A corn field long residual herbicide high-efficiency degrading bacterium and application thereof

Abstract

A corn field long residual herbicide degrading bacterium HK-1 is classified as Nocardia (Nocardia sp.) Nocardia sp. ), which was preserved in China General Microbiological Culture Collection Center on January 31, 2023, with a preservation number of CGMCC No. 26497 and a preservation address of No. 1, Xibaixili, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The preparation method is simple, the production cost is low, the high-efficiency degrading bacterium HK-1 can efficiently degrade the long residual herbicides of benzofluor and atrazine under the conditions of acid, alkaline environment, lack of nitrogen source and high-concentration herbicide pollution, the degradation effect on the soil polluted by the combined benzofluor and atrazine herbicides in corn field is remarkable, the bacterium can survive in the soil for a long time in the form of saprophyte, can promote crop growth and reduce the probability of crop disease, and has remarkable economic and social benefits.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microorganisms, in particular to a corn field long residual herbicide-degrading bacterium and application thereof. BACKGROUND

[0002] Corn is the main food crop in China and is one of the most widely distributed crops in the world. With the development of modern agriculture, people use a large amount of herbicides in order to make food high-yield and stable, which leads to soil pollution and soil ecological destruction, and a series of problems such as soil compaction and reduction of yield of subsequent crops. The most commonly used herbicides in corn fields at present are benzofluoxim and atrazine and other long residual herbicides.

[0003] Benzofluoxim is a new high-quality herbicide discovered and developed by BASF Company, and the trade names of benzofluoxim include Convey, Impact, Clio, and Bao Wei. Benzofluoxim is a post-emergence herbicide, which is used for preventing and treating main grass weeds and broadleaf weeds in corn crops worldwide. It is the first benzyl pyrazolone herbicide, and is a 4-hydroxyphenyl pyruvate dioxygenase (4-HPPD) inhibitor. It has good prevention and treatment effect on weeds resistant to glyphosate, triazines, acetyl lactate synthase (ALS) inhibitors and acetyl coenzyme A carboxylase (ACCase) inhibitors, and can effectively prevent and treat annual grasses and broadleaf weeds in corn fields. High dose has a certain inhibitory effect on sedge weeds. Although the current usage is smaller than that of glyphosate and sulfonylurea produced earlier, due to its high activity, wide weed spectrum, and no harm to crops, etc., its usage is likely to become larger and larger in the future, and the harm to biology and environment caused by long-term use and residue accumulation of benzofluoxim cannot be underestimated.

[0004] At present, domestic and foreign researches mainly focus on the weed control effect and toxicology of benzofluoxim. There are only a few universities in China that have reported researches on benzofluoxim residues, but there are almost no reports on the biological degradation of benzofluoxim. How to effectively degrade benzofluoxim to improve the environmental conditions has become a technical problem concerned by the industry.

[0005] In actual production application, in order to obtain better herbicidal effect, commonly used benzofluor and atrazine compound on corn on the main grass weeds and broadleaf weeds for prevention and control. Atrazine is a triazine herbicide, also known as atrazine, the herbicidal mechanism of atrazine is to act on the electron transport chain of photosystem II, so that the normal electron transport chain cannot proceed normally, leading to cell membrane damage, and the weeds die. Control of barnyardgrass, xanthium, gooseweed, crabgrass, amaranth, ragweed and other annual grasses and broadleaf weeds. In recent years, the use area is expanding, the annual consumption is increasing at an average rate of 20%. Because of its good solubility, high migration rate and long residual period, it has caused soil and groundwater pollution in many parts of the world, thereby attracting the attention of many governments and scientists. Atrazine is relatively stable in soil, and is easy to cause phytotoxicity to subsequent crops; the residual atrazine and metabolites in the environment threaten the stability of the ecological system and human health. Atrazine can cause male animals to be feminized and have abnormal gonads, cause chromosomal abnormalities in isolated Chinese hamster ovary cells, and increase the incidence of breast cancer in female mice. The U.S. Environmental Protection Agency has listed atrazine as a substance that may cause cancer in humans. In the 1990s, some countries in the European Union and other countries banned the use of atrazine for the protection of groundwater and the environment. Currently, atrazine is mainly used in underdeveloped areas and developing countries. Microbial degradation is the main way of atrazine degradation in the environment. The history of drug use affects the degradation and mineralization of atrazine in soil, which is mainly affected by soil properties, humidity, temperature and soil depth, and its half-life is 28-128 days.

[0006] Microbial degradation is a good way to degrade organic pollutants. For example, organophosphorus herbicides, such as glyphosate, have been isolated from many strains of bacteria, most of which are different species of Pseudomonas and Ochrobacterum. For sulfonylurea herbicides, many strains of bacteria have been isolated, most of which are mainly broken urea bridge C-N bond. For chloroacetamide herbicides, many strains of bacteria have been isolated, such as Paracoccus sp. FLY-8 and Sphingoonass sp. which can degrade ammonium acetate. The bacteria that can degrade atrazine are mainly Pseudomonas, Rhodocdcus and Arthrobacter. In Nocardia, Acinetabacter, Agrobacterium and Bacillus cereus, strains that can degrade atrazine have also been isolated. Studies have shown that the degradation of atrazine by microorganisms is controlled by genes. Pseudomonas sp. ADP and Arthrobacter sp. TC1 are the most in-depth model strains for the study of microbial degradation of atrazine. Pseudomonas sp. ADP contains all the genes for mineralizing atrazine, such as atzA, B, C, D, E and F. Arthrobacter sp. TC1 contains trzN-atzB-atzC, and its degradation end product is trichloroacetic acid. In 2016, a study found that a mixed bacterial population that can use atrazine as the sole carbon and nitrogen source was isolated from contaminated soil. The mixed bacteria are composed of 10 different bacterial genera, among which the bacteria that can degrade atrazine are Comamonas, Arthrobacter, Flexibacter, Flavobacterium and Bacillus. The mixed bacteria contain the genes trzN, atzB, atzC, atzA and trzD. Rhodococcus is widely present in soil and has the ability to degrade many organic pollutants. It has been reported that the genes of the atrazine dealkylation system of Rhodococcus strain NI86 / 21 have been identified. Rhodococcus NI86 / 21 can grow on atrazine as the sole carbon source but cannot use it as the nitrogen source. It cannot dechlorinate or ring-open. Its main degradation products are desethylatrazine, deisopropylatrazine and hydroxyisopropylatrazine. Rhodococcus B30 and TE1 also cannot ring-open atrazine and are more prone to perform the deethylation reaction of atrazine. The presence of oxygen is required for the degradation of atrazine by these bacteria.Therefore, the red ball bacteria has certain limitations in degrading atrazine. The red ball bacteria belongs to the Nocardioform actinomycetes, which is classified into the red ball genus of the Nocardia family in the actinomycete phylum. The Nocardioform actinomycetes refers to a kind of actinomycetes with primary mycelium and more or less regular breaking into spherical or rod-shaped small bodies, including dozens of genera such as Nocardia, Rhodococcus, Corynebacterium and Mycobacterium. Some strains in the Rhodococcus genus have strong hydrocarbon degradation ability and can be used to repair petroleum hydrocarbon pollution. However, at present, there are few related reports on the red ball bacteria (Rhodococcus sp.) in the Nocardia family of the Rhodococcus genus which can degrade both atrazine and benzofenap. SUMMARY

[0007] In view of the above situation, in order to overcome the defects of the prior art, the purpose of the present application is to provide a corn field long residual herbicide degrading bacterium and its application, so as to solve the residual problem of the corn field long residual herbicide benzofenap and atrazine.

[0008] To achieve the above purpose, the technical scheme solved by the present application is a corn field long residual herbicide degrading bacterium HK-1, which is classified and named as Nocardia sp. and preserved in the China General Microbiological Culture Collection Center on January 31, 2023, with a preservation number of CGMCC No. 26497 and a preservation address of No. 3, Yitianxiliujian 1st Court, Beijing Chaoyang District, Institute of Microbiology, Chinese Academy of Sciences.

[0009] The application of the corn field long residual herbicide degrading bacterium HK-1 in preparing a benzofenap and atrazine corn field long residual herbicide degrading agent.

[0010] The preparation method of the present application is simple, the production cost is low, it is beneficial to large-scale fermentation production, the high-efficiency degrading bacterium HK-1 can efficiently degrade benzofenap and atrazine long residual herbicides under conditions of acid, alkaline environment, nitrogen source deficiency and high concentration of herbicide pollution, has a significant degradation effect on the soil polluted by the corn field herbicides benzofenap and atrazine, and can survive in the soil for a long time in the form of saprophytic bacteria, promote crop growth, reduce the probability of crop disease, improve the disease resistance, and has significant economic and social benefits. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 is a phylogenetic tree of the HK-1 strain of the present application.

[0012] Figure 2 is the growth of HK-1 of the present application on PDA medium.

[0013] Figure 3 is the residual amount of herbicide in the soil at different days after inoculation of the present application.

[0014] Figure 4 is the degradation rate of herbicide in the soil at different days after inoculation of the present application.

[0015] Figure 5 is the residual amount of atrazine in the soil treated with different inoculation doses 15 days after inoculation of the present application.

[0016] Figure 6 is the degradation rate of atrazine in the soil treated with different inoculation doses 15 days after inoculation of the present application. DETAILED DESCRIPTION

[0017] The specific embodiments of the present application are described in detail below in conjunction with the accompanying drawings and examples.

[0018] The high-efficiency degrading bacteria HK-1 provided by the present application is domesticated and screened from herbicide-polluted corn field soil samples in Nanning, Guangxi, and is classified and named as Nocardia sp. Its specific classification and naming is Rhodococcus ruber HK-1 of the Nocardia family, and it was preserved in the China General Microbiological Culture Collection Center on January 31, 2023, with the preservation number CGMCC No. 26497 and the preservation address being No. 3, Yikhina, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0019] I. Isolation and identification of atrazine-degrading bacteria

[0020] Three strains of bacteria that can grow with 300 mg / L atrazine as the sole nitrogen source were domesticated and isolated from soil contaminated with atrazine using a shake flask enrichment method. The three strains of actinomycetes were inoculated into 50 mL inorganic salt medium containing 100 mg / L atrazine, and cultured at 30°C and 180 r / min for 7 days. The residual concentration of atrazine in each sample was quantitatively determined by high-performance liquid chromatography, and the degradation rate was calculated. Atrazine-degrading bacteria were screened from the strains. The degradation rates of the strains on atrazine are shown in Table 1. As can be seen from Table 1, the degradation rate of the HK-1 strain is the highest, reaching 32.67% on atrazine.

[0021] Table 1 degradation effect of different strains on atrazine

[0022]

[0023] The efficient degradation bacterium HK-1 is isolated from the herbicide contaminated corn field soil sample in Nanning, Guangxi. The colony of HK-1 on nutrient agar is round, and the edge is wavy. The surface has wrinkles and protrusions, the center of the colony is concave, and is relatively dry, with poor luster. With the increase of culture time, the color is deepened from melon flesh pink to orange red. It is gram-positive. It is not acid-fast. It grows well on potato agar (PDA) medium, and the colony surface is dry, wrinkled, golden yellow to orange orange (see attached Figure 2 ). No soluble pigment. It does not grow well on Gause synthetic agar No. 1, and the color is very poor. The colony on synthetic glucose agar is orange, dry and wrinkled. No soluble pigment. It grows vigorously in nutrient broth, and is clear. The bacterial film is flocculent, pink, and sinks. Gelatin is not liquefied. Litmus milk is alkaline. Starch is not hydrolyzed. It does not grow on cellulose. Nitrate is reduced. It does not produce H2S. It grows well on xylose, raffinose, glycerol, inositol, mannitol, sorbitol, sodium acetate, paraffin, and phenol. It grows weakly on arabinose, rhamnose, inulin, and asclepiol; it grows at 28-37℃; it grows slowly or not at all below 10℃ or above 40℃. It grows at pH 6.5-9.5, and the optimum pH is 7-8. It can grow on the medium with 100mg / L benzoxazolinone as the only nitrogen source and atrazine as the only carbon source, respectively.

[0024] Molecular biology identification of HK-1: After extracting the DNA of HK-1 strain by bacterial DNA extraction kit, PCR amplification was carried out, and the primers were as follows: 27F: 5'8AGAGTTTGATCCTGGCTCAG83';

[0025] 1492R: 5'8GGTTACCTTGTTACGACTT83'.

[0026] The reaction system was 50μL: ddH2O 21μL, 1492R 1.5μL, 27F 1.5μL, TaqMix enzyme 25μL, and sample bacterial template DNA 1μL. The PCR program was as follows: 94℃ pre-denaturation for 4min, 96℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 60s, 32 cycles, and 72℃ extension for 10min. Then the PCR product was sent to Huada Gene Company for sequencing, and the sequence information is shown in the sequence list. The 16S rDNA sequence was subjected to BLAST comparison in the database of NCBI website, and the phylogenetic tree is shown in the attached Figure 1As shown, homology comparison analysis was performed and it was found that the strain has 100% homology with Rhodococcus sp. JC435 and Rhodococcus ruber strain DSM43338T strain, therefore, the HK-1 strain, in combination with its morphological characteristics and its physiological and biochemical properties, was identified as Rhodococcus ruber.

[0027] Its 16S rDNA sequence is as follows:

[0028] >CTTCGGGTGTTACCGACTTTCATGACGTGACGGGCGGTGTGTACAAGGCC

[0029] CGGGAACGTATTCACCGCAGCGTTGCTGATCTGCGATTACTAGCGACTCC

[0030] GACTTCACGGGGTCGAGTTGCAGACCCCGATCCGAACTGAGACCGGCTTT

[0031] AAGGGATTCGCTCCACCTCGCGGTATCGCAGCCCTCTGTACCGGCCATTG

[0032] TAGCATGTGTGAAGCCCTGGACATAAGGGGCATGATGACTTGACGTCGTC

[0033] CCCACCTTCCTCCGAGTTGACCCCGGCAGTCTCCTGCGAGTCCCCACCAT

[0034] TACGTGCTGGCAACACAGGACAAGGGTTGCGCTCGTTGCGGGACTTAACC

[0035] CAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTACACCG

[0036] ACCACAAGGGAAACCCCATCTCTGGGGCGGTCCGGTGTATGTCAAACCCA

[0037] GGTAAGGTTCTTCGCGTCGCATCGAATTAATCCACATGCTCCGCCGCTTG

[0038] TGCGGGCCCCCGTCAATTCCTTTGAGTTTTAGCCTTGCGGCCGTACTCCC

[0039] CAGGCGGGGCGCTTAATGCGTTAGCTACGGCACGGATCCCGTGGAAGGAA

[0040] ACCCACACCTAGCGCCCACCGTTTACGGCGTGGACTACCAGGGTATCTAA

[0041] TCCTGTTCGCTACCCACGCTTTCGCTCCTCAGCGTCAGTTACTGCCCAGA

[0042] GACCCGCCTTCGCCACCGGTGTTCCTCCTGATATCTGCGCATTTCACCGC

[0043] TACACCAGGAATTCCAGTCTCCCCTGCAGTACTCAAGTCTGCCCGTATCG

[0044] CCTGCAAGCCCGCAGTTGAGCTGCGGGTTTTCACAGACGACGCGACAAAC

[0045] CGCCTACGAGCTCTTTACGCCCAGTAATTCCGGACAACGCTCGCACCCTA

[0046] CGTATTACCGCGGCTGCTGGCACGTAGTTGGCCGGTGCTTCTTCTGTACC

[0047] TACCGTCACTTGCGCTTCGTCGGTACTGAAAGAGGTTTACAACCCGAAGG

[0048] CCGTCATCCCTCACGCGGCGTCGCTGCATCAGGCTTGCGCCCATTGTGCA

[0049] ATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCC

[0050] AGTGTGGCCGGTCGCCCTCTCAGGCCGGCTACCCGTCGTCGCCTTGGTGG

[0051] GCCGTTACCCCACCAACAAGCTGATAGGCCGCGGGCCCATCCTGCACCGG

[0052] AAAACCTTTCCACCCCGGAACATGCATCCCGAGGTCCCTATCCGGTATTAG

[0053] ACCCAGTTTCCCAGGCTTATCCCGAAGTGCAGGGCAGATCACCCACGTGT

[0054] TACTCACCCGTTCGCCACTAATCCACCCAGCAAGCTGGGCTTCATCGTTC

[0055] GACTGC

[0056] II. Isolation and Screening of Highly Efficient Benzophenone-Degrading Bacteria

[0057] The three atrazine-degrading actinomycete strains selected above were inoculated into 50 mL of inorganic salt medium containing 100 mg / L benzoyl permethrin and cultured at 30 °C and 180 rpm for 7 days. The residual concentration of benzoyl permethrin in each sample was quantitatively determined by high-performance liquid chromatography (HPLC), and the degradation rate was calculated to screen for highly efficient benzoyl permethrin-degrading bacteria. Table 2 shows that the actinomycete strain HK-1 showed the best benzoyl permethrin degradation effect, with a degradation rate of 26.53%.

[0058] Table 2. Degradation effects of different strains on benzoxazine

[0059]

[0060] III. Application of Highly Efficient Degrading Bacteria HK-1 in Indoor Simulation Tests of Atrazine and Benzophenone in Soil Degradation

[0061] The test soils were collected from farmland at Lvyou Family Farm in Runan County and divided into treatment and control groups. A certain amount of atrazine and benzoxazole were added to the test soils to ensure that the content of atrazine and benzoxazole in both groups reached 10 mg / kg. A certain amount of highly efficient degrading bacteria HK-1 was inoculated into the treatment soils, while no bacteria were inoculated into the control soils. Both groups of samples were placed in an incubator at 25℃. To prevent errors caused by photolysis, the Erlenmeyer flasks were wrapped with tin foil, and sterile water was added regularly during the remediation experiment to maintain soil moisture at 25%. At 7, 14, 21, and 28 days, 5g soil samples were taken from each experimental group, extracted with twice the amount of methanol, and then the residues of atrazine and benzoxazole in the soil were detected by high-performance liquid chromatography (HPLC). The experimental results are shown below. Figure 3 and Figure 4 ,from Figure 3 andFigure 4 It can be seen that with the increase of inoculation days, the residual amounts of atrazine and carfentrazone-ethyl in the soil also decrease, but the degradation rates of atrazine and carfentrazone-ethyl in the soil increase, and the residual amounts of herbicides in the soil are very low after the application of high-efficiency degrading bacteria HK-121d, and the degradation rates are all above 99%, and the residual amounts of atrazine and carfentrazone-ethyl in the soil are both 0 mg / kg after 28 d, and the degradation rates reach 100%.

[0062] Four, the degradation effect of high-efficiency degrading bacteria HK-1 on herbicide residues in corn field

[0063] The field test was carried out in the farmland after corn harvesting in Lvyou Family Farm in Runan County, and the following crop was wheat. Atrazine herbicide (containing 17% of atrazine) was uniformly sprayed at 80 mL / mu before wheat sowing, soil samples were collected, and then plots were divided. The test had a total of 4 treatments, and the specific test design was as follows:

[0064] Treatment 1: control, without any inoculant.

[0065] Treatment 2: spray HK-1 fermentation liquid 10 mL / m 2 .

[0066] Treatment 3: spray HK-1 fermentation liquid 20 mL / m 2 .

[0067] Treatment 4: spray HK-1 fermentation liquid 30 mL / m 2 .

[0068] Each treatment had 3 repetitions, and the treatments were randomly arranged. The area of each plot was 30 m 2 . After sowing wheat with different doses of high-efficiency degrading bacteria HK-1, soil samples were collected again after 15 d for detection, and the results are shown in Figure 5 and Figure 6 It can be seen from Figure 5 that the residual content of atrazine in the soil sample after 15 d is very low, and the treatment group is significantly lower than the control group (p<0.05), and it can be seen from Figure 6 that the degradation rates of each dose treatment group are all above 96%, and there is no significant difference between each dose treatment group (p>0.05). Therefore, the test results show that the application of HK-1 inoculant 10-30 mL / m 2 treatment for 15 d or more can basically achieve complete remediation of soil contaminated by atrazine herbicide.

[0069] In summary, the high-efficiency degrading bacteria HK-1 can grow on the inorganic salt culture medium with the highest 300 mg / L metoxuron as the sole nitrogen source and with atrazine as the sole carbon source. In the conditions of pH 6.0-8.0 and lack of carbon or nitrogen source, the initial concentration of metoxuron and atrazine is 100 mg / L, the shaking table is 180 r / min, 30 DEG C, and after 7 days of degradation, liquid chromatography detection shows that the degradation rate of metoxuron is 20-40%, and the degradation rate of atrazine is 30-40%. However, in the soil degradation simulation test, the application of the high-efficiency degrading bacteria HK-1 is detected to be very low in the herbicide residues in the soil after 21 days, and the degradation rate is more than 99%, and the residues of atrazine and metoxuron in the soil are both 0 mg / kg after 28 days, and the degradation rate reaches 100%. In the field test, the degradation rate of atrazine in the soil is more than 96% after 15 days of application of the bacteria, and the degradation is almost complete.

[0070] The present application has the advantages of simple preparation method, low production cost, and is beneficial to large-scale fermentation production. The high-efficiency degrading bacteria HK-1 can efficiently degrade the long-residue herbicides of metoxuron and atrazine in the conditions of acidic, alkaline environment, lack of nitrogen source, and high-concentration herbicide pollution. The present application provides an effective method for microbial repair of herbicide pollution in corn fields, and has a significant degradation effect on the soil polluted by the combined pollution of the herbicides metoxuron and atrazine in corn fields. The high-efficiency degrading bacteria HK-1 disclosed in the present application is a kind of gram-positive bacteria widely distributed in soil, which can be used to prepare a degrading bacteria agent, and can survive in the soil for a long time in the form of saprophytic bacteria, promote crop growth, reduce the probability of crop disease, and improve the disease resistance, thereby achieving significant economic and social benefits.

[0071] The above examples are used to illustrate the disclosed embodiments of the present application, and should not be construed as limiting the present application. In addition, various modifications and changes in the method of the present application are obvious to those skilled in the art without departing from the scope and spirit of the present application. Although the present application has been specifically described in combination with various specific embodiments of the present application, it should be understood that the present application should not be limited to these specific embodiments. In fact, various modifications as described above to obtain the present application are obvious to those skilled in the art and should be included in the protection scope of the present application.

Claims

1. A highly efficient herbicide-degrading bacterium, HK-1, that survives long periods of cornfield residues has been classified and named Nocardia (…). Nocardia sp. It was deposited on January 31, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26497. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

2. The use of the corn field long residual herbicide high-efficiency degrading bacteria HK-1 in claim 1 in the preparation of a corn field long residual herbicide degrading bacteria agent for degrading metribuzin and atrazine.

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