Streptomyces griseorubens and application of streptomyces griseorubens in phthalate pollution remediation

Abstract

The invention discloses streptomyces griseorubens and an application of the streptomyces griseorubens in phthalate pollution remediation. The bacterial strain is Streptomyces griseorubens FZ202, the preservation number of the bacterial strain is GDMCC No: 61372, the bacterial strain has good environmental adaptability and efficient degradation capacity, DEHP can be effectively degraded within 100-800 mg / L, the highest degradation efficiency can reach 94.1%, and complete degradation can be achieved. The strain is inoculated into PAEs contaminated soil and interacts with rice, DEHP residues in the soil and crop bodies can be effectively reduced, various PAEs can be synergistically degraded, and the strain has the broad-spectrum degradation characteristic. Meanwhile, through multiple mechanisms of degrading pollutants, promoting plant growth, improving the soil environment and regulating and controlling microbial communities, the strain remarkably improves the growth performance of rice and the self-cleaning capacity of soil, and has a wide agricultural application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of soil new pollutant treatment and agricultural environmental protection technology, and particularly relates to a Streptomyces griseorubens and application thereof in phthalate pollution remediation. BACKGROUND

[0002] Phthalic acid esters (PAEs) are a class of typical endocrine disruptors (EDCs) and belong to the category of new pollutants. Common PAEs mainly include diethyl phthalate (DEP), dimethyl phthalate (DMP), di(2-ethylhexyl) phthalate (DEHP) and di-n-butyl phthalate (DBP) and the like. [1] Due to industrial “three wastes” discharge, agricultural mulching, sewage irrigation and misuse of pesticides and fertilizers, PAEs have been continuously entering farmland system for a long time and accumulated in environmental media such as soil and water. [2] PAEs can enter the human body through food chain intake, respiration or skin contact and the like [3] , posing a serious threat to the safety of agricultural products and human health [4] . Studies have shown that PAEs have endocrine disrupting, reproductive toxicity, hepatotoxicity and carcinogenic risk, and can affect the reproductive system, immune system and respiratory system of the body [5-7] . Due to its significant biological toxicological effect and slow natural degradation rate in the environment, PAEs are listed as typical persistent organic pollutants (POPs). How to reduce the PAEs pollution in soil and crops and realize the coordinated development of agricultural production and environmental remediation has become a technical problem to be solved.

[0003] The migration and transformation of PAEs in the environment mainly include abiotic processes and biological processes. The abiotic processes such as photochemical degradation and hydrolysis have low degradation efficiency; while the biological processes mainly rely on the metabolic action of microorganisms, plants or animals. Compared with traditional chemical or physical remediation technologies (such as adsorption, chemical oxidation), microbial remediation has become a research hotspot for PAEs pollution treatment due to its environmental friendliness, low cost, simple operation, strong adaptability and high degradation efficiency [7-9] . Microorganisms are rich in resources and widely distributed, and have strong adaptability and high degradation capacity to pollutants [10-11]In recent years, a variety of PAEs-degrading microorganisms have been isolated from soil, activated sludge, sediments and plant rhizosphere, including Sphingomonas, Gordonia, Arthrobacter, Pseudomonas and Rhodococcus [12-14] However, most of these reported microorganisms can only degrade a single or limited types of PAEs, have poor environmental adaptability, are easily inactivated in competition with indigenous microorganisms, and are difficult to play a long-term stable repair role. Therefore, screening functional strains with broad-spectrum degradation ability and strong environmental adaptability is a key technical requirement for microbial remediation of PAEs pollution.

[0004] Actinomycetes are widely distributed in soil, reproduce by asexual spores, and have strong terrestrial adaptability. The genome of actinomycetes is large (8-9 Mbp) and contains a variety of secondary metabolic gene clusters, which can produce antibiotics, auxins, cytokinins and other bioactive substances [15-17] Streptomyces is widely distributed in nature and has strong environmental adaptability, and is commonly used in biological fertilizers in China. More than half of the 16,500 known antibiotics are produced by Streptomyces, and their secondary metabolites have important application value in environmental management and agricultural production

[18] Actinomycetes are excellent plant biocontrol and rhizosphere growth-promoting bacteria, and their research in agriculture mainly focuses on plant growth promotion, biological control and pollutant remediation, but there are few studies on the remediation of PAEs-contaminated soil, which needs further exploration.

[0005] Currently, the research on the degradation of organic pollutants by Streptomyces mainly focuses on the remediation of organic pollutants such as petroleum, polycyclic aromatic hydrocarbons and pesticide residues

[19] Through the search of existing invention patents, it was found that actinomycete-related technologies are mainly applied in the fields of agricultural waste composting, food waste treatment, straw decomposition and pesticide residue degradation, and the types of microbial agents are mainly complex microbial agents constructed by actinomycetes and fungi or other bacteria. There are few reports on the application of Streptomyces strains in the bioremediation of PAEs-contaminated soil.

[0006] References:

[0007] [1] A, Kończak M, Oleszczuk P, et al. Environmental and food contamination by phthalic acid esters (PAEs): overview[J]. Water, Air, & Soil Pollution, 2024, 235(5): 313.

[0008] [2] Kumar M, Singh N K, Varma S K, et al. Biodegradation and removal of phthalate esters from wastewater[M]. Current Developments in Biotechnology and Bioengineering. Current Developments in Biotechnology and Bioengineering, 2023: 103-126.

[0009] [3] Lu WY, Wang ZM, Zuo XY, et al. Degradation efficiency and pathway of phthalate esters by immobilized bacteria[J]. China Environmental Science, 2024, 44(3): 1584-1591.

[0010] [4] Li X, Wang Q, Jiang N, et al. Occurrence, source, ecological risk, and mitigation of phthalates (PAEs) in agricultural soils and the environment: a review[J]. Environmental Research, 2023, 220: 115196.

[0011] [5] Xiong HY, He CC, Jiao XY, et al. Occurrence and behavior of phthalate plasticizers (PAEs) in aquatic ecological environment[J]. Journal of South-Central University for Nationalities (Science Edition), 2021, 40(3): 238-245.

[0012] [6]Zhou B, Zhao L, Sun Y, et al. Contamination and human health risks of phthalate esters in vegetable and crop soils from the Huang-Huai-Hai region of China[J]. Science of the Total Environment, 2021, 778: 146281.

[0013] [7]Xu L, Zhang Y, Zhou X, et al. Functional endophytic bacteria for the reduction of phthalate esters in crops[J]. China Environmental Science, 2024, 44(11): 6442-6452.

[0014] [8]Ye D M, Yang H, Xu T T, et al. Underlying degradation of phthalates via microbials in dust from different microenvironments[J]. Environmental Science & Technology, 2023, 57(26): 9744-9753.

[0015] [9]Ruiwen H, Haiming Z, Xihui X, et al. Bacteria-driven phthalic acid ester biodegradation: Current status and emerging opportunities[J]. Environment International, 2021, 154106560-106560.

[0016]

[10] Lü H X, Huang X J. Antibiotic resistance of phthalate-degrading endophytic bacteria[J]. Journal of Henan Normal University(Natural Science Edition), 2024, 52(06): 20-26+2.

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[11] Ren L, Lin Z, Liu H, et al. Bacteria-mediated phthalic acid esters degradation and related molecular mechanisms [J]. Applied Microbiology and Biotechnology, 2018, 102: 1085-1096.

[0018]

[12] Chen F, Chen Y, Chen C, et al. High-efficiency degradation of phthalic acid esters (PAEs) by Pseudarthrobacter defluvii E5: Performance, degradative pathway, and key genes [J]. Science of The Total Environment, 2021, 794: 148719.

[0019]

[13] Xu Y, Zhao J, Huang H, et al. Biodegradation of phthalate esters by Pantoea dispersa BJQ0007 isolated from Baijiu [J]. Journal of Food Composition and Analysis, 2022, 105: 104201.

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[14] Olanrewaju O S, Babalola O O. Streptomyces: implications and interactions in plant growth promotion [J]. Applied microbiology and biotechnology, 2019, 103: 1179-1188.

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[15] Cao P, Li C, Wang H, et al. Community structures and antifungal activity of root-associated endophytic actinobacteria in healthy and diseased cucumber plants and Streptomyces sp. HAAG3-15 as a promising biocontrol agent [J]. Microorganisms, 2020, 8(2): 236.

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[16] Flardh K, Buttner M J. Streptomyces morphogenetics: Dissecting differentiation in a filamentous bacterium [J]. Nature Reviews Microbiology, 2009, 7(1): 36-49.

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[17] Wang L, Liu S, Liu LH, et al. Advances in secondary metabolism and sporulation regulation mechanisms of Streptomyces [J]. Journal of Henan Institute of Education, 2024, 44(7): 95-102.

[0024]

[18] Benimeli, C. S., Castro, G. R., Chaile, A. P., Amoroso, M. J. Lindane uptake and degradation by aquatic Streptomyces sp. strain M7. International Biodeterioration & Biodegradation, 2007, 59(2), 148-155.

[0025]

[19] Balachandran, C., Duraipandiyan, V., Balakrishna, K., Ignacimuthu, S. (2012). Petroleum and polycyclic aromatic hydrocarbons (PAHs) degradation and naphthalene metabolismin Streptomyces sp. (ERI-CPDA-1) isolated from oil contaminated soil. Bioresource Technology, 2012, 112, 83-90. SUMMARY

[0026] In order to overcome the shortcomings and deficiencies of the prior art, such as poor environmental adaptability, short survival period and easy inactivation of PAEs degrading bacteria, the purpose of the present application is to provide a Streptomyces griseorubens and its application in phthalate ester pollution remediation. The strain is isolated from the surface of rice roots and has good environmental adaptability and stable physiological metabolic characteristics, and can efficiently degrade DEHP and other phthalate ester (PAEs) pollutants. The strain FZ202 can effectively utilize short-chain and long-chain PAEs, has a broad substrate spectrum, and can effectively degrade DEHP (100-800 mg / L) of different concentrations. In addition, the strain reproduces in the form of mycelium and spores, has outstanding environmental adaptability, and can effectively solve the technical problems of short survival period and easy inactivation of current PAEs degrading bacteria.

[0027] The purpose of the present application is achieved by the following technical solutions:

[0028] The present application provides a Streptomyces griseorubens, named Streptomyces griseorubens FZ202, which is isolated from the surface of rice roots.

[0029] The preservation information of the Streptomyces griseorubens FZ202 is as follows: the preservation unit is Guangdong Microbial Culture Collection Center (GDMCC), the preservation time is December 16, 2020, the preservation address is 5th Floor, No. 59 Building, Guangdong Microbial Institute, 100 Middle Martyrs Road, Guangzhou, Guangdong Province, and the preservation number is GDMCC No: 61372.

[0030] The single colony formed by the Streptomyces griseorubens FZ202 on the MS solid culture medium is flat, the colony surface is dark gray, has obvious concave-convex texture and protrusions, the overall distribution is irregular, the colony edge is wavy, and local branch extension can be seen, which has typical Streptomyces morphological characteristics.

[0031] MS solid medium (g / L): soybean powder 20.0, mannitol 20.0, calcium carbonate 3.0, agar powder 20.0; pH 7.2.

[0032] The application provides a biological agent, which comprises the Streptomyces griseorubiginosus FZ202.

[0033] The application also provides application of the Streptomyces griseorubiginosus FZ202 or the biological agent in degradation of PAEs and / or PAEs intermediates.

[0034] Preferably, the PAEs include at least one of dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), diisooctyl phthalate (DEHP) and n-octyl phthalate (DOP), and the PAEs intermediates include at least one of mono(2-ethylhexyl) phthalate (MEHP), phthalic acid (PA) and protocatechuic acid (PCA); the strain FZ202 can grow with the PAEs compounds and the intermediates as carbon source and energy source, improve accumulation of biomass, and can degrade the PAEs compounds.

[0035] The application also provides application of the Streptomyces griseorubiginosus FZ202 or the biological agent in remediation of a medium polluted by PAEs.

[0036] Preferably, the medium is soil or water.

[0037] As a preferred embodiment, mature spores of the Streptomyces griseorubiginosus FZ202 are prepared into a spore suspension and inoculated into soil polluted by PAEs, so as to degrade the PAEs in the soil.

[0038] Preferably, the Streptomyces griseorubiginosus FZ202 is cultured in MS solid medium, and the mature spores are collected.

[0039] The concentration of the spores in the spore suspension is 1×10 8 ~ 1×10 11 spores / mL; preferably 1×10 11 spores / mL.

[0040] The inoculation amount of the mature spores is 2×10 11 spores / kg of soil.

[0041] Preferably, the application method is: mature spores of Streptomyces griseorubens FZ202 are prepared into spore suspension and inoculated into liquid medium containing PAEs for shaking culture or mycelium of Streptomyces griseorubens FZ202 is inoculated into liquid medium containing PAEs for shaking culture, and Streptomyces griseorubens FZ202 degrades PAEs during growth.

[0042] The liquid medium is preferably Gao's No.1 medium.

[0043] The shaking culture condition is 28-30℃, 200-220rpm shaking culture for 1-6 days; preferably 28℃, 220rpm shaking culture for 1-6 days.

[0044] The inoculation amount of the mature spores is 1×10 8 ~1×10 11 spores / mL.

[0045] The inoculation amount of the mycelium is 1-1.5g / L (wet weight); preferably 1.5g / L (wet weight).

[0046] The preparation method of the mycelium comprises the following steps:

[0047] The spore suspension is inoculated into TSB liquid medium for shaking culture for 3 days, and the mycelium is collected by centrifugation.

[0048] The TSB liquid medium (g / L) is as follows: soybean papain digest 3.0, typticase 17.0, NaCl 5.0, K2HPO4 2.5, glucose 2.5, pH 7.3.

[0049] The centrifugation speed is 8000rpm.

[0050] The application further provides the application of the Streptomyces griseorubens FZ202 or the biological inoculant in combined phytoremediation of PAEs contaminated soil.

[0051] Preferably, the plant is food crop rice.

[0052] Preferably, the application method is: mature spores of Streptomyces griseorubens FZ202 are prepared into spore suspension and inoculated into PAEs contaminated soil, and the inoculation amount of the mature spores is 2×10 11 spores / kg soil, and the soil is stirred uniformly, 3-4 pieces of rice seedlings with true leaves are transplanted, and the culture is performed for 30 days.

[0053] The application further provides the application of the Streptomyces griseorubens FZ202 or the biological inoculant in reducing PAEs pollution in plants.

[0054] Preferably, the plant is the food crop rice.

[0055] The application also provides the use of the Streptomyces griseorubens FZ202 or the bioinoculant in promoting plant growth.

[0056] Preferably, the plant is the food crop rice.

[0057] The application has the following advantages and effects relative to the prior art:

[0058] (1) The application screens a strain of Streptomyces griseorubens FZ202 from the root surface of rice, which has high efficiency in degrading PAEs, grows rapidly, and reproduces in the form of asexual spores and mycelium, and has strong adaptability. By adding the strain to PAEs contaminated soil and interacting with rice plants, the strain can effectively degrade DEHP (100-800 mg / L) of different concentrations, and the degradation process conforms to the first-order kinetic model. When the initial concentration of DEHP is 300 mg / L, the degradation efficiency of the strain FZ202 on DEHP is the highest, reaching 94.1%, and complete degradation can be achieved, which has good application potential. In actual application, the strain is inoculated into PAEs contaminated soil and interacts with rice, which can effectively reduce the DEHP residues in soil and crops. FZ202 not only can degrade DEHP, but also can synergistically degrade various PAEs pollutants, and has broad-spectrum degradation characteristics. At the same time, through the multiple mechanisms of "degrading pollutants-promoting plant growth-improving soil environment-regulating microbial community", the strain significantly improves the growth performance of rice and the self-purification ability of soil. The application is simple and economical, easy to industrialize, and has no biological safety problem, which has important significance for efficient use of low-concentration contaminated soil to produce safe agricultural products and realize "production and remediation simultaneously".

[0059] (2) The strain FZ202 of the application has high efficiency in degrading DEHP and other PAEs compounds, has strong environmental adaptability, can promote the growth of rice, and effectively reduce the PAEs pollution in soil and rice, which has important significance for using Streptomyces griseorubens to reduce the PAEs pollution in soil and crops and ensure the safety of agricultural products. The application provides a green remediation technology which is simple and convenient to operate, low in cost, easy to scale up, avoids biological safety risk, and is especially suitable for the synchronous implementation of ecological remediation and safe production of agricultural products in medium and low concentration PAEs contaminated soil, and has broad agricultural application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0060] Figure 1 is a colony morphology diagram of the strain FZ202 on MS solid medium.

[0061] Figure 2 is the 16S rDNA phylogenetic tree of strain FZ202.

[0062] Figure 3 is the siderophore production ability analysis of strain FZ202; wherein, (A): the color development of strain FZ202 cultured for 3 days; (B): the quantitative determination of siderophore production of strain FZ202.

[0063] Figure 4 is the basic growth-promoting characteristics of strain FZ202; wherein, (A): DF medium; (B): ADF medium; (C): modified Ashby medium; (D): potassium-dissolving medium; (E): universal CAS detection plate; (F): phosphorus-dissolving medium.

[0064] Figure 5 is the best fitting curve of strain FZ202 to different initial concentrations of DEHP.

[0065] Figure 6 is the utilization of various substrates by strain FZ202.

[0066] Figure 7 is the influence of strain FZ202 on the DEHP content in rice roots (A), rice aboveground parts (B) and soil (C).

[0067] Figure 8 is the root surface colonization of strain FZ202; wherein, FZ202 + day4: the 4th day after strain FZ202-PnitR treatment; FZ202 + day8: the 8th day after strain FZ202-PnitR treatment. DETAILED DESCRIPTION

[0068] The present application will be further described in conjunction with the following examples and drawings, but the embodiments of the present application are not limited thereto. The test methods in the following examples, unless otherwise specified, are usually carried out according to the conventional experimental conditions or according to the experimental conditions suggested by the manufacturers. The materials, reagents and the like used, unless otherwise specified, are reagents and materials obtained from commercial channels.

[0069] Example 1 Isolation and identification of strain

[0070] The present application collects rice root system sample, retains rhizosphere soil attached to the root system, and fills into a sterile bag. Subsequently, root surface microorganisms are collected from the washed root surface thereof, the obtained microbial suspension is gradient diluted with sterile water, and is inoculated on an inorganic salt culture medium containing PAEs as the sole carbon source to culture at 28℃ for 5-7 days. When single colonies grow, single colonies with fast growth speed and strong degradation ability are picked and transferred to Gause No. 1 nutrient plate to purify and obtain isolated actinomycetes. The PAEs are DEHP, and the final concentration in the inorganic salt culture medium is 300 mg / L. The inorganic salt culture medium (MSM, g / L) is as follows: K2HPO4 5.8, KH2PO4 4.5, (NH4)2SO4 2.0, MgCl2 0.16, CaCl2 0.02, Na2MoO4·2H2O 0.0024, FeCl3 0.0018, MnCl2·2H2O 0.0015, and pH 7.0. Agar powder 15-20 g / L is added to the solid culture medium.

[0071] The above separated, purified and identified microorganism is named FZ202.

[0072] (1) Morphological identification of strain FZ202

[0073] Strain FZ202 is inoculated on MS solid culture medium and cultured at 28℃ for 4 days, and the colony morphology is observed. As shown in the figure, Figure 1 strain FZ202 is a single colony on the MS solid culture medium, which is flat, dark gray, has concave and convex textures and protrusions on the surface, irregularly distributed, and has a wavy edge with a branched shape.

[0074] The MS solid culture medium (g / L) is as follows: mannitol 20.0, soybean powder 20.0, calcium carbonate 3.0, and agar powder 20.0; and pH 7.2.

[0075] (2) Physiological and biochemical identification of strain FZ202

[0076] Physiological and biochemical tests and identification are performed on strain FZ202. The strain FZ202 with good growth on the MS solid culture medium is selected, is inoculated on TSB culture medium, is cultured at 28℃ and 220 rpm for 3 days, and the salt stress tolerance, carbon source utilization, hydrogen sulfide production ability, gelatin liquefaction ability, and pigment production of the strain FZ202 are determined.

[0077] Strain FZ202 is inoculated into different physiological and biochemical culture media to perform gelatin liquefaction test, pigment production, H2S test, carbon source test, and inorganic carbon test. The experimental results are shown in Table 1.

[0078] Table 1 Physiological and biochemical characteristics of strain FZ202

[0079] Physiological and biochemical characteristics Results Gelatin liquefaction test + H2S test - Melanin production + Growth salinity +(1-8％) Indole test + Glucose + Sucrose + Fructose + Lactose + Mannitol + Inorganic carbon +

[0080] Note: + means positive; - means negative.

[0081] (3) Molecular biological identification of strain FZ202

[0082] According to the Bergey's Manual of Systematic Bacteriology (9th edition) and BLAST alignment of the 16S rDNA sequence of strain FZ202 (the sequence is shown as SEQ ID NO: 1), the alignment result shows that the 16S rDNA sequence of strain FZ202 has the highest similarity with that of strain Streptomyces griseorubens, the sequence coverage is 100%, and the sequence similarity is 99.84%. The phylogenetic tree of the 16S rDNA sequence of strain FZ202 is constructed, and the result is shown as Figure 2

[0083] In summary, according to the morphological characteristics, physiological and biochemical characteristics and 16S rDNA sequence analysis results, strain FZ202 belongs to Streptomyces griseorubens, named Streptomyces griseorubens FZ202, the preservation information is as follows: the preservation unit is Guangdong Microbial Culture Collection Center (GDMCC), the preservation time is December 16, 2020, the preservation address is Guangdong Institute of Microbiology, 5th floor, No. 59 building, Guangzhou, Guangdong, the preservation number is GDMCC No: 61372.

[0084] The 16S rDNA sequence of the Streptomyces griseorubens FZ202 is shown as SEQ ID NO: 1:

[0085] ​

[0086] Example 2: Determination of the siderophore production capacity of *Streptomyces griseus* FZ202

[0087] Qualitative detection: Select well-grown MS medium plates, pick up bacterial pellets with an inoculation loop, and inoculate them onto universal CAS test plates. Incubate in the dark at 28°C for 2–7 days. Determine whether the strain has the ability to produce siderophores based on whether it produces a distinct orange-yellow siderophore chelation zone on the universal test plate.

[0088] Quantitative determination: The strain was cultured at 28℃ with shaking at 220 rpm for 6 days at a 2% inoculum. Every 24 hours, 1 mL of culture medium was taken, centrifuged at 8000 rpm for 4 min to obtain the supernatant. This supernatant was then mixed with an equal volume of CAS detection solution and allowed to stand for 30 min in the dark. The absorbance was then measured at 630 nm and recorded as As. The absorbance of the uninoculated culture medium mixed with the CAS detection solution was recorded as Ar. Siderophore production was expressed as siderophore activity units (SU).

[0089] Calculation formula: SU(%)=[1-As / Ar]×100.

[0090] The results are as follows Figure 3 and Figure 4 As shown in (E), in the qualitative analysis of the siderogenic vector of strain FZ202 on a plate, after 3 days of culture, an orange-yellow transparent zone appeared on the CAS detection medium. Figure 4 (E) in the middle). Identified by FeCl3 colorimetric reaction ( Figure 3 In (A) of the study, strain FZ202 produced siderophores with a significant color difference compared to the control, identifying it as a hydroxamic acid-type siderophore. With increasing culture time, the siderophore content of strain FZ202 gradually increased, reaching its maximum on day 5, at 61.03%. Figure 3 (B) indicates that strain FZ202 is a high-side-carrier-producing strain.

[0091] Example 3: Identification of growth-promoting characteristics of Streptomyces griseus FZ202, including phosphorus solubilization, potassium solubilization, nitrogen fixation, and deaminase activity.

[0092] Using an inoculation loop, select vigorous FZ202 spores from MS medium and inoculate them onto modified Assumption medium, phosphorus-solubilizing medium, potassium-solubilizing medium, ADF medium, and DF medium. After sealing the plates, invert them and incubate them at 28°C for 3–7 days. Each treatment group was replicated in triplicate. Observe the growth of the strain in the medium and whether a clear zone is formed. Determine whether strain FZ202 has the ability to fix nitrogen, dissolve minerals, and produce deaminase.

[0093] The phosphorus solubilizing medium (inorganic phosphorus) (g / L): C6H 12 O6 10.0, yeast extract 0.5, MnSO4 0.03, NaCl 0.3, (NH4)2SO4 0.5, KCl 0.3, MgSO4·7H2O 0.3, FeSO4·7H2O 0.03, Ca3(PO4)2 5.0, agar powder 18, pH 7.0±0.2.

[0094] The potassium solubilizing medium (g / L): C 12 H 22 O 11 5.0, C6H 12 O6 5.0, (NH4)2SO4 0.5, yeast powder 0.5, MgSO4·7H2O 0.3, Na2HPO4 2.0, FeSO4·7H2O 0.03, MnSO4·H2O 0.03, NaCl 0.3, potassium feldspar 2.0, agar powder 15, pH 7.0±0.2.

[0095] The modified Ashby medium (nitrogen fixation medium, g / L): KH2PO4 0.2, NaCl 0.2, MgSO4 0.2, CaCO3 5.0, K2SO4 0.1, glucose 10, agar powder 15.0, pH 7.4±0.2.

[0096] The DF medium (g / L): KH2PO4 4.0, Na2HPO4 6.0, MgSO4·7H2O 0.2, C6H 12 O6 2.0, C6H 11 O7Na2.0, citric acid 2.0, (NH4)2SO4 2.0, agar powder 15.0, trace element solution 1 mL, ferrous sulfate heptahydrate solution 0.1 mL each, pH 7.2±0.2.

[0097] The trace element solution (mg / L): H3BO3 0.01, MnSO4·H2O 0.0112, ZnSO4·7H2O 0.0778, CuSO4 0.05, MoO3 0.01, FeSO4·7H2O 1.0.

[0098] The ADF medium (g / L): DF medium without ammonium sulfate (pH 7.2±0.2) is added with 6 mL of sterilized 0.5M ACC (1-aminocarbonyl-1-cyclopropane carboxylic acid) solution after 20 min at 121℃.

[0099] The results, as shown in Table 1, show that the strain FZ202 can grow on the DF and ADF media. Figure 4 Figure 4 ​(A), (B) in FIG. 1, indicating that strain FZ202 has the ability to secrete ACC deaminase. The nitrogen fixation ability of strain FZ202 was detected by using modified Ashby medium, and it was found that it could grow on the medium Figure 4 (C) in FIG. 1, indicating that the strain can fix N2 into ammonia. Strain FZ202 was subjected to iron carrier production qualitative plate, and after 3 days of culture, orange transparent circles appeared on the CAS detection medium Figure 4 (E) in FIG. 1, and strain FZ202 also has the ability to dissolve phosphorus and potassium Figure 4 (D), (F) in FIG. 1.

[0100] Example 4 Degradation effect of Streptomyces grayi FZ202 on PAEs

[0101] (1) Streptomyces grayi FZ202 was inoculated on MS solid medium and cultured in a 28°C incubator. Then, spores of Streptomyces FZ202 were collected to prepare a spore suspension with a concentration of 1×10 11 spores / mL, which was then inoculated in TSB liquid medium and cultured for 3 days. The mycelium was collected by centrifugation at 4°C and 8000 rpm.

[0102] (2) Then, the above mycelium was inoculated into 150 mL triangular flasks containing 50 mL of Gause No. 1 liquid medium containing DEHP at an inoculation amount of 1.5 g / L (wet weight), and the exposure concentration of DEHP was 100, 200, 300, and 800 mg / L. The culture temperature was 28°C, the rotation speed was 220 rpm, and the culture was carried out in the dark for 6 days. Every 24 h, samples were taken, and the residual amount of DEHP in the medium was determined by gas chromatography-mass spectrometry (GC-MS) method. The specific method steps are referred to the reference literature Feng N X, et al., 2024 (Feng N X, Li DW, Zhang F, et al. Biodegradation of phthalate acid esters and whole-genome analysis of a novel Streptomyces sp. FZ201 isolated from natural habitats [J]. Journal of Hazardous Materials, 2024, 469: 133972.).

[0103] Gao's liquid medium (g / L): soluble starch 20.0, KNO3 1.0, NaCl 0.5 g, K2HPO4 0.5 g, MgSO4·7H2O 0.5 g, FeSO4·7H2O 0.01 g, pH adjusted to 7.2-7.4. Solid medium was added with agar powder 15-20 g / L.

[0104] The results are shown in Table 1. Figure 5 As shown in Table 1, strain FZ202 can effectively degrade DEHP (100, 200, 300, 800 mg / L). After 144 h of culture, when the initial concentration of DEHP is 300 mg / L, the degradation efficiency of strain FZ202 on DEHP is the highest, reaching 94.1%.

[0105] Example 5

[0106] The Streptomyces griseoruberrimus FZ202 was inoculated into MS solid medium, and when the spores covered the plate, the spores were collected and inoculated into TSB liquid medium. After 3 d of culture at 28°C, 220 rpm, the mycelium was collected by centrifugation at 4°C, 8000 rpm. The PAEs compounds DEHP, DBP, DOP, DEP, DMP with different length of side chain and the intermediate metabolites PA, PCA, MEHP of PAEs were selected as test substrates. After sterilization of 50 mL Gao's No. 1 medium, a certain amount of the above substrates was added to make the final concentration of each substrate 200 mg / L, and the mycelium biomass was 1.5 g / L (wet weight). The culture was carried out at 28°C, 220 rpm for 6 d, and the biomass change of strain FZ202 was determined. The medium without the above test substrates was used as negative control, and 3 replicates were set for each treatment. The utilization of the above PAEs compounds and intermediate metabolites by strain FZ202 was evaluated.

[0107] The results are shown in Table 2. Figure 6 As shown in Table 2, strain FZ202 can utilize DEHP, DBP, DOP, DEP, PA, DMP, PCA, MEHP as carbon source for its growth, and the utilization rate of strain FZ202 on PA is the highest.

[0108] Example 6

[0109] The Streptomyces griseoruberrimus FZ202 was inoculated on MS solid medium and cultured in a 28°C incubator. Then the spores of Streptomyces FZ202 were collected to prepare a spore suspension with a concentration of 1×10 11 The garden soil was dried and passed through a 2 mm sieve, and the DEHP concentration in the soil was set to 20, 100 mg / kg. Then the spore suspension of Streptomyces griseoruberrimus FZ202 was added to the contaminated soil, and the amount of spores added was 2×10 11The rice seedlings with 3-4 true leaves were transplanted, and the rhizosphere soil and plant samples were collected after 30 days of culture. The content of DEHP was determined by gas chromatography-mass spectrometry (GC-MS) method.

[0110] The chlorophyll fluorescence parameters were collected by a chlorophyll fluorescence instrument, and the fluorescence imaging was determined by using an LED light source and a CCD probe. The determination was performed from 8:00 to 12:00 in the morning, and the rice leaves were fixed on the sample stage. The fluorescence value was determined after 20 min of dark adaptation, and the average value of 3 times of determination for each sample was taken as the final fluorescence parameter. For details, refer to the literature: Zhang T, Ma B, Wang L. Phthalic acid esters in grains, vegetables, and fruits: concentration, distribution, composition, bio-accessibility, and dietary exposure [J]. Environmental Science and Pollution Research, 2023, 30(2): 2787-2799.

[0111] The above-ground part of the collected rice was treated under fresh-keeping state, and the chlorophyll concentration Chlab was determined. 0.1 g of rice leaf sample was weighed and cut into pieces, and was placed in a test tube containing 100% acetone and shaken well. It was placed in a cool and dark place until the leaf color changed to white and the extract was clear. The absorbance (A 645 and A 663 ) at 645 nm and 663 nm wavelengths was determined by using a spectrophotometer. The chlorophyll concentration (mg / L) was calculated according to the following formula: chlorophyll a content = (12.7A 663 -2.69A 645 );

[0112] Chlorophyll b content = (22.9A 645 -4.68A 663 );

[0113] Total chlorophyll content = (20.21A 645 + 8.02A 663 );

[0114] Wherein, A 645 and A 663 are the absorbance values at 645 nm and 663 nm wavelengths, respectively.

[0115] Table 2 DEHP content in soil inoculated with strain FZ202, rice roots and above-ground part of rice

[0116]

[0117] Note: "-" indicates not detected; different lowercase letters in the same column indicate significant differences between groups, P < 0.05. "a" indicates the maximum value, and "b" and "c" decrease in that order.

[0118] The results are as follows Figure 7 As shown in Table 2, based on the initial addition amount, strain FZ202 achieved a degradation efficiency of 87.6% for DEHP in rice soil contaminated with low concentrations of DEHP (L_DEHP: 20 mg / kg). In rice roots, the DEHP content after inoculation was below the detection limit (not detected). In rice aboveground parts, the content significantly decreased to 0.41 ± 0.13 mg / kg after inoculation. Under high concentration DEHP (H_DEHP: 100 mg / kg) treatment, based on the initial addition amount, the removal rates of DEHP in soil by uninoculated and inoculated treatments were 60.6% and 76.2%, respectively, with the inoculated strain increasing the degradation efficiency by 15.6%. In rice roots, the inoculated treatment significantly reduced the DEHP content to 19.2 ± 2.02 mg / kg. In rice aboveground parts, the inoculated treatment significantly reduced the DEHP content to 1.27 ± 0.24 mg / kg. This shows that after inoculation with strain FZ202, the cumulative concentration of DEHP in plants increases with the increase of pollutant concentration in the soil. Adding strain FZ202 to the soil can effectively reduce the DEHP content in the soil-rice system.

[0119] Table 3. Biomass and chlorophyll content of rice inoculated with strain FZ202

[0120] Treatment Biomass (g) Chla (mg / L) Chlb (mg / L) Chlab (mg / L) L_DEHP 12.4 ± 1.587 ab ]] 1.01 ± 0.022 bc ]] 1.48 ± 0.070 c ]] 2.49 ± 0.083 b ]] H_DEHP 9.600 ± 1.400 c ]] 0.990 ± 0.004 c ]] 1.40 ± 0.150 c ]] 2.35 ± 0.15 3 b ]] L_DEHP + FZ202 15.6 ± 2.254 a ]] 1.56 ± 0.028 a ]] 2.16 ± 0.197 a ]] 3.22 ± 0.217 a ]] H_DEHP + FZ202 12.1 ± 0.577 ab ]] 1.03 ± 0.027 ab ]] 1.94 ± 0.054 ab ]] 2.97 ± 0.080 ab ]]

[0121] Note: If the superscript letters of the values ​​in the table are the same, it means there is no significant difference between the groups (P > 0.05). If the letters are different, it means there is a significant difference between the groups (P < 0.05). "a" represents the maximum value, and "b" and "c" represent decreasing values ​​in that order.

[0122] The results are shown in Table 3. Regardless of whether the DEHP stress was low or high, rice biomass significantly increased after inoculation with strain FZ202, indicating that strain FZ202 can enhance crop resistance to PAEs stress and promote crop growth. Compared with the high DEHP stress group, the chlorophyll content of rice inoculated with strain FZ202 increased. Compared with the low DEHP stress group, the chlorophyll content of rice inoculated with strain FZ202 was significantly higher (p<0.05).

[0123] Example 7: Analysis of root colonization of strain FZ202

[0124] 1. To maintain stable expression, the green fluorescent marker strain FZ202-PnitR was continuously added with the same concentration (Apr: 50 μg / mL, Kan: 50 μg / mL) of antibiotics during the liquid pre-culture process. The strains FZ202 and FZ202-PnitR were inoculated into MS solid medium, respectively, and the spores were collected after the spores covered the plate. The mycelium was collected by centrifugation at 4°C, 8000 rpm after being cultured in TSB liquid medium at 28°C, 220 rpm for 3d.

[0125] The construction process of the green fluorescent marker strain FZ202-PnitR includes the following steps: the E. coli ET12567 / pUZ8002 transformed with the overexpression vector PnitR (SEQ ID NO: 2) is used as the donor strain and the recipient strain (S. griseoviridis FZ202) is co-cultured by solid medium to realize plasmid introduction, and the recombinant S. griseoviridis FZ202-PnitR, i.e. the green fluorescent marker strain FZ202-PnitR, is obtained by antibiotic (Apramycin (Apr) and Kanamycin (Kan)) selection and fluorescence screening verification.

[0126] The sequence of the overexpression vector PnitR is shown in SEQ ID NO: 2, wherein oriT: 455-564bp, traJ: 597-968bp, JPFHBNEE_06690_PRO: 1012-1138bp, eGFP: 1167-1829bp.

[0127] 2. The green fluorescent marker strain FZ202-PnitR was used in soil culture method to analyze the rhizosphere colonization ability. After surface sterilization, the rice seeds were germinated, and the seedlings with uniform growth were selected when they grew to 3-4 true leaves. The seedlings were transplanted into pottery pots containing a proper amount of stirred soil, and the green fluorescent marker strain FZ202-PnitR spore suspension with a proper concentration (10 11 spores / mL) was inoculated on the roots of the rice, which was cultured in a 28°C artificial climate incubator. The roots were taken out after 4d and 8d, respectively, and the fluorescence was observed by laser confocal scanning microscope (CLSM) to observe the rhizosphere colonization.

[0128] The results are shown in Figure 8 The pot colonization experiment showed that the strain FZ202-PnitR colonized on the surface of the rice roots, which could be clearly observed by laser confocal scanning microscope at 4d and 8d.

[0129] The MS solid medium (g / L) includes calcium carbonate 3.0, soybean powder 20.0, mannitol 20.0, agar powder 20.0, and pH 7.3.

[0130] In the formula, the tryptone soybean broth medium (TSB, g / L) is composed of glucose 2.5, NaCl 5.0, K2HPO4 2.5, soybean papain digest 3.0, and tryptone 17.0, and has a pH of 7.3.

[0131] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application should be equivalent replacement manners and should be included in the protection scope of the present application.

Claims

1. A strain of *Streptomyces glaucus*, characterized by: The fungus named Streptomyces griseorubens FZ202 was deposited on December 16, 2020, at the Guangdong Provincial Microbial Culture Collection Center of the Guangdong Institute of Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, with accession number GDMCC No: 61372.

2. A biological agent, characterized in that: It includes the *Streptomyces griseus* FZ202 as described in claim 1.

3. The application of *Streptomyces griseus* as described in claim 1 or the biological agent as described in claim 2, characterized in that: The application is at least one of the following: (1) Application in the degradation of PAEs and / or PAE intermediates; (2) Application in media for remediation of PAE contamination; (3) Application in combined phytoremediation of PAE-contaminated soil; (4) Application in reducing PAEs pollution in plants; (5) Application in promoting plant growth.

4. The application according to claim 3, characterized in that: In application (2), the medium is soil or water; In applications (3), (4) or (5), the plant is rice.

5. The application according to claim 4, characterized in that: In application (2) or (3), mature spores of Streptomyces griseus FZ202 are prepared into a spore suspension and inoculated into PAEs-contaminated soil for the degradation of PAEs in the soil.

6. The application according to claim 5, characterized in that: The concentration of spores in the spore suspension was 1×10⁻⁶. 8 ~1×10 11 spores / mL; The inoculation amount of the mature spores was 2 × 10⁻⁶. 11 spores / kg soil.

7. The application according to claim 4, characterized in that: In application (1) or (2), mature spores of Streptomyces griseus FZ202 are prepared into a spore suspension and inoculated into a liquid culture medium containing PAEs for shaking culture, or mycelia of Streptomyces griseus FZ202 are inoculated into a liquid culture medium containing PAEs for shaking culture. Streptomyces griseus FZ202 degrades PAEs during its growth.

8. The application according to claim 7, characterized in that: The concentration of spores in the spore suspension was 1×10⁻⁶. 8 ~1×10 11 spores / mL; The inoculum amount of the mature spores was 1×10⁻⁶. 8 ~1×10 11 spores / mL; The inoculation amount of the mycelium is 1–1.5 g / L, wet weight; The conditions for the shaking culture are 28–30℃ and 200–220 rpm for 1–6 days.

9. The application according to claim 4, characterized in that: In application (3), (4) or (5), mature spores of *Streptomyces griseus* FZ202 are prepared into a spore suspension and inoculated into PAE-contaminated soil, with the inoculation amount of the mature spores being 2 × 10⁻⁶. 11 Mix spores / kg of soil thoroughly, transplant rice seedlings with 3-4 true leaves, and cultivate for 30 days.

10. The application according to any one of claims 3 to 9, characterized in that: The PAEs include at least one of dimethyl phthalate, diethyl phthalate, dibutyl phthalate, diisooctyl phthalate, and n-octyl phthalate; The PAE intermediates include at least one of mono(2-ethylhexyl) phthalate, phthalic acid, and protocatechuic acid.

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