Enterobacter cancerogenic EcCB2 strain and application thereof

Abstract

The invention discloses an enterobacter cancerogenic EcCB2 strain and an application thereof. According to the present invention, the enterobacter cancerogenus EcCB2 strain is separated from lasioderma serricorne, and is preserved in Guangdong Province Microbial Culture Collection Center on May 31, 2024, and the preservation number is GDMCC No: 64709; the EcCB2 strain not only can effectively kill vegetable pests such as beet armyworms, spodoptera litura, prodenia litura, plutella xylostella and cabbage caterpillars which are mainly harmful to brassicaceous vegetables, but also can remarkably inhibit the growth of plant pathogenic fungi such as fusarium oxysporum, sclerotinia sclerotiorum, colletotrichum hijinensis and alternaria solani; the compound can be used for preventing and treating plant diseases caused by plant pathogenic fungi and cruciferous vegetable insect pests.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and more specifically relates to a cancer-causing Enterobacterium EcCB2 strain and its application. Background Art

[0002] The gut microbiome, comprised of bacteria, fungi, viruses, and archaea, is widely involved in numerous key physiological processes in host insects, including growth and development, immunity and homeostasis, pesticide resistance, stress tolerance, and social behavior. For example, when the gut microbiome of Drosophila melanogaster is depleted, its pupation time is prolonged and its wings become smaller. Other studies have shown that the gut microbiome influences the growth and development of host insects by regulating the insulin signaling pathway. However, the gut microbiome is not entirely beneficial to the host insect. When the host insect's gut homeostasis is disrupted, these symbiotic microorganisms can become pathogenic, known as opportunistic pathogens. For example, in studies of the Drosophila melanogaster gut microbiome, altered gut microbial composition has been associated with intestinal pathology and shortened lifespan. With the deepening of research on the relationship between insects and their gut symbionts, the use of insect gut symbionts for pest control is considered a promising approach. One of the most successful examples is the use of Wolbachia bacteria for mosquito control. Wolbachia has been shown to induce cytoplasmic incompatibility, which prevents mosquito eggs from fertilizing, thereby effectively controlling mosquito populations.

[0003] Enterobacter cancerogenus has been reported to have insecticidal activity against pests such as Spodoptera litura and Hyphantria cuneiformis, and can be used to control these pests. However, there are currently no reports of Enterobacter cancerogenus' insecticidal activity against cruciferous vegetable pests such as Plutella xylostella, nor are there any reports of its antagonistic effects against plant pathogenic fungi such as Sclerotinia sclerotiorum. Summary of the Invention

[0004] In order to enrich the microorganisms that can be used to control vegetable pests such as cruciferous vegetables, the present invention provides a strain of Enterobacter cancerogenus (EcCB2), which can not only kill vegetable pests such as beet armyworm, fall armyworm, Spodoptera litura, diamondback moth and cabbage looper, but also inhibit the growth of plant pathogenic fungi such as Fusarium oxysporum, Sclerotinia sclerotiorum, Higgins Anthrax and Alternaria solanacearum.

[0005] The first object of the present invention is to provide an Enterobacter cancerogenus EcCB2 strain.

[0006] The second object of the present invention is to provide a microbial preparation containing the EcCB2 strain.

[0007] The third object of the present invention is to provide the use of the strain or the microbial preparation in inhibiting plant pathogenic fungi.

[0008] The fourth object of the present invention is to provide use of the strain or the microbial preparation in preparing a product for inhibiting plant pathogenic fungi.

[0009] The fifth object of the present invention is to provide the use of the strain or the microbial preparation in preventing and controlling plant diseases caused by plant pathogenic fungi.

[0010] The sixth object of the present invention is to provide use of the strain or the microbial preparation in preparing products for preventing and controlling plant diseases caused by plant pathogenic fungi.

[0011] The seventh object of the present invention is to provide the use of the strain or the microbial preparation in preventing and controlling vegetable pests.

[0012] The eighth object of the present invention is to provide the use of the strain or the microbial preparation in preparing products for controlling vegetable pests.

[0013] A ninth object of the present invention is to provide a method for preventing and controlling vegetable pests.

[0014] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0015] The tobacco beetle (Lasioderma serricorne) is a common stored-food pest. The present invention has isolated a strain of Enterobacter cancerogenus (Enterobacter cancerogenus CB2) from the intestinal symbionts of tobacco beetles. This strain, designated CB2 (EcCB2), is not only effective in killing vegetable pests such as the beet armyworm, fall armyworm, diamondback moth, cabbage looper, and Spodoptera litura, but also significantly inhibits the growth of plant pathogenic fungi such as Fusarium oxysporum, Sclerotinia sclerotiorum, Colletotrichum higgins, and Alternaria solani. Therefore, the present invention seeks protection for the EcCB2 strain.

[0016] Specifically, the cancer-causing Enterobacter EcCB2 strain described in the present invention was deposited in the Guangdong Provincial Microbiological Culture Collection Center on May 31, 2024, with the deposit number GDMCC No: 64709.

[0017] The present invention also provides a microbial preparation containing the cancer-causing Enterobacterium EcCB2 strain.

[0018] The present invention also claims to protect the use of the cancer-causing Enterobacter EcCB2 strain or the microbial preparation in inhibiting plant pathogenic fungi.

[0019] The present invention also claims to protect the use of the cancer-causing Enterobacter EcCB2 strain or the microbial preparation in preparing a product for inhibiting plant pathogenic fungi.

[0020] Specifically, the plant pathogenic fungi are Fusarium oxysporum, Sclerotinia sclerotiorum, Colletotrichum higginsianum and / or Alternaria solani.

[0021] More specifically, the Fusarium oxysporum is Fusarium oxysporum f.sp.cubense, which is the banana wilt pathogen.

[0022] The present invention also claims protection for the use of the cancer-causing Enterobacter EcCB2 strain or the microbial preparation in preventing and controlling plant diseases caused by plant pathogenic fungi.

[0023] The present invention also claims protection for the use of the cancer-causing Enterobacter EcCB2 strain or the microbial preparation in preparing products for preventing and controlling plant diseases caused by plant pathogenic fungi.

[0024] Specifically, the plant pathogenic fungi are Fusarium wilt, Sclerotinia sclerotiorum, Colletotrichum higgins and / or Alternaria solani.

[0025] The present invention also claims protection for the use of the cancer-causing Enterobacter EcCB2 strain or the microbial preparation in preventing and controlling vegetable pests.

[0026] The present invention also claims protection for the use of the cancer-causing Enterobacter EcCB2 strain or the microbial preparation in preparing products for preventing and controlling vegetable pests.

[0027] Specifically, the pests are pests of the Noctuidae, Plutellae and / or Pieridae families.

[0028] More specifically, the Noctuidae pests are Spodoptera litura, Spodoptera exigua and / or Spodoptera frugiperda.

[0029] The Plutella xylostella pest is the diamondback moth (Plutella xylostella).

[0030] The Pieridae pest is Pieris rapae.

[0031] The present invention also provides a method for preventing and controlling vegetable pests, which comprises mixing the cancer-causing Enterobacterium EcCB2 strain or the microbial preparation with bait to induce the pests to feed.

[0032] Specifically, the pests are pests of the Noctuidae, Plutellae and / or Pieridae families.

[0033] More specifically, the Noctuidae pests are Spodoptera litura, Spodoptera exigua and / or Spodoptera frugiperda; the Plutella xylostella pests are Plutella xylostella; and the Pieridae pests are Pieris rapae.

[0034] The present invention has the following beneficial effects:

[0035] The present invention provides a strain of Enterobacterium carcinogenes EcCB2 isolated from tobacco beetles. The strain was deposited in the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on May 31, 2024, with a deposit number of GDMCC No: 64709. The EcCB2 strain can not only effectively kill vegetable pests that mainly harm cruciferous vegetables, such as beet armyworm, fall armyworm, cutworm, diamondback moth and cabbage looper, but also significantly inhibit the growth of plant pathogenic fungi such as Fusarium oxysporum, Sclerotinia sclerotiorum, Higgins Anthracnose and Alternaria solanacearum. It can be used to prevent and control plant diseases caused by plant pathogenic fungi and cruciferous vegetable pests. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The colony morphology of EcCB2 strain on LB and SS plates.

[0037] Figure 2 The results of scanning electron microscopy observation of EcCB2 strain bacteria; Figure A is the EcCB2 strain bacteria under an electron microscope at approximately 20,000 times magnification; Figure B is the EcCB2 strain bacteria under an electron microscope at approximately 100,000 times magnification.

[0038] Figure 3 This is the test result of the starch degradation ability of the EcCB2 strain (the colony growth position that does not change color after iodine staining is a starch-degrading bacteria, and turns blue-black is a non-starch-degrading bacteria).

[0039] Figure 4 This is a phylogenetic tree constructed based on the 16S rDNA gene sequence of the EcCB2 strain (each branch is marked with: GenBank sequence number + strain name).

[0040] Figure 5 The toxicity bioassay and Log-rank Mantel-Cox test results of EcCB2 strain against Plutella xylostella, Spodoptera frugiperda, Spodoptera exigua, Pieris rapae, and Spodoptera litura were presented.

[0041] Figure 6 These are the results of the toxicity test of the EcCB2 strain against Plutella xylostella.

[0042] Figure 7 Figure 2 shows the inhibition of EcCB2 strain on the growth of Higgins Anthracnose, Fusarium wilt, Sclerotinia sclerotiorum and Alternaria solani; A in the figure is the Higgins Anthracnose control plate; B in the figure is the confrontation plate of EcCB2 strain and Higgins Anthracnose; C in the figure is the Fusarium wilt control plate; D in the figure is the confrontation plate of EcCB2 strain and Fusarium wilt; E in the figure is the Sclerotinia sclerotiorum control plate; F in the figure is the confrontation plate of EcCB2 strain and Sclerotinia sclerotiorum; G in the figure is the Alternaria solani control plate; H in the figure is the confrontation plate of EcCB2 strain and Alternaria solani. DETAILED DESCRIPTION

[0043] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0044] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0045] The preparation method of the culture medium used in the embodiment of the present invention is as follows:

[0046] LB medium: 10 g peptone, 5 g yeast extract, 10 g NaCl, 15 g agar, dissolved in 1 L sterile ddH2O water, adjusted to pH 7.0, and sterilized at 121°C for 20 min.

[0047] SS medium: 5g beef extract powder, 5g Dissolve peptone, 10g lactose, 8.5g choline 3, 8.5g sodium citrate, 8.5g sodium thiosulfate, 1g ferric citrate, 0.025g neutral red, 0.00033g brilliant green and 17g agar in 0.8L distilled water, adjust the pH to 7.0, make up to 1L with distilled water, and sterilize by autoclaving at 121℃ for 20min

[0048] Enterobacter culture medium: 17 g tryptone, 3 g beef extract powder, 5 g yeast powder, 10 g ox bile powder, 5 g NaCl, 1 g esculin, 0.5 g ammonium ferric citrate, 0.25 g sodium azide, 1 g sodium citrate and 13.5 g agar, dissolved in 0.8 L distilled water, adjusted to pH 7.1, and diluted to 1 L with distilled water. Autoclave at 121°C for 20 min.

[0049] The formula of the artificial feed used in the embodiment of the present invention is: 40g yeast powder, 75g wheat germ powder, 2g multivitamins, 2g sorbic acid, 2g paraben, 2g ascorbic acid, 20g sucrose, 6g radish seeds, 12g agar, 2mL rapeseed oil, 3-4 drops of linoleic acid and 500mL water.

[0050] The taxonomic name of the EcCB2 strain described in the embodiment of the present invention is Enterobacter cancerogenus, which was deposited in the Guangdong Provincial Microbial Culture Collection Center on May 31, 2024, with a deposit number of GDMCC No: 64709, and the deposit address is 5th Floor, Laboratory Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province; the name of the biological material EcCB2 strain in the deposit certificate is Enterobacter cancerogenus CB2.

[0051] Example 1 Isolation and identification of EcCB2 strain

[0052] The EcCB2 strain of the present invention is isolated from the intestinal tract of the tobacco beetle (Lasioderma serricorne) and is isolated and purified from the intestinal symbiotic bacteria of the tobacco beetle. The acquisition process is as follows:

[0053] 1. Isolation and purification of intestinal symbiotic bacteria of tobacco beetles

[0054] The third-instar tobacco beetle larvae in good growth condition were selected and starved for 24 h. The food debris adhering to the beetles after starvation was first cleaned with a brush, and then soaked in 75% alcohol for 30 s, and then soaked in distilled water for 15 s, repeated twice, and the surface moisture of the beetles was dried with absorbent paper for later use. The intestines of the tobacco beetles were dissected out in a clean bench and quickly placed in a 1.5 mL centrifuge tube containing 150 μL PBS on ice, with 15 complete tobacco beetle intestines in each tube. They were ground into a homogenate with a grinding rod on ice to obtain the grinding solution. The grinding solution was diluted 10 4 ~10 8 The strain was then spread on different plates, and single colonies with growth densities suitable for the plates were selected and streaked on fresh LB plates for purification five times. The streaked plates of the isolated strains were photographed and numbered. The isolated strain numbered CB2 was the EcCB2 strain described in the present invention. The EcCB2 strain was transferred to LB liquid culture medium, shaken until the exponential growth phase, preserved with 15% glycerol aqueous solution, and frozen in a -80°C refrigerator for later use.

[0055] 2. Observation of colony and bacterial morphology of EcCB2 strain

[0056] (1) Colony morphology

[0057] The preserved EcCB2 strain was streaked onto LB and SS culture media, respectively, and the colony morphology characteristics on the culture media were observed.

[0058] The colony morphology of EcCB2 strain on LB and SS plates is as follows Figure 1 As shown. Figure 1 It can be seen that the colonies of the EcCB2 strain are round with neat edges, and the colonies on the LB medium are light yellow.

[0059] (2) Bacterial morphology

[0060] ① Collection of bacteria

[0061] The preserved EcCB2 strain was inoculated into LB liquid medium at a volume ratio of 1:100, and activated at 30°C and 220 rpm for 12 hours to obtain a seed bacterial solution; the seed bacterial solution was inoculated into LB liquid medium at a volume ratio of 1:100, and cultured at 30°C and 220 rpm for 4 hours to obtain a bacterial solution with strong activity; 45 mL of the bacterial solution of the EcCB2 strain (OD 600 The cell pellet was resuspended in 1 mL of LB liquid medium and centrifuged at 3000 g for 10 minutes. The supernatant was discarded and the cell pellet was resuspended in PBS with a pH of 7.2 (this step was repeated three times).

[0062] ②Sample preparation

[0063] After the last wash is completed, the PBS is completely aspirated, and then a small amount of PBS is used to rinse the bacteria onto a coverslip placed on a 12-well plate and let it stand for 3 minutes; the bacteria not on the coverslip are washed with PBS, and then glutaraldehyde is added to cover the coverslip for fixation and sealing, and it is kept at 4°C overnight; the fixed coverslip is washed three times with PBS to remove glutaraldehyde, and 0.5% osmium acid is added for infiltration for 30 minutes; it is washed three times with PBS to completely remove osmium acid, and dehydrated using a gradient alcohol method, sequentially soaking in 30% ethanol for 10 minutes and aspirating; soaking in 50% ethanol for 10 minutes and aspirating; soaking in 70% ethanol for 10 minutes and aspirating; soaking in 80% ethanol for 10 minutes and aspirating; soaking in 90% ethanol for 10 minutes and aspirating; soaking in 100% ethanol for 10 minutes and aspirating (this step is repeated twice); after dehydration is completed, the coverslip is placed in a zero-point dryer for drying; after drying is completed, the coverslip is attached to a foam board with the front side facing up, and then photographed in a field emission scanning electron microscope.

[0064] ③Experimental results

[0065] The results of scanning electron microscopy observation of EcCB2 strain were as follows: Figure 2 As shown. Figure 2 It was found that the bacterial cells of the EcCB2 strain were rod-shaped.

[0066] 3. Physiological and biochemical assays of EcCB2 strain

[0067] The preserved EcCB2 strain was streaked and inoculated, and a single colony was picked and inoculated on an SS plate to test sugar metabolism and acid production. A single EcCB2 colony was picked and inoculated on a starch culture medium. After culturing for 12 hours, the colonies growing on the culture medium were gently scraped off with a spoon, and the colony growth position was stained with 1% iodine / potassium iodide solution. The color change was observed to determine whether the strain could degrade starch.

[0068] The test results showed that the colony morphology of EcCB2 strain on SS plate was normal white, indicating that it would not produce acid or hydrogen sulfide. After EcCB2 strain grew on starch medium, the staining of its colony growth position was as follows: Figure 3 As shown. Figure 3 It can be seen that the colony growth position of the EcCB2 strain turned blue-black after iodine staining, indicating that the EcCB2 strain cannot directly degrade starch (those that do not change color after iodine staining are starch-degradable bacteria, and those that turn blue-black are non-starch-degradable bacteria).

[0069] 4. Molecular identification of EcCB2 strains

[0070] (1) Amplification of strain 16S rDNA

[0071] The TIANamp Bacteria DNA Kit was used to extract the genomic DNA of the EcCB2 strain. The extracted DNA was used as a template to amplify the 16S rDNA of the EcCB2 strain using the 16S rDNA universal primers 27F (5'-AGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The PCR amplification reaction system is shown in Table 1. After the PCR amplification reaction system was prepared according to Table 1, it was gently mixed and briefly centrifuged. The PCR amplification reaction was performed on a PCR instrument. The reaction conditions were as follows: pre-denaturation at 98°C for 2 min; denaturation at 98°C for 10 s, annealing at 55°C for 15 s, and extension at 72°C for 15 s, for 35 cycles; and 72°C for 5 min. The PCR products were detected on a 1.5% agarose gel, excised, recovered, purified, and sent to Qingke Biotechnology Co., Ltd. (Guangzhou) for sequencing. After sequencing, the 16S rDNA gene sequence of the EcCB2 strain of the present invention is shown as SEQ ID NO.1.

[0072] Table 1 Bacterial 16S rDNA PCR amplification system

[0073]

[0074] (2) Phylogenetic analysis

[0075] After the sequencing results were returned, SeqMan software was used to analyze the sequencing quality and splice the forward and reverse sequences. After splicing, the obtained 16S rDNA gene sequences were Blast aligned at NCBI, and strain sequences with close genetic relationships were selected. Subsequently, MEGA 7 software was used to construct a phylogenetic tree using the Neighbour-Joining method, and the bootstrap value was adjusted to test the reliability of the evolutionary tree.

[0076] The phylogenetic tree constructed based on the 16S rDNA gene sequence of the EcCB2 strain is shown in Figure 2. Figure 4 As shown. Figure 4 It can be seen that the EcCB2 strain has the highest similarity with Enterobacter cancerogenus.

[0077] Combined with the above-mentioned colony and bacterial morphology, physiological and biochemical test results and Figure 4 The results show that the EcCB2 strain of the present invention is Enterobacter cancerogenus.

[0078] The present invention was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on May 31, 2024, with the deposit number GDMCC No: 64709, and the deposit address is 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province.

[0079] Example 2 Toxicity test of EcCB2 strain against vegetable pests

[0080] The present invention uses beet armyworm (Spodoptera exigua), fall armyworm (Spodoptera frugiperda), diamondback moth (Plutella xylostella), cabbage worm (Pieris rapae) and fall armyworm (Spodoptera litura) as test insect species, and tests the toxicity of the EcCB2 strain to the pests respectively.

[0081] 1. Preparation of EcCB2 strain suspension

[0082] The EcCB2 strain glycerol stock stored at -80°C was inoculated into fresh LB liquid medium at a volume ratio of 1:100 and activated at 220 rpm and 30°C for 12 hours to obtain a seed bacterial solution. The obtained seed bacterial solution was inoculated into fresh LB liquid medium at a volume ratio of 1:100 and cultured at 220 rpm and 30°C for 4 hours to obtain a bacterial solution with strong activity (OD600 = 1.0). The bacterial solution was centrifuged at 10000g for 5 minutes, and the cells were resuspended and washed with PBS (pH 7.2) and diluted to 1×10 8 cfu / mL bacterial suspension for later use.

[0083] 2. Pest breeding

[0084] Larvae of the aforementioned pests were reared on an artificial diet at a temperature of 25±1°C, a photoperiod of 16 hours light:8 hours dark, and a relative humidity of 75%. Adults were fed an artificial diet and supplemented with 10% honey water. Eggs were collected using egg cards soaked in radish juice.

[0085] 3. Virulence test

[0086] The 2nd instar beet armyworm larvae, fall armyworm larvae, diamondback moth larvae, cabbage looper larvae, and Spodoptera litura larvae with relatively consistent growth were selected; each type of insect was divided into 6 groups, with three replicates in each group and 24 insects in each replicate, and starved for 2 hours; the prepared EcCB2 bacterial suspension was mixed with artificial diet at a ratio of 1:4 (1 mL bacterial suspension was mixed with 4 g feed, and 80% water was used when preparing the feed), and the final mixture contained 1×10 7 The experimental group was fed a feed containing EcCB2 strains, while the control group was fed a control feed containing PBS. After adding the feed, the animals were placed in an incubator at 26°C. After two days of treatment with the contaminated feed, they were fed a normal diet. The feed was changed every 12 hours, and the mortality rate was recorded and analyzed using SPSS statistical software.

[0087] 4. Experimental results

[0088] The results of toxicity test and Log-rank Mantel-Cox test of EcCB2 strain against Noctuidae pests are shown in the figure. Figure 5 As shown. Figure 5 The EcCB2 strain had a mortality rate of over 94% against Plutella xylostella and 85.2% against Spodoptera exigua. It also had a similar mortality rate of 75.63% against Spodoptera litura, Pieris rapae, and Spodoptera frugiperda. The EcCB2 strain was most effective against Plutella xylostella, with statistically significant differences compared to other Noctuidae pests (p < 0.05).

[0089] Example 3 Toxicity test of EcCB2 strain against Plutella xylostella

[0090] 1. Preparation of EcCB2 strain suspension

[0091] Refer to Example 2.

[0092] 2. Virulence test

[0093] The 2nd instar diamondback moth larvae with the same developmental period were selected and divided into 6 groups, with three replicates in each group and 24 diamondback moths in each replicate. The experimental groups were starved for 2 h. 5 cfu / g~1×10 8 The diamondback moth was fed a diet containing EcCB2 at a concentration of 100 cfu / g, while the control group was fed a control diet containing PBS. After the addition of the diet, the moths were placed in an incubator at 26°C. Two days after the treatment with the contaminated diet, the normal diet was resumed, and the diet was changed every 24 hours. The mortality rate was counted every 12 hours, the data were recorded, and the data were analyzed using SPSS statistical software.

[0094] 3. Experimental results

[0095] The results of the toxicity test of EcCB2 strain against diamondback moth are as follows: Figure 6 As shown. Figure 6 It can be seen that the survival rate of diamondback moth larvae showed a gradient effect. With the increase of EcCB2 strain treatment concentration, the mortality rate of diamondback moth larvae also increased. The mortality rate of larvae was closely related to the treatment concentration. 8 After treatment with the same concentration of cfu / g for 36h, 48h, 60h, 72h, 84h, 96h and 108h, the mortality rates of the diamondback moth larvae were 40.667%, 46.889%, 53.333%, 58.889%, 79.7778, 91.444% and 93.32%, respectively, indicating that the EcCB2 strain has the ability to effectively kill the diamondback moth and can be used as a new type of biological pesticide for the prevention and control of diamondback moth pests.

[0096] Example 4 Inhibitory Effect of EcCB2 Strain on Plant Pathogenic Fungi

[0097] The present invention uses Sclerotinia sclerotiorum, Colletotrichum higginsianum, Alternaria solani and Fusarium oxysporum f.sp.cubense race 4 as test strains to test whether the EcCB2 strain has an inhibitory effect on the plant pathogenic fungi.

[0098] 1. Preparation of EcCB2 bacterial suspension

[0099] The preparation method of bacterial suspension described in Example 2 was followed. The obtained seed bacterial suspension was inoculated into fresh LB liquid medium at a volume ratio of 1:100. The suspension was cultured at 220 rpm and 30°C for 4 hours to obtain a bacterial suspension with strong activity. The OD value was measured and the suspension was prepared into 1×10 7 CFU / mL of bacterial solution (Shi Junfeng et al., 2018).

[0100] 2. Inhibition rate test

[0101] The plate confrontation method was used to determine the inhibition rate of the EcCB2 strain against the plant pathogenic fungi (Jin et al., 2011).

[0102] The experiment used PDA medium as a solid culture medium. The specific procedure was to inoculate an activated test strain cake in the center of the PDA medium. Then, symmetrically at a distance of 2.5 cm from the cake, the EcCB2 strain solution was inoculated via streaking. A plate inoculated with only the test strain cake served as a control. The plates were incubated inverted in a constant temperature incubator maintained at 28±1°C and 75±5% humidity. Three biological replicates were used for the experiment. Following co-incubation for a specified period of time, based on the growth cycle of the test strain cake, the colony diameters of the treated and control groups were measured, and the inhibition rate was calculated to evaluate the antibacterial effect of EcCB2.

[0103] Inhibition rate (%) = [(pathogen colony diameter of the control group - pathogen colony diameter of the treatment group) / pathogen colony diameter of the control group] × 100%.

[0104] The present invention provides the inhibition of the growth of Higgins anthracnose, banana wilt pathogen, sclerotinia sclerotiorum and Alternaria solani by strain EcCB2, as shown in FIG. Figure 7 As shown; Figure 7 A and B in the figure are the Higgins anthrax control plate and the confrontation plate of EcCB2 strain and Higgins anthrax respectively; Figure 7 C and D in the figure are the banana wilt pathogen control plate and the confrontation plate of EcCB2 strain and banana wilt pathogen; Figure 7 E and F in the figure are the S. sclerotiorum control plate and the confrontation plate of EcCB2 strain and S. sclerotiorum; Figure 7G and H represent the control plate for Alternaria solani and the confrontation plate between the EcCB2 strain and Alternaria solani. The inhibition rates of the EcCB2 strain against S. sclerotiorum, C. higginsianum, A. solani, and Fusarium oxysporum f.sp. cubensis are shown in Table 2. As shown in Table 2, the EcCB2 strain of the present invention has inhibitory effects on all four plant pathogenic fungi; the inhibitory effect on Fusarium oxysporum f.sp. cubensis is the best, with an inhibition rate exceeding 90.88%, while the inhibitory effect on Fusarium oxysporum f.sp. cubensis is the weakest, with an inhibition rate of only 47.39%. These results demonstrate that the EcCB2 strain can affect the normal growth of plant pathogenic fungi and effectively inhibit their reproduction, thereby preventing and controlling corresponding plant diseases by inhibiting the growth of plant pathogenic fungi.

[0105] Table 2 Inhibition rate of EcCB2 strain on plant pathogenic fungi

[0106]

[0107] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. An Enterobacter cancerogenus EcCB2 strain, characterized in that: The strain was deposited in Guangdong Provincial Microbiological Culture Collection Center on May 31, 2024, with the deposit number GDMCC No: 64709.

2. A microbial preparation, characterized in that Contains the cancer-causing Enterobacterium EcCB2 strain described in claim 1.

3. Use of the strain according to claim 1 or the microbial preparation according to claim 2 in inhibiting plant pathogenic fungi, characterized in that: The plant pathogenic fungi are Fusarium sclerotiorum, Sclerotinia sclerotiorum, Colletotrichum higgins and / or Alternaria solani.

4. Use of the strain according to claim 1 or the microbial preparation according to claim 2 in the preparation of a product for inhibiting plant pathogenic fungi, characterized in that: The plant pathogenic fungi are Fusarium sclerotiorum, Sclerotinia sclerotiorum, Colletotrichum higgins and / or Alternaria solani.

5. Use of the strain according to claim 1 or the microbial preparation according to claim 2 in preventing and treating plant diseases caused by plant pathogenic fungi, characterized in that: The plant pathogenic fungi are Fusarium sclerotiorum, Sclerotinia sclerotiorum, Colletotrichum higgins and / or Alternaria solani.

6. Use of the strain according to claim 1 or the microbial preparation according to claim 2 in the preparation of a product for preventing and controlling plant diseases caused by plant pathogenic fungi, characterized in that: The plant pathogenic fungi are Fusarium sclerotiorum, Sclerotinia sclerotiorum, Colletotrichum higgins and / or Alternaria solani.

7. Use of the strain according to claim 1 or the microbial preparation according to claim 2 in preventing and controlling vegetable pests, characterized in that: The pests are pests of the Noctuidae, Plutellae and / or Pieridae families.

8. Use of the strain according to claim 1 or the microbial preparation according to claim 2 in preparing a product for controlling vegetable pests, characterized in that: The pests are pests of the Noctuidae, Plutellae and / or Pieridae families.

9. A method for preventing and controlling vegetable pests, characterized in that: The strain according to claim 1 or the microbial preparation according to claim 2 is mixed with bait to induce pests to feed; the pests are Noctuidae, Plutellae and / or Pieridae pests.

10. The use according to claim 7 or 8 or the method according to claim 9, characterized in that The Noctuidae pests are Spodoptera litura, Spodoptera exigua and / or Spodoptera frugiperda; the Diamondback moth pests are Plutella xylostella; and the Pieridae pests are Pieris rapae.

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