Streptomyces asenjonii R02 strain and its application

Abstract

The present invention relates to the field of microbial technology, and in particular to Streptomyces asenjonii R02 strain and application thereof. The strain R02 of the present invention is a microorganism screened by the applicant. After testing, the strain has good growth-promoting and disease-resistant effects, specifically, it can produce iron carriers, secrete cellulase, and degrade cellulose; at the same time, the antibacterial activity test proves that the bacteria has good antibacterial properties against Gram-negative bacteria, especially against Ralstonia solanacearum; pot experiments and field experiments prove that the microorganism has significant disease-resistant effects on soil-borne bacterial wilt of plants caused by Ralstonia solanacearum, has good growth-promoting effects on plants, especially ginger, and can be used to prepare biological fertilizers to effectively prevent and control the occurrence of soil-borne bacterial wilt.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to Streptomyces asenjonii R02 strain and its application. Background Art

[0002] Ralstonia solanacearum Ralstonia solanacearum ) has the characteristics of a large host range and a wide area of ​​occurrence. The disease has been found in about 450 crops in 44 families, including tomatoes, corn, potatoes, tobacco, peanuts and peppers, seriously endangering the yield and quality of food and cash crops around the world. The prevention and control measures for soil-borne bacterial wilt include fumigation with chemicals, cultivation management, breeding of disease-resistant varieties and biological control. Among them, soil drug fumigation, although it can effectively kill pathogens lurking in the soil and has significant effects in a short period of time, it will indiscriminately kill soil microorganisms and seriously damage the balance of soil microecology. Chemical residues will also damage the environment and ultimately affect human health. Cultivation management mainly includes measures such as crop rotation and water and fertilizer management, but it is difficult to eradicate the disease. The breeding of disease-resistant varieties takes a lot of time, has a long cycle and faces huge uncertainties. In contrast, biological control is green and environmentally friendly, and the main idea is to prevent and control the occurrence of diseases from multiple angles by screening beneficial microorganisms, including pathogen antagonists, plant growth-promoting bacteria, microbial strains that induce plant systemic resistance and compete with pathogens for limited resources, etc. For example, by screening actinomycetes that antagonize Ralstonia solanacearum, preparing actinomycete agents, and using the active substances secreted by actinomycetes to inhibit the growth of Ralstonia solanacearum and its interaction with rhizosphere microorganisms, the community structure of rhizosphere microorganisms is regulated, thereby preventing and controlling the occurrence of soil-borne bacterial wilt. Summary of the invention

[0003] In view of the above, it is necessary to screen for antagonistic Ralstonia solanacearum ( Ralstonia solanacearum ) and developed the probiotics into biological fertilizer to effectively prevent and control the occurrence of soil-borne bacterial wilt.

[0004] In order to achieve the above purpose, the present invention screened out a new strain: Streptomyces asenjonii R02, whose classification is named as: Streptomyces asenjonii , the deposit number is CGMCC NO.32267; the strain is deposited in the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit date is October 21, 2024.

[0005] The present invention also includes the Streptomyces asenjonii Inoculum of strain R02.

[0006] The present invention also includes the Streptomyces asenjoniiApplication of R02 strain or the bacterial agent in the preparation of biological organic fertilizer.

[0007] The present invention also includes the Streptomyces asenjonii Application of the R02 strain or the bacterial agent in promoting ginger growth, producing siderophores and / or degrading cellulose.

[0008] The present invention also includes the Streptomyces asenjonii Application of the R02 strain or the bacterial agent in the preparation of a biocontrol agent.

[0009] Furthermore, the bacteria inhibited by the biocontrol agent are Gram-negative bacilli.

[0010] Furthermore, the microorganism inhibited by the biocontrol agent is Ralstonia solanacearum ( Ralstonia solanacearum ), Escherichia coli ( Escherichia coli ), Citrobacter freundii ( Citrobacter frazier ), Klebsiella oxytoca, Fusarium oxysporum, watermelon-specific type ( Fusarium oxysporum f.sp. niveum), Fusarium oxysporum cucumber-specific type ( Fusarium oxysporum f. sp. cucumerinum) and / or Fusarium graminearum ( Fusarium graminearum ).

[0011] The present invention also includes the Streptomyces asenjonii Application of R02 strain or the bacterial agent in resisting soil-borne bacterial wilt of plants.

[0012] Furthermore, the pathogen of bacterial wilt is Ralstonia solanacearum ( Ralstonia solanacearum ).

[0013] The present invention also includes applying the Streptomyces asenjonii The method for planting tomatoes using the R02 strain or the bacterial agent is characterized in that the method comprises: applying the bacterial agent containing Streptomyces asenjonii The R02 strain conidia were inoculated and then planted for cultivation.

[0014] The present invention also includes applying the Streptomyces asenjonii The method for planting ginger using the R02 strain or the bacterial agent is characterized in that the method comprises: applying a bacterial agent containing Streptomyces asenjonii The R02 strain conidia were inoculated and then planted for cultivation.

[0015] The present invention has the following beneficial effects: the strain R02 of the present invention is a microorganism selected by the applicant. After testing, the strain has good growth-promoting and disease-resistant effects, which are specifically manifested in that it can produce iron carriers, secrete cellulase, and degrade cellulose; at the same time, it is proved by antibacterial activity tests that the bacteria has good antibacterial properties against Gram-negative bacteria, especially has a good antibacterial effect on Ralstonia solanacearum; pot experiments and field experiments have proved that the microorganism has a significant disease-resistant effect on soil-borne bacterial wilt of plants caused by Ralstonia solanacearum, has a good growth-promoting effect on plants, especially ginger, and can be used to prepare biological bacterial fertilizers to effectively prevent and control the occurrence of soil-borne bacterial wilt. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the colony morphology of strain R02.

[0017] Figure 2 This is the morphology of R02 spore hyphae and conidia under a scanning electron microscope.

[0018] Figure 3 This is the phylogenetic tree of strain R02 constructed based on the 16S rRNA sequence.

[0019] Figure 4 This is a diagram showing the antagonistic effects of strains R02 and W10 on Ralstonia solanacearum Rs1115; in the figure, A is strain R02, and B is the control strain W10.

[0020] Figure 5 The inhibitory effect of intracellular or extracellular active substances of strain R02 on Ralstonia solanacearum; in the figure, A is the supernatant and B is the mycelium.

[0021] Figure 6 This is the hydrolysis circle diagram formed by strain R02 on the culture medium (CAS).

[0022] Figure 7 This is a diagram showing the control effect of strain R02 on tomato bacterial wilt; A in the figure is a control experiment inoculated only with the bacterial wilt pathogen Rs1115; B is an experimental group inoculated with the bacterial wilt pathogen Rs1115 and strain R02; C is a blank control group not inoculated with any strain.

[0023] Figure 8 This is a graph showing the effect of strain R02 on ginger growth.

[0024] Biomaterial Deposit Information

[0025] The strain information deposited in this application is: Streptomyces asenjonii Strain R02, whose taxonomic designation is: Streptomyces asenjonii, the deposit number is CGMCC NO.32267; the strain is deposited in the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit date is October 21, 2024. DETAILED DESCRIPTION

[0026] All features disclosed in this specification, or steps in all methods or processes disclosed, except mutually exclusive features and / or steps, can be combined in any manner.

[0027] Any feature disclosed in this specification (including any appended claims and abstract), unless otherwise stated, is merely an example of a series of equivalent or similar features. Example 1

[0028] This example is the screening, identification and biological characteristics of strain R02.

[0029] Strain screening: Healthy tomato plants were collected from a tomato plantation in Houcun, Qilin Town, eastern suburbs of Nanjing (118°57'E, 32°03'N), dug up with roots, keeping the roots intact, and gently shaken to remove the soil. Then, they were placed in sterile ziplock bags and stored at 4°C. Before the experiment, the samples were taken out, and the soil samples attached to the rhizosphere were scraped with a sterile scraper. The rhizosphere soil samples were placed in a sterile mortar and ground into powder. About 5g of the soil sample was weighed and placed in a 250ml conical flask filled with 45ml sterile water and a certain amount of sterile glass beads, and placed in a shaker (280rpm, 12-20min, 28°C) for shaking. At this time, the soil sample was diluted by 10 -1 , dilute step by step, and choose the dilution multiple of 10 -3 , 10 -4 , 10 -5 200μl of the soil suspension diluted above was taken and evenly coated on the surface of Gao's No. 1 medium containing 0.1% actinomycin (preparation method: 50mg + 1ml anhydrous ethanol) and 0.1% nalidixic acid (preparation method: 10mg + 1ml sterile water). After evenly coating and drying, it was placed in a constant temperature incubator at 28℃ for 5-7 days, and the growth of the colony was observed regularly. The mature single colony of actinomycetes was picked in time, streaked and purified, and transferred to a slant for storage.

[0030] Identification of antagonistic actinomycetes R02.

[0031] Morphological observation of strain R02: Strain R02 was streaked on ISP3 plates and cultured at 28℃ for 5 days. The colonies of R02 were round or oval, with a slight convexity in the center and serrated edges. The colony color was compared with "The ISCC-NBS COLOR CHARTS Standard samples NO. 2106", and the colony color was light green-gray (see Figure 1 ). Use a sterile thin blade to cut the culture medium of the R02 colony that has been freshly cultured for 5 days into observation areas of ≤6×6mm. 2 , sample thickness ≤2mm. Fix the sample with 2.5% glutaraldehyde for more than 8h, then rinse with phosphate buffer 3 times, 10min each time, then dehydrate with 50%, 70%, 80%, 90% ethanol gradient for 15min each, and dehydrate with 100% ethanol 3 times, 30min each time. Replace with tert-butanol 3 times, 30min each time, dry the sample with a freeze dryer, stick the sample on the sample stage with double-sided tape, with the observation surface facing up, and use an ion sputtering instrument to coat the sample with a 10nm gold film. Finally, observe the morphology of spore hyphae and conidia of the antagonistic strain R02 using a scanning electron microscope. Electron microscopy revealed that R02 conidia were columnar with spines on the surface, and the spore chains were straight or open spirals (see for details). Figure 2 ).

[0032] Among them, ISP3 plate was prepared with the following components: 3 g / L oats, 0.2 g / L potassium nitrate, 0.5 g / L potassium hydrogen phosphate, 0.2 g / L magnesium sulfate, 10 g / L agar, natural pH. Note: Before sterilization, weigh an appropriate amount of oatmeal, boil it in deionized water, filter it, add the filtrate to the culture medium, make up to 1 L with deionized water, add other drugs, and finally sterilize and use it.

[0033] Molecular biological identification of strain R02: strain R02 was sent to Shanghai Lingen Biotechnology Co., Ltd. for 16SrRNA sequencing. The sequencing results were analyzed by BLAST comparison of related sequences using the EzBioCloud platform. After sequencing, the R02 gene sequence was 1429bp in total. It was logged in to NCBI (accession number MZ254790.1) for sequence comparison, and the first 9 strains with high homology to R02 were selected. The phylogenetic tree was constructed using MEGA 7.0 software using the neighbor-joining method (NJ). The results showed that strain R02 was Streptomyces asenjonii (LT621750.1) similarity reaches 99.57% ( Figure 3 ). Combined with the results of bacterial morphology, physiological and biochemical indicators and 16S rRNA gene sequence analysis, strain R02 was identified as Streptomyces asenjonii .

[0034] Physiological and biochemical characteristics of strain R02: The physiological and biochemical indicators of Streptomyces were determined by referring to the "Streptomyces Identification Manual" and the results are shown in Table 1.

[0035] Example 2

[0036] This example is a verification of the antagonism of strain R02 against Ralstonia solanacearum.

[0037] 1. Antagonism experiment: Use an inoculation loop to pick up tomato solanacearum preserved in the laboratory ( R. solanacearum )The colony of QL-Rs1115 (GenBank accession GU390462) was streaked on SMSA medium and cultured at 30℃ for 36h. The red cells with mobility were picked and inoculated into NB liquid medium. The cells were placed in a shaker at 30℃ and 170rpm for overnight culture to obtain a suspension of Ralstonia solanacearum. The bacterial suspension was centrifuged at 6000rpm for 10min, the supernatant was discarded, the cells were collected and resuspended in 0.9% saline, and the absorbance (OD 600 The concentration of bacterial suspension was adjusted to 10 7 cfu / mL. The single-point spray method was used to detect the antagonistic ability of actinomycete strains to Ralstonia solanacearum Rs1115. First, a sterile toothpick was used to pick the selected actinomycete spores and place them on the center of the NA plate. After culturing at 28°C for about 3-5 days, a single actinomycete colony was formed. Then, the plate was filled with Ralstonia solanacearum Rs1115 suspension (10 7 cfu / mL) spray bottle evenly spray Rs1115 solution once (about 0.2mL) on the NA plate with a single actinomycete colony. After culturing at 30℃ for 16h, observe the appearance and size of the antagonistic zone, and measure the diameter D of the inhibition zone and the diameter d of the colony. Repeat three times for each strain.

[0038] This example verifies two strains of the same genus: specifically, Streptomyces asenjonii R02 and Streptomyces herbicola isolated and preserved in the laboratory ( Streptomyces pratensis ) The antagonistic effect of W10 on Ralstonia solanacearum is shown in Figure 4 As shown in the figure, A is strain R02 and B is control strain W10; it can be seen that strain R02 has a good antagonistic effect on Ralstonia solanacearum, with an average inhibition zone diameter of 5.97 cm. S. pratensis )W10 has no antibacterial effect.

[0039] The SMSA medium in this example consists of the following ingredients: 50 mg / L crystal violet, 50 mg / L 1% TTC, 100 mg / L polymyxin, 20 mg / L bacitracin, 5 mg / L chloramphenicol, 50 mg / L actinomycin, 0.5 mg / L penicillin, pH 7.2.

[0040] The NB liquid culture medium in this example is composed of the following ingredients: 5 g / L peptone, 0.5 g / L yeast extract, 3 g / L beef extract, 10 g / L glucose, pH 7.2.

[0041] 2. Intracellular and extracellular distribution of antibacterial active substances: Spores of strain R02 were picked up with an inoculation loop and inoculated into the seed solution. The culture was shaken at 28°C and 170 r / min for 2 days. The inoculation amount was transferred to the liquid fermentation medium at 5%, and the culture was shaken at 28°C and 170 r / min for 4 days. After the fermentation was completed, the culture was centrifuged at 4°C and 8000 r / min for 10 min. The supernatant was filtered with a microfilter with a diameter of 0.22 μm, and the mycelium was ultrasonically broken. An Oxford cup was placed in the center of the NA culture medium. 200 μL of the fermentation supernatant and precipitate were added to the cup respectively. Repeat 3 times and use a suspension of R. solanacearum Rs1115 (10 7 cfu / mL) spray bottle evenly spray Rs1115 bacterial solution once (about 0.2mL) on the NA plate. Observe the growth of Rs1115 and determine whether the active substance is secreted into the fermentation liquid or in the mycelium based on the antibacterial effect. The experimental results are shown in Figure 5 As shown in the figure, A is the supernatant and B is the mycelium; it can be seen from the figure that the fermentation supernatant has a more obvious antibacterial effect, and the active substances produced by the strain are mainly distributed in the fermentation supernatant.

[0042] The liquid seed culture medium contains the following ingredients: 4 g / L yeast extract powder, 10 g / L malt extract powder, 4 g / L glucose, and pH 7.5.

[0043] The liquid fermentation medium contains the following ingredients: 2 g / L tryptone, 10 g / L glucose, 5 g / L soluble starch, 2 g / L yeast extract, 4 g / L sodium chloride, 0.5 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate, 2 g calcium carbonate, pH 7.5.

[0044] NA medium contains the following ingredients: peptone 5g / L, yeast extract powder 0.5g / L, beef extract 3g / L, glucose 10g / L, agar 1g, pH 7.2. Example 3

[0045] strain Streptomyces asenjonii R02 (abbreviation S. asenjonii R02) antibacterial experiment.

[0046] 1. S. asenjonii R02 strain antagonistic bacteria experiment: S. asenjoniiThe R02 spore suspension was spread on Gao's No. 1 plate and cultured for 5-7 days. When the spores covered the entire plate, a sterile punch (Φ=6 mm) was used to punch the spores into cakes to prepare the R02 cakes for later use. Ralstonia solanacearum ( Ralstonia solanacearum ), Escherichia coli ( Escherichia coli ), Citrobacter freundii ( Citrobacter frazier ), Klebsiella oxytoca ( Klebsiella oxytoca )、Bacillus polymyxa( Paenibacillus polymyxa )、Bacillus subtilis( Bacillus subtilis ), the OD600 value was measured after 24 h, and the bacterial suspension was prepared with sterile water (OD 600 =1.0), take 100 μl of bacterial suspension respectively, spread on NA medium plates, transplant strain R02 bacterial cake to the center of each plate containing indicator bacteria, do three replicates for each group, culture at 30℃ for 24-48 h, and measure the diameter of the inhibition zone.

[0047] 2. S. asenjonii Antagonistic fungi experiment of R02 strain: detection by plate confrontation method S. asenjonii R02 antagonistic fungal activity. Use an inoculation needle to pick Fusarium oxysporum watermelon special type ( Fusarium oxysporum f. sp.niveum), Fusarium oxysporum cucumber-specific type ( Fusarium oxysporum f. sp. cucumerinum), Fusarium graminearum ( Fusarium graminearum ) were inoculated in the center of the PDA plate and cultured for 5 days. When the mycelium of the pathogen covered the plate, a puncher (Φ=6 mm) was used to punch the pathogen cake. The pathogen cake was placed in the center of the PDA plate, and two were placed symmetrically on the left and right sides 2 cm away from the cake. S. asenjonii R02 bacterial cake, only pathogenic bacteria cake was placed as blank control, three replicates were made for each group, and the cells were cultured at 28°C for 5-7 days, and the diameter of the inhibition zone was measured.

[0048] Among them, Gao's medium No. 1 includes the following ingredients: soluble starch 20g, KNO3 1.0g, K2HP04.7H2O0.5g, MgSO4·7H2O 0.5g, NaCl 0.5g, FeSO4·7H2O 0.01g, agar 20g, deionized water 1000mL, pH 7.2.

[0049] The PDA medium includes the following ingredients: 200 g potato, 20 g glucose, 20 g agar, 1000 mL deionized water, and natural pH. The antibacterial results obtained from the above antibacterial experiment are shown in Table 2 below.

[0050]

[0051] As shown in Table 2, strain R02 has antibacterial effects on Ralstonia solanacearum, Escherichia coli, Citrobacter freundii, Klebsiella oxytoca, Fusarium oxysporum watermelon-specific type, Fusarium oxysporum cucumber-transformed type and Fusarium graminearum, especially has the strongest antibacterial activity against Ralstonia solanacearum, and has no antibacterial activity against Bacillus polymyxa and Bacillus subtilis. In the future, strain R02 can be compounded with biocontrol Bacillus to prepare a composite bacterial agent; this shows that strain R02 inhibits Gram-negative bacilli, has no antagonistic activity against Gram-positive Bacillus, and has an antagonistic effect on Fusarium oxysporum that causes wilt, but the effect is relatively weak. Example 4

[0052] This example studies the siderophore-producing ability of strain R02.

[0053] Qualitative detection of iron carrier production by strains: Inoculate the strain R02 cake on the iron carrier screening medium and culture at 28℃ for 7 days. The results are as follows Figure 6 As shown in the figure, transparent hydrolysis circles appeared around the colony, indicating that the strain can produce iron carriers. Iron carriers can chelate the insoluble trivalent iron ions in the soil for absorption and utilization by microorganisms and plants, thereby promoting plant growth.

[0054] Among them, the preparation method of iron carrier identification culture medium is as follows: CAS detection culture medium 10.87 g, H2O 1000 mL, pH 7.0-7.2, sterilization at 115 ℃ for 30 min. Example 5

[0055] This example studies the cellulase activity of strain R02.

[0056] Filter paper enzyme activity can reflect the synergistic ability between different cellulase activities. Therefore, filter paper enzyme activity is often used to characterize the size of total cellulase activity. The value can more accurately reflect the ability of microorganisms to decompose cellulose. Using rice straw as an inducer, the 3.5 dinitrosalicylic acid method (DNS) was used to determine S. asenjonii The filter paper enzyme activity of R02 was measured after the strain was shaken in a flask for 5 days and the supernatant was centrifuged. The results showed that the filter paper enzyme was 28.12 U / mL, indicating that strain R02 has a good ability to degrade lignocellulose. Example 6

[0057] This example studies the control effect of strain R02 on tomato bacterial wilt.

[0058] (1) S. asenjonii Preparation of R02 conidia: Spread R02 on ISP3 solid plates and culture at 28°C for 5-7 days. Scrape off R02 spores with a sterile spatula, mix with sterile water, count with a hemocytometer, and adjust the spore suspension concentration to 10 8 pcs / mL for future use.

[0059] (2) Potted experiment of controlling tomato bacterial wilt with R02 fungicide: The tomato variety was "Red Dwarf". Before the experiment, the seeds were surface disinfected. First, the seeds were soaked in sterile water for 16 hours, then disinfected with 75% ethanol for 30 seconds, rinsed with sterile water 4 times, soaked in 10% NaOCl for 20 minutes, and finally rinsed with sterile water at least 4 times and dried with sterile filter paper. The surface-sterilized seeds were transferred to 1 / 2MS culture medium and cultured in the dark at 28°C for 3 days. After the seeds germinated, they were sown in seedling trays containing 50g of sterile seedling medium in each hole at a temperature of 22°C-32°C, a relative humidity of 70%, a photoperiod of 16 / 8h, and 200µmol·m -2 ·s -1 PPFD (photosynthetic photon flux density) and watered daily with sterile water.

[0060] Around the 17th day after sowing (three-leaf stage), the tomato seedlings with uniform growth were transplanted into 200 g of yellow-brown soil (with an organic carbon content of 8.1 g kg -1 , the total nitrogen content is 0.79g·kg -1 , pH 6.5) in pots and the culture conditions were the same as those for seedling cultivation. Experimental settings: blank control (no microorganisms were inoculated), R02 treatment (R02+Rs1115), Rs1115 (only Ralstonia solanacearum Rs1115 was inoculated). Seven days after the tomato seedlings were transplanted, the R02 spore suspension was inoculated with 10 7 spores / g soil were inoculated into the roots of tomato seedlings. Seven days after inoculation with R02 spore suspension, the bacterial suspension of Rs1115 was inoculated with 10 6 cfu / g soil was applied to the roots of tomato seedlings. Each treatment included three biological replicates, each containing six tomato plants (a total of 18 plants per treatment). Tomato plants were grown under greenhouse conditions (as described above) and watered regularly with sterile water. The order of plants in the same treatment was randomly changed every 2 days during the treatment period. This greenhouse experiment ended 50 days after inoculation with R. solanacearum Rs1115 (flags indicate diseased plants). Disease index (DI) and biocontrol efficiency (BE) were used to quantify the severity of bacterial wilt. The disease level ranged from 0 to 4, with 0 indicating healthy plants; 1 indicating ≤25% of the leaves with wilting; 2 indicating 26%-50% of the leaves with wilting; 3 indicating 51%-75% of the leaves with wilting; and 4 indicating 76%-100% of the leaves with wilting. The formulas for disease index (DI) and biocontrol efficiency (BE) are as follows.

[0061] The disease index calculation formula is: .

[0062] In the formula, Nd represents the number of diseased plants with a given disease level, Cd represents the corresponding disease level, Ts represents the total number of surveyed plants, and Ad represents the actual highest disease level.

[0063] The calculation formula of biological control efficiency is: .

[0064] Where, DI CK Meaning: Disease index of Rs1115 treatment, DI T express: S. asenjonii Disease index of R02 treatment.

[0065] The pot test results obtained by the above method are shown in Table 3 and Figure 7 shown.

[0066]

[0067] As shown in Table 3, the disease index of Rs1115 treatment was 66.67, the incidence rate was 72.22%, the disease index of R02 (R02+Rs1115) treatment was 0, the incidence rate was 0, and the biological control rate was 100%. S. asenjonii R02 has excellent control effect on tomato bacterial wilt.

[0068] Depend on Figure 7 As can be seen in the figure, A is a control experiment in which only the bacterial wilt pathogen Rs1115 is inoculated; B is an experimental group inoculated with the bacterial wilt pathogen Rs1115 and the strain R02; C is a blank control group inoculated with no strain; Figure 7 In Figure A, the tomato seedlings show signs of yellowing and death, while the growth of the tomato seedlings in Figure B is significantly better than that in Figure A. There is little difference in growth compared with the blank control in Figure C, indicating that strain R02 has a good preventive and therapeutic effect on tomato bacterial wilt. Example 7

[0069] This example studies the control effect of strain R02 on ginger bacterial wilt.

[0070] (1) S. asenjonii Preparation of R02 spores: Spread R02 on ISP3 solid plates and culture at 28°C for 5-7 days. Scrape off R02 spores with a sterile spatula, mix with sterile water, count with a hemocytometer, and adjust the spore suspension concentration to 10 8 pcs / mL for future use.

[0071] (2) Potted experiment of R02 fungus agent to control ginger bacterial wilt: Ginger variety: Shandong ginger. Soil is Anhui Linquan sand ginger black soil, with organic matter content of 16.8 g / kg and pH value of 6.5. Experimental settings: CK (only inoculated with ginger bacterial wilt), R02 treatment (R02+ginger bacterial wilt), pesticide treatment (pesticide+ginger bacterial wilt). Pesticide treatment used 77% copper calcium sulfate wettable powder, with a dosage of 7500 g / hm 2 , 0.05g / Kg soil, mix with soil.

[0072] Select a medium pot (300mm×300mm×350mm), fill each pot with 1.5kg of sieved sand ginger black soil, select the sprouted ginger seedlings, and transplant them into the pot, with 2 ginger seedlings per pot. Ten days after transplanting and planting, the ginger seedlings treated with R02 were inoculated with 100mL of 10 7 / ml R02 spores, 2 weeks later, 50mL was used per pot for each treatment, 10 7 cfu / mL ginger solanacearum solution was used for root irrigation, and all treatments were repeated 8 times. The leaf disease was investigated 20 days after the inoculation of solanacearum. The investigation method was carried out in accordance with the "Guidelines for Field Efficacy Tests of Pesticides. Part 31: Fungicides for Control of Ginger Blight". The results of the pot test are shown in Table 4.

[0073]

[0074] As shown in Table 4, the disease index of the control was 58.50, the incidence rate was 100%, the disease index of the pesticide treatment was 41.23, the incidence rate was 65.32%, the disease index of the R02 treatment was 0, the incidence rate was 0, and the biological control rate was 100%.

[0075] (3) Field trial of R02 fungus agent to control ginger bacterial wilt: A ginger field trial was conducted in Linquan County Agricultural Science and Technology Park, Anhui Province. The soil was sand ginger black soil with an organic matter content of 16.8 g / kg and a pH value of 6.5. A total of 2 mu of land was cultivated in the open air. Each mu was divided into 4 areas, each area was 20 meters long, the ground was loosened on April 8, 2024, the furrows were dug on April 9, and the planting was carried out on April 10. The area of ​​each area was 90 m 2 , plant spacing 25cm×65cm, 3800 plants / mu, organic fertilizer was uniformly applied as base fertilizer, 1 ton / mu, control (only organic fertilizer) and treatment (organic fertilizer + R02 agent) were set up, R02 was fermented with millet solid for 7 days, and the spore content of R02 was 5.0×10 9 Before planting ginger, R02 was mixed with fine soil and then applied in the furrow. 60g of R02 was applied to each treatment area. Four replicates were set for each treatment and conventional management was performed. The harvest was conducted on November 5, 2024, and the yield was counted. The control group had a yield of 2999kg / mu, and the R02 treatment group had a yield of 3230kg / mu, which was 7.7% higher than the control group. Figure 8As shown in the figure, it can be seen that after inoculation with strain R02, the growth of ginger plants was significantly better than that of the uninoculated control group, indicating that strain R02 has a good growth-promoting effect on ginger.

[0076] In summary, the present invention applicants screened Streptomyces asenjonii The R02 strain has good antibacterial, disease-resistant and growth-promoting effects. It is a rare microbial agent and can be used in the preparation of bacterial fertilizer in the later stage.

[0077] The above-described embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. Streptomyces asenjonii R02 strain, its deposit number is CGMCC NO.32267.

2. Containing the method described in claim 1 Streptomyces asenjonii Inoculum of strain R02.

3. As claimed in claim 1 Streptomyces asenjonii Application of the R02 strain or the bacterial agent as claimed in claim 2 in the preparation of biological organic fertilizer.

4. As claimed in claim 1 Streptomyces asenjonii Use of the R02 strain or the bacterial agent as claimed in claim 2 in promoting ginger growth, producing siderophores and / or degrading cellulose.

5. As claimed in claim 1 Streptomyces asenjonii The use of the R02 strain or the bacterial agent as claimed in claim 2 in the preparation of a biocontrol agent, wherein the microorganism inhibited by the biocontrol agent is Ralstonia solanacearum ( Ralstonia solanacearum ), Escherichia coli ( Escherichia coli ), Citrobacter freundii ( Citrobacter frazier ), Klebsiella oxytoca ( Klebsiella oxytoca ), Fusarium oxysporum watermelon transformation type ( Fusarium oxysporum f.sp. niveum), Fusarium oxysporum cucumber transformation type ( Fusarium oxysporum f. sp. cucumerinum) and / or Fusarium graminearum ( Fusarium graminearum ).

6. As claimed in claim 1 Streptomyces asenjonii The use of the R02 strain or the bacterial agent as claimed in claim 2 in resisting plant bacterial wilt, wherein the pathogen of the bacterial wilt is Ralstonia solanacearum ( Ralstonia solanacearum ).

7. Application as claimed in claim 1 Streptomyces asenjonii The method for planting tomatoes using the R02 strain or the bacterial agent as claimed in claim 2 is characterized in that: The method comprises: applying a Streptomyces asenjonii R02 strain and / or bacterial agent, and then plant and cultivate.

8. Application as claimed in claim 1 Streptomyces asenjonii The method for planting ginger using the R02 strain or the bacterial agent as claimed in claim 2 is characterized in that: The method comprises: applying a Streptomyces asenjonii R02 strain and / or bacterial agent, and then plant and cultivate.

Images