absolute ethanol was added for dissolution, and OD was measured after 30 minutes _530nm Values.

Biofilm formation ability of the different strains As shown in FIG. 8, OD of strains 6036-R2A-26 and G3-KB-15 _530nm 1.16 and 0.98, respectively, and has certain biofilm formation capacity.

2. Detection of ability of endogenous antagonistic bacteria to secrete extracellular enzymes (proteases, cellulases and amylases)

The ability to secrete proteases, cellulases and amylases was tested based on whether a transparent loop was produced. The activated strain is respectively inoculated to the centers of an enzyme culture medium, a CMC culture medium and a starch agar culture medium, after inversion culture is carried out for 3d at 28 ℃, thalli are scraped off, 1% Congo red is used as an indicator for dyeing for 30min, and then 5% NaCl is used for repeatedly washing for 2-3 times and then soaking for 60min, and the formation of transparent rings around the colony indicates that the strain can secrete protease and cellulase; after inversion culture at 28℃for 3d, the cells were scraped off, the gram-iodine solution was soaked for 15min,5% NaCl was washed off to remove the flooding, and the formation of a transparent ring around the colony indicated that the strain was able to secrete amylase.

The diameter of the transparent circles formed by the extracellular enzymes secreted by the different strains is shown in Table 6, and the transparent circles are shown in FIG. 9. The strains YY97-KB-20, G3-NA-3, G3-KB-15 and 6036-KB-29 can produce protease, and the transparent circles of the protease produced by the strains YY97-KB-20 and G3-KB-15 are the largest and are respectively 4.20cm and 4.14cm on average; strains 6036-KB-29, 6036-R2A-26 and G3-NA-3 can produce cellulase, and the transparent circle produced by strain 6036-KB-29 is maximum and is 2.91cm on average; strain 6036-R2A-26 was able to produce amylase with an average of 1.25cm in transparent circles.

Table 6 endophytic antagonistic bacteria transparent circle diameter

3. Determination of phosphorus-decomposing capacity of endophytic antagonistic bacteria

Reference Xu Mingshuang (diversity analysis of endophytic bacteria and function study of promoting bacteria of tomato and rice seed [ D ]. University of agriculture in China, 2014.) et al, and improving, picking single colony spot, inoculating to NBRIP plate center, inoculating sterile water as control, and culturing at 30deg.C for 10D. The phosphate dissolving effect is determined by the diameter ratio of the phosphate dissolving ring to the bacterial colony, the larger the ratio is, the stronger the phosphate dissolving capability is, and the ratio is 1, the non-phosphate dissolving capability is indicated. The ratio of the phosphate solubilizing circles of the different antagonistic bacteria to the diameters of the colonies is shown in Table 7, and the phosphate solubilizing capacities of the different antagonistic bacteria are greatly different. The ratio of the strain YY97-KB-20 to the strain G3-KB-15 is 2.20 and 1.81 respectively, and the phosphate solubilizing capability is high.

TABLE 7 different endogenous antagonistic bacterial phosphate solubilizing circles

4. Determination of endogenous antagonistic bacteria nitrogen fixation ability

Reference Liu Huiying (pineapple endophyte screening and identifying and antagonistic effect evaluation [ J ]. Lake North university journal (Nature science edition), 2020,42 (2): 158-164.) et al, inoculating the strain on Ashby medium by streaking, and culturing at 30deg.C for 15d. If the strain grows well and can produce transparent growth bands, the strain has stronger nitrogen fixing capability. The growth of different antagonistic bacteria on Ashby medium is shown in FIG. 10, and the strains YY97-KB-20, G3-NA-3 and G3-KB-15 grow normally on Ashby medium and obvious transparent areas are generated at streak culture positions, so that the strain has strong nitrogen fixation capacity.

EXAMPLE 6 study of the Effect of seed endophytic antagonistic bacteria on tobacco growth

1. Effect of endogenous antagonistic bacteria on tobacco seed germination

(1) Preparing a bacterial suspension: selecting single colony of different endophytic antagonistic bacteria, inoculating into LB liquid medium, culturing at 30deg.C and 180r/min to logarithmic phase, centrifuging, washing with sterile water to obtain thallus with residual culture medium removedPrecipitation, dilution to OD with sterile water _600nm ＝0.1(10 ⁸ CFU/mL)。

(2) Effects of bacterial suspension on tobacco seed germination: the sterilized seeds were placed in sterile petri dishes (30 grains/dish) with filter paper laid on the bottom, 5mL of bacterial suspension was added and each treatment was repeated 3 times. And (3) sealing and culturing for 7d and 10d at 25 ℃, and respectively calculating the relative germination vigor and the relative germination rate.

The effect of different antagonistic bacterial treatments on seed germination is shown in table 8, and different antagonistic bacterial suspensions have promotion effects on the relative germination vigor and germination rate of tobacco seeds. In the 7d after treatment, the average germination potential of the strain G3-NA-3 is 70.88%, and the strain is not obviously different from CK, and other treatments can obviously promote seed germination; at 10d after treatment, other treatments have remarkable promotion effect on seed germination except for the strain G3-NA-3 and YY 97-KB-20. Therefore, the relative germination vigor and relative germination rate of seeds treated by the strains G3-KB-15, 6036-KB-29 and 6036-R2A-26 are obviously higher than those of CK, and the germination of tobacco seeds can be obviously promoted.

TABLE 8 Effect of endogenous antagonistic bacteria on seed germination

Note that: relative germination vigor and relative germination rate: mean ± standard error, letter differences indicate significant differences at the 0.05 level for each treatment.

2. Effect of endogenous antagonistic bacteria on tobacco growth

(1) Preparing bacterial fermentation liquid: picking single colony of each endogenous antagonistic bacteria, inoculating into LB liquid medium, culturing at 30deg.C for 180r/min to logarithmic phase, diluting with sterile water to OD _600nm ＝0.1(10 ⁸ CFU/mL)。

(2) Root irrigation of bacterial fermentation liquor: and (3) sterilizing the surface of the bare seed of the Yunyan 87, sowing the bare seed on a sterilized seedling substrate, and after about 35d, picking seedlings with the same size, and transplanting the seedlings to a seedling pot with the caliber of 9 cm. After 1d of seedling growth, root irrigation (10) of the endophytic antagonistic bacteria fermentation broth is carried out ⁸ CFU/mL, 10 mL/strain), 3 replicates per treatment, 5 strains per replicate. Root irrigation is repeated every 7 days, and the root irrigation dosage is the same as above, and is 3 times in total. Tobacco seedlings are placed in an artificial incubator with alternating illumination (5000 LX) for 14 hours and darkness for 10 hours at 25 ℃ for cultivation.

(3) Tobacco seedling biomass determination

And respectively measuring the biomass of the tobacco seedlings of different treatment groups 10d after the last inoculation. Firstly, selecting leaves at the same part of each tobacco seedling to measure the SPAD value, then carefully shaking off root matrixes, cleaning residual matrixes by clean water, and using water absorbing paper to absorb water on the surface of the plant, and respectively counting root length, fresh weight of overground parts and fresh weight of underground parts. After drying (15 min at 105 ℃ and 4h at 75 ℃), the dry weight of the aerial part and the dry weight of the underground part are counted.

The effect of different endophytic antagonistic bacterial treatments on tobacco seedling biomass is shown in table 9, figure 11. Compared with CK, the fresh weight of the overground part, the fresh weight of the underground part, the dry weight of the overground part and the chlorophyll SPAD value of tobacco seedlings can be improved by different strains, and for tobacco plants, the higher the dry weight of the overground part is, the larger the biomass of tobacco is represented, so that the growth promoting effect of the strain is better. The strain G3-NA-3, 6036-KB-29 and 6036-R2A-26 can obviously improve the fresh weight of the overground part of the tobacco seedling, the strain 6036-R2A-26 can obviously improve the fresh weight of the underground part of the tobacco seedling, and different strains can obviously improve the dry weight of the overground part of the tobacco seedling and the chlorophyll SPAD value; the root length of 6036-KB-29, 6036-R2A-26 is longer than CK. Therefore, different antagonistic bacteria can effectively promote tobacco growth by continuous root irrigation treatment in the tobacco seedling stage, and the effect of early growth and rapid hair growth is achieved.

TABLE 9 Effect of endogenous antagonistic bacteria on tobacco growth

Note that: biomass: mean ± standard error, letter differences indicate significant differences at the 0.05 level for each treatment.

Example 7

Endophytes selected for example 4

1. Preparation of bacterial wilt and endophytic antagonistic bacteria fermentation liquor

Scribing the bacterial wilt and endogenous antagonistic bacteria on Ying Ping plates, respectivelyActivating, selecting single colony of endophytic antagonistic bacteria, transferring to LB culture medium for shake culture, selecting single colony of bacterial wilt, transferring to B liquid culture medium for shake culture, and shake culturing to OD respectively _600nm =0.8-1.0, diluted with sterile water to OD _600nm ＝0.1(1×10 ⁸ CFU/mL).

2. Evaluation of control effect of endogenous antagonistic bacteria on tobacco bacterial wilt

First, root irrigation treatment (10) is performed on the endophytic antagonistic bacteria fermentation liquor ⁸ CFU/mL, 10 mL/strain), 3d bacterial wilt-root (10) ⁸ CFU/mL, 10 mL/strain). 3 replicates were set for each treatment, 8 plants each, and incubated in a greenhouse at 30℃and 75% relative humidity at a light to dark time ratio of 14/10 h. The disease condition of the tobacco bacterial wilt is investigated from the initial stage of the disease, and the investigation is carried out once every two days, and the disease investigation method is the same as before. After the disease investigation is finished, the morbidity, the disease index and the prevention effect are calculated. Disease investigation time was based on CK survival time.

The effect of different endogenous antagonistic bacterial fermentation broth rooting treatments on bacterial wilt disease index is shown in figure 12. After 5d inoculation with ralstonia solanacearum, each treatment started to develop disease gradually, and then the disease index of each treatment gradually increased over time, but the disease index of CK was always much higher than that of antagonistic bacteria treatment. The disease index between antagonistic bacteria treatments is analyzed to be obtained, and the average disease index of G3-KB-15, G3-NA-3 and 6036-R2A-26 is lower than that of 6036-KB-29 and YY97-KB-20, so that the G3-KB-15, G3-NA-3 and 6036-R2A-26 treatments have better biological control effect on tobacco bacterial wilt.

The control effect of the root irrigation treatment of different endophytic antagonistic bacteria fermentation broths on bacterial wilt is shown in fig. 13, and table 10 shows specific data. The prevention effect of different treatments on bacterial wilt is different. The average control effect of the 6036-R2A-26 treatment after the inoculation is 7-15d is more than 60%, and the average control effect after the inoculation is 19d (the end stage of the disease) can still reach 51.09%; the average control effect of the G3-KB-15 treatment after the inoculation is over 60 percent and the average control effect of the G3-KB-15 treatment after the inoculation is 19d can still reach 52.17 percent, so that the overall control effect of the strain 6036-R2A-26 and the strain G3-KB-15 is higher than that of the other three strains. The average control effect of the G3-NA-3 treatment after inoculation at 19d is 53.26%, and the average control effect of the YY97-KB-20 and 6036-KB-29 treatment after inoculation at 19d is 44.57% and 36.96% respectively. Therefore, different endophytic antagonistic bacteria treatment biocontrol effects have certain difference, wherein the bacterial strains 6036-R2A-26 and G3-KB-15 have relatively high control effects, the ralstonia solanacearum CQPS-1 adopted in the research is an extremely strong pathogenic bacterial strain, and serious infection harm is caused to general tobacco plants, so that the bacterial strains 6036-R2A-26 and G3-KB-15 have higher control effects on other ralstonia solanacearum, are biocontrol bacteria with good effects for preventing and treating tobacco bacterial wilt, have good promotion effects on tobacco plants, can obviously improve tobacco seedling biomass, can be used singly or in a compound way, have wide application prospect for biological prevention and treatment of the tobacco bacterial wilt, and have important significance for protecting ecological environment.

Table 10 corresponding control data

Finally, it is noted that the above-mentioned preferred embodiments are only intended to illustrate rather than limit the invention, and that, although the invention has been described in detail by means of the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as defined by the appended claims.

Claims

2. The tobacco seed endophytic antagonistic bacterium according to claim 1, wherein the strain of the tobacco seed endophytic antagonistic bacterium has a yellow circular shape, a moist and smooth surface and micro-ridges.

3. The tobacco seed endophytic antagonistic bacterium of claim 1, wherein the strain is capable of producing a protease.

4. Use of a tobacco seed endophytic antagonistic bacterium according to any one of claims 1 to 3 for the preparation of a biological agent for inhibiting and/or controlling bacterial wilt.

5. The use according to claim 4, wherein the concentration of the endophytic antagonistic bacteria in the tobacco seed in the biological agent is 1 x 10 ⁶ cfu/mL-1×10 ¹² cfu/mL。

6. The use according to claim 4, wherein the preparation method of the biological agent comprises the following steps: activating the endophytic antagonistic strain DW15 strain of tobacco seeds, selecting single colony, inoculating to LB liquid culture medium, culturing at 30deg.C and 180r/min to logarithmic phase, diluting with sterile water to 1×10 ⁶ cfu/mL-1×10 ¹² cfu/mL root irrigation treatment is carried out on plants.

7. Use of a tobacco seed endophytic antagonistic bacterium according to any one of claims 1 to 3 for the preparation of a biological agent for promoting the growth of tobacco plants.

8. The use according to claim 4, wherein the concentration of the endophytic antagonistic bacteria in the tobacco seed in the biological agent is 1 x 10 ⁶ cfu/mL-1×10 ¹² cfu/mL。

9. A biological bacterial agent comprising the tobacco seed endophytic antagonistic bacterial strain of any of claims 1 to 3.