Tobacco bacterial wilt antagonistic bacterium and fungicide thereof

Abstract

The invention relates to a strain of Glutamicobacter soli for antagonizing bacterial wilt, which is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number of the Glutamicobacter soli is CCTCC NO: M 20232211. The antagonistic bacterium has a good antagonistic effect on ralstonia solanacearum strains with relatively strong pathogenicity, and shows an excellent prevention and control effect in indoor potting. Field experiments prove that the bacillus subtilis has an excellent prevention and control effect, is a potential biological agent for preventing and controlling the bacterial wilt disease, has a good development and application prospect, has a greatly improved prevention and control effect compared with a chemical reagent for the tobacco bacterial wilt disease, and is expected to be applied to providing a new green and safe prevention and control scheme in the prevention and control of the tobacco bacterial wilt disease.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to a tobacco bacterial wilt antagonistic bacterium and a bacterial agent thereof. Background Art

[0002] Tobacco bacterial wilt is a soil-borne bacterial disease caused by Ralstonia solanacearum. It seriously harms the growth of tobacco plants and causes huge economic losses. It is regarded as a devastating disease that can infect more than 50 families and hundreds of plant species. There have been reports of tobacco bacterial wilt in my country, among which tobacco bacterial wilt has occurred widely in 14 of the 22 major tobacco-growing areas, causing great harm to the tobacco industry. Ralstonia solanacearum mainly enters plant cells by infecting plant root wounds, root tips or secondary roots, develops between cells, and then enters the xylem to multiply throughout the plant, producing a large amount of extracellular polysaccharides that block the vascular tissue, causing the plant to wilt and eventually die. After the disease occurs, the yield of tobacco leaves is significantly reduced, which usually causes huge economic losses to tobacco farmers.

[0003] At present, the prevention and control of tobacco bacterial wilt is mainly based on chemical agents. The commonly used chemical agents on the market mainly include inorganic copper, organic chlorine, inorganic sulfur and organic copper. Although chemical control has a good control effect on tobacco bacterial wilt, the large-scale and continuous application of chemical agents is prone to cause difficult-to-overcome problems such as pathogen resistance and environmental pollution, which is not conducive to the protection of the ecological environment and the sustainable development of agriculture. It will also increase the resistance of pathogens, reduce the diversity of soil biological communities in tobacco fields, and affect the environmental quality of tobacco fields. Considering the problems of low tobacco leaf quality and deterioration of the ecological environment of tobacco-growing soil, tobacco green control technology has received more and more attention. Among them, biological control has become a more common method of bacterial wilt control in recent years, and there are more and more studies in this area, which is the key development direction in the future. Biological control of tobacco bacterial wilt is to use the antagonism, competition, predation, parasitism and other relationships between microorganisms to inhibit the growth, development and reproduction of pathogens, thereby reducing the occurrence of diseases. It is known that tobacco wilt in Guangdong tobacco areas has become more serious year by year due to the influence of multiple factors such as long-term continuous cropping, increased chemical inputs, and abnormal climate change. The symptoms of bacterial wilt in Guangdong tobacco areas are different from those in the southwest tobacco areas. The bacterial wilt in Guangdong tobacco areas is concentrated after topping and occurs seriously in a short period of time. Guangdong tobacco areas are one of the important production areas of special tobacco leaves in my country, with typical characteristics and regional advantages. However, rhizome diseases such as tobacco bacterial wilt have brought severe challenges to the healthy and stable development of tobacco leaves, causing serious losses in local areas, and have become an important factor restricting tobacco production. In the process of plant health cultivation, microorganisms play an important role in the crop microecological system, and different rhizosphere microecological community compositions determine the health of tobacco. At present, a large number of research results have been achieved in the microecological research on bacterial wilt, but the antagonistic indigenous bacteria related to the occurrence of bacterial wilt in Guangdong tobacco areas are still unclear. Summary of the invention

[0004] In view of this, the object of the present invention is to provide an antagonistic bacterium to tobacco bacterial wilt, and also to provide a microbial agent containing the strain.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] 1. A soil glutamicum Bacillus resistant to bacterial wilt, the soil glutamicum Bacillus is DW8, classified and named: Glutamicibacter soli, preserved in China Center for Type Culture Collection, address: Wuhan, preservation date: November 13, 2023, and preservation number: CCTCC NO: M 20232211.

[0007] Furthermore, the colonies of the soil glutamicum bacteria are milky yellow, round, with smooth, raised, shiny edges and moist.

[0008] 2. A microbial agent is also provided, wherein the active ingredient of the microbial agent comprises soil glutamicum Bacillus DW8.

[0009] Furthermore, the microbial agent comprises one or more selected from the group consisting of bacterial bodies, bacterial powder, and bacterial suspension of soil Bacillus glutamicum DW8.

[0010] Furthermore, in the microbial agent, soil glutamicum Bacillus DW8 is cultured to obtain a bacterial suspension, which is a liquid microbial agent.

[0011] Furthermore, the microbial agent also includes auxiliary materials acceptable for the preparation of the microbial agent.

[0012] Furthermore, the auxiliary material is a loaded solid, and the loaded solid includes one or more of rice bran, straw powder, diatomaceous earth, sodium alginate, zeolite, and biochar.

[0013] The beneficial effects of the present invention are as follows: the present invention isolates and screens functional microbial strains that have antagonistic effects on tobacco bacterial wilt pathogens from the soil in tobacco areas with bacterial wilt in Meizhou, Guangdong. The antagonistic bacteria have good antagonistic effects on bacterial wilt strains with strong pathogenicity, and not only show excellent prevention and control effects in indoor potted plants. In addition, field tests have also confirmed its excellent prevention and control effects. It is a potential biological agent for the prevention and control of bacterial wilt diseases, has a good development and application prospect, and has greatly improved the prevention and control effects compared to chemical agents for tobacco bacterial wilt. It is expected to be applied to the prevention and control of tobacco bacterial wilt to provide a new solution for green and safe prevention and control. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0015] Figure 1The rhizosphere soil of tobacco plants infected with tobacco bacterial wilt and healthy tobacco plants in Guangdong tobacco-growing areas was collected and antagonistic bacteria were isolated and purified to obtain the rhizosphere antagonistic bacteria.

[0016] Figure 2 To evaluate the antagonistic activity of tobacco rhizosphere bacteria against Ralstonia solanacearum in Guangdong tobacco-growing areas.

[0017] Figure 3 To investigate the antibacterial activity of antagonistic bacteria isolated from the rhizosphere soil of healthy tobacco plants in Longwen Town against Ralstonia solanacearum.

[0018] Figure 4 The antibacterial activity of antagonistic bacteria against Ralstonia solanacearum was investigated by isolating them from the rhizosphere soil of healthy tobacco plants in Wuxing Village, Songyuan Town.

[0019] Figure 5 The antibacterial activity of antagonistic bacteria against Ralstonia solanacearum was investigated by isolating them from the rhizosphere soil of healthy tobacco plants in Xinnan Village, Songyuan Town.

[0020] Figure 6 This is the phylogenetic tree of the four strains constructed based on 16S rDNA sequences.

[0021] Figure 7 The antagonistic activity of 30 bacterial strains against Ralstonia solanacearum (A), production of protease (B), cellulase (C) and siderophore (D) are shown.

[0022] Figure 8 The four bacterial strains antagonized Ralstonia solanacearum (A), produced protease (B), cellulase (C) and siderophore (D) activities.

[0023] Fig. 9 To evaluate the control effect of antagonistic bacteria on tobacco bacterial wilt. (A) The effect of antagonistic bacteria on the disease index of tobacco bacterial wilt; (B) The control effect of antagonistic bacteria on tobacco bacterial wilt; (C) The potted photos of antagonistic bacteria on tobacco bacterial wilt (20 days after inoculation).

[0024] Fig.10 Evaluation of the growth-promoting effects of four antagonistic bacteria on tobacco. (A) Tobacco plant height; (B) Tobacco plant root length; (C) Tobacco plant fresh weight; (D) Tobacco plant dry weight; (E) Photo of the growth-promoting potted plant (20 days after inoculation).

[0025] Fig.11 To evaluate the outdoor control effects of four antagonistic bacteria on tobacco.

[0026] Fig.12 This is the colony morphology of soil glutamicum Bacillus MZ8-15.

[0027] Fig.13 This is a picture of the field trial planting of strains MZ3-12, MZ8-15, MZ9-28 and a mixture (Mix) of these three bacteria. DETAILED DESCRIPTION

[0028] The preferred embodiment of the present invention will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. The experimental methods for which specific conditions are not specified in the embodiments are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0029] Example 1

[0030] 1 Materials and methods

[0031] 1.1 Materials

[0032] Test soil samples: In the tobacco growing areas of Meizhou, rhizosphere soil from tobacco plants that are not infected with bacterial wilt in tobacco fields where bacterial wilt occurs all year round was collected and brought back to the Natural Product Pesticide Research Laboratory of Southwest University for subsequent processing.

[0033] Test bacterial wilt strain: CQPS-1 was isolated from diseased tobacco plant samples collected by the Natural Product Pesticide Research Laboratory in Pengshui, Chongqing. It was identified as a bacterial wilt strain with strong pathogenicity after indoor identification. After isolation and purification, it was stored in the laboratory's -80℃ refrigerator.

[0034] Test instruments:

[0035]

[0036]

[0037] 1.2 Methods

[0038] 1.2.1 Soil collection and processing

[0039] The test soil was taken from the tobacco-growing area of ​​Meixian County, Meizhou City, Guangdong Province. The rhizosphere soil of diseased and normal tobacco plants in tobacco fields with bacterial wilt were collected 60 days and 80 days after tobacco transplanting. Six replicates were collected from healthy and diseased plants, with one replicate for each tobacco plant (six tobacco plants were collected for each sample).

[0040] In this tobacco area, three plots where bacterial wilt has occurred for many years were randomly selected, and the rhizosphere soil of diseased and healthy tobacco plants in the diseased plots were collected. The sampling and subsequent processing methods were as follows: about 60 days and 80 days after tobacco transplanting (harvest period), 4-6 diseased and healthy tobacco plants were selected, the surface soil was dug with a hoe, the roots were dug out, and the larger soil clumps adhering to the roots were removed. Then 50g of soil with a thickness of 0-4mm adhering to the root surface of the tobacco plants was collected, mixed, and packed in self-sealing bags and sealed. The soil was brought back to the laboratory and sieved through a 2mm standard sieve and stored in a refrigerator at 4℃ for subsequent experiments. Figure 1 The rhizosphere soil of tobacco plants infected with tobacco bacterial wilt and healthy tobacco plants in Guangdong tobacco-growing areas was collected and antagonistic bacteria were isolated and purified to obtain the rhizosphere antagonistic bacteria.

[0041] 1.2.2 Isolation and purification of soil bacteria

[0042] 1.2.2.1 Isolation of bacteria: using plate dilution method

[0043] Weigh 5g of fresh soil into a 100mL Erlenmeyer flask, add 45mL of sterile water, and then shake it in a constant temperature shaker at a speed of 180r / min for 20min to evenly disperse the soil particles in the water to form a soil suspension;

[0044] 1. Use a pipette to draw 1 mL of soil suspension into a centrifuge tube containing 9 mL of sterile water. After multiple aspirations, mix well, and then dilute the soil suspension in 10-fold manner until it is 10 -6 ;

[0045] 2. Set the dilution factor to 10 -4 -10 -6 100 μL of soil suspension was respectively pipetted onto TSB medium, and then immediately coated with sterile glass beads until the surface of the medium was dry and no scratches from the rolling of the glass beads appeared. Each sample was repeated on 2 plates.

[0046] 3. Seal the plate and invert it in a 30℃ constant temperature incubator for 2-3 days until colonies grow.

[0047] 1.2.2.2 Bacterial purification

[0048] 1. Observe the growth of single colonies and number them according to their morphology and color;

[0049] 2. Pick a single colony according to the number and streak it on the TSA plate. Repeat three times for each single colony. After streaking, seal the plate with sealing film and invert it in a constant temperature incubator at 30℃ for 2-3 days;

[0050] 3. Take out the plate, pick a single colony again, repeat the above steps, and purify 2-3 times;

[0051] 4. After the purification step is completed, pick a single colony from each sample and place it in a flask containing 20 mL of sterile TSB medium. Place it in a constant temperature shaker at 30°C, 180 r / min, and culture overnight until the bacterial solution OD600 = 0.8-1.0;

[0052] 5. Take 1mL of bacterial solution into a sterile centrifuge tube and centrifuge at 8000r / min for 5min; discard the supernatant, add 1mL of sterile water, shake up and down to form a uniform suspension, repeat three times in sequence, store at room temperature for later use;

[0053] 6. Use a pipette to draw 100 μL of bacterial suspension into a 96-well plate, then add an equal volume of 50% glycerol, mix by repeatedly pipetting, seal the 96-well plate with sealing film, and store it in a -80°C refrigerator for later use.

[0054] 1.2.3 Preliminary screening of bacteria for antagonism of Ralstonia solanacearum

[0055] 1. Take out the preserved rhizosphere bacteria, draw 1 ml of bacterial solution and streak it on TSA medium (small), place it in a 30℃ incubator for 2 days, and streak CQPS-1 on B medium and place it in a 30℃ incubator for 2 days;

[0056] 2. Pick a single colony from the cultured rhizosphere bacteria and place it at the center of 1 / 16 of a 14cm square culture dish. Repeat twice and culture for 1 day. At the same time, pick a single colony of Ralstonia solanacearum and mix it with LB medium (liquid, 15ml). Place the triangular flask containing the bacterial solution in a shaker at 170r / min and 30℃ for 12h (OD value can reach 0.8-1.0);

[0057] 3. Transfer the solanacearum to a sterilized spray bottle and spray evenly on the square dish;

[0058] 4. After one day of cultivation, observe the inhibition zone to determine whether there is an antibacterial effect, measure the diameter of the inhibition zone using the cross method to evaluate the antibacterial effect, and take photos.

[0059] 1.2.4 Antagonism test of Ralstonia solanacearum

[0060] 1. Activation of Ralstonia solanacearum and antagonistic bacteria: Add the preserved bacterial solution to TSB medium (B medium for Ralstonia solanacearum) at a ratio of 1:200 and culture overnight until OD 600 =1.0, take 1mL of bacterial solution into a sterile centrifuge tube, centrifuge at 8000r / min for 5min; discard the supernatant, add 1mL of sterile water, shake up and down to make a suspension, repeat three times, and set aside;

[0061] 2. Pick 3 single colonies from the cultured rhizosphere bacteria and place them on a 90mm round culture dish in an equilateral triangle with a moderate distance. Repeat twice for each and culture for 1 day. At the same time, pick a single colony of Ralstonia solanacearum and mix it with 15ml of LB liquid medium. Place the triangular flask containing the bacterial solution in a shaker at 170r / min and 30℃ for 12h (OD value can reach 0.8-1.0);

[0062] 3. Transfer the solanacearum solution to a sterilized spray bottle, spray evenly on the culture dish, and culture for 1 day;

[0063] 4. Cross-cross method to measure the antibacterial zone: take the average of the three radii of the measured transparent circle as the radius of the plate antibacterial zone, and the radius of the two repeated antibacterial zones is the radius of the sample. Note: Subsequent measurements of transparent zones are all measured using the cross-cross method.

[0064] 1.2.5 Determination of bacterial hydrolase activity

[0065] 1.2.5.1 Protease activity assay

[0066] On a protease plate (A: 8 g skim milk powder, dissolved in 300 mL of water, sterilized at 115°C for 10 min; B: 8 g agar, made up to 300 mL, sterilized at 121°C for 20 min, A and B are sterilized separately and then mixed), three single colonies are plated in an equilateral triangle with a moderate distance between them. Each colony is treated twice. After culturing at 30°C for 3 days, observe the presence of a transparent circle and record its size.

[0067] 1.2.5.2 Cellulose activity assay

[0068] Referring to the method of Ghose, the prepared strain was inoculated on a cellulase activity assay plate (10 g of peptone, 10 g of yeast powder, 10 g of sodium carboxymethyl cellulose, 5 g of sodium chloride, 1 g of potassium dihydrogen phosphate, 18 g of agar, pH = 7.0). Three single colonies were inoculated into an equilateral triangle with a moderate distance. One was treated twice. After culturing at 30°C for 3 days, the plate was stained with 1 g / L Congo red for 1 hour, the stain was discarded, and the plate was soaked in 1 mol / L NaCl solution for 1 hour. The presence of a transparent circle was observed and the size was recorded.

[0069] 1.2.6 Determination of siderophore production activity

[0070] Inoculate the prepared strain onto the CAS solid culture medium purchased and prepared in advance, and inoculate three single colonies into an equilateral triangle with a moderate distance. Treat each colony twice. After culturing at 30℃ for 1 day, observe the presence of transparent circles and record their sizes.

[0071] 1.2.7 Evaluation of bacteria

[0072] According to the method of Faltin et al., the biocontrol potential of antagonistic bacteria was evaluated and scored as follows: 3 points each for protease production, siderophore production, and inhibition of Ralstonia solanacearum, among which the radius of the inhibition zone or transparent zone was 0-3 mm, 1 point, 3-6 mm, 2 points, and >6 mm, 3 points; 1 point for cellulase production, and the total score was 10 points.

[0073] 1.2.8 Molecular identification of antagonistic bacteria

[0074] The antagonistic bacterial DNA was extracted using a kit, and then the 16S specific variable region was amplified using the PCR primers 27F / 1492R. The primers were 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGCTACCTTGTTACGAC-3'). The total volume of the PCR reaction system was 25 μL:

[0075]

[0076] 1. PCR amplification conditions were: pre-denaturation at 94°C for 5 min, 30 cycles of denaturation at 94°C for 1 min, annealing at 51°C for 1 min, extension at 72°C for 3 min, and finally extension at 72°C for 10 min.

[0077] 2. The PCR products obtained by the reaction were detected by 1% agarose gel electrophoresis and finally entrusted to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0078] 3. The sequencing results were compared with the 16S rRNA gene sequences in the Genbank database for homology, and the strain name with a QueryCover value greater than 99% and the highest Per.Ident value was selected as the genus name for identifying the unknown strain. The sequenced sequences were used to construct a phylogenetic tree using the software MEGAX, and the sequences were aligned using the MUSCLE method to calculate the optimal model. The phylogenetic tree was constructed using the ML method, with a bootstrap value of 1000.

[0079] 1.2.9 Potted Planting Effect Verification

[0080] 1.2.9.1 Verification of anti-disease effect

[0081] According to the results of the evaluation and molecular identification of antagonistic bacteria, four strains of antagonistic bacteria with a score greater than 6 were selected for potted plant effect verification. Four-leaf and one-heart tobacco seedlings of varieties Yunyan 87 and K326 were prepared for standby use. The isolated antagonistic bacteria were activated, the antagonistic bacterial solution was cultured to the logarithmic phase, and then diluted to 10 8 cfu / mL, and the plants were irrigated with 10 mL of water per plant. After 3 days of antagonistic bacteria treatment, the pathogenic Ralstonia solanacearum was inoculated. The bacterial solution cultured to the logarithmic phase was diluted to 108 cfu / mL, and the plants were irrigated with 10mL per plant. The inoculated plants were placed in a greenhouse, and the culture conditions were 14h light / 10h dark, 30±1℃, and 75% relative humidity. After the pathogen was inoculated, the investigation was conducted once a day until 20 days. The grading standard for tobacco bacterial wilt in the room is divided into "0-4 levels", as follows (based on a single plant):

[0082] Level 0: whole plant with healthy leaves;

[0083] Level 1: 1 to 2 leaves are semi-wilted;

[0084] Level 2: 2 to 3 leaves wilt;

[0085] Level 3: Only 1 to 2 healthy leaves remain;

[0086] Level 4: No healthy leaves.

[0087] The disease severity was calculated according to the disease grade standard proposed by Kempe et al.

[15] . The formula for disease severity and control effectiveness is as follows:

[0088]

[0089] 1.2.9.2 Verification of growth-promoting effects

[0090] The method of treating tobacco seedlings with antagonistic strains refers to 1.2.9.1. The control group was treated with an equal volume of sterile water. Each treatment was repeated 10 times. The treated tobacco seedlings were placed in a greenhouse with 14h light / 10h dark, 30±1℃, and 75% relative humidity. Their growth was observed every day. After 21 days, the fresh weight, plant height, root length, and dry weight were counted. Each treatment was repeated 3 times.

[0091] 1.2.10 Field effect verification

[0092] Based on the results of indoor disease resistance and growth promotion effect tests, antagonistic bacteria that can significantly resist bacterial wilt and promote tobacco plant growth were screened for field effect verification.

[0093] (1) Test location

[0094] The experimental site was selected from the plot with high incidence of continuous cropping bacterial wilt. The experimental site was arranged in the Meizhou tobacco area of ​​Guangdong Province.

[0095] (2) Test methods

[0096] The experiment has 5 treatments, 3 replicates, 15 plots, and a plot area of ​​about 0.1 mu. A protection row is set up, and the experimental area is about 1.8 mu. Root irrigation treatment is carried out 25 days after transplanting and at the early stage of disease.

[0097] Process 1: MZ3-12 10 6cfu / mL 100mL;

[0098] Processing 2: MZ8-15 10 6 cfu / mL 100mL;

[0099] Process 3: MZ9-28 10 6 cfu / mL 100mL;

[0100] Treatment 4: three bacterial agents were mixed in equal volumes;

[0101] Treatment 5: Blank control

[0102] (3) Disease investigation

[0103] The occurrence of tobacco diseases is investigated according to the national standard GB / 23222-2008 "Grading and Investigation Methods of Tobacco Diseases and Insect Pests". Combined with the local characteristics of disease occurrence, a systematic investigation is mainly conducted on various diseases of concern, and the number of diseased plants and the disease level in each plot are investigated to calculate the incidence rate. The disease investigation can be carried out simultaneously with the determination of tobacco agronomic traits. According to the occurrence of different diseases, the investigation begins at the early stage of the disease, and the investigation is conducted every 5 days for more than 4 consecutive times.

[0104]

[0105] Example 2 Results and Analysis

[0106] 2.1 Isolation of bacteria from rhizosphere soil of healthy tobacco plants

[0107] A total of 9 samples of rhizosphere soil from healthy tobacco plants in tobacco fields with tobacco bacterial wilt disease that occurred all year round were collected from Songyuan Town and Longwen Town, Meixian County, Meizhou City, Guangdong Province, and antagonistic bacteria were isolated and purified. After separation and purification on TSA medium, 274 strains of tobacco rhizosphere bacteria were finally obtained.

[0108] 2.2 Preliminary screening of rhizosphere bacteria with antagonistic effects on tobacco solanacearum

[0109] The antagonistic activity of the square culture dish was evaluated by culturing the antagonistic bacteria on TSA medium 24 hours in advance, and then spraying the Ralstonia solanacearum suspension on the square culture dishes where different antagonistic bacteria grew. The antagonistic activity of the rhizospheric bacteria was evaluated according to the size of the inhibition zone. The antibacterial activity of 274 rhizospheric bacteria against Ralstonia solanacearum was evaluated. Figure 2 The results show that the rhizosphere bacteria isolated from tobacco areas in Guangdong have certain antibacterial activity against Ralstonia solanacearum. There are 84 strains with antagonistic activity, and 25 strains of antagonistic bacteria isolated from Longwen Town ( Figure 3 The antibacterial activity of these 25 antagonistic bacteria against Ralstonia solanacearum), 30 antagonistic bacteria isolated from Wuxing Village, Songyuan Town ( Figure 4 The antibacterial activity of these 30 antagonistic bacteria against Ralstonia solanacearum was investigated), and 29 antagonistic bacteria were isolated from Xinnan Village, Songyuan Town ( Figure 5 The antibacterial activity of these 29 antagonistic bacteria against Ralstonia solanacearum was measured. There were 30 strains with an inhibition zone diameter of more than 10 mm, including MZ2-3, MZ2-14, MZ2-30, MZ3-12, MZ3-30, MZ4-2, MZ4-8, MZ4-9, MZ4-13, MZ4-25, MZ4-26, MZ5-11, MZ5-26, MZ5-28, MZ6-8, MZ6-15, MZ6-19, MZ6-21, MZ7-4, MZ7-8, MZ7-11, and MZ7-13 MZ7-27, MZ7-30, MZ8-13, MZ8-15, MZ8-21, MZ8-27, MZ9-22 and MZ9-28 had good antagonistic activity against Ralstonia solanacearum. These 30 bacterial strains were further tested for their anti-Ralstonia solanacearum activity, hydrolase activity and siderophore production activity.

[0110] 2.3 Molecular identification of antagonistic bacteria isolated from tobacco-producing areas in Guangdong

[0111] The 16S rRNA gene sequence analysis was used to molecularly identify the 30 antagonistic bacteria. The target gene sequences of the antagonistic bacteria were compared in the GenBank database by BLAST and a phylogenetic tree was constructed. Some of the results are shown in Table 1. Figure 6 This is a phylogenetic tree of the four strains based on 16S rDNA sequences. The results showed that strain MZ3-12 was Bacillus cereus, strain MZ4-13 was Bacillus subtilis, strain MZ8-15 was Glutamicibacter soli, and strain MZ9-28 was Bacillus amyloliquefaciens. Fig.12 The colony morphology of strain MZ8-15 is milky yellow, round, with smooth, convex, shiny and moist edges. Soil glutamicum Bacillus MZ8-15 was classified and named: Glutamicibacter soli DW8, and preserved in China Center for Type Culture Collection, address: Wuhan, preservation date: November 13, 2023, and preservation number: CCTCCNO: M 20232211.

[0112] Table 1 Partial results of molecular identification of antagonistic bacteria isolated from tobacco growing areas in Guangdong

[0113]

[0114] 2.4 Assignment Evaluation Results

[0115] Through the experiments of measuring the antagonistic activity, hydrolase activity and siderophore production ability of these 30 antagonistic bacteria, the value of each antagonistic bacteria was obtained according to the size of the transparent zone, and the scores were as follows: 3 points each for protease production, siderophore production, and inhibition of solanacearum, among which the radius of the inhibition zone or transparent zone was 0-3mm, 1 point, 3-6mm, 2 points, and >6mm, 3 points; 1 point for cellulase production, and the total score was 10 points. Figure 7 The results are shown in Table 1. Among the strains with a total assignment score greater than 6, strains with different molecular identification results were selected for indoor pot experiments, namely MZ3-12, MZ4-13, MZ8-15, and MZ9-28. Figure 8 These are the test graphs of the antagonism of these four bacterial strains against Ralstonia solanacearum (A), production of protease (B), cellulase (C) and siderophore (D).

[0116] Table 2 Antagonistic bacteria assignment evaluation results

[0117]

[0118] 2.5 Study on the indoor control effect of four antagonistic bacteria on tobacco bacterial wilt

[0119] To verify the control effect of the selected antagonistic bacteria on tobacco bacterial wilt, we selected MZ3-12, MZ4-13, MZ8-15, and MZ9-28 with different molecular identification results from rhizospheric bacteria with a score greater than 6 for potted experiments. The control effect of these four antagonistic bacteria on tobacco bacterial wilt was evaluated as follows: Fig. 9 As shown, (A) the effect of antagonistic bacteria on the disease index of tobacco bacterial wilt; (B) the control effect of antagonistic bacteria on tobacco bacterial wilt; (C) the potted photos of antagonistic bacteria on tobacco bacterial wilt (20 days after inoculation). The incidence and disease index of tobacco bacterial wilt caused by each strain are shown in Tables 3 and 4. The results showed that compared with the control, the application of antagonistic bacteria significantly reduced the incidence of tobacco bacterial wilt. Antagonistic bacteria MZ3-12, MZ8-15, and MZ9-28 all had relatively obvious control effects on tobacco bacterial wilt, among which strain MZ8-15 had the best control effect, with relative control effects of 85.61% and 67.00% at 10 days and 15 days after inoculation, respectively. These results preliminarily prove that the Guangzhou indigenous microorganisms screened in this study have a relatively good control effect on tobacco bacterial wilt.

[0120] Table 3 The incidence of tobacco bacterial wilt caused by each strain

[0121]

[0122] Table 4 shows the disease index of each strain for tobacco bacterial wilt

[0123]

[0124]

[0125] 2.6 Study on the growth-promoting effects of four antagonistic bacteria on tobacco indoors and in the field

[0126] While verifying the control effect of the four selected antagonistic bacteria on tobacco bacterial wilt, we also conducted a study on whether the antagonistic bacteria had a growth-promoting effect on tobacco plants indoors and in the field. The results showed that the four strains had different degrees of growth-promoting effects on tobacco plants. Fig.10 Evaluation of the growth-promoting effect of four antagonistic bacteria on tobacco. (A) Tobacco plant height; (B) Tobacco plant root length; (C) Tobacco plant fresh weight; (D) Tobacco plant dry weight; (E) Growth-promoting potted plant photo (20 days after inoculation); Specific data are shown in Table 5. As shown in the figure, the four antagonistic bacteria can significantly increase tobacco plant height, root length, fresh weight, dry weight ( Fig.10 In addition, in terms of tobacco plant growth, the size and quantity of tobacco leaves in the treatment group were significantly better than those in the control group ( Fig.10 Table 6 shows the effect of different strains on the agronomic shape of tobacco in the field. Fig.13 This is a picture of the field trial planting of strains MZ3-12, MZ8-15, MZ9-28 and a mixture (Mix) of these three bacteria.

[0127] Table 5 Effects of different strains on tobacco growth and development

[0128]

[0129] Table 6 Effects of different strains on agronomic shape of tobacco (field)

[0130]

[0131]

[0132] 2.7 Study on the outdoor control effect of antagonistic bacteria on tobacco bacterial wilt

[0133] The occurrence of tobacco bacterial wilt is accompanied by a complex natural microecological environment. The efficacy of a single antagonistic bacterial potted strain against bacterial wilt is not enough to fully reflect its value in prevention and control. In order to further explore the field efficacy of the local antagonistic bacteria screened indoors in the tobacco area of ​​Meizhou, Guangdong, we conducted an outdoor prevention and control study of antagonistic bacteria in the tobacco bacterial wilt fields in Meizhou, Guangdong. Based on the results of 2.5, we selected the treatment groups of strains MZ3-12, MZ8-15, MZ9-28 and a mixture of three bacteria (Mix). The results are as follows Fig.11The incidence and disease index of tobacco bacterial wilt in the field test are shown in Table 7 and Table 8. The results showed that the four treatments had different degrees of relative protection effect, among which MZ8-15 had the best effect, with relative protection effects of 86.72% and 60.19% at 25d and 40d after inoculation, respectively.

[0134] Table 7 The incidence of tobacco bacterial wilt caused by each strain

[0135]

[0136] Table 8 shows the disease index of each strain for tobacco bacterial wilt

[0137]

[0138] 3 Discussion and Conclusion

[0139] There are many factors that affect the yield and quality of cash crops and the rhizosphere microecology of crops, among which soil-borne diseases are the main biological factors and restrict agricultural production. The occurrence of soil-borne diseases is largely due to the imbalance of soil microbial ecology caused by the enrichment of some fungi, bacteria and other microorganisms in the soil. In addition, there are many types of soil-borne pathogens, mainly divided into fungi, bacteria, viruses, etc. Among them, the bacterial soil-borne diseases caused by Ralstonia solanacearum are the most serious. Bacterial wilt mainly harms Solanaceae crops, such as tobacco and tomatoes, and often causes huge losses to their yield and quality. We isolated 274 strains of bacteria from the healthy rhizosphere soil of the diseased tobacco fields in the Guangdong tobacco area (Meizhou City). There were 84 strains with antagonistic activity, and 30 strains with inhibition zone diameters exceeding 10 mm. Subsequently, these 30 strains of bacteria were subjected to independent plate antagonistic activity assays against Ralstonia solanacearum, hydrolase activity assays, siderophore production ability assays and molecular identification. According to the above experimental results, we screened out four antagonistic bacteria, MZ3-12, MZ4-13, MZ8-15, and MZ9-28, which had different molecular identification results and had good antagonistic activity, enzyme activity, and siderophore production ability. In order to further confirm and explore the indoor disease resistance or growth-promoting effects of these four antagonistic bacteria, we also carried out indoor potted experiments. The results showed that MZ3-12, MZ8-15, and MZ9-28 had good control effects on tobacco solanacearum, and MZ8-15 had the best control effect. The relative control effects at 10d and 15d after inoculation were 85.61% and 67.00%, respectively; MZ3-12, MZ4-13, MZ8-15, and MZ9-28 treatments had significant growth-promoting effects on tobacco plants compared with the control, which were reflected by plant height, root length, fresh weight, and dry weight, respectively. Except for MZ4-13, MZ3-12, MZ8-15, and MZ9-28 all have obvious control effects. Therefore, we used these three antagonistic bacteria to carry out field control experiments in tobacco bacterial wilt disease-affected tobacco areas in Meizhou, Guangdong. The results showed that MZ3-12, MZ8-15, MZ9-28 and the combination of the three antagonistic bacteria all had certain control effects on tobacco bacterial wilt, among which MZ8-15 had the best effect, with relative control effects of 86.72% and 60.19% at 25d and 40d after inoculation, respectively. It was reported in the existing literature (Hydroxycoumarins: New, effective plant-derived compounds reduce Ralstonia pseudosolanacearumpopulations and control tobacco bacterial wilt) that the control effect of the conventional chemical control agent-thiophanate-copper on tobacco bacterial wilt was about 38.36%, as confirmed by indoor pot experiments.The three Guangdong native antagonistic bacteria finally screened in this experiment showed good disease prevention effects on tobacco bacterial wilt in indoor pot tests and outdoor field tests, and it is worthy of in-depth research on the biological control mechanism.

[0140] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A soil glutamicum strain antagonistic to bacterial wilt, characterized in that: The soil glutamicum bacteria is DW8, classified and named: Glutamicibacter soli, preserved in the China Center for Type Culture Collection, address: Wuhan, preservation date: November 13, 2023, and preservation number: CCTCC NO: M 20232211.

2. The soil glutamicum resistant to bacterial wilt according to claim 1, characterized in that: The soil glutamicum colonies are creamy yellow, round, with smooth, raised, shiny and moist edges.

3. The soil glutamicum resistant to bacterial wilt according to claim 1, characterized in that: The bacterial wilt disease is tobacco bacterial wilt disease.

4. A microbial preparation, characterized in that: The active ingredients of the microbial preparation include one or more selected from the bacterial bodies, bacterial powders and bacterial suspensions of the soil glutamicum Bacillus DW8 according to claim 1.

5. The microbial preparation according to claim 4, characterized in that Soil glutamicum Bacillus DW8 was cultured to obtain a bacterial suspension, which is a liquid microbial agent.

6. The microbial preparation according to claim 4, characterized in that The microbial agent also includes auxiliary materials acceptable for the preparation of the microbial agent.

7. The microbial preparation according to claim 6, characterized in that The auxiliary material is a loaded solid, and the loaded solid includes one or more of rice bran, straw powder, diatomaceous earth, sodium alginate, zeolite, and biochar.

8. The method for preparing the microbial preparation according to claim 4 or 5, characterized in that After the soil glutamicum DW8 strain was activated, it was inoculated into LB liquid medium and cultured at 28-30℃. 600nm To 0.6-1.0.

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