CN114437943B - High-efficiency pathogenic biocontrol strain Aspergillus fijiensis and application thereof - Google Patents

Abstract

The invention discloses a high-efficiency pathogenic biocontrol strain Aspergillus fijis strain and application thereof, and belongs to the technical field of biological control. The invention relates to a method for preparing a strain of Aspergillus feijiensis GDIZM-1, which is deposited in the microorganism strain collection of Guangdong province at 12 months and 17 days of 2021, wherein the deposited number is as follows: GDMCC No:62135. the long-term infection biology and indoor bioassay show that the strain has a strong insecticidal effect on diaphorina citri; the strain has the characteristics of high growth rate and large spore yield. The aspergillus fijis is a biocontrol fungus, is used as a living biological pesticide, has a brand-new action mechanism different from the existing chemical pesticide, has no pollution and no residue on the environment, can solve the drug resistance problem and the pesticide residue problem in the process of controlling the diaphorina citri, meets the requirements of organic food production, and has very good application potential in biological control of citrus pests.

Description

High-efficiency pathogenic biocontrol strain Aspergillus fijiensis and application thereof

Technical Field

The invention relates to the technical field of microbial control, in particular to a high-efficiency pathogenic biocontrol strain Aspergillus fijis strain and application thereof.

Background

The diaphorina citri (Diaphorina citri Kuwayama) is an important pest in the new shoot stage of Rutaceae plants, and is collected on tender shoots, buds and tender leaves by adult nymphs, phloem juice of the host plants is sucked through a piercing-sucking mouth gag, and the plant is subject to yellowing, wasaki shape, wilting and even death by harmful leaves, and a large amount of white honeydew is discharged after feeding, so that soot diseases are caused, photosynthesis of the plants is influenced, and growth and development of the plants are seriously influenced. And is also an important transmission medium for citrus yellow dragon disease. The diaphorina citri feeds on the yellow dragon disease plants of the citrus, spawns and breeds, a large number of adult bacteria can be generated, and the adult bacteria can be transferred into new plants to spread the yellow dragon disease. At present, the main method for preventing and controlling the diaphorina citri still depends on chemical agents, but the long-term and large-scale use of chemical pesticides causes a series of negative problems of death of natural enemy insects in citrus orchards, loss of biodiversity, generation of different degrees of drug resistance, pollution to the ecological environment of the orchards and the like, and the problem of exceeding of pesticide residues of fruits also occurs. In the face of such a passive situation, research and application of non-chemical means to control diaphorina citri have been urgent problems facing the citrus industry. Therefore, under the large background of the national advocate of 'pesticides and fertilizers', the search for an efficient and environment-friendly control means is particularly important.

Biological control is a control measure which uses the interdependence or restriction relationship among species to inhibit one type of organism and enable the dynamic balance relationship among organisms to incline to the favorable direction of agricultural production. Compared with other control methods, the biological control method is safe to human, livestock and ecological environment, has high efficiency and durability, and is the most promising control means in the comprehensive pest control measures. Therefore, the search for effective biological control products and application techniques is one of the main research directions for sustainable pest control in the future. The research and development of the microbial pesticide capable of safely, efficiently and continuously controlling the diaphorina citri has important social and economic benefits and ecological benefits.

The entomopathogenic fungi are excellent chemical pesticide environment-friendly substitutes, and are widely used for biological control of agricultural pests. The entomogenous fungi has the same control effect on the diaphorina citri with drug resistance and pesticide sensitivity, and the control prospect is widely seen. Research shows that the pathogenic mechanism of insect pathogenic fungi on pests is complex and different from the insecticidal mechanism of the existing chemical pesticides. The insect pest which generates drug resistance to the insect pathogenic fungi is not discovered so far, so the application and development potential of the insect pathogenic fungi on the problem of controlling the drug resistance of the insect pest are huge.

Disclosure of Invention

The invention aims to overcome the defects of the existing diaphorina citri control technology and provides a biocontrol bacterium with high pathogenicity for diaphorina citri and a control method thereof. Entomogenous fungi have a variety in genetic, ecological, biological aspects, and the like. Screening and obtaining of high-yield high-quality strains are the primary precondition for obtaining better control effect. The strain screening mainly considers 3 indexes including spore yield, colony growth rate and pathogenicity. The invention is based on these indexes to screen excellent strains.

A first object of the invention is to provide a strain of Aspergillus flavus GDIZM-1, deposited under the accession number: GDMCC No:62135.

the strain GDIZM-1 of the invention has compact texture on PDA culture medium, flat colony and radial groove on the back. White hypha is formed at the front of the colony at the early stage, and is yellow brown at the later stage, and the colony is powdery; the color of the back surface is gradually deepened and is earthy yellow. The conidiophores have different lengths and smooth walls, have podocytes, have the size of 200-1100 mu m multiplied by 8-16 mu m, expand to form a top sac nearly spherical shape with the size of 20-45 mu m multiplied by 10-15 mu m, cover a layer of small peduncles on the top sac, have the small peduncles arranged in a radial manner, have the size of 3-6 mu m multiplied by 2-3 mu m and have the surface 3/4 fertility. The pedicel is nearly spherical, has rough wall and full thorns, is arranged in a chain shape and has the diameter of about 3-5 mu m.

Different concentrations of conidium suspensions of Aspergillus feiji GDIZM-1 of the invention (1X 10) ⁴ 、1×10 ⁵ 、1×10 ⁶ 、1×10 ⁷ 、1×10 ⁸ The corrected mortality rates for 6 instar diaphorina citri (1-5 instar nymphs and adults) differed significantly from the control, indicating that the feijiale mold was highly pathogenic to diaphorina citri nymphs and adults. Meanwhile, GDIZM-1 is used for citrusLC of 1-2 years (low age), 3-4 years (high age), 5 years (last age) nymphs and adults of diaphorina citri ₅₀ And LT ₅₀ The values of (2) are 0.43×10 respectively ⁴ 、1.41×10 ⁴ 、2.41×10 ⁴ 、3.79×10 ⁴ The spores per mL and 2.30, 2.86, 3.43, 4.25 days. Shows that the feijiale has important application potential in the biological control of diaphorina citri.

It is a second object of the present invention to provide a microbial preparation comprising the culture of GDIZM-1 or a fermentation broth thereof as an active ingredient.

Preferably, GDIZM-1 is attached to an SDAY plate at a temperature of 24-26℃under light of 14L:10D, thereby obtaining a culture of GDIZM-1.

A third object of the invention is to provide the use of aspergillus feijiensis (aspergillus fijiensis) in any of the following:

(1) Preventing and treating diaphorina citri or diseases caused by the diaphorina citri;

(2) Preparing biological control medicine.

Preferably, the feijia is Aspergillus fijiensis GDIZM-1, which is deposited under the accession number: GDMCC No:62135.

preferably, the disease caused by the diaphorina citri is a disease caused by the diaphorina citri as a transmission medium.

Preferably, the disease caused by diaphorina citri includes, but is not limited to, citrus yellow-long disease.

Preferably, the feijia fungus is used for controlling diaphorina citri nymphs and adults.

Preferably, the biocontrol agent comprises a feijia conidium or suspension thereof.

Further, the preparation of the feijia conidia comprises the following steps: taking out and activating the inclined plane strain of the aspergillus fijis, inoculating the inclined plane strain to an SDAY plate, culturing for 10d in a constant temperature incubator, scraping conidium on the surface of a culture medium, and filtering and collecting the conidium by using sterile water containing 0.1% of Tween 80; the suspension is prepared by preparing collected conidium into conidium suspension.

Compared with the prior art, the invention has the following beneficial effects:

(1) The invention screens and obtains the Aspergillus feiji GDIZM-1 with high pathogenicity to the diaphorina citri, and the LC of 1-2 years old (low age), 3-4 years old (high age), 5 years old (last age) nymphs and adults of the diaphorina citri is measured through long-term infection biology and indoor bioassay ₅₀ And LT ₅₀ The values of (2) are 0.43×10 respectively ⁴ 、1.41×10 ⁴ 、2.41×10 ⁴ 、3.79×10 ⁴ The individual spores/mL and 2.30, 2.86, 3.43 and 4.25 days show that the strain has strong infection insecticidal effect on the diaphorina citri and has strong application potential in biological control of the diaphorina citri;

(2) The Aspergillus fijis GDIZM-1 has large spore yield, and after 10d culture on SDAY medium, the spore yield can reach 3.86×10 ⁸ Individual spores/mL;

(3) The growth rate of the colony of the Aspergillus fijis GDIZM-1 is high, and the diameter of the colony can reach 83mm after the colony is cultured on an SDAY culture medium for 5 days;

(4) The aspergillus fijis can be used as a living organism pesticide, has no pollution and no residue on the environment, can solve the problem of drug resistance and pesticide residue in the process of controlling the diaphorina citri, and meets the requirements of organic food production.

Aspergillus fijiensis GDIZM-1 of the invention was deposited at the microbiological bacterial collection center (GDMCC) of Guangdong in 12 months 17 of 2021, and addressed to building 5 of national institute No. 59 of Mitsui 100 of View district of Guangzhou, guangdong, and post code: 510070, accession number is: GDMCC No:62135.

drawings

FIG. 1 shows infection of a. Fijia (1×10) with diaphorina citri nymphs and adults ⁷ Individual spores/mL) symptoms at day 10, graphical representation A, B, C, D, E, F is 1, 2, 3, 4, 5-year-old nymphs and adults, respectively; the scale values of the graph A, B, C, D, E, F are 100, 200, 500 μm, respectively.

FIG. 2 is a colony morphology of an Aspergillus feiji isolate; graphical representation A, B is of the front and back sides of a colony of Aspergillus fijis on day 3; graphical representation C, D is of the front and back sides of a colony of Aspergillus fijis on day 10; graph E, F, G, H is a process of gradient of a colony of aspergillus fijis; the scale values of plot A, B, C, D were 10mm, and the scale values of plot E, F, G, H were 1mm, 500 μm, 200 μm, and 500 μm, respectively.

FIG. 3 is a morphology diagram of the sporangia and conidia of the Aspergillus feij strain GDIZM-1, with graphic A being the top cyst and conidia of Aspergillus feij GDIZM-1; panel B is a Aspergillus fijis GDIZM-1 podocyte; panel C is Aspergillus feiji GDIZM-1 conidium; the scale values of plot A, B, C are all 10 μm.

FIG. 4 is a molecular characterization phylogenetic tree of the Aspergillus fijis strain GDIZM-1.

FIG. 5 shows different concentrations of Aspergillus feiji spore suspension (1X 10) ⁴ ，1×10 ⁵ ，1×10 ⁶ ，1×10 ⁷ ，1×10 ⁸ Individual conidia/ml) mortality of diaphorina citri at different developmental stages.

Detailed Description

The present invention is further illustrated below with reference to specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.

Example 1 isolation, purification and identification of Phyllostachys citri pathogenic strains

1.1 sources of materials

(1) Sample: the diaphorina citri insect corpses are collected after the lemon garden is infected by the entomogenous fungi.

(2) Test insects: diaphorina citri is bred and propagated for multiple generations in the environmental insect research center net room of the animal research institute of the academy of sciences of Guangdong.

(3) PDA medium: 6g/L potato powder, 20g/L glucose and 20g/L agar; SDAY medium: 10g/L yeast extract powder, 40g/L glucose, 10g/L peptone and 20g/L agar; the solvent of the culture medium is water, and the preparation method comprises the steps of uniformly mixing the components and sterilizing for later use.

(4) Aseptic operating conditions: all vessels and appliances are subjected to high-pressure sterilization (121 ℃ C., 30 min), inoculation and other operations are performed in an ultra-clean workbench.

1.2 isolation and purification of Strain

Slave quiltAnd (3) separating target bacteria from the diaphorina citri cadaver sample infected by the entomogenous fungi. The method comprises the following steps: and (3) carrying out surface sterilization on the sample by using a sodium hypochlorite solution with the concentration of 5%, washing the sterilized sample in sterilized water for 3 times, placing the sterilized sample in a PDA flat plate, culturing the sample in an incubator with the temperature of 25+/-1 ℃ and the illumination (14L: 10D) in an inverted manner, transferring the sample into a PDA inclined plane after colony formation, and then transferring the sample into a refrigerator with the temperature of 4 ℃ for storage. Culturing the strains on PDA culture medium for 10d, and collecting conidium to obtain 1×10 ³ The conidium/mL suspension was dropped onto a slide glass with a cover glass, and the slide glass with only one conidium in one drop was inserted into a culture medium and cultured in an incubator to obtain an isolated strain.

Under natural conditions, typical pedicel bundles grow on diaphorina citri bodies parasitized by fungi. A fungus strain is obtained after separation and purification, and the number of the fungus strain is GDIZM-1. The tieback shows that the strain can successfully infect the diaphorina citri of each age, white mycelium can be observed to grow on the body surface after inoculation for 2 days, and meanwhile, symptoms such as slow response, slow action, body color change and the like are shown, and typical spore peduncles grow on the body after inoculation for 10 days, and the infection form is shown in figure 1. The separation result shows that the culture characteristics and morphological characteristics of the thalli grown on the inoculated bodies are the same as those of the original inoculated strain, and the strain is the entomogenous fungi infecting the diaphorina citri.

1.3 identification of strains

(1) Morphological identification:

strains were inoculated on PDA plates and cultured in a constant temperature incubator at 25+ -1deg.C, and the front and back sides of the colonies were observed daily for color, texture, size, height, surface texture, edges, exudates, etc. and photographed. A small amount of spores are picked by an inoculating needle to be planted on a PDA flat plate to manufacture a glass slide when the spores are produced and mature, transparent adhesive tapes with fine texture are selected, small adhesive tapes which are cut into proper sizes are clamped by forceps, the small adhesive tapes are gently and slowly adhered from the edge of a colony to the center, then are fixed by 95% alcohol and invade a cotton basket for dyeing, the transparent adhesive tapes adhered with the spore producing structure are placed face up, and after 2-3min, a cover glass is put down gently, and air bubbles are carefully avoided. Observing under a low power mirror to find out the visual field, then dripping cedar oil or paraffin oil, and observing under an oil mirror the structural characteristics of conidiophores, sporogenic cells and spores. The finished pictures were taken with a IMAGING MP 5.0.0-RTV-CLR-10-A camera at 1000 times and then synthesized.

The strain had dense texture on PDA medium, flat colony and radial grooves on the back. The bacterial colony morphology of the bacterial strain on the PDA culture medium is shown in figure 2, the front initial stage of the bacterial colony is white hypha, and the later stage is yellow brown, and the bacterial colony is powdery; the color of the back surface is gradually deepened and is earthy yellow. As shown in FIG. 3, the conidiophores are different in length, smooth in wall, have podocytes, are 200-1100 mu m multiplied by 8-16 mu m in size, expand at the top end of the conidiophores to form a top sac nearly spherical shape, are 20-45 mu m multiplied by 10-15 mu m in size, cover a layer of small peduncles on the top sac, are arranged in a radial manner, are 3-6 mu m multiplied by 2-3 mu m in size, and are 3/4 fertile in surface. The pedicel is nearly spherical, has rough wall and full thorns, is arranged in a chain shape and has the diameter of about 3-5 mu m.

(2) Molecular identification:

the DNA sequence specific for this study consisted of three genes including a transcriptional spacer, elongation factors and a large subunit of RNA polymerase II. Taking 100mg of the cultured mature strain, extracting hyphal genome DNA by using a fungus genome DNA extraction kit, taking the strain genome DNA as a template, taking ITS4 (5'-TCCTCCGCTTATTGATATGC-3') and ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3'), EF-1983F (5 '-GC (Y) CC (Y) GG (H) CA (Y) G GTGA (Y) TT (Y) AT-3') and EF-12218R (5 '-ATAC (R) TG (R) GC (R) GT (Y) TG-3'), fRPB2-5F (5 '-GA (Y) GA (Y) (M) G (W) GATCA (Y) TT (Y) GG-3') and fRPB2-7cR (5 '-CCCAT (R) GCTTG (Y) TT (R) GCTTG-3') as primers for amplification, and then performing BLAST comparison in GENBANK. The symbols (Y), (H), (R), (W) above represent degenerate bases, i.e., one symbol replaces some two or more bases, where (Y) represents C/T; (H) represents A/T/C; (R) represents A/G; (W) represents A/T.

PCR 50. Mu.L reaction system: the upstream and downstream primers were 1. Mu. L, ddH2, 2O 22. Mu. L, DNA template 1. Mu.L, 2 XMaster Mix 25. Mu.L each. PCR amplification procedure for amplification of rDNA-ITS sequences: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 55℃for 30s, extension at 72℃for 45s for 35 cycles; finally, the extension is carried out for 10min at 72 ℃. PCR amplification procedure for amplifying EF-1. Alpha. Gene sequence: pre-denaturation at 95℃for 10min; denaturation at 94℃for 30s, annealing at 55℃for 30s, extension at 72℃for 1min for 40 cycles; finally, the extension is carried out for 10min at 72 ℃. PCR amplification procedure for amplifying fRPB2 Gene sequence: pre-denaturation at 95℃for 10min; denaturation at 94℃for 30s, annealing at 50℃for 30s, extension at 72℃for 1min for 40 cycles; finally, the extension is carried out for 10min at 72 ℃. The amplified product was detected by electrophoresis on a 1.5% agarose gel to see if the target band was present, and the amplified product with the correct band size was sequenced on Shenzhen big Gene Co. And (3) splicing the sequencing result through a letter generation software MEGA7.0 to remove partial sequences with uneven head and tail, carrying out gene homology similarity comparison through NCBI website online program BLAST, and downloading corresponding sequences of related published mode strains. Based on ITS, EF-1 alpha and fRPB2 marker genes, a phylogenetic tree is constructed by adopting evolution analysis software MEGA7.0 for fungi to be identified. A tree is built by using a Neighbor method (adjacent) and a step size test (Bootstrap method) is adopted, wherein a selection parameter is 1000, and a genetic distance calculation model (substationmodel) is adopted to select p-distance.

The PCR products of GDIZM-1 strain were purified, recovered and sequenced to obtain three gene fragments of ITS (SEQ ID NO. 1), EF-1 alpha (SEQ ID NO. 2) and fRPB2 (SEQ ID NO. 3), which were spliced, and then constructed Neighbor Joining Tree by the adjacent method (FIG. 4). The resulting DNA sequences were Blast aligned at NCBI. The similarity between the strain GDIZM-1 and the Aspergillus fijis is found to be 99-100 percent after comparison. Meanwhile, the strain GDIZM-1 and the strain related to the aspergillus fijis are gathered into one strain, and the genetic distance from the outer group is far. And by combining with strain morphological identification of GDIZM-1, the strain GDIZM-1 can be determined to be Aspergillus feiji (Aspergillus fijiensis).

Thus, in combination with morphology and molecular biology, the GDIZM-1 strain was designated as Aspergillus fijii (Aspergillus fijiensis) GDIZM-1 and was deposited at the Guangdong province microbiological culture Collection center (GDMCC) at 12 months and 17 days 2021, at floor 5 of the first university, mitsui No. 100, guangzhou City, guangdong province, post code: 510070, accession number is: GDMCC No:62135.

EXAMPLE 2 research on hypha growth and spore-forming Properties of Aspergillus feiji GDIZM-1 Strain

Determination of colony growth rate and sporulation: activating the slant strain of Aspergillus fijis GDIZM-1, inoculating onto SDAY plate, culturing in constant temperature incubator for 10d, scraping off conidium on the surface of the culture medium, filtering with sterile water containing 0.1% Tween 80 to collect conidium, and preparing into 1×10 ⁷ Spore suspension of individual spores/mL. 1mL of spore suspension is sucked into the center of an SDAY plate, smeared evenly and sealed in a constant temperature incubator for culture, 10d is observed, and the diameter of each colony is recorded every day for 5 repetitions. After 10d, conidia were filtered with sterile water containing 0.1% tween 80 and the spore yield was determined using a hemocytometer.

The colony growth rate and spore yield of the isolated strain of Aspergillus fijis are shown in Table 1, and the data are mean.+ -. Standard error. The research shows that after the aspergillus fijis is cultivated for 5 days, the colony diameter reaches 83.7+/-0.47 mm (average value+/-standard error); after 10 days of cultivation, the sporulation yield of the strain in the SDAY medium is 3.86 multiplied by 10 ⁸ Individual spores/mL.

TABLE 1 colony growth Rate and spore yield of A.fijia isolate GDIZM-1

Example 3 determination of pathogenicity of Aspergillus feiji GDIZM-1 Strain to diaphorina citri

(1) Preparation of conidium suspension of Aspergillus feiji GDIZM-1 strain comprises taking out and activating the slant strain of Aspergillus feiji GDIZM-1, inoculating onto SDAY plate, culturing in constant temperature incubator for 10d, scraping conidium on the surface of culture medium, filtering with sterile water containing 0.1% Tween 80 to collect conidium, and preparing into 1×10 ⁴ ，1×10 ⁵ ，1×10 ⁶ ，1×10 ⁷ ，1×10 ⁸ Spore suspension of individual spores/mL.

(2) Selecting healthy lemon plants, cleaning leaves with clear water, completely soaking 1-5-year-old diaphorina citri nymphs and adults in 5 spore suspensions with different concentrations for 2 minutes, drying with filter paper, transferring onto young leaves of lemon seedlings, bagging at 25+ -2deg.C and humidity of 75+ -5%RH, and photoperiodThe culture was carried out in a climatic chamber at a ratio of 14:10 (L: D), and each treatment was repeated 3 times for 20 heads. Continuous observation recorded 8d mortality, and LC was assessed for young nymphs (1-2 years old), old nymphs (3-4 years old), last nymphs (5 years old) and adults (3 days of incubation) ₅₀ Value and LT ₅₀ Values. The negative control was double sterile water. All the test data are processed on the data processing software SPSS system.

The pathogenicity studies are shown in FIG. 5, where 5 conidium concentrations of Aspergillus feij GDIZM-1 spore suspension (1X 10) ⁴ 、1×10 ⁵ 、1×10 ⁶ 、1×10 ⁷ 、1×10 ⁸ The corrected mortality rate of individual spores/mL) for 6 ages of diaphorina citri (1-5 years nymphs and adults) was significantly different from the control. The concentration is 1 multiplied by 10 ⁸ The individual spores/mL of Aspergillus fijis GDIZM-1 were highly pathogenic to diaphorina citri nymphs (88.67% -98.14%) and adults (87.32%). At the same time, it has no effect on LC of 1-2 years (low age), 3-4 years (high age), 5 years (last age) nymphs and adults of diaphorina citri ₅₀ And LT ₅₀ The values of (2) are 0.43×10 respectively ⁴ 、1.41×10 ⁴ 、2.41×10 ⁴ 、3.79×10 ⁴ The spores per mL (Table 2) and 2.30, 2.86, 3.43, 4.25 days (Table 3). Wherein, X in tables 2 and 3 represents the logarithmic value of the spore suspension concentration, and Y represents the value corresponding to the logarithmic value of the spore suspension concentration.

TABLE 2 virulence of Aspergillus fijis against diaphorina citri at different developmental stages after 8d infection

TABLE 3 infection of Aspergillus fijis (1X 10) ⁸ Individual spores/mL) virulence against diaphorina citri at different stages of development

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Claims (7)

1. Aspergillus feijiai strainAspergillus fijiensis) GDIZM-1 with deposit number: GDMCC No:62135.

2. a microbial preparation comprising the culture of Aspergillus feiji GDIZM-1 as defined in claim 1 as an active ingredient.

3. The microbial preparation of claim 2, wherein the feijia strain GDIZM-1 is attached to an SDAY plate at a temperature of 24-26 ℃ under light of 14L:10D, thereby obtaining a culture of Aspergillus feiji GDIZM-1.

4. Use of a feijia strain GDIZM-1 of claim 1 in any one of the following:

(1) Preventing and treating diaphorina citri or diseases caused by the diaphorina citri;

(2) Preparing biological control medicine.

5. The use according to claim 4, wherein the disease caused by diaphorina citri is citrus yellow-long disease.

6. The use according to claim 4, wherein the biocontrol agent comprises conidia of feijia or a suspension thereof.

7. The use of claim 6, wherein preparing said conidia of feijia comprises the steps of: taking out and activating the inclined plane strain of the aspergillus fijis, inoculating the inclined plane strain to an SDAY plate, culturing for 10d in a constant temperature incubator, scraping conidium on the surface of a culture medium, and filtering and collecting the conidium by using sterile water containing 0.1% of Tween 80;

the suspension is prepared by preparing collected conidium into conidium suspension.

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