Application of sphingomonas melonis spz1 and its complex microbial inoculant in promoting photosynthetic physiology of peanut plants

Abstract

The application discloses a kind of cucurbitaceae sphingomonas SPZ1 and its complex microbial inoculant in application of promoting peanut plant photosynthetic physiology and nitrogen metabolism, belong to the field of microbial technology.The active bacteria of the present application is first composed of plant Arthrobacter ARZ1 (Arthrobacter oryzae) and cucurbitaceae sphingomonas SPZ1 (Sphingomonas melonis) complex microbial inoculant is applied to peanut planting field, the two bacteria in the complex microbial inoculant have obvious improvement on the maximum photochemical efficiency of peanut plant and leaf chlorophyll SPAD value through synergistic effect, especially can improve the activity of whole peanut leaf nitrogen metabolism enzyme (nitrate reductase and glutamine synthetase), so as to be applied to promote peanut plant photosynthetic physiology and nitrogen metabolism.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically, it relates to the application of a sphingomyelin-monosporobacter SPZ1 strain of cucurbits and its compound inoculum in promoting photosynthetic physiology and nitrogen metabolism in peanut plants. Background Technology

[0002] Nitrogen is an indispensable nutrient element for peanut growth, influencing its growth, development, and yield. Within appropriate application ranges, nitrogen significantly promotes peanut plant growth, fosters healthy development of lateral branches and roots, and enhances root absorption capacity and leaf photosynthetic efficiency. Simultaneously, the efficient conversion and utilization of nitrogen promotes the efficient accumulation and translocation of nutrients within the plant, thereby increasing dry matter accumulation and improving peanut yield. However, excessive nitrogen application in the context of intensive agriculture may inhibit the nitrogen-fixing function of peanut root nodules, reduce nitrogen fertilizer utilization, lead to excessive vegetative growth and premature aging, inhibit nutrient translocation and metabolism, and ultimately affect peanut yield and quality.

[0003] Therefore, exploring ways to improve peanut photosynthetic physiology and promote nutrient transport and metabolism without increasing nitrogen fertilizer application has significant value for sustainable development. Previous studies have primarily focused on improving fertilization methods and optimizing fertilizer properties to enhance peanut photosynthetic physiology and metabolism, but have to some extent overlooked the crucial role of microorganisms in regulating peanut growth and physiology. Therefore, it is necessary to develop suitable microbial agents to improve peanut photosynthetic physiology and nitrogen metabolism, enhance nitrogen use efficiency, and lay the foundation for increasing peanut yield. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to provide an application of Sphingomonas sp. SPZ1 of cucurbitaceae and its compound inoculant in promoting photosynthetic physiology and nitrogen metabolism in peanut plants.

[0005] To achieve the above objectives, the inventors conducted extensive experiments to investigate the effects of different bacterial strains on the maximum photochemical efficiency, leaf chlorophyll SPAD value, leaf glutamine synthase activity, and leaf nitrate reductase activity of two peanut varieties. The final results yielded the following technical solutions: a *Sphingomonas melonis* SPZ1 strain, with the strain preservation number CCTCC No: M20241884; and an *Arthrobacter oryzae* ARZ1 strain, with the strain preservation number CCTCC No: M20241883. The depository address is Wuhan University, Wuhan, China.

[0006] The *Arthrobacter oryzae* strain used in this invention was isolated from peanut rhizosphere soil. It possesses excellent nitrogen-fixing ability and can survive in the peanut rhizosphere and soil. It grows well in an environment with a pH of 7.0-9.0 and survives well at pH 8.0. Based on morphological characteristics, culture properties, and physiological and biochemical characteristics, this strain was identified as *Arthrobacter oryzae* ARZ1. This strain has been deposited at the China Center for Type Culture Collection (CCTCC) on September 2, 2024, with accession number CCTCCNo: M20241883. The address of the depository is Wuhan University, Wuhan, China.

[0007] The *Sphingomonas melonis* SPZ1 used in this invention was also isolated from peanut rhizosphere soil. It possesses excellent nitrogen-fixing ability and can survive in the peanut rhizosphere and soil. It grows well in an environment with a pH of 6.0-9.0 and survives well at pH 8.0. Based on morphological characteristics, culture properties, and physiological and biochemical characteristics, this strain was identified as *Sphingomonas melonis* SPZ1. This strain was deposited at the China Center for Type Culture Collection (CCTCC) on September 2, 2024, with accession number CCTCC No: M20241884.

[0008] When used alone or in combination, the two strains mentioned above significantly and positively promote the maximum photochemical efficiency, leaf chlorophyll SPAD value, leaf glutamine synthase activity, and leaf nitrate reductase activity of the two peanut varieties, Qinghua 6 and Qinghua 22. Therefore, this invention also provides the following applications:

[0009] (1) Application of ARZ1 strain or SPZ1 strain in improving photosynthetic physiology and nitrogen metabolism in peanut plants.

[0010] (2) Application of ARZ1 and SPZ1 composite strain in improving the maximum photochemical efficiency of peanut plants during the pod-setting and maturity stages.

[0011] (3) Application of ARZ1 strain or SPZ1 strain or ARZ1 and SPZ1 compound strain in improving the SPAD value of chlorophyll in peanut plants.

[0012] (4) Application of ARZ1 strain or SPZ1 strain or ARZ1 and SPZ1 compound strain in improving the activity of glutamine synthase in peanut plant leaves.

[0013] (5) Application of ARZ1 strain or SPZ1 strain or ARZ1 and SPZ1 compound strain in improving the activity of nitrate reductase in peanut plant leaves.

[0014] In the above applications, the ARZ1 strain is Arthrobacter oryzae ARZ1CCTCCNo:M20241883, and the SPZ1 strain is Sphingomonas melonis SPZ1CCTCCNo:M20241884.

[0015] More preferably, when the compound strain is used, the ratio of viable bacteria of Sphingomonasmelonis SPZ1 to Arthrobacter oryzae ARZ1 is 1:(1-2).

[0016] It should be noted that, during the screening of bacterial strains that enhance peanut photosynthetic physiology and nitrogen metabolism, the inventors unexpectedly discovered that a compound bacterial solution containing *Sphingomonas melonis* SPZ1 and *Arthrobacter oryzae* ARZ1 significantly improves the maximum photochemical efficiency of peanut plants and noticeably increases the SPAD value of chlorophyll in peanut leaves at both growth stages. It also enhances the activity of nitrogen metabolism enzymes (glutamine synthase and nitrate reductase) in the entire peanut leaf. Based on this discovery, the inventors prepared a compound bacterial agent from these two bacteria through cultivation and fermentation.

[0017] The aforementioned compound microbial agent can be prepared by the following method: A culture of Sphingomonas melonis SPZ1 bacteria, cultured to the logarithmic growth phase, and Arthrobacter oryzae ARZ1 bacteria are mixed thoroughly. A protectant is added to the mixed culture, and the viable cell count is adjusted to (1.0–2.0) × 10⁻⁶. 10 cfu / mL, pH 6-9; the protective agent consists of: 90-120 g / L skim milk powder, 25-35 mL / L glycerol, 90-120 g / L maltodextrin, 120-180 g / L laccase and 8-13 g / L L-glutamate sodium.

[0018] The fermentation culture method for *Sphingomonas melonis* SPZ1 is as follows: *Sphingomonas melonis* SPZ1 is inoculated into beef extract peptone broth at an inoculum size of 4%-10%, and cultured at 25-27℃ for 24-72 hours. After centrifugation and washing, a mixture containing 0.5 g / L yeast extract, 20 g / L mannitol, 0.2 g / L K₂HPO₄, 0.8 g / L KH₂PO₄, 0.2 g / L MgSO₄·7H₂O, 0.1 g / L CaSO₄·2H₂O, 0.01 g / L FeCl₃, 0.002 g / L Na₂MoO₄·2H₂O, and 15 g / L agar is added to each bacterial precipitate. The pH is adjusted accordingly. 6-9, and adjust the viable count of *Sphingomonas melonis* SPZ1 (a type of cucurbit) to (1.0–2.0) × 10⁻⁹. 10 CFU / mL, mix well and pour into culture.

[0019] The fermentation culture method for Arthrobacter oryzae ARZ1 is as follows: Arthrobacter oryzae ARZ1 is fermented... Arthrobacter oryzae ARZ1 was inoculated at a rate of 4%-10% into beef extract peptone broth and incubated at 25-27℃ for 24-72 hours. After centrifugation and washing, a mixture containing 0.5 g / L Yeast Extract, 20 g / L Mannitol, 0.2 g / L K₂HPO₄, 0.8 g / L KH₂PO₄, 0.2 g / L MgSO₄·7H₂O, 0.1 g / L CaSO₄·2H₂O, 0.01 g / L FeCl₃, 0.002 g / L Na₂MoO₄·2H₂O, and 15 g / L Agar was added to each bacterial precipitate. The pH was adjusted to 6-9, and the viable count of Arthrobacter oryzae ARZ1 was adjusted to (1.0-2.0) × 10⁻⁶. 10 CFU / mL, mix well and pour into culture.

[0020] More preferably, the compound strain is prepared to have a total viable count of (1.0–2.0) × 10⁻⁶. 10 A compound microbial agent with a concentration of CFU / mL; applied for the first time at the initial flowering stage of peanuts, at a rate of 2.5-3.5 L / 667m³. 2 The second application is during the peanut pod-setting stage, at a rate of 2.5-3.5 L / 667 m³. 2 .

[0021] More preferably, if applied manually, the solution is diluted with water and sprayed onto the peanut roots using an agricultural sprayer; if sprayed by drone, the bacterial solution is diluted with water and placed in a spray tank before rain.

[0022] In a further preferred embodiment, when peanuts are grown under mulch, drip irrigation with bacterial solution is used, with the water depth moistening the soil to a depth of 8-10cm.

[0023] More preferably, the bacterial solution and pesticides cannot be used together, and the time interval between their use should be more than 2 days.

[0024] Compared with the prior art, the active bacteria in the compound microbial agent of this invention are composed of Arthrobacter oryzae ARZ1 and Sphingomonas melonis SPZ1. The two bacteria in this compound microbial agent have a synergistic effect, which significantly improves the maximum photochemical efficiency of peanut plants and the SPAD value of leaf chlorophyll. In particular, it can improve the activity of nitrogen metabolism enzymes (nitrate reductase and glutamine synthase) in the whole peanut leaves, so it can be used to promote the photosynthetic physiology and nitrogen metabolism of peanut plants. Attached Figure Description

[0025] Figure 1 Plate culture and scanning electron microscope image of Arthrobacter oryzae ARZ1;

[0026] Figure 2 Plate culture and scanning electron microscope image of Sphingomonas melonis SPZ1;

[0027] Figure 3 Effects of various treatments on the maximum photochemical efficiency of peanuts;

[0028] Figure 4 Effects of various treatments on chlorophyll SPAD in peanut leaves;

[0029] Figure 5 Effects of different inoculants on the activity of glutamine synthase in peanut leaves;

[0030] Figure 6 Effects of different inoculants on nitrate reductase activity in peanut leaves;

[0031] Figures 3-6 Different letters in the text represent significant differences between different treatments within the same group (p<0.05). Detailed Implementation

[0032] The following embodiments further illustrate the above-described content of the present invention in detail. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Furthermore, unless otherwise specified, the experimental methods in the following embodiments are conventional methods.

[0033] Example 1: Screening and isolation of ARZ1 and SPZ1 strains

[0034] (1) Discovery, screening, isolation, and identification of bacterial strains. Based on the screening experiment of adding specific root exudates in maize-peanut intercropping, the inventors found that adding root exudates can improve the maximum photochemical efficiency and nitrogen metabolism enzyme activity of peanut plants, and stimulate the photosynthetic physiology and nitrogen metabolism of peanut plants. Therefore, we speculate that the addition of root exudates reassembles the soil microbial community, thereby promoting the photosynthetic physiology and nitrogen metabolism of peanuts. On this basis, we prepared a suspension of peanut rhizosphere soil samples under the above root exudate treatment, spread the bacterial suspension on bacterial culture medium, and isolated and purified the bacterial strains. The 16S rRNA sequence of the isolated strains was obtained using high-throughput sequencing technology. The 16S rRNA sequence was compared with the NCBI nr database. Sequence clustering analysis was performed based on the degree of comparison and similarity to construct a phylogenetic tree. The isolated and purified strains were compared with known strains, and comprehensive analysis of morphology, physiology, and biochemistry was performed to determine the bacterial species. To further verify the function of the strains, peanut cultivation addition experiments were carried out using the isolated and purified strains.

[0035] (2) Microbiological properties of the microorganisms. Arthrobacter oryzae ARZ1 colonies are white, round, and moist. Figure 1 It can grow on nitrogen-fixing medium, with an optimal growth pH of 6-9 and a temperature of 25-27℃. *Sphingomonas melonis* SPZ1 colonies are yellow, round, with small, moist, smooth, opaque mycelial growths, regular edges, and no halo. Figure 2 It can grow on nitrogen-fixing medium, with a suitable pH of 6-9 and a temperature of 25-27℃.

[0036] Example 2: Preparation of ARZ1, SPZ1 inoculants and compound inoculants

[0037] (1) Arthrobacter oryzae ARZ1 was inoculated into beef extract peptone broth (3 g / L beef extract, 10 g / L peptone, 5 g / L NaCl, 15 g / L agar) at an inoculation rate of 4%-10%, pH 10. 7.4-7.6, incubate at 25-27℃ for 24-72 hours. After centrifugation and washing, add a nitrogen-fixing medium containing 0.5 g / L Yeast Extract, 20 g / L Mannitol, 0.2 g / L K₂HPO₄, 0.8 g / L KH₂PO₄, 0.2 g / L MgSO₄·7H₂O, 0.1 g / L CaSO₄·2H₂O, 0.01 g / L FeCl₃, 0.002 g / L Na₂MoO₄·2H₂O, and 15 g / L Agar to the bacterial precipitate. Adjust the pH to 6-9 and adjust the viable count of *Arthrobacter oryzae* ARZ1 to approximately 1.0 × 10⁻⁶. 10 CFU / mL, mix well and then pour culture; the specific operation method of pour culture is as follows: dilute the Arthrobacter oryzae ARZ1 suspension and add it to sterile nitrogen-fixing medium. After solidification, invert the culture and culture. A single strain will proliferate to form a colony. Take a single colony to make a suspension. Repeat the process multiple times to obtain a pure culture.

[0038] (2) Inoculate *Sphingomonas melonis* SPZ1 at a concentration of 4%-10% into beef extract peptone broth (3 g / L beef extract, 10 g / L peptone, 5 g / L NaCl, 15 g / L agar), and incubate at 25-27℃ for 24-72 hours. After centrifugation and washing, add a mixture containing 0.5 g / L Yeast Extract, 20 g / L Mannitol, 0.2 g / L K₂HPO₄, 0.8 g / L KH₂PO₄, 0.2 g / L MgSO₄·7H₂O, 0.1 g / L CaSO₄·2H₂O, 0.01 g / L FeCl₃, 0.002 g / L Na₂MoO₄·2H₂O, and 15 g / L Agar to each bacterial precipitate. pH [missing value]. 6-9, and adjust the viable count of Sphingomonas melonis SPZ1 to approximately 1.0 × 10⁻⁹. 10cfu / mL, mix well and then pour culture; the specific operation method of pour culture is as follows: dilute the suspension of Sphingomonas melonis SPZ1 and add it to sterile nitrogen-fixing medium. After solidification, invert the culture and culture. A single strain will proliferate to form a colony. Take a single colony to make a suspension. Repeat the process multiple times to obtain a pure culture.

[0039] (3) Prepare a protective agent with the following composition: 100g / L skim milk powder, 30mL / L glycerol, 100g / L maltodextrin, 150g / L sea saccharide and 10g / L L-glutamate sodium, pH 6-9.

[0040] (4) Add the protectant prepared in step (3) to the Arthrobacter plantarum ARZ1 bacterial suspension obtained in step (1) to adjust the viable count to approximately 1.0 × 10⁻⁶. 10 cfu / mL ARZ1 bacterial agent was obtained. Add the protectant prepared in step (3) to the Sphingomonas cucurbita SPZ1 bacterial suspension obtained in step (2) to adjust the viable count to approximately 1.0 × 10⁻⁶. 10 cfu / mL SPZ1 bacterial agent was obtained. .

[0041] The bacterial solutions obtained in steps (1) and (2) were mixed at a viable cell ratio of Sphingomonas SPZ1:Arthrobacter plantarum ARZ1 of 1:(1-2), and the protective agent prepared in step (3) was added to the mixed bacterial solution to adjust the total effective viable cell count to approximately 1.0 × 10⁻⁶. 10 cfu / mL To obtain compound microbial agents .

[0042] Example 3: Trial of ARZ1 inoculant in potted peanut cultivation

[0043] Experiment type: Pot experiment

[0044] Experimental location: Qingdao Agricultural University (Qingdao, China)

[0045] Test period: May 2023 - September 2023

[0046] Experimental crop: Peanuts (varieties: Qinghua 6 and Qinghua 22)

[0047] Test soil: Sterilized soil (soil pH 6.70, organic matter 11.92 g / kg, available nitrogen 99.86 mg / kg, available phosphorus 35.22 mg / kg, available potassium 89.79 mg / kg).

[0048] Experimental groups: CK was the blank control, ARZ1-F and ARZ1-T were both treated with Arthrobacter plantarum ARZ1 inoculum, with 3 replicates for each treatment, arranged in randomized block design.

[0049] Experimental design: PVC pots with an inner diameter of 16cm and a height of 23.5cm were used for potted plants. Inoculant was added 25 days after peanut sowing (the inoculant solution was mixed with 200ml of water and poured onto the peanut roots). Samples were taken at the pod-forming and maturity stages after the inoculant solution was added.

[0050] Dosage and method: For ARZ1-F, apply 0.05 mL / pot of ARZ1 inoculant prepared in Example 2; for ARZ1-T, apply 0.1 mL / pot of ARZ1 inoculant prepared in Example 2. Dilute the inoculant to 200 mL and pour it onto the peanut roots. CK is 200 mL of blank aqueous solution.

[0051] Table 1. Maximum photochemical efficiency of plants treated with Arthrobacterium plantarum ARZ1

[0052]

[0053]

[0054] Note: Different letters indicate significant differences (p<0.05) between different treatments within the same group, and the same applies below.

[0055] The experimental results in Table 1 show that the maximum photochemical efficiency of the Qinghua 6 peanut variety during the pod-setting stage was significantly increased by 5.19% under ARZ1-T treatment compared to the control (CK), while the maximum photochemical efficiency at maturity showed no significant difference among the different treatments. For the Qinghua 22 peanut variety, the maximum photochemical efficiency during the pod-setting stage was significantly increased by 3.80% and 1.27% under ARZ1-F and ARZ1-T treatments, respectively, compared to the control (CK), while the maximum photochemical efficiency at maturity showed no significant difference among the different treatments. In conclusion, both ARZ1-F and ARZ1-T treatments with Arthrobacter plantarum have a significant effect on improving the maximum photochemical efficiency of peanut plants during the pod-setting stage.

[0056] Example 4: Trial of SPZ1 inoculant in potted peanut cultivation

[0057] Experiment type: Pot experiment

[0058] Experimental location: Qingdao Agricultural University (Qingdao, China)

[0059] Test period: May 2023 - September 2023

[0060] Experimental crop: Peanuts (varieties: Qinghua 6 and Qinghua 22)

[0061] Test soil: Sterilized soil (soil pH 6.70, organic matter 11.92 g / kg, available nitrogen 99.86 mg / kg, available phosphorus 35.22 mg / kg, available potassium 89.79 mg / kg).

[0062] Experimental groups: CK was the blank control, SPZ1-F and SPZ1-T were both treated with SPZ1 inoculum of Sphingomonas cucurbita, with 3 replicates per treatment, arranged in randomized block.

[0063] Experimental design: PVC pots with an inner diameter of 16cm and a height of 23.5cm were used for potted plants. Inoculant was added 25 days after peanut sowing (the inoculant solution was mixed with 200ml of water and poured onto the peanut roots). Samples were taken from the pod-setting and ripening areas after the inoculant solution was added.

[0064] Dosage and method: SPZ1-F was treated with 0.05 mL of SPZ1 bacterial agent prepared in Example 2 per pot, and SPZ1-T was treated with 0.1 mL of SPZ1 bacterial agent prepared in Example 2 per pot. The bacterial agents were diluted to 200 mL and poured onto the peanut roots. CK was 200 mL of blank aqueous solution.

[0065] Table 2. Maximum photochemical efficiency of plants treated with Sphingomonas sp. SPZ1 in cucurbitaceous plants.

[0066]

[0067]

[0068] The experimental results in Table 2 show that the maximum photochemical efficiency of the Qinghua 6 peanut variety during the pod-setting stage was significantly increased by 5.19% under the SPZ1-F treatment compared to the control (CK), while the maximum photochemical efficiency at maturity showed no significant difference among the different treatments. Similarly, the maximum photochemical efficiency of the Qinghua 22 peanut variety during the pod-setting stage was significantly increased by 2.53% under the SPZ1-F treatment compared to the control (CK), while the maximum photochemical efficiency at maturity showed no significant difference among the different treatments. In conclusion, the SPZ1-F treatment with plant straw fungi has a significant effect on improving the maximum photochemical efficiency of peanut plants during the pod-setting stage.

[0069] Example 5: Trial of Compound Microbial Agent in Peanut Field Planting

[0070] Test location: Pingdu City, Qingdao, Shandong Province

[0071] Test period: May 2024 - September 2024

[0072] Experimental crop: Peanuts (varieties: Qinghua 6 and Qinghua 22)

[0073] Test soil: The soil was sandy black soil (soil pH 7.06, organic matter 16.18 g / kg, available nitrogen 77.76 mg / kg, available phosphorus 45.58 mg / kg, available potassium 78.69 mg / kg).

[0074] Experimental group:

[0075] CK is a blank aqueous solution control.

[0076] ARZ1-F group was treated with the ARZ bacterial agent prepared in Example 2 (total viable count approximately 1.0 × 10⁻⁶). 10 The dosage was 1.5 L / 667 mL (cfu / mL). 2 ;

[0077] The ARZ1-T group was treated with the ARZ bacterial agent prepared in Example 2 (total viable count approximately 1.0 × 10⁻⁶). 10 cfu / mL), the application rate was 3L / 667m 2 ;

[0078] Group SPZ1-F was treated with the SPZ1 bacterial agent prepared in Example 2 (total viable count approximately 1.0 × 10⁻⁶). 10 The dosage was 1.5 L / 667 mL (cfu / mL). 2 ;

[0079] The SPZ1-T group was treated with the SPZ1 bacterial agent prepared in Example 2 (total viable count approximately 1.0 × 10⁻⁶). 10 cfu / mL), the application rate was 3L / 667m 2 ;

[0080] The SF+AF group was treated with the compound microbial agent prepared in Example 2, which was a 1:1 mixture of *Sphingomonas cucurbita* SPZ1 and *Arthrobacter plantarum* ARZ1 (total viable count approximately 1.0 × 10⁻⁶). 10 cfu / mL), the application rate was 3L / 667m 2 ;

[0081] The SF+AT group was treated with the compound microbial agent prepared in Example 2, which was a mixture of *Sphingomonas cucurbita* SPZ1 and *Arthrobacter plantarum* ARZ1 in a 1:2 ratio (total viable count approximately 1.5 × 10⁻⁶). 10 cfu / mL), the application rate was 3L / 667m 2 .

[0082] Application method: For the first application, at the initial flowering stage of peanuts, dilute the amount of microbial agent per acre as designed in the above-mentioned experimental groups with water to 15L, and apply manually using an agricultural sprayer to the peanut roots. For the second application, at the pod-setting stage of peanuts, use the same dosage and method as the first application. Note that the microbial agent should not be used with pesticides, and the application time for both should be at least 2 days apart.

[0083] Planting Management: Peanuts are planted using the mulching and ridging method, with a ridge spacing of 90cm, 2 rows per ridge, a small row spacing of 35cm on the ridge, and a hole spacing of 11cm (2 seeds per hole). Apply 50 kg of compound fertilizer (N-P2O5-K2O: 15-15-15) per mu, and no further topdressing is required later.

[0084] Experimental results:

[0085] 1) Effect of bacterial strains on the maximum photochemical efficiency of peanut

[0086] pass Figure 3 The experimental results showed that, during the pod-setting stage, compared with the CK group, the maximum photochemical efficiency of Qinghua 6 was significantly increased by 7.70% and that of Qinghua 22 was significantly increased by 6.28% under the treatment of the compound strain SF+AT group; under the treatment of the compound strain SF+AF group, the maximum photochemical efficiency of Qinghua 6 was significantly increased by 7.56% and that of Qinghua 22 was significantly increased by 6.32%.

[0087] At maturity, compared with the CK group, the maximum photochemical efficiency of Qinghua 6 was significantly increased by 3.15% and 3.00% under the treatment of the compound strain SF+AT group and SF+AF group, and that of Qinghua 22 was significantly increased by 1.42% and 1.70%. At the same time, through statistical comparison, in terms of the maximum photochemical efficiency of the two peanut varieties, at both growth stages, the ST+AT and ST+AF treatment groups showed significant improvement compared with the SPZ1-F, SPZ1-T, ARZ1-F and ARZ1-T groups.

[0088] 2) Effects on the SPAD value of peanut plant chlorophyll

[0089] pass Figure 4 The experimental results showed that, during the pod-setting stage, compared with the CK group, the SPAD value of chlorophyll in Qinghua 6 was significantly increased by 18.22% and that of Qinghua 22 was significantly increased by 18.41% under the treatment of the compound strain SF+AT group; under the treatment of the compound strain SF+AF group, the SPAD value of chlorophyll in Qinghua 6 was significantly increased by 20.20% and that of Qinghua 22 was significantly increased by 15.91%.

[0090] At maturity, compared with the CK group, the SPAD value of chlorophyll in Qinghua 6 was significantly increased by 43.70% and 46.47% under the treatment of the compound strain SF+AT group and SF+AF group, and significantly increased by 7.72% and 8.73% for Qinghua 22. At the same time, through statistical comparison, under the two growth stages and the two peanut varieties, the SF+AF treatment group had a significant improvement effect compared with the SPZ1-F, SPZ1-T, ARZ1-T and ARZ1-F groups.

[0091] 3) Effects on the activity of glutamine synthase in peanut leaves

[0092] pass Figure 5 The experimental results showed that, during the pod-setting stage, compared with the CK group, the SF+AT group of the compound strain significantly increased the glutamine synthase activity of peanut leaves of Qinghua 6 by 25.44% and Qinghua 22 by 39.47%; while the SF+AF group of the compound strain significantly increased the glutamine synthase activity of peanut leaves of Qinghua 6 by 28.23% and Qinghua 22 by 38.37%.

[0093] At maturity, compared with the CK group, the SF+AT and SF+AF treatments significantly increased the glutamine synthase activity in the leaves of Qinghua 6 peanut by 61.07% and 68.97%, and significantly increased it by 48.80% and 44.03% in Qinghua 22 peanut by 48.80% and 44.03%, respectively. Statistical comparisons also showed that, in terms of leaf glutamine synthase activity in both peanut varieties, the SF+AT and SF+AF treatments significantly improved the activity compared with the SPZ1-F, SPZ1-T, ARZ1-T, and ARZ1-F groups at both growth stages.

[0094] 4) Effects on nitrate reductase activity in peanut leaves

[0095] pass Figure 6 The experimental results showed that, during the pod-setting stage, compared with the CK group, the SF+AT group of the compound strain significantly increased the nitrate reductase activity of peanut leaves of Qinghua 6 by 88.86% and Qinghua 22 by 88.58%; the SF+AF group of the compound strain significantly increased the nitrate reductase activity of peanut leaves of Qinghua 6 by 64.20% and Qinghua 22 by 80.87%.

[0096] At maturity, compared with the CK group, the nitrate reductase activity in the leaves of Qinghua 6 peanuts was significantly increased by 83.35% and 61.63% under the treatment of the compound strain SF+AT group and SF+AF group, and significantly increased by 99.28% and 94.52% under the treatment of Qinghua 22 group. At the same time, through statistical comparison, under the two peanut varieties, at both growth stages, the nitrate reductase activity of the ST+AT and ST+AF treatment groups was significantly increased compared with the SPZ1-F, SPZ1-T, ARZ1-F and ARZ1-T groups.

[0097] In summary, through statistical comparison, the SF+AT and SF+AF treatment groups of the compound bacterial strains significantly improved the maximum photosynthetic efficiency, chlorophyll SPAD value, nitrate reductase activity, and glutamine synthetase activity in peanut plants across multiple aspects of photosynthetic physiology and nitrogen metabolism. This indicates that the compound bacterial agent prepared by combining *Sphingomonasmelonis* SPZ1 and *Arthrobacter oryzae* ARZ1 at a ratio of 1:(1-2) (with an effective viable count of approximately 1-2 × 10⁻⁶) 10 cfu / mL), at 3L / 667m 2 Applying the appropriate amount of the product during the pod-setting and ripening stages of peanut plants can significantly promote the maximum photochemical efficiency, chlorophyll SPAD value, and the activities of nitrate reductase and glutamine synthase in peanut leaves, which is beneficial for improving the photosynthetic physiology and nitrogen metabolism of peanut plants.

Claims

1. A type of cucurbitaceous sphingosomal bacterium ( Sphingomonas melonis SPZ1, whose strain preservation number is CCTCC No:M20241884.

2. Application of ARZ1 strain or SPZ1 strain in improving the maximum photochemical efficiency of peanut plants during the pod-setting stage, wherein the ARZ1 strain is *Arthrobacter plantarum*. Arthrobacter oryzae ARZ1 CCTCC No:M20241883, the SPZ1 strain mentioned is a cucurbit sphingosine monoclonal antibody (Calamus sphingosine monoclonal antibody). Sphingomonas melonis SPZ1 CCTCC No:M20241884.

3. Application of the ARZ1 and SPZ1 compound strain in improving the maximum photochemical efficiency of peanut plants during the pod-setting and maturity stages, wherein the ARZ1 strain is *Arthrobacter plantarum*. Arthrobacter oryzae ARZ1 CCTCC No:M20241883, the SPZ1 strain mentioned is *Sphingomonas cucurbita* (…). Sphingomonas melonis SPZ1 CCTCC No:M20241884.

4. Application of ARZ1 strain, SPZ1 strain, or a combination of ARZ1 and SPZ1 strain in improving the SPAD value of chlorophyll in peanut leaves, wherein the ARZ1 strain is *Arthrobacter plantarum*. Arthrobacter oryzae ARZ1 CCTCC No:M20241883, the SPZ1 strain mentioned is *Sphingomonas cucurbita* (…). Sphingomonas melonis SPZ1 CCTCC No:M20241884.

5. Application of ARZ1 strain, SPZ1 strain, or a combination of ARZ1 and SPZ1 strain in improving the activity of glutamine synthase in peanut plant leaves, wherein the ARZ1 strain is *Arthrobacter plantarum*. Arthrobacter oryzae ARZ1 CCTCC No:M20241883, the SPZ1 strain mentioned is *Sphingomonas cucurbita* (…). Sphingomonas melonis SPZ1 CCTCCNo:M20241884.

6. Application of ARZ1 strain, SPZ1 strain, or a combination of ARZ1 and SPZ1 strain in improving nitrate reductase activity in peanut plant leaves, wherein the ARZ1 strain is *Arthrobacter plantarum*. Arthrobacter oryzae ARZ1 CCTCC No:M20241883, the SPZ1 strain mentioned is *Sphingomonas cucurbita* (…). Sphingomonas melonis SPZ1 CCTCCNo:M20241884.

7. The application according to any one of claims 3-6, characterized in that, The aforementioned compound strain, when used, contains Sphingosine monocytogenes of Cucurbita ( Sphingomonas melonis SPZ1 and Plantar Arthrobacter Arthrobacter oryzae The live bacteria ratio of ARZ1 is 1:(1-2).

8. The application according to claim 7, characterized in that, The aforementioned compound strain was prepared to have a total viable count of (1.0–2.0) × 10⁻⁶. 10 The bacterial solution was prepared at cfu / mL; the first application was made at the initial flowering stage of peanuts, with a dosage of 2.5-3.5 L / 667 m³. 2 The second application is during the peanut pod-setting stage, at a rate of 2.5-3.5 L / 667 m³. 2 .

9. The application according to claim 7, characterized in that, If applied manually, dilute with water and spray onto the peanut roots using an agricultural sprayer; if using a drone, dilute the bacterial solution with water and place it in a spray tank before rain.

10. The application according to claim 7, characterized in that, When peanuts are grown under plastic film, drip irrigation with bacterial solution is used, with the water depth moistening the soil to a depth of 8-10 cm.

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