New species of photosynthetic nitrogen-fixing red bacteria and its application

Abstract

The present invention discloses a new species of photosynthetic nitrogen-fixing red bacteria and its application. The present invention provides photosynthetic nitrogen-fixing red bacteria (Rhodobacter photoazotoformans) S2205, whose deposit number in the General Microbiological Center of China Microorganism Culture Collection Administration Committee is CGMCC No.26164. The strain can produce 5-aminolevulinic acid (ALA), and when co-cultured with algae cells, it can inhibit algae growth. 5-ALA can be used as a natural herbicide, pesticide and plant growth promoter. Therefore, the strain S2205 of the present invention is an excellent strain resource for preparing microbial agents for improving the crop ecological environment and promoting the healthy growth of crops.

Description

Technical Field

[0001] This invention relates to the field of microbiology, specifically to a new species of photosynthetic nitrogen-fixing red bacteria and its applications. Background Technology

[0002] Most members of the domain Bacteria, phylum Pseudomonadota, class Alphaproteobacteria, order Rhodobacterales, and family Rhodobacteraceae are capable of photosynthesis, and are therefore often referred to as an important group of photosynthetic bacteria. Photosynthetic bacteria play a crucial role in the fixation and cycling of carbon, nitrogen, and sulfur, and possess the ability to produce physiologically active substances involved in metabolic processes. Therefore, the research, application, and commercialization of photosynthetic bacteria in agriculture, animal husbandry, environmental protection, food, medicine, and other fields have received increasing attention and become a research hotspot. For example, Kenji et al. [Kenji T, Sasaki K, Masanori W, et al. Removal of phosphorus from Oyster Farm mud sediment using a photosynthetic bacterium, Rhodobacter sphaeroides IL106. Journal of Bioscience and Bioengineering, 1999, 88(4):410-415] used the sphaerobacterium IL106 to remove oyster farm mud sediment containing high concentrations of phosphorus, with significant results. The total phosphorus concentration was reduced from 26.8 mg / L to 0.73 mg / L in 4 days. The genus *Rhodopseudomonas* was proposed by Imhoff et al. (Imhoff J, Trüper H, Pfennig N. Rearrangement of the species and genera of the phototrophic “purple nonsulfur bacteria”[J]. International Journal of Systematic and Evolutionary Microbiology, 1984, 34(3):340-343.) in 1984 when they reclassified some species of the genus *Rhodopseudomonas*. It was subsequently revised several times in 1994, 2014, and 2019 (Wang D, Liu H, Zheng S, Wang G. *Paenirhodobacter enshiensisgen.nov., sp. nov.)., a non-photosynthetic bacterium isolated from soil, and emended descriptions of the genera Rhodobacter and Haematobacter. Int J SystEvol Microbiol 2014;64:551–558; Hiraishi A, Ueda Y. Intrageneric structure of the genus Rhodobacter: transfer of Rhodobacter sulfidophilus and relatedmarine species to the genus Rhodovulum gen.nov[J].International Journal of Systematic and Evolutionary Microbiology,1994,44(1):15-23;Suresh G,Lodha TD,Indu B,Sasikala C,Ramana CV.Taxogenomics resolves conflict in the genusRhodobacter:a two and half decades pending thought to reclassify the genusRhodobacter.Front Microbiol 2019; 10:2480.). Currently, the genus Rhodobacter contains 15 valid descriptive species (https: / / lpsn.dsmz.de / genus / Rhodobacter). The type strains of these species are widely derived, including estuarine areas in mangrove habitats (Venkata Ramana V, Anil Kumar P, Srinivas TN, et al. Rhodobacteraestuarii sp.nov., a phototrophic alphaproteobacterium isolated from anestuarine environment[J]. Int J Syst Evol Microbiol, 2009, 59(Pt 5):1133-6.) and alkaline brown mangroves (Gandham S, Lodha T, Chintalapati S, et al. Rhodobacter alkalitolerans sp.nov.).Arch Microbiol, 2018, 200(10):1487-1492.) [Sheu C,Li ZH,Sheu SY,etal.Tabrizicola oligotrophicasp.nov.and Rhodobacter tardus sp.nov.,two new Int JSyst Evol Microbiol,2020,70(12):6266-6283.) [Suresh G,Sailaja B,Ashif A,et al.Description ofRhodobacter azollae sp.nov.and Xian WD, Liu ZT, Li MM, et al.Rhodobacterflagellatus sp.nov., a thermophilic bacterium isolated from a hot spring[J].Int J Syst EvolMicrobiol,2017,67(9):3289-3295 Microbiol,2020,70(3):1541-1546;Khan IU,Habib N,Xiao M,etal.Rhodobacter thermarum sp.nov.,a novel phototrophic bacterium isolated fromsediment of a hot spring[J].Antonie Van Leeuwenhoek,2019,112(6):867-875.) Int J Syst Evol. Rhodobacter maris sp.nov., an aphototrophic alphaproteobacterium isolated from a marine habitat of India[J].Int J Syst Evol Microbiol,2008,58(Pt 7):1719-22.), lagoon sediments (Subhash Y, Lee SS. Rhodobacter sediminis sp. nov., isolated from lagoon sediments [J]. Int J SystEvol Microbiol, 2016, 66 (8): 2965-2970.), creek mud (Raj PS, Ramaprasad EVV, VaseefS, et al. Rhodobacter viridis sp. nov., a phototrophic bacterium isolated frommud of a stream[J].Int J Syst Evol Microbiol,2013,63(Pt 1):181-186.), soil (Arunasri K, Venkata Ramana V,. C, et al. *Rhodobacter megalophilus* sp. nov., a phototroph from the Indian Himalayas possessing a wide temperature range for growth[J]. *Int J Syst Evol Microbiol*, 2008, 58(Pt 8): 1792-6.). Additionally, Kurahashi et al. (Kurahashi M, Yokota A. *A preliminary report of phylogenetic diversity of bacterial strains isolated from marine creatures[J]. *J Gen Appl Microbiol*, 2002, 48(5): 251-9.) isolated strains belonging to the genus *Rhodobacter* from the gut microbiota of various marine organisms. *Rhodobacter* strains have oval or rod-shaped cells, most of which exhibit unipolar flagellar motility. They are positive for catalase and oxidase. They are photoheterotrophic. The cells possess a complex cytoplasmic endomembrane system and a natural carotenoid biosynthesis system, exhibiting both photoautotrophic and dark chemotrophic (aerobic / anaerobic) capabilities, and can effectively assimilate various organic substances as carbon sources for cell growth. Most strains in this genus are aerobic mesophilic bacteria. The dominant fatty acid composition includes C. 18:1 ω7c, most of which also contain fatty acid component C 10:0 3OH and C 18:03OH. Phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and phosphatidylcholine (PC) are the main polar lipid components of this genus, and Q-10 is the only respiratory quinone contained in this genus. The DNA G+C content of the genome is 62.9-70.6%.

[0003] The type species of the genus Rhodophyta, Rhodophyta capsulatus, can convert atmospheric nitrogen into ammonia to supplement the nitrogen needs of plants. Because these bacteria have nitrogen-fixing properties, inoculating Rhodobacter capsulatus in rice cultivation can effectively improve plant nitrogen absorption, plant growth, and yield (Maeda I. Potential of Phototrophic Purple Nonsulfur Bacteria to Fix Nitrogen in Rice Fields[J]. Microorganisms, 2021, 10(1); Elbadry M, Gamal-Eldin H, Elbanna K. Effects of Rhodobacter capsulatus inoculation in combination with graded levels of nitrogen fertilizer on growth and yield of rice in pots and lysimeter experiments[J]. World Journal of Microbiology and Biotechnology, 1999, 15(3): 393-395; Gamal-Eldin H, Elbanna K. Field evidence for the potential of Rhodobacter capsulatus as Biofertilizer for flooded rice[J]. Curr Microbiol, 2011, 62(2): 391-5.). Min et al. (Min K, Park YS, Park GW, et al. Elevated conversion of CO(2) to versatile formate by a newly discovered formate dehydrogenase from Rhodobacter aestuarii[J]. Bioresour Technol, 2020, 305(123155.) discovered an oxygen-tolerant CO2 reductase from Rhodobacter aestuarii, which can be used to catalyze the conversion of carbon dioxide to a versatile formate. Its significant advantage in reducing carbon dioxide emissions allows for the construction of a feasible enzyme system capable of producing a versatile formate from carbon dioxide as a cheap, abundant, and renewable resource. Furthermore, Rhodobacter strains are also commonly used in the production of environmentally friendly fuel hydrogen through photofermentation.This is an anaerobic process that requires light as an energy source and organic substrates (such as lactic acid) as a carbon source to maintain the growth of Rhodophyte strains and thus produce hydrogen. Turon et al. (Turon V, Ollivier S, Cwicklinski G, et al. H(2) production by photofermentation in an innovative plate-type photobioreactor with meandering channels[J]. Biotechnol Bioeng, 2021, 118(3): 1342-1354.) found that using a plate-type photobioreactor, the maximum hydrogen production flow rate of capsular Rhodophyte can reach 157.7 ± 9.3 mL H2 / L / h. Rhodophyte strains have also been found to have the ability to degrade carcinogenic polycyclic aromatic hydrocarbons and pollutants such as benzene (Oberoi AS, Philip L, Bhallamudi SM. Biodegradation of Various Aromatic Compounds by Enriched Bacterial Cultures: Part A-Monocyclic and Polycyclic Aromatic Hydrocarbons[J]. Appl Biochem Biotechnol, 2015, 176(7):1870-88.). They can also degrade various pollutants in soil, sediment, and wastewater. Ponsano et al. found that Rubrivivax gelatinosus can treat fat in poultry slaughterhouse wastewater (Ponsano EH, Paulino CZ, Pinto MF. Phototrophic growth of Rubrivivax gelatinosus in poultry slaughterhouse wastewater[J]. Bioresour Technol, 2008, 99(9):3836-42.); Wu et al. found that Rubrivivax gelatinosus can treat protein and starch in soybean protein wastewater (Wu P, Li Jz, Wang Yl, et al. Promoting the growth of Rubrivivax gelatinosus in sewage purification by the addition of magnesium ions[J]. Biochemical engineering journal, 2014, 91(66-71.).Kong et al. found that these strains could dechlorinate and mineralize tetrachlorophenol (Kong F, Wang A, Ren HY, et al. Improved dechlorination and mineralization of 4-chlorophenol in a sequential biocathode-bioanode-bioelectrochemical system with mixed photosynthetic bacteria[J]. Bioresour Technol, 2014, 158(32-8.), while Ouchane et al. found the pufL gene in Rubrivivax gelatinosus, which exhibits a terbutryn resistance phenotype (Ouchane S, Picaud M, Astier CA new mutation in the pufL gene responsible for the terbutryn resistance phenotype in Rubrivivax gelatinosus[J]. FEBS Lett, 1995, 374(1):130-4.). Wu et al. found that adding Rubrivivax gelatinosus to soybean processing wastewater could effectively remove carbaryl residues from the soil (Wu P, Li Jz, Wang Yl, et al. Promoting the growth of Rubrivivax gelatinosus in sewage purification by the addition of magnesium ions[J]. Biochemical Engineering Journal, 2014, 91(66-71). Furthermore, due to intracellular nutrients (vitamin B, coenzyme Q10, pigments, folic acid), the capsular red bacterium in soybean processing wastewater can also improve soil quality and fertility. All of the above information suggests that strains of this genus have strong bioremediation potential for environmental pollution.A 5-aminolevulinic acid synthase (ALAS) with excellent enzymatic properties extracted from Rhodobacter capsulatus was evaluated and applied to the production of 5-aminolevulinic acid (ALA) (Lou JW, Zhu L, Wu MB, et al. High-level soluble expression of the hemA gene from Rhodobacter capsulatus and comparative study of its enzymatic properties[J]. J Zhejiang Univ Sci B, 2014, 15(5):491-9.). 5-Aminolevulinic acid (ALA) is a common precursor in the biosynthesis of compounds such as heme, vitamin B12, cytochromes, and chlorophyll tetrapyrrole. It has been practically applied in agriculture as a herbicide, insecticide, and plant growth promoter. Moreover, it can enable plants to have salt tolerance and low temperature tolerance, playing a key role in plant growth promotion, stress resistance, and disease prevention. Meanwhile, these substances have also been applied in medical fields such as tumor diagnosis and cancer treatment (Sasaki K, Watanabe M, Tanaka T, et al. Biosynthesis, biotechnological production and applications of 5-aminolevulinic acid[J]. Appl Microbiol Biotechnol, 2002, 58(1):23-9.). Currently, many different genes encoding 5-aminolevulinic acid synthase have been cloned in bacterial strains and expressed in Escherichia coli strains. By combining overexpression and optimizing the culture process, researchers have increased ALA production to 7.34 g / L. -1(Lin J, Fu W, Cen P. Characterization of 5-aminolevulinate synthase from Agrobacterium radiobacter, screening new inhibitors for 5-aminolevulinated ehydratase from Escherichia coli and their potential use for high 5-aminolevulinate production[J]. Bioresour Technol, 2009, 100(7): 2293-7.). Therefore, Rhodopsin strains have good application prospects in industry, agriculture, food and medicine. In-depth exploration of new Rhodopsin resources is of great significance. Summary of the Invention

[0004] The purpose of this invention is to provide a new species of photosynthetic nitrogen-fixing red bacteria and its applications.

[0005] In a first aspect, the present invention claims protection for a new species of photosynthetic nitrogen-fixing red bacteria.

[0006] The novel species of photosynthetic nitrogen-fixing red bacteria claimed in this invention is Rhodobacter photoazotoformans S2205, which has the accession number CGMCC No. 26164 at the China General Microbiological Culture Collection Center.

[0007] The Rhodobacter photoazotoformans S2205 strain is a Gram-negative, mesophilic, and facultative aerobic bacterium. After culturing at 30°C on NA medium for 48 hours, the strain forms moist, smooth, and pale yellow colonies. The optimal growth conditions are 20-37°C, 0-3% NaCl, and pH 6.0-8.0, with the optimal conditions being 30°C, 0-1% NaCl, and pH 7.0. This strain is positive for oxidase and catalase tests, and positive for esculin hydrolysis; it is negative for gelatin liquefaction and cannot hydrolyze starch or cellulose.

[0008] Secondly, this invention claims protection for a culture.

[0009] The culture claimed in this invention is the culture of Rhodobacter photoazotoformans S2205 described in the first aspect above, which is a substance obtained by culturing Rhodobacter photoazotoformans S2205 described in the first aspect above in a bacterial culture medium.

[0010] The substances in the above-mentioned culture include the *Shinella algicida* SD203 (the bacterial cell itself) and the metabolites of the *Shinella algicida* SD203.

[0011] In the above-mentioned cultures, the bacterial culture medium can be a solid culture medium or a liquid culture medium.

[0012] The term "culture" refers to any liquid or solid culture medium that has grown a microbial community after artificial inoculation and cultivation. It is the product obtained by growing and / or amplifying microorganisms; it 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 cultivation process. The term "culture" also includes passaged cultures obtained by subculturing microorganisms; these can be cultures of a single generation or mixtures of several generations.

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

[0014] Thirdly, the present invention claims protection for a metabolite.

[0015] The metabolites claimed in this invention are those of the photosynthetic nitrogen-fixing red bacterium (Rhodobacter photoazotoformans) S2205 described in the first aspect above.

[0016] The term "metabolite" refers to the primary and / or secondary metabolites produced during microbial metabolism. Primary metabolism refers to the process by which microorganisms absorb various nutrients from the external environment and, through catabolism and anabolism, generate substances and energy to sustain life activities. The products of primary metabolism are called primary metabolites, such as monosaccharides or monosaccharide derivatives, nucleotides, vitamins, amino acids, fatty acids, and various macromolecular polymers composed of them, such as proteins, nucleic acids, polysaccharides, and lipids. Secondary metabolism refers to the process by which microorganisms, at a certain growth stage, use primary metabolites as precursors to synthesize substances that have no clearly defined function for their life activities. The products of secondary metabolism are called secondary metabolites, and are mostly compounds with relatively complex molecular structures. Based on their functions, they can be classified into types such as antibiotics, hormones, alkaloids, and toxins.

[0017] Fourthly, this invention claims protection for a microbial agent.

[0018] The bacterial agent claimed in this invention contains Rhodobacter photoazotoformans S2205 as described in the first aspect above, the culture as described in the second aspect above, and / or the metabolites as described in the third aspect above.

[0019] The microbial agent is a microbial agent that inhibits algae growth and / or a microbial agent used for weed control and / or a microbial agent used for insect control and / or a microbial agent used for promoting plant growth.

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

[0021] The above-mentioned microbial agents can be in various formulations, such as liquid, emulsion, suspension, powder, granules, wettable powder or water-dispersible granules.

[0022] Depending on the requirements, surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH adjusters, etc. may also be added to the bacterial agent.

[0023] Fifthly, the present invention claims protection for the use of Rhodobacter photoazotoformans S2205 as described in the first aspect above, or the culture as described in the second aspect above, or the metabolite as described in the third aspect above, or the bacterial agent as described in the fourth aspect above, in any of the following:

[0024] (A1) Inhibits algal growth;

[0025] (A2) Prepare products for inhibiting algal growth;

[0026] (A3) Controlling algal blooms;

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

[0028] (A5) Weeding;

[0029] (A6) Prepare products for weed control;

[0030] (A7) Insecticide;

[0031] (A8) Prepare products for insecticidal use;

[0032] (A9) Promotes plant growth;

[0033] (A10) Prepare products for promoting plant growth;

[0034] (A11) Production of 5-aminolevulinic acid (5-ALA);

[0035] (A12) Prepare a product for the production of 5-aminolevulinic acid;

[0036] (A13) Improve the ecological environment of crops;

[0037] (A14) Prepare products for improving the ecological environment of crops.

[0038] Sixthly, the present invention claims protection for a product used to inhibit algae growth.

[0039] The product for inhibiting algal growth claimed in this invention has the active ingredient being Rhodobacter photoazotoformans S2205 as described in the first aspect above, or the culture as described in the second aspect above, or the metabolite as described in the third aspect above, or the bacterial agent as described in the fourth aspect above.

[0040] Seventhly, the present invention claims protection for a product used to control algal blooms.

[0041] The product for controlling algal blooms claimed in this invention has the active ingredient being Rhodobacter photoazotoformans S2205 as described in the first aspect above, or the culture as described in the second aspect above, or the metabolite as described in the third aspect above, or the bacterial agent as described in the fourth aspect above.

[0042] Eighthly, the present invention claims protection for a product used for weed control.

[0043] The product for weed control claimed in this invention has an active ingredient that is Rhodobacter photoazotoformans S2205 as described in the first aspect above, or a culture as described in the second aspect above, or a metabolite as described in the third aspect above, or a microbial agent as described in the fourth aspect above.

[0044] Ninthly, the present invention claims protection for a product used for insecticidal purposes.

[0045] The product for insecticide claimed in this invention has an active ingredient that is Rhodobacter photoazotoformans S2205 as described in the first aspect above, or a culture as described in the second aspect above, or a metabolite as described in the third aspect above, or a bacterial agent as described in the fourth aspect above.

[0046] In a tenth aspect, the present invention claims a product for promoting plant growth.

[0047] The product for promoting plant growth claimed in this invention has an active ingredient that is Rhodobacter photoazotoformans S2205 as described in the first aspect above, or a culture as described in the second aspect above, or a metabolite as described in the third aspect above, or a bacterial agent as described in the fourth aspect above.

[0048] Eleventhly, the present invention claims protection for a method for inhibiting algal growth.

[0049] The method for inhibiting algal growth claimed in this invention may include the following steps: treating the algal sample to be treated with the Rhodobacter photoazotoformans S2205 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.

[0050] In a twelfth aspect, the present invention claims a method for controlling algal blooms.

[0051] The method for controlling algal blooms claimed in this invention may include the following steps: treating the algal bloom water body to be controlled with Rhodobacter photoazotoformans S2205 as described in the first aspect above, or the culture as described in the second aspect above, or the metabolite as described in the third aspect above, or the bacterial agent as described in the fourth aspect above.

[0052] In a thirteenth aspect, the present invention claims a method for weeding and / or insecticidal and / or promoting plant growth.

[0053] The method for weeding and / or insecticidal and / or promoting plant growth claimed in this invention may include the following steps: treating the plant to be treated or its growth substrate with the Rhodobacter photoazotoformans S2205 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.

[0054] In all the above aspects, the algae is cyanobacteria; the water bloom is a cyanobacterial bloom.

[0055] Furthermore, the cyanobacteria mentioned are *Anabaena hygrophora*, a type of algae that blooms.

[0056] In a specific embodiment of the present invention, the algae is Anabaena facsifolia FACHB-245.

[0057] In a fourteenth aspect, the present invention claims the use of the photosynthetic nitrogen-fixing red bacterium (Rhodobacter photoazotoformans) S2205 described in the first aspect above in the preparation of the culture described in the second aspect above, the metabolite described in the third aspect above, or the bacterial agent described in the fourth aspect above.

[0058] Experiments have demonstrated that strain S2205 of this invention represents a new species of the genus Rhodobacter, named *Rhodobacter photoazotoformans*. This strain can produce 5-aminolevulinic acid (ALA) and, when co-cultured with algal cells, can inhibit algal growth. 5-ALA can serve as a natural herbicide, insecticide, and plant growth promoter. Therefore, strain S2205 of this invention is an excellent microbial resource for preparing microbial inoculants to improve the crop ecological environment and promote healthy crop growth.

[0059] Preservation Instructions

[0060] Classification and nomenclature: Rhodobacter photoazotoformans;

[0061] Biomaterial of ginseng: S2205;

[0062] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures;

[0063] The abbreviation for the depository institution is CGMCC.

[0064] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;

[0065] Deposit date: December 7, 2022;

[0066] Registration number at the Preservation Center: CGMCC No. 26164. Attached Figure Description

[0067] Figure 1 The results show the polar lipid components of strain S2205.

[0068] Figure 2A phylogenetic tree was constructed based on the 16S rRNA gene sequences of strain S2205 and related strains. The phylogenetic tree was used with Rhodospirillum rubrum ATCC 11170T (D30778) as the outgroup. Detailed Implementation

[0069] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0070] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0071] Example 1: Isolation, screening and identification of strain S2205

[0072] I. Isolation of strain S2205

[0073] Strain S2205 was isolated from a water sample from Erhai Lake in Dali, Yunnan Province. The isolation medium was BG-11, the composition of which is shown in Table 1. The methods and procedures for strain isolation and purification were performed according to the methods described in the literature by Deng Y et al. (Deng Y, Han XF, Jiang ZM, Yu LY, Li Y and Zhang YQ (2022) Characterization of three Stenotrophomonas strains isolated from different ecosystems and proposal of Stenotrophomonas mori sp.nov. And Stenotrophomonas lacuserhaiisp.nov..Front.Microbiol.13:1056762.doi:10.3389 / fmicb.2022.1056762.). The obtained pure strain was preserved in liquid nitrogen and cryopreserved at -80℃ using 20% ​​(v / v) glycerol as a protectant.

[0074] In this experiment, strain S2205 was isolated and purified.

[0075] Table 1. Composition and final concentration of BG-11 culture medium

[0076]

[0077]

[0078] II. Cell morphology observation and physiological and biochemical characteristics detection of strain S2205

[0079] The following experiments were generally conducted on NA solid medium (with agar added) or NA liquid medium (without agar added) and BG-11 medium. Exceptions will be noted separately.

[0080] NA medium composition: 10g peptone, 3g beef extract, 5g sodium chloride, dissolved in distilled water and brought to a final volume of 1000mL, pH 7.0.

[0081] The growth temperature range for strain S2205 was 10-45℃, with 11 detection points (10, 15, 20, 25, 28, 30, 32, 35, 37, 40, and 45℃). The growth salt concentration (NaCl) range was 0-8% (0-8 g / 100 ml) across 9 concentration gradients (0, 1, 2, 3, 4, 5, 6, 7, and 8%). The growth pH range was 4-11 across 8 gradients (4, 5, 6, 7, 8, 9, 10, and 11). The physiological and biochemical functions of the strain were assessed using API 50CH and API ZYM assay kits, as well as BiOLOG GEN III plates and corresponding procedures. Other physiological and biochemical characteristics of the strain, including Gram staining properties, oxygen requirement, catalase activity, oxidase activity, gelatin hydrolysis activity, starch hydrolysis activity, and cellulose hydrolysis activity, were mainly determined with reference to the "Handbook of Systematic Identification of Common Bacteria" (Dong Xiuzhu, Cai Miaoying. 2001. Handbook of Systematic Identification of Common Bacteria. Beijing: Science Press).

[0082] Identification results showed that strain S2205 is a Gram-negative bacterium, mesophilic, and facultative aerobic. After culturing at 30°C on NA medium for 48 hours, the strain forms moist, smooth, and pale yellow colonies. The optimal growth conditions for the strain are 20-37°C, 0-3% NaCl, and pH 6.0-8.0, with the optimal growth conditions being 30°C, 0-1% NaCl, and pH 7.0.

[0083] Strain S2205 was positive for oxidase and catalase tests, and positive for esculin hydrolysis; it was negative for gelatin liquefaction and could not hydrolyze starch and cellulose. Phenotypic differences between strain S2205 and its close relative Rhodobacter tardus CYK-10 are shown in Table 2.

[0084] Table 2. Differential phenotypic characteristics of strain S2205 and its closest relative, Rhodobacter tardus CYK-10

[0085]

[0086]

[0087] Note: In the table, + indicates positive, - indicates negative, and w indicates weak positive.

[0088] III. Detection of cytochemical characteristics of strain S2205

[0089] The cytochemical components of strain S2205, including fatty acids, quinone types, and polar lipids, were detected by gas chromatography (GC), high-performance liquid chromatography (HPLC), and thin-layer chromatography (TLC) (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 main fatty acid of strain S2205 is C. 18:1 ω7c. As shown in Table 3, the content of many fatty acid components in strain S2205 is also different from that of closely related species of Rhodobacteria (Sheu C, Li ZH, Sheu SY, Yang CC, Chen WM. Tabriziacola oligotrophica sp. nov. and Rhodobacter tardus sp. nov., two new species of bacteria belonging to the family Rhodobacteraceae. Int J Syst Evol Microbiol 2020; 70:6266-6283.). The only respiratory quinone of strain S2205 is Q10. In strain S2205, polar lipid components include diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), and unknown components of phospholipids (PL) and glycolipids (GL) such as Figure 1As shown in the figure. The dominant fatty acids, quinones, and major polar lipid components confirm that strain S2205 is a member of the Rhodobacterium genus; at the same time, the trace components and their contents of the fatty acid composition of strain S2205 can distinguish strain S2205 from other species in the Rhodobacterium genus.

[0090] Table 3. Fatty acid composition of strain S2205

[0091] fatty acid(%) S2205 <![CDATA[anteiso-C 14:0 ]]> 1.4 <![CDATA[C 16:0 ]]> 9.8 <![CDATA[C 18:0 ]]> 3.9 <![CDATA[C 10:0 2OH]]> 1.4 <![CDATA[C 10:0 3OH]]> 2.1 <![CDATA[C 18:1 2OH]]> 0.9 <![CDATA[C 18:0 3OH]]> 5.7 <![CDATA[C 16:1 ω7c]]> 4.7 <![CDATA[C 18:1 ω7c]]> 59.7 <![CDATA[11-MethylC 18:1 ω7c]]> 4.3

[0092] IV. Determination of the phylogenetic position of strain S2205

[0093] Genomic DNA was extracted from strain S2205 and sequenced. The 16S rRNA gene sequence (SEQ ID No. 1) was then compared online in an internationally authoritative bacterial taxonomy 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 strain S2205 of this invention had the highest similarity to species in the Rhodobacteriaceae family. A phylogenetic tree was constructed by retrieving the 16S rRNA genes of closely related strains and strain S2205. In the phylogenetic tree of Rhodobacteriaceae, strain S2205 clustered on the evolutionary branch of the Rhodobacteria genus, adjacent to Rhodobactertardus CYK-10 ( Figure 2The 16S rRNA gene similarity between strain S2205 and the typical strain of *Rhodobacter tardus*, *Rhodobacter cykaloids* CYK-10, is 95.2%, which is far below the threshold of 98.65% for distinguishing prokaryotic species (Kim M, Oh HS, Park SC, Chun J. Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. *Int J Syst Evol Microbiol* 2014; 64:346–351). Therefore, strain S2205 is determined to be a member of the *Rhodobacteria* genus that is distinct from known species.

[0094] To further clarify the taxonomic position of the strain, the average nucleotide similarity (ANI) between the whole genome sequence of strain S2205 and the nearest-neighbor control strain Rhodobacter tardus CYK-10 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.). Comparative analysis of the whole genome sequences showed that the average nucleotide similarity (ANI) between the whole genome sequence of strain S2205 and the nearest-neighbor control strain Rhodobacter tardus CYK-10 was 71.9%. These values ​​are all well below the 95% ANI threshold for distinguishing prokaryotic gene species (Kim, M., Oh, HS, Park, SC, and Chun, J. (2014). Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. Int. J. Syst. Evol. Microbiol. 64, 346–351.). This result further supports the conclusion that strain S2205 represents a new species within the genus Rhodobacteria. Based on the complete genome sequence of strain S2205, the GC content of the genome was calculated to be 65.6%.

[0095] Based on the taxonomic data of strain S2205 in terms of phenotype and genotype, and its comparison with closely related strains, we determined that strain S2205 represents a new species in the genus Rhodobacter. Because it contains the rate-limiting enzyme gene for the C4 pathway of 5-ALA synthesis in photosynthetic bacteria (see "VI. Test of 5-Aminolevulinic Acid Production Capacity of Strain S2205" below), it is named *Rhodobacter photoazotoformans*.

[0096] Rhodobacter photoazotoformans was deposited at the China General Microbiological Culture Collection Center on December 7, 2022, with accession number CGMCC No. 26164.

[0097] V. Detection Experiment of Inhibitory Effect of Straw S2205 on Anabaena facsimile FACHB-245

[0098] Under aseptic conditions, add 40 mL of sterile BG-11 culture medium to a 250 mL Erlenmeyer flask. Inoculate with *Anabaena f. sp.* FACHB-245 solution. The final inoculum concentration is approximately 2.0 × 10⁻⁶. 5 cells·mL -1 Three replicates were set up for both the control and treatment groups. 1.0 mL of S2205 bacterial suspension was added to the treatment group until its concentration reached 2.0 × 10⁻⁶. 5 Bacterial cells·mL -1 Add 1.0 mL of BG-11 medium to the control group and shake well. Incubate at 30°C in a light incubator (12 h light: 12 h no light) for 5 days.

[0099] The experimental results of the inhibitory effect of strain S2205 on the growth of *Anabaena facsimile* FACHB-245 are shown in Table 4. Table 4 shows that when bacteria and algae were co-cultured, strain S2205 significantly inhibited the biomass growth of *Anabaena facsimile* FACHB-245, with an inhibition rate of up to 67.5%. Therefore, it can be determined that strain S2205 has a significant inhibitory effect on the growth of *Anabaena facsimile* FACHB-245 (Yuan Junfeng, Meng Zhifang, Chen Dehui, et al. Allelopathy of neutral citric acid bacteria on the growth of several common algae [J]. Freshwater Fisheries, 1999, 29(4):12-15.).

[0100] Table 4. Experimental results of growth inhibition of Anabaena facsimile FACHB-245 by strain S2205

[0101]

[0102] Note: 1, 2, and 3 represent repeated experiments under the same conditions. The reduction rate refers to the percentage decrease in cell concentration in the treated sample compared to the control sample.

[0103] VI. Test on the ability of strain S2205 to produce 5-aminolevulinic acid

[0104] The experiment to test the ability of strain S2205 to produce 5-aminolevulinic acid (5-ALA) was carried out according to the method in the literature (Sun Yong, Wu Ren, Zhang Li, et al. Cloning and sequence analysis of hemA gene of high-yielding 5-aminolevulinic acid photosynthetic strain [J]. Journal of Jinan University, 2007, 28(5): 518-523.). In summary: (1) Strain S2205 was cultured in a Molisch medium at 30℃ in a light incubator for 72h (12h light: 12h no light). The components of Molisch medium were: peptone 10g / L; glycerol 5g / L; MgSO4 0.5g / L; KH2PO4 0.5g / L; FeSO4 0.03g / L, and deionized water to 1L; pH 7.2. (2) Under aseptic conditions, take 5 mL of bacterial culture and centrifuge at 5000 rpm for 15 min. Take 2 mL of the supernatant and transfer it to another test tube. Add 2 mL of 2 mol / L sodium acetate (pH 4.6) buffer and 0.5 mL of levulinic acid. Heat in a boiling water bath for 15 min. After cooling to room temperature, take 2 mL of the reaction solution and mix it with 2 mL of Ehrlich's reagent. After stabilizing for 15 min, determine its OD value. 553nm The absorbance was used to calculate the 5-ALA production value of the strain based on the standard curve. The experiment was repeated in triplicate, and the average value was taken. Distilled water was used as a blank control. The experimentally determined 5-ALA production of strain S2205 was approximately 5.21 mg / L.

[0105] The preparation of the standard curve is summarized as follows: Precisely prepare 5-ALA·HCl standard sample solutions (Sigma products) with mass concentrations of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, and 6.0 mg / L. Take 2 mL of each concentration of 5-ALA·HCl standard sample solution and transfer it to another tube. Add 2 mL of 2 mol / L sodium acetate buffer (pH 4.6) and 0.5 mL of levulinic acid. Heat in a boiling water bath for 15 min and then cool to room temperature. Mix 2 mL of the above reaction solution with 2 mL of Ehrlich's reagent. After stabilizing for 15 min, measure the absorbance of the mixture at 553 nm using a spectrophotometer with a cuvette. Use distilled water as a blank control. Preparation of Ehrlich's reagent: In a 50mL graduated cylinder, add 30mL of glacial acetic acid, 1g of p-dimethylaminobenzoic acid, and 8mL of 70% perchloric acid in sequence. After dissolving, dilute to 50mL with glacial acetic acid and store in a brown bottle at 4℃ for later use.

[0106] The rate-limiting enzyme gene for the C4 pathway of 5-ALA synthesis in photosynthetic bacteria is 5-ALA synthase, which is encoded by the hemA gene. We retrieved the hemA gene (C8J29_RS06575) from the genome of strain S2205.

[0107] The genetic and phenotypic characteristics of strain S2205 mutually confirm that the strain has a strong ability to produce 5-ALA.

[0108] 5-ALA can be used as a natural herbicide, insecticide, and plant growth promoter (Sasikalac, Ramamav, Raghuveerrp. 5-Aminolevulinic acid: a potential herbicide / insecticide from microorganisms[J]. Biotechnol Prog, 1994, 10: 451-459.). Therefore, the strain of this invention is an excellent microbial resource for preparing microbial inoculants to improve the crop ecological environment and promote healthy crop growth.

[0109] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Photosynthetic nitrogen-fixing Rhodobacter Rhodobacter photoazotoformans ) S2205, whose accession number in the General Microbiological Center of China Culture Collection Administration is CGMCC No.26164.

2. The photosynthetic nitrogen-fixing red bacteria of claim 1 ( Rhodobacter photoazotoformans ) The culture of S2205 is the photosynthetic nitrogen-fixing red bacteria described in claim 1 ( Rhodobacter photoazotoformans ) Biologically pure culture of S2205.

3. A bacterial agent, characterized in that: The bacterial agent contains the photosynthetic nitrogen-fixing red bacteria according to claim 1 ( Rhodobacter photoazotoformans ) S2205 and / or the culture described in claim 2.

4. The bacterial agent according to claim 3, characterized in that: The microbial agent is a microbial agent for inhibiting algae growth and / or a microbial agent for weed control and / or a microbial agent for killing insects and / or a microbial agent for promoting plant growth; The algae is cyanobacteria; The cyanobacteria is Anabaena blooms.

5. The photosynthetic nitrogen-fixing red bacteria according to claim 1 ( Rhodobacter photoazotoformans ) Use of S2205 or the culture according to claim 2 or the bacterial agent according to claim 3 or 4 in any of the following: (A1) Inhibit algae growth; (A2) preparing a product for inhibiting algal growth; (A3) Control of algal blooms; (A4) Preparation of products for use in controlling algal blooms; (A5) Weed control; (A6) Preparation of products for use in killing weeds; (A7) insecticide; (A8) Preparation of products for use as insecticides; (A9) Promote plant growth; (A10) Preparation of products for promoting plant growth; (A11) producing 5-aminolevulinic acid; (A12) preparing a product for producing 5-aminolevulinic acid; (A13) Improve the ecological environment of crops; (A14) Preparation of products for improving the ecological environment of crops; The algae are cyanobacteria; the bloom is cyanobacteria bloom; The cyanobacteria is Anabaena blooms.

6. A product for inhibiting algae growth, the active ingredient of which is the photosynthetic nitrogen-fixing red bacteria ( Rhodobacter photoazotoformans ) S2205 or the culture of claim 2 or the bacterial agent of claim 3 or 4; The algae is cyanobacteria; The cyanobacteria is Anabaena blooms.

7. A product for controlling algal blooms, the active ingredient of which is the photosynthetic nitrogen-fixing red bacteria ( Rhodobacter photoazotoformans ) S2205 or the culture of claim 2 or the bacterial agent of claim 3 or 4; The bloom is a cyanobacterial bloom; The cyanobacteria is Anabaena blooms.

8. A product for weed control, the active ingredient of which is the photosynthetic nitrogen-fixing red bacteria ( Rhodobacter photoazotoformans ) S2205 or the culture described in claim 2 or the bacterial agent described in claim 3 or 4.

9. A product for insecticide, the active ingredient of which is the photosynthetic nitrogen-fixing red bacteria ( Rhodobacter photoazotoformans ) S2205 or the culture described in claim 2 or the bacterial agent described in claim 3 or 4.

10. A product for promoting plant growth, the active ingredient of which is the photosynthetic nitrogen-fixing red bacteria ( Rhodobacter photoazotoformans ) S2205 or the culture described in claim 2 or the bacterial agent described in claim 3 or 4.

11. A method for inhibiting algae growth, comprising the following steps: using the photosynthetic nitrogen-fixing red bacteria ( Rhodobacter photoazotoformans ) S2205 or the culture described in claim 2 or the bacterial agent described in claim 3 or 4 to treat the algae sample to be treated; The algae is cyanobacteria; The cyanobacteria is Anabaena blooms.

12. A method for controlling algal blooms, comprising the following steps: using the photosynthetic nitrogen-fixing red bacteria ( Rhodobacter photoazotoformans ) S2205 or the culture described in claim 2 or the bacterial agent described in claim 3 or 4 is used to treat the algal bloom water body to be treated; The bloom is a cyanobacterial bloom; The cyanobacteria is Anabaena blooms.

13. A method for weeding and / or insecticide and / or promoting plant growth, comprising the following steps: using the photosynthetic nitrogen-fixing red bacteria ( Rhodobacter photoazotoformans ) S2205 or the culture described in claim 2 or the fungal agent described in claim 3 or 4 is used to treat the plant to be treated or its growth medium.

14. The photosynthetic nitrogen-fixing red bacteria of claim 1 ( Rhodobacter photoazotoformans ) Use of S2205 in the preparation of the culture described in claim 2 or the bacterial agent described in claim 3 or 4.

Images