CN112694999B - Achromobacter xylosoxidans, microbial inoculum comprising same, and preparation method and application thereof - Google Patents
Achromobacter xylosoxidans, microbial inoculum comprising same, and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a strain of Achromobacter xylosoxidans (Achromobacter xylosoxidans) SL-6 with the preservation number as follows: CGMCC No.20949 belongs to the technical field of microbial preparations. The achromobacter xylosoxidans SL-6 has high tolerance to high-concentration diuron, can grow in a culture medium with the diuron as a unique carbon source, can degrade diuron medicaments in soil, and can degrade more than 90% of diuron with the concentration of 500mg/kg within 72 hours. The method for degrading diuron by using the xylose oxidation achromobacter SL-6 strain belongs to a biological metabolism method, and does not produce secondary pollution. The invention provides an efficient and excellent strain for efficiently degrading diuron and provides a high-quality strain resource for repairing and treating diuron-polluted soil in agricultural areas for a long time.
Description
Technical Field
The invention relates to the technical field of microbial preparations, in particular to an achromobacter xylosoxidans strain, a microbial inoculum comprising the achromobacter xylosoxidans strain, a preparation method and application thereof.
Background
Diuron (diuron) is a low-toxicity substituted urea herbicide, and has a systemic conduction effect and a certain contact action. After the medicament is absorbed by roots or leaves of plants, photosynthesis is inhibited, so that the leaves are green, the leaf tips and edges are faded, and the plants die due to lack of nutrition. Is mainly used for preventing and killing annual gramineous weeds in crops such as cotton, soybean, tomato and the like. The degradation mechanism of diuron is mainly biodegradation and photodegradation, and biodegradation is an important strategy for reducing harmful compounds. At present, the microbial method for degrading pesticides mainly comprises the following steps: the method comprises the steps of screening and separating strains with excellent properties and good growth from soil seriously polluted by pesticides for a long time, carrying out directional culture, and carrying out artificial mutation breeding, genetic engineering, construction of engineering strains and the like on the basis. However, related researches on diuron microbial degradation at home and abroad are few, and no report or record is provided for degrading diuron by using achromobacter xylosoxidans.
Disclosure of Invention
The invention aims to provide an achromobacter xylosoxidans strain, a microbial inoculum comprising the achromobacter xylosoxidans strain, a preparation method and application.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a strain of Achromobacter xylosoxidans (Achromobacter xylosoxidans) SL-6 with the preservation number as follows: CGMCC No. 20949.
In the present invention, the Achromobacter xylosoxidans SL-6 belongs to Achromobacter (Achromobacter sp.); in 2018, the Podocon is collected in a cotton field of a general stone field of the northern spring town of Stone river, and the achromobacter xylosoxidans SL-6 has high tolerance to high-concentration diuron and can grow in a culture medium with the diuron as a unique carbon source.
The invention also provides a microbial inoculum comprising the achromobacter xylosoxidans in the scheme.
Preferably, the effective viable count of the achromobacter xylosoxidans in the microbial inoculum is 3.15 multiplied by 10 8 CFU/mL。
The invention also provides a preparation method of the microbial inoculum in the scheme, which comprises the following steps:
inoculating the achromobacter xylosoxidans into an LB liquid culture medium, and performing amplification culture to obtain a microbial inoculum;
the initial concentration of the achromobacter xylosoxidans is (1-9) multiplied by 10 9 CFU/mL; the inoculation amount of the achromobacter xylosoxidans is 10-20% of the volume of the LB liquid culture medium.
Preferably, the temperature of the amplification culture is 28-32 ℃; the rotation speed of the amplification culture is 150-200 rpm; the time of the amplification culture is 3-5 days.
The invention also provides application of the xylose oxidation achromobacter or the microbial inoculum prepared by the preparation method in degrading diuron.
The invention also provides application of the xylose oxidation achromobacter or the microbial inoculum prepared by the preparation method of the microbial inoculum in improving the degradation rate of diuron.
The invention also provides application of the xylose oxidation achromobacter or the microbial inoculum prepared by the preparation method in reducing plant damage caused by diuron and/or promoting plant growth.
Preferably, when the diuron is diuron in soil, the application comprises the following steps:
inoculating a microbial inoculum containing the achromobacter xylosoxidans in the soil;
adjusting the water content of the soil to be 55-65%.
Preferably, the inoculation mode comprises spraying or drip irrigation of the microbial inoculum with water.
The invention has the beneficial effects that: the invention provides a strain of Achromobacter xylosoxidans (Achromobacter xylosoxidans) SL-6 with the preservation number as follows: CGMCC No. 20949. The achromobacter xylosoxidans SL-6 has high tolerance to high-concentration diuron, can grow in a culture medium with the diuron as a unique carbon source, can degrade diuron medicaments in soil, and can degrade more than 90% of diuron with the concentration of 500mg/kg within 72 hours. The method for degrading diuron by using the achromobacter xylosoxidans SL-6 strain belongs to a biological metabolism method, and does not produce secondary pollution. The invention provides an efficient and excellent strain for efficiently degrading diuron and provides a high-quality strain resource for repairing and treating diuron-polluted soil in agricultural areas for a long time.
Biological preservation Instructions
Achromobacter xylosoxidans (Achromobacter xylosoxidans) SL-6) is preserved in the common microorganism center of China general microbiological culture Collection management Committee in 26 months 10 and 2020, and is addressed to Xilu No.1 Hospital No.3 of Beijing market facing Guangyang district, with the preservation number: CGMCC No. 20949.
Drawings
FIG. 1 shows actinomycetes SL-6 isolated in example 1;
FIG. 2 is a graph showing the gram staining results of the strain of example 1: gram staining is negative;
FIG. 3 is a phylogenetic tree;
FIG. 4 shows the growth of cotton seedlings under different treatments; wherein A is CK clear water contrast; b is diuron content of 500mg/kg, and no strain is inoculated; d is the medicament + SL-6.
Detailed Description
The invention provides a strain of Achromobacter xylosoxidans (Achromobacter xylosoxidans) SL-6 with the preservation number as follows: CGMCC No. 20949.
The invention also provides a microbial inoculum comprising the achromobacter xylosoxidans in the scheme.
In the invention, the effective viable count of the achromobacter xylosoxidans in the microbial inoculum is preferably 3.15 multiplied by 10 8 CFU/mL。
The invention also provides a preparation method of the microbial inoculum in the scheme, which comprises the following steps:
inoculating the achromobacter xylosoxidans into an LB liquid culture medium, and performing amplification culture to obtain a microbial inoculum; the initial concentration of the achromobacter xylosoxidans is (1-9) multiplied by 10 9 CFU/mL; the inoculation amount of the achromobacter xylosoxidans is 10-20% of the volume of the LB liquid culture medium. In the present invention, the initial concentration of the Achromobacter xylosoxidans is preferably 5.6X 10 9 CFU/mL; the inoculation amount of the achromobacter xylosoxidans is 15 of the volume of the LB liquid culture medium.
In the invention, the LB liquid culture medium takes water as a solvent and comprises the following components in mass concentration: 8-12 g/L of tryptone, 3-8 g/L of yeast extract and 8-12 g/L of NaCl.
In the invention, the temperature of the amplification culture is preferably 28-32 ℃, and more preferably 30 ℃; the rotation speed of the amplification culture is preferably 150-200 rpm, and more preferably 180 rpm; the time for the amplification culture is preferably 3-5 d, and more preferably 4 d; the enlarged culture is preferably dark culture; the humidity of the scale-up culture is preferably 55% to 65%, and more preferably 60%.
The invention also provides application of the xylose oxidation achromobacter or the microbial inoculum prepared by the preparation method in degrading diuron.
The invention also provides application of the xylose oxidation achromobacter or the microbial inoculum prepared by the preparation method of the microbial inoculum in improving the degradation rate of diuron.
The invention also provides application of the xylose oxidation achromobacter or the microbial inoculum prepared by the preparation method in reducing plant damage caused by diuron and/or promoting plant growth.
In the invention, the concentration of the diuron is preferably less than or equal to 1000mg/kg, because the diuron with high concentration in the soil can inhibit the growth of degradation strains, thereby reducing the degradation efficiency.
In the present invention, when the diuron is diuron in soil, the application includes the steps of: inoculating a microbial inoculum containing the achromobacter xylosoxidans in the soil;
adjusting the water content of the soil to be 55-65%.
In the present invention, the water content of the soil is preferably 60%.
In the present invention, the inoculation mode preferably includes spraying or dripping the microbial inoculum with water.
In the present invention, the inoculation amount of the microbial inoculum is 8% to 12% of the volume of the soil, and more preferably 10%.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It should be apparent that the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The test methods used in the following examples are all conventional methods unless otherwise specified.
Materials, reagents and the like used in the following examples were commercially available unless otherwise specified.
The media used in the following examples are as follows:
A. inorganic salt culture medium: MgSO (MgSO) 4 ·7H 2 O 0.2g，KH 2 PO 4 0.5 g，(NH 4 ) 2 SO 4 1 g，NaCl 0.5g，K 2 HPO 4 1.5 g, 1000mL of distilled water, and pH 7.0-7.2.
B. Beef extract peptone medium: 3.0g of beef extract, 10.0g of peptone, 5.0g of NaCl, 1000mL of distilled water and pH 7.0-7.2.
C. Enrichment culture medium: diuron is added into the inorganic salt culture medium at the required concentration.
LB medium: 10g of tryptone, 5g of yeast extract, 10g of NaCl and 1000mL of distilled water.
Solid culture medium 18g/L agar powder was added to the above medium.
Example 1
In 2018, soil continuously applied with diuron is collected in a cotton field of a general rocky town stone farm in northern spring town of the river city for more than 5 years, the soil is inoculated into an inorganic salt culture medium for shaking culture and is continuously transferred into an enrichment culture medium containing the diuron, and the formula of the enrichment nutrient solution is as follows: 10g/L of tryptone, 5g/L, NaCl 10g/L of yeast extract, 15g/L of agar powder and 1L of distilled water. Adding 10g of test soil sample into 90mL of enrichment culture solution with diuron concentration of 100mg/L, culturing for 7 days at 30 ℃ at 180r/min, taking liquid with 10% inoculation volume (v: v), inoculating the liquid into the enrichment culture solution with diuron concentration of 500mg/L, continuously culturing for 7 days, then inoculating the enrichment culture solution into the enrichment culture solution with diuron content of 1000mg/L according to 10% inoculation amount, continuously transferring to 1500mg/L, 1800mg/L and 2000mg/L in the way, carrying out plate coating, finding that bacterial colonies do not appear on the culture solution coated plate when the diuron content is 2000mg/L, and thus taking the single bacterial colony coated when the final diuron content is 1800mg/L to carry out subsequent tests. Before each operation, the microorganism plate coating is needed before the transfer to ensure that the strain is in a survival state (the steps are all carried out on an ultra-clean workbench).
Selecting strains with good growth numbers for storage, and taking the strains as alternative strains and naming the strains as SL-1 to SL-12. Inoculating single colony of 12 candidate strains with serial number into a culture medium of beef extract peptone liquid, placing on a shaker at 30 deg.C and 180r/min, and culturing for 1d to obtain seed liquid. Inoculating 10% inoculation volume of seed liquid into an inorganic salt degradation culture medium with diuron concentration of 100mg/L, placing on a shaking bed for shaking culture, taking degradation bacterial liquid after 3 days, 6 days, 9 days, 12 days and 15 days after shaking culture to detect the diuron content in the degradation bacterial liquid, and calculating the degradation efficiency. And then screening to obtain the target strain SL-6.
And performing morphological identification and physiological and biochemical experiments on the separated and purified bacterial strain SL-6, extracting genomic DNA of the bacterial strain, performing PCR amplification, sequencing a recovered product, performing sequence analysis on functional genes of related species, searching homologous sequences of the obtained sequence in GenBank, and constructing a phylogenetic tree by using DNMAN software.
SL-6 was identified based on multiple identification analyses of cell morphology (see fig. 1, fig. 1 is a colony morphology map of strain SL-6), physiological and biochemical data (see fig. 2, a gram stain result map of strain SL-6, gram stain negative), 16S rDNA sequence and nrdA functional gene sequence data: achromobacter xylosoxidans (Achromobacter xylosoxidans). FIG. 3 is a phylogenetic tree of the SL-6 strain.
The separation and identification process is as follows:
culturing and separating strains: adding 10g of test soil sample into 90mL of enrichment culture solution with diuron concentration of 100mg/L, culturing for 7 days at 30 ℃ at 180r/min, taking liquid with 10% inoculation volume (v: v), inoculating the liquid into the enrichment culture solution with diuron concentration of 500mg/L, continuously culturing for 7 days, then inoculating the enrichment culture solution into the enrichment culture solution with diuron content of 1000mg/L according to 10% inoculation amount, continuously transferring to 1500mg/L, 1800mg/L and 2000mg/L in the way, carrying out plate coating, finding that bacterial colonies do not appear on the culture solution coated plate when the diuron content is 2000mg/L, and thus taking the single bacterial colony coated when the final diuron content is 1800mg/L to carry out subsequent tests.
Conventional morphological identification: the test strains were inoculated on LB plates, cultured at 30 ℃ and observed for the basic morphological characteristics of the colonies, such as external morphology, size, color, etc., after different days.
Molecular biological identification:
PCR primer
27F: 5'-agagtttgatcctggctcag-3', as shown in SEQ ID NO. 1;
1492-R: 5'-ggttaccttgttacgactt-3', as shown in SEQ ID NO. 2;
nrdA-F: 5'-actgattc ccgacctgttc-3', as shown in SEQ ID NO. 3;
nrdA-R: 5'-ttcgatttgacgtac aagttc tgg-3', as shown in SEQ ID NO. 4;
strain genome DNA extraction
1) Activating the strain.
2) 1.0mL of the bacterial solution was put in a 1.5mL tube at 12000r/min for 2min, and the supernatant was discarded.
3) The cells were collected and resuspended in 1.0mL of 0.85% saline.
4)12,000 r/min 2min, and discarding the supernatant.
5) The cells were collected in 550. mu.L of 1 XTE.
6) Add 17. mu.L lysozyme (35mg/mL) and let stand at 38 ℃ for 0.5 h.
7) Add 3. mu.L proteinase K (20mg/mL) and let stand at 38 ℃ for 0.5 h.
8) Add 30. mu.L 10% SDS at 38 ℃ for 30 min.
9) Add 100. mu.L of 5M NaCl and mix well.
10) Adding 80 μ of LCTAB/NaCl solution, mixing, and water bathing at 65 deg.C for 10 min.
11) Adding chloroform/isoamyl alcohol (24:1) with the same volume (0.7-0.8 mL) and shaking gently until the mixture is uniform.
12) Treating at 12,000 r/min for 10 min.
13) The upper layer was transferred to another 1.5mL tube and mixed by gentle shaking with the same volume of phenol/chloroform/isoamyl alcohol (25:24: 1).
14) Repeating step 12).
15) The treatment is carried out at 20 ℃ and 12,000 r/min for 7 min.
16) The supernatant was discarded, 500. mu.L of 70% ethanol was added, and the mixture was gently shaken to wash the salt.
17) The treatment is carried out at 4 ℃ and 12,000 r/min for 7 min.
18) The salt washing was repeated twice.
19) The centrifuge tube was inverted and the DNA pellet was dried for 7 min.
20) The DNA pellet was dissolved in 100. mu.L of 1 XTE buffer and stored at-20 ℃ until use.
PCR amplification system
And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 1min, annealing at 57 ℃ for 1min, extension at 72 ℃ for 1.5min (30 cycles above); post-extension at 72 ℃ for 5 min.
16SrDNA reaction system (100. mu.L system): taq (5U/. mu.L) 0.8. mu.L; 10 × PCR Buffer (Mg) 2+ Plus) 10. mu.L; dNTP mix (2.5mM/each) 8. mu.L; 2.5 ng of template DNA; primer F 1 (10. mu. mol/L) 2. mu.L, primer R 1 (10μmol/L)2μL；ddH 2 O make up to 100. mu.L.
nrdA gene sequence reaction conditions: pre-denaturation 95 ℃ for 5min, denaturation 95 ℃ for45 s (33 cycles), annealing 52 ℃ for45 s; extension at 72 ℃ for 60 s; after extension 10min at 72 ℃.
nrdA reaction system (100 μ L system): mu.L of upstream primer (50 mu mol/L) and mu.L of downstream primer (50 mu mol/L); taq (2.5U/ml) 0.8. mu.L; 10 × PCR Buffer (Mg) 2+ Plus) 10. mu.L; dNTP mix (2.5. mu.M/each) 8. mu.L; 10ng of template DNA; ddH 2 O make up to 100. mu.L.
And (3) after sequencing the product, comparing the result in a GenBank database through BLAST to construct a phylogenetic tree.
Physiological and biochemical identification: refer to BIOLOG GEN III, API 50CH, API ZYM, etc.
As a result: and (3) gradually increasing the diuron content in the culture medium through plate primary screening, performing pressure screening, screening 12 strains, naming the strains as SL-1-12, and coating for storage. Wherein the degradation effect of the bacterial strain SL-6 on diuron is obviously higher than that of the rest strains. Extracting DNA of the SL-6 diuron degrading strain, taking the DNA as a template, performing PCR amplification by using a 16S rRNA gene universal primer to respectively obtain fragments of about 1500bp, recovering PCR products, connecting and transforming, and then sequencing. It was preliminarily determined that the strain SL-6 was Achromobacter xylosoxidans of Achromobacter sp.
The colony morphology of the LB medium of the strain SL-6 is shown in figure 1, and the gram stain of the SL-6 is negative (figure 2); the temperature tolerance range of the strain is 10-40 ℃, and the optimal growth temperature is 28 ℃; the pH tolerance range is pH5.0-8.0, and the optimum pH is 7.0; can tolerate NaCl below 4%. Culturing for4 days at 28 ℃ on an LB culture medium to form a bacterial colony with the diameter of 0.8-1.2 mm, wherein the bacterial colony is light yellow and has a wet and smooth surface; the cells are in the shape of short rods, have the size of about (0.8-1.0 multiplied by 0.9-1.1 mu m), have no motility and do not form endospores.
The results of physiological and biochemical characteristic tests of the strain SL-6 comprise an API ZYM enzymatic test, an API 50CH acidogenic test and a Biolog G3 test (Table 1).
TABLE 1 physiological and biochemical characteristics of the Strain SL-6
+: positive; -: negative; w: and (4) weak positive.
The 16s rDNA sequence of the strain SL-6 is shown in SEQ ID NO. 5 through determination.
The MEGA7 software is adopted, the temporary connection method shows that the strain SL-6 and a 16S rDNA phylogenetic tree of related species are subjected to similarity repeated calculation for 1000 times, the nodes of the phylogenetic tree in the graph only show that the Bootstrap value is more than 50% of the numerical value, and the superscript 'T' represents a model strain.
As can be seen from FIG. 3, the homology between the strain SL-6 and the strain of Achromobacter xylosidases is as high as 99.65%; the homology with the model species under Achromobacter xylosoxidans is as high as 99.51%; the homology with the model species under Achromobacter xylosoxidans is as high as 98.68%.
Example 2
By controlling the initial concentration (25, 50, 100, 200, 500mg/L) and the inoculation amount (1%, 3%, 5%, 10%) of diuron in the culture medium% 15% (v: v), initial concentration of Achromobacter xylosoxidans 5.6X 10 9 cfu/mL), temperature (25, 28, 30, 32, 35 ℃), pH (adjusted to pH5.0, 6.0, 7.0, 8.0, 9.0), and an external carbon source (0.0%, 0.5%, 1.0%, 1.5%, 2.0% (m: m) sucrose as an external carbon source), compared the degradation of SL-6 strain under different conditions.
The results show that:
the bacterial strain SL-6 has the advantages that the concentration of the medicament is 500mg/L, the inoculation amount is 15%, the temperature is 30 ℃, the pH value is 8, no external carbon source is added, and the degradation rate of the bacterial strain to the medicament is up to 93.2% on the third day.
Example 3
And selecting a single colony of the bacterial strain SL-6 with clear morphology and no pollution in LB, and treating at 30 ℃ and 180r/min for 48 hours until the liquid is turbid to obtain a bacterial suspension of SL-6.
Adding diuron into soil to make the concentration of diuron in the soil 100, 200, 500, 1000mg/kg respectively, after uniformly mixing the medicament and the soil, inoculating SL-6 bacterial suspension with the inoculum size of 10% by volume fraction respectively, and then adding water into the soil to make the water content in the soil reach 60% (v: m). After the soil treatment is finished, the flowerpot filled with the soil is placed in a constant-temperature incubator at 30 ℃ for dark culture, soil is taken after 0, 3, 6, 9, 12 and 15 days, and the content of diuron in the soil is detected.
The test results are as follows:
with the increase of the diuron content in the soil, the degradation rate of the bacterial strain to the medicament is gradually reduced, the degradation effect in the soil with the lowest diuron content (100mg/kg) is the fastest, the degradation rate is up to 77.3%, the degradation effect of the bacterial strain in the soil with the highest diuron content (1000mg/kg) is the slowest, the degradation rate is only 57.1%, and compared with a control group without the bacterial strain, the degradation rate of the diuron in the soil after inoculation is obviously increased. Therefore, the bacterial strain can accelerate the degradation of the diuron, and the diuron with high concentration in the soil can inhibit the growth of the degrading bacterial strain, thereby reducing the degradation efficiency.
Example 4
1) CK soil without diuron and without degrading strains served as control;
2) so that the diuron content in the soil sample reaches 100mg/kg, and the bacterial strain SL-6 (medicament + SL-6) is inoculated according to the addition amount of 10 percent (v: m);
3) the concentration of diuron in the soil sample is 100mg/kg, but no strain is inoculated.
The three groups of soil are repeatedly arranged, soil treatment is carried out in an incubator at 30 ℃ in a dark place for 15 days, 10 cotton seeds are sown in each pot of soil (the cotton seeds are soaked for 12 hours in advance), the emergence rate is observed, and the physiological indexes (fresh weight, dry weight, root length and the like) are measured and counted at 21 days after inoculation, and the plant growth condition is observed. See table 2 and fig. 4 for results.
The results show that SL-6 has better restoration capability to soil containing diuron and can relieve phytotoxicity to plants to a certain extent. The cotton seedlings treated by the clear water control and the strain grow well, while the cotton seedlings treated by the diuron have lower emergence rate, short and small plants and wilting cotton seedlings at the later stage (shown in figure 4). While the phytotoxicity phenomenon of cotton seedlings in the soil treated by the bacterial strain SL-6 is relieved and lightened.
Table 2 shows the effect of SL-6 treatment on diuron residual soil on cotton seedlings. As can be seen from Table 2, the rate of emergence of cotton is low, only 50%, when the soil is sown after the soil is subjected to diuron treatment for 15 days and only the diuron treatment is carried out; while the emergence rate of cotton treated by the strain is increased to 63.3 percent and 70 percent. And for the data results of the measurement results of fresh weight, dry weight, plant height, root length, root number and the like of the cotton seedlings cultured for 21d, the growth vigor of the cotton seedlings in the clear water control group is the best, and the growth vigor of the cotton seedlings in the application soil treated by the bacterial strain SL-6 is inferior to that of the clear water control group, but is obviously superior to that of the application treatment group only. The fresh weight of the diuron-treated cotton seedlings is only 0.65g and is 66.32%, and after the diuron-treated cotton seedlings are treated by the bacterial strain SL-6, the fresh weight inhibition rate is reduced by 40.41%; the plant height of the diuron-treated cotton seedlings is only 6.53cm, the inhibition rate of the plant height is 62.96 percent, and the inhibition rate of the plant height is reduced by 38.12 percent after the diuron-treated cotton seedlings are treated by a bacterial strain SL-6; the root length of the cotton seedlings treated by the diuron is only 3.88cm, the number of beard roots is 14.67, the inhibition rate of the root length is 58.85 percent, and the inhibition rate of the root length is reduced by 26.51 percent after the cotton seedlings are treated by SL-6; and the number of fibrous roots increased to 22.67.
TABLE 2 Effect of treatment of diuron residual soil with Strain SL-6 on Cotton
As can be seen from Table 2, the bacterial strain SL-6 has better repairing capability on the drug-containing soil and can also reduce the damage of diuron to plants in a certain sense, the data results of the measurement results of fresh weight, dry weight, plant height, root length, root number and the like of cotton seedlings all show that the cotton seedlings in the clear water control group have the best growth vigor, and the cotton seedlings in the drug-applying soil treated by the bacterial strain grow inferior to the cotton seedlings in the clear water control group, but the growth vigor is obviously superior to that of the drug-applying treatment group only.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> river university
<120> achromobacter xylosoxidans, microbial inoculum comprising same, preparation method and application
<160> 5
<170> SIPOSequenceListing 1.0
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ggttaccttg ttacgactt 19
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ttcgatttga cgtacaagtt ctgg 24
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<213> Achromobacter xylosoxidans
<400> 5
attgaacgct agcgggatgc cttacacatg caagtcgaac ggcagcacgg acttcggtct 60
ggtggcgagt ggcgaacggg tgagtaatgt atcggaacgt gcccagtagc gggggataac 120
tacgcgaaag cgtagctaat accgcatacg ccctacgggg gaaagcaggg gatcgcaaga 180
ccttgcacta ttggagcggc cgatatcgga ttagctagtt ggtggggtaa cggctcacca 240
aggcgacgat ccgtagctgg tttgagagga cgaccagcca cactgggact gagacacggc 300
ccagactcct acgggaggca gcagtgggga attttggaca atgggggaaa ccctgatcca 360
gccatcccgc gtgtgcgatg aaggccttcg ggttgtaaag cacttttggc aggaaagaaa 420
cgtcgcgggt taatacctcg cgaaactgac ggtacctgca gaataagcac cggctaacta 480
cgtgccagca gccgcggtaa tacgtagggt gcaagcgtta atcggaatta ctgggcgtaa 540
agcgtgcgca ggcggttcgg aaagaaagat gtgaaatccc agagcttaac tttggaactg 600
catttttaac taccgggcta gagtgtgtca gagggaggtg gaattccgcg tgtagcagtg 660
aaatgcgtag atatgcggag gaacaccgat ggcgaaggca gcctcctggg ataacactga 720
cgctcatgca cgaaagcgtg gggagcaaac aggattagat accctggtag tccacgccct 780
aaacgatgtc aactagctgt tggggccttt cgggccttgg tagcgcagct aacgcgtgaa 840
gttgaccgcc tggggagtac ggtcgcaaga ttaaaactca aaggaattga cggggacccg 900
cacaagcggt ggatgatgtg gattaattcg atgcaacgcg aaaaacctta cctacccttg 960
acatgtctgg aatgccgaag agatttggca gtgctcgcaa gagaaccgga acacaggtgc 1020
tgcatggctg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa 1080
cccttgtcat tagttgctac gaaagggcac tctaatgaga ctgccggtga caaaccggag 1140
gaaggtgggg atgacgtcaa gtcctcatgg cccttatggg tagggcttca cacgtcatac 1200
aatggtcggg acagagggtc gccaacccgc gagggggagc caatcccaga aacccgatcg 1260
tagtccggat cgcagtctgc aactcgactg cgtgaagtcg gaatcgctag taatcgcgga 1320
tcagcatgtc gcggtgaata cgttcccggg tcttgtacac accgcccgtc acaccatggg 1380
agtgggtttt accagaagta gttagcctaa ccgcaagggg ggcgattacc acggtaggat 1440
tcatgactgg ggtg 1454
Claims (10)
1. Achromobacter xylosoxidans strainAchromobacter xylosoxidans) SL-6, accession number: CGMCC No. 20949.
2. An agent comprising the achromobacter xylosoxidans of claim 1.
3. The microbial inoculum according to claim 2, wherein the effective viable count of achromobacter xylosoxidans in the microbial inoculum is 3.15 x 10 8 CFU/mL。
4. A method for producing the microbial agent according to claim 2 or 3, comprising the steps of:
inoculating the achromobacter xylosoxidans into an LB liquid culture medium, and performing amplification culture to obtain a microbial inoculum;
the initial concentration of the achromobacter xylosoxidans is (1-9) multiplied by 10 9 CFU/mL; the inoculation amount of the achromobacter xylosoxidans is 10-20% of the volume of the LB liquid culture medium.
5. The method according to claim 4, wherein the temperature of the scale-up culture is 28-32 ℃; the rotation speed of the amplification culture is 150-200 rpm; the time of the amplification culture is 3-5 days.
6. Use of the achromobacter xylosoxidans according to claim 1, the microbial inoculum according to claim 2 or 3 or the microbial inoculum prepared by the preparation method according to claim 4 or 5 for degrading diuron.
7. Use of the achromobacter xylosoxidans strain of claim 1, the microbial inoculum of claim 2 or 3 or the microbial inoculum prepared by the preparation method of claim 4 or 5 for improving the degradation rate of diuron.
8. Use of the achromobacter xylosoxidans according to claim 1, the microbial inoculum according to claim 2 or 3 or the microbial inoculum prepared by the preparation method according to claim 4 or 5 for reducing the damage of diuron to plants.
9. The use according to any one of claims 6 to 8, wherein when the diuron is diuron in soil, the use comprises the steps of: inoculating a microbial inoculum containing the achromobacter xylosoxidans in the soil; and adjusting the water content of the soil to be 55-65%.
10. The use according to claim 9, wherein the means of inoculation comprises spraying or drip irrigation of the inoculum with water.
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