CN115044488A

CN115044488A - Bacterial strain capable of degrading diuron and imidacloprid and application thereof

Info

Publication number: CN115044488A
Application number: CN202210863096.9A
Authority: CN
Inventors: 吴秀丽; 郭伶娜; 马鹏生; 白长财; 周丽; 刘成; 徐克�; 焦美娟; 林文星
Original assignee: Ningxia Medical University
Current assignee: Ningxia Medical University
Priority date: 2022-07-20
Filing date: 2022-07-20
Publication date: 2022-09-13
Anticipated expiration: 2042-07-20
Also published as: CN115044488B

Abstract

The invention discloses a bacterial strain capable of degrading diuron and imidacloprid and application thereof. The strain is a sclerotium cladosporium (Acremoumsp.) strain QHSH-33 with the preservation number of CGMCCNo.23259. The strain can be used for degrading pesticides diuron and imidacloprid, is beneficial to solving the residue problem of the pesticides diuron and imidacloprid and improving the quality safety of agricultural products.

Description

Bacterial strain capable of degrading diuron and imidacloprid and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and relates to a bacterial strain capable of degrading diuron and imidacloprid and application thereof.

Background

Diuron (Diuron) is a selective herbicide that is poorly soluble in water and also poorly soluble in most organic solvents. With the Chinese development of the great country for pesticide production, the domestic production and use of diuron are rapidly increased, and diuron is applied to cotton, corn, peanut, sugarcane and fruit trees; preventing and killing annual weeds such as marsupa, Echinochloa stolonifera, green bristlegrass, sedge, polygonum, chenopodium album and the like, and also has good preventing and controlling effect on perennial weeds such as cynodon dactylon, cyperus rotundus and the like. At present, the demand of diuron for sugarcane in China is about 1500 tons. Due to the large use of diuron, diuron residue exists in underground water and soil, and although the diuron is a low-toxicity herbicide, toxic symptoms can still appear after the diuron is taken by mistake, and the diuron has irritant effects on the upper respiratory tract. Diuron has been studied as an endocrine disrupter and has been found to adversely affect the human body after contact.

Imidacloprid (Imidacloprid) is a nitromethylene systemic insecticide, also known as a neonicotinoid insecticide. The action mechanism of the neonicotinoid insecticides is that the neonicotinoid insecticides are used as nicotinic acetylcholine receptor (nAChR) agonists to be selectively combined with acetylcholine receptors to block normal conduction of central nerves, so that the nerves are over-stimulated. Poisoned insects exhibit symptoms of convulsions, paralysis and eventual death. The insecticidal mechanism is different from the previous generations of traditional insecticides, namely organophosphorus, carbamate, pyrethroid and the like, has better physicochemical properties, has high efficiency, broad spectrum, good root systemic property and low toxicity to mammals, is widely applied to rice, wheat and cotton, can effectively control homoptera, coleoptera, diptera, lepidoptera and other pests, and can also control aphididae, aleyrodidae, cicadae, compositae, nematodes, plant hoppers, mealybugs and phytophagous mites. Since the emergence of neonicotinoid insecticides, however, the number of a large number of pollinating insects has been reduced suddenly, researchers find that the acute toxicity of neonicotinoid insecticides such as imidacloprid to bees is high or severe, and have a sub-lethal effect. The effective components and metabolites of the bee pollen are remained in pollen and nectar, so that the bee population is reduced, the survival and reproductive capacity is reduced, the general attention of growers all over the world is aroused, and various countries are caused to dispute policies to deal with the bee pollen and nectar.

In the use of the pesticide, most of the pesticide enters air, water and soil, and a large amount of pesticide residue is caused in the water and the soil because only a small part of the pesticide is volatilized. In the current pesticide residue degradation method, compared with the traditional degradation method, the microbial degradation method has unique advantages, can carry out bioremediation and decomposition on pesticides to convert the pesticides into simple inorganic matters, and has the characteristics of safety, high efficiency, rapidness, low price and no secondary pollution. And the separation and screening of the bacterial strains with the degradation effect is beneficial to solving the problem of pesticide residue, improving the quality of agricultural products and promoting the agricultural development.

Disclosure of Invention

In order to solve the problems, the invention aims to provide a bacterial strain with degradation effect on diuron and imidacloprid. The strain can effectively degrade pesticide diuron and imidacloprid, lays a foundation for solving the problem of diuron and imidacloprid residue, and can improve the environment of soil, water and the like.

To achieve the above objects, the present invention provides a strain QHSH-33 of Verticillium pleioides (Acremonium sp.) as specified in the following. The verticillium lecanii is also called as acremonium.

The strain name is as follows: pleated branches of mushroom

Latin name: acremonium sp.

The strain number is as follows: QHSH-33

The preservation organization: china general microbiological culture Collection center

The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)

Address: xilu No. 1 Hospital No. 3 of Beijing market facing Yang district

The preservation date is as follows: 24 days 9 and 2021

Registration number of the preservation center: CGMCC No.23259

The mycobacterium plebeium (Acremonium sp.) strain QHSH-33 is screened by the following method:

a single microbial strain is separated and purified from pesticide-polluted soil by methods of gradient dilution, plate streaking and the like and is used for subsequent experiments.

Inoculating single microorganism strains into culture medium, respectively, performing shaking culture at 28 deg.C in a shaking table at 180r/min to obtain experimental group, and setting self control group and standard control group. When the strain grows to the logarithmic phase, diuron and imidacloprid are added into the experimental group and the standard control group, the concentration is 50mg/L, samples are respectively taken at 28 ℃ and 180r/min, the thin layer analysis is carried out to observe the degradation effect of the strain on the pesticide, and the strain with the degradation effect is selected to be QHSH-33 and used as the next research object.

Furthermore, the invention carries out morphological and molecular biological identification on the screened strain QHSH-33. The method specifically comprises the following steps:

1) morphological identification

The strain QHSH-33 is respectively inoculated on a solid plate of a commercial Martin, a potato and a Chao's medium and cultured for 5d at 28 ℃, and the colony morphology is observed.

2) Molecular biological identification

Extracting the genome DNA of the strain, and carrying out PCR amplification and sequencing by taking the extracted genome as a template. And comparing and analyzing ITS sequences measured by the strains in an NCBI database by using BLAST, selecting related sequences with higher homology, constructing a phylogenetic tree by using MAGE 7.0 software, and analyzing an evolutionary relationship.

Besides, the invention also specifically measures the growth curve, transformation products and degradation capability of QHSH-33.

1) Growth Curve determination

The purified strain QHSH-33 was inoculated into commercial Martin fungus medium, 3 sets of replicates were set, and growth curves were measured by dry weight method after

sampling cultures

1, 2, 3,4, 5, 6, 7, 8, 9, 10d, respectively.

2) Conversion product

And (3) qualitatively analyzing an intermediate product generated by degrading diuron and imidacloprid by using a liquid chromatography-mass spectrometer.

3) Determination of degradation Capacity

The degradation efficiency of the diuron and the imidacloprid of the QHSH-33 is determined by adding the diuron and the imidacloprid in the strain QHSH-33, sampling at regular time, and then carrying out HPLC-DAD measurement.

Based on the above measurement results, the present invention also provides:

(1) the application of the strain or the culture solution thereof or the fermentation liquid thereof or the microbial inoculum containing the strain in degrading diuron and imidacloprid.

(2) The application of the strain or the culture solution thereof or the fermentation liquid thereof or the microbial inoculum containing the strain in reducing the residual quantity of diuron and imidacloprid after use.

(3) The application of the strain or the culture solution thereof or the fermentation liquid thereof or the microbial inoculum containing the strain in preparing products for degrading diuron and imidacloprid.

(4) A product for degrading diuron and imidacloprid, which comprises the strain or a culture solution thereof or a fermentation solution thereof or a microbial inoculum containing the strain.

Based on the technical scheme, the screened verticillium Acremonium sp.QHSH-33 with diuron and imidacloprid degradation effects can be used for degrading pesticide diuron and imidacloprid, is beneficial to solving the problem of diuron and imidacloprid residue in agricultural production, and simultaneously improves the quality safety of agricultural products.

Drawings

FIG. 1 is a schematic view ofColony morphology characteristic map of bacteria Pleurotus ostreatus strain QHSH-33. In FIG. 1, A ₁ Is a colony morphology chart (front) of the mycorrhiza pleioides strain QHSH-33 cultured for 5d on a martin culture medium; a. the ₂ Is a colony morphology chart (reverse side) of the mycorrhiza pleioides strain QHSH-33 cultured on a martin culture medium for 5 d; b is ₁ Is a colony morphology chart (front) of the mycorrhiza pleioides strain QHSH-33 cultured on a potato culture medium for 5 d; b is ₂ Is a colony morphology diagram (reverse side) of the Verticillium pleioides strain QHSH-33 cultured on a potato culture medium for 5 d; c ₁ Is a colony morphology chart (front) of the mycorrhiza pleioides strain QHSH-33 cultured on a Chachi's culture medium for 5 d; c ₂ Is a colony morphology graph (reverse side) of the Verticillium pleioides strain QHSH-33 cultured on a Chachi culture medium for 5 d; d is a morphological diagram of the Verticillium pleioides strain QHSH-33 under an optical microscope.

FIG. 2 shows ITS phylogenetic tree of Mycobacterium fortuitum strain QHSH-33.

FIG. 3 is a graph showing the growth of the Verticillium gilsonii strain QHSH-33.

FIG. 4 is a liquid quality diagram of diuron degradation by Verticillium mycorrhizae strain QHSH-33 (diuron).

FIG. 5 is a liquid-mass diagram of diuron 1 degradation product of Verticillium mycoides strain QHSH-33.

FIG. 6 is a liquid-mass diagram of diuron degradation product 2 by Verticillium mycorrhizae strain QHSH-33.

FIG. 7 is a liquid-mass diagram of degradation of imidacloprid (imidacloprid) by the Verticillium plericum strain QHSH-33.

FIG. 8 is a liquid-mass diagram of degradation product 1 of imidacloprid by Verticillium habeni strain QHSH-33.

FIG. 9 is a liquid-mass diagram of imidacloprid degradation product 2 by using Verticillium habeni strain QHSH-33.

FIG. 10 is a diuron standard chromatogram

FIG. 11 shows the results of the degradation experiment of the Verticillium mycorrhizae strain QHSH-33 to diuron.

FIG. 12 is a chromatogram of imidacloprid standard.

FIG. 13 shows the results of the degradation experiment of imidacloprid by the Verticillium habeni strain QHSH-33.

Detailed Description

The present invention is further illustrated by the following detailed description, which is given by way of illustration only, and not by way of limitation. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1

This example is a method for separating, purifying and screening the strain QHSH-33.

The commercial Martin medium used in this example had a composition comprising peptone 5.0g, dipotassium hydrogenphosphate 1.0g, magnesium sulfate 0.5g, yeast extract powder 2.0g, glucose 20.0g, distilled water 1000mL, and pH 7.0. Solid medium: 15.0g of agar powder was added per 1L.

The specific test of this example includes:

1. isolation and purification of the strains

In this example, the pesticide contaminated soil was collected from Pingxu county, Ningxia Shizushan city in 7 months of 2020, 2g of the soil was mixed in 50mL of sterile water, and after the solution was settled and clarified, the supernatant was diluted to 10% by gradient dilution method ^-1 、10 ^-2 、10 ^-3 、10 ^-4 、10 ^-5 Uniformly coating the mixture in a commercial Martin solid culture medium in an equal gradient manner, culturing at 28 ℃, and purifying by a plate streaking separation method to obtain a plurality of single purified microbial strains.

2. Screening of diuron and imidacloprid degrading bacteria

Preparing commercial Martin liquid culture medium (without agar), inoculating single purified strains into the culture medium in a 250mL triangular flask containing 150mL culture medium, and performing shaking culture at 28 ℃ and 180r/min by a shaking table. When the strain grows to the logarithmic phase, adding substrates diuron and imidacloprid with the concentration of 50mg/L respectively, continuously culturing for 5 days, 7 days and 9 days under the same conditions, respectively sampling, carrying out thin layer detection and analyzing the result.

Meanwhile, a standard control group was set: adding diuron and imidacloprid (concentration is 50mg/L) into the liquid culture medium with the same amount respectively; self-control group: the above strains were inoculated into an equal amount of liquid medium in the same manner as above, except that diuron and imidacloprid were not added.

The thin-layer analysis result is influenced by the interference of self secondary metabolism of self control group elimination bacteria; the standard control group excluded medium interference with the compound diuron. According to the thin layer information, the degradation of different bacteria to the pesticides diuron and imidacloprid is observed, and the bacterial strain with obvious degradation effect is selected as the next research object. Obtaining a bacterial strain with degrading effect on diuron and imidacloprid, and numbering is QHSH-33.

Example 2

This example is the identification of the strain QHSH-33, including colony morphology and strain biological identification methods.

The potato medium referred to in this example comprises 12.0g of potato extract powder, 20.0g of glucose, 15.0g of agar, 1000mL of distilled water, pH 5.6. + -. 0.2. The Chao's medium comprises 3.0g of sodium nitrate, 1.0g of dipotassium hydrogen phosphate, 0.5g of magnesium sulfate, 0.5g of potassium chloride, 0.01g of ferrous sulfate, 30.0g of sucrose, 15.0g of agar and 1000mL of distilled water. Martin medium was composed of the same medium as in example 1.

The embodiment specifically includes:

1. identification of colony morphology

Inoculating strain QHSH-33 to Martin medium, potato medium, and Chao's medium, culturing at 28 deg.C for 5d, observing colony morphology, and observing morphological characteristics of strain under optical microscope by means of insert sheet method as shown in figure 1.

The observation shows that the QHSH-33 is on Martin medium, the colony is concentric circle in a dry white villus shape, the center is provided with a cotton-shaped bulge, the back of the colony is corrugated concentric circle, and the diameter of the colony can reach 54.7mm when the colony grows for 5 d. QHSH-33 is on potato culture medium, the colony is in the form of dry white villus, is in the form of concentric circle, and grows slowly, the centre has cotton-like projection, the back of colony is in the form of fold concentric circle, the centre colour is yellow, and the diameter of colony can reach 46.7mm when growing for 5 d. QHSH-33 is on the Chaudou's culture medium, the colony is in the form of fold round, grows slowly, the centre has bigger projection, the back of the colony is also in the form of fold, the centre is yellow, the diameter of the colony can reach 46mm when growing for 5 d.

2. Molecular biological identification

1) The QHSH-33 strain is inoculated into a liquid commercial Martin culture medium, and is cultured for 3d by shaking at 28 ℃ and 180 r/min.

2) After centrifugation, a small amount of the cells were collected, and the obtained product was subjected to "fungal genome DNA extraction kit" (Stannless Beck Biotechnology Ltd., product number: b004009020) and extracting QHSH-33 genome DNA, wherein the extraction steps refer to the specification of a fungus genome DNA extraction kit.

3) PCR amplification was performed using QHSH-33 genomic DNA as a template and primers ITS1 and ITS 4.

Fungal primer sequences were selected as follows:

ITS1(5'-TCCGTAGGTGAACCTGCGG-3')；

ITS4(5'-TCCTCCGCTTATTGATATGC-3')。

the PCR amplification reaction system (50. mu.L) included: 5 × buffer 10 μ L; dNTP 4. mu.L; 1 μ L of forward primer (10 μ M); reverse primer (10. mu.M) 1. mu.L; prime STAR 0.5 μ L; template (1. mu.M) 1. mu.L; ddH ₂ O 32.5μL。

And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 2min, and 30 cycles; extension at 72 ℃ for 5 min.

4) The amplification product (i.e., ITS of QHSH-33) was submitted to Yogji technologies, Beijing for sequencing.

5) The obtained sequence was subjected to homology alignment analysis with NCBI nucleic acid database using BLAST, a part of the strain having high similarity was selected and subjected to phylogenetic analysis with QHSH-33 strain, and phylogenetic tree was constructed using Neighbor-Joining in MEGA7.0 software. The phylogenetic tree is shown in FIG. 2.

Finally, QHSH-33 was determined to be a Pleurotus (Acremonium sp.) according to morphological characteristics, homology distance and phylogenetic tree.

Example 3

In this example, the pesticide degradation performance of QHSH-33 was mainly measured, including the growth curve, degradation product and degradation rate.

The Martin medium referred to in this example had the same composition as in example 1.

1. Growth Curve determination

1) Inoculating a single bacterial colony of the strain into a triangular flask filled with 150mL of liquid Martin medium, and performing shake activation culture in a shaking table at 28 ℃ and 180 r/min.

2) Then inoculating 2% of the inoculum size into a 27-bottle triangular flask containing 150mL of liquid Martin medium, taking out 3 triangular flasks every 24h from 48h (i.e. 2d) of the culture time, reserving mycelium, drying at 60 ℃ to constant weight, weighing and calculating the average value, and measuring the growth curve by a dry weight method as shown in figure 3.

The growth curve shows that the growth of the QHSH-33 mycelium is slow between 0d and 2d, and the growth of the mycelium requires a process of adaptation. The growth of hyphae is accelerated from the 2 nd to the logarithmic growth phase, and after the 7 th, the growth of hyphae gradually becomes stable along with the consumption of nutrient substances in the liquid culture medium.

2. Degradation products

1) The purified and cultured strain QHSH-33 is inoculated in 150mL of liquid commercial Martin medium and cultured with shaking at 28 ℃ and 180r/min until logarithmic growth phase.

2) Adding diuron and imidacloprid as substrates respectively at a concentration of 100mg/L, and continuously culturing for 5 days under the same conditions.

3) And (4) passing the fermentation liquor through a small ODS C18 column to obtain a detection sample. And (3) determining the condition of the degradation product of the strain by adopting a liquid chromatography-mass spectrometer.

The used instruments are: agilent 1200-6530HPLC-Q-TOF liquid chromatography mass spectrometer (Agilent, USA).

HPLC conditions: column CAPCELL PAK C18AQ (250 mm. times.4.6 mm, 5.0 μm); mobile phase: acetonitrile (A) -water (B), and gradient elution (0-40 min, 10-70% of A); flow rate: 1.0 mL/min; column temperature: 20 ℃; sample introduction volume: 5 μ L.

MS conditions: the time-of-flight mass spectrometry adopts an electrospray negative ion mode: capillary voltage 4.5kV, atomizer pressure 2.0bar, dry gas (N) ₂ ) The flow rate is 4.0L/min, the temperature of the drying gas is 180 ℃, the ion energy of the quadrupole is 3.0eV, the ion transmission time is 80 mus, the pre-pulse storage time is 5 mus, the collision gas is argon, and the collision energy is 30 eV. The mass spectrometry data are collected in a full scanning mode, and the collection range is 100-2000 m/z.

The liquid quality data shows that the strain QHSH-33 is p-diuron, the degradation product of which is shown in figure 4 and the degradation product of which is shown in figure 7 are obtained. The bacterial strain QHSH-33 degrades diuron to generate two degradation intermediate products: 3- (3, 4-dichlorophenyl) -1-methylurea is shown in figure 5, and has a molecular formula of C ₈ H ₈ Cl ₂ N ₂ O; and 3, 4-dichlorophenylurea shown in figure 6, and formula C ₇ H ₆ Cl ₂ N ₂ And O. Degradation of imidacloprid produces 2 degradation intermediates: nitrosoimidacloprid is shown in figure 8, and molecular formula C ₉ H ₁₀ ClN ₅ O; and 5-hydroxy imidacloprid shown in figure 9, and its molecular formula C ₉ H ₁₀ ClN ₅ O ₃ . Experimental results further show that the strain QHSH-33 has the capacity of degrading diuron and imidacloprid and has the potential of environmental remediation.

3. Determination of degradation Rate

1) Washing with sterile normal saline to preserve degradation bacteria QHSH-33, mixing, diluting with normal saline to 10 ^-2 And doubling to obtain a bacterial suspension of QHSH-33 with the concentration of 106 CFU/mL.

2) Treatment groups were inoculated in liquid commercial Martin medium at an inoculum size of 10%, with 3 groups in parallel.

3) Carrying out constant temperature shaking culture at 28 ℃ and 180r/min, and adding 100mg/L diuron and imidacloprid respectively when the culture is carried out until logarithmic growth phase.

4) Sampling is carried out on

days

1, 2, 4, 6, 8 and 10 after the diuron and the imidacloprid are added, the degradation amount in the process of degrading the diuron and the imidacloprid is dynamically monitored, and the content change of the diuron and the imidacloprid in the culture solution is detected by adopting HPLC-DAD.

5) A liquid commercial Martin culture medium under the same conditions is not inoculated with strains, and diuron and imidacloprid are added as a blank control group, and the time of the peak emergence of the diuron and imidacloprid standard substances (Shanghai Aladdin Biotechnology, Ltd., D101260, I109898) is used as a control to judge the diuron and the imidacloprid in the culture solution, which is shown in the attached figure 10 and the attached figure 12. FIG. 10 is a liquid chromatogram of diuron, and FIG. 12 is an imidacloprid chromatogram.

6) Centrifuging the fermentation liquor to remove thallus, extracting the fermentation liquor for 3 times by adopting dichloromethane with equal volume, concentrating and drying by a rotary evaporator, dissolving by using chromatographic methanol to constant volume, filtering by using a 0.22 mu m organic filter membrane, and placing in a brown chromatographic bottle. Detecting the content by high performance liquid chromatography, and calculating the degradation rate.

The degradation rate calculation formula is as follows:

rate of degradation

Note: c _X : detected content of pesticide in soil (mg/kg) after a certain number of days

C ₀ : initial content of pesticide applied (mg/kg) in soil

The analysis adopts a high performance liquid chromatograph: shimadzu LC2030C chromatograph, Megers C18 chromatograph (250X 4.6mm, 10 μm). The mobile phases are respectively:

diuron: methanol-water (volume ratio 65:35), flow rate 0.8mL/min, detection wavelength 254nm, column temperature 30 ℃, injection volume 10 uL.

Imidacloprid: methanol-water (volume ratio 65:35), flow rate 0.8mL/min, detection wavelength 254nm, column temperature 30 ℃, injection volume 5 uL.

The degradation experiment result shows that after the strain is cultured for 10 days, the degradation rate of the strain QHSH-33 to diuron is 57.85 percent, and is shown in figure 11, and the degradation rate of the strain QHSH-33 to imidacloprid is 47.58 percent, and is shown in figure 13.

As described above, the present invention can be preferably implemented, and the above-mentioned embodiments only describe the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various changes and modifications of the technical solution of the present invention made by those skilled in the art without departing from the design spirit of the present invention shall fall within the protection scope defined by the present invention.

Claims

1. The strain QHSH-33 is characterized in that the preservation number of the strain in China general microbiological culture Collection center is CGMCC No. 23259.

2. The strain of claim 1, wherein the primers used for PCR amplification of the genomic DNA of the strain are:

ITS1(5'-TCCGTAGGTGAACCTGCGG-3')；

ITS4(5'-TCCTCCGCTTATTGATATGC-3')。

3. the strain of claim 1, or a culture broth of the strain of claim 1, or the strain of claim 1 and its culture broth for use in degradation of pesticides.

4. The use as claimed in claim 3, wherein the pesticide is any one of diuron and imidacloprid.

5. The use of a strain according to claim 1, or a culture broth of a strain according to claim 1, or a strain according to claim 1 and a culture broth thereof for reducing the amount of residual pesticides after use.

6. The use as claimed in claim 5, wherein the pesticide is any one of diuron and imidacloprid.

7. An article for degrading a pesticide, comprising the strain of claim 1, or a culture broth of the strain of claim 1, or the strain of claim 1 and a culture broth thereof.

8. The article of claim 7, wherein the article is configured to degrade any one of diuron and imidacloprid or to reduce the amount of diuron and imidacloprid remaining after use.