Formula of liquid culture medium for entomopathogenic nematodes and culture method

Abstract

The invention discloses a liquid culture medium formula and a culture method for entomopathogenic nematodes, and the formula is obtained by adding isopropylstilbene (3, 5-dihydroxy-4-isopropyl stilbene) and insulin into a basic culture medium together. When the culture medium formula is used for culturing the entomopathogenic nematodes, the quality of the entomopathogenic nematodes is obviously improved, and the yield of the entomopathogenic nematodes is increased to 20 * 10 < 4 > IJs / mL from 12.6 * 10 < 4 > infection period (IJs) / mL of a basic culture medium; the survival rate is increased from 31.42% (basic culture medium) to 56.83% after the culture medium is stored at low temperature for 1 month, and the shelf life is prolonged. Compared with traditional solid culture, the culture medium formula and the culture method have the advantages that the culture period is shortened, the quality of the entomopathogenic nematodes is improved, the production cost of raw materials is reduced, commercial production and development of the entomopathogenic nematodes can be greatly promoted, and new opportunities and wide development prospects are brought to related industries.

Description

Technical Field

[0001] The invention belongs to the technical field of insect pathogenic nematode cultivation, and particularly relates to an insect pathogenic nematode liquid culture medium formula and a cultivation method. Background Art

[0002] Entomopathogenic nematodes (EPNs) are obligate parasitic natural enemies of insects and have been used to control various pests. Insect pathogenic nematodes can kill pests mainly because of the symbiotic bacteria in their bodies. The symbiotic relationship begins when the insect host infects the free-living third-stage infective larvae. These larvae house mutualistic symbiotic bacteria in their intestines, which makes them toxic to insects. EPNs can actively seek out hosts through chemical stimulation of the host itself or other substances, and use mechanical and enzymatic means to enter the insect hemocoel, release bacteria, and kill the insect host within 48 hours. Symbiotic bacteria produce various antimicrobial substances and decompose host tissues during metabolism, providing a suitable environment and essential nutrients for nematode proliferation.

[0003] Since EPNs have the advantages of actively searching for hosts, high efficiency, wide host range, and environmental friendliness, they have very good effects in pest biological control. Globally renowned companies, such as Germany's E-nema, the Netherlands' Koppert, the United Kingdom's Becker Underwood, and Japan's SDS, are committed to the research and development and commercialization of such biological pesticides. However, due to the immaturity of domestic commercialization technology, in vitro solid culture is still the main method, and the high production cost makes the use of insect pathogenic nematodes not widespread. The feasibility of low-cost industrial mass production of IJs, as well as maintaining infectivity and extending storage life are the main challenges for the successful commercialization of EPNs as biological pesticide products. Due to its economies of scale, many authors believe that in vitro liquid culture is generally considered to be the preferred technology for large-scale commercial mass production of EPNs.

[0004] The current insect pathogenic nematode cultivation technologies mainly include:

[0005] (1) In vivo culture:

[0006] The nematodes are inoculated on a petri dish or tray containing a wax moth lined with absorbent paper (e.g., filter paper), or another substrate that is conducive to nematode infection. After the wax moth dies, it is transferred to a collection plate (a set of a large and a small plate), the small plate is wrapped with filter paper, the worm body is placed on top, and the large plate is filled with sterile water (not covering the small plate in the middle). The emerging daughter infectious larvae migrate to the surrounding water and are captured and then harvested. The whole process takes about 14 days.

[0007] (2) In vitro culture:

[0008] Solid culture: Use sterilized polypropylene tubes, conical flasks, etc. as breeding containers, which must be anti-contamination and well ventilated. Use small pieces of sponge as carriers and fill in the homogenate of chicken viscera or other animal viscera as the culture medium. Inoculate the symbiotic bacteria and nematodes into the prepared culture medium. Before inoculation, the bacteria and nematodes need to be treated to ensure their activity and purity. Place the inoculated breeding container under appropriate conditions for cultivation, observe the growth of the nematodes regularly, and harvest when the number of nematodes reaches a certain level.

[0009] Liquid culture:

[0010] Prepare a liquid culture medium suitable for the growth of insect pathogenic nematodes and symbiotic bacteria, which contains necessary nutrients such as carbon source, nitrogen source, inorganic salts, etc., inoculate the purified and activated symbiotic bacteria and nematodes into the liquid culture medium, and culture them in containers such as Erlenmeyer flasks or fermentation tanks, and control the temperature, stirring speed, ventilation volume, etc. After culturing for a period of time, harvest the nematode culture solution and obtain the nematodes by centrifugation and filtration.

[0011] Research on liquid culture of nematodes has mainly focused on improving the yield, quality and cost of nematodes. The maximum yield can be obtained by optimizing the culture medium formulation and culture conditions. Lipids are the main energy source for nematodes during the infection period. Since parasitic nematodes have limited ability to synthesize lipids, they rely on the host to provide the necessary lipids. Therefore, the core components of the liquid culture medium are lipid content and composition. The in vitro liquid culture process can be divided into two growth stages: symbiotic bacteria and nematodes. Currently, EPNs are produced in large bioreactors using liquid-based culture media. However, liquid culture methods still need to be fine-tuned and optimized to reduce production costs, reduce production time and cultivate large quantities of high-quality nematodes, promoting the commercial production and development of insect pathogenic nematodes. Summary of the invention

[0012] Under the current technical background, the existing insect pathogenic nematode cultivation technology faces many severe challenges. On the one hand, its high cost makes related enterprises bear a heavy burden on production input. At the same time, the long cultivation cycle greatly reduces production efficiency and cannot meet the rapid market demand. Furthermore, the low yield leads to insufficient product supply, making it difficult to form economies of scale. In addition, the low pathogenicity greatly reduces its effectiveness in pest control and disease prevention, making it difficult to play its due role. Moreover, the short delivery time also brings great uncertainty to production arrangements and market supply. These factors work together to make it impossible for the cultivation technology of insect pathogenic nematodes to be successfully commercialized and promoted in my country. The technical formula of this application has a good effect on improving the yield, growth rate and survival rate of insect pathogenic nematodes. Compared with a single optimized ingredient, this formula successfully overcomes the problem of incomplete nutrition and provides a richer and more balanced nutritional environment for the growth of insect pathogenic nematodes. Compared with the traditional solid culture method, this new technical formula can not only greatly shorten the culture cycle and significantly improve production efficiency, but also effectively reduce the production cost of raw materials, greatly promoting the commercial production and development of insect pathogenic nematodes, and bringing new opportunities and broad development prospects to related industries.

[0013] In view of this, one of the purposes of the present invention is to provide a liquid culture medium formula for insect pathogenic nematodes, which comprises a basal culture medium and Isopropylstilbene (3,5-dihydroxy-4-isopropyl diphenylethylene) and insulin. That is, Isopropylstilbene and insulin are added together to the basal culture medium for liquid culture of insect pathogenic nematodes.

[0014] Preferably, the composition and content of the basic culture medium are: yeast extract 15g / L, soybean powder 15g / L, sunflower oil 25mL / L, KCl 4g / L, NaCl 0.3g / L, CaCl2 0.2g / L, MgSO4 0.35g / L.

[0015] Preferably, the Isopropylstilbene is added to the basal medium in the following manner: Isopropylstilbene is diluted to 2 nM with 0.1% dimethyl sulfoxide and then added to the basal medium, so that the concentration of the added Isopropylstilbene is 0.02 nM;

[0016] The insulin is added to the basal culture medium in the following manner: 1 mL of pH = 2 hydrochloric acid solution is added to 1 mg of insulin dry powder to prepare an insulin mother solution, and then an insulin optimization solution is prepared at a volume ratio of 1:500 between the insulin mother solution and Ringer's solution, and finally the insulin optimization solution is added to the basal culture medium to a concentration of 1 μL / mL.

[0017] Preferably, the insect pathogenic nematode species is the insect pathogenic nematode Steinernema carpocapsae All.

[0018] The second object of the present invention is to provide a method for culturing insect pathogenic nematodes in liquid culture, comprising the following steps:

[0019] 1) Preparing a bacterial suspension of symbiotic bacteria of insect pathogenic nematodes;

[0020] 2) Add the bacterial suspension to the above formula at a volume ratio of 1:25 and culture at 25°C and 180 rpm for 48 hours;

[0021] 3) Add insect pathogenic nematodes, continue culturing for 10-15 days, and harvest the insect pathogenic nematodes. The amount of insect pathogenic nematodes added can be 1000 or 2000 IJs / mL per 50 mL of culture medium. The yield of nematodes inoculated with 1000 or 2000 IJs / mL is similar, so 1000 IJs / mL can be selected.

[0022] Preferably, the symbiotic bacteria is Xenorhabdus.

[0023] Preferably, the method for preparing the bacterial suspension comprises: activating the symbiotic bacteria to obtain a single colony without contamination, and then inoculating the single colony into LB liquid culture medium and culturing on a shaker at 25° C. and 180 rpm for 48 hours to obtain a bacterial suspension.

[0024] Preferably, the continuing culturing for 10-15 days in step 3) is continuing culturing for 10 days.

[0025] Preferably, the insect pathogenic nematodes harvested in step 3) are stored in a low temperature environment.

[0026] The third object of the present invention is to provide the use of the above-mentioned insect pathogenic nematode liquid culture medium formula in increasing the culture quantity and / or survival rate of insect pathogenic nematodes.

[0027] The advantages of the present invention are:

[0028] (1) The culture medium formula of the present invention is used to cultivate nematodes, which has the advantages of high yield and strong pathogenicity.

[0029] (2) The nematodes produced using the culture medium formula of the present invention have a high survival rate after storage and their pathogenicity is not weakened.

[0030] (3) The culture medium formula of the present invention is used to produce worms, which has the advantages of a shorter growth cycle than traditional solid and liquid cultures and low cost.

[0031] The culture medium formula of the present invention not only improves the quality of insect pathogenic nematodes, but also reduces the production cost of raw materials, which can greatly promote the commercial production and development of insect pathogenic nematodes and bring new opportunities and broad development prospects to related industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The change of bacterial mass during the fermentation process of colonies with different diameters in LB liquid medium;

[0033] Figure 2 The effect of different liquid culture media on nematode production;

[0034] Figure 3 The results of the survival rate test of nematode sponges cultured in different media after being stored for one month;

[0035] Figure 4 The effects of different culture media and storage for one month on the pathogenicity of nematodes. DETAILED DESCRIPTION

[0036] The present invention will be described in detail below in conjunction with examples, which are merely illustrative and are not intended to limit the scope of application of the present invention. The present invention is not limited to the following embodiments or examples, and any modifications and variations that do not violate the spirit of the present invention should be included within the scope of the present invention.

[0037] In this embodiment, Isopropylstilbene (3,5-dihydroxy-4-isopropyl diphenylethylene) and insulin were added to the basal medium for liquid culture of insect pathogenic nematodes; the scientific name of the insect pathogenic nematode species used was Steinernema carpocapsae All (Sc), which was purchased from Weifang Hongrun Agricultural Technology Co., Ltd. The following steps were included:

[0038] 1. Fermentation growth curve of Sc nematode symbiotic bacteria

[0039] Take out the Xenorhabdus strain (purchased from Weifang Hongrun Agricultural Technology Co., Ltd.) from the -80℃ refrigerator and streak it on the NBTA plate for culture. After 48 hours, pick up blue-green single colonies with diameters of 1.1mm, 1.5mm, and 1.9mm, respectively, and inoculate them into a 250mL Erlenmeyer flask filled with 150mL LB liquid culture medium at 25℃ and 180rpm. Take samples every 6 hours from 0h, and take 1mL of bacterial solution each time and add it to the cuvette to measure the OD of the bacterial solution at different culture times. 600 , repeated three times, and measured continuously for 7 days. The growth curve of bacterial fermentation process was drawn with time as the horizontal axis and the OD value of the bacterial solution as the vertical axis. 1 mL of fresh bacterial solution was taken from each time period and diluted 10, 102 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , from the diluted bacterial solution, draw 100 μL of bacterial solution into NBTA medium, spread it evenly with a glass rod, and then invert it in a 28°C incubator. When colonies grow on the plate, start counting. Repeat 3 times for each dilution concentration. With time as the horizontal axis and the number of live bacteria in the bacterial solution as the vertical axis, draw a curve of the number of live bacteria during the bacterial fermentation process.

[0040] The results are as follows Figure 1 (Among them, (A) bacterial fermentation process growth curve, (B) bacterial fermentation process live bacteria count) As shown: With the extension of fermentation time, the amount of bacteria gradually increased. The results of fermentation time, total bacteria count and live bacteria count showed that the logarithmic phase live bacteria count and total bacteria count (OD 600 It is positively correlated with the fermentation time, indicating that with the increase of fermentation time, the bacteria in the logarithmic phase multiply rapidly.

[0041] Combined with OD 600 The growth trends of the three colonies with different diameters were roughly similar, with the colony with d = 1.9 mm entering the logarithmic growth phase (12 h) first and the stable phase being 42 ± 6 h; the other two entered the logarithmic growth phase at 30 h and the stable phase being 42 ± 6 h. During the culture of 1.1-1.9 mm colonies, the highest colony forming units per milliliter could reach 82.72 × 10 6 cells、75.60×10 6 cells, 79.71×10 6 cells. 42-78h(OD 600 =1.4-1.7) is the optimal time for adding bacteria to nematode culture medium and subsequent additions.

[0042] 2. Liquid culture of single-strain EPNs

[0043] The Xenorhabdus bacterial suspension was taken out from the -80°C ultra-low temperature freezer and streaked on an NBTA plate for activation culture (28°C). After about 48 hours, it was tested for contamination using a hydrogen peroxide solution (no contamination if no bubbles appeared), and independent and full colonies (primary type) were selected and inoculated into LB liquid culture medium and cultured on a shaker at 25°C and 180 rpm for about 48 hours to obtain a bacterial preculture (i.e., bacterial suspension).

[0044] 2 mL of the above bacterial pre-culture was inoculated into each 250 mL Erlenmeyer flask containing 50 mL of sterilized nematode liquid culture medium, and continued to be placed in a shaker for 48 hours (parameters are the same as above). Then, 1000 or 2000 IJs / mL (the initial concentration of nematodes in each basic culture medium is 2000 IJs / mL, and the other different optimized culture media are 1000 IJs / mL) of sterilized Sc single bacterial nematodes (infective nematodes: IJs) were inoculated in each flask, and culture was continued (shaking parameters are the same as above), and observation was made every 5 days until the number of infective nematodes or the overall population showed a downward trend, and observation was stopped and harvested. The components of each liquid culture medium are shown in Tables 1 and 2. Table 1 is the basic culture medium (liquid culture medium, the solvent is water); Table 2 is the optimized components, which can be added to the basic culture medium to optimize the formula.

[0045] Table 1 Basic culture medium

[0046]

[0047]

[0048]

[0049] Table 2 Optimized ingredients

[0050]

[0051] The specific method of adding the optimized components in Table 2 to the basal medium is:

[0052] (1) Insulin: 1 mL of pH = 2 hydrochloric acid solution was added to 1 mg of insulin dry powder to prepare an insulin mother solution, and then the insulin mother solution was mixed with Ringer's solution at a volume ratio of 1:500 or 1:5000 to obtain an insulin optimized solution. Finally, the insulin optimized solution was added to the basal culture medium so that the concentration of the insulin optimized solution after addition was 1 μL / mL;

[0053] (2) ISO: ISO stock was diluted to 2 nM with 0.1% dimethyl sulfoxide and added to the basal medium to a concentration of 0.02 nM.

[0054] (3) ascr#5: the ascr#5 stock solution was prepared with sterile 0.1% PBS solution to a concentration of 2 nM, and then added to the basal culture medium to make the concentration of ascr#5 0.02 nM after addition; ascr#7 was added in the same manner;

[0055] (4) In addition to the above ingredients, the dosages of the remaining ingredients in Table 2 (chlorogenic acid, dimethyl sulfoxide and insect powder) are all concentrations after being directly added to the basal culture medium without any special treatment.

[0056] 3. Determination of EPNs yield

[0057] The nematode sample was diluted 5-100 times, 100 μL of the dilution was placed in a 96-well plate, and a 5-15 second video was recorded using a computer with video recording software connected to a stereo microscope. The nematodes were then counted using automatic counting software, and the whole process was repeated 4 times.

[0058] The results are as follows Figure 2 (Among them, (A) different basal culture media, (B) adding an optimized culture media with different optimized components to the basal culture media, (C) adding two optimized culture media with different optimized components to the basal culture media) As shown: The best harvest time for nematode culture is 10 days. Among them, the F basal culture media has the highest yield, about 130,000 IJs / mL, followed by C and E culture media, with a yield of about 90,000 IJs / mL ( Figure 2 A). After adding optimized components to F as the basic medium, the nematode yield in Fc medium (Fc means that the F basic medium in Table 1 is added with the optimized component numbered c in Table 2, and the meanings of the following symbols such as Fe, Fa, Fcd, and Fbf refer to Fc) was the highest (157,000 IJs / mL), followed by Fe medium (138,400 IJs / mL); the nematode yields in Fa and Fd medium were the lowest, both at 90,000 IJs / mL ( Figure 2 B), and significantly lower than F medium, it is speculated that insulin may have an antagonistic effect with a certain component in the medium. The medium with insect powder added will cause nematodes to grow slowly or even fail to grow. When adding the two optimized components, such as Figure 2 As shown in C, the yield of Fcd was the highest at 10 days, reaching 196,300 IJs / mL, which was about 60,000 IJs / mL higher than F medium, about 40,000 IJs / mL higher than Fc medium, and much higher than Fd medium; and the other mediums with two optimized components had poor effects, among which Fbf did not even have a higher yield than F, Fb, and Ff medium, even though the yield of Fb and Ff medium was higher than F. This indicates that in the in vitro culture of EPNs, the simultaneous addition of Isopropylstilbene and insulin has a synergistic effect on the culture of nematodes.

[0059] 4. EPNs survival rate determination

[0060] The EPN was washed and stored in a shallow water layer and a sponge at 4±1°C, and the survival rate of the nematodes was determined after 1 month of storage. The sponge storage method was carried out using a 6-well plate, with an equal amount of sponge and nematode suspension (7 mL) (nematodes harvested after 10 and 15 days of culture) placed in each well. After the storage time reached the target time, it was washed out and fixed to 20-50 mL with pure water, and the amount of nematodes per milliliter was calculated. Because the basal culture medium was stored in a shallow water layer and a sponge, it was found that the mortality rate of nematodes stored in the shallow water layer was as high as 95%, which was much higher than that of sponge storage, so subsequent experiments used sponge storage.

[0061] The survival rate of nematodes was Figure 3 As shown: after the nematode sponge harvested after 10 days of culture was stored for one month, the survival rate of nematodes in the culture medium with two optimized ingredients, Fcd, was the highest, reaching 56.83%, followed by Fed (55.41%); overall, the survival rate of nematodes harvested after 15 days of culture was higher than that of nematodes harvested after 10 days, but the survival rate of nematodes in Fcd culture medium after 10 days and 15 days of culture was almost the same; in addition, the survival rate of nematodes in the culture medium with optimized ingredients was higher than that without optimized ingredients, especially for nematodes cultured for 10 days, the survival rate of nematodes was significantly improved by adding optimized ingredients.

[0062] 5. Determination of pathogenicity of EPNs

[0063] The toxicity evaluation was carried out at a temperature of 27±1°C and a relative humidity of 65% in an artificial climate box. Two layers of filter paper were placed in a 90 mm culture dish, 1.5 mL of pure water (including nematode suspension) was added to keep the filter paper moist, and the EPNs cultured in different culture media were dripped into the culture dish using a micropipette at a number of 120 nematodes per mealworm or greater wax moth larvae, and a control group was established by adding an equivalent volume of pure water, with 10 mealworms or greater wax moth larvae per dish, and each treatment was repeated three times.

[0064] The nematodes were harvested and tested for pathogenicity to the larvae of Tenebrio molitor (T) or G. mellonella (G). Figure 4 (Among them, (A) the pathogenicity of nematodes harvested after 10 days of culture in the basic culture medium before preservation, (B) and (C) the pathogenicity of nematodes harvested after 10 and 15 days of culture in the optimized culture medium with two different optimized components added to the basic culture medium before preservation, (D) the pathogenicity of nematodes harvested after 10 days of culture in the basic culture medium after preservation, (E) and (F) the pathogenicity of nematodes harvested after 10 and 15 days of culture in the optimized culture medium with two different optimized components added to the basic culture medium after preservation). Overall, for the basic culture medium, the pathogenicity of nematodes cultured in F culture medium was the best overall effect; for the culture medium with two optimized components added, the pathogenicity of each remained at a high level at 48 hours, indicating that the addition of optimized components had no significant effect on pathogenicity.

[0065] In summary, it is shown that the combined addition of Isopropylstilbene and insulin to the basal culture medium of the present invention has a better effect on the culture of nematodes, not only greatly increasing the number of cultures, but also improving the survival rate, without affecting the pathogenicity of the nematodes.

[0066] The conventional techniques and schemes not described in detail in the above embodiments are well known in the art, so they will not be described in detail here. The above embodiments and / or experimental examples describe the preferred embodiments of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, the technical scheme of the present invention can be subjected to a variety of simple modifications, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A liquid culture medium formulation for insect pathogenic nematodes, characterized in that: Includes basal medium and isopropylstilbene and insulin.

2. The formulation according to claim 1, characterized in that The composition and content of the basic culture medium are: yeast extract 15g / L, soybean powder 15g / L, sunflower oil 25g / L, NaCl 4g / L, CaCl2 0.3g / L, MgSO4 0.2g / L, KCl 0.35g / L.

3. The formulation according to claim 1, characterized in that The Isopropylstilbene is added to the basal medium in the following manner: Isopropylstilbene is diluted to 2 nM with 0.1% dimethyl sulfoxide and then added to the basal medium, so that the concentration of the added Isopropylstilbene is 0.02 nM; The insulin is added to the basal culture medium in the following manner: 1 mL of pH = 2 hydrochloric acid solution is added to 1 mg of insulin dry powder to prepare an insulin mother solution, and then an insulin optimization solution is prepared at a volume ratio of 1:500 between the insulin mother solution and Ringer's solution, and finally the insulin optimization solution is added to the basal culture medium to a concentration of 1 μL / mL.

4. The formulation according to claim 1, characterized in that The insect pathogenic nematode species is the insect pathogenic nematode Steinernema carpocapsae All.

5. A method for culturing insect pathogenic nematodes in liquid culture, characterized in that: The following steps are involved: 1) Preparing a bacterial suspension of symbiotic bacteria of insect pathogenic nematodes; 2) adding the bacterial suspension to the formulation of claim 1 at a volume ratio of 1:25, and culturing at 25°C and 180 rpm on a shaker for 48 hours; 3) Add insect pathogenic nematodes, continue culturing for 10-15 days and then harvest the insect pathogenic nematodes.

6. The method according to claim 5, characterized in that The symbiotic bacteria is Xenorhabdus.

7. The method according to claim 5, characterized in that The preparation method of the bacterial suspension comprises: activating the symbiotic bacteria to obtain a single colony without contamination, and then inoculating the single colony into an LB liquid culture medium and culturing on a shaker at 25° C. and 180 rpm for 48 hours to obtain a bacterial suspension.

8. The method according to claim 5, characterized in that The continuing culturing for 10-15 days in step 3) means continuing culturing for 10 days.

9. The method according to claim 5, characterized in that The insect pathogenic nematodes harvested in step 3) are stored in a low temperature environment.

10. Use of the formulation according to any one of claims 1 to 4 in increasing the culture quantity and / or survival rate of insect pathogenic nematodes.

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