Fermentation supernatant preparation of insect pathogenic nematode symbiotic bacteria, preparation method and application

Abstract

The present application belongs to the field of biotechnology, and particularly relates to a kind of insect pathogenic nematode symbiotic bacteria fermentation supernatant preparation, including Enterobacter nematophilus LY2W fermentation supernatant and adjuvant.The preparation method includes S1, picking single colony on NBTA differential culture medium;S2, picking single colony on NA culture medium;S3, LB liquid culture medium culture;S4, shake flask fermentation culture;S5, centrifugation, obtain supernatant;S6, the supernatant is mixed with adjuvant evenly, obtain insect pathogenic nematode symbiotic bacteria fermentation supernatant preparation.The present application also provides a kind of insect pathogenic nematode symbiotic bacteria fermentation supernatant preparation in the application of preventing and treating leek gray mold and tomato gray mold.The insect pathogenic nematode symbiotic bacteria fermentation supernatant preparation of the present application has significant prevention and treatment effect on leek gray mold and tomato gray mold.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a fermentation supernatant preparation of symbiotic bacteria of entomopathogenic nematodes, its preparation method, and its application. Background Technology

[0002] Gray mold in chives is caused by *Botrytis squamosal*, *Botrytis allii*, and *Botrytis chinensis*, and it can cause damage during the growth, softening, storage, and transportation stages of chives. Gray mold in tomatoes is caused by *Botrytis cinerea*, and it can infect plants from growth to flowering and fruiting. Both generally lead to a 20%–30% reduction in yield for chives and tomatoes, and in severe cases, up to 50%, or even total crop failure. With increasing awareness and demand for food safety, the search for new, safe, effective, and sustainable methods for pest control—biological control—has become a hot research topic and a key area of ​​focus.

[0003] Symbiotic bacteria of entomopathogenic nematodes have attracted significant attention both domestically and internationally due to their rapid insecticidal action, safety for humans and animals, wide host range, and strong resistance. With the deepening research into entomopathogenic nematode symbiotic bacteria, various biological functions of these bacteria have been discovered. Their secondary metabolites possess multiple biological activities, including insecticidal, antibacterial, antitumor, and nematicidal effects, making them a novel biological resource with development potential and application prospects, and showing good commercial potential in agriculture. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria for the prevention and control of gray mold in leeks and tomatoes.

[0005] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0006] A fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria, characterized in that it comprises fermentation supernatant of Enterobacter nematode LY2W and adjuvants.

[0007] In the entomopathogenic nematode symbiotic bacteria fermentation supernatant preparation provided by the present invention, preferably, by weight percentage, it includes 80-93% of Enterobacter nematode LY2W fermentation supernatant and 7-20% of adjuvants.

[0008] In the entomopathogenic nematode symbiotic bacteria fermentation supernatant preparation provided by the present invention, more preferably, by weight percentage, it includes 90.8% of Enterobacter nematode LY2W fermentation supernatant and 9.2% of adjuvants.

[0009] In the fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria provided by the present invention, the adjuvants include emulsifier OP-10, potassium sorbate and ethylene glycol, in a weight ratio of 20:1:25.

[0010] In this invention, the additives include surfactants, preservatives, and antifreeze agents. The surfactant may be NP-10, emulsifier OP-10, or emulsifier OP-15; the preservative may be potassium sorbate, benzoic acid, or sodium benzoate; and the antifreeze agent may be glycerol or ethylene glycol.

[0011] On the other hand, the present invention also provides a method for preparing a fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria to improve its antibacterial activity, which includes the following steps:

[0012] S1. Take the suspension of Enterobacter lysine LY2W preserved in a low-temperature glycerol tube, streak it on NBTA differential medium, and incubate it at 28℃ for 24-36 h. Pick the primary single colony from the differential medium.

[0013] S2. Streak the single colonies picked in step S1 onto NA medium plates and incubate at 28°C for 24–36 h, then pick single colonies.

[0014] S3. Inoculate the single colony picked in step S2 into LB liquid medium and culture it at 28℃ and 180r / min for 12-14h to obtain seed culture.

[0015] S4. Inoculate the seed culture cultured in step S3 into the shake flask fermentation medium at an inoculation rate of 6% by volume, and culture at 28°C with shaking for 24-28 hours to obtain the fermentation broth.

[0016] S5. Centrifuge the fermentation broth obtained in step S4 to obtain the fermentation supernatant, and filter it with a sterile filter to remove bacteria, thus obtaining the fermentation supernatant of Enterobacter nematodes LY2W.

[0017] S6. By weight percentage, mix 80-93% of the fermentation supernatant of Enterobacter nematodes LY2W obtained in step S5 with 7-20% of the adjuvant to obtain the fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria.

[0018] In the entomopathogenic nematode symbiotic bacteria fermentation supernatant preparation provided by the present invention, preferably, by weight percentage, the shake flask fermentation culture medium comprises: 1.5-2.9% peptone, 0.4-1.5% glucose, 0.008-0.02% ginsenosides, 0.1-0.3% MgSO4, 0.08-0.4% Na2SO4, 0.1-0.5% (NH4)2SO4, 0.06-0.08% KH2PO4, 0.04-0.065% K2HPO4, with the balance being water, and the pH adjusted to 7.2-7.4.

[0019] In the entomopathogenic nematode symbiotic bacteria fermentation supernatant preparation provided by the present invention, more preferably, the shake flask fermentation culture medium, by weight percentage, comprises: 1.8-2.2% peptone, 0.85-1.1% glucose, 0.008-0.015% ginsenosides, 0.1-0.2% MgSO4, 0.1-0.25% Na2SO4, 0.18-0.3% (NH4)2SO4, 0.07-0.08% KH2PO4, 0.05-0.06% K2HPO4, with the balance being water, and the pH adjusted to 7.2-7.4.

[0020] More preferably, in the fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria provided by the present invention, the shake flask fermentation culture medium, by weight percentage, comprises: 2.1% peptone, 0.91% glucose, 0.012% ginsenosides, 0.13% MgSO4, 0.15% Na2SO4, 0.24% (NH4)2SO4, 0.072% KH2PO4, 0.05% K2HPO4, with the balance being water, and the pH adjusted to 7.2-7.4.

[0021] The NBTA plate culture medium, by weight percentage, consists of: 0.5% peptone, 1.6% agar powder, 0.3% beef extract, 0.0025% bromothymol blue, 0.004% triphenyltetrazolium chloride, with the balance being water, and the pH adjusted to 7.2–7.4.

[0022] The composition of NA liquid culture medium by weight percentage is: 0.5% peptone, 1.6% agar powder, 0.3% beef extract, with the remainder being water, adjusted to pH 7.2–7.4.

[0023] The composition of LB liquid medium by weight percentage is: 0.5% yeast extract, 1% peptone, 1% sodium chloride, with the balance being water, and the pH adjusted to 7.2-7.4.

[0024] This invention also provides the application of a fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria in the prevention and control of gray mold in leeks and tomatoes.

[0025] In the application of the fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria provided by the present invention in the prevention and control of gray mold in leeks and tomato, preferably, when the preparation is applied to gray mold in leeks, it targets *Botrytis cinerea*; when the preparation is applied to gray mold in tomatoes, it targets *Botrytis cinerea*.

[0026] The Enterobacter LY2W strain in this invention was isolated from the intestine of Heterorhabditis sp., an entomopathogenic nematode, collected from Pingdingshan.

[0027] Using the above technical solution, the fermentation supernatant of *Enterobacter nematode* LY2W and the adjuvant combination result in a fermentation supernatant preparation that exhibits significant control effects against gray mold in leeks and tomatoes. The addition of 0.008–0.02% (by weight) of notoginseng saponins to the shake-flask fermentation medium promotes the production of metabolites by *Enterobacter nematode* LY2W that kill and inhibit *Botrytis cinerea* and *Botrytis bacillus*, thereby enhancing the control effect of the fermentation supernatant preparation on gray mold in leeks and tomatoes. The fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria provided by this invention shows better preventative effects than curative effects against gray mold in leeks and tomatoes. Attached Figure Description

[0028] Figure 1 The inhibition zone diagram of the fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria against Botrytis cinerea (diagram of parallel experiment 1);

[0029] Figure 2 The inhibition zone diagram of the fermentation supernatant preparation of worm pathogenic nematode symbiotic bacteria inhibiting tomato gray mold (diagram of parallel experiment 1);

[0030] Figure 3 Image showing the protective effect of fermentation supernatant preparation of worm pathogen nematode symbiotic bacteria on living leek tissue;

[0031] Figure 4 The therapeutic effect of fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria on living leek tissue is shown in the figure.

[0032] Figure 5 Image showing the protective effect of fermentation supernatant preparation of worm pathogenic nematode symbiotic bacteria on living tomato tissue;

[0033] Figure 6 The therapeutic effect of the fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria on living tomato tissue is shown in the figure. Detailed Implementation

[0034] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0035] Example 1

[0036] This embodiment provides a fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria, including 930g of fermentation supernatant of Enterobacter nematode LY2W and 70g of adjuvants, including emulsifier OP-10, potassium sorbate and ethylene glycol, in a weight ratio of 20:1:25.

[0037] Its preparation method includes the following steps:

[0038] S1. Take the suspension of Enterobacter lysine LY2W preserved in a low-temperature glycerol tube, streak it on NBTA differential medium, incubate at 28℃ for 36h, and pick the primary single colony from the differential medium.

[0039] S2. Streak the single colonies picked in step S1 onto NA medium plates and incubate at 28°C for 24 hours. Pick single colonies.

[0040] S3. Inoculate the single colony picked in step S2 into LB liquid medium and culture it at 28℃ and 180r / min for 14h to obtain seed culture.

[0041] S4. The seed culture cultured in step S3 is inoculated into the shake flask fermentation medium at an inoculation rate of 6% by volume, and the culture is shaken at 28°C for 28 hours to obtain the fermentation broth.

[0042] S5. Centrifuge the fermentation broth obtained in step S4 to obtain the fermentation supernatant, and filter it with a sterile filter to remove bacteria, thus obtaining the fermentation supernatant of Enterobacter nematodes LY2W.

[0043] S6. Weigh 930g of the fermentation supernatant of Enterobacter nematodes LY2W obtained in step S5 and mix it evenly to obtain the fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria.

[0044] The formulations for each culture medium are as follows:

[0045] The shake-flask fermentation medium consisted of: 290g peptone, 40g glucose, 2g ginsenosides, 10g MgSO4, 40g Na2SO4, 50g (NH4)2SO4, 8g KH2PO4, and 4g K2HPO4, with water added to a final volume of 10kg and the pH adjusted to 7.2.

[0046] The composition of NBTA plate medium is as follows: 0.5g peptone, 1.6g agar powder, 0.3g beef extract, 0.0025g bromothymol blue, 0.004g triphenyltetrazol chloride, water added to 100g, and pH adjusted to 7.2.

[0047] The composition of NA liquid culture medium is: 5g peptone, 16g agar powder, 3g beef extract, add water to 1000g, and adjust the pH to 7.4.

[0048] The composition of LB liquid medium is: 0.5g yeast extract, 1g peptone, 1g sodium chloride, water added to 100g, and pH adjusted to 7.4.

[0049] Example 2

[0050] This embodiment provides a fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria, including 800g of fermentation supernatant of Enterobacter nematode LY2W and 200g of adjuvants, including emulsifier OP-10, potassium sorbate and ethylene glycol, in a weight ratio of 20:1:25.

[0051] Its preparation method includes the following steps:

[0052] S1. Take the suspension of Enterobacter lysine LY2W preserved in a low-temperature glycerol tube, streak it on NBTA differential medium, incubate at 28℃ for 24h, and pick the primary single colony from the differential medium.

[0053] S2. Streak the single colonies picked in step S1 onto NA medium plates and incubate at 28°C for 36 hours. Pick single colonies.

[0054] S3. Inoculate the single colony picked in step S2 into LB liquid medium and culture it at 28℃ and 180r / min for 12h to obtain seed culture.

[0055] S4. The seed culture cultured in step S3 is inoculated into the shake flask fermentation medium at an inoculation rate of 6% by volume, and the culture is shaken at 28°C for 24 hours to obtain the fermentation broth.

[0056] S5. Centrifuge the fermentation broth obtained in step S4 to obtain the fermentation supernatant, and filter it with a sterile filter to remove bacteria, thus obtaining the fermentation supernatant of Enterobacter nematodes LY2W.

[0057] S6. Weigh 800g of the fermentation supernatant of Enterobacter nematodes LY2W obtained in step S5 and mix it evenly to obtain the fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria.

[0058] The formulations for each culture medium are as follows:

[0059] The shake-flask fermentation medium consisted of: 150g peptone, 150g glucose, 0.8g ginsenosides, 30g MgSO4, 8g Na2SO4, 10g (NH4)2SO4, 6g KH2PO4, and 6.5g K2HPO4, with water added to a final volume of 10kg and the pH adjusted to 7.4.

[0060] The composition of NBTA plate medium is as follows: 0.5g peptone, 1.6g agar powder, 0.3g beef extract, 0.0025g bromothymol blue, 0.004g triphenyltetrazol chloride, water added to 100g, and pH adjusted to 7.4.

[0061] The composition of NA liquid culture medium is: 5g peptone, 16g agar powder, 3g beef extract, add water to 1000g, and adjust the pH to 7.2.

[0062] The composition of LB liquid medium is: 0.5g yeast extract, 1g peptone, 1g sodium chloride, water added to 100g, and pH adjusted to 7.2.

[0063] Example 3

[0064] This embodiment provides a fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria, including 908g of fermentation supernatant of Enterobacter nematode LY2W and 92g of adjuvants, including emulsifier OP-10, potassium sorbate and ethylene glycol, in a weight ratio of 20:1:25.

[0065] Its preparation method includes the following steps:

[0066] S1. Take the suspension of Enterobacter lysine LY2W preserved in a low-temperature glycerol tube, streak it on NBTA differential medium, incubate at 28℃ for 30h, and pick the primary single colony from the differential medium.

[0067] S2. Streak the single colonies picked in step S1 onto NA medium plates, incubate at 28°C for 32 hours, and then pick single colonies.

[0068] S3. Inoculate the single colony picked in step S2 into LB liquid medium and culture it at 28℃ and 180r / min for 12h to obtain seed culture.

[0069] S4. The seed culture cultured in step S3 is inoculated into the shake flask fermentation medium at an inoculation rate of 6% by volume, and the culture is shaken at 28°C for 25 hours to obtain the fermentation broth.

[0070] S5. Centrifuge the fermentation broth obtained in step S4 to obtain the fermentation supernatant, and filter it with a sterile filter to remove bacteria, thus obtaining the fermentation supernatant of Enterobacter nematodes LY2W.

[0071] S6. Weigh 908g of the fermentation supernatant of Enterobacter nematodes LY2W obtained in step S5 and mix it evenly to obtain the fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria.

[0072] The formulations for each culture medium are as follows:

[0073] The shake-flask fermentation medium consisted of: 180g peptone, 110g glucose, 1.5g ginsenosides, 20g MgSO4, 10g Na2SO4, 30g (NH4)2SO4, 7g KH2PO4, and 5g K2HPO4, with water added to a final volume of 10kg and the pH adjusted to 7.4.

[0074] The composition of NBTA plate medium is as follows: 0.5g peptone, 1.6g agar powder, 0.3g beef extract, 0.0025g bromothymol blue, 0.004g triphenyltetrazol chloride, water added to 100g, and pH adjusted to 7.4.

[0075] The composition of NA liquid culture medium is: 5g peptone, 16g agar powder, 3g beef extract, add water to 1000g, and adjust the pH to 7.2.

[0076] The composition of LB liquid medium is: 0.5g yeast extract, 1g peptone, 1g sodium chloride, water added to 100g, and pH adjusted to 7.2.

[0077] Example 4

[0078] This embodiment provides a fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria, including 908g of fermentation supernatant of Enterobacter nematode LY2W and 92g of adjuvants, including emulsifier OP-10, potassium sorbate and ethylene glycol, in a weight ratio of 20:1:25.

[0079] Its preparation method includes the following steps:

[0080] S1. Take the suspension of Enterobacter lysine LY2W preserved in a low-temperature glycerol tube, streak it on NBTA differential medium, incubate at 28℃ for 30h, and pick the primary single colony from the differential medium.

[0081] S2. Streak the single colonies picked in step S1 onto NA medium plates, incubate at 28°C for 32 hours, and then pick single colonies.

[0082] S3. Inoculate the single colony picked in step S2 into LB liquid medium and culture it at 28℃ and 180r / min for 12h to obtain seed culture.

[0083] S4. The seed culture cultured in step S3 is inoculated into the shake flask fermentation medium at an inoculation rate of 6% by volume, and the culture is shaken at 28°C for 25 hours to obtain the fermentation broth.

[0084] S5. Centrifuge the fermentation broth obtained in step S4 to obtain the fermentation supernatant, and filter it with a sterile filter to remove bacteria, thus obtaining the fermentation supernatant of Enterobacter nematodes LY2W.

[0085] S6. Weigh 908g of the fermentation supernatant of Enterobacter nematodes LY2W obtained in step S5 and mix it evenly to obtain the fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria.

[0086] The formulations for each culture medium are as follows:

[0087] The shake-flask fermentation medium consisted of: 220g peptone, 85g glucose, 1.5g ginsenosides, 18g MgSO4, 25g Na2SO4, 18g (NH4)2SO4, 7.5g KH2PO4, and 5.2g K2HPO4, with water added to a final volume of 10kg and the pH adjusted to 7.4.

[0088] The composition of NBTA plate medium is as follows: 0.5g peptone, 1.6g agar powder, 0.3g beef extract, 0.0025g bromothymol blue, 0.004g triphenyltetrazol chloride, water added to 100g, and pH adjusted to 7.4.

[0089] The composition of NA liquid culture medium is: 5g peptone, 16g agar powder, 3g beef extract, add water to 1000g, and adjust the pH to 7.2.

[0090] The composition of LB liquid medium is: 0.5g yeast extract, 1g peptone, 1g sodium chloride, water added to 100g, and pH adjusted to 7.2.

[0091] Example 5

[0092] This embodiment provides a fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria, including 908g of fermentation supernatant of Enterobacter nematode LY2W and 92g of adjuvants, including emulsifier OP-10, potassium sorbate and ethylene glycol, in a weight ratio of 20:1:25.

[0093] Its preparation method includes the following steps:

[0094] S1. Take the suspension of Enterobacter lysine LY2W preserved in a low-temperature glycerol tube, streak it on NBTA differential medium, incubate at 28℃ for 30h, and pick the primary single colony from the differential medium.

[0095] S2. Streak the single colonies picked in step S1 onto NA medium plates, incubate at 28°C for 32 hours, and then pick single colonies.

[0096] S3. Inoculate the single colony picked in step S2 into LB liquid medium and culture it at 28℃ and 180r / min for 12h to obtain seed culture.

[0097] S4. The seed culture cultured in step S3 is inoculated into the shake flask fermentation medium at an inoculation rate of 6% by volume, and the culture is shaken at 28°C for 25 hours to obtain the fermentation broth.

[0098] S5. Centrifuge the fermentation broth obtained in step S4 to obtain the fermentation supernatant, and filter it with a sterile filter to remove bacteria, thus obtaining the fermentation supernatant of Enterobacter nematodes LY2W.

[0099] S6. Weigh 908g of the fermentation supernatant of Enterobacter nematodes LY2W obtained in step S5 and mix it evenly to obtain the fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria.

[0100] The formulations for each culture medium are as follows:

[0101] The shake-flask fermentation medium consisted of: 210g peptone, 91g glucose, 1.2g ginsenosides, 13g MgSO4, 15g Na2SO4, 15g (NH4)2SO4, 7.2g KH2PO4, and 5g K2HPO4, with water added to a final volume of 10kg and the pH adjusted to 7.4.

[0102] The composition of NBTA plate medium is as follows: 0.5g peptone, 1.6g agar powder, 0.3g beef extract, 0.0025g bromothymol blue, 0.004g triphenyltetrazol chloride, water added to 100g, and pH adjusted to 7.4.

[0103] The composition of NA liquid culture medium is: 5g peptone, 16g agar powder, 3g beef extract, add water to 1000g, and adjust the pH to 7.2.

[0104] The composition of LB liquid medium is: 0.5g yeast extract, 1g peptone, 1g sodium chloride, water added to 100g, and pH adjusted to 7.2.

[0105] Example 6

[0106] This embodiment provides a fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria, including 908g of fermentation supernatant of Enterobacter nematode LY2W and 92g of adjuvants, including emulsifier OP-10, potassium sorbate and ethylene glycol, in a weight ratio of 20:1:25.

[0107] Its preparation method includes the following steps:

[0108] S1. Take the suspension of Enterobacter lysine LY2W preserved in a low-temperature glycerol tube, streak it on NBTA differential medium, incubate at 28℃ for 30h, and pick the primary single colony from the differential medium.

[0109] S2. Streak the single colonies picked in step S1 onto NA medium plates, incubate at 28°C for 32 hours, and then pick single colonies.

[0110] S3. Inoculate the single colony picked in step S2 into LB liquid medium and culture it at 28℃ and 180r / min for 12h to obtain seed culture.

[0111] S4. The seed culture cultured in step S3 is inoculated into the shake flask fermentation medium at an inoculation rate of 6% by volume, and the culture is shaken at 28°C for 25 hours to obtain the fermentation broth.

[0112] S5. Centrifuge the fermentation broth obtained in step S4 to obtain the fermentation supernatant, and filter it with a sterile filter to remove bacteria, thus obtaining the fermentation supernatant of Enterobacter nematodes LY2W.

[0113] S6. Weigh 908g of the fermentation supernatant of Enterobacter nematodes LY2W obtained in step S5 and mix it evenly to obtain the fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria.

[0114] The formulations for each culture medium are as follows:

[0115] The shake-flask fermentation medium consisted of: 210g peptone, 91g glucose, 13g MgSO4, 15g Na2SO4, 15g (NH4)2SO4, 7.2g KH2PO4, and 5g K2HPO4, with water added to a final volume of 10kg and the pH adjusted to 7.4.

[0116] The composition of NBTA plate medium is as follows: 0.5g peptone, 1.6g agar powder, 0.3g beef extract, 0.0025g bromothymol blue, 0.004g triphenyltetrazol chloride, water added to 100g, and pH adjusted to 7.4.

[0117] The composition of NA liquid culture medium is: 5g peptone, 16g agar powder, 3g beef extract, add water to 1000g, and adjust the pH to 7.2.

[0118] The composition of LB liquid medium is: 0.5g yeast extract, 1g peptone, 1g sodium chloride, water added to 100g, and pH adjusted to 7.2.

[0119] Example 7

[0120] This embodiment provides a fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria, including 908g of fermentation supernatant of Enterobacter nematode LY2W and 92g of adjuvants, including emulsifier OP-10, potassium sorbate and ethylene glycol, in a weight ratio of 20:1:25.

[0121] Its preparation method includes the following steps:

[0122] S1. Take the suspension of Enterobacter lysine LY2W preserved in a low-temperature glycerol tube, streak it on NBTA differential medium, incubate at 28℃ for 30h, and pick the primary single colony from the differential medium.

[0123] S2. Streak the single colonies picked in step S1 onto NA medium plates, incubate at 28°C for 32 hours, and then pick single colonies.

[0124] S3. Inoculate the single colony picked in step S2 into LB liquid medium and culture it at 28℃ and 180r / min for 12h to obtain seed culture.

[0125] S4. The seed culture cultured in step S3 is inoculated into the shake flask fermentation medium at an inoculation rate of 6% by volume, and the culture is shaken at 28°C for 25 hours to obtain the fermentation broth.

[0126] S5. Centrifuge the fermentation broth obtained in step S4 to obtain the fermentation supernatant, and filter it with a sterile filter to remove bacteria, thus obtaining the fermentation supernatant of Enterobacter nematodes LY2W.

[0127] S6. Weigh 908g of the fermentation supernatant of Enterobacter nematodes LY2W obtained in step S5 and mix it evenly to obtain the fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria.

[0128] The formulations for each culture medium are as follows:

[0129] The shake-flask fermentation medium consisted of: 210g peptone, 91g glucose, 0.6g ginsenosides, 13g MgSO4, 15g Na2SO4, 15g (NH4)2SO4, 7.2g KH2PO4, and 5g K2HPO4, with water added to a final volume of 10kg and the pH adjusted to 7.4.

[0130] The composition of NBTA plate medium is as follows: 0.5g peptone, 1.6g agar powder, 0.3g beef extract, 0.0025g bromothymol blue, 0.004g triphenyltetrazol chloride, water added to 100g, and pH adjusted to 7.4.

[0131] The composition of NA liquid culture medium is: 5g peptone, 16g agar powder, 3g beef extract, add water to 1000g, and adjust the pH to 7.2.

[0132] The composition of LB liquid medium is: 0.5g yeast extract, 1g peptone, 1g sodium chloride, water added to 100g, and pH adjusted to 7.2.

[0133] Example 8

[0134] This embodiment provides a fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria, including 908g of fermentation supernatant of Enterobacter nematode LY2W and 92g of adjuvants, including emulsifier OP-10, potassium sorbate and ethylene glycol, in a weight ratio of 20:1:25.

[0135] Its preparation method includes the following steps:

[0136] S1. Take the suspension of Enterobacter lysine LY2W preserved in a low-temperature glycerol tube, streak it on NBTA differential medium, incubate at 28℃ for 30h, and pick the primary single colony from the differential medium.

[0137] S2. Streak the single colonies picked in step S1 onto NA medium plates, incubate at 28°C for 32 hours, and then pick single colonies.

[0138] S3. Inoculate the single colony picked in step S2 into LB liquid medium and culture it at 28℃ and 180r / min for 12h to obtain seed culture.

[0139] S4. The seed culture cultured in step S3 is inoculated into the shake flask fermentation medium at an inoculation rate of 6% by volume, and the culture is shaken at 28°C for 25 hours to obtain the fermentation broth.

[0140] S5. Centrifuge the fermentation broth obtained in step S4 to obtain the fermentation supernatant, and filter it with a sterile filter to remove bacteria, thus obtaining the fermentation supernatant of Enterobacter nematodes LY2W.

[0141] S6. Weigh 908g of the fermentation supernatant of Enterobacter nematodes LY2W obtained in step S5 and mix it evenly to obtain the fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria.

[0142] The formulations for each culture medium are as follows:

[0143] The shake-flask fermentation medium consisted of: 210g peptone, 91g glucose, 2.2g ginsenosides, 13g MgSO4, 15g Na2SO4, 15g (NH4)2SO4, 7.2g KH2PO4, and 5g K2HPO4, with water added to a final volume of 10kg and the pH adjusted to 7.4.

[0144] The composition of NBTA plate medium is as follows: 0.5g peptone, 1.6g agar powder, 0.3g beef extract, 0.0025g bromothymol blue, 0.004g triphenyltetrazol chloride, water added to 100g, and pH adjusted to 7.4.

[0145] The composition of NA liquid culture medium is: 5g peptone, 16g agar powder, 3g beef extract, add water to 1000g, and adjust the pH to 7.2.

[0146] The composition of LB liquid medium is: 0.5g yeast extract, 1g peptone, 1g sodium chloride, water added to 100g, and pH adjusted to 7.2.

[0147] Test case

[0148] 1. Inhibitory effect of fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria on Botrytis cinerea.

[0149] Add the cryopreserved Botrytis cinerea to PDB medium and culture at 28℃ and 150r / min for 48h with constant temperature shaking. Spread the cultured Botrytis cinerea seed culture onto PDB plates, adding 1mL of seed culture to each plate and incubating at 28℃ for 48h. Select the cultured plates for mycelial cake preparation.

[0150] This experiment included a blank control group, a shake-flask fermentation medium control group, an adjuvant control group, an adjuvant-free group, and groups from Examples 1 to 8. The blank control group was prepared by mixing sterile water with an equal volume of PDA medium and pouring the mixture into a petri dish. The shake flask fermentation medium from Example 5 was diluted 10 times and mixed with an equal volume of PDA medium, then poured into a petri dish to prepare the shake flask fermentation medium control group plate. The adjuvant control group was prepared by diluting an equal volume of adjuvant (emulsifier OP-10, potassium sorbate, and ethylene glycol in a weight ratio of 20:1:25) 10 times and mixing it with PDA medium, then pouring the mixture into a petri dish to prepare the adjuvant control group plate. The adjuvant-free group was prepared by diluting the Enterobacter nematode LY2W fermentation supernatant obtained in Example 5 10 times and mixing it with an equal volume of PDA medium, then pouring the mixture into a petri dish to prepare the adjuvant-free group plate. The entomopathogenic nematode symbiotic bacteria fermentation supernatant preparations obtained in Examples 1 to 8 were diluted 10 times and mixed with an equal volume of PDA medium, then poured into petri dishes to obtain the plates of Examples 1 to 8.

[0151] Using a 1 cm diameter fungal punch, several 1 cm diameter mycelial cakes were punched from the edge of the culture medium containing *Botrytis cinerea*. These cakes were then inverted and placed in the center of the plates for each experimental group, and marked accordingly. After treatment, the edges of the culture dishes were sealed with a breathable filter membrane and incubated at 28℃ for 60 h. The colony diameter was measured using the cross-sectional method, and the inhibition efficiency was calculated. Inhibition rate (%) = [(blank control colony diameter - mycelial cake diameter) - (treated colony diameter - mycelial cake diameter)] / (blank control colony diameter - mycelial cake diameter) × 100%. The results are shown in Table 1 and... Figure 1 .

[0152] Table 1. Antibacterial rate of fermentation supernatant preparations containing entomopathogenic nematodes

[0153]

[0154]

[0155] Table 1 shows that the inhibition rate of the shake-flask fermentation medium control group was 21.15%, indicating that the shake-flask fermentation medium had no inhibitory effect on Botrytis cinerea. The inhibition rate of the adjuvant control group was 59.62%, indicating that the adjuvant (emulsifier OP-10, potassium sorbate, and ethylene glycol in a weight ratio of 20:1:25) had a certain inhibitory effect on Botrytis cinerea. The inhibition rate of the group without adjuvant was 89.04%, indicating that the fermentation supernatant of Enterobacter nematodes LY2W obtained according to Example 5 had a good inhibitory effect on Botrytis cinerea. The inhibition rates of groups 1 to 5 were all higher than 99.42%, and the inhibition rates of Examples 3 to 5 reached 100%, indicating that the fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria obtained according to Examples 1 to 5 had a very good inhibitory effect on Botrytis cinerea. The inhibition rate of the 6th group was 68.65%, the 7th group was 94.87%, and the 8th group was 93.65%. The shake-flask fermentation medium in the 6th group did not contain ginsenosides, and the rest was the same as in the 5th group. The shake-flask fermentation medium in the 7th group contained 0.6g of ginsenosides, and the rest was the same as in the 5th group. The shake-flask fermentation medium in the 8th group contained 2.2g of ginsenosides, and the rest was the same as in the 5th group. This indicates that adding 0.008–0.02% (by weight) of ginsenosides to the shake-flask fermentation medium promotes the inhibition rate of the fermentation supernatant preparation of the entomopathogenic nematode symbiotic bacteria. 2. Effect of the fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria on inhibiting tomato gray mold.

[0156] Add the cryopreserved Botrytis cinerea to PDB medium and culture at 28℃ and 150 r / min for 48 h with constant temperature shaking. Spread the cultured pathogen seed culture onto PDB plates, adding 1 mL of seed culture to each petri dish and incubating at 28℃ for 48 h. Select the cultured plates for mycelial cake preparation.

[0157] This experiment included a blank control group, a shake-flask fermentation medium control group, an adjuvant control group, an adjuvant-free group, and groups from Examples 1 to 8. The blank control group was prepared by mixing sterile water with an equal volume of PDA medium and pouring the mixture into a petri dish. The shake flask fermentation medium from Example 5 was diluted 10 times and mixed with an equal volume of PDA medium, then poured into a petri dish to prepare the shake flask fermentation medium control group plate. The adjuvant control group was prepared by diluting an equal volume of adjuvant (emulsifier OP-10, potassium sorbate, and ethylene glycol in a weight ratio of 20:1:25) 10 times and mixing it with PDA medium, then pouring the mixture into a petri dish to prepare the adjuvant control group plate. The adjuvant-free group was prepared by diluting the Enterobacter nematode LY2W fermentation supernatant obtained in Example 5 10 times and mixing it with an equal volume of PDA medium, then pouring the mixture into a petri dish to prepare the adjuvant-free group plate. The entomopathogenic nematode symbiotic bacteria fermentation supernatant preparations obtained in Examples 1 to 8 were diluted 10 times and mixed with an equal volume of PDA medium, then poured into petri dishes to obtain the plates of Examples 1 to 8.

[0158] Using a 1 cm diameter fungal punch, several 1 cm diameter mycelial cakes were punched from the edge of the culture medium containing *Botrytis cinerea*. These cakes were then inverted and placed in the center of the plates in each experimental group, and marked accordingly. After treatment, the edges of the culture dishes were sealed with a breathable filter membrane and incubated at 28℃ for 60 h. The colony diameter was measured using the cross-sectional method, and the inhibition efficiency was calculated. Inhibition rate (%) = [(blank control colony diameter - mycelial cake diameter) - (treated colony diameter - mycelial cake diameter)] / (blank control colony diameter - mycelial cake diameter) × 100%. The results are shown in Table 2 and... Figure 2 .

[0159] Table 2. Inhibition rate of fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria against Botrytis cinerea

[0160]

[0161] Table 2 shows that the inhibition rate of the shake-flask fermentation medium control group was 6.67%, indicating that the shake-flask fermentation medium had no inhibitory effect on *Botrytis cinerea*. The inhibition rate of the adjuvant control group was 28.89%, indicating that the adjuvant (emulsifier OP-10, potassium sorbate, and ethylene glycol in a weight ratio of 20:1:25) had a certain inhibitory effect on *Botrytis cinerea*, but the effect was small. The inhibition rate of the group without adjuvant was 37.78%, indicating that the fermentation supernatant of *Enterobacter nematodes* LY2W obtained in Example 5 had a certain inhibitory effect on *Botrytis cinerea*, but the effect was not good. The inhibition rates of groups 1 to 5 were all higher than 82.89%, and the inhibition rate of Example 5 reached 82.89%, indicating that the fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria obtained in Examples 1 to 5 had a very good inhibitory effect on *Botrytis cinerea*. The combination of the fermentation supernatant of entomopathogenic nematode symbiotic bacteria obtained in Examples 1 to 5 and the adjuvant has the effect of improving the inhibitory effect on *Botrytis cinerea*. The inhibition rate of the group in Example 6 was 59.56%, the inhibition rate of the group in Example 7 was 62.22%, and the inhibition rate of the group in Example 8 was 56.67%. In Example 6, the shake-flask fermentation medium did not contain ginsenosides, and the rest was the same as in Example 5. In Example 7, the shake-flask fermentation medium contained 0.6g of ginsenosides, and the rest was the same as in Example 5. In Example 8, the shake-flask fermentation medium contained 2.2g of ginsenosides, and the rest was the same as in Example 5. This indicates that adding 0.008-0.02% (by weight) of ginsenosides to the shake-flask fermentation medium has a promoting effect on improving the inhibition rate of the fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria against Botrytis cinerea.

[0162] 3. Protective effect of fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria on living leek tissues.

[0163] This experiment included a blank control group, a shake-flask fermentation medium control group, an adjuvant control group, an adjuvant-free group, and groups from Examples 1 to 8.

[0164] Spray the pesticide onto freshly picked, disease-free chive leaves in groups until the pesticide drips onto the leaves.

[0165] The blank control group was sprayed with sterile water; the shake flask fermentation medium control group was sprayed with the shake flask fermentation medium from Example 5; the adjuvant control group was sprayed with emulsifier OP-10, potassium sorbate, and ethylene glycol in a weight ratio of 20:1:25; the adjuvant-free group was sprayed with the fermentation supernatant of Enterobacter nematodes LY2W obtained in Example 5; and the groups in Examples 1 to 8 were sprayed with fermentation supernatant preparations of worm pathogenic nematode symbiotic bacteria obtained in Examples 1 to 8, respectively.

[0166] After spraying with the pesticide, the chive leaves were placed in moist filter paper petri dishes and incubated at a constant temperature and humidity of 28°C. After 48 hours, *Botrytis cinerea* seed solution was inoculated onto the chive leaves, with three inoculation points on each leaf. After 60 hours, the presence or absence of *Botrytis cinerea* mycelium formation on the surface of the chive leaves in each experimental group was observed. The results are shown in Table 3. Figure 3 .

[0167] Table 3. Protective effect of fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria on live leek tissue.

[0168]

[0169] Table 3 shows that: the shake-flask fermentation medium had no protective effect against infection of leeks by *Botrytis cinerea*, the pathogen of gray mold of leeks; the adjuvant (emulsifier OP-10, potassium sorbate, and ethylene glycol in a weight ratio of 20:1:25) had a relatively small protective effect against infection of leeks by *Botrytis cinerea*, the pathogen of gray mold of leeks; the fermentation supernatant of *Enterobacter nematodes* LY2W had a protective effect against infection of leeks by *Botrytis cinerea*, the pathogen of gray mold of leeks, but the effect was not ideal; the fermentation supernatant preparations of entomopathogenic nematode symbiotic bacteria obtained in Examples 1 to 5 had a very good protective effect against infection of leeks by *Botrytis cinerea*, the pathogen of gray mold of leeks, with Example 5 showing the best effect; the fermentation supernatant preparations of entomopathogenic nematode symbiotic bacteria obtained in Examples 6 to 8 had a protective effect against infection of leeks by *Botrytis cinerea*, the pathogen of gray mold of leeks, but the effect was not ideal. In Example 6, the shake-flask fermentation medium did not contain ginsenosides, and the rest was the same as in Example 5; in Example 7, the shake-flask fermentation medium contained 0.6 g of ginsenosides, and the rest was the same as in Example 5; in Example 8, the shake-flask fermentation medium contained 2.2 g of ginsenosides, and the rest was the same as in Example 5. This indicates that adding 0.008–0.02% (by weight) of ginsenosides to the shake-flask fermentation medium has a promoting effect on improving the protection of the fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria on live leek tissue infected with Botrytis cinerea.

[0170] 4. The therapeutic effect of fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria on living leek tissue.

[0171] This experiment included a blank control group, a shake-flask fermentation medium control group, an adjuvant control group, an adjuvant-free group, and groups from Examples 1 to 8.

[0172] Botrytis cinerea, the pathogen of gray mold, was inoculated onto freshly harvested, disease-free chive leaves. The leaves were placed in moistened filter paper petri dishes and incubated at 28°C with constant temperature and humidity. After 48 hours, the experimental groups were sprayed with the appropriate fungicide, ensuring the chive leaves were moist. Spraying was repeated every 24 hours. After 60 hours, the differences in Botrytis cinerea colonies on the chive leaves compared to the untreated control group were observed to determine if the fungicide inhibited the growth of the gray mold pathogen. Results are shown in Table 4. Figure 4 .

[0173] The blank control group was sprayed with sterile water; the shake flask fermentation medium control group was sprayed with the shake flask fermentation medium from Example 5; the adjuvant control group was sprayed with emulsifier OP-10, potassium sorbate, and ethylene glycol in a weight ratio of 20:1:25; the adjuvant-free group was sprayed with the fermentation supernatant of Enterobacter nematodes LY2W obtained in Example 5; and the groups in Examples 1 to 8 were sprayed with fermentation supernatant preparations of worm pathogenic nematode symbiotic bacteria obtained in Examples 1 to 8, respectively.

[0174] Table 4. Results of the treatment of Botrytis cinerea infection in live leek tissue by fermentation supernatant of entomopathogenic nematodes.

[0175]

[0176]

[0177] Table 4 shows that: the shake-flask fermentation medium had no therapeutic effect on chives infected with Botrytis cinerea, the pathogen of gray mold of chives; the adjuvant (emulsifier OP-10, potassium sorbate, and ethylene glycol in a weight ratio of 20:1:25) had only a minor therapeutic effect on chives infected with Botrytis cinerea, the pathogen of gray mold of chives; the fermentation supernatant of Enterobacter nematodes LY2W had a certain therapeutic effect on chives infected with Botrytis cinerea, the pathogen of gray mold of chives, but the effect was not significant; the fermentation supernatant preparations of entomopathogenic nematode symbiotic bacteria obtained in Examples 1 to 5 had a therapeutic effect on chives infected with Botrytis cinerea, the pathogen of gray mold of chives, with Example 5 showing the best effect; the fermentation supernatant preparations of entomopathogenic nematode symbiotic bacteria obtained in Examples 6 to 8 had a partial therapeutic effect on chives infected with Botrytis cinerea, the pathogen of gray mold of chives, but the effect was significantly lower than that of Example 5. In Example 6, the shake-flask fermentation medium did not contain ginsenosides, and the rest was the same as in Example 5; in Example 7, the shake-flask fermentation medium contained 0.6 g of ginsenosides, and the rest was the same as in Example 5; in Example 8, the shake-flask fermentation medium contained 2.2 g of ginsenosides, and the rest was the same as in Example 5. This indicates that adding 0.008–0.02% (by weight) of ginsenosides to the shake-flask fermentation medium enhances the therapeutic effect of the fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria on leeks infected with Botrytis cinerea.

[0178] 5. Protective effect of fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria on living tomato tissues.

[0179] Add the cryopreserved Botrytis cinerea to PDB medium and culture at 28℃ and 150 r / min for 48 h with constant temperature shaking. Spread the cultured pathogen seed culture onto PDB plates, adding 1 mL of seed culture to each petri dish and incubating at 28℃ for 48 h. Select the cultured plates for mycelial cake preparation.

[0180] This experiment included a blank control group, a shake-flask fermentation medium control group, an adjuvant control group, a no-adjuvant group, and groups 1 to 8. Each group contained three tomatoes, with no significant differences in size or appearance among the tomatoes. A 1*1cm incision was made on the surface of each tomato using a sterile blade. The blank control group was sprayed with sterile water; the shake-flask fermentation medium control group was sprayed with the shake-flask fermentation medium from Example 5; the adjuvant control group was sprayed with emulsifier OP-10, potassium sorbate, and ethylene glycol in a weight ratio of 20:1:25; the no-adjuvant group was sprayed with the fermentation supernatant of *Enterobacter nematodes* LY2W obtained in Example 5; and groups 1 to 8 were sprayed with fermentation supernatant preparations of symbiotic bacteria of entomopathogenic nematodes obtained in Examples 1 to 8, respectively.

[0181] The corresponding fungicide was sprayed onto tomatoes in each experimental group. 24 hours later, *Botrytis cinerea* fungal cakes were punched from the agar plate using a 1cm fungal punch and inoculated onto the cut surfaces of the tomatoes. After incubation at 28℃ for 72 hours, the changes in the tomatoes were observed. The results are shown in Table 5. Figure 5 .

[0182] Table 5. Protective effect of fermentation supernatant preparations made from worm-borne nematode symbiotic bacteria on tomato tissues

[0183]

[0184]

[0185] As shown in Table 5, the fermentation supernatant preparations of the entomopathogenic nematode symbiotic bacteria in Examples 1 to 5 showed significantly better protective effects on live tomato tissues than the group without adjuvants and Examples 6 to 8. The shake-flask fermentation medium in the control group showed no protective effect against *Botrytis cinerea* infection in live tomatoes. In Example 6, the shake-flask fermentation medium did not contain ginsenosides, and the rest was the same as in Example 5; in Example 7, the shake-flask fermentation medium contained 0.6 g of ginsenosides, and the rest was the same as in Example 5; in Example 8, the shake-flask fermentation medium contained 2.2 g of ginsenosides, and the rest was the same as in Example 5. This indicates that adding 0.008–0.02% (by weight) of ginsenosides to the shake-flask fermentation medium promotes the protection of live tomatoes infected with *Botrytis cinerea* by the entomopathogenic nematode symbiotic bacteria fermentation supernatant.

[0186] 6. Therapeutic effects of fermentation supernatant preparations of entomopathogenic nematode symbiotic bacteria on living tomato tissues.

[0187] Add the cryopreserved Botrytis cinerea to PDB medium and culture at 28℃ and 150 r / min for 48 h with constant temperature shaking. Spread the cultured pathogen seed culture onto PDB plates, adding 1 mL of seed culture to each petri dish and incubating at 28℃ for 48 h. Select the cultured plates for mycelial cake preparation.

[0188] This experiment included a blank control group, a shake-flask fermentation medium control group, an adjuvant control group, a no-adjuvant group, and groups 1 to 8. Each group contained three tomatoes, with no significant differences in size or appearance among the tomatoes. A 1*1cm incision was made on the surface of each tomato using a sterile blade. The blank control group was sprayed with sterile water; the shake-flask fermentation medium control group was sprayed with the shake-flask fermentation medium from Example 5; the adjuvant control group was sprayed with emulsifier OP-10, potassium sorbate, and ethylene glycol in a weight ratio of 20:1:25; the no-adjuvant group was sprayed with the fermentation supernatant of *Enterobacter nematodes* LY2W obtained in Example 5; and groups 1 to 8 were sprayed with fermentation supernatant preparations of symbiotic bacteria of entomopathogenic nematodes obtained in Examples 1 to 8, respectively.

[0189] Using a 1cm fungal punch, Botrytis cinerea cakes were created from the edge of the agar plate and inoculated onto the cut surfaces of the tomatoes. The plates were then incubated at 28°C. After 48 hours, the corresponding fungicide for each group was sprayed every 24 hours. Changes in the tomatoes were observed after 72 hours. The results are shown in Table 6. Figure 6 .

[0190] Table 6. Therapeutic effects of fermentation supernatant preparations containing entomopathogenic nematode symbiotic bacteria on living tomato tissues

[0191]

[0192]

[0193] Table 6 shows that the shake-flask fermentation medium control group had no therapeutic effect on live tomatoes infected with *Botrytis cinerea*. The adjuvant in the adjuvant group had a therapeutic effect on live tomatoes infected with *Botrytis cinerea*, but the effect was not significant. The fermentation supernatant preparations of the entomopathogenic nematode symbiotic bacteria in Examples 1 to 5 showed excellent therapeutic effects on live tomato tissues. The therapeutic effects of the fermentation supernatant preparations of the entomopathogenic nematode symbiotic bacteria in Examples 6 to 9 on live tomato tissues were significantly less than those in Examples 1 to 5. Therefore, adding 0.008–0.02% (by weight) of Panax notoginseng saponins to the shake-flask fermentation medium promotes the therapeutic effect of the fermentation supernatant preparations of the entomopathogenic nematode symbiotic bacteria on live tomatoes infected with *Botrytis cinerea*.

[0194] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria, characterized in that, By weight percentage, it consists of 80-93% fermentation supernatant of Enterobacter nematodes LY2W and 7-20% adjuvants; The additives are emulsifier OP-10, potassium sorbate and ethylene glycol, in a weight ratio of 20:1:

25.

2. The fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria according to claim 1, characterized in that, The supernatant of the *Enterobacter nematodes* LY2W fermentation contains 90.8% by weight and the adjuvant contains 9.2%.

3. A method for preparing a fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria, characterized in that, Includes the following steps: S1. Take the suspension of Enterobacter lysine LY2W preserved in a low-temperature glycerol tube, streak it on NBTA differential medium, and incubate it at 28℃ for 24-36 h. Pick the primary single colony from the differential medium. S2. Streak the single colonies picked in step S1 onto NA medium plates and incubate at 28°C for 24–36 h, then pick single colonies. S3. Inoculate the single colony picked in step S2 into LB liquid medium and culture it at 28℃ and 180r / min for 12-14h to obtain seed culture. S4. Inoculate the seed culture cultured in step S3 into the shake flask fermentation medium at an inoculation rate of 6% by volume, and culture at 28°C with shaking for 24-28 hours to obtain the fermentation broth. S5. Centrifuge the fermentation broth obtained in step S4 to obtain the fermentation supernatant, and filter it with a sterile filter to remove bacteria, thus obtaining the fermentation supernatant of Enterobacter nematodes LY2W. S6. By weight percentage, mix 80-93% of the fermentation supernatant of Enterobacter nematodes LY2W obtained in step S5 with 7-20% of the adjuvant to obtain the fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria.

4. The method for preparing the fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria according to claim 3, characterized in that, The shake flask fermentation medium, by weight percentage, comprises: 1.5–2.9% peptone, 0.4–1.5% glucose, 0.008–0.02% ginsenosides, 0.1–0.3% MgSO4, 0.08–0.4% Na2SO4, 0.1–0.5% (NH4)2SO4, 0.06–0.08% KH2PO4, 0.04–0.065% K2HPO4, with the balance being water, and the pH adjusted to 7.2–7.

4.

5. The method for preparing the fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria according to claim 4, characterized in that, The shake flask fermentation medium, by weight percentage, comprises: 1.8–2.2% peptone, 0.85–1.1% glucose, 0.008–0.015% ginsenosides, 0.1–0.2% MgSO4, 0.1–0.25% Na2SO4, 0.18–0.3% (NH4)2SO4, 0.07–0.08% KH2PO4, 0.05–0.06% K2HPO4, with the balance being water, and the pH adjusted to 7.2–7.

4.

6. The method for preparing the fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria according to claim 5, characterized in that, The shake flask fermentation medium, by weight percentage, consists of: 2.1% peptone, 0.91% glucose, 0.012% ginsenosides, 0.13% MgSO4, 0.15% Na2SO4, 0.24% (NH4)2SO4, 0.072% KH2PO4, 0.05% K2HPO4, with the remainder being water, and the pH adjusted to 7.2–7.

4.

7. The application of the fermentation supernatant preparation of entomopathogenic nematode symbiotic bacteria according to claim 1 or 2 in the prevention and control of gray mold in leeks and tomato.

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