Solid fermentation culture medium, culture, method and application of arthrobotrys nematophagi

Abstract

The invention discloses a solid fermentation culture medium for arthrobotrys nematophagi. The culture medium is composed of a basic culture medium, additional nutrient substances and a proper amount of water. The basic culture medium takes a mixture of rice husks and corn flour as a matrix, the additional nutrient substances comprise a carbon source, a nitrogen source and vitamins, the carbon source can be selected from cane sugar and / or white sugar, and the nitrogen source is soy peptone. The invention also introduces a method for culturing the arthrobotrys nematophagi by using the solid fermentation culture medium, and also provides a related fermentation culture and application thereof. The solid fermentation culture medium can replace a rice culture medium to be used for solid fermentation of arthrobotrys nematophila chlamydospore, the production cost can be reduced, and large-scale popularization and application are easy.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a solid fermentation medium, culture, method and application of Arthrospora nematophila. Background Art

[0002] The long-term and large-scale use of chemical anthelmintics and pesticides has caused a series of problems in animal husbandry and agriculture, such as the increased resistance of animal parasites and agricultural pests to chemical synthetic drugs, the biological accumulation and residues of drugs in agricultural products (fruits, vegetables, grains, crops) and livestock products (meat, eggs, milk), environmental pollution, and the cost increase caused by the frequent and large-scale use of chemical drugs by farmers and herdsmen. Therefore, finding alternative methods to chemical drugs such as biological control has become an inevitable trend in the control of crop diseases and insect pests and domestic animal parasites in the future. Arthrobotrys flagrans is the most promising biological control strain for animal parasitic nematodes or plant nematodes. This fungus has a unique characteristic, that is, it can produce a large number of thick-walled spores during artificial cultivation. It is superior to other fungi in the preparation of biocontrol agents. Other fungi produce very few thick-walled spores or even have to be produced under artificial induction. This fungus begins to produce thick-walled spores after 3 days of cultivation on solid culture medium. This spore has a double-layer thick wall, is intercalated or terminal, and is an asexual spore formed by the concentration and expansion of the protoplasm of one or more cells during the growth of vegetative hyphae. They are different from other asexual spores such as conidia, because the important function of these spores is that they have strong resistance to external environment and can survive under adverse conditions. Therefore, they are an important survival structure of fungi, often existing in the form of structural dormancy to maintain long-term survival. They can also spread and reproduce fungi like conidia, so they are also a reproductive structure. Due to the above characteristics, the spores of A. flagrans have a long shelf life when prepared into various preparations for easy application. When thick-walled spores are orally administered to animals, they are able to resist the adverse environment of the digestive tract such as gastric acid, enzymes, high osmotic pressure, relatively high temperature and anaerobic conditions in the front part of the digestive tract, and thus pass through the digestive tract without being inactivated. The thick-walled spores are excreted with the feces and develop and germinate synchronously with the worm eggs to become nutritional hyphae and then produce predatory organs, which kill the parasitic nematode larvae in the feces as their own nutrition, thereby exerting an insecticidal effect.

[0003] At present, feed additives made from thick-walled spores of A. flagrans reduce the number of infective larvae (L3) on pastures after being eaten by domestic animals, minimizing the risk of reinfection of animals. It has been proven to have a reliable effect on Cyathostomins sp. in horses, and can effectively reduce the infection of Haemonchus contortus in sheep and goats; A. flagrans thick-walled spore preparations are effective in biologically controlling gastrointestinal nematodes in cattle, such as Ostertagia spp., Cooperia spp., Oesophagostomum spp., and Trichostrongylus spp. At present, Australian and Brazilian A. flagrans isolates have been registered internationally in 2018 and 2021, and commercial products have been launched. However, the high production cost of traditional fermentation of thick-walled spores has seriously restricted the large-scale application of this biocontrol agent. The bottleneck of producing thick-walled spores of nematophilic fungi A. flagrans is still the yield of spores, the selection of production medium and the scale-up process. The solid-state fermentation system is still a reliable method for the large-scale production of different spores of fungi, because many fungi do not produce spores in liquid fermentation tanks or even if they produce spores, their survival ability is very poor. By using solid-state fermentation, the spores produced, including thick-walled spores, can obtain good survival performance. Further, the solid-state fermentation system is simple in process, requires lower water and electricity costs and simple fermentation substrates, and downstream processing is cheaper than liquid fermentation, but it is not easy for large-scale production compared with liquid fermentation, and is easy to pollute, and the production process is not well controlled. In addition to selecting suitable culture medium, solid-state fermentation also considers many variables such as temperature, pH, humidity, particle size, culture medium thickness, removal of fermentation heat and ventilation conditions.

[0004] At present, the commonly used culture medium for solid-state fermentation of A. flagrans is various grains, such as corn, wheat, oats, barley, rice, etc. Studies have shown that rice has the highest spore production. Under laboratory conditions, it has been reported that after 30 days of solid-state fermentation of rice, the yield of thick-walled spores produced by A. flagrans ARSEF strain is 1.5×10 6 / per gram of dry matter (Santurio et al., Vet Parasitology, 2009, 164:344-346). In recent years, Castillo-Saldarriaga et al. reported (Biocontrol Science and Technology, 2020, 30(1):1100-1115) that 35g of rice grains were placed in a special aluminum tray with ventilation holes at the bottom of the tray. It is said that after 14 days of culture, the yield of rice can reach 108 Thick-walled spores / gram of dry matter, which is the highest yield so far. We have never exceeded this yield in laboratory culture with rice. In this study, only 35 grams of rice were placed in a 11.2×8.7×5cm tray. Because the bacteria are strictly aerobic, the surface area of ​​rice in contact with air after spreading is larger than that of ordinary containers. In addition, there are holes at the bottom of the tray, which increases the amount of air contact for each grain of rice, thus obtaining a high yield. However, due to the small amount of loading, it is difficult to achieve in actual production. Osorio et al. used this type of tray device in 2020 and used rice as a substrate to carry out pilot production in the solid-state fermentation system of A. flagrans thick-walled spores (Applied Biochemistry and Biotechnology, 2020, 192: 1044-1059). After conditions were optimized, 4.96×10 thick-walled spores per gram of dry matter could be produced per day within 14 days. 6 After 14 days, it can reach 6.9×10 7 / g dry matter, which is the highest yield in pilot production at present. On the other hand, according to our previous studies, the culture time is positively correlated with the yield of chlamydospores. Generally speaking, within the range of 40 days of culture, the spore yield increases with the extension of culture time, especially within 21 days of culture. The culture humidity and water content of the culture medium are also closely related to the yield of chlamydospores. Therefore, in addition to the culture medium formula, the culture conditions also need to be optimized to obtain good culture effects. In addition, according to the study of agar plates, adding inositol and mannose to Sabouraud medium can increase the yield of chlamydospores (Sagues et al. 2013; Junco et al. 2024), but agar medium can only be limited to laboratory research and will be difficult to use in production in the future. Since the diameter of chlamydospores can reach 30 microns, which is much larger than conidia, their yield cannot reach the level of bacteria or conidia per gram of dry matter, which can reach 100 million. However, chlamydospores have a very long survival life due to their large size and thick cell wall, making them an ideal component for the preparation of fungal biocontrol agents. Although the spore yield of A. flagrans thick-walled spores cultured with rice is high, the high cost of rice, which is a staple food for humans, may lead to an increase in the price of the final product. In addition, rice is prone to clumps after adding water and high-pressure sterilization, and needs to be redispersed during inoculation, which is cumbersome, time-consuming and labor-intensive. Summary of the invention

[0005] In view of all or part of the deficiencies of the above-mentioned prior art, the object of the present invention is to provide a solid fermentation medium, culture, method and application of Arthrospora nematophila. The provided solid fermentation medium of Arthrospora nematophila can replace rice culture medium for solid-state fermentation of thick-walled spores of Arthrospora nematophila, which not only reduces production costs but also is easy to promote and apply on a large scale.

[0006] In order to achieve the above-mentioned invention object, the present invention provides the following technical solutions:

[0007] The invention provides a solid fermentation medium for Arthrospora nematophila, which consists of a basic medium, added nutrients and an appropriate amount of water. The matrix of the basic medium is a mixture of rice husks and corn flour, the added nutrients include a carbon source, a nitrogen source and vitamins, the carbon source is sucrose and / or white sugar, and the nitrogen source is soy peptone.

[0008] The weight ratio of the rice husk to the corn flour is 1:2-3, and the ratio of the total weight of the rice husk and the corn flour to the weight of water is 1:1.2.

[0009] The Arthrospora nematophila is Arthrospora nematophila isolate NBS063. Arthrospora nematophila isolate NBS063 has good spore production performance.

[0010] The particle size of the corn flour is 0.5-1 mm, and the particle size of the rice husk is 2-10 mm.

[0011] Based on the mass of the basic culture medium, the added amount of sucrose is 4%, the added amount of soy peptone is 0.05-0.1%, and the vitamin is vitamin B1, and the added amount of vitamin B1 is 0.0002-0.0003%.

[0012] The present invention also provides a method for culturing Arthrospora nematophila, comprising the following steps:

[0013] Step 1, preparation of seed solution: inoculating Arthrospora nematophila in a PDA plate, transferring the mycelium grown on the PDA plate to a culture bottle containing Sabouraud broth for culture, and obtaining seed solution;

[0014] Step 2, solid-state culture: the solid fermentation culture medium of the Arthrospora nematophila is placed in a culture container, sterilized and cooled, and then the seed liquid is inoculated into the culture container for culture.

[0015] In step 2, the inoculation amount of the seed solution is 10-15% (v / m); in step 1, the PDA plate is a plate with a diameter of 9 cm, 4 squares of 4×4 cm are cut from the edge of the growing colony, transferred to 100 mL of Sabouraud broth, placed in a shaker at 28°C and 200 rpm for 7 days to obtain the seed solution. In step 2, the sterilization and cooling process is: high-pressure steam sterilization, the sterilization temperature is 121°C, the sterilization time is 20 minutes, and cooling to room temperature after sterilization.

[0016] In step 2, the process of inoculating the seed liquid into the culture container for culturing is as follows: after the solid fermentation medium is stirred evenly, it is placed at 26° C. for culturing, wherein the relative humidity is adjusted to 90-95% for the first 2 days and 50-60% for the rest of the time, and the culturing is terminated after 14 days of culturing.

[0017] The present invention also provides a fermentation culture obtained by culturing the above-mentioned Arthrospora nematophila culture method, wherein the fermentation culture comprises chlamydospores of Arthrospora nematophila.

[0018] The present invention also provides an application of the fermentation culture in preparing a nematode biological control preparation.

[0019] Compared with the prior art, the present invention has at least the following beneficial effects: after screening tests, the present invention finally found that corn feed powder fed to livestock plus an appropriate amount of rice husk as a combined basic culture medium, after adding a small amount of carbon and nitrogen sources and vitamins, A. flagrans thick-walled spores can be fermented by a solid-state method to obtain a yield similar to that of rice fermentation, and at the same time, the fermentation conditions such as water content are also tested. At present, there is no report on the formula of this culture medium, which can be used as a culture medium to replace rice for solid-state fermentation of A. flagrans thick-walled spores, which can reduce production costs and is easy to promote in large-scale production in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the specific embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 It is the preliminary selection result of the basic culture medium screening in Example 1;

[0022] Figure 2 is the screening result of the ratio of rice husk to corn flour in Example 2;

[0023] Figure 3 is the screening result of adding carbon source and nitrogen source in Example 3;

[0024] Figure 4 It is the screening result of seed inoculation amount in Example 5;

[0025] Figure 5 The effect of the amount of water added to the material (material-water ratio) on the yield of chlamydospores in Example 6;

[0026] Figure 6 The effect of culture humidity on chlamydospore yield in Example 6;

[0027] Figure 7 The stability of the culture medium formulation and culture method screened in Example 7 on the production of chlamydospores. DETAILED DESCRIPTION

[0028] The technical solutions in the specific embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] It should be noted that, in order to describe the technical solution more specifically, the steps described in the following embodiments do not strictly correspond to the steps described in the content of the invention.

[0030] Example 1A. Single factor screening of basal medium for solid-state fermentation of flagrans

[0031] 1. Preparation of seed solution: Inoculate the A. flagrans isolate NBS063 in the center of a 9-cm diameter plate containing potato dextrose agar (PDA). When the mycelium grows to about 2 / 3 of the plate, cut 4×4 cm cubes from the edge of the colony with a sterilized scalpel and transfer them to a 500-mL high-temperature plastic culture bottle containing 100 mL Sabouraud broth. Place the bottle in a shaker at 28°C and 200 rpm for 7 days. After a purity inspection, use the resulting cubes as seed solution for inoculation.

[0032] 2. Basic culture medium screening: Prepare 30 grams of each of the following culture media:

[0033] Table 1 Composition of basal medium

[0034] Group Matrix type Weight Ratio Group 1 Bran flour: Rice husk 1:1 Group 2 Bran flour: Rice husk 3:1 Group 3 Peanut flour: bran flour 1:2 Group 4 Peanut cake powder: bran powder: rice husk 3:12:5 Group 5 Rice Husk: Corn Flour 3:1 Group 6 Peanut flour: corn flour 1:2 Group 7 Bran flour: corn flour 3:1 Group 8 Bran flour: corn flour 1:1 Group 9 Rice Husk: Corn Flour 1:1 Group 10 Peanut flour: corn flour: rice husk 2:5:3 Control group Pure hulled rice -

[0035] The corn flour is of feed grade particle size (0.5-1mm), the rice husk particle size is 2-10mm, the peanut cake powder and bran powder are of the particle size when purchased on the market, and are not reprocessed. The above-mentioned various culture media prepared in proportion are added with distilled water at a material-water ratio of 1:0.55, stirred evenly, and then divided into 500mL plastic culture bottles, each culture medium is filled with 3 bottles, and sterilized at 121℃ for 20min. After cooling, 3mL of the seed liquid of the strain is inoculated into each bottle at 10% (v / w), stirred evenly with a gun tip, and then the bottle cap is covered. The culture was cultured in a 28°C ordinary constant temperature incubator for 12 days and then stopped. The culture was then dried in a 65°C oven for 48 hours. Each bottle of dried culture was crushed and evenly mixed in a grinder. 1 gram of dry powder containing thick-walled spores in each bottle was weighed, 9 mL of 0.05% Tween was added and mixed in a centrifuge tube. Then, the diluted suspension containing thick-walled spores was placed in a hemocytometer for counting. Each bottle of culture was sampled and counted three times in a row. Finally, the average of 9 consecutive counts of 3 bottles of each culture medium was calculated, and the number of thick-walled spores per gram of dry matter of each culture medium was counted.

[0036] The preliminary results of the basic culture medium are as follows Figure 1 As shown, it can be seen that the ninth group, namely the rice husk + corn flour (1:1) group, had the highest chlamydospore yield, which was significantly different from the control rice and the other groups (p < 0.05).

[0037] Example 2 Screening test of the ratio of rice husk to corn flour

[0038] The ratio of rice husk to corn flour was designed to be 1:1 and 1:2, and another group of bran flour + corn flour + rice husk (2:2:1) was added. The feed-water ratio was 1:0.55, the seed inoculation amount was 10%, and the culture was carried out at 28℃ for 12 days. The chlamydospore counts were carried out according to the same method as above, and the results are shown in Figure 2 .Depend on Figure 2 It can be seen that the optimal ratio of rice husk to corn flour is 1:2, which is significantly different from the other two groups (p < 0.05).

[0039] Example 3 Screening test of adding carbon source and nitrogen source

[0040] A 1:2 ratio of rice husk + corn flour was selected as the basic culture medium. Based on the previous agar culture medium test that produced thick-walled spores, sucrose and commercially available white sugar with better spore production were selected as carbon sources at a concentration of 4%. Soybean peptone and ammonium sulfate were used as nitrogen sources at a concentration of 0.1%. After adding to the basic culture medium, water was added according to the aforementioned material-water ratio of 1:0.55, routine sterilization was performed, and seed liquid was inoculated. After culturing at 28°C for 12 days, the thick-walled spores were counted according to the same method as above. The results are shown in Figure 3 .Depend on Figure 3It can be seen that there is no significant difference in the production of chlamydospores between the sucrose group and the white sugar group among carbon sources (p>0.05), and soy peptone can significantly increase the production of chlamydospores more than ammonium sulfate among nitrogen sources (p<0.05).

[0041] Example 4 Orthogonal experiment of culture medium formulation

[0042] The ratio of rice husk + corn flour, the concentration of sucrose and soy peptone (w / w) were designed as factors affecting the yield of thick-walled spores. Based on the previous experience that adding vitamin B1, namely VB1, to agar medium can increase the yield of thick-walled spores, VB1 (w / w) was added as one factor, and a total of 4 factors and 3 levels were used for orthogonal experiments (Table 2). According to Table 2, 9 culture media such as A1B1C1D1 were prepared, with 3 parallels for each culture medium. 30 grams of each culture medium was divided into 500mL plastic bottle culture bottles, with a material-water ratio of 1:0.65. In addition, 3 500mL plastic culture bottles were taken, each bottle was divided into 30 grams of rice, and water was added at a material-water ratio of 1:0.65 as a control. The above culture bottles were sterilized at 121℃ for 20min, and after cooling, seed liquid was inoculated according to the above method, with an inoculation amount of 10%.

[0043] Table 24 Factor 3 level orthogonal experimental design

[0044]

[0045] Then the above culture bottles were placed in a constant temperature incubator at 28°C for 12 days and then the culture was stopped. The thick-walled spores were counted according to the above method. The results are shown in Table 3. As can be seen from Table 3, the culture medium formula of group A2B3C1D2 has the best spore production, that is, rice husk + corn flour is 1:2, sucrose is added in an amount of 4%, soy peptone is added in an amount of 0.05%, and VB1 is added in an amount of 0.0002%. There are significant differences (p < 0.05) with other groups, including the control group, except for experimental group No. 9, namely A3B3C2D1 (rice husk + corn flour is 1:2.5, sucrose is added in an amount of 4%, soy peptone is added in an amount of 0.1%, and VB1 is added in an amount of 0.0001%). The amount of thick-walled spores can reach 1.04×10 per gram of dry matter during the 12-day culture period. 7 , which was 7.28×10 higher than the rice control. 6 indivual.

[0046] Table 3 Results of orthogonal experiment on the production of thick-walled spores of A. flagrans NBS063

[0047]

[0048] *Rice control

[0049] Example 5 Seed inoculation test

[0050] The above orthogonal experiment optimized the culture formula and selected an inoculation amount of 10%. However, there have been no reports on the effect of seed inoculation amount on the yield of thick-walled spores. Therefore, the above formula was selected, that is, rice husk + corn flour was 1:2, sucrose was added at 4%, soy peptone was added at 0.05%, VB1 was added at 0.0002%, 30 grams of culture medium was weighed, the material-water ratio was 1:0.75, and it was divided into 500mL plastic culture bottles, a total of 9 bottles. After sterilization and cooling according to the above method, 3 bottles each were inoculated with seed inoculation amounts of 5%, 10%, and 15% (v / w), and the culture was stopped after culturing at 28°C for 14 days. The yield of thick-walled spores was counted according to the above method. The results are shown in Figure 4 As can be seen from the figure, during the culture period, the yield of chlamydospores increased with the increase of seed inoculum. When the inoculum increased from 5% to 10%, the yield of chlamydospores increased significantly, while when it increased from 10% to 15%, the increase in spore yield slowed down. There were significant differences among the inoculums of 5%, 10%, and 15% as the inoculum increased (p < 0.05). Considering that excessive inoculum in actual production will increase production costs, it is not meaningful to increase the inoculum further. The inoculum of 15% is already the upper limit, so no relevant experiments were conducted on inoculums above 15%.

[0051] Example 6 Effect of water addition amount (material-water ratio) and culture humidity on chlamydospore yield

[0052] According to the optimized formula of basal culture medium rice husk + corn flour 1:2, the material-water ratio is designed to be 1:0.75, 1:1.2, and 1:1.5, that is, the amount of water added is 75%, 120%, and 150%. Take 50g of basal culture medium material and add tap water instead of distilled water according to the above ratio. In order to increase the air contact surface, it is divided into a large plate with a diameter of 20cm, and the basal culture medium material and water are mixed evenly, and sterilized at 121℃ for 20min. Take the same mass of rice and soak it in water overnight according to the above ratio, then divide it into the above plate, and use it as a control after high-pressure sterilization. After the culture medium is cooled to room temperature, the seed liquid is inoculated at a seed inoculation rate of 15% (v / w). Then it is placed in a 26℃ constant temperature incubator for culture. Two days before culture, put an appropriate amount of water at the bottom of the constant temperature incubator, adjust the relative humidity of the constant temperature incubator to 90-95%, and then withdraw the water at the bottom of the constant temperature incubator to maintain its relative humidity at 50-60%. The culture was terminated after 14 days of continuous cultivation in the plate, and dried according to the above method, and the production of chlamydospores was counted.

[0053] At the same time, the same number of basal culture medium test groups and rice control groups were designed, prepared, sterilized and inoculated according to the same method as above, placed in a 26°C constant temperature incubator without humidity adjustment (relative humidity of about 50%), cultured for 14 days, dried according to the same method, and chlamydospore counts were performed. The test results are shown in Figure 5 , Figure 6 , Figure 5The figure shows the yield of chlamydospores without humidity adjustment under different material-water ratio conditions. Figure 6 The figure shows the yield of thick-walled spores under humidity-regulated conditions at different material-water ratios.

[0054] Depend on Figure 5 It can be seen that for the unadjusted culture humidity, during the 14-day culture period, the yield of thick-walled spores was the highest in both the experimental group and the control group when the feed-water ratio was 1:1.2, i.e., the water addition level was 120%, which was 7.12×10 per gram and 1.2×10 per gram, respectively. 7 , 6.98×10 7 , which was significantly different from the other water addition groups (p<0.05). Figure 6 It can be seen that after adjusting the culture humidity, under the same culture time and temperature, the yield of thick-walled spores in the experimental group and the control group was still the best when the feed-water ratio was 1:1.2, that is, the water addition amount was 120%, reaching 8.30×10 7 ,8.22×10 7 , which was significantly different from the other water addition groups (p<0.05). Compared with the unadjusted humidity, the chlamydospores increased significantly after humidity adjustment (p<0.05).

[0055] Example 7 Verification Culture

[0056] After the above tests, in order to verify the stability of the culture medium formula and culture method selected in the present invention for the production of thick-walled spores, the best formula and culture conditions were selected for the final culture test. The basic formula is rice husk + corn flour 1:2 (material-water ratio 1:1.2), added with 4% sucrose, 0.05% soy peptone, and 0.0002% VB1, divided into large plates with a diameter of 20 cm, three parallel plates, sterilized at 121°C for 20 minutes, inoculated at a seed inoculation rate of 15% (v / w) when cooled to room temperature, stirred evenly, and cultured at 26°C. The relative humidity was adjusted to 90-95% for the first 2 days, and the relative humidity was 50-60% for the rest of the time. The culture time was 14 days, and then the thick-walled spores were counted according to the above method. The control group used rice (material-water ratio 1:1.2), and the culture conditions and methods, spore counts, etc. were the same as those of the test group. The results are as follows Figure 7 ,Depend on Figure 7 It can be seen that the verification result of the chlamydospore yield in the experimental group is basically consistent with the best result of the previous experiment, which is 8.45×10 7 / g, while the control group rice was 8.41×10 7 The results of the two groups were similar, and the yield of the test group was higher than that of the control group (p < 0.05), which further demonstrated that the culture medium formula and culture method for culturing A. flagrans chlamydospores of the present invention can stably produce a large number of chlamydospores, and the effect of the new formula in producing chlamydospores under the same culture conditions is basically equivalent to or slightly higher than that of rice, and can completely replace rice.

[0057] The raw materials of the basic culture medium of the present invention are rice husks and corn flour for feed, which are cheaper than rice and suitable for solid fermentation. The obtained fermentation culture can be directly used as an animal feed additive, or can be used as a biological pesticide by crushing and separating spores, thereby reducing the cost of the final product. In addition, rice is easy to clump after high-pressure sterilization, which hinders ventilation and affects the fermentation effect, and can only be cultured in a tray. Rice husks are easy to absorb water and are not easy to clump, which is conducive to air circulation and can produce thick-walled spores in a solid fermentation tank. Even in a tray, efficient solid-state fermentation of A. flagrans can be carried out in a thicker culture layer, which is conducive to large-scale industrial production. The new culture medium formula and culture method for solid-state fermentation of thick-walled spores of Arthrospora nematophila provided by the present invention can produce 8.45×10 thick-walled spores after 14 days of fermentation. 7 / g dry matter, with an average daily spore production of 6.04×10 6 This exceeds the 4.96×10 per gram per day produced by Osrio et al. in 2020 using a metal tray pilot test. 6 , with the prospect of reducing costs and mass production.

[0058] That is, the present invention provides a solid fermentation medium for Arthrospora nematophila, which is composed of a basic medium, additional nutrients and an appropriate amount of water. The matrix of the basic medium is a mixture of rice husks and corn flour, and the additional nutrients are carbon sources, nitrogen sources and vitamins. The carbon source is sucrose, and the nitrogen source is soy peptone. The weight ratio of the rice husks and corn flour is 1:2, and the ratio of the total weight of the rice husks and corn flour to the weight of water is 1:1.2. The amount of sucrose added is 4%, the amount of soy peptone added is 0.05%, and the vitamin is vitamin B1, and the amount of vitamin B1 added is 0.0002%. The fermentation culture obtained by the above-mentioned culturing method of Arthrospora nematophila includes thick-walled spores of Arthrospora nematophila. The use of the fermentation culture in the preparation of nematode biological control agents is also provided.

[0059] The above embodiments are only used to help understand the method and core idea of ​​the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A solid fermentation medium for Arthrospora nematophila, characterized in that: The solid fermentation medium consists of a basic medium, additional nutrients and an appropriate amount of water. The matrix of the basic medium is a mixture of rice husks and corn flour. The additional nutrients include a carbon source, a nitrogen source and vitamins. The carbon source is sucrose and / or white sugar, and the nitrogen source is soy peptone.

2. The solid fermentation medium of Arthrospora nematophila according to claim 1, characterized in that: The weight ratio of the rice husk to the corn flour is 1:2-3, and the ratio of the total weight of the rice husk and the corn flour to the weight of water is 1:1.

2.

3. The solid fermentation medium of Arthrospora nematophila according to claim 1, characterized in that: The Arthrospora nematophila is the Arthrospora nematophila isolate NBS063.

4. The solid fermentation medium of Arthrospora nematophila according to claim 1, characterized in that: The particle size of the corn flour is 0.5-1 mm, and the particle size of the rice husk is 2-10 mm.

5. The solid fermentation medium of Arthrospora nematophila according to claim 1, characterized in that: Based on the mass of the basic culture medium, the added amount of sucrose is 4%, the added amount of soy peptone is 0.05-0.1%, and the vitamin is vitamin B1, and the added amount of vitamin B1 is 0.0002-0.0003%.

6. A method for culturing Arthrospora nematophila, characterized in that: The steps include: Step 1, preparation of seed solution: inoculating Arthrospora nematophila in a PDA plate, transferring the mycelium grown on the PDA plate to a culture bottle containing Sabouraud broth for culture, and obtaining seed solution; Step 2, solid-state culture: the solid fermentation medium of the Arthrospora nematophila according to any one of claims 1 to 5 is placed in a culture container, sterilized and cooled, and then the seed liquid is inoculated into the culture container for culture.

7. The method for culturing Arthrospora nematophila according to claim 6, characterized in that: In the step 2, the inoculation amount of the seed solution is 10-15%; in the step 1, the PDA plate is a plate with a diameter of 9 cm, 4 squares of 4×4 cm are cut from the edge of the growing colony, transferred to 100 mL of Sabouraud broth, placed in a shaker at 28° C., 200 rpm and cultured for 7 days to obtain the seed solution.

8. The method for culturing Arthrospora nematophila according to claim 6, characterized in that: In step 2, the process of inoculating the seed liquid into the culture container for culturing is as follows: after the solid fermentation medium is evenly stirred, it is cultured at 26° C., wherein the relative humidity is adjusted to 90-95% for the first 2 days and 50-60% for the rest of the time, and the culture is terminated after 14 days of culturing.

9. A fermentation culture obtained by culturing the method for culturing Arthrospora nematophila according to any one of claims 6 to 8, wherein the fermentation culture comprises chlamydospores of Arthrospora nematophila.

10. Use of the fermentation culture according to claim 9 in preparing a nematode biological control agent.

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