An indoor cultivation method for tobacco root-knot nematodes

Abstract

The present invention discloses a method for indoor cultivation of tobacco root-knot nematodes, which relates to the technical field of tobacco root-knot nematode cultivation. It solves the technical problems in the prior art that the reproduction and cultivation of tobacco root-knot nematodes rely on living host plants, require greenhouses, soil, and susceptible plant varieties, and involve a large number of experimental materials, cumbersome operations, long cycles, and difficult management. The indoor cultivation method of tobacco root-knot nematodes of the present invention comprises the following steps: isolation, purification, and cultivation of Fusarium graminearum, preparation of Fusarium graminearum seed liquid, preparation of Fusarium graminearum plates for cultivating tobacco root-knot nematodes, screening to obtain tobacco root-knot nematode eggs and tobacco root-knot nematodes by a sieving method, and inoculation and cultivation of tobacco root-knot nematodes. The indoor cultivation method of tobacco root-knot nematodes of the present invention has the advantages of being simple and quick, and being able to obtain a large number of tobacco root-knot nematodes from plants, and can provide test subjects for indoor screening of tobacco root-knot nematode biocontrol bacteria and chemical pesticides to a large extent.

Description

Technical Field

[0001] The invention relates to the technical field of tobacco root-knot nematode cultivation, in particular to an indoor cultivation method of tobacco root-knot nematodes. Background Art

[0002] The tobacco root-knot nematode is a type of nematode that is a sedentary endoparasite widely distributed within the roots of tobacco plants. Classified as Nematoda, M. nicotianae, and M. nicotianae, it is one of the ten most important plant-parasitic nematode genera worldwide. Over 90 species of tobacco root-knot nematodes have been reported, with the four most widespread and widespread dominant groups being the southern tobacco root-knot nematode (CM. incognita), the peanut tobacco root-knot nematode (M. arenaria), the Javan tobacco root-knot nematode (M. javanica), and the northern tobacco root-knot nematode (M. hapla). The tobacco root-knot nematode genus has a wide range of parasites, infecting over 3,000 plant species belonging to 114 families, including monocots, dicots, herbaceous plants, and woody plants. It can infect vegetables, grains, cash and fruit crops, ornamental plants, and weeds, earning it the nickname "hidden enemy" of plants.

[0003] The tobacco root-knot nematode's life cycle consists of three distinct stages: egg, larva, and adult. The fertilized egg undergoes mitosis and develops into a first-instar larva (J1). After molting, it becomes a second-instar larva (J2), which then sheds its eggshell and enters the soil, beginning to infect young plant roots. The second-instar larva, once inside the root, undergoes a second molt to become a third-instar larva (J3). Subsequently, the male and female gonads swell and elongate, and a distinct "root knot" appears on the plant's roots. Once inside the plant's root system, the tobacco root-knot nematode feeds on the roots, preventing mitosis in the root's soft tissue protoplasmocytes, destroying conductive tissues and altering host physiology. While mechanically damaging the host plant, it secretes toxic substances that attract other pathogens, resulting in slowed plant growth, stunted development, and, in severe cases, complete plant withering and death. It has been classified as a significant plant pathogen that is both highly harmful and difficult to control.

[0004] Tobacco root-knot nematodes are a major pest in tobacco production worldwide, causing severe damage in tropical, subtropical, and warm temperate zones. Common in Yunnan, they can cause complex infections with blackleg pathogens and bacterial wilt pathogens, leading to reduced tobacco leaf resistance, decreased yield and quality, and a vicious cycle. To stabilize tobacco yield and quality, crop rotation is often used to mitigate the damage caused by tobacco root-knot nematodes, but the effect is minimal. Coupled with the ban on the use of numerous chemical pesticides on tobacco, the development and utilization of new environmentally friendly measures (such as biological control) to control tobacco root-knot nematodes has become an urgent task for the tobacco industry.

[0005] Since tobacco root-knot nematodes are obligate parasites, their growth and reproduction under natural conditions cannot be separated from plant roots and soil. Therefore, the cultivation and preservation of their populations have always been one of the main problems that have plagued in-depth research. Conventional reproduction and cultivation of tobacco root-knot nematodes have always relied on living host plants, requiring greenhouses, soil, and susceptible plant varieties. This involves a large number of experimental materials, cumbersome operations, long cycles, and difficulty in management. Currently, research on tobacco root-knot nematodes has been very in-depth, and biological control research has been widely carried out in countries around the world. However, there are relatively few research and application reports on the cultivation and reproduction of tobacco root-knot nematodes. Tobacco root-knot nematodes cannot be artificially cultivated, and they rely on host plants for a long reproduction cycle. Under natural conditions, their life cycle must go through two unified but different microenvironments, from the soil to the host roots, to complete an infection process. When conditions are suitable, an infection cycle takes about 30 days, which constitutes an inconvenience factor for artificially propagating tobacco root-knot nematodes.

[0006] To date, research reported domestically and internationally has shown a trend toward culturing tobacco root-knot nematodes in laboratories using nutrient solutions or culture media, away from greenhouses and soil. However, a soil-cultured host plant, tissue-cultured seedling, or root segment explant is essential for the nematode's feeding and parasitism. Aseptically culturing such plants or explants requires significant time and effort. If tobacco root-knot nematodes could complete their growth and reproduction cycle by simply providing a food source rather than a host plant, this would greatly simplify the nematode's culture procedures and shorten the generation time. This would significantly facilitate research into the reproduction, preservation, and biochemical control of tobacco root-knot nematodes. Summary of the Invention

[0007] The present invention aims to provide a method for indoor cultivation of tobacco root-knot nematodes. This method addresses the existing technical issues of tobacco root-knot nematode propagation and cultivation, which rely on living host plants, require greenhouses, soil, and susceptible plant varieties, and involve numerous experimental materials, complex procedures, long cycles, and difficult management. The various technical benefits of the preferred technical solution of the present invention are detailed below.

[0008] To achieve the above objectives, the present invention provides the following technical solutions:

[0009] The indoor cultivation method of tobacco root-knot nematodes of the present invention comprises the following steps:

[0010] Step 1: Isolation, purification and cultivation of Fusarium graminearum: using tissue separation and single spore purification culture method to obtain purified strains of Fusarium graminearum from the straw of plants infected with fusarium graminearum;

[0011] Step 2: Preparation of Fusarium graminearum seed liquid: The purified Fusarium graminearum strain and its carrier obtained in step 1 were punched with a puncher and then picked and inoculated into liquid PDA culture medium. The culture was carried out at a temperature of 27°C-28°C and a rotation speed of 180 r / min for 72 h to obtain Fusarium graminearum seed liquid, i.e., Fusarium graminearum suspension. The effective viable bacterial count of the bacterial suspension was controlled at 0.50×10 8 cfu / g or above;

[0012] Step 3: Preparation of Fusarium graminearum plates for culturing tobacco root-knot nematodes: 100-200 μL of the obtained Fusarium graminearum seed solution is evenly spread on solid PSA culture medium, and then placed in a constant temperature incubator at 28°C for 5-7 days until the mycelium fills the plate for later use;

[0013] Step 4: Tobacco root-knot nematode eggs and tobacco root-knot nematodes were screened by a sieving method, the tobacco root-knot nematodes were collected into a beaker using a pipette, and the suspension was diluted with distilled water to prepare a tobacco root-knot nematode suspension with a content of 200 / 10 mL;

[0014] Step 5: Under sterile conditions, 1 mL of the bacterial suspension was inoculated onto a solid PSA plate grown with Fusarium graminearum, and the solid PSA medium grown with Fusarium graminearum was shaken left and right to completely penetrate the mycelium and mix with the mycelium, and the tobacco root-knot nematode suspension droplets were immersed in the PSA plate;

[0015] Step 6: Seal the culture dish with a sealing film, with the front of the culture dish facing up, and culture the culture dish in a 28°C constant temperature incubator in dark conditions for 7-14 days. Rinse the PSA plate with sterile water to obtain the tobacco root-knot nematode suspension, and collect the tobacco root-knot nematode suspension.

[0016] According to a preferred embodiment, the preparation method of the liquid PDA culture medium includes the following steps: weighing 200 g of potatoes and 20 g of glucose in a preparation amount of 1 L, washing and chopping the potatoes and boiling them for 30 minutes, filtering them through at least two layers of gauze to obtain a filtrate, dissolving the weighed glucose in the filtrate, and packaging them in triangular flasks for sterilization to obtain a liquid PDA culture medium for culturing Fusarium graminearum.

[0017] According to a preferred embodiment, the preparation method of solid PSA culture medium includes the following steps: taking 200 g of potatoes, boiling for 30 minutes and filtering, adding 14 g of sucrose to the potatoes, adding purified water to make the volume 1 L, then adding 18 g / L of agar powder, and adjusting the pH to 7.0-7.4.

[0018] According to a preferred embodiment, the method of obtaining a tobacco root-knot nematode suspension by screening comprises the following steps:

[0019] Collect field soil samples where tobacco root-knot nematodes are infested and root tissues infected with tobacco root-knot nematodes. Wash the field soil samples and root tissues, cut them into pieces, and place them in a container. Cut the root tissue into pieces of 0.5-1 cm.

[0020] Add pure water to the container to submerge the sample and stir thoroughly to suspend the tobacco root-knot nematodes. Alternatively, place the container in a 28°C constant temperature shaker and shake for 0.5-1 hour. Let it stand for 3-5 minutes to allow the soil or plant tissue to sink. Collect the suspension containing tobacco root-knot nematodes. The content of the tobacco root-knot nematode suspension is 100-300 tobacco root-knot nematodes per 10 ml.

[0021] The suspension is injected into a filter sieve with 200 mesh, 400 mesh and 500 mesh, and the sieve is rinsed with clean water. The residue in the sieve is collected into a beaker or a culture dish. The 500 mesh sieve can collect tobacco root-knot nematode eggs, and the 400 mesh sieve can collect tobacco root-knot nematodes. The collected tobacco root-knot nematode eggs and tobacco root-knot nematodes are set aside.

[0022] According to a preferred embodiment, the dilution with distilled water to prepare a bacterial suspension with a content of 200 tobacco root-knot nematodes / 10 mL includes the following steps: aspirating 5 mL of the collected tobacco root-knot nematodes into a clean culture dish plate with a diameter of 6 cm, placing it under a dissecting microscope to observe the number of tobacco root-knot nematodes, and adding water to dilute the suspension to a content of 200 nematodes / 10 mL according to the observed number.

[0023] According to a preferred embodiment, 1 mL of the bacterial suspension is aspirated under sterile conditions and inoculated onto a solid PSA plate covered with Fusarium graminearum, comprising the following steps: irradiating with ultraviolet light for 30 minutes in a sterile clean bench, and aspirating 1 mL of the bacterial suspension with a 1 mL pipette and inoculating it onto a PSA plate covered with Fusarium graminearum.

[0024] According to a preferred embodiment, collecting the tobacco root-knot nematode suspension comprises the following steps: washing the PSA plate from which the Fusarium graminearum hyphae have disappeared with sterile water, and collecting the washing liquid to obtain the tobacco root-knot nematode suspension.

[0025] The indoor cultivation method of tobacco root-knot nematodes provided by the present invention has at least the following beneficial technical effects:

[0026] The present invention provides an indoor cultivation method for tobacco root-knot nematodes, which screens out Fusarium graminearum, a fungus that can effectively provide a food source for tobacco root-knot nematodes. By isolating and collecting tobacco root-knot nematodes, a tobacco root-knot nematode suspension is obtained. This suspension is then inoculated onto potato sucrose (PSA) plates covered with Fusarium graminearum. After culturing in the dark at 28°C for 7-14 days, a large number of second-instar larvae of tobacco root-knot nematodes can be collected. This method can provide test subjects for indoor screening of tobacco root-knot nematode biocontrol bacteria and chemical pesticides. The present invention also significantly reduces the cycle of indoor laboratory cultivation and the number of culture media required.

[0027] The indoor cultivation method of tobacco root-knot nematodes of the present invention is characterized in that a potato glucose culture medium can provide screening and cultivation of Fusarium graminearum, which can rapidly reproduce and grow and meet the food source required during the reproduction cycle of tobacco root-knot nematodes, while a potato sucrose culture medium can greatly increase the number of tobacco root-knot nematodes within 7-14 days and shorten the cultivation cycle of tobacco root-knot nematodes. Therefore, the indoor cultivation method of tobacco root-knot nematodes of the present invention is simple and fast and can obtain a large number of tobacco root-knot nematodes from plants. This solves the technical problems in the prior art that the reproduction and cultivation of tobacco root-knot nematodes rely on living host plants, require a greenhouse, soil, and susceptible plant varieties, involve a large number of experimental materials, are complicated to operate, have a long cycle, and are difficult to manage. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. 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 any creative work.

[0029] Figure 1 This is a schematic diagram of Fusarium graminearum hyphae growing all over the plate;

[0030] Figure 2 This is a schematic diagram showing the disappearance of some Fusarium graminearum hyphae 5 days after inoculation with tobacco root-knot nematodes;

[0031] Figure 3 This is a schematic diagram showing the disappearance of all Fusarium graminearum hyphae 14 days after inoculation with tobacco root-knot nematodes;

[0032] Figure 4 This is a microscopic examination of tobacco root-knot nematodes after reproduction on a PSA plate. DETAILED DESCRIPTION

[0033] To make the objectives, technical solutions, and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other implementations obtained by those of ordinary skill in the art without inventive effort are within the scope of protection of the present invention.

[0034] The following is attached with the instruction manual Figures 1 to 4 And Examples 1 to 7 describe in detail the indoor culture method of tobacco root-knot nematodes of the present invention.

[0035] Example 1

[0036] This example provides a method for preparing a culture medium for Fusarium graminearum.

[0037] Preparation of test tube slant culture medium: Weigh 200g of potatoes, peel them, and add 20g of glucose. Wash and chop the potatoes, then boil them for about 30 minutes. Then filter them through double-layer gauze into a measuring cup containing 20g of glucose. Add 24g of agar, heat and boil until the agar is dissolved, and make up the volume to 1000mL. After cooling appropriately, divide the mixture into test tubes, with the filling amount being 1 / 5-1 / 4 of the test tube height. Plug the test tube mouth with a test tube stopper, and sterilize it by high pressure at 121℃ for 30 minutes. Remove it while it is hot and swing the slant to cool for use. The slant height should not exceed 1 / 3 of the test tube height.

[0038] Preparation of liquid PDA culture medium: Weigh 200g of potatoes, peel them, and add 20g of glucose. Wash and chop the potatoes, then boil them for about 30 minutes. Filter them through double-layer gauze into a measuring cup containing 20g of glucose, make up the volume to 1000mL, and then divide the volume into 20-30ml / 250ml Erlenmeyer flasks. Plug the flasks with cotton plugs, sterilize them under high pressure at 121℃ for 30min, and cool them for later use.

[0039] Preparation of solid PDA culture medium: Weigh 200 g of potatoes, peel them, chop them and boil them for half an hour, filter them through two layers of gauze, add 20 g of glucose and 24 g of agar powder (for culturing Fusarium graminearum spores), add water to 1000 mL after dissolving, and sterilize them under high pressure at 121°C for 30 min.

[0040] Example 2

[0041] This embodiment provides a method for preparing a spore solution of Fusarium graminearum.

[0042] The Fusarium graminearum of this embodiment is a strain preserved by the laboratory of Yunnan Microbial Fermentation Engineering Research Center Co., Ltd. using the slant cold storage method.

[0043] The preparation method of Fusarium graminearum spore liquid comprises the following steps:

[0044] a. Activation of Fusarium graminearum strains.

[0045] The activation method of the slant-preserved bacterial strain is as follows: a piece of bacterial strain with a diameter of about 5 mm is picked from the slant-preserved bacterial strain and inoculated into the test tube slant culture medium for activation, and then cultured in a constant temperature incubator at 28°C for 3-5 days for use.

[0046] b. Cultivation of seed liquid.

[0047] Pick three bacterial blocks from the activated slant culture medium in the test tube and inoculate them into three 250 mL Erlenmeyer flasks containing 25 mL of liquid seed culture medium. Place them on a shaker with a speed of 180 r / min and continue culturing in a constant temperature shaker at 28°C for 3-4 days.

[0048] c. Preparation of spore solution.

[0049] Take 200uL of cultured Fusarium graminearum seed liquid, inoculate it into multiple solid culture medium plates with a diameter of 9cm, spread it evenly, and culture it in a constant temperature incubator at 28℃ for 5-10 days. Select the plate with plump and pure mycelium growth, use 15mL of sterile water for each plate, add it to the mycelium of the plate three times, gently scrape it with a coating stick, and use a pipette to transfer the spore liquid into a 100ml sterilized triangular flask, seal it, and store it in a refrigerator at 4℃ for later use.

[0050] All the above inoculation and spore fluid preparation processes must be performed under sterile conditions, that is, sterile operations must be performed next to an alcohol lamp on a clean bench that has been sterilized by ultraviolet light for half an hour.

[0051] Example 3

[0052] This example provides a method for preparing a tobacco root-knot nematode inoculation culture medium.

[0053] Melt the prepared potato sucrose PSA solid medium while it's still hot or by heating. Pour 20 mL into 9 cm diameter sterile Petri dishes in a sterilized clean bench. Too little will restrict the nematodes' living space, while too much will hinder their isolation. After the medium cools and solidifies, add 200 μL of the prepared Fusarium graminearum spore solution to each plate. Spread evenly with a glass spreader, seal with parafilm, and incubate in a 28°C incubator for 5-7 days, until white mycelium has grown throughout the plate. This is the inoculation medium for tobacco root-knot nematodes. Figure 1 A schematic diagram showing white hyphae growing over the entire plate.

[0054] Example 4

[0055] This example provides a method for preparing a tobacco root-knot nematode suspension.

[0056] Tobacco roots or rhizosphere soil infested with tobacco root-knot nematodes and showing obvious root knots were collected from the greenhouse test site of Yunnan Microbial Fermentation Engineering Research Center Co., Ltd., and the tobacco root-knot nematodes were isolated and collected using the Kappa sieving method. The specific operation method is: wash the tobacco rhizosphere soil sample and tobacco plant root tissue used for separation, cut them into pieces and put them into a container (2L triangular flask), add clean water to submerge the sample and stir it thoroughly to suspend the nematodes as much as possible (or place it in a 28°C constant temperature shaker and shake it for 1-2 hours), then let it stand for 3-5 minutes to allow the soil or plant root tissue to sink, then inject the suspension into a set of filter screens with 200 mesh, 400 mesh and 500 mesh, and then slowly rinse the screen with clean water. After sieving, collect the residue in the 400 mesh fine mesh into a beaker. Under normal circumstances, most tobacco root-knot nematodes are collected in the 400 mesh screen, about 100-300 / 10mL, and finally prepare a tobacco root-knot nematode suspension of about 200 / 10mL for use.

[0057] Example 5

[0058] This example provides a method for culturing and breeding root-knot nematodes by inoculating a 200 / 5 mL tobacco root-knot nematode suspension into a potato sucrose PSA culture medium.

[0059] In a sterile clean bench, take 0.1 mL of the prepared root-knot nematode suspension and inoculate it onto a PSA medium plate covered with Fusarium graminearum hyphae. Allow the inoculation droplet to completely penetrate the hyphae until it is immersed in the plate. Seal the plate with a sealing film and place it in a 28°C incubator for 7-14 days. During this period, it was observed that the hyphae on the plate remained unchanged in the first 3-5 days, and the hyphae at the inoculation site began to disappear after 5 days (such as Figure 2 ), and gradually extended to the surrounding areas. After 14 days, the hyphae completely disappeared (as shown in Figure 3 As shown), the dark red moist culture medium remains. Take 50 mL of distilled water to rinse the surface of the culture medium five times and collect them in a beaker. After they are completely collected, use a 5 mL pipette to draw 5 mL of washing solution from the beaker into a 6 cm diameter plate. Count the number of second-instar larvae of the root-knot nematode under an inverted microscope (as shown). Figure 4 The results showed that second-instar larvae of a tobacco root-knot nematode suspension incubated on PSA medium at 28°C began to hatch after 5 days and began feeding on Fusarium graminearum hyphae. Their numbers began to increase after 14 days and continued to multiply, reaching a peak of approximately 800 larvae per mL on the 30th day.

[0060] Example 6

[0061] This example provides a method for propagating and preserving tobacco root-knot nematodes by subculture via plate transfer.

[0062] Using the culture plates described in Example 5, cultured to day 30, 10 ml of the culture medium was placed in a 250 mL Erlenmeyer flask, 90 mL of distilled water was added, and the flask was sealed and mixed thoroughly. The mixed liquid consisted mostly of second-instar nematodes. 1 mL was taken for microscopic examination and counting, and a suspension of approximately 50 second-instar nematodes / mL was prepared. 1 mL was then inoculated onto a freshly prepared PSA culture plate covered with Fusarium graminearum. The plate was then incubated at 28°C for 7-14 days as described in Example 5. Observation records showed that the rate of consumption of Fusarium graminearum hyphae and conidia in the PSA culture plate and the rate of nematode proliferation were roughly the same as those described in Example 5. Further plate-changing experiments demonstrated that the propagation of tobacco root-knot nematodes through plate-changing subculture could be continued without restriction.

[0063] On the 30th day of culture, when the nematode reproduction density in the plate reached its peak, the plates were transferred to a constant temperature box at 10°C for storage. After 3 months, the plates were randomly removed for washing, collection and microscopic examination. The plates were counted under a microscopic examination according to the method described in Example 5, and it was found that the average live worm density in the plates was about 50-60% of that at the time of transfer. According to the method described in Example 5, the plates were transferred to a newly prepared medium grown with Fusarium graminearum and cultured again. The same results as those described in Example 5 were obtained, demonstrating that under a constant temperature of 10°C, the plate culture time can be as long as 3 months. If the purpose is only to preserve the population of tobacco root-knot nematodes, the plates need to be changed and cultured 4-5 times a year to ensure the source of tobacco root-knot nematodes.

[0064] Example 7

[0065] This example provides the ability of tobacco root-knot nematodes cultured in a Fusarium graminearum propagation medium to infect tomato plants.

[0066] Sandy loam soil was collected, sterilized at 121°C for 30 minutes, and left exposed indoors for two days with frequent stirring. A portion of the sterilized soil was used to grow tomato seedlings (tomato variety: Hezuo 903, Shanghai Changzhong Tomato Seed Co., Ltd.) in a floating seedling culture in a greenhouse. Four weeks later, seedlings with three or more true leaves were transplanted into pots containing sterilized soil and placed in a greenhouse. Water was applied every five days until the seedlings survived. Thirty days after transplanting, robust plants were selected and their roots were inoculated with a three-times-replaced tobacco root-knot nematode solution (100 mL / plant). Three tomato plants were inoculated with the solution, and three additional tomato plants were inoculated with water as a control, for a total of six potted treatments. Watering was carried out lightly daily. Thirty days after inoculation, each treated plant was gently removed from its pot with soil, its roots rinsed clean, and the root knot index was calculated.

[0067] The root knot index adopts a five-level classification method.

[0068] Level 0: No root knots.

[0069] Grade 1: trace root knots (1%-10%).

[0070] Grade 2: A small amount of root knots (11%-30%).

[0071] Grade 3: Moderate root knots (31%-50%).

[0072] Grade 4: Severe root knot (51%-70%).

[0073] Level 5: Root knots are very serious (more than 71%).

[0074] The statistical results are shown in Table 1. As shown in Table 1, tobacco root-knot nematodes cultured in the Fusarium graminearum propagation medium maintained their ability to infect host plants, re-infecting host roots and forming numerous root knots. This demonstrates that the method of the present invention is feasible for the cultivation and propagation of tobacco root-knot nematodes and that tobacco root-knot nematodes can be artificially cultured and maintained in the laboratory.

[0075] Table 1 Root knot index survey of potted tomato seedlings inoculated with tobacco root knot nematodes

[0076] Processing number Root knot index (unit: level) Process 1 3 Process 2 3 Process 3 2 Control 1 0 Control 2 0 Control 3 0

[0077] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A method for indoor cultivation of tobacco root-knot nematodes, characterized in that: The steps include: Step 1: Isolation, purification and cultivation of Fusarium graminearum: using tissue separation and single spore purification culture method to obtain purified strains of Fusarium graminearum from the straw of plants infected with fusarium graminearum; Step 2: Preparation of Fusarium graminearum seed liquid: The purified Fusarium graminearum strain and its carrier obtained in step 1 were punched with a puncher and then picked and inoculated into liquid PDA culture medium. The culture was carried out at a temperature of 27°C-28°C and a rotation speed of 180 r / min for 72 h to obtain Fusarium graminearum seed liquid, i.e., Fusarium graminearum suspension. The effective viable bacterial count of the bacterial suspension was controlled at 0.50×10 8 cfu / g or above; Step 3: Preparation of Fusarium graminearum plates for culturing tobacco root-knot nematodes: 100-200 μL of the obtained Fusarium graminearum seed solution is evenly spread on solid PSA culture medium, and then placed in a constant temperature incubator at 28°C for 5-7 days until the mycelium fills the plate for later use; Step 4: Tobacco root-knot nematode eggs and tobacco root-knot nematodes were screened by a sieving method, the tobacco root-knot nematodes were collected into a beaker using a pipette, and the suspension was diluted with distilled water to prepare a tobacco root-knot nematode suspension with a content of 200 / 10 mL; Step 5: Under sterile conditions, 1 mL of the bacterial suspension was inoculated onto a solid PSA plate grown with Fusarium graminearum, and the solid PSA medium grown with Fusarium graminearum was shaken left and right to completely penetrate the mycelium and mix with the mycelium, and the tobacco root-knot nematode suspension droplets were immersed in the PSA plate; Step 6: Seal the culture dish with a sealing film, with the front of the culture dish facing up, and culture the culture dish in a 28°C constant temperature incubator in dark conditions for 7-14 days. Rinse the PSA plate with sterile water to obtain the tobacco root-knot nematode suspension, and collect the tobacco root-knot nematode suspension.

2. The indoor cultivation method of tobacco root-knot nematodes according to claim 1, characterized in that: The preparation method of the liquid PDA culture medium includes the following steps: weighing 200g of potatoes and 20g of glucose in a preparation amount of 1L, washing and chopping the potatoes and boiling them for 30 minutes, filtering them through at least two layers of gauze to obtain a filtrate, dissolving the weighed glucose in the filtrate, and packaging the filtrate in triangular flasks for sterilization to obtain a liquid PDA culture medium for culturing Fusarium graminearum.

3. The indoor cultivation method of tobacco root-knot nematodes according to claim 1, characterized in that: The preparation method of solid PSA culture medium includes the following steps: taking 200g of potatoes, boiling for 30 minutes and filtering, adding 14g of sucrose to the potatoes, adding purified water to make the volume 1L, then adding 18g / L of agar powder, and adjusting the pH to 7.0-7.

4.

4. The indoor cultivation method of tobacco root-knot nematodes according to claim 1, characterized in that: The steps of obtaining a tobacco root-knot nematode suspension by screening include: Collect field soil samples where tobacco root-knot nematodes are infested and root tissues infected with tobacco root-knot nematodes. Wash the field soil samples and root tissues, cut them into pieces, and place them in a container. Cut the root tissue into pieces of 0.5-1 cm. Add pure water to the container to submerge the sample and stir thoroughly to suspend the tobacco root-knot nematodes. Alternatively, place the container in a 28°C constant temperature shaker and shake for 0.5-1 hour. Let it stand for 3-5 minutes to allow the soil or plant tissue to sink. Collect the suspension containing tobacco root-knot nematodes. The content of the tobacco root-knot nematode suspension in the tobacco root-knot nematode suspension is 100-300 tobacco root-knot nematodes per 10 ml. The suspension is injected into a filter sieve with 200 mesh, 400 mesh and 500 mesh, and the sieve is rinsed with clean water. The residue in the sieve is collected into a beaker or a culture dish. The 500 mesh sieve can collect tobacco root-knot nematode eggs, and the 400 mesh sieve can collect tobacco root-knot nematodes. The collected tobacco root-knot nematode eggs and tobacco root-knot nematodes are set aside.

5. The indoor cultivation method of tobacco root-knot nematodes according to claim 1, characterized in that: The preparation of a bacterial suspension containing 200 tobacco root-knot nematodes per 10 mL by dilution with distilled water includes the following steps: 5 mL of the collected tobacco root-knot nematodes is pipetted into a clean culture dish plate with a diameter of 6 cm, the plate is placed under a dissecting microscope to observe the number of tobacco root-knot nematodes, and water is added to dilute the suspension according to the observed number to a bacterial suspension containing 200 nematodes per 10 mL.

6. The indoor cultivation method of tobacco root-knot nematodes according to claim 1, characterized in that: Under sterile conditions, 1 mL of the bacterial suspension was aspirated and inoculated onto a solid PSA plate covered with Fusarium graminearum, comprising the following steps: irradiating with an ultraviolet lamp for 30 minutes in a sterile clean bench, and aspirating 1 mL of the bacterial suspension with a 1 mL pipette and inoculating onto the PSA plate covered with Fusarium graminearum.

7. The indoor cultivation method of tobacco root-knot nematodes according to claim 1, characterized in that: The method of collecting tobacco root-knot nematode suspension comprises the following steps: washing the PSA plate from which the hyphae of Fusarium graminearum disappear with sterile water, and collecting the washing liquid to obtain the tobacco root-knot nematode suspension.

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