CN117025462B - Enterococcus strain for degrading insecticide and application thereof - Google Patents

Abstract

The invention discloses an enterococcus casei strain for degrading pesticides and application thereof, wherein the preservation name of the enterococcus casei strain is: EMBL-3, accession number: china center for type culture Collection, date of preservation: 2023, 17, 07, accession number: cctccc M20231305. The strain is identified by gene sequence analysis as enterococcus buttermidis of enterococcus, named EMBL-3, and belongs to gram-positive bacteria. The strain can be used for degrading chlorantraniliprole and has the prospect of further research and development and engineering application in the fields of agricultural pest control, novel green pesticide development, environmental pesticide pollutant restoration and the like.

Description

A pesticide-degrading enterococcus strain and its use

Technical field

The present invention relates to a bacterial strain, in particular to an Enterococcus caseinus strain and its use.

Background technique

Spodoptera Frugiperda is an extremely destructive and important pest that has been issued a global warning by the United Nations Food and Agriculture Organization. Since it first invaded southern my country from Myanmar in December 2018, it has rapidly spread to many provinces in my country and continues to expand northward, which is harmful to many provinces in China. The production of important crops poses a serious threat. In order to ensure the safe production of crops, the use of pesticides is particularly important. Bisamide pesticides represented by chlorantraniliprole are currently the primary recommended pesticides for the control of Spodoptera Frugiperda. played a key role in the process. Environmental problems and pesticide resistance problems caused by the unreasonable and unscientific use of pesticides have become increasingly serious. According to resistance monitoring data in recent years, high levels of chlorantraniliprole in Spodoptera frugiperda have been detected in parts of Puerto Rico, the United States. Although the domestic field population of Spodoptera Frugiperda is still at a low level of resistance to chlorantraniliprole, it continues to increase, and the situation is not optimistic. The development of Spodoptera Frugiperda's resistance to new insecticides has seriously hampered the efficient control of Spodoptera Frugiperda, posing a great threat to the quality and quantity of crops. The increased amount of pesticides required to successfully control the pests seeps into the environment, causing environmental damage. The problem of pollution cannot be ignored. Therefore, it is particularly important to discover and develop pesticide-degrading bacteria that can be used to "overcome" targets and remediate pesticide-polluted environments.

In recent years, the insect intestine has been considered to be a natural reactor containing active genes for various nutrient synthesis and xenobiotic metabolism. It accommodates multifunctional and diverse microorganisms and is an important resource library in nature. An important reason why pests develop resistance is their enhanced ability to detoxify and metabolize insecticides. More and more evidence has confirmed that symbiotic bacteria are important participants in the detoxification and metabolism of insecticides. In pests such as the stink bug and Bactrocera dorsalis, the colonization of some symbiotic bacteria can directly degrade pesticides and enhance the host's resistance. In addition, a large number of insecticide-degrading bacteria derived from the intestinal tract of insects have also been used. It was found that these bacterial isolates can be effectively exploited for transformation in environmental remediation and degradation of xenobiotics. However, there are still no studies reporting the involvement of enterococcal strains in the degradation and transformation of pesticides. We isolated an Enterococcus casseliflavus EMBL-3 strain from the intestinal microorganisms of Spodoptera frugiperda larvae and confirmed the strain's ability to degrade the pesticide chlorantraniliprole. E.casseliflavus exists in almost all lepidopteran pests, so it has great application prospects in the fields of pest control technology and environmental remediation.

Contents of the invention

The purpose of the present invention is to provide an enterococcus strain and its use in view of the shortcomings of the existing technology, which can provide new strategies and methods for solving pest resistance target selection and pesticide chlorantraniliprole pollution in the environment. .

The enterococcus strain of the present invention is Enterococcus casseliflavus. The preservation information: the preservation name is EMBL-3, the preservation unit: China Typical Culture Collection Center, the preservation date: July 17, 2023, the preservation number: CCTCC M 20231305.

The source of the strain in this study is novel. It was isolated from the intestine of the larvae of the invasive alien pest Spodoptera frugiperda. It is a beneficial strain isolated from the intestine of the pest. It has been verified through research that this strain can be used for the degradation of chlorantraniliprole. , is the first member of the genus Enterococcus found to be able to degrade chlorantraniliprole.

The strain is the bacterium Enterococcus caseinate EMBL-3. Enterococcus caseinate is a common symbiotic bacterium in the intestinal tract of lepidopteran pests and is a very important tryptophan producer in silkworms. The above studies indicate that this bacterium is a beneficial symbiotic bacterium for most lepidopteran pests. However, there are no reports on the ability of this bacterium to degrade and transform pesticides.

In addition, the present invention also provides a pesticide pollution remediation agent, which contains the bacterial strain or its culture solution or dry powder preparation, and the bacterial strain or its culture can be used for the degradation of pesticides. The present invention can also be based on a method for identifying active proteins for microorganisms degrading pesticides, and can preliminarily screen and identify potentially active proteins that degrade chlorantraniliprole, which is conducive to research on the selection of pest resistance targets.

The beneficial effects of the present invention are:

The present invention found that the strain can significantly degrade chlorantraniliprole under short-term liquid culture conditions (3 days), and can reduce the concentration of chlorantraniliprole by 25%. The proteome results of chlorantraniliprole pre-exposure of intracellular and extracellular proteins can be screened to identify potential active proteins that degrade chlorantraniliprole, and further speculate on the degradation mechanism. The chemical bonds of chlorantraniliprole molecules are broken, which may involve Amide bond cleavage and dehalogenation reactions.

The specific liquid culture conditions are: the test is divided into four groups: ① Minimum inorganic salt liquid medium (MSM) added with 10 mg/L chlorantraniliprole as a control group; ② Minimum inorganic salt liquid medium (MSM) added with 10 mg/L Chlorantraniliprole and EMBL-3 strain culture; ③ Minimum inorganic salt liquid medium (MSM) added with 100 mg/L intracellular protein extract of EMBL-3; ④ Minimum inorganic salt liquid medium (MSM) added 100mg/L extracellular protein extract of EMBL-3. All treatments were placed at 30°C and cultured at 150 rpm/min in a temperature-controlled shaker for 3 days, during which detection was performed at set time points (1d, 2d and 3d).

Description of drawings

Figure 1 Growth status of EMBL-3 strain on LB plate;

Figure 2 Strain growth curve of EMBL-3 strain during 3 days of culture with chlorantraniliprole;

Figure 3 Degradation efficiency of chlorantraniliprole by strain EMBL-3;

Figure 4 Degradation efficiency of chlorantraniliprole by intracellular and extracellular proteins of EMBL-3 strain;

Figure 5 Response of intracellular and extracellular proteins of EMBL-3 strain to chlorantraniliprole;

Figure 6 Potential degradation proteins of EMBL-3 strain towards chlorantraniliprole;

Figure 7 The mass spectrum of the degradation products of chlorantraniliprole by strain EMBL-3;

Figure 8 Potential degradation process of chlorantraniliprole by strain EMBL-3.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

Example 1. Isolation, culture and identification of Enterococcus casseliflavus EMBL-3:

Surviving Spodoptera frugiperda larvae 2 days after feeding with sublethal concentration (LC ₅₀ ) of chlorantraniliprole were dissected under sterile conditions to obtain their intestines. Combine 15 intestines into one group, add physiological saline, grind and shake to obtain a mixed solution of intestinal contents, dilute the mixed solution of intestinal contents gradiently from 10 ^-1 to 10 ^-7 times and apply it to chlorantraniliprole. In the solid inorganic salt medium plate, place it for culture at 30°C. After a single colony of a certain size has grown, select a single colony to expand the culture in a nutrient-rich LB liquid medium, and further draw lines to pick single colonies for purification. After visible colonies grow on the plate, subculture again until pure strains are obtained. Then the pure strain is again inoculated into a liquid inorganic salt medium containing chlorantraniliprole for culture. If the strain can grow with chlorantraniliprole as the only energy substance, it is a chlorantraniliprole-degrading strain, recorded as EMBL-3 strain.

Example 2. Identification of strain EMBL-3

After obtaining the EMBL-3 strain, perform strain identification and determine its species classification. Therefore, 16S rDNA sequencing technology was used to identify the species classification of the EMBL-3 strain. Primers 27F and 1492R were used to amplify the full length of its gene. After 16S rRNA sequencing and ribosome comparison with the National Center for Biotechnology Information (NCBI), it was determined to be Enterococcus caseinate (see additional material 1), named EMBL-3, is a Gram-positive bacterium. The colonies are off-white, with translucent edges, smooth and moist surfaces, regular edges, round, no halos, and a raised center. The colonies are smooth and mucus-like (Figure 1).

This strain is Enterococcus casei, and has been deposited as follows. The deposit name is: EMBL-3, the deposit unit: China Type Culture Collection Center, the deposit date: July 17, 2023, the deposit number: CCTCC M20231305.

Example 3. Degradation of chlorantraniliprole by strain EMBL-3

The culture fluid obtained by liquid culture of EMBL-3 strain can be used to degrade chlorantraniliprole.

The liquid culture conditions are to insert the EMBL-3 strain into a 50 mL Erlenmeyer flask containing 20 mL of inorganic salt liquid culture medium at an inoculation amount of 10%, add chlorantraniliprole to the culture medium, and culture on a shaker at 220 rpm and 30°C. Set up a control group, that is, only chlorantraniliprole is added to the liquid inorganic salt culture medium without adding the EMBL-3 strain. Samples are taken on the 1st, 2nd and 3rd days of culture. EMBL-3 can be added with chlorantraniliprole. Grow slowly in inorganic salt medium as the sole carbon source (Figure 2). Chlorantraniliprole in the culture medium was extracted with ethyl acetate and detected by high-performance liquid chromatography. It was found that the concentration of chlorantraniliprole could be significantly reduced by 25% after 3 days of cultivation, indicating that EMBL-3 can be used in a short time. Effectively degrade chlorantraniliprole within 10 seconds (Figure 3).

Example 4. Identification of active protein for chlorantraniliprole degradation by strain EMBL-3

By comparing the response patterns of EMBL-3 intracellular and extracellular proteins to chlorantraniliprole and compared with controls, active proteins potentially related to chlorantraniliprole degradation can be screened and identified.

Protein degradation experiments showed that extracellular secreted proteins had a better degradation effect on chlorantraniliprole than intracellular proteins, about 18.6% (Figure 4). Through proteomic analysis of the response patterns of intracellular proteins and extracellular secreted proteins of EMBL-3 during the degradation process of chlorantraniliprole, it was found that extracellular proteins are more responsive to chlorantraniliprole than intracellular proteins, among which There were as many as 543 differentially expressed proteins (283 down-regulated and 260 up-regulated). Compared with intracellular proteins (30 down-regulated and 44 up-regulated), more up-regulated proteins were detected extracellularly (Figure 5), among which 18 proteins were screened out, including four N-acetyltransferases, methyltransferases, dehalogenases, etc., defined as potential chlorantraniliprole-degrading proteins (Figure 6). Through the prediction of the function of the protein and the mass spectrometry results of the binding degradation products (Figure 7), it can be initially proposed that the potential degradation process of EMBL-3 to chlorantraniliprole is the cleavage and dehalogenation reaction of the amide bond, producing benzene ring derivatives and pyridine cyclic compounds (Figure 8).

The specific detection method is: intracellular and extracellular protein extraction: EMBL-3 is cultured in an inorganic salt medium containing chlorantraniliprole for 3 days, centrifuged at 5000 rpm for 10 minutes, and the supernatant and precipitate are collected separately. First, the supernatant is Add an equal volume of -20°C pre-cooled acetone to the solution, stir in an ice bath for 2 hours, collect all the liquid and centrifuge at 14,000 rpm for 20 minutes, and redissolve the precipitate with PBS. This is extracellular protein. The bacterial cells collected above were disrupted based on the ultrasonic disruption method, and the supernatant was collected by centrifugation to obtain intracellular proteins.

In summary, Enterococcus casei EMBL-3 and its culture solution of the present invention can effectively degrade the pesticide chlorantraniliprole, and can be applied in the remediation of pesticide pollution. Therefore, the present invention also provides a pesticide pollution repair agent, which contains the bacterial strain or the culture solution of the bacterial strain, and can be sprayed on the object to be repaired during use. With further research on Enterococcus caseinate EMBL-3, it was discovered that it is also likely to have a degradation effect on other types of insecticides or pesticides, and is worthy of more extensive and in-depth exploration.

The above are only some of the embodiments of the present invention. Those skilled in the art can easily implement various embodiments according to the text description, drawings and claims provided by the present invention without departing from the spirit and scope of the present invention as defined by the claims. , many variations and modifications can be made. Any modifications, modifications or equivalent changes made to the above embodiments based on the technical ideas and essence of the present invention shall fall within the scope of protection defined by the claims of the present invention.

Additional material 1

16S rRNA gene sequence of EMBL-3 strain

AGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGCTTTTTCTTTCACCGGAGCTTGCTCCACCGAAAGAAGTGGCGAACGGGTGAGTAACACGTGGGTAACCTGCCCATCAGAAGGGGATAACACTTGGAAACAGGTGCTAATACCGTATAACACTATTTTCCGCATGGAAGAAAGTTGAAAGGCGCTTTTGCGTCACTGATGGATGGACCCGCGGTGCATTAGCTAGT TGGTGAGGTAACGGCTCACCAAGGCAACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGAGAAGAACAAGGATGAGAGTAAAATGTTCATCCCTTGACGGTATCTAACCAGAAAGCCACGGCTAACTAC GTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCG TGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCAAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGACCACTCTAGAGATAGAGCTTCCCCTTCGGGGGCAAAGT GACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTGTTAGTTGCCATCATTTAGTTGGGCACTCTAGCGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGAAGTACAACGAGTTGCGAAGTCGCGAGGCTAAGCTAATCTCTTAAAGCTTCTCTCAGTTCGGATT GTAGGCTGCAACTCGCCTACATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTGGAGCCAGCCGCCTAAGGTGGGATAGATGATTGGGGTGAAGTCGTAACAAGGTAACC

Claims (7)

1. Enterococcus casseliflavus strain, characterized in that the preservation name is: EMBL-3, the preservation unit: China Type Culture Collection Center, the preservation date: July 17, 2023, the preservation number: CCTCC M20231305. 2. Use of the Enterococcus caseinate strain according to claim 1, characterized in that it is used to degrade the pesticide chlorantraniliprole. 3. A method for degrading chlorantraniliprole, characterized in that the method includes culturing the bacterial strain as claimed in claim 1, and contacting the chlorantraniliprole to be degraded with the strain culture solution. 4. The method for degrading chlorantraniliprole according to claim 3, characterized in that the temperature required for degradation is 20~40°C. 5. The method for degrading chlorantraniliprole according to claim 3, characterized in that the concentration of chlorantraniliprole can be reduced by 25% within 3 days of contact. 6. The method for degrading chlorantraniliprole according to claim 3, characterized in that the potential degradation process of EMBL-3 to chlorantraniliprole is the cleavage of the amide bond and the dehalogenation reaction to produce a benzene ring. Derivatives and pyridine ring compounds. 7. A pesticide pollution remediation agent, characterized in that it contains the strain according to claim 1 or the culture solution or dry powder preparation of the strain.