Insect pathogenic nematode symbiotic bacteria preparation, preparation method and application thereof

Abstract

The present invention discloses an insect pathogenic nematode symbiotic bacteria preparation, a preparation method and an application thereof, wherein the symbiotic bacteria comprises the following components: Enterobacter nematophilus NK, an adjuvant and sterile water. In its preparation process, the shake flask fermentation medium is composed of: 1.8-2.5% peptone, 0.7-1.5% glucose, 0.005-0.015% houttuynia cordata extract, 0.1-0.2% MgSO4, 0.08-0.25% Na2SO4, 0.18-0.34% (NH4)2SO4, 0.068-0.078% KH2PO4, 0.02-0.13% K2HPO4, the remainder is water, and the pH is adjusted to 7.2-7.4. The insect pathogenic nematode symbiotic bacteria preparation is used to prevent and control leek late eye fungus gnat and leek gray mold, and has excellent prevention and control effects. The preparation method of the present invention also increases the yield of Enterobacter nematophilus NK.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to an insect pathogenic nematode symbiotic bacteria preparation, a preparation method and applications thereof, and specifically relates to an insect pathogenic nematode symbiotic bacteria preparation for preventing and controlling leek bradya fungus gnat and gray mold, a preparation method and applications thereof. Background Art

[0002] In the prevention and control of agricultural pests and diseases, it is necessary to reduce chemical control and increase biological control. Therefore, seeking new safe, effective and sustainable methods of pest control - biological control, has become a hot topic and key area of ​​research.

[0003] Bradysia odoriphaga Yang et Zhang, the larvae of which are commonly known as leek maggots, and the pathogens of leek gray mold (Botrytis cinerea, Botrytis cinerea, and Botrytis cinerea) are the main pests of leek. At present, in order to eliminate leek maggots and leek gray mold, vegetable farmers continue to increase the dosage of pesticides, and even use highly toxic or extremely toxic chemical pesticides, resulting in serious excess of pesticide residues in leek, which makes people "fear leek", seriously affecting the market and development prospects of leek.

[0004] The main biological control of leek maggots is Bacillus thuringiensis (Bt), which has an insecticidal effect on more than 3,000 pests in 16 orders, but it has caused the problem of drug resistance. The development of new biological insecticide toxins has become a top priority. The main biological control of leek gray mold is Bacillus subtilis from the United States and Trichoderma from Israel, as well as the chemical pesticide procymidone, but there are high costs and pesticide residues. Therefore, breeding new leek varieties resistant to gray mold and developing green control technology for leek gray mold are the most effective ways to solve the problem of excessive procymidone residues.

[0005] In the pathogenic process of the insect pathogenic nematode-symbiotic bacteria complex, the protein toxins synthesized and secreted by the symbiotic bacteria play a vital role and are a research hotspot in the field of biological control of field pests. Insect pathogenic nematode symbiotic bacteria are used for the control of pests, and they need to form a certain form of preparation before they can be used. Therefore, strengthening the application research of insect pathogenic nematode live bacteria preparations will play an important role in the integrated control of agricultural pests in the future. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide an insect pathogenic nematode symbiotic bacteria preparation for preventing and controlling leek bradya fungus gnat and gray mold.

[0007] In order to solve the above technical problems, the technical solution of the present invention is:

[0008] An insect pathogenic nematode symbiotic bacteria preparation includes the following components: Enterobacter nematophila NK, an adjuvant and sterile water. The Enterobacter nematophila NK is isolated from the intestine of Heterorhabditis sp. insect pathogenic nematodes collected from Pingdingshan. The 16S rRNA of Enterobacter nematophila NK is detected for bacterial species identification. The PCR amplification primers used are: the upstream primer is: 5′-GAGCGGATAACAATTTCACACAGG-3′ and the downstream primer is: 5′-CGCCAGGGTTTTCCCAGTCACGAC-3′. The PCR amplification product is connected with the PMD18-T vector vector and transformed into E. coli DH5α competent cells for 16S rRNA sequence alignment.

[0009] In the insect pathogenic nematode symbiotic bacteria preparation provided by the present invention, preferably, the following components are included by weight: 50-75 parts of Enterobacter nematophila NK, 8-20 parts of auxiliary agents and 18-35 parts of sterile water.

[0010] In the insect pathogenic nematode symbiotic bacteria preparation provided by the present invention, more preferably, the following components are included in parts by weight: 58-70 parts of Enterobacter nematophila NK, 10-15 parts of auxiliary agents and 25-30 parts of sterile water.

[0011] In the insect pathogenic nematode symbiotic bacteria preparation provided by the present invention, more preferably, the following components are included by weight: 60 parts of Enterobacter nematophila NK, 13 parts of auxiliary agents and 27 parts of sterile water.

[0012] The auxiliary agents include wetting and dispersing agents, thickeners, preservatives, antifreeze agents, defoaming agents and ultraviolet light protective agents.

[0013] The wetting and dispersing agent is one or more of sodium lignin sulfonate, emulsifier OP-10, Tween 80, emulsifier 500# and emulsifier 600#; the thickener is one or more of sodium carboxymethyl cellulose (CMC), xanthan gum (XG), polyvinyl alcohol (PVG) and gum arabic; the preservative is one or more of potassium sorbate, methyl paraben and sodium dehydroacetate; the antifreeze is ethylene glycol; the defoaming agent is silicone; and the ultraviolet light protector is one or more of whole milk, activated carbon and sodium lignin sulfonate.

[0014] In the insect pathogenic nematode symbiotic bacteria preparation provided by the present invention, it is further preferred that the auxiliary agent is composed of the following ingredients in parts by weight: 4 parts of emulsifier OP-10, 0.2 parts of potassium sorbate, 5 parts of ethylene glycol, 3 parts of whole milk powder, 0.2 parts of xanthan gum and 0.6 parts of silicone.

[0015] The present invention also provides a method for preparing an insect pathogenic nematode symbiotic bacteria preparation to increase yield, which comprises the following steps:

[0016] S1. Pick out Enterobacter nematophila and streak it on the NKNBTA identification medium, culture it at 28℃ for 24-36h, and pick out the primary single colony on the identification medium;

[0017] S2, streaking the single colony picked in step S1 on a NA plate medium, culturing at 28°C for 24-36 hours, and picking a single colony slant for storage;

[0018] S3, inoculating the single colony picked in step S2 into LB liquid culture medium, and culturing for 12 to 14 hours at 28°C and 180 r / min to obtain a seed bacterial solution;

[0019] S4, taking the seed bacterial liquid cultured in step S3 and inoculating it into a shake flask fermentation medium at a volume ratio of 6%, and shaking and culturing it at 28° C. for 24 to 28 hours to obtain a fermentation liquid;

[0020] S5, centrifuging the fermentation liquid obtained in step S4 to obtain Enterobacter nematophila NK bacterial precipitate;

[0021] S6. Weigh the following components by weight: 50-75 parts of Enterobacter nematophila NK, 8-20 parts of adjuvants and 18-35 parts of sterile water, and mix the components to obtain an insect pathogenic nematode symbiotic bacteria preparation.

[0022] In the preparation method of the insect pathogenic nematode symbiotic bacteria preparation provided by the present invention, preferably, the shake flask fermentation medium is composed of, by weight percentage, 1.8-2.5% peptone, 0.7-1.5% glucose, 0.005-0.015% houttuynia cordata extract, 0.1-0.2% MgSO4, 0.08-0.25% Na2SO4, 0.18-0.34% (NH4)2SO4, 0.068-0.078% KH2PO4, 0.02-0.13% K2HPO4, and the balance is water, and the pH is adjusted to 7.2-7.4.

[0023] In the preparation method of the insect pathogenic nematode symbiotic bacteria preparation provided by the present invention, preferably, the shake flask fermentation medium is composed of, by weight percentage, 2.0-2.5% peptone, 0.9-1.2% glucose, 0.007-0.012% houttuynia cordata extract, 0.1-0.15% MgSO4, 0.1-0.2% Na2SO4, 0.2-0.25% (NH4)2SO4, 0.07-0.075% KH2PO4, 0.04-0.1% K2HPO4, and the balance is water, and the pH is adjusted to 7.2-7.4.

[0024] In the preparation method of the insect pathogenic nematode symbiotic bacteria preparation provided by the present invention, more preferably, the shake flask fermentation medium is composed of, by weight percentage, 2.1% peptone, 0.91% glucose, 0.009% houttuynia cordata extract, 0.13% MgSO4, 0.15% Na2SO4, 0.24% (NH4)2SO4, 0.072% KH2PO4, 0.05% K2HPO4, and the balance is water, and the pH is adjusted to 7.2-7.4.

[0025] In the preparation method of the insect pathogenic nematode symbiotic bacteria preparation provided by the present invention, the composition of the NBTA plate culture medium is, by weight percentage, 0.5% peptone, 1.6% agar powder, 0.3% beef extract, 0.0025% bromomusk thymol blue, 0.004% triphenyltetrazolium chloride, and the balance is water, and the pH is adjusted to 7.2-7.4.

[0026] In the preparation method of the insect pathogenic nematode symbiotic bacteria preparation provided by the present invention, the composition of the NA liquid culture medium is as follows, by weight percentage: 0.5% peptone, 1.6% agar powder, 0.3% beef extract, and the balance is water, and the pH is adjusted to 7.2-7.4.

[0027] In the preparation method of the insect pathogenic nematode symbiotic bacteria preparation provided by the present invention, the composition of the LB liquid culture medium is as follows by weight percentage: 0.5% yeast powder, 1% peptone, 1% sodium chloride, and the balance is water, and the pH is adjusted to 7.2-7.4.

[0028] The present invention also provides an application of an insect pathogenic nematode symbiotic bacteria preparation in preventing and controlling leek Bradysia fungi and leek gray mold. The pathogen targeted by the symbiotic bacteria preparation when applied to leek gray mold is Botrytis cinerea.

[0029] Compared with the prior art, the present invention has the following beneficial technical effects:

[0030] 1. Insecticidal activity tests show that the insect pathogenic nematode symbiotic bacteria preparation obtained by the present invention has an excellent effect of killing the larvae of Bradysia leek (leek maggots), and the killing rate reaches more than 90%, and can reach 100%.

[0031] 2. Tests on the inhibition of hyphae growth of Botrytis cinerea by symbiotic bacteria preparations, the protective effect of symbiotic bacteria preparations on living tissues of leek, the therapeutic effect of symbiotic bacteria preparations on gray mold of leek, and the inhibitory effect of symbiotic bacteria preparations on Botrytis cinerea of ​​potted leek show that the insect pathogenic nematode symbiotic bacteria preparation obtained by the present invention has a good effect on the prevention and treatment of gray mold of leek.

[0032] 3. The yield test of Enterobacter nematophila NK showed that adding 0.005-0.015% by weight of Houttuynia cordata extract to the shake flask fermentation medium has a promoting effect on increasing the yield of Enterobacter nematophila NK, thereby increasing the yield of Enterobacter nematophila NK. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a bar graph showing the insecticidal activity test results of the symbiotic bacteria preparation against the larvae of Bradysia oleracea (leek maggots);

[0034] Figure 2 This is the inhibition zone diagram of the inhibitory effect of the symbiotic bacteria preparation on the hyphae of Botrytis cinerea;

[0035] Figure 3 The protective effect of symbiotic bacteria preparation on the tissues of living leek;

[0036] Figure 4 This is a diagram showing the treatment results of leek gray mold with symbiotic bacteria preparation;

[0037] Figure 5 The bar graph is a graph showing the yield of Enterobacter nematophila NK in Examples 1 to 9. DETAILED DESCRIPTION

[0038] The specific embodiments of the present invention are further described below in conjunction with the accompanying drawings. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, 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.

[0039] Example 1

[0040] This embodiment provides an insect pathogenic nematode symbiotic bacteria preparation, which includes the following components in parts by weight: 50g of Enterobacter nematophila NK, 8g of an auxiliary agent and 18g of sterile water.

[0041] The auxiliary agent is composed of the following ingredients by weight: 2.5 g of emulsifier OP-10, 0.12 g of potassium sorbate, 3 g of ethylene glycol, 1.9 g of whole milk powder, 0.13 g of xanthan gum and 0.35 g of silicone.

[0042] The preparation method of the symbiotic bacteria preparation comprises the following steps:

[0043] S1. Pick out Enterobacter nematophila NK and streak it on NBTA identification medium, culture it at 28℃ for 36h, and pick out the primary single colony on the identification medium;

[0044] S2, streak the single colony picked in step S1 on the NA plate medium, culture at 28°C for 36 hours, and pick a single colony slant for storage;

[0045] S3, inoculating the single colony picked in step S2 into LB liquid culture medium, and culturing for 12 hours at 28°C and 180r / min to obtain a seed bacterial solution;

[0046] S4, taking the seed bacterial liquid cultured in step S3 and inoculating it in a shake flask fermentation medium at a volume ratio of 6%, and shaking and culturing it at 28° C. for 28 h to obtain a fermentation liquid;

[0047] S5, centrifuging the fermentation liquid obtained in step S4 to obtain Enterobacter nematophila NK bacterial precipitate;

[0048] S6. Weigh the following components: 50 g of Enterobacter nematophila NK, 8 g of an auxiliary agent, and 18 g of sterile water, and mix the components to obtain an insect pathogenic nematode symbiotic bacteria preparation.

[0049] The shake flask fermentation medium consists of: 250 g peptone, 70 g glucose, 0.5 g Houttuynia cordata extract, 20 g MgSO4, 8 g Na2SO4, 18 g (NH4)2SO4, 6.8 g KH2PO4, 13 g K2HPO4, add water to 10 kg, and adjust the pH to 7.2.

[0050] The composition of the NBTA plate medium is: 5 g of peptone, 16 g of agar powder, 3 g of beef extract, 0.025 g of bromomusk thymol blue, 0.04 g of triphenyltetrazolium chloride, add water to 1000 g, and adjust the pH to 7.2.

[0051] The composition of NA liquid culture medium is: 5g peptone, 16g agar powder, 03g beef extract, add water to 1000g, and adjust the pH to 7.4.

[0052] The composition of LB liquid culture medium is: 5 g yeast powder, 10 g peptone, 10 g sodium chloride, add water to 1000 g, and adjust the pH to 7.2.

[0053] Example 2

[0054] This embodiment provides an insect pathogenic nematode symbiotic bacteria preparation, which includes the following components by weight: 75g of Enterobacter nematophila NK, 20g of an auxiliary agent and 35g of sterile water.

[0055] The auxiliary agent is composed of the following ingredients by weight: 6.2 g of emulsifier OP-10, 0.3 g of potassium sorbate, 7.6 g of ethylene glycol, 4.6 g of whole milk powder, 0.3 g of xanthan gum and 1 g of silicone.

[0056] The preparation method of the symbiotic bacteria preparation comprises the following steps:

[0057] S1. Pick out Enterobacter nematophila NK and streak it on NBTA identification medium, culture it at 28°C for 24 hours, and pick out a primary single colony on the identification medium;

[0058] S2, streak the single colony picked in step S1 on NA medium and plate medium, culture at 28°C for 24h, and pick a single colony slant for storage;

[0059] S3, inoculating the single bacterial colony picked in step S2 into LB liquid culture medium, and culturing for 14 hours at 28°C and 180r / min to obtain a seed bacterial solution;

[0060] S4, taking the seed bacterial liquid cultured in step S3 and inoculating it in a shake flask fermentation medium at a volume ratio of 6%, and shaking and culturing it at 28° C. for 28 h to obtain a fermentation liquid;

[0061] S5, centrifuging the fermentation liquid obtained in step S4 to obtain Enterobacter nematophila NK bacterial precipitate;

[0062] S6. Weigh the following components: 50-75 parts of Enterobacter nematophila NK, 8-20 parts of auxiliary agent and 18-35 parts of sterile water, mix the components and obtain the insect pathogenic nematode symbiotic bacteria preparation. The shake flask fermentation medium consists of: 180g of peptone, 150g of glucose, 1.5g of Houttuynia cordata extract, 10g of MgSO4, 25g of Na2SO4, 34g of (NH4)2SO4, 7.8g of KH2PO4, 2g of K2HPO4, add water to 10kg, and adjust the pH to 7.4.

[0063] The composition of the NBTA plate medium is: 5 g of peptone, 16 g of agar powder, 3 g of beef extract, 0.025 g of bromomusk thymol blue, 0.04 g of triphenyltetrazolium chloride, add water to 1000 g, and adjust the pH to 7.4.

[0064] The composition of NA liquid culture medium is: 5g peptone, 16g agar powder, 03g beef extract, add water to 1000g, and adjust the pH to 7.2.

[0065] The composition of LB liquid culture medium is: 5 g yeast powder, 10 g peptone, 10 g sodium chloride, add water to 1000 g, and adjust the pH to 7.4.

[0066] Example 3

[0067] This embodiment provides an insect pathogenic nematode symbiotic bacteria preparation, which includes the following components by weight: 58g of Enterobacter nematophila NK, 15g of an auxiliary agent and 25g of sterile water.

[0068] The auxiliary agent is composed of the following ingredients in parts by weight: 4.6 g of emulsifier OP-10, 0.24 g of potassium sorbate, 5.77 g of ethylene glycol, 3.46 g of whole milk powder, 0.23 g of xanthan gum and 0.7 g of silicone.

[0069] The preparation method of the symbiotic bacteria preparation comprises the following steps:

[0070] S1. Pick out Enterobacter nematophila NK and streak it on NBTA identification medium, culture it at 28℃ for 25h, and pick out the primary single colony on the identification medium;

[0071] S2, streak the single colony picked in step S1 on NA medium and plate medium, culture at 28°C for 30h, and pick a single colony slant for storage;

[0072] S3, inoculating the single bacterial colony picked in step S2 into LB liquid culture medium, and culturing for 14 hours at 28°C and 180r / min to obtain a seed bacterial solution;

[0073] S4, taking the seed bacterial liquid cultured in step S3 and inoculating it in a shake flask fermentation medium at a volume ratio of 6%, and shaking and culturing it at 28° C. for 26 hours to obtain a fermentation liquid;

[0074] S5, centrifuging the fermentation liquid obtained in step S4 to obtain Enterobacter nematophila NK bacterial precipitate;

[0075] S6. Weigh the following components: 58 g of Enterobacter nematophila NK, 15 g of an auxiliary agent, and 25 g of sterile water, and mix the components to obtain an insect pathogenic nematode symbiotic bacteria preparation.

[0076] The shake flask fermentation medium consists of: 190 g peptone, 130 g glucose, 1.3 g Houttuynia cordata extract, 15 g MgSO4, 10 g Na2SO4, 30 g (NH4)2SO4, 7.1 g KH2PO4, 4 g K2HPO4, add water to 10 kg, and adjust the pH to 7.4.

[0077] The composition of the NBTA plate medium is: 5 g of peptone, 16 g of agar powder, 3 g of beef extract, 0.025 g of bromomusk thymol blue, 0.04 g of triphenyltetrazolium chloride, add water to 1000 g, and adjust the pH to 7.4.

[0078] The composition of NA liquid culture medium is: 5g peptone, 16g agar powder, 03g beef extract, add water to 1000g, and adjust the pH to 7.2.

[0079] The composition of LB liquid culture medium is: 5 g yeast powder, 10 g peptone, 10 g sodium chloride, add water to 1000 g, and adjust the pH to 7.4.

[0080] Example 4

[0081] This embodiment provides an insect pathogenic nematode symbiotic bacteria preparation, which includes the following components in parts by weight: 70 g of Enterobacter nematophila NK, 10 g of an auxiliary agent and 30 g of sterile water.

[0082] The auxiliary agent is composed of the following ingredients by weight: 3.07 g of emulsifier OP-10, 0.15 g of potassium sorbate, 3.86 g of ethylene glycol, 2.3 g of whole milk powder, 0.15 g of xanthan gum and 0.47 g of silicone.

[0083] The preparation method of the symbiotic bacteria preparation comprises the following steps:

[0084] S1. Pick out Enterobacter nematophila NK and streak it on NBTA identification medium, culture it at 28℃ for 30h, and pick out the primary single colony on the identification medium;

[0085] S2, streak the single colony picked in step S1 on NA medium and plate medium, culture at 28°C for 35h, and pick a single colony slant for storage;

[0086] S3, inoculating the single colony picked in step S2 into LB liquid culture medium, and culturing for 13 hours at 28°C and 180r / min to obtain a seed bacterial solution;

[0087] S4, taking the seed bacterial liquid cultured in step S3 and inoculating it into a shake flask fermentation medium at a volume ratio of 6%, and shaking and culturing it at 28° C. for 25 hours to obtain a fermentation liquid;

[0088] S5, centrifuging the fermentation liquid obtained in step S4 to obtain Enterobacter nematophila NK bacterial precipitate;

[0089] S6. Weigh the following components: 70 g of Enterobacter nematophila NK, 10 g of an auxiliary agent, and 30 g of sterile water, and mix the components to obtain an insect pathogenic nematode symbiotic bacteria preparation.

[0090] The shake flask fermentation medium consists of: 230 g peptone, 85 g glucose, 0.7 g Houttuynia cordata extract, 17 g MgSO4, 22 g Na2SO4, 25 g (NH4)2SO4, 7.6 g KH2PO4, 11 g K2HPO4, add water to 10 kg, and adjust the pH to 7.2.

[0091] The composition of the NBTA plate medium is: 5 g of peptone, 16 g of agar powder, 3 g of beef extract, 0.025 g of bromomusk thymol blue, 0.04 g of triphenyltetrazolium chloride, add water to 1000 g, and adjust the pH to 7.2.

[0092] The composition of NA liquid culture medium is: 5g peptone, 16g agar powder, 03g beef extract, add water to 1000g, and adjust the pH to 7.4.

[0093] The composition of LB liquid culture medium is: 5 g yeast powder, 10 g peptone, 10 g sodium chloride, add water to 1000 g, and adjust the pH to 7.2.

[0094] Example 5

[0095] This embodiment provides an insect pathogenic nematode symbiotic bacteria preparation, which includes the following components by weight: 60g of Enterobacter nematophila NK, 13g of an auxiliary agent and 27g of sterile water.

[0096] The auxiliary agent is composed of the following ingredients by weight: 4 g of emulsifier OP-10, 0.2 g of potassium sorbate, 5 g of ethylene glycol, 3 g of whole milk powder, 0.2 g of xanthan gum and 0.6 g of silicone.

[0097] The preparation method of the symbiotic bacteria preparation comprises the following steps:

[0098] S1. Pick out Enterobacter nematophila NK and streak it on NBTA identification medium, culture it at 28℃ for 33h, and pick out the primary single colony on the identification medium;

[0099] S2, streak the single colony picked in step S1 on NA medium and plate medium, culture at 28°C for 28h, and pick a single colony slant for storage;

[0100] S3, inoculating the single bacterial colony picked in step S2 into LB liquid culture medium, and culturing for 14 hours at 28°C and 180r / min to obtain a seed bacterial solution;

[0101] S4, taking the seed bacterial liquid cultured in step S3 and inoculating it in a shake flask fermentation medium at a volume ratio of 6%, and shaking and culturing it at 28° C. for 26 hours to obtain a fermentation liquid;

[0102] S5, centrifuging the fermentation liquid obtained in step S4 to obtain Enterobacter nematophila NK bacterial precipitate;

[0103] S6. Weigh the following components: 60 g of Enterobacter nematophila NK, 13 g of an auxiliary agent and 27 g of sterile water, and mix the components to obtain an insect pathogenic nematode symbiotic bacteria preparation.

[0104] The shake flask fermentation medium consists of: 220 g peptone, 110 g glucose, 0.7 g Houttuynia cordata extract, 18 g MgSO4, 10 g Na2SO4, 20 g (NH4)2SO4, 7.6 g KH2PO4, 12 g K2HPO4, add water to 10 kg, and adjust the pH to 7.2.

[0105] The composition of the NBTA plate medium is: 5 g of peptone, 16 g of agar powder, 3 g of beef extract, 0.025 g of bromomusk thymol blue, 0.04 g of triphenyltetrazolium chloride, add water to 1000 g, and adjust the pH to 7.3.

[0106] The composition of NA liquid culture medium is: 5g peptone, 16g agar powder, 03g beef extract, add water to 1000g, and adjust the pH to 7.4.

[0107] The composition of LB liquid culture medium is: 5 g yeast powder, 10 g peptone, 10 g sodium chloride, add water to 1000 g, and adjust the pH to 7.3.

[0108] Example 6

[0109] This embodiment provides an insect pathogenic nematode symbiotic bacteria preparation, which includes the following components by weight: 60g of Enterobacter nematophila NK, 13g of an auxiliary agent and 27g of sterile water.

[0110] The auxiliary agent is composed of the following ingredients by weight: 4 g of emulsifier OP-10, 0.2 g of potassium sorbate, 5 g of ethylene glycol, 3 g of whole milk powder, 0.2 g of xanthan gum and 0.6 g of silicone.

[0111] The preparation method of the symbiotic bacteria preparation comprises the following steps:

[0112] S1. Pick out Enterobacter nematophila NK and streak it on NBTA identification medium, culture it at 28℃ for 33h, and pick out the primary single colony on the identification medium;

[0113] S2, streak the single colony picked in step S1 on NA medium and plate medium, culture at 28°C for 28h, and pick a single colony slant for storage;

[0114] S3, inoculating the single bacterial colony picked in step S2 into LB liquid culture medium, and culturing for 14 hours at 28°C and 180r / min to obtain a seed bacterial solution;

[0115] S4, taking the seed bacterial liquid cultured in step S3 and inoculating it in a shake flask fermentation medium at a volume ratio of 6%, and shaking and culturing it at 28° C. for 26 hours to obtain a fermentation liquid;

[0116] S5, centrifuging the fermentation liquid obtained in step S4 to obtain Enterobacter nematophila NK bacterial precipitate;

[0117] S6. Weigh the following components: 60 g of Enterobacter nematophila NK, 13 g of an auxiliary agent and 27 g of sterile water, and mix the components to obtain an insect pathogenic nematode symbiotic bacteria preparation.

[0118] The shake flask fermentation medium consists of: 210 g peptone, 91 g glucose, 0.9 g Houttuynia cordata extract, 13 g MgSO4, 15 g Na2SO4, 24 g (NH4)2SO4, 7.2 g KH2PO4, 5 g K2HPO4, add water to 10 kg, and adjust the pH to 7.2.

[0119] The composition of the NBTA plate medium is: 5 g of peptone, 16 g of agar powder, 3 g of beef extract, 0.025 g of bromomusk thymol blue, 0.04 g of triphenyltetrazolium chloride, add water to 1000 g, and adjust the pH to 7.3.

[0120] The composition of NA liquid culture medium is: 5g peptone, 16g agar powder, 03g beef extract, add water to 1000g, and adjust the pH to 7.4.

[0121] The composition of LB liquid culture medium is: 5 g yeast powder, 10 g peptone, 10 g sodium chloride, add water to 1000 g, and adjust the pH to 7.3.

[0122] Example 7

[0123] This embodiment provides an insect pathogenic nematode symbiotic bacteria preparation, which includes the following components in parts by weight: 60g of Enterobacter nematophila NK, 13g of an auxiliary agent and 27g of sterile water.

[0124] The auxiliary agent is composed of the following ingredients by weight: 4 g of emulsifier OP-10, 0.2 g of potassium sorbate, 5 g of ethylene glycol, 3 g of whole milk powder, 0.2 g of xanthan gum and 0.6 g of silicone.

[0125] The preparation method of the symbiotic bacteria preparation comprises the following steps:

[0126] S1. Pick out Enterobacter nematophila NK and streak it on NBTA identification medium, culture it at 28℃ for 33h, and pick out the primary single colony on the identification medium;

[0127] S2, streak the single colony picked in step S1 on NA medium and plate medium, culture at 28°C for 28h, and pick a single colony slant for storage;

[0128] S3, inoculating the single bacterial colony picked in step S2 into LB liquid culture medium, and culturing for 14 hours at 28°C and 180r / min to obtain a seed bacterial solution;

[0129] S4, taking the seed bacterial liquid cultured in step S3 and inoculating it in a shake flask fermentation medium at a volume ratio of 6%, and shaking and culturing it at 28° C. for 26 hours to obtain a fermentation liquid;

[0130] S5, centrifuging the fermentation liquid obtained in step S4 to obtain Enterobacter nematophila NK bacterial precipitate;

[0131] S6. Weigh the following components: 60 g of Enterobacter nematophila NK, 13 g of an auxiliary agent and 27 g of sterile water, and mix the components to obtain an insect pathogenic nematode symbiotic bacteria preparation.

[0132] The shake flask fermentation medium consists of: 210 g peptone, 91 g glucose, 0.4 g Houttuynia cordata extract, 13 g MgSO4, 15 g Na2SO4, 24 g (NH4)2SO4, 7.2 g KH2PO4, 5 g K2HPO4, add water to 10 kg, and adjust the pH to 7.2.

[0133] The composition of the NBTA plate medium is: 5 g of peptone, 16 g of agar powder, 3 g of beef extract, 0.025 g of bromomusk thymol blue, 0.04 g of triphenyltetrazolium chloride, add water to 1000 g, and adjust the pH to 7.3.

[0134] The composition of NA liquid culture medium is: 5g peptone, 16g agar powder, 03g beef extract, add water to 1000g, and adjust the pH to 7.4.

[0135] The composition of LB liquid culture medium is: 5 g yeast powder, 10 g peptone, 10 g sodium chloride, add water to 1000 g, and adjust the pH to 7.3.

[0136] Example 8

[0137] This embodiment provides an insect pathogenic nematode symbiotic bacteria preparation, which includes the following components by weight: 60g of Enterobacter nematophila NK, 13g of an auxiliary agent and 27g of sterile water.

[0138] The auxiliary agent is composed of the following ingredients by weight: 4 g of emulsifier OP-10, 0.2 g of potassium sorbate, 5 g of ethylene glycol, 3 g of whole milk powder, 0.2 g of xanthan gum and 0.6 g of silicone.

[0139] The preparation method of the symbiotic bacteria preparation comprises the following steps:

[0140] S1. Pick out Enterobacter nematophila NK and streak it on NBTA identification medium, culture it at 28℃ for 33h, and pick out the primary single colony on the identification medium;

[0141] S2, streak the single colony picked in step S1 on NA medium and plate medium, culture at 28°C for 28h, and pick a single colony slant for storage;

[0142] S3, inoculating the single bacterial colony picked in step S2 into LB liquid culture medium, and culturing for 14 hours at 28°C and 180r / min to obtain a seed bacterial solution;

[0143] S4, taking the seed bacterial liquid cultured in step S3 and inoculating it in a shake flask fermentation medium at a volume ratio of 6%, and shaking and culturing it at 28° C. for 26 hours to obtain a fermentation liquid;

[0144] S5, centrifuging the fermentation liquid obtained in step S4 to obtain Enterobacter nematophila NK bacterial precipitate;

[0145] S6. Weigh the following components: 60 g of Enterobacter nematophila NK, 13 g of an auxiliary agent and 27 g of sterile water, and mix the components to obtain an insect pathogenic nematode symbiotic bacteria preparation.

[0146] The shake flask fermentation medium consists of: 210 g peptone, 91 g glucose, 1.7 g Houttuynia cordata extract, 13 g MgSO4, 15 g Na2SO4, 24 g (NH4)2SO4, 7.2 g KH2PO4, 5 g K2HPO4, add water to 10 kg, and adjust the pH to 7.2.

[0147] The composition of the NBTA plate medium is: 5 g of peptone, 16 g of agar powder, 3 g of beef extract, 0.025 g of bromomusk thymol blue, 0.04 g of triphenyltetrazolium chloride, add water to 1000 g, and adjust the pH to 7.3.

[0148] The composition of NA liquid culture medium is: 5g peptone, 16g agar powder, 03g beef extract, add water to 1000g, and adjust the pH to 7.4.

[0149] The composition of LB liquid culture medium is: 5 g yeast powder, 10 g peptone, 10 g sodium chloride, add water to 1000 g, and adjust the pH to 7.3.

[0150] Example 9

[0151] This embodiment provides an insect pathogenic nematode symbiotic bacteria preparation, which includes the following components by weight: 60g of Enterobacter nematophila NK, 13g of an auxiliary agent and 27g of sterile water.

[0152] The auxiliary agent is composed of the following ingredients by weight: 4 g of emulsifier OP-10, 0.2 g of potassium sorbate, 5 g of ethylene glycol, 3 g of whole milk powder, 0.2 g of xanthan gum and 0.6 g of silicone.

[0153] The preparation method of the symbiotic bacteria preparation comprises the following steps:

[0154] S1. Pick out Enterobacter nematophila NK and streak it on NBTA identification medium, culture it at 28℃ for 33h, and pick out the primary single colony on the identification medium;

[0155] S2, streak the single colony picked in step S1 on NA medium and plate medium, culture at 28°C for 28h, and pick a single colony slant for storage;

[0156] S3, inoculating the single bacterial colony picked in step S2 into LB liquid culture medium, and culturing for 14 hours at 28°C and 180r / min to obtain a seed bacterial solution;

[0157] S4, taking the seed bacterial liquid cultured in step S3 and inoculating it in a shake flask fermentation medium at a volume ratio of 6%, and shaking and culturing it at 28° C. for 26 hours to obtain a fermentation liquid;

[0158] S5, centrifuging the fermentation liquid obtained in step S4 to obtain Enterobacter nematophila NK bacterial precipitate;

[0159] S6. Weigh the following components: 60 g of Enterobacter nematophila NK, 13 g of auxiliary agent and 27 g of sterile water, mix the components and obtain the insect pathogenic nematode symbiotic bacteria preparation. The shake flask fermentation medium consists of: 210 g of peptone, 91 g of glucose, 13 g of MgSO4, 15 g of Na2SO4, 24 g of (NH4)2SO4, 7.2 g of KH2PO4 and 5 g of K2HPO4. Add water to 10 kg and adjust the pH to 7.2.

[0160] The composition of the NBTA plate medium is: 5 g of peptone, 16 g of agar powder, 3 g of beef extract, 0.025 g of bromomusk thymol blue, 0.04 g of triphenyltetrazolium chloride, add water to 1000 g, and adjust the pH to 7.3.

[0161] The composition of NA liquid culture medium is: 5g peptone, 16g agar powder, 03g beef extract, add water to 1000g, and adjust the pH to 7.4.

[0162] The composition of LB liquid culture medium is: 5 g yeast powder, 10 g peptone, 10 g sodium chloride, add water to 1000 g, and adjust the pH to 7.3.

[0163] Test Example 1

[0164] In order to verify the insecticidal activity of the insect pathogenic nematode symbiotic bacteria preparation of the present invention, this test set up a blank control group, an adjuvant control group, a non-adjuvant control group and groups of Examples 1 to 5. The test animals were larvae of Bradysia oleracea (leek maggots).

[0165] The feed for feeding the larvae of the leek bradya fungus gnat (leek maggots) is prepared by weight percentage: 9.2% of oyster mushroom powder, 7.7% of leek powder, 0.76% of yeast powder, 1.9% of agar powder, 0.08% of benzoic acid, 0.06% of sorbic acid, 76.5% of water and 3.8% of vitamin C.

[0166] The larvae of Bradysia vegetabilis (leek maggots) were packed in plates, with 20 maggots on each plate. Three parallel tests were set up for each group, for a total of 24 plates.

[0167] The blank control group was directly fed with feed; the adjuvant control group was fed with feed containing 5% of the weight of adjuvant, and the weight ratio of the adjuvant was 4 parts of emulsifier OP-10, 0.2 parts of potassium sorbate, 5 parts of ethylene glycol, 3 parts of whole milk powder, 0.2 parts of xanthan gum, 0.6 parts of silicone and 27 parts of sterile water; the adjuvant-free control group was fed with feed containing 5% of the weight of symbiotic bacteria suspension, and the weight ratio of the symbiotic bacteria suspension was 60 parts of Enterobacter nematophila NK and 27 parts of sterile water; the groups of Examples 1 to 5 were fed with feed containing 5% of the weight of the symbiotic bacteria preparation obtained in Examples 1 to 5.

[0168] The animals were observed once 6 hours after the first feeding, and then every 12 hours. The animals were fed once every 12 hours. The mortality of the larvae of the leek black eye fungus gnat (leek maggots) was calculated after 48 hours. The results are shown in Tables 1 and Figure 1 .

[0169] Table 1. Insecticidal activity test of symbiotic bacteria preparations against larvae of Bradysia chinensis (leek maggots)

[0170]

[0171] Note: Average values ​​are rounded down

[0172] As shown in Table 1, the killing rate of the symbiotic bacteria preparations obtained in Examples 1 to 5 on the leek Bradysia fungi larvae (leek maggots) reached more than 90%, and the killing rate of the symbiotic bacteria preparations obtained in Examples 4 and 5 on the leek Bradysia fungi larvae (leek maggots) reached 100%. The insect pathogenic nematode symbiotic bacteria preparation obtained by the present invention has an excellent effect of killing the leek Bradysia fungi larvae (leek maggots).

[0173] Test Example 2

[0174] The low-temperature preserved Botrytis cinerea pathogen was cultured at 28°C and 150r / min with constant temperature shaking for 48h, and the seed liquid was collected for later biopsy and pot test. At the same time, the collected seed liquid was spread on PDA plates, and the cultured plates were selected and several fungus cakes were punched out from the edge of the culture medium with a fungus puncher with a diameter of 1cm in preparation for the experiment of inhibiting mycelial growth.

[0175] 1. Symbiotic bacteria preparations inhibit the growth of Botrytis cinerea hyphae

[0176] In order to verify the inhibitory effect of the symbiotic bacteria preparation obtained in the present invention on the mycelial growth of Botrytis cinerea, this experiment was conducted. A blank control group, an adjuvant control group, a non-adjuvant control group and groups 1 to 5 of Examples were set up in this experiment.

[0177] No other ingredients were added to the PDA culture medium of the blank control group; 5% (weight ratio) of the additive was added to the PDA culture medium of the adjuvant control group, and the weight ratio of the adjuvant was 4 parts of emulsifier OP-10, 0.2 parts of potassium sorbate, 5 parts of ethylene glycol, 3 parts of whole milk powder, 0.2 parts of xanthan gum, 0.6 parts of silicone and 27 parts of sterile water; 5% (weight ratio) of the symbiotic bacteria suspension was added to the PDA culture medium of the no-adjuvant control group, and the weight ratio of the symbiotic bacteria suspension was 60 parts of Enterobacter nematophila NK and 27 parts of sterile water; 5% (weight ratio) of the symbiotic bacteria preparation obtained according to Examples 1 to 5 was added to the PDA culture medium of Examples 1 to 5.

[0178] The PDA medium of each group was poured into a culture dish. After cooling, a 1-cm diameter cake of Botrytis cinerea was inverted in the center of the culture dish. The edge of the culture dish was sealed with a breathable filter membrane and cultured at 28°C. After 60 hours, the colony diameter was measured by the cross method and the inhibition efficiency was calculated. The results are shown in Table 2 and Figure 2 Bacterial inhibition rate (%) = [(control colony diameter - bacterial cake diameter) - (treated colony diameter - bacterial cake diameter)] / (control colony diameter - bacterial cake diameter) × 100%. Three parallel tests were performed in each group.

[0179] Table 2. Inhibitory effect of symbiotic bacteria preparations on the hyphae of Botrytis cinerea

[0180]

[0181] It can be seen from Table 2 that the antibacterial rates of Examples 1 to 5 all reached 100%, the antibacterial rate of the adjuvant control group was 23.08%, and the antibacterial rate of the non-adjuvant control group was 76.92%. It can be seen that the combination of Enterobacter nematophila NK and adjuvants is beneficial to improving the antibacterial effect.

[0182] 2. Protective effect of symbiotic bacteria preparation on living tissue of leek

[0183] Spray the pesticide on the freshly picked disease-free leek leaves in groups until the pesticide drips on the leek leaves.

[0184] The agent sprayed by the blank control group was sterile water; the agent components sprayed by the adjuvant control group were arranged by weight as 4 parts of emulsifier OP-10, 0.2 parts of potassium sorbate, 5 parts of ethylene glycol, 3 parts of whole milk powder, 0.2 parts of xanthan gum, 0.6 parts of silicone and 27 parts of sterile water; the agent components sprayed by the no-adjuvant control group were arranged by weight as 60 parts of Enterobacter nematophila NK and 27 parts of sterile water; the agents sprayed by Examples 1 to 5 were the symbiotic bacteria preparations obtained according to Examples 1 to 5, respectively.

[0185] The leek leaves sprayed with the pesticide were placed in a moist filter paper culture dish at 28°C for constant temperature and moisture preservation. After 48 hours, the leek leaves were inoculated with the seed solution of Botrytis cinerea, with three inoculation points on each leek leaf. After 60 hours, the surface of the leek leaves in each test group was observed for the formation of Botrytis cinerea hyphae. The results are shown in Table 3 and Figure 3 .

[0186] Table 3. The protective effect of symbiotic bacteria preparation on the tissues of living leek

[0187]

[0188] As shown in Table 3, the symbiotic bacteria preparations of Examples 1 to 5 have a protective effect on the infection of leeks by Botrytis cinerea, the pathogen of leek gray mold.

[0189] 3. The therapeutic effect of symbiotic bacteria preparations on leek gray mold

[0190] The pathogen of gray mold, Botrytis cinerea, was inoculated on freshly picked disease-free leek leaves, placed in a moist filter paper culture dish, and cultured at a constant temperature and humidity of 28°C. After 48 hours, the test group of agents was sprayed until the surface of the leek leaves was moistened. The spraying was done once every 24 hours. After 60 hours, the colonies of Botrytis cinerea on the surface of the leek leaves were observed to see if they were different from those of the leek leaves of the control group that were not sprayed with agents, and whether they had an inhibitory effect on the growth of the gray mold pathogen. The results are shown in Tables 4 and Figure 4 .

[0191] The agent sprayed by the blank control group was sterile water; the agent components sprayed by the adjuvant control group were arranged by weight as 4 parts of emulsifier OP-10, 0.2 parts of potassium sorbate, 5 parts of ethylene glycol, 3 parts of whole milk powder, 0.2 parts of xanthan gum, 0.6 parts of silicone and 27 parts of sterile water; the agent components sprayed by the no-adjuvant control group were arranged by weight as 60 parts of Enterobacter nematophila NK and 27 parts of sterile water; the agents sprayed by Examples 1 to 5 were the symbiotic bacteria preparations obtained according to Examples 1 to 5, respectively.

[0192] Table 4. Treatment results of symbiotic bacteria preparations against gray mold of leek

[0193]

[0194] As shown in Table 4, the symbiotic bacteria preparations of Examples 1 to 5 have a therapeutic effect on leeks infected by Botrytis cinerea, the pathogen of leek gray mold.

[0195] 4. The inhibitory effect of symbiotic bacteria preparation on Botrytis cinerea of ​​potted leeks

[0196] The healthy potted leeks in the experimental base were selected for the test. The tissue determination method was adopted. The wound was gently scratched on the leek leaves with an inoculation needle but the leaves could not be pierced. The gray mold pathogen Botrytis cinerea was inoculated on the leaves. This test set up a blank control group, an adjuvant control group, a non-adjuvant control group and groups 1 to 5 of Examples. The agent was sprayed 48 hours after inoculation of Botrytis cinerea, and the spraying was continued for one week and the results were observed and recorded. The results are shown in Table 5.

[0197] The agent sprayed by the blank control group was sterile water; the agent components sprayed by the adjuvant control group were arranged by weight as 4 parts of emulsifier OP-10, 0.2 parts of potassium sorbate, 5 parts of ethylene glycol, 3 parts of whole milk powder, 0.2 parts of xanthan gum, 0.6 parts of silicone and 27 parts of sterile water; the agent components sprayed by the no-adjuvant control group were arranged by weight as 60 parts of Enterobacter nematophila NK and 27 parts of sterile water; the agents sprayed by Examples 1 to 5 were the symbiotic bacteria preparations obtained according to Examples 1 to 5, respectively.

[0198] Table 5. Inhibitory effect of symbiotic bacteria preparation on Botrytis cinerea in potted leeks

[0199]

[0200] It can be seen from Table 5 that the symbiotic bacteria preparations obtained in Examples 1 to 5 have a significant inhibitory effect on Botrytis cinerea.

[0201] Test Example 3

[0202] This test example sets up Examples 1 to 9, and each set up three parallel tests to investigate the yield of Enterobacter nematophila NK. Examples 1 to 9 were respectively cultured according to steps S1 to S5 in the preparation method of the symbiotic bacteria preparation in Examples 1 to 9, and the bacterial precipitate obtained by centrifugation was weighed, and the bacterial output per liter of shake flask fermentation medium was calculated. The results are shown in Tables 6 and Figure 5 .

[0203] Table 6. Yield of Enterobacter nematophila NK in Examples 1 to 9

[0204]

[0205] As can be seen from Table 6, the average output of Example 1 to Example 6 is higher than 111.6 g / L, among which the average output of Example 6 is the highest, reaching 137.5 g / L. The average output of Example 8 is only 59.4 g / L. In the shake flask fermentation medium of Examples 1 to 6, 0.005-0.015% of Houttuynia cordata extract was added, while the weight percentage of Houttuynia cordata extract added to the shake flask fermentation medium of Example 7 was 0.004%, the weight percentage of Houttuynia cordata extract added to the shake flask fermentation medium of Example 8 was 0.017%, and no Houttuynia cordata extract was added to the shake flask fermentation medium of Example 9. It can be seen that adding 0.005-0.015% of Houttuynia cordata extract to the shake flask fermentation medium has a promoting effect on increasing the yield of Enterobacter nematophila NK.

[0206] The embodiments of the present invention are 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 of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the scope of protection of the present invention.

Claims

1. A method for preparing an insect pathogenic nematode symbiotic bacteria preparation, characterized in that: The steps include: S1. Pick out Enterobacter nematophila NK and streak it on NBTA identification medium, culture it at 28℃ for 24-36h, and pick out the primary single colony on the identification medium; S2, streaking the single colony picked in step S1 on the NA plate medium, culturing at 28°C for 24-36h, and picking a single colony slant for storage; S3, inoculating the single colony picked in step S2 into LB liquid culture medium, and culturing at 28°C and 180 r / min for 12-14 h to obtain a seed bacterial solution; S4, taking the seed bacterial liquid cultured in step S3 and inoculating it into a shake flask fermentation medium at a volume ratio of 6%, and shaking and culturing it at 28° C. for 24 to 28 hours to obtain a fermentation liquid; S5, centrifuging the fermentation liquid obtained in step S4 to obtain a precipitate of Enterobacter nematophila NK bacteria; S6. Weigh the following components by weight: 50-75 parts of Enterobacter nematophila NK, 8-20 parts of adjuvants and 18-35 parts of sterile water, and mix the components to obtain an insect pathogenic nematode symbiotic bacteria preparation; The shake flask fermentation medium is composed of 1.8-2.5% peptone, 0.7-1.5% glucose, 0.005-0.015% houttuynia cordata extract, 0.1-0.2% MgSO4, 0.08-0.25% Na2SO4, 0.18-0.34% (NH4)2SO4, 0.068-0.078% KH2PO4, 0.02-0.13% K2HPO4, and the balance is water, and the pH is adjusted to 7.2-7.

4.

2. The method for preparing the insect pathogenic nematode symbiotic bacteria preparation according to claim 1, characterized in that: The shake flask fermentation medium is composed of 2.1% peptone, 0.91% glucose, 0.009% houttuynia cordata extract, 0.13% MgSO4, 0.15% Na2SO4, 0.24% (NH4)2SO4, 0.072% KH2PO4, 0.05% K2HPO4, and the balance is water, and the pH is adjusted to 7.2-7.4.

Images