An Enterococcus casei phage, its application and a method for controlling pests with a target bacterium

Abstract

The present invention discloses an Enterococcus casseliflavus phage, its application and a method for controlling pests using the target bacteria. Enterococcus casseliflavus is a type of probiotic bacteria that commonly coexists in lepidopteran pests; the preservation name of the phage is: Enterococcus casseliflavus phage SP-1, the preservation unit: China Center for Type Culture Collection, the preservation date: July 22, 2024, the preservation number: CCTCC M 20241620. This phage was identified as a Caudoviricetes phage through genome sequencing and named SP-1. The technology utilizes the targeted elimination ability of this phage against the probiotic bacteria of pests to reduce the load of probiotic bacteria in pests, thereby increasing the control effect of pesticides on pests. This technology is helpful for further research and field application in the fields of agricultural pest control, development of new green biological pesticides, etc.

Description

Technical Field

[0001] The present invention relates to a technology for controlling pests by targeting bacteria, and particularly to an Enterococcus casseliflavus phage, its application, and a method for controlling pests by targeting bacteria. Background Art

[0002] The fall armyworm (Spodoptera frugiperda) has been listed by the Food and Agriculture Organization of the United Nations as a major agricultural pest under global early warning. Its spread speed is astonishing, posing a severe threat to agricultural production. If effective prevention and control measures are not taken in a timely manner, it may cause huge economic losses. Therefore, the prevention and control of the fall armyworm is extremely urgent and has become an important part of ensuring food security. In the face of this challenge, traditionally, highly effective diamide insecticides such as chlorantraniliprole have been relied on for emergency treatment. However, with the widespread and improper use of these chemical agents, the fall armyworm has begun to show increasingly enhanced resistance. This not only weakens the insecticidal effect but may also force the agricultural department to increase the dosage of pesticides, forming a vicious cycle and posing potential risks to the ecological environment and human health.

[0003] Previous studies have found that a symbiotic bacterium of the fall armyworm, Enterococcus casseliflavus EMBL-3, can enhance the host's resistance to pesticides by degrading chlorantraniliprole and is commonly infective to wild fall armyworm populations. Therefore, EMBL-3 has the potential to become a potential target for the management of fall armyworm resistance. The present invention targets and reduces beneficial symbiotic bacteria of the pest host so as to maintain or enhance the biological efficacy of pesticides without significantly increasing the dosage of chemical agents. In this way, not only can the control cost of the fall armyworm be effectively reduced, but more importantly, it greatly slows down the generation rate of pest resistance, providing a strong guarantee for long-term and sustainable pest management. For this purpose, a phage Caudoviricetes SP-1 was isolated from sewage, and this phage demonstrated the ability to precisely eliminate a specific symbiotic bacterium in the intestines of fall armyworm larvae, namely the Enterococcus strain EMBL-3. Through in vivo and in vitro experiments, it was confirmed that phage SP-1 can act on EMBL-3 efficiently and specifically, effectively reducing its quantity in the fall armyworm body. At the same time, it can significantly reduce the insecticide resistance of pest hosts infected with EMBL-3. This achievement not only reveals the great potential of phages in the biological control of agricultural pests but also provides a scientific basis and technical support for reducing the use of chemical insecticides, alleviating the problem of pest resistance, and protecting the ecological environment. Summary of the Invention

[0004] The purpose of the present invention is to provide an Enterococcus casseliflavus phage, its application, and a method for controlling pests by targeting bacteria in view of the deficiencies of the prior art, and this method can effectively increase the control effect of pesticides on pests.

[0005] The technical solution adopted by the present invention is as follows:

[0006] An Enterococcus casseliflavus phage, named Enterococcus casseliflavus phage SP-1, is deposited at the China Center for Type Culture Collection on July 22, 2024, with the deposit number CCTCC M20241620. It can be used in the preparation of pesticides or pesticide synergists.

[0007] A pesticide synergist contains the above-mentioned Enterococcus casseliflavus phage.

[0008] A pesticide contains, in addition to the pesticidal active ingredient, the above-mentioned pesticide synergist.

[0009] Furthermore, the pesticidal active ingredient is a diamide pesticide.

[0010] A method for controlling pests by targeting bacteria uses an isolate of a pest probiotic as the host bacterium to directionally isolate a phage targeting the pest probiotic, and uses the phage in combination with a pesticide to control the pest.

[0011] The beneficial effects of the present invention are as follows:

[0012] The phage of the present invention is identified as a Caudoviricetes phage named SP-1 through genome sequencing. By utilizing the phage's ability to target and eliminate pest probiotics, the load of probiotics in pests is reduced, thereby increasing the control effect of pesticides on pests, and significantly increasing the mortality rate of pests by more than 50%. This technology has prospects for further research and field application in the fields of agricultural pest control and the development of new green biological pesticides. Description of the Drawings

[0013] Figure 1 It is an image of phage SP-1 under a scanning transmission microscope.

[0014] Figure 2 It is a double-layer plate plaque map of phage SP-1.

[0015] Figure 3 It is an electrophoresis map of nucleic acid extraction and identification of phage SP-1.

[0016] Figure 4 It is the optimal multiplicity of infection map of phage SP-1.

[0017] Figure 5 It is the one-step growth curve of phage SP-1.

[0018] Figure 6 It is the inhibition of the growth of EMBL-3 by phage SP-1.

[0019] Figure 7 The inhibitory ability of phages in Spodoptera frugiperda against EMBL-3.

[0020] Figure 8 The effect of phage SP-1 on the drug resistance of Spodoptera frugiperda infected with EMBL-3. Detailed implementation manners

[0021] The technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. For those not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0022] Example 1 Isolation and identification of phages

[0023] Phage isolation: The phages were isolated from untreated domestic sewage, and the sewage source was Hangzhou West City Sewage Treatment Plant. The sewage sample was centrifuged at 10000 rpm for 10 minutes at 4 °C to remove impurities. Then the supernatant was filtered through 0.45 μm and 0.22 μm polyethersulfone (PES) membranes in sequence to further remove bacterial cells, and the sample filtrate was stored at 4 °C. Take 1 mL of the host bacterium EMBL-3 cultured to the logarithmic phase (OD600 ~ 0.5), add 7 mL of the sample filtrate and 4 mL of 3-fold BHI fresh liquid medium, mix well, and culture overnight at 37 °C and 180 r / min. The phage enrichment solution cultured overnight was centrifuged at 12000 rpm for 5 min at 4 °C, and the supernatant was filtered through a 0.22 μm PES membrane to obtain a fresh phage lysate. The enrichment sample filtrate of the phages of EMBL-3 was serially diluted with SM buffer. At each dilution gradient, 100 μL of the filtrate and 400 μL of the host bacterium were added to 4.5 mL of 0.5% BHI semi-solid medium, mixed evenly, poured onto the solid medium, and leveled. After the semi-solid medium solidified, it was cultured overnight in an inverted manner in a 37 °C incubator.

[0024] Phage purification: The phages were isolated and purified from single plaques using the double-layer plate method. According to the plaque morphology, a sterile toothpick or a 10 μL pipette tip was used to pick the plaque and transfer it to 1 mL of SM buffer. Take 0.1 mL of the host bacterium EMBL-3 cultured to the logarithmic phase (OD600 ~ 0.5), add 0.7 mL of the SM buffer containing phages and 0.4 mL of 3-fold BHI fresh liquid medium, mix well, and culture overnight at 37 °C and 180 r / min. Repeat the steps of phage enrichment and isolation, and perform 3 rounds of purification to obtain a single phage with consistent plaque characteristics on the plate.

[0025] Phage preservation: The fresh lysate of phage was mixed with 50% sterilized glycerol at a ratio of 1:1 and stored at -80 °C, while the fresh lysate was stored at 4 °C.

[0026] Titer determination: The double-layer plate method was used to evaluate the titer of the phage. First, LB solid medium was poured into a sterile petri dish as the bottom layer. Then, the appropriately diluted phage was mixed with the host bacteria cultured to the logarithmic phase. After incubation and adsorption, semi-solid BHI medium cooled to about 45 °C was added, and after rapid mixing, it was spread on the plate as the upper layer. After it cooled and solidified, it was cultured in an inverted position. Observe the number of plaques appearing on plates with different dilution factors, and select the plate with 30 - 300 plaques on the petri dish to calculate the titer of the original phage solution. The phage titer calculation formula is: plaque-forming units (PFU / mL) = number of plaques × dilution factor × 10.

[0027] Observation of phage morphology: The morphology of the phage was observed using a transmission electron microscope (TEM). Select a phage lysate with a titer of about 10 8 (PFU / mL), and pipette 10 μL of it onto the carbon film of the copper grid. After standing still for 5 min on the front side, use a filter paper to suck away the excess sample. Then, pipette 10 μL of 2% (W / V) phosphotungstic acid staining solution onto the front side of the copper grid for negative staining for 1 min, immediately use a filter paper to suck away the excess sample, and wait for the copper grid to dry naturally. Use a Talos L120C G2 120 kV transmission electron microscope to observe the phage morphology.

[0028] After the phage was stained with 2% phosphotungstic acid, Figure 1 transmission electron microscope observation showed that its overall morphology was tadpole-shaped, with a head that was a regular polyhedron, about 60 - 80 nm in diameter, a tail length of about 150 nm, and a relatively long tail, belonging to the order Caudoviricetes of tailed phages.

[0029] Plaque morphology: The double-layer plate was used to observe the plaque morphology of the phage. The results were as Figure 2 shown. The plaques were round, transparent empty spots with a halo around the edge, about 2 mm in diameter.

[0030] Phage nucleic acid extraction, identification, and genome determination: Take 20 mL of phage lysate (centrifuged to filter bacteria), add PEG8000 and NaCl for precipitation, let it stand at 4°C for 16 h, centrifuge at 10000 r / min for 20 min to obtain the precipitate, and resuspend the phage precipitate with 400 μL of SM buffer (Tris-HCl / MgSO4 / NaCl / H2O, pH 7.5). Add 8 U of DNaseI, 20 U of RNase A, and 40 μL of 10-fold DNA buffer to the resuspended sample, mix well, and incubate at 37°C for 1 h for enzymatic digestion. The treated sample is used to extract the genome using the MiniBEST Viral RNA / DNA Extraction Kit Ver.5.0 (TaKaRa). The genomic DNA is digested and identified using DNase I and RNase I, and 1% agarose gel electrophoresis is used to detect the composition of the phage's genetic material. As Figure 3 , the results showed that DNase I could completely digest the genetic material of this phage, proving that its genetic material is DNA.

[0031] The concentration of the extracted genome was quantified by fluorescence using a Qubit fluorometer (Thermo Fisher Scientific). The whole-genome library was constructed using the Nextera TM DNA Library Preparation Kits (Illumina). The concentration of the library was quantified using Qubit, and the quality of the library construction was inspected using the Agilent 4150 TapeStation system. The library samples were sequenced using the Illumina MiSeq sequencing platform. The sequencing results were assembled using the software SPAdes.

[0032] Based on the online website PhageScope(deepomics.org) , it was predicted that this phage is a Caudoviricetes phage, named SP-1. The described phage was preserved, and the preservation name is: Enterococcus casseliflavusphage SP-1, the preservation unit: China Center for Type Culture Collection, the preservation date: July 22, 2024, the preservation number: CCTCC M 20241620.

[0033] Example 2 Determination of the optimal multiplicity of infection (MOI)

[0034] The optimal multiplicity of infection was used to determine the infection conditions under which the phage could produce the maximum number of progeny phages when infecting the host bacteria. SP-1 (~10 9 PFU / mL) and the host bacterium EMBL-3 cultured to the logarithmic phase (~10 8CFU / mL) were mixed at ratios of SP-1 / EMBL-3 = 0.001, 0.01, 0.1, 1, 10, 100, cultured overnight in an incubator at 37°C and 180 rpm / min, and the titer of the final phage was determined using a double-layer plate. The one with the highest titer was the optimal multiplicity of infection of the phage. As Figure 4 shown, the experimental results indicated that the optimal multiplicity of infection was 0.1.

[0035] Example 3 One-step growth curve determination:

[0036] Mix 2 mL each of SP-1 with an MOI ratio of 0.1 / 0.01 and fresh EMBL-3 growth medium thoroughly. Incubate at 37°C for 10 min, centrifuge at 11000 g for 1 min, discard the supernatant, wash once with 10 mL of LB broth, and discard the supernatant. Suspend the precipitate with pre-warmed 10 mL of LB broth and mix thoroughly, then quickly place it in a shaker at 37°C and shake at 180 rpm. Take out 150 μL at 0, 2, 5, 10, 20, 30, 40, 50, 60, 90, 120, 180, 240 min, centrifuge at 10000 rpm for 1 min, and dilute 100 μL of the supernatant and determine the phage titer using the double-layer plate method. Using the infection time as the abscissa and the phage titer as the ordinate, plot the one-step growth curve, as Figure 5 shown in a, b. Under the optimal MOI (MOI = 0.1), the latent period of the phage was shorter, and the breakthrough period was 0 - 90 min. At an MOI of 0.01, the latent period of the phage was 2 - 20 min.

[0037] Example 4 Inhibition of EMBL-3 growth by phage:

[0038] Mix 100 μL each of the purified phage SP-1 solution and 10 8 CFU / mL logarithmic-phase EMBL-3 at MOI = 100, 10, 1, 0.1, 0.01, 0.001 in a 96-well plate. At the same time, mix 100 μL of 10 8 CFU / mL logarithmic-phase EMBL-3 with 100 μL of LB liquid medium as a positive control, 200 μL of LB liquid medium as a negative control, and 200 μL of sterile water as a blank control. Each group was set with three replicates. Incubate in an enzyme-labeled instrument at 37°C and 180 rpm / min for 12 h, and detect OD600 every 0.5 h. The results are as Figure 6 shown.

[0039] The results showed that when the MOI was greater than 10, SP-1 could significantly inhibit the proliferation of EMBL-3. When the MOI was between 1 - 5, the logarithmic growth phase of EMBL-3 was postponed, while when the MOI was below 1, the effect on the proliferation of EMBL-3 was relatively small.

[0040] Example 5: In vivo inhibition of EMBL-3 by phage

[0041] A sterile Spodoptera frugiperda strain was constructed by surface disinfection of eggs. When the sterile Spodoptera frugiperda reached the 2nd - 3rd instar stage, it was fed with feed containing EMBL-3 for 2 days to allow EMBL-3 to colonize in the Spodoptera frugiperda body until a steady state was reached. Subsequently, the experimental subjects were divided into two groups. One group of Spodoptera frugiperda was fed with feed containing SP-1 for 2 days, while the remaining group was fed with phage-free feed for 2 days as well. After the feeding experiment ended, these two groups of Spodoptera frugiperda were dissected, and intestinal tissues and total DNA were extracted from them. The qRT-PCR method was used to detect the content of EMBL-3. As Figure 7 shown, the experimental results indicated that compared with the control group fed with phage-free feed, the growth of the symbiotic bacterium EMBL-3 in the experimental group of Spodoptera frugiperda fed with SP-1 was significantly inhibited.

[0042] Example 6: Effect of phage on the insecticide resistance of Spodoptera frugiperda

[0043] The same as in Example 5, a sterile Spodoptera frugiperda strain was constructed using the surface disinfection method. When it reached the 2nd - 3rd instar stage, it was fed with feed containing EMBL-3 for 2 days. Subsequently, it was divided into two groups. One group continued to be fed with phage-free feed for 2 days, and the other group was fed with phage-containing feed for 2 days. After feeding, both groups of Spodoptera frugiperda were fed with feed containing the same concentration of chlorantraniliprole, and a sterile normal feeding group and a non-sterile normal feeding group were set up for bioassays. The Control group was the normal Spodoptera frugiperda group, the Sterilized group was the constructed sterile Spodoptera frugiperda strain, the EMBL-3 group was the sterile Spodoptera frugiperda strain fed with feed supplemented with EMBL-3, and the SP-1 group was the sterile Spodoptera frugiperda strain fed with feed containing SP-1 after being fed with feed containing EMBL-3. The results were as Figure 8 shown. The experimental results showed that it was found that SP-1 could significantly reduce the host insecticide resistance increased by EMBL-3.

[0044] In the present invention, the probiotic Enterococcus caseliflavus isolate of the pest Spodoptera frugiperda was used as the host bacterium, and phages targeting this bacterium were isolated directionally. Using this phage in combination with insecticides to control pests can effectively reduce the insecticide resistance of Spodoptera frugiperda benefited from Enterococcus caseliflavus and significantly increase the mortality rate of pests caused by insecticides. Obviously, this phage can be prepared as an insecticide synergist for use in pest insecticides or target-bacterium pest control biological pesticides. The inventive concept can also be extended to the control of other pests.

[0045] The above-described embodiments are only some of the better solutions of the present invention, but they are not intended to limit the present invention. Those of ordinary skill in the relevant technical field can still make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, all technical solutions obtained by means of equivalent replacement or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A Enterococcus casei phage, named: ( Enterococcus casseliflavus phage ) SP-1, deposited with the China Center for Type Culture Collection on July 22, 2024, deposit number: CCTCC M20241620.

2. Use of the Enterococcus casseliflavus phage according to claim 1 in the preparation of a synergist for an insecticide, wherein the insecticidal active ingredient of the insecticide is a diamide insecticide.

3. An insecticide synergist, characterized in that, Containing the Enterococcus casseliflavus phage according to claim 1, wherein the insecticidal active ingredient of the insecticide is a diamide insecticide.

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