A new species of Micromonospora and its application

Abstract

The present invention discloses a new species of the genus Micromonospora and its use, belonging to a technical solution for microorganisms. The present invention provides a new species of the genus Micromonospora. The present invention provides a Micromonospora sp. 181, which has been deposited with the China General Microbiological Culture Collection under the accession number CGMCC No. 16626 at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Micromonospora sp. 181 has a significant inhibitory effect on the pathogen causing pecan dry rot.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a new species of Micromonospora and application thereof. Background Art

[0002] Micromonospora ( 小单孢菌属 ) belongs to the domain Bacteria, phylum Actinobacteria, class Actinobacteria_c, order Actinomycetales, suborder Micromonosporineae, family Micromonosporaceae.

[0003] The first strain of Micromonas was discovered in 1905 when Foulerton isolated a strain from the air and named it 铜色链丝菌 In 1923, Ørskov established the genus Micromonospora by analyzing the phenotypic characteristics of this strain and named it 铜色小单孢菌 , currently covering 110 validly published species (https: / / www.bacterio.net / genus / 小单孢菌 ). Micromonospora ( 小单孢菌属 ) taxonomically belongs to the Actinobacteria, Actinobacteria_c, Actinomycetales, Micromonosporineae, Micromonosporaceae. Micromonospora is a typical genus of Micromonospora, and the typical species of Micromonospora is Micromonospora bronze ( 铜色小单孢菌 ).

[0004] Micromonospora has a wide range of living environments and is found in soil and aquatic environments, low-temperature environments, and alkaline environments. Micromonospora has been isolated from different types of soil, plant roots, leaves, nodules, and marine environments (sediments, sea sand, and sponges). Among them, micromonospora from mangroves account for a higher proportion, such as Micromonospora 小单孢菌属 帕塔隆小单孢菌 , 小单孢菌属 利福霉素小单孢菌 , 小单孢菌属 根球小单孢菌 , 小单孢菌属 文昌小单孢菌 , ​ ​ , ​ ​ , ​ , ​ , ​All of them were isolated from mangrove soil, and Micromonospora was isolated from the roots of mangrove plants. ​ .

[0005] Pecans are mainly distributed in Zhejiang and Anhui provinces. They are not only used as shelterbelts and furniture materials, but their nuts can also be eaten directly and used as ingredients for pastries and cooking oils. They have high nutritional value and certain health benefits. However, the outbreak of pecan dry rot has not only significantly reduced pecan production, but also weakened the trees, leading to tree death, posing a serious threat to the sustainable development of the pecan economy and environment. ​ ) is also called ulcer disease, the main pathogen of which is Botrytis cinerea ( ​ ) strains, with ​ ​ Tian Tian et al. found that two fungi (Trichoderma acanthosporum and Penicillium oxalicum) are potential biocontrol bacteria for pecan dry rot, of which Penicillium oxalicum ( ​ ) mainly works through the production of antibiotics through metabolism. Cheng Min et al. found that Bacillus amyloliquefaciens subsp. amyloliquefaciens has an inhibitory effect on pecan dry rot pathogens. Ge Kangkang et al. found a biocontrol bacterium BS111 with antibacterial activity, which was identified as Bacillus subtilis ( ​ ).

[0006] Micromonospora produces a diverse array of bioactive metabolites, including macrolides, anthraquinones, and alkaloids. Some strains of the genus Micromonospora have been reported to produce metabolites that inhibit plant pathogens. For example, the antibiotic produced by the carbonaceous Micromonospora strain JXNU-1 has inhibitory effects on various plant pathogens, including tobacco brown spot pathogen, wheat fusarium head blight, and rice sheath blight. Micromonospora metabolites have shown promising application potential in agriculture and forestry. Summary of the Invention

[0007] The purpose of the present invention is to provide a new species of Micromonospora.

[0008] The present invention provides a micromonospora ​ sp., the Micromonospora ​ Micromonospora sp. ​ sp.181 is deposited in the China General Microorganism Culture Collection Center with the accession number CGMCC No.16626. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China General Microorganism Culture Collection Center.

[0009] The present invention provides the above-mentioned Micromonospora ​ Microbial preparations of sp.181.

[0010] Further defined, Micromonospora​ sp.181 was inoculated into the fermentation medium at an inoculum rate of 6% and fermented at 28°C for 7 days.

[0011] It is further defined that the fermentation medium is: 0.4% yeast extract, 1.0% malt extract, 0.4% glucose, 2.0% soluble starch by mass and 0.2% CaCO3, pH 7.2-7.4.

[0012] It is further defined that the fermentation medium is: 1.0% soluble starch, 1.0% cottonseed meal, 0.5% yeast extract, 0.5% malt extract, 0.2% MgSO4·7H2O and 0.2% CaCO3, pH 7.2-7.4.

[0013] It is further defined that the fermentation medium is: 2.0% soybean flour, 2.0% glucose, 1.0% soluble starch, 0.2% beef extract, 0.2% peptone and 0.5% K2HPO4, pH 7.2-7.4.

[0014] The present invention provides the above-mentioned Micromonospora ​ Use of sp.181 or the above-mentioned microbial preparation in the preparation of an agent for inhibiting walnut dry rot.

[0015] The present invention provides the above-mentioned Micromonospora ​ Use of sp.181 or the above-mentioned microbial preparation in the preparation of a medicament for inhibiting walnut dry rot.

[0016] It is further defined that the dosage form of the drug or agent is any one of tablets, capsules, granules, powders, and liquid preparations.

[0017] The present invention provides a method for inhibiting walnut dry rot, characterized in that the above-mentioned Micromonospora ​ sp. were inoculated into Petri dishes containing walnut dry rot and cultured for 7 days.

[0018] Further limited, culture Micromonospora ​ The culture medium for sp.181 is: ISP2 solid culture medium.

[0019] It is further defined that the culture medium for culturing walnut dry rot is: PDA culture medium.

[0020] Beneficial Effects: Micromonospora ​ sp. 181 is a newly discovered species of the genus Micromonospora. ​ sp. 181 has a good inhibitory effect on the pathogen of pecan dry rot.

[0021]

Biological Deposit Information

[0022] ​ Micromonospora ​ Transmission electron micrograph of sp. 181 cultured in ISP2 medium at 28 °C for 21 days;

[0023] ​ Micromonospora ​ Polar lipid diagram of sp. 181, where DPG, diphosphatidylglycerol; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PG, phosphatidylglycerol; PGL, phosphoglycolipid; GL, glycolipid; PL, phospholipid; AL, amino lipid;

[0024] ​ Micromonospora ​ 16S rRNA gene phylogenetic tree of sp. 181 and similar strains (NJ). Bootstrap values ​​are based on 1000 replicates; only values ​​≥50% are shown. Bar, 0.005 substitution per nucleotide position. ​ IFO 12514 T As an external species;

[0025] ​ Micromonospora ​ sp. 181 pairs ​ antagonistic effect, where a is the experimental group and b is the control group inoculated with pathogens only. DETAILED DESCRIPTION

[0026] Through polyphasic taxonomic research, Micromonospora 181 was identified from the aspects of phenotypic, genotypic and chemical classification characteristics, and its taxonomic status was determined.

[0027] (1) Phenotypic studies

[0028] References for observation of the growth, colony morphology and cell morphology of the strain on different culture media (ISP1, ISP2, ISP3, ISP4, ISP5, ISP6, ISP7, nutrient agar and Gao's solid medium No. 1). Growth restriction factor experiment: Micromonospora ​Sp. 181 was grown at temperatures of 4, 10, 15, 20, 25, 28, 30, 32, 35, 37, 40, 45, and 50°C. The NaCl concentration range was 0-10% with 12 concentration gradients (0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10%). The pH range was 4-10 with 12 pH gradients (4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, and 10.0). Anaerobic tests were performed by inoculating the strain onto solid anaerobic culture medium and sealing the culture in sealed anaerobic bags. Gram staining, oxidase activity, catalase activity, esterase activity, and starch, cellulose, and gelatin hydrolysis activities, as well as nitrate reduction, were performed. The substrate utilization of the strains was detected using the Biolog GN2 kit.

[0029] (2) Chemical classification characteristics

[0030] 1) Fatty acids

[0031] The Sherlock (MIDI) fully automatic bacterial identification system was used to automatically detect and analyze the fatty acid composition of the strains.

[0032] 2) Respiratory quinones

[0033] Respiratory quinones were extracted according to the method described in [K. Komagata, and KI Suzuki. (1988) 4 Lipid and Cell-Wall Analysis in Bacterial Systematics. Methods in Microbiology 16, 161-207], and their types were identified by HPLC-MS analysis.

[0034] 3) Polar lipids

[0035] The extraction, chromatography and color development procedures of polar lipids were referred to [Jia YY, Sun C, Pan J, et al. (2014) ​ sp nov., isolated from deep-sea sediment. International Journal of Systematic and Evolutionary Microbiology 64, 2637-2641].

[0036] (3) Genotypic characteristics

[0037] Micromonospora ​The genomic DNA of Micromonospora sp. 181 was sequenced, and the 16S rRNA gene sequence of Micromonospora sp. 181 (shown in SEQ ID NO.1:

[0038] ​ sp. nov., a thermophilic species from a geothermal area. International Journal of Systematic and Evolutionary Microbiology 60, 834-839].

[0039] Example 1. Isolation and identification of bacterial strains

[0040] 1. Micromonospora ​ sp. 181 separation and screening

[0041] Micromonospora ​ sp. 181 was isolated from the root soil of Tung Blossom Tree in Longhai City, Fujian Province.

[0042] The specific isolation operation is as follows: after the soil is naturally air-dried and ground, 10 g of soil is weighed and added to 90 mL of sterile water and shaken to mix; under a sterile environment, the soil is inoculated onto a glucose-asparagine medium (10 g of glucose, 0.5 g of asparagine, 0.5 g of K2HPO4, 1000 mL of distilled water, pH 7.2-7.4, and 20 g of agar) using the doubling dilution method, and cultured at 28°C for one week. A single colony is picked from the plate and transferred to a newly prepared ISP2 plate, and purified repeatedly to obtain pure bacteria.

[0043] 2. Strain Identification Method

[0044] Micromonospora ​ sp. 181 is a Gram-positive bacterium that is aerobic and forms well-developed intrabasal hyphae. Single spores are formed on the matrix hyphae, with a diameter of 0.5-0.8 µm and a warty appearance on the spore surface ( ​ However, the strain lacks aerial hyphae and produces no soluble pigment on agar plates. When incubated on ISP2 plates at 30°C, the colonies change color from orange to dark brown. With increasing incubation time, the colonies become black with raised surfaces. Table 1 shows the growth characteristics of the strain on different media. The strain grows well on ISP2, moderately well on ISP3, ISP4, ISP6, and Gao's No. 1, and poorly on ISP2, ISP5, ISP7, and nutrient agar.

[0045] Table 1 Characteristics of Micromonospora sp. 181 cultured on different media at 28 °C for 14 days

[0046]

[0047] Micromonospora ​The temperature range for the growth of sp. 181 is 15-45 °C (optimum growth temperature is 28-35 °C); the pH range for growth is 5.5-9.0 (optimum growth pH is 7.0-8.0); the NaCl salinity range for growth is 0-4.0% (optimum is 1.0%). ​ Sp. 181 can hydrolyze starch, esculin, Tween 40, Tween 60, and Tween 80, but cannot hydrolyze cellulose or gelatin. It does not produce H2S and is negative in both the milk peptone and milk coagulation tests.

[0048] Micromonospora ​ sp. 181 and three closely related reference strains M. ​ AM105 T 、 ​ CCTCC AA2012002 T and ​ 2803GPT1-18 T In contrast, some characteristics can be compared with Micromonospora ​ sp. 181 were distinguished from the reference strains. For example, unlike the three reference strains, Micromonospora ​ sp. 181 nitrate reduction test results were negative; Micromonospora ​ sp. 181 can grow at pH 5.5 and 9.0, but ​ AM105 T and ​ 2803GPT1-18 T No; unlike Micromonospora Micromonospora sp. 181 catalase was positive. In the substrate utilization experiment, Micromonospora Micromonospora sp. 181 can utilize raffinose, which is different from the three reference strains; in terms of the utilization of D-xylose, lactose and melibiose, Micromonospora Micromonospora sp.181 and M. wenchangensis CCTCC AA2012002 T Different; in terms of the utilization of D-fructose, maltose and sucrose, the strains M. rifamycinica AM105 T The results were different; Micromonospora Micromonospora sp. 181 can utilize D-fructose, but the strain M. mangrovi 2803GPT1-18 T No, the results are shown in Table 2.

[0049] Table 2 Micromonospora sp. 181 and MicromonosporaCharacteristic differences among reference strains within the genus

[0050] ;

[0051] (Strain 1,181;2, M. wenchangensis CCTCC AA2012002 T ; 3, M. rifamycinica AM105 T ; 4, M. mangrovi 2803GPT1-18 T +, positive reaction; -, negative reaction; w, weakly positive reaction.

[0052] (2) Chemical classification characteristics

[0053] In Micromonospora Micromonospora The main respiratory quinones detected in sp. 181 were MK-10(H4), MK-9(H6) and MK-10(H6), with contents of 83.1%, 10.7% and 6.2%, respectively. Micromonospora The main component of sp. 181 is MK-10(H4), while the reference strains are all MK-10(H6). Micromonospora The main fatty acid in sp. 181 (accounting for ≥5.0% of the total fatty acids) is iso-C 16:0 (29.7%), iso-C 15:0 (16.9%), 10-methyl-C 18:0 (11.5 %) and C 18:1 ω 9 c (6.3%), the specific components and contents are shown in Table 3. From the perspective of the proportion of some major contents, there are some characteristics that Micromonospora Micromonospora sp. 181 was distinguished from the reference strains by 10-methyl-C 18:0 Micromonospora Micromonospora sp. 181, but not in the reference strain M. rifamycinica AM105 T and M. mangrovi 2803GPT1-18 T Not in Micromonospora Micromonospora sp. 181 and M. wenchangensis CCTCC AA2012002 T In comparison, C 18:1 ω 9 c is a major component, but C 18:0Not a major ingredient. Micromonospora Micromonospora The major polar lipids of sp. 181 are diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylinositol phospholipids (PI), phospholipids (PL), phosphoglycolipids (PGL), glycolipids (GL) and amino lipids (AL), see Figure 2 Micromonospora Micromonospora sp. 181 does not contain phosphatidylinositol mannoside (PIM), and is different from the reference strain M. wenchangensis CCTCC AA2012002 T and M. rifamycinica AM105 T Different; phosphatidylglycerol (PG) and phosphoglycolipid (PGL) can distinguish Micromonospora Micromonospora sp. 181 and M. mangrovi 2803GPT1-18 T , the results are shown in Table 3.

[0054] Table 3 Comparison of fatty acids between Micromonospora sp. 181 and reference strains

[0055] ;

[0056] (Strains: 1,181;2, M. wenchangensis CCTCC AA2012002 T ; 3, M. rifamycinica AM105 T ; 4, M. mangrovi 2803GPT1-18 T Fatty acids with a content of less than 1% in all four strains are not shown (ND, not detected).

[0057] (3) Genotype analysis

[0058] Micromonospora Micromonospora The 16S rRNA gene sequence of sp. 181 was submitted to the EzBioCloud website for comparison and obtained the accession number MK108078 on GeneBank. Micromonospora sp.181 and Micromonospora The 16S rRNA gene of the genus is very similar to [[ID=3i]]M. wenchangensis CCTCCAA2012002 T , M. rifamycinica AM105 T and M. mangrovi 2803GPT1-18 T The strains have 99.5%, 99.4% and 99.1% similarity.Micromonospora sp. 181 in NJ tree M. rifamycinica AM105 T Form a cluster ( Figure 3 ). Micromonospora Micromonospora sp. 181 had a G + C content of 72.8 mol%, which was similar to that of the reference strain. DNA-DNA hybridization results showed that Micromonospora Micromonospora sp. 181 and M. wenchangensis CCTCC AA2012002 T , M. rifamycinica AM105 T and M. mangrovi 2803GPT1-18 T The hybridization rates of the present invention were 63.5%, 55.9% and 52.8%, respectively. These results are lower than the threshold value (70%) for determining bacterial species. / / 这里原文有误,推测是你多打了一个字母,应为M. mangrovi a sp. 181 is a newly discovered genospecies of the genus Micromonospora.

[0059] Example 2. Preparation of a biological preparation containing Micromonospora sp. 181

[0060] In vitro antibacterial activity rescreening: Micromonospora Micromonospor Sp. 181 was inoculated onto ISP2 solid medium and cultured at 28°C for 7 days. The cultured strain was then inoculated into seed culture medium and cultured at 28°C and 200 rpm for 2-3 days. Three different fermentation media (FA-FC) were selected (see Table 4). The seed culture was inoculated at a 6% inoculum into each of the three fermentation media and cultured at 28°C and 200 rpm for 7 days. The fermentation broth was added with an equal volume of ethanol and sonicated for 60 minutes. The supernatant was filtered and concentrated by rotary evaporation until approximately 5 mL remained.

[0061] Table 4 Formulas of three fermentation media

[0062]

[0063] Example 3. Micromonospora Micromonospora Inhibitory activity of sp. 181 against hickory stem rot

[0064] pecan dry rot pathogens by plate confrontation method Micromonospora (The pathogen was provided by Zhejiang Agricultural and Forestry University) and preliminary activity screening was carried out, and Micromonospora Botryosphaeria dothidea sp. 181 was inoculated on ISP2 solid medium. Micromonospora Inoculate on PDA and culture MicromonosporaBotryosphaeria dothidea sp. 181 inoculated in Micromonospora The fungus was measured by cross-cross method in the culture medium. Botryosphaeria dothidea diameter, the antibacterial rate calculation formula is as follows, indicating that Micromonospora Botryosphaeria dothidea sp.181 has a good inhibitory effect on pecan dry rot pathogens ( Micromonospora ).

[0065] Inhibition rate (%) = (fungal diameter of the control group - fungal diameter of the experimental group) / fungal diameter of the control group × 100%.

[0066] The control group was inoculated with 5mm pecan dry rot pathogen in the middle of the PDA medium. Figure 4 Botryosphaeria The experimental group was inoculated in the center of the PDA culture medium. dothidea Inoculate Micromonospora at four points 2.5 cm from the center of the cake. Botryosphaeria dothidea The bacterial cakes (5 mm in diameter) of sp. 181 were cultured at 28°C for 7 days in both the control and experimental groups, and then the inhibition rates were determined.

[0067] The concentrated fermentation filtrate obtained in Example 2 was subjected to activity rescreening test, and the rotary evaporation filtrate of blank culture medium was used as control, and the activity was measured by cross-cross method. Micromonospora diameter, and calculate the Micromonospora Botryosphaeria dothidea The inhibition rate of the fermentation liquid of sp. 181 on pecan dry rot was calculated using the same formula as above. The results are shown in Table 5.

[0068] Table 5 Micromonospora Micromonospora sp. 181 different fermentation broths Micromonospora Botryosphaeria dothidea The inhibitory effect

[0069] .

Claims

1. A Micromonospora Micromonospora sp., characterized in that Micromonospora Micromonospora sp. named Micromonospora Micromonospora sp.181, deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number CGMCC No. 16626, the deposit date is October 24, 2018, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. Containing the Micromonospora according to claim 1 Micromonospora Microbial preparations of sp.

181.

3. The microbial preparation according to claim 2, characterized in that Micromonospora Micromonospora sp.181 was inoculated at a 6% inoculum into a fermentation medium and fermented at 28°C for 7 days. The fermentation medium had the following formula: 0.4% yeast extract, 1.0% malt extract, 0.4% glucose, 2.0% soluble starch, and 0.2% CaCO3, with a pH of 7.2-7.

4. Alternatively, the fermentation medium has the following formula: 1.0% by mass of soluble starch, 1.0% by mass of cottonseed meal, 0.5% by mass of yeast extract, 0.5% by mass of malt extract, 0.2% by mass of MgSO4·7H2O, and 0.2% by mass of CaCO3, with a pH of 7.2-7.4; Alternatively, the fermentation medium has the following formula: 2.0% soybean powder by mass, 2.0% glucose by mass, 1.0% soluble starch by mass, 0.2% peptone by mass and 0.5% K2HPO4 by mass, pH 7.2-7.

4.

4. Micromonospora according to claim 1 Micromonospora Use of sp.181 or the microbial preparation described in claim 2 in the preparation of an agent for inhibiting pecan dry rot.

5. Micromonospora according to claim 1 Micromonospora Use of sp.181 or the microbial preparation described in claim 2 in the preparation of a drug for inhibiting pecan dry rot.

6. The use according to claim 5, characterized in that The pharmaceutical dosage form is any one of tablets, capsules, granules, powders and liquid preparations.

7. The use according to claim 4, characterized in that The dosage form of the reagent is any one of tablets, capsules, granules, powders and liquid preparations.

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