Algicidal Bacillus schenkelii and its application

Abstract

The present invention discloses algicidal Shinella algicida and its application. The present invention provides a new species of Shinella algicida, specifically Shinella algicida SD203, whose deposit number in the General Microbiological Center of China Microbiological Culture Collection Administration Committee is CGMCC No.25922. Shinella algicida SD203 can lyse cyanobacterial cells, and can be applied to biological control of cyanobacterial growth, and has broad application prospects in the treatment of eutrophic sewage (cyanobacterial bloom).

Description

Technical Field

[0001] The invention relates to the field of microorganisms, in particular to algicidal Bacillus schenkelii and applications thereof. Background Art

[0002] Bacteria of the Rhizobiaceae family are widely distributed in a variety of ecological environments, such as agricultural farmland, lakes, and forests. They can effectively promote the cycle of elements in the environment, especially the nitrogen fixation function of some strains, which has made an irreplaceable contribution to the effective utilization of nitrogen sources by crops. Shinella is a major branch of the Rhizobiales. The genus Shinella was proposed by Dong-Shan An and other researchers in 2006 (An, DS, Im, WT, Yang, HC & Lee, ST (2006). Shinella granuli gen.nov., sp.nov., and proposal of the reclassification of Zoogloea ramigera ATCC 19623 as Shinella zoogloeoides sp.nov. Int J Syst Evol Microbiol 56, 443–448.), with the strain Shinella granuli ATCC 19623 as the model bacteria. Subsequently, the description of Shinella was revised and improved in 2009. The data on morphology, physiological and biochemical characteristics and cellular fatty acid composition were mainly supplemented (Matsui, T., Shinzato, N., Tamaki, H., Muramatsu, M. & Hanada, S. (2009). Shinella yambaruensis sp.nov., a3-methyl-sulfolane-assimilating bacterium isolated from soil. Int J Syst Evol Microbiol 59, 536–539.). At present, Shinella covers 8 species of strains isolated from a variety of microecological environments (https: / / lpsn.dsmz.de / genus / Shinella). Most of the strains of Shinella were isolated from an environment rich in organic matter. The influence of environmental pressure on the phenotypic evolution of microbial strains has enabled these strains to develop characteristics that can adapt to multiple ecological environments. Therefore, the ability of Shinella strains to degrade organic matter and their applications have also attracted much attention. For example: Degradation of 4-aminobenzenesulfonate (Biala, S., Chadha, P., & Saini, HS (2014).Biodegradation of 4-aminobenzenesulfonate by indigenous isolate Shinellayambaruensis SA1 and its validation by genotoxic analysis.Biotechnol.Bioprocess.Eng.19,1034–1041.), degradation of p-chlorophthalide (Liang, B., Wang, G., Zhao, Y., Chen, K., Li, S., & Jiang, J. (2011). Facilitation of bacterial adaptation to chlorothalonil-contaminated sites by horizontal transfer of the chlorothalonil hydrolyticdehalogenase gene.Appl.Environ.Microbiol.77,4268–4272.), degradation of phenol (SepehrS.,Shahnavaz B.,Asoodeh A.&Karrabi M(2019).Biodegradation of phenol by cold-tolerant bacteria isolated from alpine soils of Binaloud Mountains in Iran.JEnviron Sci Health A Tox Hazard Subst Environ Eng 54,367-379.), the degradation of nornicotine (Qiu J., Li N., Lu Z., Yang Y., Ma Y., Niu L., He J. & Liu W (2016). Conversion of nornicotine to 6-hydroxy-nornicotine and 6-hydroxy-myosmine by Shinella sp. strain HZN7. Appl Microbiol Biotechnol 100,10019-10029.) and the degradation of pyridine (Bai, Y., Sun, Q., Zhao, C., Wen, D. & Tang, X. (2009). Aerobic degradation of pyridine by a new bacterial strain, Shinella zoogloeoides BC026. J. Ind.Microbiol.Biotechnol.36,1391–1400.). .

[0003] With global warming and the increasing eutrophication of freshwater ecosystems, the overgrowth of cyanobacteria has led to frequent outbreaks of cyanobacterial blooms around the world. Cyanobacterial blooms have seriously affected human production and life, and even caused harm to human life (Wang Jun, Zhang Yisheng. Chemical pollutants and ecological effects. Beijing, China Environmental Science Press, 1993; Perron MC, Qiu BS, Boucher N, et al.. Use of chlorophyll a fluorescence to detect the effect of microcystins on photosynthesis and photosystem II energy fluxes of green algae. Toxicon, 2012, 59: 567-577). In recent years, my country's inland lakes have also become the hardest hit areas of cyanobacterial blooms. Eutrophication and cyanobacterial bloom control are urgent (Yang Liuyan, Xiao Lin. Lake cyanobacterial bloom outbreaks, hazards and control. Beijing, Science Press, 2011). Although physical and chemical methods play an important role in controlling the harm of cyanobacterial blooms, most of these methods have disadvantages such as high capital investment, short prevention period, and potential secondary pollution risks, making it difficult to completely solve the bloom problem. We hope to be able to effectively control the biomass of harmful algae with the help of biological means to achieve the purpose of prevention and control. Biological control technology has the advantages of high efficiency, strong specificity and environmental friendliness (Qin Boqiang, Wang Xiaodong, Tang Xiangming, et al. Drinking water crisis caused by eutrophication of Taihu Lake and cyanobacterial blooms - causes and countermeasures. Progress in Earth Sciences, 2007, 22(9): 896-906; Shi Shunyu. Study on the physiological and ecological effects and mechanism of algae-lysing bacteria on algae. Wuhan Institute of Hydrobiology, Chinese Academy of Sciences, 2006). Summary of the invention

[0004] The purpose of the present invention is to provide a new species of Bacillus shenkelii with algae-lysing function.

[0005] In a first aspect, the present invention claims a new species of the genus Shenkel.

[0006] The new species of Shinella genus claimed for protection in the present invention is specifically Shinella algicida SD203, and its deposit number in the General Microbiological Center of China National Microbiological Culture Collection Committee is CGMCC No.25922.

[0007] The algicidal Shinella algicida SD203 is a gram-negative bacterium that is aerobic and can form moist, smooth, cream-colored to light yellow colonies after being cultured on R2A medium at 30°C for 72 hours. The cells are short rod-shaped, and the growth temperature, salt tolerance, and acid-base tolerance ranges of the strain are 15-32°C, 0-3% NaCl, and pH 6.0-8.0, respectively, and the optimal growth conditions are 30°C, 0-1% NaCl, and pH 7. The strain is positive for oxidase detection and negative for nitrate reduction. It can utilize maltose, L-rhamnose, sucrose, and cellobiose as the sole carbon source. The main fatty acids of the strain are C 18:1 ω7c / C 18:1 ω6c,C 19: 0cycloω8c,C 18:1 ω7c 11-methyl and C 14:0 3OH / iso-C 16:1 I; The respiratory quinone component is Q10; The polar lipid components include diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), hydroxyphosphatidylethanolamine (OH-PE), and phosphatidylcholine (PC).

[0008] In a second aspect, the invention claims a culture.

[0009] The culture claimed in the present invention is the culture of Shinella algicida SD203 described in the first aspect above, specifically a substance obtained by culturing the Shinella algicida SD203 in a bacterial culture medium.

[0010] In the above culture, the substances include the Shinella algicida SD203 (the bacteria itself) and metabolites of the Shinella algicida SD203.

[0011] In the above culture, the bacterial culture medium may be a solid culture medium or a liquid culture medium.

[0012] The term "culture" refers to a general term for liquid or solid culture media that have a microbial population after artificial inoculation and culture. That is, the product obtained by growing and / or amplifying microorganisms, which can be a biologically pure culture of microorganisms, or it can contain a certain amount of culture medium, metabolites or other components produced during the culture process. The term "culture" also includes a subculture obtained by subculturing microorganisms, which can be a culture of a certain generation or a mixture of several generations.

[0013] In a specific embodiment of the present invention, the bacterial culture medium is specifically R2A culture medium.

[0014] In a third aspect, the present invention claims a metabolite.

[0015] The metabolite claimed in the present invention is the metabolite of Shinella algicida SD203 described in the first aspect above.

[0016] The term "metabolite" refers to the primary metabolites and / or secondary metabolites produced during the metabolism of microorganisms. Primary metabolism refers to the process in which microorganisms absorb various nutrients from the outside world and generate substances and energy to maintain life activities through catabolism and anabolism. The products of primary metabolism are primary metabolites, such as monosaccharides or monosaccharide derivatives, nucleotides, vitamins, amino acids, fatty acids and other monomers, as well as various macromolecular polymers composed of them, such as proteins, nucleic acids, polysaccharides, lipids, etc. Secondary metabolism refers to the process in which microorganisms use primary metabolites as precursors to synthesize some substances that have no clear function in the life activities of microorganisms during a certain growth period. The products of secondary metabolism are secondary metabolites, most of which are compounds with relatively complex molecular structures. According to their functions, they can be divided into types such as antibiotics, hormones, alkaloids, toxins, etc.

[0017] In a fourth aspect, the present invention claims protection for a bacterial agent.

[0018] The bacterial agent claimed in the present invention contains the algicidal Shinella algicida SD203 described in the first aspect above, the culture described in the second aspect above and / or the metabolite described in the third aspect above.

[0019] The bacterial agent is an algae-dissolving bacterial agent and can be used to control algal blooms.

[0020] In the above-mentioned microbial agent, in addition to the active ingredient, the microbial agent also contains a carrier. The carrier may be a carrier commonly used in the field of pesticides and biologically inert. The carrier may be a solid carrier or a liquid carrier; the solid carrier may be a mineral material, a plant material or a polymer compound; the mineral material may be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica and diatomaceous earth; the plant material may be at least one of corn flour, soybean flour and starch; the polymer compound may be polyvinyl alcohol and / or polyglycol; the liquid carrier may be an organic solvent, vegetable oil, mineral oil or water; the organic solvent may be decane and / or dodecane.

[0021] The above-mentioned bacterial agents may be in various dosage forms, such as liquid, emulsion, suspension, powder, granule, wettable powder or water dispersible granule.

[0022] As required, a surfactant (such as Tween 20, Tween 80, etc.), a binder, a stabilizer (such as an antioxidant), a pH adjuster, etc. may also be added to the bacterial agent.

[0023] In a fifth aspect, the present invention claims the use of the algicidal Shinella algicida SD203 described in the first aspect above, or the culture described in the second aspect above, or the metabolite described in the third aspect above, or the bacterial agent described in the fourth aspect above in any of the following:

[0024] (A1) Algicidal;

[0025] (A2) preparing a product for algae lysis;

[0026] (A3) Control of algal blooms;

[0027] (A4) preparing products for controlling algal blooms;

[0028] (A5) Soil improvement;

[0029] (A6) Production of bacterial fertilizer preparations.

[0030] In a sixth aspect, the present invention claims a product for algae lysis.

[0031] The active ingredient of the algae-lysing product claimed in the present invention is the algicidal Shinella algicida SD203 described in the first aspect above, or the culture described in the second aspect above, or the metabolite described in the third aspect above, or the bacterial agent described in the fourth aspect above.

[0032] In a seventh aspect, the present invention claims a product for controlling algal blooms.

[0033] The product for controlling algal blooms claimed in the present invention has as its active ingredient the algicidal Shinella algicida SD203 described in the first aspect above, or the culture described in the second aspect above, or the metabolite described in the third aspect above, or the bacterial agent described in the fourth aspect above.

[0034] In an eighth aspect, the present invention claims a method for algae lysis.

[0035] The algae lysing method claimed in the present invention may include the following steps: treating the algae sample to be treated with the algicidal Shinella algicida SD203 described in the first aspect above, or the culture described in the second aspect above, or the metabolite described in the third aspect above, or the bacterial agent described in the fourth aspect above.

[0036] In a ninth aspect, the present invention claims a method for controlling algal blooms.

[0037] The method for controlling algal blooms claimed in the present invention may include the following steps: treating the algal bloom water body to be controlled with the algicidal Shinella algicida SD203 described in the first aspect above, or the culture described in the second aspect above, or the metabolite described in the third aspect above, or the bacterial agent described in the fourth aspect above.

[0038] In the above aspects, the algae may be cyanobacteria; and the water bloom may be a cyanobacterial bloom.

[0039] In a specific embodiment of the present invention, the cyanobacteria is Anabaena blooms.

[0040] In the tenth aspect, the present invention claims protection for the use of Shinella algicida SD203 described in the first aspect above in the preparation of the culture described in the second aspect above or the metabolite described in the third aspect above or the bacterial agent described in the fourth aspect above.

[0041] Experiments have shown that the strain SD203 of the present invention has some obvious differences from the effective described strains of the genus Shinella that have been scientifically studied, including phenotype, physiological and biochemical and cytochemical components. At the same time, the systematic evolution analysis based on the gene level further illustrates that the strain SD203 can be distinguished from the existing effective species of the genus Shinella, which fully proves that the strain SD203 of the present invention represents a new species of the genus Shinella, named Shinella algicida. At the same time, it was found that the strain SD203 can lyse cyanobacterial cells, proving the ability of the strain of the present invention to biologically control the growth of cyanobacteria, and has a wide range of application prospects in the treatment of eutrophic sewage (cyanobacterial blooms). It can be used for sewage treatment, soil improvement, production of bacterial fertilizer preparations, etc.

[0042] Collection Instructions

[0043] Classification name: Shinella algicida;

[0044] Reference biological materials: SD203;

[0045] Depository: General Microbiology Center, China Microbiological Culture Collection Administration;

[0046] Abbreviation of depository institution: CGMCC;

[0047] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0048] Deposit date: October 17, 2022;

[0049] The registration number of the Collection Center is: CGMCC No.25922. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is a transmission electron microscopy photograph of cells of strain SD203 cultured in R2A medium at 30℃ for 72h.

[0051] Figure 2 This is the detection result of polar lipid components of strain SD203.

[0052] Figure 3 The diagram shows the algicidal activity of strain SD203 on Anabaena aquatica FACHB-245. a, shows the algicidal zone around strain SD203; b, shows the lysis of algal cells by the culture solution of strain SD203: green is normal algal cells; yellow is lysed algal cells.

[0053] Figure 4 A phylogenetic tree was constructed based on the 16S rRNA gene sequences of strain SD203 and all described species of the genus Rhizobium. T (KF551141) is the outgroup.

[0054] Figure 5 This is a phylogenetic tree constructed based on the core genes of the genus Schenckia and related species, showing the taxonomic status of strain SD203. DETAILED DESCRIPTION

[0055] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0056] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0057] Example 1: Isolation and identification of Shinella algicida SD203

[0058] 1. Isolation and screening of strain SD203

[0059] The strain SD203 of the present invention was isolated from a water sample of Sancha Lake in Jianyang, Sichuan. The strain was isolated using R2A medium as the isolation medium, and the medium components were: 0.5 g glucose, 0.5 g soluble starch, 0.5 g of peptone, 0.5 g of yeast extract, 0.5 g of acid-hydrolyzed casein, 0.3 g of sodium pyruvate, 0.3 g of dipotassium hydrogen phosphate, 0.05 g of magnesium sulfate heptahydrate, 15 g of agar, and deionized water were dissolved and made up to 1 L, pH value 7.2.

[0060] Separation and screening method: Take 2L of surface water from the center of Sancha Lake in Jianyang, Sichuan, filter it with a sterile filter membrane with a pore size of 3μm to remove large particles such as algae, and collect the filtrate in a sterile container; then filter the filtrate with a sterile filter membrane with a pore size of 0.22μm to enrich the bacteria on the filter membrane; cut the filter membrane into pieces, immerse it in 20mL of sterile saline, and shake it for 30min to prepare a bacterial suspension. Dilute the bacterial suspension in the previous step to 10 -3 , mix by blowing and aspirating, then aspirate 0.3 mL of the mixed bacterial suspension and spread it on the R2A medium plate, and culture at 30°C for 3 weeks. Pick the plates with better microbial diversity, and inoculate the single colonies with complete colonies and different morphological characteristics on the R2A plates for streak culture to obtain pure cultures for subsequent research. At the same time, the pure strains obtained were preserved in liquid nitrogen and frozen at -80°C with 20% (v / v) glycerol as a protective agent. In this experiment, a new species that is most closely related to the strains of the genus Shinella (16S rRNA gene similarity is 95.87-98.79%) was obtained, and the strain number is SD203.

[0061] 2. Identification of strain SD203

[0062] The strain SD203 was grown on R2A medium (BD medium, USA) at 30°C. The strain was studied at the morphological, physiological and biochemical, cytochemical and genetic levels. Other special cases will be explained.

[0063] 1. Cell morphology observation and physiological and biochemical characteristics detection of strain SD203

[0064] The growth temperature detection range of strain SD203 is 4, 10, 15, 28, 30, 32, 37, 40 and 45°C; the growth salt concentration (NaCl) detection range is 12 (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15) concentration gradients of 0-15% (0-15g / 100ml); the growth pH detection range is 8 (4, 5, 6, 7, 8, 9, 10, 11) gradients between pH4-11. The physiological and biochemical characteristics of the strain were detected using API 50CH, API ZYM, and BIOLOG GEN III carbon source detection kits. Other strain physiological characteristics, including Gram staining properties, oxygen demand, catalase activity, oxidase activity, gelatin hydrolysis activity, starch hydrolysis activity and cellulose hydrolysis activity, were mainly carried out with reference to the "Common Bacteria System Identification Manual" (Dong Xiuzhu et al., edited and published: Beijing: Science Press, 2001.2).

[0065] The identification results showed that strain SD203 was a Gram-negative bacterium and was aerobic. After culturing on R2A medium at 30°C for 72 hours, the strain formed moist, smooth, cream-colored to light yellow colonies; the cells were short rod-shaped, such as Figure 1 The growth temperature, salt tolerance and acid-base tolerance range of the strain are 15-32℃, 0-3% NaCl and pH6.0-8.0, and the optimal growth conditions are 30℃, 0-1% NaCl, pH 7. The strain SD203 was positive for oxidase detection and negative for nitrate reduction; it can use maltose, L-rhamnose, sucrose and cellobiose as the sole carbon source. The phenotypic differences between strain SD203 and representative strains of closely related species Shinella curvata JCM 31239, Shinella kummerowiae JCM 14778, Shinella granuli JCM 13254 and Shinella zoogloeoides DSM 287 are shown in Table 1.

[0066] Table 1. Phenotypic differences between strain SD203 and closely related control strains of the genus Schenckia

[0067]

[0068]

[0069]

[0070] Note: +, indicates positive; -, indicates negative; w, indicates weak positive.

[0071] From the results shown in Table 1, it can be seen that the strain SD203 of the present invention has obvious differences in some phenotypic characteristics such as growth conditions, physiological and biochemical characteristics and the like between the closely related strains of Bacillus shenkelii that have been published.

[0072] 2. Detection of cytochemical composition of strain SD203

[0073] The cellular chemical components of strain SD203, such as fatty acids, quinone types, and polar lipids, were detected by GC gas chromatography, HPLC liquid chromatography, and TLC thin-layer chromatography (Sasser M. Identification of bacteria by gasghromatography of cellular fatty acids, MIDI Technical Note 101. Newark, DE: MIDIinc; 1990. Minnikin DE, O'Donnell AG, Goodfellow M, Alderson G, Athalye M et al. An integrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 1984; 2: 233–241.). The fatty acid composition of strain SD203 is shown in Table 2. The results show that the main fatty acids of strain SD203 are C 18:1 ω7c / C 18:1 ω6c,C 19:0 cycloω8c,C 18:1 ω7c 11-methyl and C 14:0 3OH / iso-C 16:1 I. In strain SD203, the respiratory quinone component is Q10; the polar lipid components include diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), hydroxyphosphatidylethanolamine (OH-PE), phosphatidylcholine (PC), such as Figure 2 shown.

[0074] Table 2. Fatty acid composition of strain SD203

[0075] Fatty acid composition (%) SD203 Straight-chain fatty acid <![CDATA[C 18:0 ]]> 1.6 <![CDATA[C 13:1 at 12-13]]> 1.4 <![CDATA[C 17:0 cyclo]]> 2.2 <![CDATA[C 18:1 ω7c 11-methyl]]> 10.8 <![CDATA[C 19:0 cycloω8c]]> 16.5 <![CDATA[C 20:2 ω6.9c]]> 2.7 Hydroxy fatty acids <![CDATA[C 16:0 3-OH]]> 2.3 Others <![CDATA[C 14:0 3OH / iso-C 16:1 I]]> 11.1 <![CDATA[C 16:1 ω7c / C 16:1 ω6c]]> 3.3 <![CDATA[C 18:1 ω7c / C 18:1 ω6c]]> 34.6

[0076] 3. Algicidal activity detection of strain SD203

[0077] (1) Agar block method for screening algae-lytic bacteria

[0078] The Anabaena flos-aquae FACHB-245 strain was purchased from the Freshwater Algae Seed Bank of Wuhan Institute of Hydrobiology, Chinese Academy of Sciences as the test algae. The strain SD203 was inoculated on R2A medium by the spot grafting method. After culturing at 30°C for 7 days, the agar block with colonies was taken out with a sterile cork puncher and placed on an algae plate (algae culture medium: NaNO3 496mg, KH2PO4 39mg, MgSO4·7H2O 75mg, CaCl2·2H2O 36mg, Na2SiO3·9H2O58mg, composite salt 3ml (composition formula of composite salt: Na2-EDTA 75mg, FeCl3·6H2O 9.7mg, MnCl2·4H2O 4.1mg, ZnCl2 0.5mg, CoCl2·6H2O 0.2mg, Na2MoO4·2H2O 0.4mg, agar powder 15g, deionized water was added to make the volume to 1000mL, and the pH was adjusted to pH 5 with sodium hydroxide and hydrochloric acid. 8-8.5), culture at 25℃, observe for 7 days continuously to see if there is algae lysis ring, and judge the algae lysis activity of the strain according to the transparency, generation time and size of the algae lysis ring. Use an optical microscope to observe the morphology and integrity of the algae cells to further confirm the algae lysis activity of the strain. The initial screening results are as follows Figure 3 In a, strain SD203 showed an obvious algal lysis zone around it; under the microscope, most algal cells were obviously ruptured, such as Figure 3 Middle b.

[0079] (2) Chlorophyll a method for screening algae-lysing bacteria

[0080] The strain SD203 was activated and a slant strain was prepared. The well-growing slant strain was inoculated into a seed culture medium (R2A liquid culture medium) (the volume of the culture medium in a 500ml triangular flask was 200ml), and cultured in a shaking incubator at 30°C and 180r / min for about 48h. Then, the slant strain was inoculated into R2A liquid culture medium (the volume of the culture medium in a 500ml triangular flask was 200ml) for expansion culture. The inoculation amount was about 10% (v / v). After inoculation, the slant strain was cultured in a shaking incubator at 30°C and 180r / min for 6d. The fermentation broth was then centrifuged in a sterile centrifuge tube. At a cell concentration of 5×10 6 / ml algae solution (250ml triangular flask algae solution volume is 100ml) was added with 2% (v / v) supernatant of strain SD203 liquid culture medium, the culture temperature was 25°C, the light intensity was 30-50μmol photon / m 2 / s, the light-dark ratio was 12 hours of light: 12 hours of no light, and the culture was carried out for 3-5 days. The color change of the algae solution was observed during the culture process. After 3 days, the chlorophyll a (Chl-a) content was determined by the hot ethanol method (Chen Yuwei, Chen Kaining, Hu Yaohui. Discussion on the "hot ethanol method" for phytoplankton chlorophyll determination and its measurement error. Lake Science, 2006, 18 (5): 550-552), and the algae lysis efficiency was calculated using the following formula:

[0081]

[0082] The experimental group was inoculated with the culture fluid of strain SD203 and Anabaena flos-aquae FACHB-245; the control group was not inoculated with the culture fluid of strain SD203 but only with the culture fluid of Anabaena flos-aquae FACHB-245.

[0083] The experiment was repeated three times and the results were expressed as mean ± standard deviation.

[0084] After substituting the formula into calculation, the algae-lysing rate of strain SD203 on Anabaena flos-aquae FACHB-245 was 83.35±0.29%, confirming that strain SD203 has good algae-lysing activity.

[0085] 4. Determination of the phylogenetic status of strains

[0086] The genomic DNA of strain SD203 was extracted for sequencing, and the 16S rRNA gene sequence (SEQ ID No. 1) was compared online in the international authoritative bacterial taxonomy analysis database (http: / / www.ezbiocloud.net / ) (Kim OS, Cho YJ, Lee K, et al. 2012, Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol, 62: 716-721.). The results showed that the strain SD203 of the present invention had the highest similarity with the species of Shinella. The highest similarity of the 16S rRNA gene sequence of the strain SD203 of the present invention with the known species was: Shinella curvata JCM 31239 T (98.79%),Shinella kummerowiae JCM 14778 T (97.81%),Shinella granuli JCM 13254 T(97.80%) and Shinella zoogloeoides DSM 287 T (97.23%) and the 16S rRNA gene sequences of other strains were all lower than 97.0%. The 16S rRNA gene sequences of all valid strains of the genus Shen'e and some strains of the genera adjacent to the genus Shen'e in the family Rhizobiaceae were selected to construct a phylogenetic tree ( Figure 4 ). The results showed that strain SD203 was a member of the genus Shinella and clustered in a subclade with Shinella curvata JCM 31239, Shinellaakummerowiae JCM 14778, Shinella granuli JCM 13254, and Shinella zoogloeoides DSM287.

[0087] To further clarify the phylogenetic status of the strain, the average nucleotide similarity (ANI value) of the whole genome sequence of strain SD203 and the whole genome sequence of closely related control bacteria was compared and calculated on EZbiocloud (Yoon SH, Ha SM, Lim J, Kwon S, Chun JA large-scale evaluation of algorithms to calculate average nucleotide identity. Antonie van Leeuwenhoek 2017; 110: 1281–1286.). The whole genome sequence comparison analysis showed that the average nucleotide similarity (ANI) of strain SD203 with Shinella curvata JCM 31239, Shinella akummerowiae JCM 14778, Shinella granuli JCM 13254, and Shinella zoogloeoides DSM 287 was 89.0%, 85.4%, 85.4%, and 85.2%, respectively. These values ​​are far below the ANI threshold of 95% for distinguishing prokaryotic microbial gene species. In the phylogenetic tree based on the core genes of the whole genome, strain SD203 occupies a taxonomic position of a species within the genus Schenckia ( Figure 5 ).

[0088] These results indicate that the strain SD203 of the present invention is a newly discovered genospecies of the genus Schenckia.

[0089] In summary, the data analysis of the polyphasic taxonomic research shows that the strain SD203 of the present invention has some obvious differences from the effectively described strains of the genus Shinella that have been scientifically studied, including phenotype, physiological and biochemical and cytochemical components. At the same time, the systematic evolutionary analysis based on the gene level further illustrates that the strain SD203 can be distinguished from the existing effective species of the genus Shinella, which fully proves that the strain SD203 of the present invention represents a new species of the genus Shinella, named Shinella algicida. At the same time, it was found that the strain SD203 can lyse cyanobacterial cells, proving the ability of the strain of the present invention to biologically control the growth of cyanobacteria, and has broad application prospects in the treatment of eutrophic sewage. It can be used for sewage treatment, soil improvement, production of bacterial fertilizer preparations, etc.

[0090] Shinella algicida SD203 was deposited in the China General Microbiological Culture Collection Center on October 17, 2022, and its deposit number is CGMCC No.25922.

[0091] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Algicidal Bacillus schenkelii ( Shinella algicida )SD203, whose accession number in the General Microbiological Center of China Culture Collection Administration is CGMCC No.25922.

2. The algicidal Bacillus schenkelii of claim 1 ( Shinella algicida ) The culture of SD203 is obtained by mixing the algicidal Bacillus schenkelii described in claim 1 ( Shinella algicida ) The substance obtained by culturing SD203 in bacterial culture medium.

3. A microbial agent, characterized in that: The bacterial agent contains the algicidal Bacillus schenkelii described in claim 1 ( Shinella algicida ) SD203 and / or the culture described in claim 2.

4. The bacterial agent according to claim 3, characterized in that: The bacterial agent is an algae-dissolving bacterial agent.

5. The algicidal Bacillus schenkelii of claim 1 ( Shinella algicida ) Use of the culture described in SD203 or claim 2 or the bacterial agent described in claim 3 or 4 in any of the following: (A1) algae lysis; (A2) preparing products for algae lysis; (A3) Control of algal blooms; (A4) Preparation of products for use in controlling algal blooms; The algae are cyanobacteria; the bloom is cyanobacteria bloom; The cyanobacteria is Anabaena blooms.

6. A product for algae dissolution, wherein the active ingredient is the algicidal Bacillus schenkelii described in claim 1 ( Shinella algicida ) SD203 or the culture described in claim 2 or the bacterial agent described in claim 3 or 4; The algae is cyanobacteria; The cyanobacteria is Anabaena blooms.

7. A product for controlling algal blooms, wherein the active ingredient is the algicidal Bacillus schenkelii described in claim 1 ( Shinella algicida ) SD203 or the culture described in claim 2 or the bacterial agent described in claim 3 or 4; The bloom is a cyanobacterial bloom; The cyanobacteria is Anabaena blooms.

8. A method for lysing algae, comprising the following steps: using the algicidal Bacillus schenkelii of claim 1 ( Shinella algicida ) The algae sample to be treated is treated with the culture described in SD203 or claim 2 or the bacterial agent described in claim 3 or 4; The algae is cyanobacteria; The cyanobacteria is Anabaena blooms.

9. A method for controlling algal blooms, comprising the following steps: using the algicidal Bacillus schenkelii of claim 1 ( Shinella algicida ) Treating the algal bloom water body to be treated with the culture described in SD203 or claim 2 or the bacterial agent described in claim 3 or 4; The bloom is a cyanobacterial bloom; The cyanobacteria is Anabaena blooms.

10. The algicidal Bacillus schenkelii of claim 1 ( Shinella algicida ) Use of SD203 in the preparation of the culture described in claim 2 or the bacterial agent described in claim 3 or 4.

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