AI New Species of Rhodopseudomonas and Its Application

Abstract

The present invention discloses a new species of AI Rhodopseudomonas and its application. The new species of Rhodopseudomonas provided by the present invention is specifically Rhodopseudomonas agrinica, whose strain number is Y009, and its preservation number in the General Microbiological Center of the China Microbiological Culture Collection Administration is CGMCC No.26446. Through the study of the biological characteristics of strain Y009, it was found that strain Y009 can tolerate CrCl2 with a concentration of 15%; and has a strong ability to produce 5-ALA. Accordingly, strain Y009 of the present invention can be used for environmental remediation of heavy metal pollution, improvement of farmland environment, promotion of crop growth, etc.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a new species of AI Rhodopseudomonas and applications thereof. Background Art

[0002] Rhodopseudomonas belongs to the Bacteria domain, Pseudomonas phylum, Alphaproteobacteria, Hyphomicrobiales, Nitrobacteraceae, and is one of the oldest prokaryotic organisms on Earth with a primitive photosynthesis system. This type of bacteria is purple non-sulfur photosynthetic bacteria, most of which are anaerobic photoautotrophic bacteria, but some species can carry out oxidative metabolism under lightless microaerobic or aerobic conditions (Mcgrath JE, Harfoot CG. Reductive dehalogenationg of halocarboxylic acids By the phototrophicgenera Rhodosirillum and Rhodopseudomonas. Appl. Environ. Microbiol. 1997, 63(8): 3333-3335; Yu JA, Ye YJ, Lin ZX, et al. Effect of carotenoids in photosynthetic bacteria on inhibiting lipid per-oxidation. Shanghai Traffic Univ. 1998, 32(3): 107-109). Early studies found that this type of photosynthetic bacteria (PSB) plays an important role in the material cycle, energy conversion and water purification of aquatic ecosystems (Kobayashi Masayu. Treatment of high-concentration organic wastewater by photosynthetic bacteria. Fermentation and Industry (Japan). 1978, 36(9): 753-760). Therefore, Rhodopseudomonas plays many roles in aquaculture and sewage treatment (Lv Hong, Zhou Jitui, Wang Jing. Research progress on photosynthetic bacteria degrading organic pollutants. Industrial Water Treatment. 2003, 23 (10): 9-12), such as degrading organic matter, purifying water quality by absorbing ammonia nitrogen, hydrogen sulfide and other harmful substances in water bodies, reducing water oxygen consumption by absorbing oxygen-consuming factors in water bodies, and serving as bait and feed additives for cultivating zooplankton (Zhao Ling, Shen Chaoping, Chen Weimin. Application of photosynthetic bacteria (PSB) in juvenile turtle farming. Inland Fisheries. 2005, 6 (10): 17-19; Zhang Xindi, Chen Ying. Effects of photosynthetic bacteria on water quality of cumbria aquaculture water bodies. Freshwater Fisheries. 2007, 12 (1): 30-34). Rhodopseudomonas palustris is a typical species of this genus (https: / / lpsn.dsmz.de / genus / rhodopseudomonas) and was first identified by van Niel in 1944 (van Niel CB. THE CULTURE, GENERAL PHYSIOLOGY, MORPHOLOGY, AND CLASSIFICATION OF THE NON-SULFUR PURPLE AND BROWN BACTERIA. Bacteriol Rev 1944; 8: 1-118.). Since then, Rhodopseudomonas strains have been isolated and cultured from a variety of environments, such as freshwater ponds (Oda, Y.; Wanders, W.; Huisman, LA; Meijer, WG; Gottschal, JC; Forney, LJ Genotypic and phenotypic diversity within species of purple nonsulfur bacteria isolated from aquatic sediments. Appl. Environ. Microbiol. 2002, 68, 3467–3477; Rayyan, A.; Meyer, T.; Kyndt, J. Draft whole-genome sequence of the purple photosynthetic bacterium Rhodopseudomonas palustris XCP. Microbiol. Res. Announc. 2018, 7, e00855-18; Kim, MK; Choi, KM; Yin, CR; Lee, KY; Im, WT; Lim, JH; Lee, ST. Odorous swine wastewater treatment by purplenon-sulfur bacteria, Rhodopseudomonas palustris, isolated from eutrophicatedponds. Biotechnol. Lett. 2004, 26, 819–822), activated sludge (Hiraishi, A.; Kitamura, H. Distribution of phototropic purple nonsulfur bacteria in activated sludgesystems and other aquatic environments. Bull. Jpn. Soc. Sci.Fish 1984, 50, 1929–1937; Venkidusamy, K.; Megharaj, M. A novel electrophototrophic bacterium Rhodopseudomonas palustris strain RP2 exhibits hydrocarbonoclastic potential in anaerobic environments. Front. Microbiol. 2016, 7, 1071), soil (Akiba, T.; Usami, R.; Horikoshi, K. Rhodopseudomonas rutila, a new species of nonsulfur purple photosynthetic bacteria. Int. J. Syst. Bacteriol. 1983, 33, 551–556; Madigan, M. T.; Gest, H. Selective enrichment and isolation of Rhodopseudomonas palustris using trans-cinnamic acid as sole carbon source. FEMS Microbiol. Ecol. 1988, 53, 53–58) and alkaline water (Cetinkaya Donmez, G.; Ozturk, A.; Cakmakci, L. Properties of the Rhodopseudomonas palutris strains isolated from an alkaline lake in Turkey. Turk. J. Biol. 1999, 23, 457–464.).

[0003] The cells of Rhodopseudomonas strains are Gram-negative rods that move by a single polar flagellum. Colonies and liquid cultures grown anaerobically under light are brown-red and light peach-brown, respectively. Colonies grown aerobically in the dark are light red with white edges throughout. The cells have bacteriochlorophyll a and carotenoids of the normal spirulinaxanthin series (Weaver PF, Wall JD, Gest H. Characterization of Rhodopseudomonas capsulam [J]. Arch Microbiol, 1975, (105): 207-216.). To date, the genus Rhodopseudomonas contains 10 validly described species (https: / / lpsn.dsmz.de / genus / rhodopseudomonas).

[0004] Rhodopseudomonas is an attractive option for biotechnological applications and industrial engineering due to its metabolic versatility and ability to decompose a variety of feedstocks and convert them into several high-value products. It has strong metabolic capacity and has attracted extensive attention at home and abroad (Adessi, A., Concato, M., Sanchini, A., Rossi, F., De Philippis, R., 2016. Hydrogen production under salt stress conditions by a freshwater Rhodopseudomonas palustris strain. Appl Microbiol Biotechnol 100, 2917–2926; Bajracharya, S., Sharma, M., Mohanakrishna, G., Dominguez Benneton, X., Strik, DPBTB, Sarma, PM, Pant, D., 2016. An overview on emerging bioelectrochemical systems (BESs): Technology for sustainable electricity, waste remediation, resource recovery, chemical production and beyond. Renew. Energy 98, 153–170; Brown B, Wilkins M, Saha R. Rhodopseudomonas palustris: A biotechnology chassis. Biotechnol Adv. 2022 Nov; 60: 108001. doi: 10.1016 / j.biotechadv.2022.108001. Epub 2022 Jun 6. PMID: 35680002.). Therefore, we hope to collect more abundant bacterial resources and provide high-quality candidate strains for environmental remediation, industrial and agricultural production, and biotechnology engineering research. Summary of the invention

[0005] The purpose of the present invention is to provide a new species of AI Rhodopseudomonas and its application.

[0006] In a first aspect, the present invention claims a new species of Rhodopseudomonas.

[0007] The new species of the genus Rhodopseudomonas claimed for protection in the present invention is specifically AI Rhodopseudomonas agrinica Y009, and its preservation number in the General Microbiological Center of China National Microbiological Culture Collection Committee is CGMCC No. 26446.

[0008] The AI ​​Rhodopseudomonas agrinica Y009 is a Gram-negative bacterium, mesophilic, and facultatively aerobic. After the strain is cultured on NA medium at 30°C for 48 hours, moist, smooth and light yellow colonies can be formed. The growth conditions of strain Y009 are 22-37°C, 0-3% NaCl and pH 6.0-10.0, and the optimal growth conditions are 30-32°C, 0% NaCl, pH 7.0-9.0. Strain Y009 can grow on a CrCl2 medium containing 0-15g / 100ml, that is, the tolerance of strain Y009 to CrCl2 reaches 15%. The oxidase and catalase tests of strain Y009 are positive, and the hydrolysis of esculin is positive; the gelatin liquefaction test is negative, and it can hydrolyze starch and cellulose.

[0009] In a second aspect, the invention claims a culture.

[0010] The culture claimed in the present invention is the culture of AI Rhodopseudomonas agrinica Y009 described in the first aspect above, which is a substance obtained by culturing the AI ​​Rhodopseudomonas agrinica Y009 in a bacterial culture medium.

[0011] In the above culture, the substances include the culture of AI Rhodopseudomonas agrinica Y009 (the bacteria itself) and metabolites of AI Rhodopseudomonas agrinica Y009.

[0012] In the above culture, the bacterial culture medium may be a solid culture medium or a liquid culture medium.

[0013] 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.

[0014] In a specific embodiment of the present invention, the bacterial culture medium is specifically NA medium.

[0015] In a third aspect, the present invention claims a metabolite.

[0016] The metabolite claimed in the present invention is the metabolite of AI Rhodopseudomonas agrinica Y009 described in the first aspect above.

[0017] 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.

[0018] In a fourth aspect, the present invention claims protection for a bacterial agent.

[0019] The bacterial agent claimed in the present invention contains the AI ​​Rhodopseudomonas agrinica Y009 described in the first aspect above, the culture described in the second aspect above and / or the metabolites described in the third aspect above.

[0020] Wherein, the microbial agent is a microbial agent for weed control and / or a microbial agent for insecticide and / or a microbial agent for promoting plant growth.

[0021] 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.

[0022] The above-mentioned bacterial agents may be in various dosage forms, such as liquid, emulsion, suspension, powder, granule, wettable powder or water dispersible granule.

[0023] 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.

[0024] In a fifth aspect, the present invention claims the use of the AI ​​Rhodopseudomonas agrinica Y009 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:

[0025] (A1) producing 5-aminolevulinic acid;

[0026] (A2) preparing a product for producing 5-aminolevulinic acid;

[0027] (A3) weed control;

[0028] (A4) preparing products for use in weed control;

[0029] (A5) insecticide;

[0030] (A6) preparing products for use in killing insects;

[0031] (A7) promoting plant growth;

[0032] (A8) preparing products for promoting plant growth;

[0033] (A9) Repair heavy metal polluted environment;

[0034] (A10) preparing products for repairing heavy metal polluted environments;

[0035] (A11) Improve the ecological environment of crops;

[0036] (A12) Preparing products for improving the ecological environment of crops.

[0037] The above-mentioned applications may be applications under normal conditions or applications under heavy metal stress.

[0038] Wherein, the heavy metal is cadmium. The AI ​​Rhodopseudomonas agrinica Y009 can tolerate 150 g / L CrCl2.

[0039] In a sixth aspect, the present invention claims a product for producing 5-aminolevulinic acid.

[0040] The product for producing 5-aminolevulinic acid claimed in the present invention has the active ingredient of the AI ​​Rhodopseudomonas agrinica Y009 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.

[0041] In a seventh aspect, the present invention claims a product for weed control and / or insect killing and / or promoting plant production.

[0042] The product for weed control and / or insecticide control and / or plant production promotion claimed in the present invention has the active ingredient of the AI ​​Rhodopseudomonas agrinica Y009 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.

[0043] In an eighth aspect, the present invention claims a method for producing 5-aminolevulinic acid.

[0044] The method for producing 5-aminolevulinic acid claimed in the present invention may include the following steps: culturing the AI ​​Rhodopseudomonas agrinica Y009 described in the first aspect above, and obtaining 5-aminolevulinic acid from the culture.

[0045] In a ninth aspect, the present invention claims a method for weeding and / or insecticidal and / or promoting plant production.

[0046] The method for weeding and / or insecticide and / or promoting plant production claimed in the present invention may include the following steps: treating the plant to be treated or its growth substrate with the AI ​​Rhodopseudomonas agrinica Y009 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.

[0047] In the tenth aspect, the present invention claims protection for the use of the AI ​​Rhodopseudomonas agrinica Y009 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.

[0048] Experiments have shown that the strain Y009 of the present invention represents a new species of the genus Rhodopseudomonas, named AI Rhodopseudomonas agrinica. Through the study of the biological characteristics of strain Y009, it was found that strain Y009 can tolerate CrCl2 at a concentration of 15% (w / v); and has a strong ability to produce 5-ALA. Accordingly, the strain of the present invention, AI Rhodopseudomonas agrinica Y009, can be used for environmental remediation of heavy metal pollution, improvement of farmland environment, promotion of crop growth, etc.

[0049] Collection Instructions

[0050] Taxonomic nomenclature: Rhodopseudomonas agrinica;

[0051] Reference biological materials: Y009;

[0052] Depository: General Microbiology Center, China Microbiological Culture Collection Administration;

[0053] Abbreviation of depository institution: CGMCC;

[0054] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0055] Deposit date: January 12, 2023;

[0056] The registration number of the Collection Center is: CGMCC No.26446. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 This is the colony morphology formed by strain Y009 after culturing on NA medium for 48 hours.

[0058] Figure 2 This is the detection result of polar lipid components of strain Y009.

[0059] Figure 3 A phylogenetic tree was constructed based on the 16S rRNA gene sequences of strain Y009 and related strains. The phylogenetic tree used Rhizobium leguminosarum USDA 2370 (U29386) as the outgroup.

[0060] Figure 4 Preparation of 5-ALA standard curve and three replicates of 5-ALA production of strain Y009. Solid circles represent samples of different concentrations of standard products used to prepare the standard curve, and solid triangles represent samples used to measure 5-ALA production of strain Y009 three times. DETAILED DESCRIPTION

[0061] 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.

[0062] 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.

[0063] Example 1. Isolation, screening and identification of strain Y009

[0064] 1. Isolation of strain Y009

[0065] Strain Y009 was isolated from the sediment sample of Nanwan Reservoir in Xinyang City, Henan Province. The isolation medium was R2A medium: 0.5g yeast extract powder, 0.5g peptone, 0.5g casamino acid (casein amino acids, casein hydrolysate), 0.5g glucose, 0.5g soluble starch, 0.3g dipotassium hydrogen phosphate, 0.05g magnesium sulfate heptahydrate, 0.3g sodium pyruvate, distilled water, mixed evenly and fixed to 1000mL, pH7.5. The strain isolation, purification method and operation process were carried out according to the method 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 pure strains obtained by separation and purification were frozen in liquid nitrogen and stored at -80°C using 20% ​​(v / v) glycerol as a protective agent.

[0066] In this experiment, strain Y009 was isolated and purified.

[0067] 2. Cell morphology observation and physiological and biochemical characteristics detection of strain Y009

[0068] The following experiments were basically carried out using NA solid medium (with agar added) or NA liquid medium (without agar added). Special cases will be explained separately.

[0069] NA medium ingredients: peptone 10g, beef extract 3g, sodium chloride 5g, distilled water, mix well and make up to 1000mL, pH 7.0.

[0070] The growth temperature detection range of strain Y009 is 10-45°C, and the detection points include 10, 15, 20, 22, 25, 28, 30, 32, 35, 37, 40, 42 and 45°C; the growth salt concentration (NaCl) detection range is 0-8% (0-8g / 100ml) with 9 concentration gradients (0, 1, 2, 3, 4, 5, 6, 7, 8); the strain's tolerance to cadmium chloride is tested by adding CrCl2 to NA medium, and the detection range is 5 concentration gradients (0, 5, 10, 15, 20g / 100ml) with 0-20g / 100ml; the growth pH detection range is 8 gradients (4, 5, 6, 7, 8, 9, 10, 11) between 4-11. The physiological and biochemical functions of the strain are detected using detection kits API 50CH, APIZYM, and BiOLOG GEN III plates and corresponding operating methods. Other physiological and biochemical characteristics of the strain, 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 Manual of Identification of Common Bacterial Systems (Dong Xiuzhu, Cai Miaoying. 2001. Manual of Identification of Common Bacterial Systems. Beijing: Science Press).

[0071] The identification results showed that strain Y009 was a Gram-negative bacterium, mesophilic, and facultatively aerobic. After culturing on NA medium at 30°C for 48 hours, the strain formed moist, smooth, and light yellow colonies ( Figure 1 ). The growth conditions of strain Y009 are 22-37°C, 0-3% NaCl and pH 6.0-10.0, and the optimal growth conditions are 30-32°C, 0% NaCl, pH 7.0-9.0. Strain Y009 can grow on a culture medium containing 0-15g / 100ml of CrCl2, that is, the tolerance of strain Y009 to CrCl2 reaches 15% (i.e. 15g / 100ml). The oxidase and catalase tests of strain Y009 were positive, and the hydrolysis of esculin was positive; the gelatin liquefaction test was negative, and it was able to hydrolyze starch and cellulose. The phenotypic distinguishing characteristics of strain Y009 and the closely related bacterium Rhodopseudomonas boonkerdiiNS23 are shown in Table 1.

[0072] Table 1. Distinguishing phenotypic characteristics of strain Y009 and its closest relative Rhodopseudomonas boonkerdii NS23

[0073] Strain characteristics Y009 NS23 Optimum growth temperature (℃) 30-32 25-32 NaCl tolerance range (%) 0-3 0-1 Optimal growth pH 7.0-9.0 6.5-8.0 pigment + - Nitrate reduction + - Urea hydrolysis w + Esculin w - Carbon source utilization D-glucose + - L-arabinose - + D-mannose + - D-mannitol - + N-acetylglucosamine w - D-maltose + - Potassium gluconate - + Adipic acid - + Malic acid - + Trisodium citrate + - Phenylacetic acid w -

[0074] Note: In the table, + indicates positive, - indicates negative, and w indicates weak positive.

[0075] 3. Cytochemical characteristics of strain Y009

[0076] The cellular chemical components of strain Y009, 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 main fatty acid of strain Y009 is C 18:1 ω7c and C 16:0. As shown in Table 2, the main fatty acid components of strain Y009 are consistent with the taxonomic characteristics of the genus Rhodopseudomonas; the specific content of each fatty acid component is different from that of the closely related species of Rhodopseudomonas (Noisangiam R, Nuntagij A, Pongsilp N, Boonkerd N, Denduangboripant J, Ronson C, Teaumroong N. Erratum to "Heavy metal tolerant Metalliresistens boonkerdii gen.nov., sp.nov., a new genusin the family Bradyrhizobiaceae isolated from soil in Thailand" [Syst.Appl.Microbiol.33(2010)374-382]. Proposal of Rhodopseudomonas boonkerdiisp.nov., a new heavy metal tolerant bacterium isolated from Thailand.Syst.Appl.Microbiol.2011;34:166-168; Ramana VV, Chakravarthy SK, Raj PS, Kumar BV, Shobha E, Ramaprasad EV, Sasikala Ch, Ramana ChV. Descriptions of Rhodopseudomonas parapalustris sp. nov., Rhodopseudomonas harwoodiae sp. nov. and Rhodopseudomonas pseudopalustris sp. nov., and emended description of Rhodopseudomonas palustris. Int J Syst Evol Microbiol 2012; 62: 1790-1798.). The only respiratory quinone of strain Y009 is Q-10. In strain Y009, the polar lipid components include diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), phospholipids of unknown composition (PL) and unknown polar lipids containing amino groups (AL) such as Figure 2Among them, the dominant fatty acids, quinones and major polar lipid components all confirmed that strain Y009 was a member of Rhodopseudomonas; at the same time, the trace components and their contents of the fatty acid composition of strain Y009 could distinguish strain Y009 from other species of Rhodopseudomonas.

[0077] Table 2. Fatty acid composition of strain Y009

[0078]

[0079]

[0080] IV. Determination of the phylogenetic status of strain Y009

[0081] The genomic DNA of strain Y009 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 Y009 of the present invention had the highest similarity with the species of the Nitrobacteraceae. The 16S rRNA gene of the closely related strain was retrieved and the 16S rRNA gene of the strain Y009 was used to construct a phylogenetic tree. In the phylogenetic tree of the Nitrobacteraceae, strain Y009 was clustered on the evolutionary branch of Rhodopseudomonas, and formed a stable sub-evolutionary branch with Rhodopseudomonas boonkerdii NS23 ( Figure 3); the 16S rRNA gene similarity between strain Y009 and the typical strain of Rhodopseudomonas boonkerdii NS23 was 98.0%. This value is lower than the limit value of 98.65% for distinguishing prokaryotic microbial species (Kim M, Oh HS, Park SC, Chun J. Towards a taxonomic coherence between average nucleotide identity and 16SrRNA gene sequence similarity for species demarcation of prokaryotes. Int J Syst Evol Microbiol 2014; 64: 346–351. Therefore, strain Y009 is a member of Rhodopseudomonas that is different from known species.

[0082] To further clarify the taxonomic status of the strain, the average nucleotide similarity (ANI value) between the whole genome sequence of strain Y009 and the whole genome sequence of the closest reference bacterium Rhodopseudomonas boonkerdii NS23 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) between the whole genome sequence of strain Y009 and the closest reference bacterium Rhodopseudomonas boonkerdii NS23 was 85.1%. The values ​​are far below 95% - the ANI threshold for distinguishing prokaryotic microbial 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 Y009 represents a new species within the genus Rhodopseudomonas. The GC content of the genome of strain Y009 was calculated to be 62.1% based on the whole genome sequence of strain Y009.

[0083] Based on the taxonomic data of strain Y009 in terms of phenotype and genotype and its comparison with closely related strains, we determined that strain Y009 represents a new species of Rhodopseudomonas. Its Latin name is named Rhodopseudomonas agrinica, and its Chinese name is AI Red Pseudomonas.

[0084] Rhodopseudomonas agrinica Y009 was deposited in the China General Microbiological Culture Collection Center on January 12, 2023, and its deposit number is CGMCC No.26446.

[0085] 5. Test on the ability of strain Y009 to produce 5-aminolevulinic acid (5-ALA)

[0086] The 5-aminolevulinic acid (5-ALA) production capacity test of strain Y009 was carried out according to the literature method (Sun Yong, Wu Ren, Zhang Li, et al. Cloning and sequence analysis of hemA gene of photosynthetic bacteria with high 5-aminolevulinic acid production [J]. Journal of Jinan University, 2007, 28 (5): 518-523.). In summary: (1) strain Y009 was cultured in a light incubator at 30°C (12 h light: 12 h no light) for 72 h. The components of Molisch medium: peptone 10 g / L; glycerol 5 g / L; MgSO4 0.5 g / L; KH2PO4 0.5 g / L; FeSO4 0.03 g / L, deionized water was added to 1 L; pH 7.2. (2) Under aseptic conditions, take 5 mL of bacterial solution 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 stabilization for 15 min, measure its OD value. 553nm The absorbance value was calculated based on the standard curve to determine the 5-ALA production of the strain. The experiment was repeated three times in parallel, with distilled water used as a blank control. The 5-ALA values ​​obtained were 4.89 mg / L, 5.12 mg / L, and 4.90 mg / L ( Figure 4 ). The average value of the three measurements was 4.97 mg / L. That is, the experimental measurement showed that the yield of 5-ALA produced by strain Y009 was approximately 4.97 mg / L.

[0087] The preparation of the standard curve is summarized as follows: accurately 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 5-ALA·HCl standard sample solutions of different concentrations into another 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 minutes, and then cool to room temperature. Take 2 mL of the above reaction solution and mix it with 2 mL of Ehrlich's reagent. After stabilization for 15 minutes, use a cuvette to detect the absorbance of the mixed solution with a spectrophotometer at 553 nm. Distilled water is used as a blank control. Preparation of Ehrlich's reagent: In a 50mL measuring cylinder, add 30mL of glacial acetic acid, 1g of p-dimethylaminobenzoic acid, and 8mL of 70% perchloric acid in sequence, dissolve them, and then dilute to 50mL with glacial acetic acid. Place them in a brown bottle and store at 4°C for later use. The standard curve prepared in this experiment is shown in Figure 4 .

[0088] The rate-limiting enzyme gene of the C4 pathway for photosynthetic bacteria to synthesize 5-ALA is 5-ALA synthase, which is encoded by the hemA gene. We retrieved the hemA gene (C8J29_RS06575) in the genome of strain Y009.

[0089] The genetic characteristics and phenotypic characteristics of strain Y009 confirm each other: strain Y009 has a strong ability to produce 5-ALA.

[0090] 5-ALA can be used as a natural herbicide, insecticide and plant growth promoter (Sasikalac, Ramamav, Raghuveer RP. 5-Aminolevulinic acid: a potential herbicide / insecticide from microorganisms [J]. Biotechnol Prog, 1994, 10: 451-459.). Therefore, the strain Y009 of the present invention is an excellent strain resource for preparing microbial agents for improving the ecological environment of crops and promoting the healthy growth of crops.

[0091] Based on the taxonomic characteristics of strain Y009, we determined that the isolated strain Y009 is a new species of the genus Rhodopseudomonas. Through the study of the biological characteristics of strain Y009, it was found that strain Y009 can tolerate CrCl2 with a concentration of 15% (w / v); and has a strong ability to produce 5-ALA. Based on this, strain Y009 of the present invention can be used for environmental remediation of heavy metal pollution, improvement of farmland environment, and promotion of crop growth.

[0092] 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. Rhodopseudomonas, characterized in that: The Rhodopseudomonas Rhodopseudomonas agrinica Its strain number is Y009, and its collection number at the General Microbiology Center of China Culture Collection Administration is CGMCC No.26446.

2. The culture of Rhodopseudomonas according to claim 1, which is obtained by culturing the Rhodopseudomonas according to claim 1 in a bacterial culture medium.

3. A bacterial agent, characterized in that: The bacterial agent contains the Rhodopseudomonas according to claim 1 and / or the culture according to claim 2.

4. The bacterial agent according to claim 3, characterized in that: The microbial agent is a microbial agent for weed control and / or a microbial agent for insecticide and / or a microbial agent for promoting plant growth.

5. Use of the Rhodopseudomonas according to claim 1, the culture according to claim 2, or the bacterial agent according to claim 3 or 4 in any of the following: (A1) Production of 5-aminolevulinic acid; (A2) preparing a product for producing 5-aminolevulinic acid; (A3) Weed control; (A4) preparing products for use in killing weeds; (A5) Insecticide; (A6) Preparation of products for use as insecticides; (A7) Promote plant growth; (A8) Preparation of products for promoting plant growth; (A9) Improvement of crop ecological environment; (A10) Preparing products for improving the ecological environment of crops.

6. A product for producing 5-aminolevulinic acid, wherein the active ingredient is the Rhodopseudomonas according to claim 1 or the culture according to claim 2 or the bacterial agent according to claim 3 or 4.

7. A product for weeding and / or insecticide and / or promoting plant growth, wherein the active ingredient is the Rhodopseudomonas according to claim 1 or the culture according to claim 2 or the bacterial agent according to claim 3 or 4.

8. A method for producing 5-aminolevulinic acid, comprising the following steps: culturing the Rhodopseudomonas according to claim 1, and obtaining 5-aminolevulinic acid from the culture.

9. A method for weeding and / or insecticide and / or promoting plant growth, comprising the following steps: treating a plant to be treated or its growth substrate with the Rhodopseudomonas according to claim 1 or the culture according to claim 2 or the bacterial agent according to claim 3 or 4.

10. Use of the Rhodopseudomonas according to claim 1 in preparing the culture according to claim 2 or the bacterial agent according to claim 3 or 4.

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