Streptomyces amylase and application thereof

Abstract

The invention discloses streptomyces diastatusYPL-2 and application thereof, the classification name is Streptomyces diastatusYPL-2, the preservation number is CGMCC NO.: 28657, the preservation date is October 24, 2023, the preservation unit is China General Microbiological Culture Collection Center, the application of the streptomyces diastatususYPL-2 in preventing and treating tobacco bacterial wilt has the advantages that the streptomyces diastatususYPL-2 can be used for preventing and treating tobacco bacterial wilt, and the streptomyces diastatususYPL-2 can be used for preventing and treating tobacco bacterial wilt; the strain streptomyces diastatosus YPL-2 is separated from rhizosphere soil of healthy tobacco plants in a tobacco bacterial wilt disease field and can be well colonized at the rhizosphere of the tobacco, and fermentation metabolites of the strain can inhibit growth of ralstonia solanacearum and have a remarkable prevention and treatment effect on the tobacco bacterial wilt caused by the ralstonia solanacearum. The bacillus subtilis has high efficiency in preventing and treating tobacco bacterial wilt, has the advantages of high colonization capability, difficulty in generating drug resistance, environment friendliness and the like, and has a relatively good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and specifically to a Streptomyces diastaticus and its application. Background Art

[0002] Plant bacterial wilt is a soil-borne bacterial disease caused by Ralstonia solanacearum. As one of the top ten plant pathogenic bacteria, Ralstonia solanacearum can infect plants of more than 200 genera in more than 50 families, seriously threatening the production and development of solanaceous crops. As a major tobacco-producing country, there have been reports of the occurrence of tobacco bacterial wilt in 30 provinces in China. Among the 22 main tobacco-growing areas, 14 areas are widely affected, and there is a trend of spreading from plains to plateaus. At present, the prevention and control of tobacco bacterial wilt mainly rely on chemical control, supplemented by agricultural control and biological control. However, chemical control will cause problems such as pesticide residues, and the long-term use of chemical agents will make the pathogenic bacteria develop drug resistance, resulting in a significant reduction in the control effect. Agricultural control methods such as mixing soil with quicklime will lead to problems such as soil microbial community imbalance and soil compaction. Crop rotation with paddy-upland rotation or rotation between tobacco and gramineous crops can not only effectively reduce the occurrence of tobacco bacterial wilt, but also avoid problems such as soil compaction, pesticide residues and drug resistance. It is a good agricultural control measure. However, the rotation time cycle is relatively long, and in some regions, due to geographical environment and cultivated land area, it is not conducive to large-scale promotion. Therefore, biological control has attracted wide attention because it can effectively control diseases while having a shorter time cycle than agricultural control and being safe without side effects.

[0003] Biological control usually uses beneficial bacteria to colonize in the rhizosphere or inside the plant, compete for ecological niche with pathogenic bacteria, or inhibit or kill pathogenic bacteria by secreting secondary metabolites, so as to achieve the effect of disease control. In addition, most of the beneficial bacteria used as biocontrol agents can directly or indirectly promote plant growth, or recruit beneficial microbial communities in the soil, enriching soil microbial diversity, thereby controlling diseases. At present, Bacillus and Pseudomonas are widely used in the biological control of tobacco bacterial wilt. Therefore, finding high-quality and efficient microbial strains for controlling tobacco bacterial wilt is an important working basis for enriching biological control. Summary of the Invention

[0004] The purpose of the present invention is to provide a Streptomyces diastaticus and its application to solve the problems raised in the above background art.

[0005] To achieve the above purpose, the present invention provides the following technical solution: A Streptomyces diastaticus, classified and named as Strepomyces diastaticus YPL-2, with a preservation number of CGMCC NO.: 28657, a preservation date of October 24, 2023, and the preservation unit is the General Microbiological Center of the China Committee for Culture Collection of Microorganisms.

[0006] Preferably, the accession number of the 16S rDNA sequence of the Streptomyces diastaticus in GenBank is OR649151

[0007] Application of Streptomyces diastaticus in preventing and controlling tobacco bacterial wilt

[0008] Preferably, the Streptomyces diastaticus is used for preparing a highly effective biocontrol agent for preventing and controlling tobacco bacterial wilt

[0009] Preferably, the highly effective biocontrol agent is prepared by the following method: inoculating Streptomyces diastaticus YPL-2 with the preservation number of CGMCC NO.: 28657 into a Gao's No. 1 liquid medium for liquid fermentation, and the fermentation conditions are: culturing at 28°C and 180 r / min for 7 d. After the culturing is completed, adjust the OD value with sterile water to make the concentration of the bacterial suspension ≥ 108 CFU / ml to form the highly effective biocontrol agent

[0010] Preferably, the preparation method of the Gao's No. 1 medium is: 20.0 g of soluble starch, 1.0 g of KNO3, 0.5 g of NaCl, 0.5 g of K2HPO4, 0.5 g of MgSO4·7H2O, 0.01 g of FeSO4·7H2O, make up the volume to 1 L with distilled water, adjust the pH to 7.3, sterilize at 121°C for 30 min

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows: The strain Streptomyces diastaticus YPL-2 of the present invention is isolated from the rhizosphere soil of healthy tobacco plants in a tobacco bacterial wilt field, can colonize well in the tobacco rhizosphere, and the fermentation metabolites of this strain can inhibit the growth of Ralstonia solanacearum, and have a significant prevention and control effect on tobacco bacterial wilt caused by Ralstonia solanacearum. The present invention has high efficiency in preventing and controlling tobacco bacterial wilt, and at the same time has the advantages of strong colonization ability, not easy to produce drug resistance, and environmental friendliness, and has a good application prospect Description of the drawings

[0012] Figure 1 It is the colony morphology diagram of Streptomyces diastaticus YPL-2 of the present invention

[0013] Figure 2 It is the phylogenetic tree constructed based on the 16S rDNA sequence of Streptomyces diastaticus YPL-2 of the present invention Detailed implementation manners

[0014] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention

[0015] Please refer to Figure 1 - Figure 2 Figure 1 - Figure 2 , the present invention provides a technical solution: The present invention relates to a Streptomyces diastaticus strain, which is Streptomyces diastaticus YPL-2, deposited in the China General Microbiological Culture Collection Center (CGMCC) on October 24, 2023, with the deposit number CGMCC NO.: 28657.

[0016] The accession number of the 16S rDNA sequence of the Streptomyces diastaticus YPL-2 on GenBank is OR649151.

[0017] The Streptomyces diastaticus YPL-2 is used for preventing and controlling tobacco bacterial wilt.

[0018] The Streptomyces diastaticus YPL-2 is used for preparing a highly effective biocontrol agent for preventing and controlling tobacco bacterial wilt.

[0019] The following further illustrates the present invention in conjunction with specific embodiments.

[0020] The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels without special instructions.

[0021] Example 1: Isolation and purification of the strain

[0022] Collect soil samples from a tobacco planting area in Yongping Town, Jinggu County, Pu'er City, Yunnan Province. Take 10 g of rhizosphere soil and put it into a 250 mL conical flask, add 90 mL of sterile water, shake at 180 r / min and 28 °C for 30 min to prepare a 10-1 dilution. Pipette 100 μL of the 10-1 dilution into 900 μL of sterile water to obtain a 10-2 dilution. Sequentially perform serial gradient dilutions of 10-3, 10-4, and 10-5 in this way. Take 100 μL of the obtained 10-3, 10-4, and 10-5 dilutions and spread them on the medium. The obtained strains are re-streaked and purified on Gause's No. 1 medium and cultured at 30 °C for 7 d. Name and preserve the cultured strains and conduct screening for antagonistic bacterial strains.

[0023] Example 2: Screening of antagonistic strains

[0024] (1) Culture medium formula: NA medium: 10.0 g of glucose, 5.0 g of peptone, 3.0 g of beef extract, 1 g of yeast extract, 18.0 g of agar powder, made up to 1 L with distilled water, pH adjusted to 7.0, sterilized at 121 °C for 30 min.

[0025] CPG medium: 1.0 g of casein hydrolysate, 10.0 g of peptone, 5.0 g of glucose, 18.0 g of agar powder, made up to 1 L with distilled water, pH adjusted to 7.0, sterilized at 121 °C for 30 min.

[0026] Gause's No. 1 medium: 20.0 g of soluble starch, 1.0 g of KNO3, 0.5 g of NaCl, 0.5 g of K2HPO4, 0.5 g of MgSO4·7H2O, 0.01 g of FeSO4·7H2O, made up to 1 L with distilled water, pH adjusted to 7.3, sterilized at 121 °C for 30 min.

[0027] (2) Strain activation: The Ralstonia solanacearum stored at room temperature was activated on the CPG solid medium; the strain isolated in Implementation Case 1 was activated on Gause's No. 1 medium.

[0028] (3) Plate confrontation test: The strains obtained in Implementation Case 1 were inoculated into Gause's No. 1 liquid medium respectively, cultured with shaking at 28 °C and 180 r / min for 7 d; single colonies of Ralstonia solanacearum were picked and cultured with shaking at 28 °C and 180 r / min for 2 d in the CPG liquid medium. The fermentation broth of the strains obtained in Implementation Case 1 and the bacterial suspension of Ralstonia solanacearum were diluted with sterile water to a bacterial suspension of 108 CFU / mL. 100 μL of the Ralstonia solanacearum bacterial suspension was evenly spread on the NA solid medium, and 200 μL of the fermentation broth of the strains obtained in Implementation Case 1 that had been cultured well was inoculated into the center of the medium through an Oxford cup. Each strain was in parallel for 3 times, and the NA medium coated only with Ralstonia solanacearum was used as a control. It was cultured in a constant temperature incubator at 28 °C for 2 d, and the size of the inhibition zone was observed and recorded. The results showed that the strain YPL-2 had the largest inhibition zone diameter, and the inhibition zone diameter was 38 mm.

[0029] Implementation Case 3: Identification of strain YPL-2

[0030] (1) Morphological identification: The strain YPL-2 was inoculated on Gause's No. 1 medium and cultured at a constant temperature of 28 °C for 7 d, and the colony morphology was observed. The results showed that after the strain YPL-2 grew on Gause's No. 1 medium for 7 d, the colony was pink, with white aerial hyphae, the pigment penetrated into the medium, the hyphae in the medium were pink, the colony was round or oval, and the colony was dry and not smooth (as Figure 1 shown).

[0031] (2) Determination of the strain 16S rDNA

[0032] a. Inoculate strain YPL-2 into Gause's No. 1 medium and culture it at 28 °C for 7 days.

[0033] b. Inoculate a single colony of strain YPL-2 obtained in step a into Gause's No. 1 liquid medium, and culture it at 28 °C with shaking at 180 r / min for 5 days to obtain a seed solution.

[0034] c. Genomic DNA extraction: Extract the DNA of strain YPL-2 according to the instructions of the genomic extraction kit of Aikerui Biotechnology Co., Ltd.

[0035] d. pCR verification: Perform PCR amplification on the DNA of YPL-2 obtained in step c using the 16S universal primers 27F / 1492R; Primer sequences: 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′; 1492R: 5′-TACGGCTACCTTGTTACGACTT-3′. After verifying the amplified PCR product by gel electrophoresis, send the PCR product to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0036] 16S rDNA sequence analysis: Perform BLAST alignment of the obtained sequence in the National Center for Biotechnology Information database, and it is found that the homology between strain YPL-2 and strain SG4 (Streptomyces diastaticus) reaches 99.18%. A phylogenetic tree was constructed based on the 16S rDNA gene sequence (as Figure 2 shown). The results show that strain YPL-2 is Streptomyces diastaticus, that is, Streptomyces diastaticus. This strain was deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee on October 24, 2023, with the deposit number CGMCC NO.: 28657.

[0037] 16S rDNA sequence of YPL-2 (Streptomyces diastaticus) (1081 bp)

[0038] GGGATGCTTTACACATGCAGTCGAACGATGAACCACCTTCGGGTGGGGATTAGTGGCGAACG

[0039] GGTGAGTA

[0040] ACACGTGGGCAATCTGCCCTGCACTCTGGGACAAGCCCTGGAAACGGGGTCTAATACCGGAT

[0041] ACTGACCT

[0042] GCCAAGGCATCTTGGCGGGTCGAAAGCTCCGGCGGTGCAGGATGAGCCCGCGGCCTATCAGC

[0043] TTGTTGGT

[0044] GAGGTAATGGCTCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCGCGTCGGTTGTGAAAGCCCGGGGCTTAACCCCGGGTCTGCAGTCGATACGGGCAGGCTAGAGTTCGGTAGGGGAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCCGATACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAACGGTGGGCACTAGGTGTGGGCAACATTCCACGTTGTCCGTGCCGCAGCTAA

[0045] CGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGCT

[0046] AAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGCTTAATTCGACGC

[0047] AACGCGAA

[0048] GAACCTTACCAAGGCTTGACATACACCGGAATCATCAGAGATGGTGCCCCCCTTGTGGTCGG

[0049] TGTACAGG

[0050] TGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAC

[0051] CCTTGTCC

[0052] CGTGTGCAGCACTCTTCGGAGGTTGGGGACTCCACGGGAGACCGCCGGGTCACTCGGAGAAGTG

[0053] Case 4 of implementation example: Greenhouse control effect of Streptomyces diastaticus YPL-2 on tobacco bacterial wilt

[0054] (1) Tested tobacco variety: Yunyan 87

[0055] (2) Preparation of bacterial suspension: After activating the strain YPL-2 on Gao's No. 1 medium, pick a single colony and inoculate it into Gao's No. 1 liquid medium. Incubate it at 28 °C with shaking at 180 r / min for 7 d. Dilute the obtained bacterial liquid with sterile water to a bacterial suspension of 108 CFU / mL for standby.

[0056] Activate Ralstonia solanacearum on CPG medium, pick a single colony and inoculate it into CPG liquid medium. Incubate it at 28 °C with shaking at 180 r / min for 2 d. Dilute the obtained bacterial liquid with sterile water to a bacterial suspension of 108 CFU / mL.

[0057] Test method: Three treatments were set up in the test, namely the YPL-2 fermentation broth treatment group, the medicament treatment group and the control group. Tobacco seedlings at the 5-6 leaf stage were selected for transplanting, and test treatments were carried out 5 days after transplanting. Strain YPL-2 treatment group: The roots were irrigated with YPL-2 fermentation broth, 100 ml per tobacco plant. After 24 hours, the roots were wounded and inoculated with the fermentation broth of Ralstonia solanacearum, 20 ml per tobacco plant, and 20 plants were used for each treatment. Medicament treatment group: 30% thiodiazole copper was diluted to 800-fold solution for root irrigation treatment, 100 ml per tobacco plant. After 24 hours, the roots were wounded and inoculated with the fermentation broth of Ralstonia solanacearum, 20 ml per tobacco plant, and 20 plants were used for each treatment. Control group: Clear water was used instead of YPL-2 fermentation broth, that is, root irrigation treatment with clear water, 100 ml per tobacco plant. After 24 hours, the roots were wounded and inoculated with the fermentation broth of Ralstonia solanacearum, 20 ml per tobacco plant, and 20 plants were used for each treatment. Each treatment was set with 3 replicates. The inoculated tobacco seedlings were placed in a constant temperature and light greenhouse, ensuring 12 hours of sunshine per day, the temperature was 30±1°C, and the humidity was above 80%. After inoculation, the disease incidence was observed and recorded, and the disease index and relative control effect were recorded and calculated 14 days after inoculating the pathogen.

[0058] Grading standard for tobacco bacterial wilt:

[0059] Grade 0: The whole plant is disease-free;

[0060] Grade 1: There are occasional chlorotic spots on the stem, or the leaves below 1 / 2 of the diseased side wither;

[0061] Grade 3: There are black stripes on the stem, but not exceeding 1 / 2 of the stem height, or the leaves on the diseased side wither from 1 / 2 to 2 / 3;

[0062] Grade 5: The black stripes on the stem exceed 1 / 2 of the stem height but do not reach the top of the stem, or the leaves on the diseased side wither more than 2 / 3;

[0063] Grade 7: The black stripes on the stem reach the top of the stem, or all the leaves of the diseased plant wither;

[0064] Grade 9: The diseased plant is basically dead.

[0065] Disease incidence = (Number of diseased plants / Total number of plants surveyed) × 100%

[0066] Disease index = 100×∑(Number of diseased plants at each level × Value at each level) / (Total number of plants surveyed × Highest level value)

[0067] Relative control effect = (Control disease index - Treatment disease index) / Control disease index × 100%

[0068] Test results: Table 1: Control effect of strain YPL-2 on tobacco bacterial wilt

[0069]

[0070]

[0071] The results showed that the tobacco plants in the control group started to get sick 7 days after inoculation with Ralstonia solanacearum, and unilateral wilting occurred in the leaves of the tobacco plants. By the 14th day after inoculation, the incidence rate of the tobacco plants in the control group was 100%. The relative control effect of the YPL-2 fermentation broth was 66.67%, which was higher than that of the 30% thiodiazole copper 800-fold liquid treatment. This indicates that Streptomyces diastaticus has a good control effect on tobacco bacterial wilt, and the test results are repeatable and stable, providing strain resources for the biological control of tobacco bacterial wilt.

[0072] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A Streptomyces diastaticus, characterized in that: It is classified as Strepomyces diastaticus YPL-2, with the preservation number of CGMCC NO.: 28657, the preservation date of October 24, 2023, and the preservation unit of the General Microbiology Center of the China Committee for Culture Collection of Microorganisms.

2. An Amycolatopsis amylolytica according to claim 1, characterized in that: The accession number of the 16S rDNA sequence of the said Streptomyces diastaticus in GenBank is OR649151.

3. Application of the Streptomyces diastaticus according to claim 1 in preventing and treating tobacco bacterial wilt.

4. The application according to claim 3, characterized in that: The said Streptomyces diastaticus is used for preparing a highly effective biocontrol agent for preventing and treating tobacco bacterial wilt.

5. The application according to claim 4, wherein: The said highly effective biocontrol agent is prepared by the following method: inoculating Streptomyces diastaticus YPL-2 with the preservation number of CGMCC NO.: 28657 into the Gao's No. 1 liquid medium for liquid fermentation, and the fermentation conditions are: culturing at 28 °C and 180 r / min for 7 d. After the culturing is completed, adjust the OD value with sterile water to make the concentration of the bacterial suspension ≥ 108 CFU / ml, thus forming the said highly effective biocontrol agent.

6. The application according to claim 5, characterized in that: The preparation method of the Gao's No. 1 medium is as follows: 20.0 g of soluble starch, 1.0 g of KNO3, 0.5 g of NaCl, 0.5 g of K2HPO4, 0.5 g of MgSO4·7H2O, 0.01 g of FeSO4·7H2O, make up the volume to 1 L with distilled water, adjust the pH to 7.3, sterilize at 121 °C for 30 min.

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