A strain of Streptomyces acidophilus C162 and its application in biological control of tomato bacterial wilt

Abstract

The application discloses a strain of Streptacidiphilus C162 for promoting growth and resisting diseases and application of the strain in biological prevention and control of tomato bacterial wilt, and belongs to the technical field of agricultural microorganisms. The strain C162 is identified as a potential new species of Streptacidiphilus, named Streptacidiphilus sp. C162, and the strain has been preserved in the Guangdong Microbial Culture Collection Center (GDMCC) with a preservation number of GDMCC No: 64914. The Streptacidiphilus disclosed in the application can produce an iron carrier, mineralize insoluble organic phosphorus and significantly reduce the incidence of tomato bacterial wilt. Therefore, the Streptacidiphilus C162 disclosed in the application has important research value and application potential in promoting the absorption of iron and phosphorus by crops and resisting tomato bacterial wilt.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agricultural microorganism technology, in particular to a strain of Streptacidiphilus sp. C162 with growth promotion and disease resistance and its application in biological prevention and control of tomato bacterial wilt. BACKGROUND

[0002] As a major tomato producing area in China, Guangdong Province has high temperature and humidity all year round, and tomato bacterial wilt occurs frequently, with a high incidence of up to 80%, seriously endangering the development of the tomato industry. Tomato bacterial wilt is a devastating soil-borne bacterial disease caused by Ralstonia solanacearum, and is known as "tomato cancer". Due to the complexity of R. solanacearum typing, it can also survive in the VBNC (viable but non-culturable) state for a long time in adverse environments, making it difficult to prevent and control tomato bacterial wilt in agricultural production. At present, chemical pesticides play an important role in the prevention and control of tomato bacterial wilt, but they pose a great risk to food safety and environmental protection, and can also lead to pathogen resistance. Therefore, developing green prevention and control technology for tomato bacterial wilt has become a consensus, and also meets the strategic needs of China's agricultural green development concept.

[0003] Soil is home to a rich variety of microbial resources, and some microbial groups are closely related to tomato bacterial wilt resistance. At present, there is a wealth of research on disease-resistant bacteria. Bacteria of the genera Bacillus and Pseudomonas are often reported as antagonistic bacteria. The mechanisms of antagonistic bacteria in inhibiting pathogenic bacteria mainly include the synthesis and secretion of various secondary metabolites, lytic enzymes, volatile organic compounds, carbon sources or niche competition, etc. In addition to antagonistic bacteria, some bacteria can improve the disease resistance of tomatoes through other ways, such as inducing systemic resistance in plants and improving the nutritional status of plants. Although there have been a large number of reports on disease-resistant microorganisms, there is still a lack of microorganisms with growth promotion and disease resistance functions. Streptomycetaceae is a microbial group often reported to have disease resistance functions, and they are most widely concerned about their ability to produce metabolites with various antibacterial functions. Streptacidiphilus is a relatively rare group, and only a few species in this genus have been named. Therefore, resource mining and functional exploration of microorganisms in this genus have strong innovation and application value. SUMMARY

[0004] The purpose of the present application is to provide a new species of Streptacidiphilus sp. C162 with growth promotion and disease resistance, and to provide new microbial resources for promoting the absorption of iron and phosphorus by crops and resisting tomato bacterial wilt.

[0005] To achieve the above objectives, the present invention provides a new species of Streptacidiphilus C162 isolated from tomato rhizosphere soil and named Streptacidiphilus sp. C162. The strain is deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, Postal Code: 510070, the deposit number is GDMCC No: 64914, and the deposit date is July 26, 2024.

[0006] Identification characteristics of the acidophilic Streptomyces:

[0007] Strain C162 is a Gram-positive bacterium. Its single colonies on TSA medium are round, bright yellow, smooth, surrounded by a transparent ring with neat edges. The 16S rRNA gene sequence of Streptacidiphilus acidophilus C162 is 1519 bp long. Sequence alignment analysis showed that this strain is similar to Streptacidiphilus monticola NEAU-SW11. T The strain C162 showed the highest similarity, at 98.61%. Based on comparative genomic analysis, the average nucleotide identity between strain C162 and the model species of the genus Streptomyces was 77.96-78.88%, and the digital DNA-DNA hybridization values ​​were 22.40-23.20%. These results indicate that strain C162 represents a new species of the genus Streptomyces.

[0008] The present invention found that the strain C162 has the ability to produce siderophores. Through quantitative analysis of bacterial siderophore production, it was found that the relative content of siderophores produced by the strain C162 was 33.31%.

[0009] The present invention found that strain C162 has the ability to mineralize insoluble organic phosphorus.

[0010] The present invention finds that inoculating the tomato root system with the strain C162 can significantly reduce the incidence of tomato bacterial wilt.

[0011] The present invention also provides a microbial fertilizer and / or microbial preparation, characterized in that it contains the acidophilic Streptomyces C162, or its fermentation liquid, or the supernatant of its fermentation liquid as an active ingredient.

[0012] The present invention also provides the use of the acidophilic Streptomyces C162 or the microbial fertilizer and / or microbial preparation in producing siderophores.

[0013] The present invention also provides the use of acidophilic Streptomyces C162 or the microbial fertilizer and / or microbial preparation in dissolving organic phosphorus.

[0014] Preferably, the organophosphorus is a poorly soluble organophosphorus.

[0015] The present invention also provides the use of acidophilic Streptomyces C162 or the microbial fertilizer and / or microbial preparation in resisting tomato bacterial wilt.

[0016] The present invention has the following advantages over the prior art:

[0017] 1) Streptomyces acidophilus C162 is a new species different from other Streptomyces acidophilus bacteria.

[0018] 2) Streptomyces acidophilus C162 can not only produce siderophores but also mineralize insoluble organophosphates.

[0019] 3) Acidophilic Streptomyces C162 can significantly reduce the incidence of tomato bacterial wilt, with an anti-disease efficiency of up to 66.66%.

[0020] Therefore, the acidophilic Streptomyces C162 disclosed in the present invention can promote the absorption of iron and phosphorus by crops and resist tomato bacterial wilt, and has broad application prospects in the preparation of crop microbial fertilizers and microbial preparations.

[0021] Streptacidiphilus sp.C162 is deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, Postal Code: 510070, the deposit number is GDMCC No: 64914, and the deposit date is July 26, 2024. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the colony morphology of Streptomyces acidophilus C162 on TSA medium.

[0023] Figure 2 This is the phylogenetic tree of the 16S rRNA gene of Streptomyces acidophilus C162.

[0024] Figure 3 It is the ability of acidophilic Streptomyces C162 to mineralize insoluble organic phosphorus. DETAILED DESCRIPTION

[0025] The following are specific implementation examples of the present invention. It should be noted that these embodiments are merely examples and do not constitute any limitation on the scope of the present invention. Modifications and replacements of the details and forms of the embodiments within the scope of the present invention fall within the scope of protection of the present invention.

[0026] Unless otherwise defined, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The experimental reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents.

[0027] Example 1: Isolation of strain C162

[0028] The soil used to isolate strain C162 was collected from a tomato field in Jintang Town, Maonan District, Maoming City, Guangdong Province. Soil bacteria were isolated using high-throughput bacterial isolation, culture, and identification techniques. The specific procedure was as follows: 1 g of fresh soil was diluted 5000-fold and then 2000-fold with sterile magnesium chloride solution and 10% TSB liquid medium, respectively. 160 μL of the dilution was added to 48 96-well cell culture plates and incubated at 30°C in the dark for 2 weeks. 10 μL of the bacterial solution was evenly aspirated from each well of the culture plate and bacterial DNA was extracted using alkaline lysis. 140 μL of 80% (v / v) glycerol was added to the remaining bacterial solution and stored as a bacterial glycerol stock for subsequent bacterial activation. PCR amplification was performed using primers 799F / 1193R (V5-V7 region of the 16S rRNA gene) and Novozymes Green Taq Mix. The product was diluted 120 times and used as a template for PCR amplification using primers 799F and 1193R with sequencing adapters (Illumina adapters) and barcodes. The second-round PCR product was electrophoresed, and the approximately 550bp band was excised for purification and its product concentration was determined. Each 96-well plate was prepared according to 100ng, and the mixed library of 48 plates totaled 4800ng. High-throughput sequencing was performed at Beijing Novogene Technology Co., Ltd. The sequencing data was analyzed using the analysis process Culturome (https: / / github.com / YongxinLiu / Culturome) to determine the position of the cultured bacteria in the 96-well plate. 25μL of bacterial solution was aspirated from the wells of the corresponding 96-well cell culture plate and the bacteria were activated on 1 / 2TSA medium. The bacteria were purified by multiple streaking method, and the purified bacterial strain was mixed with 25% glycerol (v / v) and stored in a -20℃ refrigerator.

[0029] The strain C162 was identified as a Gram-positive bacterium. The single colony on TSA medium was round, bright yellow, smooth, with a transparent circle around it and neat edges ( Figure 1 ).

[0030] Example 2: 16S rRNA gene sequence analysis of strain C162

[0031] Genomic DNA from strain C162 was extracted using the HiPure Bacterial DNA Kit from Guangzhou Meiji Biotechnology Co., Ltd., and the full-length 16S rRNA gene was amplified using the universal bacterial primers 27F / 1492R. The PCR product was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing. The sequence is shown in SEQ ID NO. 1, with a length of 1519 bp. Sequences were aligned on the EzBioCloud website (https: / / www.ezbiocloud.net / identify). Figure 2 The results showed that strain C162 had a similar strain to the model strain Streptacidiphilus monticola NEAU-SW11 T The strain C162 had the highest similarity, at 98.61%, while the 16S rRNA gene sequence similarity with other model species of the genus Streptomyces was lower than 98.60%. Based on the 16S rRNA gene sequence similarity results, strain C162 belongs to the genus Streptomyces, but its taxonomic status cannot be accurately determined.

[0032] Example 3: Full gene sequence analysis of strain C162

[0033] Strain C162 was sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. for genome sequencing using the Illumina Novaseq platform. SPAdes v3.15.3 software was used for genome assembly, and low-quality contig sequences less than 500 bp in length were removed. CheckM v1.1.3 software was used to analyze the integrity and contamination of the bacterial genome. The results showed that the total genome length of strain C162 was 7.06 Mbp, the N50 length was 103,005 bp, the G+C content of the genomic DNA was 72.46%, the genome completeness was 99.47%, and the contamination level was 0%.

[0034] Genomes of model species of the genus Streptacidiphilus were downloaded from NCBI (https: / / www.ncbi.nlm.nih.gov / datasets / genome / ?taxon=228398). Because Streptacidiphilus bacteria are rarely reported, a total of 10 reference genomes were downloaded. To further clarify the differences between these bacteria, the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values ​​between strain C162 and the genome sequences of the model species were calculated using the ANI Calculator (http: / / www.ezbiocloud.net / tools / ani) and the Genome-to-Genome Distance Calculator v3.0 (https: / / ggdc.dsmz.de / ggdc.php#), respectively. The results are shown in Table 1. The ANI values ​​between strain C162 and the model species ranged from 77.96% to 78.88%, and the dDDH values ​​ranged from 22.40% to 23.20%. When the ANI value of two bacterial genomes is 95-96%, it is equivalent to a DNA-DNA hybridization value of 70%, which is equivalent to a 16S rRNA gene similarity of 98.65%. Therefore, when the ANI value of two bacterial genomes is greater than 96%, they are the same species; when the ANI value of two bacterial genomes is less than 95%, they are different species. In addition, a whole-genome DNA-DNA hybridization value of 70% is the gold standard for prokaryotic species classification. Therefore, strain C162 is a potential new species of the genus Streptacidiphilus and is named Streptacidiphilus sp. C162. The strain is deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, Postal Code: 510070, with a deposit number of GDMCC No: 64914 and a deposit date of July 26, 2024.

[0035] Table 1 ANI and dDDH values ​​between acidophilic Streptomyces C162 and type species of the genus Acidophilus

[0036]

[0037]

[0038] Example 4: Identification of the ability of strain C162 to produce siderophores

[0039] Activate Streptomyces acidophilus C162 on TSA medium, pick a single colony and inoculate it into 50mL TSB liquid medium, culture it at 30℃ and 200rpm for 2 days, and set up 3 biological replicates. Pipette 1mL of bacterial liquid and collect the supernatant by centrifugation at 12000rpm for 5min. The iron carrier production was determined by CAS method. Take 100μL of supernatant and mix it with an equal volume of CAS detection solution. After standing for 1h in the dark, use a microplate reader to measure its absorbance at a wavelength of 630nm, and the result is expressed as As. Sterile TSB liquid medium was used as a control, and the absorbance at a wavelength of 630nm was Ar. The relative content of iron carriers (%) = (Ar-As) / Ar×100%.

[0040] The results showed that the relative content of siderophore in Streptomyces acidophilus C162 was 33.31%

[0041] Example 5: Identification of the Phosphate-Solubilizing Ability of Strain C162

[0042] Insoluble organophosphorus culture medium: glucose 10 g·L -1 , ammonium sulfate 0.5 g·L -1 , yeast extract powder 0.5g·L -1 , sodium chloride 0.3g·L -1 , potassium chloride 0.3g·L -1 , magnesium sulfate 0.3g·L -1 , ferrous sulfate 0.03g·L -1 , manganese sulfate 0.03g·L -1 , lecithin 0.2g·L -1 , calcium carbonate 1.0g·L -1 , agar 15 g·L -1 The culture medium was purchased from Qingdao Hi-Tech Industrial Park Haibo Biotechnology Co., Ltd., product number: HB8673-1.

[0043] A single colony of activated acidophilic Streptomyces C162 was inoculated into TSB liquid culture medium and cultured at 30°C and 200 rpm for 2 days. 10 μL of the bacterial liquid was added to the insoluble organophosphorus culture medium. The plate was then inverted and cultured at 30°C for 5 days. The phosphate solubility zone on the plate was observed to determine whether the strain C162 had the ability to solubilize phosphate.

[0044] according to Figure 3 The results showed that acidophilic Streptomyces C162 had a strong ability to mineralize insoluble organic phosphorus, with a ratio of the diameter of the phosphate-dissolving zone (D) to the colony diameter (d) (D / d) of 2.28. This ability ranked in the top 5% among the 52 potential new species isolated.

[0045] Example 6: Evaluation of the ability of strain C162 to resist tomato bacterial wilt

[0046] The soil from which Streptomyces acidophilus C162 was isolated was used as the test soil. The soil was air-dried, passed through a 2mm sieve, and then sterilized by high-temperature and high-pressure steam sterilization (121°C for 1 hour). Micro-Tom tomato seeds were germinated and directly sown in pots. After 3 weeks of growth, tomato seedlings with uniform growth were selected and inoculated with 5 mL of a suspension of Streptomyces acidophilus C162 in sterile water in the treatment group, while the positive and negative controls were inoculated with 5 mL of sterile water. Ten days later, the treatment and positive controls were inoculated with 5 mL of a suspension of Ralstonia solanacearum QL-Rs1115, while the negative control was inoculated with 5 mL of sterile water. Seven biological replicates were set for each treatment, and the bacterial wilt grade of the tomato plants was calculated after 3 weeks. The bacterial wilt grade of tomato plants is coded from 0 to 5, representing no symptoms (grade 0), 20% (grade 1), 40% (grade 2), 60% (grade 3), 80% (grade 4), and 100% (grade 5) of leaves showing symptoms, respectively.

[0047] According to the results in Table 2, no bacterial wilt symptoms were observed in the negative control group. Inoculation with acidophilic Streptomyces C162 significantly reduced the severity of bacterial wilt in tomatoes, achieving an anti-disease efficacy of 66.66%. Therefore, acidophilic Streptomyces C162 is a biocontrol bacterium with strong potential for application.

[0048] Table 2 Effects of acidophilic Streptomyces C162 on tomato bacterial wilt

[0049]

[0050] Note: The negative control group and the treatment group were compared with the positive control group by independent sample t-test (n=7).

[0051] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. Persons skilled in the art will appreciate that improvements and modifications may be made without departing from the spirit and scope of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention.

[0052] Full-length sequence of the 16S rRNA gene of Streptomyces acidophilus C162 (SEQ ID NO.1)

[0053] TTCACGGAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCTTAACACATGC

[0054] AAGTCGAACGGTGAAGCCCTTCGGGGTGGATCAGTGGCGAACGGGTGAGTAACACGT

[0055] GGGCAATCTGCCCTGCACTCTGGGACAAGCCCTGGAAACGGGGTCTAATACCGGATAC

[0056] GACCACGGAACGCATGTTCTGTGGTGGAAAGCTCCGGCGGTGCAGGATGAGCCCGCGG

[0057] CCTATCAGCTTGTTGGTGGGGTGATGGCCTACCAAGGCGACGACGGGTAGCCGGCCTG

[0058] AGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAG

[0059] CAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGA

[0060] TGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGAAGAAGCGCAAGTGACGGTACC

[0061] TGCAGAAGAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCG

[0062] AGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCGCGTCGGATG

[0063] TGAAAGCCCGGGGCTTAACCCCGGGTCTGCATTCGATACGGGCAGGCTGGAGTGTGGT

[0064] AGGGGAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACAC

[0065] CGGTGGCGAAGGCGGATCTCTGGGCCATTACTGACGCTGAGGAGCGAAAGCGTGGGG

[0066] AGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGTTGGGCACTAGGTGTG

[0067] GGTCGCATTCCACGCGGTCCGCGCCGTAGCTAACGCATTAAGTGCCCCGCCTGGGGAG

[0068] TACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCAGCGGAG

[0069] CATGTGGCTTAATTCGACGCAACGCGAAGAACCTTACCAAGGCTTGACATACACCGGA

[0070] AAACTCTGGAGACAGGGTCCCCCTTGTGGTCGGTGTACAGGTGGTGCATGGTTGTCGTC

[0071] AGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGTTCTGTGT

[0072] TGCCAGCGGGTTATGCCGGGGACTCACAGGAGACTGCCGGGGTCAACTCGGAGGAAG

[0073] GTGGGGATGACGTCAAATCATCATGCCCCTTATGTCTTGGGCTGCACACGTGCTACAAT

[0074] GGCCGGTACAATGAGCTGCGATACCGTGAGGTGGAGCGAATCTCAAAAAGCCGGTCTC

[0075] AGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTTGCTAGTAATCGCAG

[0076] ATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCAC

[0077] GAAAGTCGGTAACACCCGAAGCCGGTGGCCTAACCCCTTGTGGGAGGGAGCTGTCGAA

[0078] GGTGGGACCAGCGATTGGGACGAAGTCGTAACAAGGTAGCCGTACCGGAAGGTGCGG

[0079] CTGGATCACCTCCTTT。

Claims

1. Streptomyces acidophilus ( Streptacidiphilus sp.) C162, deposited with GDMCC No: 64914.

2. A microbial preparation, characterized in that: Contains the acidophilic Streptomyces C162 or the fermentation broth of acidophilic Streptomyces C162 according to claim 1 as an active ingredient.

3. Use of the acidophilic Streptomyces C162 according to claim 1 or the microbial preparation according to claim 2 in producing siderophores.

4. Use of the acidophilic Streptomyces C162 according to claim 1 or the microbial preparation according to claim 2 in dissolving organophosphorus.

5. The use according to claim 4, characterized in that The organic phosphorus is poorly soluble organic phosphorus.

6. Use of the acidophilic Streptomyces C162 or the fermentation broth of acidophilic Streptomyces C162 according to claim 1 or the microbial preparation according to claim 2 in combating tomato bacterial wilt.

Images