Nitrogen-fixing rhodobacter and application thereof

Abstract

The invention discloses nitrogen-fixing rhodobacter and application thereof. The nitrogen-fixing rhodobacter provided by the invention has the preservation name of Rhodobacter azotoformans SY5 and the preservation number of GDMCC NO: 65285, and is preserved in Guangdong Microbiological Culture Collection Center on October 14, 2024, and the preservation address is Institute of Microbiology, Guangdong Academy of Sciences, on the fifth floor, No. 59, No. 100, Xianlie Middle Road, Guangzhou. The SY5 strain has the functions of inhibiting crop pathogenic fungi, preventing crop gray mold and degrading vegetable pesticide residues at the same time, can be widely applied to crop disease control and pollution-free vegetable production, and has a relatively strong production and application prospect in reduction of crop gray mold and reduction of pesticide residues in the vegetable production process.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microorganisms and their applications, and particularly relates to a Rhodobacter azotoformans SY5 capable of inhibiting crop pathogenic fungi, preventing crop diseases and degrading pesticide residues and applications thereof. BACKGROUND

[0002] At present, agricultural diseases and pesticide residues have become one of the key bottlenecks affecting crop yield and quality. For example, gray mold is the most common and difficult to control fungal disease of crops such as grapes, leeks and lilies, which is mainly caused by infection of pathogenic fungi such as Microdochium denticulatum and Botrytis cinerea, resulting in huge losses in crop yield; pesticide residue is common in vegetables, and the over-standard rate of phoxim reaches 30% (Reference: Liao YJ. Investigation of organophosphorus pesticide residues in agricultural products in Guangzhou [J]. Ecological Science, 2009, 28(4): 352-356.). Therefore, how to effectively control the diseases caused by various crop pathogenic fungi including Microdochium denticulatum, and effectively reduce the pesticide residues in vegetables, is a technical problem to be solved.

[0003] Using beneficial microorganisms to control crop diseases and pesticide residues is an internationally recognized green and environmentally friendly technology with abundant resources, high efficiency, environmental protection and convenient operation. At present, there is no report on color-producing probiotics with both antibacterial and disease resistance and pesticide degradation functions. SUMMARY

[0004] The first object of the present application is to provide a Rhodobacter azotoformans capable of inhibiting crop pathogenic fungi, preventing crop diseases and degrading pesticide residues in vegetables. The second object of the present application is to provide an application of the Rhodobacter azotoformans in inhibiting crop pathogenic fungi, solving the problem of how to inhibit the growth of crop pathogenic fungi. The third object of the present application is to provide an application of the Rhodobacter azotoformans in preventing gray mold of crops, solving the problem of how to prevent gray mold. The fourth object of the present application is to provide an application of the Rhodobacter azotoformans in degrading pesticide residues in vegetables, solving the problem of how to degrade pesticide residues in vegetables. The fifth object of the present application is to provide a composition for preventing crop diseases and reducing pesticide residues in vegetables, solving the problem of how to reduce pesticide residues in vegetables while preventing crop diseases.

[0005] Technical solution: The Rhodobacter azotoformans according to the present application has a preservation name of Rhodobacter azotoformans SY5 and a preservation number of GDMCC NO: 65285, and was preserved in the Guangdong Microbial Culture Collection Center on October 14, 2024, at an address of 5th Floor, No. 59 Building, Institute of Microbiology, Guangdong Academy of Sciences, 100 Middle Martyrs Road, Guangzhou.

[0006] The application isolates and purifies a color single colony from the soil of apple tree roots to obtain the above-mentioned nitrogen-fixing red bacteria, the strain can inhibit the growth of Cladosporium tenuissimum, prevent blueberry gray mold, and degrade multiple pesticides such as pymetrozine, thiamethoxam, acetamiprid, propamocarb, clothianidin, fludioxonil, phoxim, etc. in Chinese cabbage, can be used to inhibit crop pathogenic fungi, reduce the occurrence of crop gray mold, and reduce vegetable pesticide residues.

[0007] The application discloses the application of the above-mentioned nitrogen-fixing red bacteria in inhibiting crop pathogenic fungi.

[0008] Preferably, the crop pathogenic fungi include at least one of Cladosporium tenuissimum and Botrytis cinerea.

[0009] The application discloses the application of the above-mentioned nitrogen-fixing red bacteria in preventing crop gray mold.

[0010] Preferably, the crops include at least one of blueberry, leek, lily, grape, cucumber, tomato, strawberry, Chinese rose, and chrysanthemum.

[0011] The application discloses the application of the above-mentioned nitrogen-fixing red bacteria in degrading vegetable pesticide residues.

[0012] Preferably, the pesticides include at least one of pymetrozine, thiamethoxam, acetamiprid, propamocarb, clothianidin, fludioxonil, and phoxim.

[0013] The application discloses a composition for reducing vegetable pesticide residues, the composition contains live bacteria of the above-mentioned nitrogen-fixing red bacteria.

[0014] Preferably, the concentration of the nitrogen-fixing red bacteria in the composition is at least 1.0x10 6 CFU / mL, preferably 4.0x10 6 -4x10 8 CFU / mL

[0015] Preferably, the dosage form of the composition includes at least one of emulsifiable concentrate, suspension concentrate, wettable powder, powder, granule, aqueous agent, mother liquor, or slow-release agent.

[0016] Beneficial effects: compared with the prior art, the application has the following obvious advantages:

[0017] The SY5 strain in the application has the functions of inhibiting crop pathogenic fungi, preventing crop gray mold, and degrading vegetable pesticide residues, can be widely applied to crop disease control and pollution-free vegetable production, and has a strong production application prospect in reducing the occurrence of crop gray mold and reducing pesticide residues in the vegetable production process. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The colony of the strain SY5 in the present application on a nutrient agar plate;

[0019] Figure 2 The cell morphology of the strain SY5 in the present application under an optical microscope oil lens;

[0020] Figure 3 The phylogenetic tree of the strain SY5 in the present application;

[0021] Figure 4 The colony of the Agrobacterium radiobacter treated by the strain SY5 in the present application for 168 h;

[0022] Figure 5 The degradation effect of the strain SY5 in the present application on the pymetrozine and thiamethoxam in the Chinese flowering cabbage;

[0023] Figure 6 The degradation effect of the strain SY5 in the present application on the clothianidin in the Chinese flowering cabbage;

[0024] Figure 7 The degradation effect of the strain SY5 in the present application on the acetamiprid in the Chinese flowering cabbage;

[0025] Figure 8 The degradation effect of the strain SY5 in the present application on the fludioxonil in the Chinese flowering cabbage;

[0026] Figure 9 The degradation effect of the strain SY5 in the present application on the propamocarb in the Chinese flowering cabbage;

[0027] Figure 10 The degradation effect of the strain SY5 in the present application on the phoxim in the Chinese flowering cabbage. DETAILED DESCRIPTION

[0028] The technical solutions of the present application will be further described below in combination with the drawings.

[0029] Example 1: The separation and identification method of the nitrogen-fixing Rhodobacter SY5 is as follows:

[0030] (1) Separation of pigment-producing strains

[0031] Apple tree root system soil, 20 g, collected from an orchard in Caojiawa Village, Zhaoyuan City, Shandong Province, and stored in a sterile sealed bag; the apple tree root system soil was diluted to 10 -1 ~10 -4, 50 μL of the extract was spotted on a nutrient agar plate and incubated at 30°C for 3-5 days, and the color of the colonies on the plate was observed. A single colony with color was picked and further purified on a nutrient agar plate. After three times of purification, the single colony was inoculated on a nutrient agar slant and incubated at 30°C for 1-2 days, and then identified. A pigment-producing strain SY5 was isolated from the soil of apple tree roots. The colonies on the nutrient agar plate were red, round, smooth, and well-defined (as shown in FIG. 1). The strain SY5 was gram-negative, and the cell was spherical under an oil immersion lens (100x) (as shown in FIG. 2). Figure 1 Figure 2

[0032] (2) Identification of the pigment-producing strain

[0033] ​​

[0034] The measured 16S rRNA gene sequences were subjected to homology comparison by BLAST in the GenBank database, and the phylogenetic tree was constructed by the neighbor-joining method using MEGA 6.0 software (Replications = 1000, bootstrap value in percentage). The results are shown in FIG. 2. Figure 3 As shown in FIG. 2, the 16S rRNA gene sequence of the SY5 strain was naturally clustered with the 16S rRNA gene sequence of the Rhodobacter azotoformans strain, and had the closest genetic relationship with the Rhodobacter azotoformans strain P24 (GenBank accession number: PP434591).

[0035] The pigmented strain SY5 was inoculated into a nutrient agar culture medium plate by sterile operation, and after 96 h of culture at 30°C, a single colony was picked and subjected to physiological and biochemical identification of the SY5 strain using a bacterial micro-biochemical identification tube. The results are as follows:

[0036] Table 1 Physiological and biochemical characteristics of the SY5 strain

[0037]

[0038] In Table 1, "+" represents positive; "-" represents negative; "±" represents positive or negative; and "ND" represents no record in the literature. a, the data are derived from the literature (Cao H P, Zhang S M, Diao Q, Xu L, Gai C L. Isolation, identification and characterization of a Rhodobacter azotoformans strain SY5 that enhances the disease resistance of Eriocheir sinensis [J]. Biotechnol Bull, 2022, 38(11): 277-285); b, the data are derived from the literature (Messner, Katia, Vladimir Yurkov. Abundance, Characterization and Diversity of Culturable Anoxygenic Phototrophic Bacteria in Manitoban Marshlands. Microorganisms, 2024, 12(5): 12051007. Sanjin, Chad Saltikov. Genome sequence and characterisation of a freshwater photoarsenotroph, Cereibacter azotoformans strain ORIO, isolated from sediments capable of cyclic light–dark arsenic oxidation and reduction. Environmental Microbiology, 2023, 25(12): 3738-3752)

[0039] The physiological and biochemical identification results in Table 1 show that the strain SY5 grows anaerobically, does not have contact enzyme activity, has oxidase activity, can utilize glucose, fructose, mannitol, sorbitol, glycerol, mannose, alanine, xylose, acetate, malonate, sulfate, and cannot utilize methanol, butyrate, and is basically the same as the physiological and biochemical characteristics of azotobacter. Therefore, the strain SY5 is azotobacter. However, the strain SY5 is different from the azotobacter reported in document a in terms of contact enzyme and sulfate utilization, and is different from the azotobacter reported in document b in terms of sulfate utilization.

[0040] Example 2: Application of strain SY5 in inhibiting crop pathogenic fungi, the method is as follows:

[0041] (1) Take Micrococcus as a representative strain of crop pathogenic fungi to carry out growth inhibition test of pigment-producing strain SY5 on crop pathogenic fungi. Before the test, sterile operation is performed to inoculate the pigment-producing strain SY5 separated in Example 1 into fermentation medium (patent number: ZL 201810182288.7) to culture at 30℃, 160r / min, light intensity 2000Lx for 96h, and then prepare the culture solution with a concentration of 4.0×10 8 CFU / mL. The culture solution is added into PDA culture medium at a volume ratio of 10%, 20%, 30%, 40% and 50% respectively to prepare PDA culture medium plates added with the culture solution of pigment-producing strain SY5;

[0042] (2) Cladosporium tenuissimum was provided by the Marine University of Aquatic Animal Pathogen Library. Then, the Cladosporium tenuissimum with a diameter of 7 mm was inoculated in the central part of the PDA culture medium plate added with the culture solution of the pigment-producing strain SY5, and placed in a constant temperature incubator at 25°C. The colony diameters (mm) of the Cladosporium tenuissimum in the PDA culture medium plate were observed at 24, 48, 72, 96, 120, 144 and 168 h after culture, respectively, and the Cladosporium tenuissimum inoculated in the blank PDA culture medium plate under the same conditions was used as a control. Each treatment had three parallel plates. The inhibition rate of the pigment-producing strain SY5 on the Cladosporium tenuissimum was calculated according to formula (1).

[0043] Inhibition rate (%) = (control colony diameter - treatment colony diameter) / control colony diameter x 100% (1)

[0044] The obtained data were expressed as mean ± deviation, and the statistical analysis was performed by using SPSS 11.5 statistical software. P < 0.05 indicated a significant difference.

[0045] Table 2 Inhibition effect of the strain SY5 on the growth of Cladosporium tenuissimum

[0046]

[0047]

[0048] In Table 2, the same row and different letters indicated a significant difference (P < 0.05), and the same below.

[0049] The results are shown in Table 2 and Table 2. Figure 4 and Table 2, Figure 4 A1: control group; B1: 10% culture solution of the strain SY5; C1: 20% culture solution of the strain SY5; D1: 30% culture solution of the strain SY5; E1: 40% culture solution of the strain SY5; F1: 50% culture solution of the strain SY5. Figure 4The results show that the 10%, 20%, 30%, 40%, 50% strain SY5 culture solution has different degrees of inhibitory effect on the growth of Cladosporium tenuis, which is specifically shown as follows: the diameters of Cladosporium tenuis inoculated on the PDA medium plates containing 10%, 20%, 30%, 40%, 50% strain SY5 culture solution for 168 h are 38.67 mm, 34.67 mm, 25.33 mm, 15.67 mm and 7.00 mm respectively, which are reduced by 0.33 mm (P>0.05), 4.33 mm (P<0.05), 13.67 mm (P<0.05), 23.33 mm (P<0.05) and 32.00 mm (P<0.05) respectively compared with the diameter (39.00 mm) of Cladosporium tenuis inoculated on the blank PDA medium plate (control group) (Table 2), and thus the 168 h inhibitory rates of strain SY5 on Cladosporium tenuis are 0.85%, 11.10%, 35.05%, 59.82% and 82.05% respectively.

[0050] Example 3: The method for applying strain SY5 to prevent the gray mold of crops is as follows:

[0051] Taking blueberry as a representative of crops, the prevention effect test of the pigment-producing strain SY5 on the gray mold of crops was carried out. One control group and one treatment group were set, each group was set with three parallels, each parallel corresponded to 50 mu of blueberry planting area, and the planting density was 170 plants / mu. The treatment group of blueberry was treated with the pigment-producing strain SY5 culture solution, and the specific method was as follows:

[0052] (1) After pruning: the new branches of 10 cm in Example 2 were treated with the pigment-producing strain SY5 culture solution, and the first spraying + drip irrigation was carried out, that is, the pigment-producing strain SY5 culture solution (4.0 x 10 8 CFU / mL) was diluted 100 times with water and uniformly sprayed on the leaf surface of blueberry, and at the same time, drip irrigation was carried out on the blueberry (500 mL of 100 times diluted solution / plant), and 100 times diluted solution was used to spray the leaves for 3 times continuously in the next 2 months.

[0053] (2) Germination period: 100 times diluted solution of the pigment-producing strain SY5 culture solution was used for drip irrigation, and the amount was 500 mL of 100 times diluted solution / plant, once every half month.

[0054] (3) Initial flowering period: 100 times diluted solution of the pigment-producing strain SY5 culture solution was used for leaf spraying + drip irrigation, once every half month, that is, the pigment-producing strain SY5 culture solution was diluted 100 times with water and uniformly sprayed on the leaf surface of blueberry, and at the same time, drip irrigation was carried out on the blueberry according to the amount of 500 mL of 100 times diluted solution / plant.

[0055] (4) Flowering stage: 100-fold diluted SY5 culture solution was used for drip irrigation, with a dosage of 500 mL of 100-fold diluted solution per plant, once every half a month. The control group was sprayed with an equal amount of water. The disease index of the control group and the treatment group was observed according to the method in the literature (Su J, Li W, Yang J, et al. Field efficacy of five pesticides for controlling blueberry gray mold. Agricultural Technology Service, 2022, 39(12): 19-21.) at the initial flowering stage and the full flowering stage, and the control efficiency of SY5 on blueberry gray mold was calculated according to formula (2).

[0056] Control efficiency (%) = (disease index of the control group - disease index of the treatment group) / disease index of the control group x 100% (2)

[0057] The obtained data are expressed as mean ± deviation, and statistical analysis was performed using SPSS 11.5 statistical software. P<0.05 indicates a significant difference. The results are as follows:

[0058] Table 3 Control efficiency of SY5 on blueberry gray mold

[0059]

[0060] The experimental results in Table 3 show that the disease index of blueberry gray mold treated with SY5 is 1.43, which is significantly lower than that of the control group (9.90), P<0.05. Therefore, the control efficiency of SY5 on blueberry gray mold is 85.56%. This indicates that SY5 has a good preventive effect on blueberry gray mold.

[0061] Example 4: Method for reducing pesticide residues in vegetables using SY5 is as follows:

[0062] Thiamethoxam-pymetrozin (total effective ingredient content 80%, pymetrozin content 40%, thiamethoxam content 40%, water dispersible granule), bifenthrin-thiamethoxam (total effective ingredient content 20%, bifenthrin 10%, thiamethoxam 10%, suspension concentrate), abamectin- acetamiprid (total effective ingredient content 4%, abamectin content 0.5%, acetamiprid content 3.5%, microemulsion), cyprodinil-fludioxonil (total effective ingredient content 62%, fludioxonil content 25%, cyprodinil content 37%, water dispersible granule), propamocarb hydrochloride (effective ingredient and content: propamocarb hydrochloride 722 g / L, water agent pesticide), phoxim (effective ingredient 40%, emulsifiable concentrate), purchased from Shanghai Aoshan Industrial Development Co., Ltd.

[0063] With green vegetables as a representative of vegetables, the degradation effect test of the pigment-producing strain SY5 on pesticide residues in vegetables was carried out. One control group and six treatment groups were set up, each group was set up three parallels, and each parallel corresponded to 10 square meters of green vegetable planting area. The thiamethoxam-pymetrozine, bifenthrin-thiamethoxam, abamectin-acetamiprid, azoxystrobin-fludioxonil, propamocarb hydrochloride, and phoxim in the treatment groups were uniformly sprayed on the surface of green vegetable leaves according to the application method and dosage in the instruction book until the leaves were fully wet but no obvious liquid drops fell off, and then after 4 hours of leaf surface drying, the culture solution (4.0×10 8 CFU / mL) of the pigment-producing strain SY5 in Example 2 was diluted 50 times with sterile water and uniformly sprayed on the green vegetable leaves, and the green vegetables were irrigated with 200-250 mL of 50 times diluted solution per plant. The thiamethoxam-pymetrozine, bifenthrin-thiamethoxam, abamectin-acetamiprid, azoxystrobin-fludioxonil, propamocarb hydrochloride, and phoxim in the control group were uniformly sprayed on the surface of green vegetable leaves according to the application method and dosage in the instruction book until the leaves were fully wet but no obvious liquid drops fell off, and then after 4 hours of leaf surface drying, the green vegetables were sprayed and irrigated with the same amount of sterile water. Three days after the start of the test, pymetrozine, thiamethoxam, thiamethoxam, acetamiprid, azoxystrobin, propamocarb, and phoxim in the green vegetables were detected according to GB / T 20769-2008 Liquid Chromatography-Tandem Mass Spectrometry Method for Determination of Residues of 450 Pesticides and Related Chemicals in Fruits and Vegetables.

[0064] The obtained data were expressed as mean ± deviation, and statistical analysis was performed using SPSS 11.5 statistical software. P<0.05 indicates significant difference.

[0065] The results are shown in Table 1. Figures 5-10 Figure 5 It is shown that the residual amount of pymetrozine and thiamethoxam in the green vegetables of the treatment group is reduced by 40.29% (P<0.05) and 43.68% (P<0.05) compared with the control group, which indicates that the strain SY5 has a significant degradation effect on the residues of pymetrozine and thiamethoxam in green vegetables.

[0066] Figure 6 It is shown that the residual amount of thiamethoxam in the green vegetables of the treatment group is reduced by 9.73% (P>0.05) compared with the control group, which indicates that the strain SY5 has a certain degradation effect on the residues of thiamethoxam in green vegetables.

[0067] Figure 7 It is shown that the residual amount of acetamiprid in the green vegetables of the treatment group is reduced by 40.54% (P<0.05) compared with the control group, which indicates that the strain SY5 has a significant degradation effect on the residues of acetamiprid in green vegetables.

[0068] Figure 8 ​The results showed that the residual amount of fludioxonil in the treated group of Chinese cabbage was reduced by 4.71% (P>0.05) compared with the control group, indicating that strain SY5 had a certain degradation effect on the residual fludioxonil in Chinese cabbage.

[0069] Figure 9 The results showed that the residual amount of fludioxonil in the treated group of Chinese cabbage was reduced by 4.71% (P>0.05) compared with the control group, indicating that strain SY5 had a certain degradation effect on the residual fludioxonil in Chinese cabbage.

[0070] Figure 10 The results showed that the residual amount of fludioxonil in the treated group of Chinese cabbage was reduced by 4.71% (P>0.05) compared with the control group, indicating that strain SY5 had a certain degradation effect on the residual fludioxonil in Chinese cabbage.

Claims

1. A Rhodobacter azotoformans strain with a preservation name of Rhodobacter azotoformans SY5 and a preservation number of GDMCC NO: 65285, preserved in the Guangdong Microbial Culture Collection Center on October 14, 2024. 2.The Rhodobacter azotoformans strain of claim 1 is applied to inhibit crop pathogenic fungi.

3. Use according to claim 2, characterized in that, The crop pathogenic fungi include at least one of Clavibacter michiganensis and Botrytis cinerea. 4.The Rhodobacter azotoformans strain of claim 1 is applied to prevent gray mold of crops. 5.The application of claim 4, wherein the crops include at least one of blueberries, leeks, lilies, grapes, cucumbers, tomatoes, strawberries, roses, and chrysanthemums. 6.The Rhodobacter azotoformans strain of claim 1 is applied to degrade pesticide residues of vegetables.

7. Use according to claim 6, characterized in that, The pesticides include at least one of pymetrozine, thiamethoxam, acetamiprid, propamocarb, clothianidin, fludioxonil, and phoxim.

8. A composition for reducing pesticide residues in vegetables, characterized by, Live bacteria comprising the Rhodobacter azotoformans strain of claim 1.

9. The composition of claim 8, wherein, The concentration of the cell bodies of the nitrogen-fixing Rhodopseudomonas in the composition is at least 1.0 x 10 6 CFU / mL.

10. The composition of claim 8, wherein, The dosage form of the composition includes at least one of emulsifiable concentrate, suspension concentrate, wettable powder, powder, granule, aqueous agent, mother liquor, or slow-release agent.

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