CN114934001A

CN114934001A - Endogenous antagonistic bacterium of tobacco seeds and application thereof

Info

Publication number: CN114934001A
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: 2022-08-23
Anticipated expiration: 2042-07-01
Also published as: CN114934001B

Abstract

The invention relates to a tobacco seed endogenetic antagonistic bacterium DW15 with a preservation number: CCTCC NO: m2022820, classified and named as Pseudomonas paraafulva, deposited in the China center for type culture Collection. The strain is yellow and round, and has a moist and smooth surface and a slight bulge. The screened endophyte DW15 has strong bacteriostatic action on Ralstonia solanacearum, is a biocontrol bacterium with good effect on tobacco bacterial wilt, can produce more proteases and has strong phosphorus dissolving and nitrogen fixing capabilities. Meanwhile, the compound bactericide has good growth promotion effect on tobacco plants, can obviously improve the biomass of tobacco seedlings, can be used singly or in a compound way, has wide application prospect on biological control of tobacco bacterial wilt, and has important significance on protecting the ecological environment.

Description

Endogenous antagonistic bacterium of tobacco seeds and application thereof

Technical Field

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

Background

Bacterial wilt is a destructive soil-borne disease caused by Ralstonia solanacearum, 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 witt) is a soil-borne disease which is seriously harmful and difficult to control in the current Tobacco production. The tobacco bacterial wilt is widely harmful in the southwest tobacco area of China, has a tendency of migrating to high-altitude cold areas, and is a plant disease to be solved urgently in practical production. In China, due to factors such as continuous cropping of tobacco, improper fertilization and management modes and the like for a long time, continuous cropping obstacles of tobacco are serious, tobacco bacterial wilt outbreaks in a large area are large, and the yield and quality of tobacco leaves in various large tobacco areas are seriously reduced.

The control of tobacco bacterial wilt is very challenging. For the prevention and treatment of tobacco bacterial wilt, measures such as breeding resistant varieties, biological prevention and chemical prevention and treatment and the like are mainly taken at present. Because the chemical control has the problems of harming human health, destroying ecological balance and the like, and the biological control has the advantages of safety to human and livestock, environmental friendliness and the like, the biological control on the soil-borne disease tobacco bacterial wilt is more and more emphasized in recent years, and the research on biocontrol bacteria for controlling the tobacco bacterial wilt is wider.

At present, the prevention and treatment research on tobacco bacterial wilt is mainly focused on the prevention and treatment effect and mechanism of the induced and resistant compound 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. Plants and endophytes co-evolve, and are classified into three categories according to the action of endophytes: first, neutral endophytic microorganisms which do not have significant deleterious or beneficial effects on plants; the biological fertilizer is beneficial to endogenous microorganisms, and can play the beneficial roles of protecting plants from being damaged by pathogens or herbivores, promoting plant growth and the like; thirdly, the potential pathogenic endophytic microorganisms produce substances which are not beneficial to the growth of plants. Therefore, finding an endogenous microorganism beneficial to tobacco plants is an urgent problem to be solved.

Disclosure of Invention

In view of the above, the present invention aims to provide a strain of endogenous antagonistic bacteria of tobacco seeds with multiple properties such as growth promoting effect, and also provides various applications of the endogenous antagonistic bacteria in agricultural industrialization.

In order to achieve the purpose, the invention provides the following technical scheme:

1. the tobacco seed endogenous antagonistic bacteria is characterized in that the tobacco seed endogenous antagonistic bacteria is DW15, and is classified and named as: pseudomonas paraflava, deposited in the China center for type culture Collection, address: wuhan, date of preservation: 7/6/2022, accession number: CCTCC NO: m2022820.

Further, the strain of the endogenous antagonistic bacteria in the tobacco seeds is yellow and round, and the surface of the strain is moist and smooth and is slightly raised.

Further, the strain of endogenous antagonistic bacteria in the tobacco seeds can produce protease.

2. The application of any one of the tobacco seed endogenous antagonistic bacteria as a biological agent for inhibiting and/or preventing bacterial wilt.

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

Further, the application of the antagonistic bacteria in the tobacco seeds as a biological agent for inhibiting and/or preventing bacterial wilt is provided, and the preparation method of the biological agent comprises the following steps: activating the strain of the antagonist bacteria DW15 in the tobacco seeds, selecting a single colony, inoculating the single colony to an LB liquid culture medium, culturing at 30 ℃ and 180r/min to logarithmic phase, and diluting to 1 × 10 by using sterile water ⁶ cfu/mL-1×10 ¹² cfu/mL plants were root irrigated.

Preferably, the dilution is carried out to 1X 10 using sterile water ⁸ cfu/mL plants were root drenched.

3. The application of any one of the antagonistic bacteria having tobacco seed endogenesis as a biological agent for promoting the growth of tobacco plant.

Further, in the application of the antagonist bacteria in the tobacco seeds as a biological agent for promoting the growth of tobacco plant plants, the using concentration of the antagonist bacteria in the tobacco seeds in the biological agent is 1 × 10 ⁶ cfu/mL-1×10 ¹² cfu/mL。

Further, in the application of the antagonist bacteria in the tobacco seeds as a biological agent for promoting the growth of tobacco plant plants, the preparation method of the biological agent comprises the following steps: planting tobaccoActivating the strain of the endogenous antagonistic bacteria, inoculating the single colony to LB liquid culture medium, culturing at 30 deg.C and 180r/min to logarithmic phase, diluting with sterile water to 1 × 10 ⁶ cfu/mL-1×10 ¹² cfu/mL plants were root drenched.

4. A biological agent comprises a tobacco seed endogenous antagonistic bacterium DW15 which is classified and named as Pseudomonas paraflava and is preserved in China center for type culture collection with the preservation number: CCTCC NO: m2022820.

The invention has the beneficial effects that: the invention has the beneficial effects that: on the basis of early-stage work in a laboratory, the disease resistance phenotype of different tobacco varieties to bacterial wilt is systematically screened by two methods of indoor root irrigation inoculation and field disease nursery; comparing the characteristics of the endophytic bacterial communities of the tobacco seeds with different resistance phenotypes by adopting an Illumina high-throughput sequencing technology system; and further separating, purifying and screening the endogenous antagonistic bacteria of the seeds of the resistant tobacco variety through 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 the endogenous antagonistic bacteria on tobacco seedlings. The invention screens 5 tobacco seed endophytic antagonistic bacteria from the middle layer of 328 seed endophytic bacteria screened and purified in the previous stage, has certain antagonistic effect on Ralstonia solanacearum, and has different growth promoting effects. The strain G3-KB-15(DW15) and the strain 6036-R2A-26(DW26) have the best biocontrol effect, under the condition of dip-dyeing of the strong pathogenic Ralstonia solanacearum CQPS-1, the average control effect of the strain G3-KB-15 in 7-15 days after inoculation treatment is more than 60%, the average control effect in 19 days (late stage of disease) after inoculation can still reach 52.17%, the strain G3-KB-15 shows good antagonistic action, and the strain G3-KB-15 has better effects of inhibiting or preventing and controlling the bacterial wilt of tobacco.

The screened seed endophyte G3-KB-15 can generate more protease, the generated protease transparent circle can reach 4.14cm on average, and the cell wall of pathogenic bacteria is damaged, so that the pathogenic bacteria are killed or inhibited, and a better biocontrol effect is synergistically played. And the strain has certain biofilm formation capability, which indicates that the strain has good colonization capability. The strain also has strong phosphorus dissolving capacity and nitrogen fixing capacity, can dissociate phosphorus which is indissolvable in soil, and convert the phosphorus into a compound which can be absorbed and utilized by plants, promotes the growth of the plants through the effects of dissolving phosphorus, fixing nitrogen and the like, and also can achieve the aim of indirectly preventing and treating diseases.

The strain G3-KB-15 not only has good Ralstonia solanacearum antagonistic action, but also has the effect of promoting the growth of 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 continuous root irrigation of the fermentation liquor can improve the tobacco seedling biomass such as the fresh weight of the overground part, the fresh weight of the underground part, the dry weight of the overground part, the dry weight of the underground part, the maximum root length, SPAD and the like, effectively promotes the growth of the tobacco seedling stage and achieves the effect of early growth and quick growth of the tobacco. The strain G3-KB-15 also has the function of influencing the functional diversity of tobacco plant rhizosphere soil microorganisms, can solve the problems of tobacco planting soil nutrient supply and soil microecological imbalance and the like caused by the increase of the continuous cropping age of tobacco, and avoids the growth and development retardation of tobacco and the reduction of quality and yield. Therefore, the screened endogenous antagonistic bacteria have wide application prospects and certain guiding significance for relieving continuous cropping obstacles in practical production application.

Preservation information

The strain name: DW15, classification name: pseudomonas parahaemolytica, deposited in the China center for type culture Collection, address: wuhan, date of preservation: 2022, 6/7, accession No.: CCTCC NO: m2022820.

Drawings

In order to make the purpose, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1 shows the disease of tobacco varieties in bacterial wilt in room.

FIG. 2 shows the index of the disease conditions in the bacterial wilt room of different tobacco varieties.

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

FIG. 4 shows the field expression of tobacco bacterial wilt for different tobacco varieties.

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

FIG. 6 shows a partially screened and purified seed endophyte of a resistant tobacco variety.

FIG. 7 is a single colony plot of different selected endogenous antagonistic bacteria.

FIG. 8 is a graph of the biofilm-forming ability of different selected endogenous antagonistic bacteria.

FIG. 9 is a transparent circle formed by secretion of extracellular enzymes from different selected endogenous antagonistic bacteria.

FIG. 10 shows growth of different selected endogenous antagonistic bacteria on Ashby medium.

FIG. 11 is a graph of the effect of different selected treatment with endogenous antagonistic bacteria on tobacco shoot biomass.

FIG. 12 shows the effect of root irrigation treatment on bacterial wilt disease index of different screened fermentation liquid of endogenous antagonistic bacteria.

FIG. 13 shows the control effect of bacterial wilt by the irrigation of the screened different endophytic antagonistic bacteria fermentation broth.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out under conventional conditions or under conditions recommended by the manufacturers.

Example 1

1. Materials and methods:

on the basis of early-stage multi-variety resistance screening and evaluation, 6 tobacco varieties are further screened out to carry out indoor root irrigation inoculation and comprehensive resistance evaluation of bacterial wilt in a field disease garden, and a foundation is laid for the analysis of the endophytic bacterial community characteristics of seeds of the subsequent anti-susceptible tobacco varieties. In the embodiment, four-leaf one-core tobacco seedlings of different tobacco varieties are taken as research objects, and the disease resistance phenotype of the different tobacco varieties to bacterial wilt is evaluated by adopting the inoculation of the rhizoctonia solani through root irrigation.

Test tobacco seeds: the tobacco variety to be tested was planted in the test plot of Runxi Xiangxi tea Tree lawn in Penghui county of Chongqing, 5.2020 (elevation 1360m, 29 degrees 7 '43' in northern latitude, 107 degrees 56 '38' in east longitude). Selecting 15-20 standard plants with typical characters of various varieties before flowering, and bagging and reserving seeds after flower and fruit thinning; harvesting when the pericarp turns brown, air drying, threshing, and sealing in refrigerator at-20 deg.C for use. The original information of each variety is shown in table 1.

TABLE 1 original material information of varieties to be tested

The pathogenic bacteria to be tested are highly pathogenic Ralstonia solanacearum CQPS-1(Ralstonia solanacearum) (which is simply called Ralstonia solanacearum) separated from pathogenic tobacco strains of Pingchun prefecture (altitude 1210m, 29 degrees 8 '12' in northern latitude and 107 degrees 56 '31' in Dongding city) in Chongqing.

BG culture medium: 10g/L of bactopeptone, 1g/L of casamino acid, 1g/L of yeast extract, 15g/L of agar powder and 5g/L of glucose. Cooling to about 55 deg.C after sterilization, adding 1% TTC (2, 3, 5-triphenyltetrazolium chloride) water solution to make its final concentration be 0.005%,

b, liquid culture medium: 10g/L of bacterial peptone, 1g/L of yeast extract and 1g/L of tyrosine peptone.

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

Tobacco cultivation: carrying out floating seedling culture by using the collected seeds of each tobacco variety, wherein the seedling culture conditions are as follows: culturing at 25 deg.C, relative humidity of 60%, and illumination-dark time ratio of 14/10h until four leaves and one heart are ready for use.

Preparing a ralstonia solanacearum inoculation liquid: activating Ralstonia solanacearum on BG plate by streaking, culturing at 30 deg.C for 48 hr, selecting single colony, inoculating to liquid culture medium B, and shake culturing (30 deg.C, 180r/min) to logarithmic phase (OD) _600nm 0.8-1.0). Then diluted to OD _600nm ＝0.1(10 ⁸ CFU/mL) for use.

Inoculating bacterial suspension by root irrigation: root irrigation and inoculation of each tobacco seedling 10 ⁸ CFU/mL of 10mL of a suspension of Ralstonia solanacearum, 4 replicates per treatment, and 8 tobacco shoots per replicate. After inoculation of the pathogenic bacteria, the cells were cultivated in a greenhouse at a temperature of 30 ℃ and a relative humidity of 75% for a ratio of light to dark of 14/10 h.

Tobacco bacterial wilt disease investigation: according to the indoor investigation grading standard of Zhengjun et al, the system investigation of the disease onset of different varieties is started at the early stage of the disease onset, the investigation is performed once every other day until the end stage of the disease onset, and the disease incidence and the disease index are calculated.

The evaluation criteria of indoor resistance 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); disease index is 41-60: medium Resistance (MR); disease index is 61-80: low Resistance (LR); the disease index is 81-90: (ii) susceptibility to disease (S); the disease index is 91-100: high feeling (HS).

And (3) data analysis: the summary data was collated using Excel 2016, analyzed for variance and significance (P <0.05) using IBM SPSS Statistics 23, plotted using Origin 2017 and GraphPad Prism 9.

2. Results and analysis:

the disease condition of bacterial wilt of different tobacco varieties: the inoculation concentration is 10 ⁸ The final stage of the disease (23 d after inoculation) of cfu/mL is shown in FIG. 1, and the disease index over the entire period of the study is shown in FIG. 2. Because the inoculation concentration is relatively high, and the pathogenic bacteria to be tested are strong pathogenic strains, the diseases of different varieties of tobaccos are serious. The onset of disease in each tobacco variety began to progress 5 days after inoculation. Over time, disease indices of each variety gradually increased, but over the entire disease survey period, disease indices of varieties HD, K326 and YY87 were all greater than those of varieties 6036, YY97 and G3. 23d after inoculation is at the end of disease, the average disease indexes of the varieties HD, K326 and YY87 are respectively 95.83, 95.83 and 91.67, and are obviously higher than the average disease indexes (respectively 66.44, 64.58 and 60.42) of the varieties 6036, YY97 and G3. The disease index analysis of the whole investigation period is integrated, and the resistance of each tobacco variety to the bacterial wilt is changed from high to lowThe following steps are carried out: g3>YY97>6036>YY87>HD≥K326。

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

TABLE 2 Classification of bacterial wilt resistance levels for different tobacco varieties

Example 2

Evaluating the field resistance of different tobacco varieties to bacterial wilt: the tobacco seeds are cultured in a floating mode in the same way as in the embodiment 1, and unified field transplanting and later-period management are carried out according to local related technical standards. The test field selects a flat land (1210 m above sea level, 29 degrees in northern latitude 8 '12' and 107 degrees in east longitude 56 '31') of the Changshui county Ruixi village of Bingqing which is continuously planted all the year round and has serious tobacco bacterial wilt. The method comprises the steps of setting 3 cells for different varieties to be repeated, totaling 18 cells, designing random block groups, and planting 40 tobacco plants in each cell.

Tobacco bacterial wilt field investigation:

the investigation of the tobacco bacterial wilt is carried out according to the reference national standard GB/T23222 and 2008 < tobacco pest classification and investigation method >. Beginning investigation at the early stage of bacterial wilt disease, and investigating every 5-7d until the later stage of harvesting. Calculating the incidence, Disease index and Area Under the Disease progression Curve (AUDPC):

(X _i the disease index at the ith survey, t _i Date of the ith survey)

The evaluation standard of the field resistance of different varieties refers to GB/T23224-2008 tobacco variety disease resistance appraisal, which is as follows: the disease index is 0: (ii) high resistance or immunity (I); the disease index is 0.1-20: disease resistance (R); disease index is 20.1-40: medium Resistance (MR); disease index is 40.1-60: (ii) a neutral feeling (MS); the disease index is 60.1-80: (ii) susceptibility to disease (S); the disease index is 80.1-100: high Sensitivity (HS).

The incidence of bacterial wilt of different tobacco varieties: when the bacterial wilt happens sporadically, the occurrence of the bacterial wilt of different tobacco varieties is systematically investigated, the incidence and disease index of the bacterial wilt are respectively shown in figure 3, wherein a: incidence, b: the index of the disease condition. The field performance at the middle stage (2021.07.28) and the middle and late stage (2021.08.07) of the onset is shown in fig. 4, wherein a: in the middle stage of onset; b: in the middle and late stage of onset. With the lapse of time, the incidence and disease index of tobacco bacterial wilt gradually increase. Under the influence of high-temperature and high-humidity climate, the morbidity and disease index of varieties HD, YY87 and K326 in the middle and later period of disease are greatly increased. The morbidity and disease indexes of different varieties are shown by integrating the morbidity situation of the whole growth period: HD > YY87> K326> G3>6036> YY 97.

Bacterial wilt resistance grades of different tobacco varieties

The area under the disease progression curve (AUDPC) results based on disease index are shown in FIG. 5. The results show that the area under the disease progression curve (AUDPC) is as follows from large to small: HD. YY87, K326, G3, 6036 and YY97, wherein varieties HD, YY87 and K326 are obviously higher than varieties G3, 6036 and YY97, the varieties HD, YY87 and K326 have obvious difference, the varieties G3, 6036 and YY97 have no obvious difference, and the resistance of different tobacco varieties to bacterial wilt is shown as follows in combination: YY97>6036> G3> K326> YY87> HD. The results of grading the resistance of tobacco according to disease indexes of different growth periods and disease onset periods are shown in Table 3. The topping period (7 months and 16 days) is in the middle of the onset of disease, the disease index of HD is the largest, the HD is shown as resistant, and other varieties are shown as resistant. The middle and later harvesting period (8 months and 16 days) is in the peak disease stage, the disease index of HD is the largest, and the disease is manifested as affection; the disease index of YY87 is inferior, showing a feeling of well being; the disease index of K326 is intermediate, and the disease index is marked as resistance; other varieties are disease resistant. The later harvesting stage (8 months and 22 days) is at the end stage of disease development, the disease index of HD is the largest and is obviously higher than that of other varieties, and the HD shows disease susceptibility; the disease index of YY87 is inferior, showing a feeling of well being; the disease indexes of K326 and G3 are indicated as resistance, and other varieties are indicated as resistance.

TABLE 3 bacterial wilt resistance rating of different tobacco varieties

Note: disease index: mean ± standard error, alphabetical differences indicate that treatments differed significantly at the 0.05 level.

Example 3

Structural feature analysis of endophytic bacteria of seeds of different resistant tobacco varieties: tobacco seeds as in example 1, the sterilized seeds were transferred to a sterile mortar and ground for 3-5min by adding liquid nitrogen until the seeds were ground. 0.5g of ground seed was weighed out in each repetition and FastDNA was used ^TM SPIN kit, DNA extraction according to protocol.

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

first, the integrity of the DNA is checked, and the concentration and purity of the DNA are determined. After quality inspection is qualified, performing PCR amplification on the V5-V7 variable region of the 16S rDNA of the endophytic bacteria of the seeds.

The PCR reaction system is 20 μ L: 4 μ L of 5 XFastPfu Buffer, 2 μ L of 2.5mM dNTPs, 0.8 μ L of Forward Primer (5 μ M), 0.8 μ L of Reverse Primer (5 μ M), 0.4 μ L of FastPfu Polymerase, 0.2 μ L of BSA, 10ng of Template DNA, complement 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'), and uses the extracted DNA as a template to perform PCR amplification on V5-V8 variable region of 16S rDNA of an endogenous bacterium, pre-denature at 95 ℃ for 3min, and perform 27 cycles (denaturation at 95 ℃ for 30S, annealing at 55 ℃ for 30S, and extension at 72 ℃ for 45S), and finally extend at 72 ℃ for 10 min; the second round takes the first round amplification product as a DNA template, and performs PCR amplification on the 16S rDNA V5-V7 variable region by using primers 799F (AACMGGATTAGATACCCKG,5 '-3') and 1193R (ACGTCATCCCCACCTTCC,5 '-3'), for 13 cycles, and the rest conditions are the same as the first round amplification. And after the quality of the amplification product is qualified, the Shanghai Meiji biological medicine science and technology limited company is entrusted to Illumina MiSeq sequencing.

According to the embodiment, the screened endophytic bacteria of the tobacco variety seeds with different bacterial wilt resistance phenotypes are subjected to high-throughput sequencing analysis, 405651 optimized sequence fragments are obtained in total, and a sufficient data basis is provided for endophytic bacteria community composition and diversity analysis and excavation of endophytic biocontrol bacteria of resistant variety seeds. High throughput sequencing analysis results: (1) beta diversity analysis results show that the structure of the endophytic bacterial community of the tobacco seeds between resistant and susceptible varieties is greatly different. (2) The relative abundance of the firmicutes of the resistant varieties G3, YY97 and 6036 is obviously higher than that of the susceptible variety, namely 2.34, 5.58 and 8.46 times of that of the susceptible variety YY87 and 1.54, 3.68 and 5.58 times of that of the susceptible variety HD; the relative abundance of actinomycetes of the resistant varieties YY97 and 6036 is obviously higher than that of the susceptible variety YY87, namely 2.77 times and 2.40 times of that of the susceptible variety YY87, and 2.59 times and 2.24 times of that of the susceptible variety HD. (3) The composition of the endogenous bacterial colonies of seeds of different tobacco varieties at the genus level is relatively similar, but the relative abundance is greatly different. Analyzing the bacterial genus with significant difference in relative abundance among the resistant varieties, wherein the unclosed _ f __ Enterobacteriaceae of the resistant variety G3 has the highest relative abundance which is significantly higher than that of the susceptible variety HD; the relative abundance of Pseudomonas (Pseudomonas) of the resistant varieties G3 and YY97 is higher and is obviously higher than that of the susceptible variety YY 87; the relative abundance of Paenibacillus (Paenibacillus) of resistant varieties G3, 6036 and YY97 is significantly higher than that of susceptible varieties; the relative abundance of Bacillus (Bacillus) of the resistant variety 6036 is the highest and is significantly higher than that of the susceptible variety.

Example 4

On the basis of evaluating the resistance of different tobacco varieties and analyzing the structural feature system of the endophytic bacteria of the seeds, the method further separates, purifies and screens the endophytic culturable bacteria of the resistant varieties G3, YY97 and 6036, and inspects the relevant characteristics of the endophytic antagonistic bacteria of the seeds to obtain the endophytic fungi-proofing resources of the seeds.

Tobacco seeds the test medium (all medium components mixed and autoclaved) of example 1 was used:

LB culture medium: 10g/L of tryptone and 5g/L, NaCl 10g/L of yeast extract.

BG culture medium: as before.

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

(Oxoid Co., UK)

TSA medium: tryptone 15g/L, soytone 5g/L, NaCl 5g/L, agar 15 g/L. (Haibo Biotechnology Co., Ltd.)

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

R2A medium: bacterial peptone 0.5g/L, soluble starch 0.5g/L, yeast extract 0.5g/L, glucose 0.5g/L, casamino acid 0.5g/L, KH ₂ PO ₄ 0.3g/L, sodium pyruvate 0.3g/L, MgSO ₄ 0.024g/L and 15g/L agar.

1. Separation and purification of endophytic bacteria

Weighing 2g of seeds of different tobacco varieties with disinfected surfaces by adopting a dilution plate coating method, fully grinding the seeds in a mortar, and adding 8mL of sterile water for uniformly mixing. Standing for 5min, sucking 100 μ L supernatant, adding 900 μ L sterile water, and diluting as 10 ^-2 In this way, the dilution was made to 10 ^-3 、10 ^-4 And 10 ^-5 . Respectively taking 100 μ L of each dilution gradient, uniformly coating with glass beads on NA, TSA, R2A and KB plate, arranging 3 times of each plate at each concentration, culturing at 30 deg.C for 2-5d, and observing growth condition continuously according to the principleSelecting different representative strains for naming (the naming rule is variety-culture medium-sequence number), selecting single colony to be on corresponding plate for continuous streak purification 3-4 times until the colony on the plate is consistent and has no mixed bacteria, and considering the purification is finished.

And (3) streak purification is carried out according to phenotype difference on different culture media, and finally the bacterial 101 strain of the variety YY97, the bacterial 115 strain of the variety 6036 and the bacterial 112 strain of the variety G3 are obtained, and all the resistant variety seed endophytic bacteria 328 strains are obtained. FIG. 6 shows a partially resistant Nicotiana species seed endophyte. The purified strain was scraped off a single colony using a sterile cotton swab, placed in a 1.5mL centrifuge tube containing 25% glycerol, and stored at-80 ℃ at ultra low temperature.

2. Antagonistic bacterial screening

(1) Screening of plate bacteriostatic activity

And performing primary screening on antagonistic strains of the purified and stored bacteria by adopting a bacterium spraying method. First, a single colony was spotted in the center of the NA plate, and each strain was repeated 3 times. Culturing at 30 deg.C by inverting overnight, and mixing OD with sterile spray can _600nm ＝0.1(10 ⁸ CFU/mL) is evenly sprayed on an NA plate, inverted culture is carried out at 30 ℃ for 24h, and then the diameter of the inhibition zone is measured by adopting a cross method. And selecting the strains with larger inhibition zones for subculture, repeatedly detecting the inhibition stability of the strains, and finally determining the potential antagonistic bacteria with better and stable plate inhibition effect. By evaluating the plate bacteriostatic activity of the endophytic bacteria of the resistant variety seeds, the bacteria with better and stable bacteriostatic circle effect are finally selected as shown in table 4. The resistant variety has more potential antagonistic bacteria in seeds, wherein 3 strains of YY97, 7 strains of G3 and 5 strains of 6036 exist.

(2) Evaluation and screening of potting effect

And (4) taking the strain with better plate bacteriostatic activity obtained by screening as a material to evaluate the potting effect. Selecting single colony of potential antagonistic bacteria, inoculating to LB culture medium, performing shake culture, selecting single colony of Ralstonia solanacearum, inoculating to B liquid culture medium, performing shake culture to OD _600nm 0.8-1.0, sterile water dilution to OD _600nm ＝0.1(10 ⁸ CFU/mL) for use.

First, the root irrigation treatment of the fermentation liquor of the potential antagonistic bacteria is carried out (10) ⁸ CFU/mL, 10 mL/strain), 3d after inoculation, irrigation of Ralstonia rhizogenes (10) ⁸ CFU/mL, 10 mL/plant), incubated in a greenhouse at 30 ℃, 75% relative humidity, and a light to dark time ratio of 14/10h, for 2 replicates per treatment, and 8 tobacco shoots per replicate. The disease investigation method is the same as the disease investigation method of the tobacco bacterial wilt in the example 1.

The pot activity evaluation is carried out on the screened 15 potential endogenous antagonistic bacteria, and the result shows that the strains with large plate bacteriostatic circle diameter have inconsistent and good greenhouse biocontrol effect, thereby showing that the biocontrol effect and the size of the plate bacteriostatic circle do not necessarily have positive correlation. Through repeated pot experiments, 5 stable endogenous antagonistic bacteria (YY97-KB-20, G3-NA-3, G3-KB-15, 6036-KB-29 and 6036-R2A-26) with good effects are finally selected for subsequent experiments.

TABLE 4 evaluation of bacteriostatic effect of potential antagonistic bacteria

4. Morphological observation of endophytic antagonistic bacteria colony

And streaking and activating the screened endogenous antagonistic bacteria on a corresponding plate, culturing for 36h at 30 ℃, and observing the appearance morphological characteristics of a single colony.

Single colonies of different endogenous antagonistic bacteria are shown in figure 7. The strain YY97-KB-20 is white and round, and the surface is wet and smooth; the strain G3-NA-3 is white and round, and has a moist and smooth surface and a micro-bulge; the strain G3-KB-15 is yellow and round, and has a moist and smooth surface and is slightly raised; the strain 6036-KB-29 is milky white, has irregular and opaque edges, unsmooth colony surface and folds and bulges; strain 6036-R2A-26 is white round, opaque, with oval spores in the expanding cyst.

5. Molecular identification of endogenous antagonistic bacteria

(1) Bacterial genomic DNA extraction

Selecting single colony, transferring to LB liquid culture medium, culturing at 30 deg.C and 180r/min for 12-14h, centrifuging to collect thallus, and extracting DNA with 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 1min), extension at 72 ℃ for 10min, 4 ℃. The PCR reaction system was 25. mu.L: 1.0 μ L of template DNA; 1.0 μ 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 a PCR product:

after the PCR product is detected to be qualified by 1 percent agarose gel electrophoresis, the sequencing of the PCR product is entrusted to Huada Gene Limited company. Sequencing results BLAST homology comparisons were performed with the NCBI (national Center for Biotechnology information) database and a phylogenetic tree was constructed using the method of Neighbor-joining (NJ) using MEGA X software, and examined with the Bootstrap method (Bootstrap) for 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. The target gene sequences obtained by different endogenous antagonistic bacteria are subjected to BLAST comparison in a GenBank database to construct a phylogenetic tree. The results showed that the strain YY97-KB-20 is defensive Pseudomonas (Pseudomonas proteins), the strain G3-NA-3 is Enterobacter avermitilis (Enterobacter alburiae), the strain G3-KB-15 is Pseudomonas paraflava (Pseudomonas paranova), the strain 6036-KB-29 is Bacillus altitudinis, and the strain 6036-R2A-26 is Paenibacillus odorifer.

The strain G3-KB-15 was named DW15, class name: pseudomonas parahaemolytica, deposited in the China center for type culture Collection, address: wuhan, date of preservation: 7/6/2022, accession number: CCTCC NO: m2022820.

The strain 6036-R2A-26 is named DW26, and the classification is as follows: paenibacillus odorifer, deposited in the chinese collection of type cultures at the address: wuhan, date of preservation: 7/6/2022, accession number: CCTCC NO: m2022821.

Example 5

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

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

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

CMC culture 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 15 g/L.

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

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

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 and agar 20 g/L.

Ashby medium: KH (natural Kill) 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 0.1g/L of O and 18g/L of agar.

1. Evaluation of biofilm Forming ability of endogenous antagonistic bacterium

The method uses crystal violet staining method, and utilizes the characteristic that the biological membrane can be combined with dye crystal violet to determine the biological membrane of different bacteria. Transferring single colonies of different antagonistic bacteria to LB liquid culture medium for culturing to logarithmic phase, centrifuging, washing with sterile water to obtain thallus precipitate with residual culture medium removed, and performing resuspension dilution (first dilution to OD) with new LB liquid culture medium _600nm 0.1, 10-fold further dilution in unison), and 200 μ L of the extract was pipetted into a 96-well plate, 6 replicates for each treatment, and incubated sealed at 30 ℃ for 48 h. Removing culture after culturing, adding 200 μ L sterile water, washing for 2 times, adding 150 μ L crystal violet solution (0.1%) for dyeing for 30min, washing each well with 200 μ L sterile water for 2 times, drying at room temperature for 30min, adding 200 μ L95% anhydrous ethanol for dissolving, and measuring OD after 30min _530nm The value is obtained.

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

2. Detection of ability of endogenetic antagonistic bacteria to secrete extracellular enzymes (protease, cellulase and amylase)

The ability to secrete proteases, cellulases and amylases was tested depending on whether a clearing zone was produced. Respectively point-grafting the activated strains to the centers of an enzyme culture medium, a CMC culture medium and a starch agar culture medium, carrying out inverted culture at 28 ℃ for 3d, scraping the thalli, dyeing for 30min by taking 1% Congo red as an indicator, repeatedly washing for 2-3 times by using 5% NaCl, and soaking for 60min, wherein the formation of a transparent ring around a bacterial colony indicates that the strains can secrete protease and cellulase; after the cells are inversely cultured for 3d at the temperature of 28 ℃, the cells are scraped, the cells are soaked in a gram iodine solution for 15min, 5 percent NaCl is used for washing away the loose color, and the formation of a transparent ring around the bacterial colony indicates that the bacterial strain can secrete amylase.

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

TABLE 6 diameter of transparent circle of endogenous antagonistic bacteria

3. Determination of phosphate-solubilizing ability of endogenous antagonistic bacteria

Referring to the method of Schwangtang (diversity analysis of tomato and rice seed culturable endophytic bacteria and research on growth-promoting bacteria function [ D ]. Chinese agriculture university, 2014.), and the like, and improving the method, single colony is selected to be inoculated to the center of an NBRIP flat plate, sterile water is inoculated as a control, and the NBRIP flat plate is cultured for 10 days at the temperature of 30 ℃. The phosphate solubilizing effect of the phosphate solubilizing ring is determined according to the diameter ratio of the phosphate solubilizing ring to the bacterial colony, the higher the ratio is, the stronger the phosphate solubilizing capability is, and the ratio is 1, the phosphate solubilizing capability is not available. The ratio of the phosphate-solubilizing ring of different antagonistic bacteria to the diameter of the colony is shown in table 7, and the phosphate-solubilizing abilities of the 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 strain has stronger phosphate solubilizing capability.

TABLE 7 phosphate solubilizing rings for different endogenous antagonistic bacteria

4. Determination of nitrogen fixation capability of endogenous antagonistic bacteria

Referring to Liuhui English (the Chinese pineapple endophyte screening identification and antagonistic effect evaluation [ J ]. Hubei university school report (Nature science edition), 2020,42(2):158-164.) and the like, strains are inoculated on an Ashby culture medium by adopting a scribing method and cultured for 15 days at the temperature of 30 ℃. If the strain grows well and can generate a transparent growth zone, the strain has stronger nitrogen fixation capacity. Growth of different antagonistic bacteria on Ashby medium is shown in FIG. 10, where strains YY97-KB-20, G3-NA-3 and G3-KB-15 grew normally on Ashby medium and streaked culture produced clear regions indicating strong nitrogen fixation.

Example 6 Effect of endogenous antagonistic bacterium in seeds on growth promotion of tobacco

1. Effect of endogenous antagonistic bacteria on tobacco seed germination

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

(2) Effect of bacterial suspension on tobacco seed germination: sterilized seeds were placed in sterile petri dishes (30/dish) with filter paper on the bottom, and 5mL of bacterial suspension was added, each treatment being repeated 3 times. And (4) hermetically culturing at 25 ℃ for 7d and 10d, and calculating the relative germination potential and the relative germination rate respectively.

The effect of different antagonistic bacterial treatments on seed germination is shown in table 8, where different antagonistic bacterial suspensions have a promoting effect on both the relative germination vigour and germination rate of tobacco seeds. At 7d after treatment, the average germination potential of the degerming strain G3-NA-3 is 70.88%, and the degerming strain has no obvious difference with CK, and other treatments can obviously promote seed germination; in addition to the degerming strains G3-NA-3 and YY97-KB-20 at the 10 th day after treatment, other treatments have obvious promotion effect on seed germination. Therefore, the relative germination vigor and relative germination rate of the 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 the tobacco seeds can be obviously promoted.

TABLE 8 Effect of endogenous antagonistic bacteria on seed Germination

Note: relative germination vigor and relative germination rate: mean ± standard error, alphabetical differences indicate that treatments differed significantly at the 0.05 level.

2. Effect of endogenous antagonistic bacteria on tobacco growth

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

(2) Irrigating roots with bacterial fermentation liquor: sterilizing the surface of the naked Yunnan 87 seed, sowing the sterilized seedling substrate, and selecting the seedlings with the same size and transplanting the seedlings to a seedling raising pot with the caliber of 9cm after about 35 days. After 1d of seedling recovery, the root irrigation of the endophytic antagonistic bacteria fermentation liquor is carried out (10) ⁸ CFU/mL, 10 mL/strain), 5 per replicate, per 3 replicates per treatment. The root irrigation is repeated every 7 days, and the root irrigation dosage is the same as above, and the total is 3 times. Tobacco seedlings were cultured in an artificial incubator at 25 ℃ under 14h light (5000LX) and 10h dark alternately.

(3) Tobacco seedling biological quantity determination

And respectively measuring the biomass of the tobacco seedlings of different treatment groups 10 days after the last inoculation. Firstly, selecting the leaves of the same part of each tobacco seedling to measure the SPAD value, then carefully shaking off the root matrix, washing the residual matrix with clear water, absorbing the water on the surface of the plant with absorbent paper, and respectively counting the root length, the fresh weight of the overground part and the fresh weight of the underground part. After drying (deactivation of enzymes at 105 ℃ for 15min, drying at 75 ℃ for 4h), the dry weight of the overground part and the dry weight of the underground part are counted.

The effect of different endogenous antagonistic bacterial treatments on tobacco shoot biomass is shown in table 9, fig. 11. Compared with CK, different strains can improve 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, and for tobacco plants, the higher the dry weight of the overground part is, the larger the tobacco biomass is, which shows that the growth promoting effect of the strains is better. The strains G3-NA-3, 6036-KB-29 and 6036-R2A-26 can obviously improve the fresh weight of the underground part of the tobacco seedling, the strains 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 underground part of the tobacco seedling and the chlorophyll SPAD value; the root length of 6036-KB-29 and 6036-R2A-26 is greater than CK. Therefore, different antagonistic bacteria are treated by continuous root irrigation in the tobacco seedling stage, so that the growth of tobacco can be effectively promoted, and the effects of early growth and quick growth are achieved.

TABLE 9 Effect of endogenous antagonistic bacteria on tobacco growth

Note: biomass: mean ± standard error, alphabetical differences indicate that treatments differed significantly at the 0.05 level.

Example 7

Endophytes selected in example 4

1. Preparation of ralstonia solanacearum and endophytic antagonistic bacterium fermentation liquor

Respectively streak-activating ralstonia solanacearum and endogenous antagonistic bacteria on corresponding plates, selecting single colony of endogenous antagonistic bacteria, inoculating to LB culture medium, shake-culturing, selecting single colony of ralstonia solanacearum, inoculating to liquid culture medium B, shake-culturing to OD _600nm 0.8-1.0, diluted to OD with sterile water _600nm ＝0.1(1×10 ⁸ CFU/mL) for use.

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

Firstly, the fermentation liquor of the endogenous antagonistic bacteria is treated by root irrigation (10) ⁸ CFU/mL, 10 mL/strain), 3d irrigated with Ralstonia rhizogenes (10) ⁸ CFU/mL, 10 mL/strain). Each treatment was set to 3 replicates, each replicate 8, and incubated in a greenhouse at 30 deg.C, 75% relative humidity, and a light to dark time ratio of 14/10 h. And (4) investigating the disease incidence of the tobacco bacterial wilt at the early spontaneous incidence, wherein the investigation is carried out once every two days, and the disease investigation method is the same as that of the above. And after disease investigation is finished, calculating the morbidity, disease index and prevention effect. The disease investigation time is based on CK survival time.

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

The control effect of the different endogenous antagonistic bacteria fermentation broth root irrigation treatment on bacterial wilt is shown in fig. 13, and table 10 is specific data. The control effect of different treatments on bacterial wilt is different. The average prevention effect of 6036-R2A-26 treatment after inoculation is over 60 percent in 7-15 days, and the average prevention effect of 19 days (late stage of disease) after inoculation still reaches 51.09 percent; the average control effect of the G3-KB-15 treated by the strain at 7-13d after inoculation is more than 60%, and the average control effect of the G3-KB-15 treated by the strain at 19d after inoculation still reaches 52.17%, so that the overall control effect of the strains 6036-R2A-26 and G3-KB-15 is higher than that of the other three strains. The average control effect of 19d after inoculation of G3-NA-3 was 53.26%, and the average control effects of YY97-KB-20 and 6036-KB-29 after inoculation of 19d were 44.57% and 36.96%, respectively. Therefore, the biocontrol effects of different endogenous antagonistic bacteria treatment are different, wherein the control effects of strains 6036-R2A-26 and G3-KB-15 are relatively high, the Ralstonia solanacearum CQPS-1 adopted in the research is a very strong pathogenic strain and has serious infection harm to general tobacco plants, so that the control effects of the strains 6036-R2A-26 and G3-KB-15 on other Ralstonia solanacearum are higher and stronger, the biocontrol bacteria have good effects on controlling the tobacco bacterial wilt, and the 2 bacteria have good growth promoting effects on the tobacco plants, can remarkably improve the biomass of tobacco seedlings, can be used singly or in a compounding way, have wide application prospects on biological control of the tobacco bacterial wilt and have important significance on protecting the ecological environment.

Table 10 corresponds to the control data

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

Claims

1. The tobacco seed endogenous antagonistic bacterium is DW15, and is classified and named as: pseudomonas parahaemolytica, deposited in the China center for type culture Collection, address: wuhan, date of preservation: 2022, 6/7, accession No.: CCTCC NO: m2022820.

2. The tobacco seed endogenous antagonist bacteria of claim 1, wherein the strain of the tobacco seed endogenous antagonist bacteria is yellow and round, has a wet and smooth surface and is micro-raised.

3. An endogenous antagonistic bacterium in tobacco seeds as claimed in claim 1, characterised in that the strain is capable of producing a protease.

4. Use of the endogenous antagonist bacteria of tobacco seeds as claimed in any one of claims 1-3 as a biological agent for inhibiting and/or controlling bacterial wilt.

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

6. The use of claim 4, wherein the preparation method of the biological agent is as follows: activating the strain of the antagonist bacteria DW15 in the tobacco seeds, selecting a single colony, inoculating the single colony to an LB liquid culture medium, culturing at 30 ℃ and 180r/min to logarithmic phase, and diluting to 1 × 10 by using sterile water ⁶ cfu/mL-1×10 ¹² cfu/mL plants were root irrigated.

7. Use of an antagonist bacterium endogeny in tobacco seeds as claimed in any one of claims 1 to 3 as a biological agent for promoting growth of tobacco plant plants.

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

9. A biological agent comprising the tobacco seed endophytic antagonist strain of any one of claims 1-3.