Composite flora capable of degrading atrazine and promoting plant growth and application thereof
By constructing complex bacterial groups, including atrazine degradation bacteria and plant proliferation bacteria, using metabolites to improve the atrazine degradation efficiency and promote plant growth, the problems of soil pollution and plant growth are solved, and the problems of soil pollution and plant growth are provided. New ideas for restoring pesticide pollution.
Patent Information
- Application Number
- CN202411568104.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-05
- Publication Date
- 2025-02-18
AI Technical Summary
Long-term use of herbicides such as atrazine has resulted in soil pollution and limited plant growth, and the efficiency of existing microbial degradation methods is limited.
Complex bacterial flora, including the atrazine-degrading bacteria Pseudart robacter sp.rb and the plant proliferation bacteria Variovorax sp.tm, were constructed to improve the atrazine degradation efficiency and promote plant growth through metabolite cross-feeding.
It significantly improves the degradation efficiency of atrazine, and promotes plant growth through the subsequent degradation of metabolites, providing new ideas for the repair of pesticide pollution.
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Figure CN119464124A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of agriculture, and in particular to a composite bacterial community capable of degrading atrazine and promoting plant growth, and also to the application of the composite bacterial community. Background Art
[0002] Atrazine has been produced in China since the 1970s and is still one of the most widely used herbicides in China. The Northeast Black Soil Region is an important agricultural production base in China and is also the area where atrazine is most widely used, especially in corn production. The Northeast Black Soil is fertile and rich in nutrients necessary for agricultural production and social benefits. However, the long-term and large-scale use of fertilizers, herbicides, and pesticides has led to increased soil pollution, degradation of soil ecological functions, and reduction of biodiversity, limiting the improvement of black soil productivity and quality.
[0003] Microbial degradation has long been considered an efficient and low-cost method to reduce atrazine residues in soil and alleviate the growth of atrazine-stressed plants. Atrazine-degrading strains have been isolated from soil or sewage, which can use atrazine as the sole nitrogen or carbon source, such as Pseudomonas sp. ADP. There are two main pathways for common atrazine microbial degradation. The first is controlled by atrazine degradation genes atzA, atzB, atzC, atzD, atzE, and atzF, which gradually degrade atrazine into ammonia and carbon dioxide. The second pathway involves trzN, atzB, and atzC, which degrades atrazine into cyanuric acid.
[0004] In recent years, the application of artificial microbial consortia has provided new prospects for environmental remediation. This method uses the "division of labor" mechanism to distribute the burden to the entire population to improve microbial resistance and environmental remediation efficiency. Compared with single microorganisms, their environmental behaviors are more complex. Summary of the invention
[0005] In view of this, one of the objects of the present invention is to provide a composite bacterial community capable of degrading atrazine and promoting plant growth, and a second object of the present invention is to provide the use of the composite bacterial community in degrading atrazine.
[0006] In order to achieve the above object, the present invention provides the following technical solutions:
[0007] 1. A composite bacterial community capable of degrading atrazine and promoting plant growth, the composite bacterial community comprising atrazine-degrading bacteria (Pseudarthrobacter sp.rb) and plant growth-promoting bacteria (Variovorax sp.tm);
[0008] The deposit number of the atrazine-degrading bacteria (Pseudarthrobacter sp.rb) is GDMCC NO: 64867, and the deposit number of the plant growth-promoting bacteria (Variovorax sp.tm) is GDMCC NO: 64866.
[0009] 2. Application of the composite bacterial community in the degradation of atrazine.
[0010] Preferably, the composite flora of atrazine-degrading bacteria (Pseudarthrobacter sp.) and plant growth-promoting bacteria (Variovorax sp.) is used to promote plant growth in an atrazine environment.
[0011] Preferably, the promoting of plant growth in the present invention is promoting the growth of taproot, total root network or root hair.
[0012] Preferably, the volume ratio of the atrazine-degrading bacteria (Pseudarthrobacter sp.rb) to the plant growth-promoting bacteria (Variovorax sp.tm) is 1:1.
[0013] The beneficial effects of the present invention are as follows: by constructing a new type of artificial microbial community, including atrazine-degrading bacteria Pseudarthrobacter sp.rb and plant growth-promoting bacteria Variovorax sp.tm, the atrazine degradation efficiency is significantly improved compared with a single bacterial community, and the subsequent degradation of atrazine metabolites is achieved through cross-feeding between metabolites, thereby promoting plant growth. This provides a new idea for pesticide pollution remediation and lays a foundation for the research of multifunctional composite strains in pesticide degradation and plant growth promotion. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:
[0015] Figure 1 The degradation mechanism of atrazine by strain rb; a, liquid chromatography determination of substrate amount and intermediate metabolite cyanuric acid content of strain rb using 100 mg / L atrazine as carbon and nitrogen source; b, growth of strain rb on inorganic salt medium containing atrazine, where the white powder is atrazine, and atrazine is degraded to produce a transparent circle around the colony growth area; c, PCR of atrazine degradation functional genes contained in strain rb; d, prediction of atrazine degradation pathway of strain rb; e, mass spectrum of atrazine identified by LC-MS; f, mass spectrum of hydroxyatrazine identified by LC-MS; g, mass spectrum of cyanuric acid identified by LC-MS.
[0016] Figure 2 Changes in substrate concentration, intermediate metabolite cyanuric acid and cell concentration within 7 days when strain rb and strain tm were cultured alone and co-cultured at v:v=1:1 and inoculated into an inorganic salt culture medium with atrazine as the carbon and nitrogen source (a, b, c, d); changes in substrate concentration and cell concentration within 7 days when strain rb and strain tm were cultured alone and co-cultured at v:v=1:1 and inoculated into an inorganic salt culture medium with cyanuric acid as the carbon and nitrogen source (e, f); a, change in atrazine concentration; b, change in cyanuric acid concentration; c, change in total cell concentration; d, the number of colonies that can be formed in each milliliter of culture medium of strain rb and strain tm during the co-culture process within 7 days; e, change in cyanuric acid concentration; f, change in cell concentration.
[0017] Figure 3 The effects of single strains and multifunctional composite strains on the root growth of Arabidopsis seedlings. a, Plate imaging of single strains and multifunctional composite strains inoculated into MM medium for planting Arabidopsis seedlings; b, Microscopic imaging of the effects of single strains and multifunctional composite strains on the growth of root hairs of Arabidopsis seedlings; c, Effects of single strains and multifunctional composite strains on the growth of the main root of Arabidopsis seedlings; d, Effects of single strains and multifunctional composite strains on the total root network of Arabidopsis seedlings; e, Effects of single strains and multifunctional composite strains on the length of root hairs of Arabidopsis seedlings; f, Effects of single strains and multifunctional composite strains on the number of root hairs per unit length of Arabidopsis seedlings.
[0018] Figure 4 The effects of single and mixed strains on the growth of corn seedlings in Northeast China black soil containing atrazine. a, the overall morphological appearance of corn seedlings under different treatments; b, the effects of different treatments on the fresh weight of the aboveground part of corn seedlings; c, the effects of different treatments on the root weight of corn seedlings; d, the effects of different treatments on the taproot length of corn seedlings; e, the effects of different treatments on the plant height of corn seedlings.
[0019] Strain preservation
[0020] The atrazine-degrading bacteria (Pseudarthrobacter sp.rb) used in the present invention are referred to as rb and the plant growth-promoting bacteria (Variovorax sp.tm) are referred to as tm, both of which are separated from the Northeast black soil by using the atrazine concentration gradient pressure acclimation method; they are sent to the Guangdong Microbial Culture Collection Center for preservation, the atrazine-degrading bacteria (Pseudarthrobacter sp.rb) is stored with a storage number of GDMCC NO: 64867, the storage address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, China, the storage date is July 12, 2024, and the classification name is atrazine-degrading bacteria (Pseudarthrobacter sp.rb).
[0021] The deposit number of the plant growth-promoting bacteria (Variovorax sp.tm) is GDMCC NO: 64866, the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, China, the deposit date is July 12, 2024, and the classification name is plant growth-promoting bacteria (Variovorax sp.tm). DETAILED DESCRIPTION
[0022] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.
[0023] Example 1
[0024] The atrazine degradation mechanism of strain rb is as follows:
[0025] Take out the test tube containing atrazine-degrading bacteria (Pseudarthrobacter sp.rb) from the -20℃ refrigerator and immediately put it on ice to revive the bacteria. Wipe the surface of the cryopreserved tube clean, disinfect the surface with alcohol, and then perform subsequent operations on the clean bench; draw 5μL of bacterial solution from the cryopreserved tube and purify it on TSB solid culture medium, then place it in a 28℃ constant temperature incubator for 2-3 days until a single colony grows. Pick the grown single colony in the clean bench and inoculate it into TSB liquid culture medium, and culture it in a constant temperature shaker at 120rpm and 28℃ for 2-3 days; measure the cell concentration with a UV spectrophotometer, wash off the residual nutrients on the bacteria with sterile PBS and adjust the OD 600 ≈1.0, and then inoculated into a basic inorganic salt medium containing 100 mg / L atrazine at a volume ratio of 1:50, and cultured in a constant temperature shaker at 120 rpm and 28°C. After 6 days, the concentrations of atrazine and cyanuric acid in the culture medium were measured by liquid chromatography.
[0026] Single rb colonies grown on TSB medium were selected and streaked on inorganic salt solid medium containing atrazine, and cultured in a 28°C biochemical incubator for 30 days.
[0027] Atrazine metabolites in the culture medium were determined by LC-MS.
[0028] PCR amplification was performed on the atrazine degradation functional genes contained in strain rb, including atzA, atzB, atzC, atzD, atzE, atzF, trzN and trzD.
[0029] The results showed that strain rb was able to degrade atrazine. The atrazine degradation functional genes contained in strain rb were trzN, atzB and atzC. The atrazine degradation pathway of strain rb was atrazine → hydroxyatrazine → isopropylamine-cyanuric acid-amide → cyanuric acid ( Figure 1 ).
[0030] Example 2
[0031] Changes in substrates, intermediate metabolites and cell concentrations during atrazine degradation by single strains and composite strains. The specific steps are as follows:
[0032] Take out the atrazine-degrading bacteria (Pseudarthrobacter sp.rb) and plant growth-promoting bacteria (Variovorax sp.tm) from the -20℃ refrigerator and immediately put them on ice to revive the bacteria. Wipe the surface of the cryopreserved tube clean, disinfect the surface with alcohol, and then perform subsequent operations on the clean bench; draw 5μL of bacterial solution from the cryopreserved tube and purify it on the TSB solid culture medium, then place it in a 28℃ constant temperature incubator for 2-3 days until a single colony grows. Pick the grown single colony in the clean bench and inoculate it into TSB liquid culture medium, and culture it in a constant temperature shaker at 120rpm and 28℃ for 2-3 days; measure the cell concentration with a UV spectrophotometer, wash off the residual nutrients on the bacteria with sterile PBS and adjust the OD 600 ≈1.0, then inoculated into a basic inorganic salt medium containing 100 mg / L atrazine at a volume ratio of 1:50, and cultured in a constant temperature shaker at 120 rpm and 28°C. Samples were taken every 24 hours to measure the atrazine, cyanuric acid content and cell concentration in the culture medium. There are four treatments in total:
[0033] Control: 100 mg / L atrazine; (2) Monoculture_rb: 100 mg / L atrazine + strain Pseudarthrobacter sp.rb; (3) Monoculture_tm: 100 mg / L atrazine + strain Variovorax sp.tm; (4) Coculture_Mix: 100 mg / L atrazine + strain Pseudarthrobacter sp.rb + strain Variovorax sp.tm; Three replicates were set for each treatment.
[0034] Then the changes of atrazine concentration, cyanuric acid concentration and cell concentration during the cultivation of single strain and multifunctional composite strain were detected. The results are as follows Figure 2As shown. The results showed that compared with the single strain rb, the co-culture of rb and tm significantly improved the atrazine degradation efficiency of rb. tm did not grow in the inorganic salt medium with atrazine as the carbon and nitrogen source, but after the addition of strain rb, tm resumed growth. Single culture rb degraded atrazine into cyanuric acid, and the amount of cyanuric acid continued to accumulate from the fourth day. During the co-culture of rb and tm, tm, which could not grow, resumed growth and achieved the subsequent degradation of cyanuric acid. Under the co-culture system, the number of colonies formed by rb per milliliter of culture medium was significantly less than that of single culture, but the atrazine degradation efficiency was significantly improved. This may be because strain tm degraded cyanuric acid, reducing the concentration of cyanuric acid in the culture medium, thereby stimulating the positive feedback regulation mechanism of strain rb, increasing the gene expression of trzN, atzB and atzC, and increasing the enzyme catalytic reaction rate, thereby improving the degradation efficiency of atrazine. Under the culture conditions with cyanuric acid as the carbon and nitrogen source, rb cultured alone could not grow and did not degrade cyanuric acid. The co-culture of rb and tm significantly improved the degradation efficiency of the substrate cyanuric acid compared with tm cultured alone.
[0035] Example 3
[0036] Single strain rb, single strain tm, and mixed strains rb and tm (ratio 1:1) were pre-cultured in TSB medium until OD 600 ≈1.0, then use an inoculation loop to dip the bacterial solution and streak it on the plate below 1 / 2MS medium, and the plate without bacteria inoculation is used as a control. All experiments included two independent replicates, each replicate included 3 plates.
[0037] Arabidopsis thaliana seeds (Arabidopsis thaliana Col-0seedlings) were first washed with 70% ethanol for 1 minute, then rinsed with 1% sodium hypochlorite for 7 minutes, and finally rinsed with sterile water for 5 times to remove the residual disinfectant on the seed surface. The sterilized Arabidopsis seeds were inoculated into a square vertical culture dish containing 1 / 2MS medium and cultured for 7 days, and then 10 seedlings with basically the same growth were selected and transferred to a new agar plate and inoculated with bacteria (rb, tm, rb and tm) and cultured for 12 days.
[0038] After 12 days of culture, the agar plates were imaged using a root scanner. The main root length, root hair length and number of root hairs were measured using Fiji-ImageJ, and the total root network was measured using WinRhizo software. Figure 3 shown.
[0039] The results showed that rb significantly inhibited the growth of the root system of Arabidopsis seedlings, while tm significantly promoted the growth of the root system of Arabidopsis seedlings. When rb and tm were co-inoculated, strain tm relieved the inhibition of strain rb on the root growth of Arabidopsis seedlings.
[0040] Example 4
[0041] The experimental setting of the effect of inoculation of single strain and multifunctional composite strain on the growth of corn seedlings in Northeast black soil containing atrazine is as follows:
[0042] The strains rb and tm were inoculated into TSB medium for enrichment culture respectively. When the cells grew to OD600≈1.0, the cells were collected, washed with sterile water, and resuspended with sterile water to OD600≈1.0.
[0043] Soak the selected corn seeds in 10g / L sodium hypochlorite solution for disinfection for 12 minutes, then soak the corn seeds in 75% ethanol for disinfection for 1 minute, repeatedly wash with sterile water to remove the disinfectant remaining on the surface of the seeds, and then soak the corn seeds in sterile water for 6 hours for use.
[0044] Northeastern black soil and sterilized vermiculite were prepared in a volume ratio of 1:1, and atrazine (atrazine was dissolved in acetone) was added to the soil. The soil was stirred evenly to make the atrazine concentration in the soil 50 mg / kg, and placed in a fume hood until the acetone was completely evaporated.
[0045] Place the sterilized corn seeds in a square culture dish with sterile gauze at the bottom, add sterile water to the culture dish until the corn seeds are just soaked, and then place the culture dish containing the seeds in a constant temperature of 25°C and dark environment to germinate for 5 days. After that, select corn seedlings with consistent root and shoot growth for subsequent potted experiments.
[0046] Select seedlings with good growth and consistent growth and move them into pots, and plant 3 seedlings in each pot. The first day is the day of transplanting, and the culture is 25 days; the method of inoculating bacteria is root irrigation, and the bacteria are inoculated twice, on the first day and the 10th day, and 5mL of bacterial suspension is poured into the root zone of the corn seedlings each time; sterile water is used for watering, and watering is done once every two days. The background is a black soil-sterile vermiculite mixed soil containing 50mg / kg atrazine, and the specific experimental settings are as follows:
[0047] (1) Mixed soil + corn seedlings; (2) Mixed soil + corn seedlings + rb; (3) Mixed soil + corn seedlings + tm; (4) Mixed soil + corn seedlings + rb + tm.
[0048] Then the effects of single strains and multifunctional composite strains on the growth of corn seedlings in Northeast China black soil containing atrazine were statistically analyzed. Figure 4The results showed that in the mixed system of black soil and vermiculite (1:1) containing atrazine, rb significantly inhibited the growth of the root system of corn seedlings and reduced the weight of the aboveground part of corn seedlings; tm significantly promoted the growth of corn seedlings. When rb and tm were co-inoculated, strain tm relieved the inhibition of strain rb on the growth of corn seedlings.
[0049] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.
Claims
1. A composite bacterial community capable of degrading atrazine and promoting plant growth, characterized in that: The composite bacterial community includes atrazine-degrading bacteria (Pseudarthrobacter sp.rb) and plant growth-promoting bacteria (Variovorax sp.tm); The deposit number of the atrazine-degrading bacteria (Pseudarthrobacter sp.rb) is GDMCC NO: 64867, and the deposit number of the plant growth-promoting bacteria (Variovorax sp.tm) is GDMCC NO: 64866.
2. Use of the composite bacterial flora according to claim 1 in degrading atrazine.
3. The use according to claim 1, characterized in that: The composite bacterial community of atrazine-degrading bacteria (Pseudarthrobacter sp.) and plant growth-promoting bacteria (Variovorax sp.) is used to promote plant growth in an atrazine environment.
4. The use according to claim 2, characterized in that: Promoting plant growth is promoting the growth of the taproot, the total root network or the root hairs.
5. The use according to claim 1 or 2, characterized in that: The volume ratio of the atrazine-degrading bacteria (Pseudarthrobacter sp.rb) and the plant growth-promoting bacteria (Variovorax sp.tm) is 1:1.