A kind of Enterobacter holmesii A3 strain and its application

Abstract

The invention discloses an Enterobacter holmesii A3 strain and its application, and the strain is preserved in the Guangdong Provincial Microbial Culture Collection Center, and the biological preservation number is: GDMCC NO: 64603. The Enterobacter holmesii A3 strain of the invention can improve the production of resistance of diamondback moth, and the strain can survive in a culture medium containing only bromofenac or chlorfenapyr as the only nutrient source, and can degrade the pesticide in vitro; the Enterobacter holmesii A3 strain of the invention provides a basis for the study of the resistance mechanism of diamondback moth, and at the same time, because it has the application in pesticide degradation, it provides a basis for the application of biological control.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural microorganisms, and in particular relates to an Enterobacter hormaechei A3 strain of the diamondback moth and an application thereof. Background Art

[0002] The diamondback moth (Plutella xylostella) belongs to the family Plutellidae of the order Lepidoptera. It is a global pest with the strongest destructive power against cruciferous vegetables and is also internationally recognized as one of the pests with the most serious pesticide resistance.

[0003] At present, the prevention and control of diamondback moth is mainly based on chemical control. Long-term irrational use of drugs and continuous cropping of cruciferous vegetables, coupled with the strong reproductive ability and detoxification adaptability of diamondback moth, have led to a sharp increase in the resistance of diamondback moth to insecticides. Research reports show that diamondback moth has developed resistance to 97 different insecticide active ingredients. The control of diamondback moth's resistance has become a global problem, so it is necessary to study the resistance of diamondback moth.

[0004] There are a huge number of microorganisms in the intestines of insects, and they are diverse. These intestinal microorganisms have co-evolved with the host insects, and have undergone a long evolutionary process of mutual influence. In this process, the intestinal microorganisms and the host insects have formed a co-evolutionary and inseparable relationship. The host provides a suitable environment for the intestinal flora, and the intestinal flora also has many effects on the host, such as participating in food digestion, nutrient supply, and improving the host's defense and detoxification capabilities. In recent years, society's attention to environmental issues has promoted the study of the detoxification function of insect intestinal microorganisms on the host. Intestinal microorganisms degrade complex organic matter into inorganic substances through mineralization and metabolic processes, and convert toxic substances in the insect body into non-toxic substances, thereby improving its resistance. Summary of the invention

[0005] The purpose of the present invention is to address the deficiencies of the prior art, to isolate and screen a strain from the intestine of Plutella xylostella that can improve the host insect's resistance to chlorantraniliprole / bromofenac, and to measure the improvement of drug resistance when introduced into the body of Plutella xylostella.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A strain of Enterobacter hormaechei, named Enterobacter hormaechei A3, deposited in Guangdong Microbiological Culture Collection Center; deposited at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; deposited on May 10, 2024; biological deposit number: GDMCC No: 64603.

[0008] The Enterobacter hormaechei A3 strain of the present invention is a round white colony with a smooth and glossy surface and is opaque, and is arranged in a single or chain shape. The optimum growth temperature is 30° C., the optimum pH is 6.0-7.0, and the strain is facultative anaerobic.

[0009] Preferably, as a preferred embodiment, the Enterobacterhormaechei A3 strain described in the present invention can improve the drug resistance of Plutella xylostella.

[0010] Preferably, as a preferred embodiment, the Enterobacterhormaechei A3 strain described in the present invention can degrade the diamide pesticides bromofenac or chlorfenapyr in vitro.

[0011] Another object of the present invention is to provide an application of Enterobacter hormaechei A3 strain for degrading pesticides.

[0012] The present invention isolates and cultures an Enterobacter hormaechei strain from the midgut of the diamondback moth of the Huizhou and Lianzhou populations of the resistant strains. The present invention conducts a feeding test on the diamondback moth with the Enterobacter hormaechei A3 strain, and feeds the diamondback moth with specific antibiotics in order to eliminate part of the Enterobacter hormaechei A3. The indoor toxicity test is performed on the feed with bacteria, the feed with antibiotics, and the blank control without treatment at the same time. The results show that: (1) Chlorantraniliprole: LC after feeding with bacteria 50 The LC value was 1.567 mg / L after adding antibiotics to feed. 50 The blank control treatment LC 50 0.848 mg / L; (2) Brofenac: LC after feeding with bacteria 50 The LC value is 0.021 mg / L after adding antibiotics to feed. 50 0.011 mg / L, blank control treatment LC 50 The resistance of the fish fed with bacteria was improved to a certain extent compared with those fed with antibiotics and blank control.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The research results of the present invention show that the Enterobacter hormaechei A3 strain has the ability to improve the resistance of diamondback moth to chlorantraniliprole / brofenac, and can degrade chlorantraniliprole / brofenac in vitro to reduce pesticide residues. The Enterobacter hormaechei A3 strain can be used as a basic biological material for resistance mechanism research, used for pest resistance mechanism research, and can also be used as a biodegradable material, applied to reducing pesticide residues, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Shown is a plate image of the separation of Enterobacter hormaechei A3 strain;

[0016] Figure 2 A line graph showing the growth of Enterobacter hormaechei A3 strain in MS medium;

[0017] Figure 3 The standard curve of chlorantraniliprole degradation by Enterobacter hormaechei A3 strain is shown;

[0018] Figure 4 The standard curve of bromofenac degradation by Enterobacter hormaechei A3 strain is shown;

[0019] Figure 5 The peak area graph of chlorantraniliprole degradation by Enterobacter hormaechei A3 strain is shown;

[0020] Figure 6 The peak area graph of the degradation of bromofenac by Enterobacter hormaechei A3 strain is shown. DETAILED DESCRIPTION

[0021] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The following specific implementation methods further describe the present invention.

[0022] Unless otherwise specified, the reagents and materials used in the embodiments of the present invention can be purchased from the market.

[0023] 1. Isolation and culture of strains

[0024] 1. Preparation of culture medium

[0025] LB medium: 10 g peptone, 5 g yeast extract, 10 g NaCl, 15 g agar, dissolved in 1 L sterile water, and adjusted to pH 7.0.

[0026] 2. Isolation and purification of strains

[0027] 2.1 Dissection and plating

[0028] Dissection and plating: Under sterile conditions, the third instar larvae of Plutella xylostella were dissected in a super working chamber and the midgut tissue was removed and put into a grinder. Five concentration gradients were diluted with sterile water and LB medium plates were spread according to the gradients. The plates were placed in a constant temperature incubator at 30°C and observed every 24 hours.

[0029] 2.2 Purification, culture and photography

[0030] When a single colony grows in the culture medium, pick the single colony according to the colony color, shape and colony size. Each single colony is streaked on the LB plate at least five times continuously, and then take a photo of the streaked plate of the isolated strain ( Figure 1 ), and then transferred to LB liquid culture medium, the bacterial liquid was stored in 25% glycerol aqueous solution and frozen in a -80℃ refrigerator for later use.

[0031] 3. Identification of strains

[0032] 3.1 Morphological identification

[0033] From the morphological point of view, it is a round white colony with a smooth, shiny and opaque surface. The optimal growth temperature is 30℃, the optimal pH is 6.0-7.0, and it is facultative anaerobic ( Figure 1 ).

[0034] 3.2 Molecular identification

[0035] The bacterial genomic DNA extraction kit of Tiangen Biotechnology was used to extract the genomic DNA of the single colony strain, and the extracted DNA was used as a template, and the universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') were used as the upper and lower primers to amplify the bacterial 16S rRNA sequence. The PCR reaction system is shown in Table 1. The PCR setting program was 95℃ pre-denaturation for 2min, 95℃ denaturation for 20s, 52℃ annealing for 20s, 72℃ extension for 30s, 35 cycles, and 72℃ final extension for 5min. The PCR product was detected by 1% agarose gel, excised, recovered, purified, and sent to Qingke Biotechnology Co., Ltd. for sequencing.

[0036] Table 1 Bacterial 16S rDNA PCR amplification system

[0037]

[0038]

[0039] According to the results of sequencing primers 27F and 1492R, the sequencing quality was analyzed and the sequence was blasted in the NCBI database. The results showed that the A3 strain had the highest similarity with Enterobacter hormaechei strain AUH-ENM30. It was named Enterobacter hormaechei A3 strain of Plutella xylostella. The classification status is Bacteria, Proteobacteria, Gammaproteobacteria, Enterobacterales, Enterobacteriaceae, Enterobacter, Enterobacter hormaechei. The strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on May 10, 2024, with the strain collection number GDMCC.No: 64603, the classification name is Enterobacter hormaecheiA3, and the collection address is No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province.

[0040] 2. In vitro degradation test of strains

[0041] 1. Determination of resistance of intestinal bacteria of Plutella xylostella to chlorantraniliprole / bromofenac

[0042] The A3 strain isolated from Plutella xylostella was inoculated into 30 ml of MS medium (1.50 g K2HPO4, 0.50 g KH2PO4, 0.50 g NaCl, 0.50 g (NH4)2SO4, 0.20 g MgSO4, 0.05 g CaCl2, 0.02 g FeSO4, 1 L H2O, and a few drops of dimethyl sulfoxide were added to aid dissolution) with chlorantraniliprole / bromofenac as the sole carbon source. The concentration of chlorantraniliprole / bromofenac was 50 mg / L. The culture was placed in a shaking incubator at 30°C and 170 rpm, and the OD was measured and recorded every 24 h. 600 The MS medium without bacteria was used as blank control ( Figure 2 ).

[0043] 2. Draw the standard curve of chlorantraniliprole and bromofenac, prepare chlorantraniliprole and bromofenac technical into 1000 mg / L mother solution, dilute them step by step to 50 mg / L, 25 mg / L, 12.5 mg / L concentration, and use high performance liquid chromatography to measure and collect data. Liquid chromatograph: Waters Ark HPLC; Chromatographic column: Kromasil 4.6 mm × 250 mm, 5 μm C 18 .

[0044] Chlorantraniliprole conditions: detection wavelength 268nm; column temperature 30℃; flow rate 1mL / min; mobile phase acetonitrile-water (65-35); injection volume 20μL; peak time 4.8min ( Figure 3 ).

[0045] Bromofenac conditions: detection wavelength 254nm; column temperature 40℃; flow rate 0.50mL / min; mobile phase acetonitrile-water (75-25); injection volume 10μL; peak time 14.6min ( Figure 4 ).

[0046] 3. Degradation of brofenac by intestinal bacteria: The A3 strain was inoculated into LB medium, cultured in a shaking incubator at 30°C and 170 rpm for 48 h, and then the medium was removed by centrifugation. The enriched bacteria were added to MS medium with brofenac as the sole carbon source and cultured in a shaking incubator.

[0047] A3 (Enterobacter hormaechei) was inoculated into liquid LB medium, cultured in a shaker at 30°C and 170 rpm for 24 h, centrifuged at 8000 rpm, washed twice in MS medium, and the enriched bacteria were added to 100 mL of MS medium with chlorfenapyr / brofenapyr as the sole carbon source, the concentration of the agent was 50 mg / L, and the OD 600 =1.0, culture in a shaker at 30°C and 140rpm; take 20mL of MS culture medium each time, centrifuge at 10000rpm for 3min, remove the supernatant, add 20mL of acetonitrile, shake, add 10mL of deionized water and sufficient NaCl to saturation in turn, shake and mix, let stand until a clear interface is formed, take the upper acetonitrile solution, dehydrate the taken acetonitrile through a purification tube filled with anhydrous magnesium sulfate, and then filter the treated liquid through a filter membrane and inject it into a sample bottle, collect data at 0h, 48h, and 144h, and use an equal amount of 50mg / L solution without inoculation of bacteria as a control, and repeat each treatment 3 times. The treated samples were sent to the Institute of Chemical Engineering of Guangdong Academy of Sciences for HPLC analysis. The chromatographic conditions were the same as those in 2( Figure 5 , 6).

[0048] The isolated strain was subjected to a degradation test in vitro, and it was found that the strain could survive in a culture medium containing only chlorantraniliprole / bromfenac as the sole nutrient source, and could degrade the pesticide in vitro. Chlorantraniliprole and bromfenac were measured by high performance liquid chromatography to obtain corresponding degradation standard curves, and the degradation of chlorantraniliprole and bromfenac by Enterobacter hormaechei A3 was detected. The results showed that the degradation rate of Enterobacter hormaechei A3 to chlorantraniliprole was 15.08% for 48h and 34.79% for 144h, and the degradation rate of bromfenac was 13.58% for 48h and 29.27% ​​for 144h. It was shown that the Enterobacter hormaechei A3 strain separated and identified by the present invention has the effect of improving the resistance of diamondback moth, and can degrade chlorantraniliprole / bromfenac in vitro.

[0049] 3. Effect of strains on insect resistance

[0050] 1. Feeding of Plutella xylostella A3 strain

[0051] Enterobacter hormaechei A3 strain was inoculated into LB liquid medium and the OD 600 =1.0, cut fresh artificial feed into small pieces and soak them in bacterial solution for 10 minutes, take out the feed and air dry it naturally, put it in a petri dish, and put it into the sensitive strain of diamondback moth of the same age and good health. The one soaked in sterile water was used as a blank control.

[0052] 2. Indoor biological activity determination of Plutella xylostella

[0053] According to the agricultural industry standard of the People's Republic of China "Technical Regulations for Monitoring Resistance of Plutella xylostella in Cruciferous Vegetables" (NY / T 2360-2013), the biological activity of Plutella xylostella larvae fed with bacteria, fed with antibiotics and blank treatment was determined. The leaf dipping method was used to prepare chlorantraniliprole / bromofenac solution in a gradient, and the sterile water soaking was used as the blank control. The cabbage leaves were soaked in the solution for 10 seconds, the leaves were taken out and dried, and placed in a culture dish. Plutella xylostella larvae of the same age and good health after different treatments were placed. After 48 hours, the results were checked and the LC50 was calculated. The insects were considered dead if they could not move normally and had no reaction when the insects were touched. The results are shown in Table 2. Chlorantraniliprole: LC after feeding with bacteria 50 The LC value was 1.567 mg / L after adding antibiotics to feed. 50 The blank control treatment LC 50 0.848 mg / L; (2) Brofenac: LC after feeding with bacteria 50The LC value is 0.021 mg / L after adding antibiotics to feed. 50 0.011 mg / L, blank control treatment LC 50 The LC of the feed with bacteria was compared with that of the feed with antibiotics and blank control. 50 The results show that the Enterobacter hormaechei A3 strain isolated and identified by the present invention has the ability to improve the resistance of Plutella xylostella to chlorfenapyr / bromofenapyr, and the Enterobacter hormaechei A3 strain isolated by the present invention can be used as a basic material for studying the resistance mechanism. It can also be used in pesticide degradation and has a good application prospect.

[0054] Table 2 Resistance levels of Plutella xylostella to chlorantraniliprole / bromofenac under different feeding methods

[0055]

[0056] The above embodiments are preferred implementation schemes of the present invention. In addition, the present invention may also be implemented in other ways. Any obvious replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. A strain, characterized in that Enterobacter hallii Enterobacter hormaechei A3, deposit unit: Guangdong Microbiological Culture Collection Center; deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; deposit date: May 10, 2024; biological deposit number: GDMCC No: 64603.

2. Use of the strain according to claim 1 for degrading brofenac or chlorfenapyr.

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