A method for culturing root-knot nematodes

Abstract

The present invention relates to a method for culturing root-knot nematodes. The method comprises the following steps: 1) adding asparagine and / or carotene to the soil planted with chard; 2) inoculating the root-knot nematodes on the roots of the chard; 3) allowing the root-knot nematodes to infect the roots of the chard at the infection temperature of the root-knot nematodes, and allowing the root-knot nematodes to develop at the development temperature, and then propagating the nematodes at the propagation temperature; 4) harvesting the root-knot nematodes.

Description

Technical Field

[0001] The present invention relates to a method for culturing root-knot nematodes. Background Art

[0002] The root-knot nematodes that cause relatively serious harm in agricultural production mainly include Meloidogyne incognita, M. hapla, M. arenaria, M. javanica, and M. enterolobii. Screening for disease-resistant varieties against root-knot nematodes, identifying resistance genes and cloning and expressing functional genes, and researching agricultural, physical, biological, and chemical control methods for plant root-knot nematodes all require a large number of root-knot nematodes as targets. Moreover, a large number of root-knot nematodes are also needed as targets to verify the research on agricultural, physical, biological, and chemical control methods for root-knot nematodes in pot experiments.

[0003] Currently, the main method for in-vitro propagation of root-knot nematodes is to use the seedlings of some Solanaceae crops (such as potatoes, tomatoes, eggplants, sweet peppers, tobacco, etc.), Cucurbitaceae crops (cucumbers, loofahs, balsam pears, etc.), and Convolvulaceae crops (water spinach) as hosts. These crops are greatly restricted by seasons, and it takes about 60 days to harvest a small amount of nematodes. There are also reports on culturing Meloidogyne incognita with the in-vitro roots of water spinach, but it also takes at least 50 days to harvest a small amount of root-knot nematodes after inoculation. The main disadvantages of the existing in-vivo culture or in-vitro root culture are as follows: 1) The time cycle is relatively long. It takes at least 50 days to obtain a small amount of nematodes, and it also consumes a large amount of time and labor; 2) Most of the plants selected for in-vivo culture are also greatly restricted by seasons. For example, some are only suitable for planting in cool conditions, and some are only suitable for planting under relatively high temperature conditions; 3) Although the plants conventionally propagated in vivo are susceptible to Meloidogyne incognita, some crops have poor resistance to other root diseases such as root rot, fusarium wilt, bacterial wilt, and basal stem rot, as well as above-ground pests and diseases such as downy mildew, leaf mold, gray mold, greenhouse whitefly, spider mites, and thrips, which is not conducive to the reproduction of Meloidogyne incognita. 4) The secondary roots of the plants used for conventional in-vivo propagation are not very developed. Even some plants need artificial support due to their relatively tall growth, which is time-consuming and laborious; 5) After 2-3 generations of root propagation of some crops for in-vivo propagation of Meloidogyne incognita, the roots rot and die, resulting in the root-knot nematodes formed on the roots being unable to parasitize and reproduce for a long time; 6) The main reason for not being able to obtain a large number of offspring in a short time is that the inoculated nematodes cannot be concentratedly infected in a short time, and the development progress of the nematodes infecting the roots is inconsistent, resulting in the non-concentrated formation time of the offspring. Summary of the Invention

[0004] One aspect of the present invention provides a method for culturing Meloidogyne nematodes, which comprises the following steps:

[0005] 1) Adding asparagine and / or carotene to the soil planted with Swiss chard;

[0006] 2) Inoculating the Meloidogyne nematodes onto the roots of Swiss chard;

[0007] 3) Allowing the Meloidogyne nematodes to infect the roots of Swiss chard at the infection temperature of the nematodes, and allowing the nematodes to develop at the development temperature, and then allowing the nematodes to reproduce at the reproduction temperature;

[0008] 4) Harvesting the Meloidogyne nematodes.

[0009] In a specific embodiment, the Meloidogyne nematode is Meloidogyne incognita.

[0010] In a specific embodiment, in step 1), the addition amount of asparagine is 0.4 g / L soil to 0.8 g / L soil.

[0011] In a specific embodiment, in step 1), the addition amount of carotene is 4 g / L soil to 6 g / L soil.

[0012] In a specific embodiment, in step 1), the mass ratio of the addition of asparagine to carotene is 1:10.

[0013] In a specific embodiment, in step 2), the inoculation amount of the Meloidogyne nematodes is 500 nematodes per plant to 2000 nematodes per plant.

[0014] In a specific embodiment, in step 2), the inoculation amount of the Meloidogyne nematodes is 1000 nematodes per plant to 2000 nematodes per plant.

[0015] In a specific embodiment, in step 2), the inoculated Meloidogyne nematodes are second-stage larvae.

[0016] In a specific embodiment, in step 3), the infection temperature is 20 to 30 °C.

[0017] In a specific embodiment, in step 3), the infection time is 6 to 9 days.

[0018] In a specific embodiment, in step 3), the development temperature is 25 to 28 °C.

[0019] In a specific embodiment, in step 3), the development time is 6 to 12 days.

[0020] In a specific embodiment, in step 3), the development time is 9 to 12 days.

[0021] In a specific embodiment, in step 3), the breeding temperature is 28 to 30 °C.

[0022] In a specific embodiment, in step 3), the breeding time is 19 to 35 days.

[0023] In a specific embodiment, in step 3), first infect at an infection temperature of 25 °C for 6 days, then transfer to a development temperature of 28 °C for 9 days, and then transfer to a breeding temperature of 30 °C for 25 days to harvest.

[0024] Advantages of the present invention:

[0025] The present invention first discovers that asparagine and / or carotene are beneficial to the infection and reproduction of root-knot nematodes. In addition, the infection temperature of root-knot nematodes is 20 to 30 °C, and the infection time is 6 to 9 days; the development temperature is 25 to 28 °C, and the development time is 6 to 12 days; the breeding temperature is 28 to 30 °C, and the breeding time is 19 to 35 days. The above culture conditions are beneficial to shortening the culture time, so a large number of nematode populations can be obtained in a short time. The above can save labor and time for subsequent use. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Shows the effects of different constant temperatures of 10, 15, 20, 25, 30, and 34 °C on the number of second-stage larvae invading the roots.

[0027] Figure 2 Shows the effects of different constant temperatures of 25, 28, 30, and 32 °C on the development of third-stage larvae.

[0028] Figure 3 Shows the effects of different constant temperatures of 25, 28, 30, and 32 °C on the development of fourth-stage larvae.

[0029] Figure 4 Shows the effect of asparagine on the infectivity of Meloidogyne incognita.

[0030] Figure 5 Shows the effect of carotene on the infectivity of Meloidogyne incognita. DETAILED DESCRIPTION OF THE INVENTION

[0031] The above content of the present invention will be further described in detail below in the form of preferred implementation cases, but it does not constitute a limitation to the present invention.

[0032] Unless otherwise specified, the reagents in the embodiments of the present invention can be purchased through commercial channels.

[0033] The variety of chard is Baifengyuan chard, purchased from the breeding base of high-quality leafy vegetables in Shandong Province.

[0034] The variety of tomato is Shuoyuan Jiali.

[0035] The variety of water spinach is Xinmei Zhuye water spinach.

[0036] The bottom of the small pots for planting chard and water spinach has 12 small round holes in two inner and outer circles by itself, 8 in the outer circle and 4 in the inner circle. The diameter of each small round hole is about 0.8 cm, and the size is Φ11 cm × 10 cm. The bottom of the small pot is placed on the tray. The upper diameter of the tray is Φ11 cm, and the height is about 2 cm.

[0037] Example

[0038] 1. Seedling raising

[0039] Put the seeds of tomato, chard or water spinach into hot water at 50 °C to 55 °C and soak for 20 minutes. Stir constantly during the soaking process to avoid scalding the seeds. Then wash them clean with clean water and soak in clean water for about 6 h. Then pour off most of the liquid, transfer the seeds to a sterilized petri dish lined with 2 layers of sterilized filter paper, and place them in a constant temperature incubator at 28 °C for germination. Seed white appears in about 2 to 3 days. When about 70% of the seeds show white, sow them on the seedling tray with pre-placed seedling soil, and place them under greenhouse conditions for cultivation. When the seedlings grow to 5 leaves and 1 core in 35 to 45 days, transplant them into small pots.

[0040] 2. Obtaining root-knot nematodes

[0041] Transplant the well-raised tomato seedlings into small pots, plant 1 plant in each pot, and plant them with sterilized (121 °C, 30 min) sandy soil (the ratio of fine sand to loam is 3:1). After planting, place them in a greenhouse at 22 to 27 °C. After 5 days of slow seedling growth, inoculate with Meloidogyne incognita. The total amount of eggs and larvae inoculated per plant is 1000. Among them, when inoculating, use a glass rod to make 3 holes at a distance of 2 - 3 cm from the base of the tomato seedling stem in a triangular shape. Use a 1 mL pipette to suck 1000 nematode suspensions of eggs and second-instar larvae, and vertically and evenly drip them into the 3 small holes, and then bury the small holes with the nearby soil. After 70 days of inoculation, obtain Meloidogyne incognita from the tomato roots.

[0042] Among them, the method of harvesting nematodes from tomato roots is as follows: First, wash the tomato roots clean, then cut them into small segments 1 cm long with scissors, add an aqueous solution of 1 wt% sodium hypochlorite to submerge the root system and treat for 4 minutes. During this period, continuously stir with a glass rod, then pour it onto a nested sieve of 60 mesh and 700 mesh. Rinse the 60-mesh sieve with running water, and then rinse the 700-mesh sieve until there is no smell of sodium hypochlorite. Then rinse with sterile water 3 times. Rinse and collect the eggs and larvae on the 700-mesh sieve into a beaker to obtain a mixed solution of eggs and larvae. Then pour the obtained mixed solution of eggs and larvae (usually mainly eggs and very few larvae when just washed out) into a sterilized petri dish with a diameter of Φ15 cm, place it in a constant temperature incubator at 28 °C for 1 week, and then separate the eggs and larvae by the shallow dish method: The outer dish is an open container that can hold liquid, the inner dish is a 60-mesh sieve, place a support on the bottom surface of the outer dish, place the inner dish on the support, then lay 2 layers of paper towels of 21 cm × 21 cm in the inner dish, pour the mixed solution of eggs and larvae into the inner dish. The water in the mixed solution quickly seeps from the inner dish to the outer dish. If the mixed solution of eggs and larvae is not enough to make the water in the outer dish cover the bottom of the inner dish, sterile water needs to be added to make the water in the outer dish cover the bottom of the inner dish. Let it stand for 8 h, so that the eggs and the dead larvae are intercepted on the paper towel, while the live second-stage larvae migrate from the 60-mesh sieve to the outer dish and settle to the bottom of the dish, thus realizing the separation of eggs and larvae. Discard the water in the outer dish carrying the second-stage larvae, and then add sterile water to the inner dish again until the water in the outer dish covers the bottom of the inner dish. After incubating the shallow dish in an incubator at 28 °C for one week, collect the concentrated second-stage larvae (J2) hatched. After washing the second-stage larvae clean, suspend the obtained nematode suspension with sterile water, then add an aqueous solution of streptomycin sulfate so that the concentration of streptomycin sulfate in the nematode suspension is 2000 ppm, disinfect for 10 hours, and then wash with sterile water 5 times. Prepare the nematodes into a nematode solution with a concentration of 500 second-stage larvae / mL with sterile water, and place it at 8 to 10 °C for subsequent inoculation of chard and water spinach.

[0043] 3. Effects of chard and water spinach on root-knot nematodes

[0044] Transplant the pre-raised seedlings of chard and water spinach into small pots with a size of Φ11 cm × 10 cm respectively. The pots are filled with 500 mL of sterilized sandy soil (the ratio of fine sand to loam is 3:1). Transplant 1 chard seedling or water spinach seedling into each pot, and transplant 6 pots of chard and 6 pots of water spinach respectively. After transplantation, place them in a climate chamber at 25 °C, with 12 h of light and 12 h of darkness every day, and keep the soil in the pot moist every day.

[0045] Five days after transplantation, 500 second-instar larvae were inoculated into each plant in the same way as inoculated onto tomato seedlings. At 21 days and 31 days after inoculation, the root lengths, the numbers of primary roots and secondary roots of 3 pots of leaf beet and 3 pots of water spinach were investigated; as well as the numbers of root knots, second-instar larvae (J2), third-instar larvae (J3) and fourth-instar larvae (J4) on the roots. Among them, the results of root lengths, the numbers of primary roots and secondary roots are shown in Table 1, and the results of the numbers of root knots, J2, J3 and J4 on the roots are shown in Table 2.

[0046] Table 1

[0047]

[0048] Note: The data in the table are mean ± standard error. Different lowercase letters after the data in the same row indicate significant differences at the P<0.05 level tested by Duncan's new multiple range method.

[0049] As can be seen from Table 1, the root lengths, the numbers of primary roots and secondary roots of leaf beet are all significantly higher than those of water spinach.

[0050] Table 2

[0051]

[0052] Note: The data in the table are mean ± standard error. Different lowercase letters after the data in the same row indicate significant differences at the P<0.05 level tested by Duncan's new multiple range method.

[0053] As can be seen from Table 2, at 21 days and 31 days after inoculation, root knots had formed on the roots, and the numbers of root knots, as well as the numbers of J2, J3 and J4 in leaf beet were all significantly higher than those in water spinach.

[0054] 4. Effects of different constant temperatures on the infection and reproduction of root-knot nematodes

[0055] The pre-raised leaf beet seedlings were transplanted into small pots with a size of Φ11 cm × 10 cm. Each pot was filled with 500 mL of sterilized sandy soil (the ratio of fine sand to loam was 3:1), and 1 leaf beet seedling was transplanted into each pot, with a total of 36×5 pots transplanted. After transplantation, 36 pots were placed in a constant temperature incubator at 10°C, 36 pots were placed in a constant temperature incubator at 15°C, 36 pots were placed in a constant temperature incubator at 20°C, 36 pots were placed in a constant temperature incubator at 25°C, 36 pots were placed in a constant temperature incubator at 30°C, and 36 pots were placed in a constant temperature incubator at 34°C. The light and darkness were each 12 h per day, and the soil in the pots was kept moist every day.

[0056] Five days after transplantation, each plant was inoculated with 500 second-instar larvae in the same inoculation manner as that for tomato seedlings. At 3, 6, 9, 12, 15, 18, and 21 days after inoculation, 3 pots were taken at each temperature. After taking out 3 chards, the roots were rinsed clean with running water and stained with acid fuchsin staining solution (3.5 g of acid fuchsin was dissolved in 250 ml of acetic acid, and after dissolution, it was made up to 1 L with distilled water), and the number of second-instar larvae invading the roots at different stages in the roots was observed under a microscope. The results are shown in Figure 1 ; At 18, 21, 24, 27, 30, 40, and 50 days after inoculation, the nematodes on the roots were washed off with 1 wt% sodium hypochlorite aqueous solution, and the nematodes on the roots were counted. The results are shown in Table 3.

[0057] According to Figure 1 the results, when infecting for different days under different constant temperature conditions of 10, 15, 20, 25, 30, and 34 °C, in the temperature range of 10 to 25 °C, the infectivity increased with the increase of temperature. Among them, at 10 °C, only 0.5 J2 / plant invaded the roots at 18 days after inoculation; at 15 °C and 20 °C, 3.2 J2 / plant and 2.4 J2 / plant invaded the roots at 6 days after inoculation respectively, and the invasion number reached the maximum of 94.9 J2 / plant at 9 days after inoculation under the condition of 20 °C; at 25 °C, a small amount of nematodes invaded the roots during the investigation at 3 days, and the infection peak was reached at 6 days, with an average of 125.4 J2 / plant per plant; while the infectivity decreased after 30 °C, and only 54.1 J2 / plant was reached at 6 days. No J2 was detected invading into the roots within 3 to 21 days after inoculation at 34 °C. It shows that the infection conditions of 20 to 30 °C for 6 to 9 days are better, among which the infection condition of 25 °C for 6 days is the best.

[0058] According to the results in Table 3, in the reproduction of Meloidogyne incognita within 50 days under different constant temperature conditions, no offspring were produced at 10 to 15 °C and 34 °C, while in the temperature range of 20 °C to 30 °C, the number of offspring produced increased with the increase of temperature. At 30 °C, a small amount of offspring were produced at 18 days, which was 18 total nematodes / plant. At 25 °C, a small amount of offspring were produced at 24 days, which was 12 total nematodes / plant, while the number of offspring produced at 30 °C at this time was 496 total nematodes / plant. At 30 days, the offspring produced at 25 °C and 30 °C exceeded 10,000, which were 16,220.3 nematodes / plant and 28,793.3 nematodes / plant respectively; at 40 days, offspring began to be produced at 20 °C, which was 193.3 nematodes / plant, while the offspring produced at 25 °C and 30 °C at this time reached 52,635.3 nematodes / plant and 71,542.7 nematodes / plant respectively.

[0059] Table 3

[0060]

[0061] Note: The data in the table are mean ± standard error. Different lowercase letters after the data in the same row indicate significant differences at the P<0.05 level by Duncan's new multiple range test.

[0062] 5. Developmental and reproductive conditions at different temperatures after inoculation at a constant temperature of 25°C

[0063] Transplant 123 pots of root spinach into a constant temperature incubator at 25°C. Others are the same as in 1.4.

[0064] At the end of the 6th day of cultivation at 25°C after inoculation, randomly select 3 pots. After rinsing the roots with running water, stain the roots using the acid fuchsin staining method, and observe the number of J2, J3, and J4 invading the roots under a microscope. Then, divide the 120 pots into portions of 30 pots each and transfer them to 4 temperature conditions of 25, 28, 30, and 32°C for cultivation. At 9, 12, 15, 18, and 21 days after inoculation (corresponding to the number of days of further cultivation at different temperatures after 6 days of cultivation and infection at 25°C, which are 3, 6, 9, 12, and 15 days respectively), randomly select 3 pots, take out 3 root spinach plants, rinse the roots with running water, stain the roots using the acid fuchsin staining method, and observe the number of J2, J3, and J4 in the roots. Among them, the number of J3 is shown in Figure 2 , and the number of J4 is shown in Figure 4 ; At 21, 24, 30, 40, and 50 days after inoculation (corresponding to the number of days of further cultivation at different temperatures after 6 days of cultivation and infection at 25°C, which are 15, 18, 24, 34, and 44 days respectively), randomly select 3 pots, take out 3 root spinach plants, wash the nematodes on the roots with 1wt% sodium hypochlorite aqueous solution, and count the nematodes on the roots. The results are shown in Table 4.

[0065] According to Figure 2 's results, it can be seen that as the temperature increases, the time taken for the peak period of J3 development becomes shorter and shorter. At 25°C, the peak period is reached 15 days after inoculation, with 152.3 nematodes per plant; at 28°C, J3 reaches the development peak after 6 days of further cultivation, that is, 12 days after inoculation, with 168.3 nematodes per plant. The number of J3 developed at 28°C is significantly higher than that at 25°C. The number of J3 developed at the peak period under these two temperature conditions is significantly higher than that at 30°C and 32°C. At 30°C and 32°C, the development peak periods are both reached on the 9th day after inoculation, with 55.6 nematodes per plant and 32 nematodes per plant respectively. It shows that the conditions for better J3 development are 6 to 9 days of further cultivation at 25 to 28°C, that is, 12 to 15 days after inoculation. Among them, the best condition for J3 development is 6 days of further development at 28°C, that is, 12 days after inoculation.

[0066] According to Figure 3It can be seen from the results that as the temperature increases, the peak time for J3 to develop into J4 becomes shorter and shorter. At 25°C, the peak is reached 18 days after inoculation, with 95 individuals per plant; at 28°C, after continued cultivation for 9 days, the development peak is reached 15 days after inoculation, with 123.5 individuals per plant; at 30°C, after continued cultivation for 6 days, the development peak is reached on the 12th day after inoculation, with 67.4 individuals per plant; at 32°C, after continued cultivation for 3 days, it is 26.3 individuals per plant on the 9th day after inoculation. Among them, the number of J4 developed at 28°C is significantly higher than that at 25°C, and the number of J4 developed at the peak under these two temperature conditions is significantly higher than that at 30°C and 32°C. It shows that continued development at 25 to 28°C for 9 to 12 days, that is, 15 to 18 days after inoculation, is a better condition for J4 development. Among them, continued development at 28°C for 9 days, that is, 15 days after inoculation, is the best condition for J4 development.

[0067] According to the results in Table 4, as the cultivation time prolongs, the number of offspring reproduced under the same temperature condition becomes more and more. Under different temperature conditions, between 25°C and 30°C, as the temperature rises, before 40 days after inoculation, the number of offspring produced within the same time is increasing and there are significant differences. Among them, at 21 days after inoculation (that is, after transferring from 25°C to each temperature and culturing for another 15 days), the number of offspring produced at 30°C reaches the maximum of 1,129 heads per plant, followed by 820 heads per plant at 28°C, 482.7 heads per plant at 32°C, and 0 heads per plant at 25°C; at 30 days, the number of offspring produced at 30°C reaches the maximum of 43,565.3 heads per plant, followed by 32,902.3 heads per plant at 28°C, 23,474.7 heads per plant at 32°C, and 16,568.7 heads per plant at 25°C. After 40 days of inoculation (that is, after transferring from 25°C to each temperature and culturing for another 34 days), the highest number of offspring is produced at 28°C, which is 146,539 heads per plant, followed by 103,481 heads per plant at 30°C. There is no significant difference in the production quantities at 32°C and 25°C, which are 51,015.3 heads per plant and 54,528 heads per plant respectively. According to the number of offspring produced from inoculation to the 30th day (that is, after transferring from 25°C to each temperature and culturing for another 24 days), 30°C is significantly higher than other temperatures, indicating that the J2 larvae that invade the roots earlier at 30°C develop faster, produce offspring earlier, and form the largest number of offspring; while at 40 days of inoculation (that is, after transferring from 25°C to each temperature and culturing for another 34 days) and 50 days of inoculation (that is, after transferring from 25°C to each temperature and culturing for another 44 days), the number of offspring produced at 28°C is significantly higher than that at 30°C. This shows that when transferred to different temperatures under the same conditions with similar invasion quantities, the reason why the number of offspring produced at 30°C is the largest in the early stage is that some of the second-stage larvae invading at 30°C can further develop and develop faster than at 28°C; while in the later stage, the number of offspring produced at 28°C is more than that at 30°C because the number of nematodes invading at 28°C that can further develop is more than that at 30°C. At 30°C, due to the relatively high temperature, some of the invading second-stage larvae will float outside the root system and cannot further develop, so the number of offspring produced in the later stage is less than that at 28°C, and even fewer J2 larvae at 32°C can further develop. It shows that continuing to develop at 28 to 30°C for 34 to 44 days is a better condition for reproducing offspring, among which, developing at 30°C for 34 to 44 days is the best reproduction condition.

[0068] Table 4

[0069]

[0070] Note: The data in the table are mean ± standard error. Different lowercase letters after the data in the same column indicate significant differences at the P<0.05 level tested by Duncan's new multiple range method.

[0071] 6. Reproduction situation after invading at 6.25℃ for 6 days, then transferring to 28℃ for 9 days and then to 30℃ for different days of development

[0072] Transplant the pre-grown root spinach seedlings into small pots with a size of Φ11 cm × 10 cm. The pots are filled with 500 mL of sterilized sandy soil (the ratio of fine sand to loam is 3:1). Transplant 1 root spinach seedling per pot, and a total of 15 pots are transplanted.

[0073] After transplantation, place them in a constant temperature incubator at 25℃, with 12 hours of light and 12 hours of darkness each day, and keep the soil in the pots moist every day.

[0074] Five days after transplantation, inoculate 500 J2 per plant, and the inoculation method is the same as that for tomato seedlings. At the end of the 6th day after inoculation, transfer them to 28℃ for 9 days and then to 30℃ for continuous development (30℃ treatment). Investigate the reproduction situation after culturing at 30℃ for 6, 9, 12, 15, 25 days, that is, corresponding to 21, 24, 27, 30, 40 days after inoculation. And use the treatment of invading at 25℃ for 6 days and then transferring to 28℃ for continuous development and reproduction as the control (28℃ treatment). When investigating, randomly select 3 pots each time. After washing the roots in the pots with running water and cutting them into small sections about 1 cm long, wash the nematodes on the roots with 1 wt% sodium hypochlorite aqueous solution, and count the nematodes on the roots. The results are shown in Table 5.

[0075] According to the results in Table 5, it can be seen that the total number of nematodes obtained in the treatment is significantly higher than that in the control. The former can obtain 33539.7 nematodes per plant and 268770.3 nematodes per plant at 27 and 30 days after inoculation, while the control only has 9839.3 nematodes per plant and 33216.7 nematodes per plant.

[0076] Table 5

[0077]

[0078] Note: The data in the table are mean ± standard error. Different lowercase letters after the data in the same row indicate significant differences at the P<0.05 level tested by Duncan's new multiple range method.

[0079] 7. Effects of additives on the infectivity and reproduction of Meloidogyne

[0080] 7.1 Effects of asparagine on the infectivity and reproduction of Meloidogyne

[0081] The pre-grown Beta vulgaris seedlings were transplanted into small pots with a size of Φ11 cm × 10 cm. Each pot was filled with 500 mL of sterilized sandy soil (the ratio of fine sand to loam was 3:1), 500 mL of sterilized sandy soil (the ratio of fine sand to loam was 3:1) added with 0.1 g of asparagine, 500 mL of sterilized sandy soil (the ratio of fine sand to loam was 3:1) added with 0.2 g of asparagine, and 500 mL of sterilized sandy soil (the ratio of fine sand to loam was 3:1) added with 0.4 g of asparagine. There were a total of 4 treatments. One Beta vulgaris seedling was transplanted into each pot, and 21 pots were transplanted for each treatment.

[0082] After transplantation, they were placed in an incubator at a constant temperature of 25 °C, with 12 h of light and 12 h of darkness each day, and the soil in the pots was kept moist every day.

[0083] Five days after transplantation, 500 J2 per plant were inoculated into each plant. The inoculation method was the same as that for tomato seedlings. After inoculation at 25 °C, at 3, 6, 9, 12, and 15 d of cultivation, 3 pots were randomly selected each time. After the roots were rinsed clean with running water, the number of second-stage larvae invading the roots was observed by staining. The results are shown in Figure 4 ; At 30, 40, and 50 d after cultivation, 3 pots of each treatment were randomly selected. After the roots were rinsed clean, they were cut into small segments about 1 cm long. The nematodes on the roots were washed off with 1 wt% sodium hypochlorite aqueous solution, and the nematodes on the roots were counted. The results are shown in Table 6.

[0084] According to Figure 4 the results, adding different doses of asparagine to the soil could significantly improve the infectivity of Meloidogyne incognita. Among them, when the dose was 0.4 g of asparagine per liter of soil, the infectivity increased the most. At 6 d after inoculation, the maximum number of invaded nematodes reached 215.6 nematodes per plant; while for the treatment without adding asparagine, it was only 121.4 nematodes per plant at 6 d after inoculation.

[0085] According to the results in Table 6, since the significantly increased number of invaded nematodes also led to an increase in the number of offspring produced. Among them, when 0.4 g of asparagine was added per liter of soil during the same cultivation time, the number of offspring produced by the roots was the largest. At 30, 40, and 50 d after inoculation, the roots produced 29067.7 offspring per plant, 96570 offspring per plant, and 128481 offspring per plant respectively; while for the control without adding asparagine, they were 15845 offspring per plant, 53877 offspring per plant, and 66789.3 offspring per plant at 30, 40, and 50 d after inoculation respectively.

[0086] Table 6

[0087]

[0088] Note: The data in the table are mean ± standard error. Different lowercase letters after the data in the same column indicate significant differences at the P<0.05 level tested by Duncan's new multiple range method.

[0089] 7.2 Effects of carotene on the infectivity and reproduction of Meloidogyne

[0090] The pre-grown root spinach seedlings were transplanted into small pots with a size of Φ11 cm × 10 cm. The pots were filled with 500 mL of sterilized sandy soil (the ratio of fine sand to loam was 3:1), 500 mL of sterilized sandy soil (the ratio of fine sand to loam was 3:1) added with 1 g of carotene, 500 mL of sterilized sandy soil (the ratio of fine sand to loam was 3:1) added with 2 g of carotene, and 500 mL of sterilized sandy soil (the ratio of fine sand to loam was 3:1) added with 3 g of carotene. There were a total of 4 treatments. One root spinach seedling was transplanted into each pot, and 21 pots were transplanted for each treatment.

[0091] After transplantation, they were placed in a constant temperature incubator at 25 °C, with 12 h of light and 12 h of darkness each day, and the soil in the pots was kept moist every day.

[0092] Five days after transplantation, each plant was inoculated with 500 J2 per plant, and the inoculation method was the same as that for tomato seedlings. After inoculation at 25 °C, at 3, 6, 9, 12, and 15 d after culture, 3 pots were randomly selected each time. After the roots were washed clean with running water, the number of second-stage larvae invading the roots was observed by staining method. The results are shown in Figure 5 ; At 30, 40, and 50 d after culture, 3 pots of each treatment were randomly selected. After the roots were washed clean, they were cut into small segments about 1 cm long. The nematodes on the roots were washed off with 1 wt% sodium hypochlorite aqueous solution, and the nematodes on the roots were counted. The results are shown in Table 7.

[0093] According to Figure 5 the results, adding different doses of carotene to the soil can also improve the infectivity to Meloidogyne. Among them, when the dose is 4 g of carotene added per liter of soil, the infectivity increases the most. The maximum number of invaded individuals reaches 194.3 individuals per plant at 6 d after inoculation; while for the control without adding carotene, it is 119.7 individuals per plant at 6 d after inoculation.

[0094] According to the results in Table 7, since the significantly increased number of invaded individuals also leads to an increase in the number of offspring produced. Among them, when the same culture time is considered, when 4 g of carotene is added per liter of soil, the number of offspring produced by the roots is the largest. At 30, 40, and 50 d after inoculation, the roots produce 26396.7 individuals per plant, 85689 individuals per plant, and 105689.3 individuals per plant respectively, while for the control without adding carotene, they are 14991.3 individuals per plant, 52365 individuals per plant, and 65440.7 individuals per plant at 30, 40, and 50 d after inoculation respectively.

[0095] Table 7

[0096]

[0097] Note: The data in the table are mean ± standard error. Different lowercase letters after the data in the same column indicate significant differences at the P < 0.05 level by Duncan's new multiple range test.

[0098] 8. Effects of the combined temperature change of adding two substances on reproduction

[0099] The pre-raised seedlings of leaf beet were transplanted into small pots with a size of Φ11 cm × 10 cm. Each pot was filled with 500 mL of sterilized sandy soil (the ratio of fine sand to loam was 3:1), 500 mL of sterilized sandy soil (the ratio of fine sand to loam was 3:1) added with 0.2 g of asparagine, 500 mL of sterilized sandy soil (the ratio of fine sand to loam was 3:1) added with 2 g of carotene, and 500 mL of sterilized sandy soil (the ratio of fine sand to loam was 3:1) added with 0.2 g of asparagine and 2 g of carotene. There were 4 treatments in total. One leaf beet seedling was transplanted into each pot, and 12 pots were transplanted for each treatment, with a total of 48 pots.

[0100] After transplantation, they were placed in a constant temperature incubator at 25 °C, with 12 h of light and 12 h of darkness every day, and the soil in the pot was kept moist every day.

[0101] Five days after transplantation, 500 J2 per plant were inoculated, and the inoculation method was the same as that for tomato seedlings. At the end of the 6th day after inoculation, they were transferred to 28 °C for 9 days of development and then transferred to 30 °C for continued development. The reproduction was investigated at 9, 12, 15, and 25 days of cultivation at 30 °C, that is, corresponding to 24, 27, 30, and 40 days after inoculation. During the investigation, 3 pots were randomly selected each time. The roots were washed clean with running water and cut into small sections about 1 cm long, and the nematodes on the roots were washed off with 1 wt% sodium hypochlorite aqueous solution, and the nematodes on the roots were counted. The results are shown in Table 8.

[0102] According to the results in Table 8, at 24 days after inoculation, the treatment with the addition of both substances had the highest number of offspring produced, which was 13,895.7 individuals per plant; followed by the treatment with the addition of asparagine, which was 11,914 individuals per plant; then the treatment with the addition of carotene, which was 9,940.3 individuals per plant; and finally the treatment without the addition of substances, which was 6,357 individuals per plant. The trends at 27 days, 30 days, and 40 days were the same as those at 24 days. This shows that the combined use of asparagine and carotene has a synergistic effect.

[0103] Table 8

[0104]

[0105] Note: The data in the table are mean ± standard error. Different lowercase letters after the data in the same column indicate significant differences at the P<0.05 level by Duncan's new multiple range test.

[0106] 9. Effect of inoculation density on offspring production

[0107] The pre-grown root spinach seedlings were transplanted into small pots with a size of Φ11 cm × 10 cm. The pots were filled with 500 mL of sterilized sandy soil (the ratio of fine sand to loam is 3:1), 500 mL of sterilized sandy soil (the ratio of fine sand to loam is 3:1) added with 0.2 g of asparagine, 500 mL of sterilized sandy soil (the ratio of fine sand to loam is 3:1) added with 2 g of carotene, and 500 mL of sterilized sandy soil (the ratio of fine sand to loam is 3:1) added with 0.2 g of asparagine and 2 g of carotene, a total of 4 soil treatments. Each soil treatment was further divided into three inoculation amount treatments, namely inoculating 500, 1000, and 2000 second-instar larvae per plant. The investigation was carried out at 27 and 30 days after inoculation, with a total of 24 treatments. One root spinach seedling was transplanted into each pot, and 3 pots were transplanted for each treatment, with a total of 72 pots.

[0108] After transplantation, it was placed in a constant temperature incubator at 25 °C, with 12 h of light and 12 h of darkness every day, and the soil in the pot was kept moist every day.

[0109] Five days after transplantation, root-knot nematodes were inoculated respectively. When inoculating, small holes were pricked with a thin glass rod (about 1 cm in diameter) at a distance of 2-3 cm from the base of the root spinach seedling stem. Among them, when inoculating 500 second-instar larvae per plant, 3 holes were pricked in a triangular shape; when inoculating 1000 second-instar larvae per plant, 4 holes were pricked; when inoculating 2000 second-instar larvae per plant, 6 holes were pricked. The nematode suspension was prepared into 1 mL containing 500 second-instar larvae. The nematode suspension was sucked with a 1 mL pipette and vertically dropped into the small holes, and then the small holes were buried with the nearby soil. At the end of the 6th day after inoculation, it was transferred to 28 °C for 9 days of development and then transferred to 30 °C for continued development. At 30 °C, it was continued to be cultured for 12 and 15 days respectively, that is, corresponding to 27 and 30 days after inoculation. Randomly select 3 pots, and after washing the roots of the root spinach in them with running water and cutting them into small sections about 1 cm long, the nematodes on the roots were washed off with 1 wt% sodium hypochlorite aqueous solution, and the nematodes on the roots were counted. The results are shown in Table 9.

[0110] According to the results in Table 9, inoculation densities of 500 to 2000 second-instar larvae per plant can harvest a large number of root-knot nematodes in the later stage. Among them, there are significant differences in the number of offspring produced by the inoculation density, and with the increase of the inoculation density, the number of offspring produced is more; and the treatment with the addition of asparagine and carotene at the same time produces the highest number of offspring, significantly higher than the other three treatments, which is consistent with the trend in Table 8.

[0111] Table 9

[0112]

[0113] Note: The data in the table are mean ± standard error. Different lowercase letters after the data in the same column or different uppercase letters in the same row at the same number of days with different inoculation densities indicate significant differences at the P<0.05 level by Duncan's new multiple range test.

Claims

1. A method for culturing Meloidogyne, comprising the following steps: 1) Adding asparagine and / or carotene to the soil planted with Swiss chard, wherein the addition amount of asparagine is 0.4 g / L soil to 0.8 g / L soil, and the addition amount of carotene is 4 g / L soil to 6 g / L soil; 2) Inoculating the Meloidogyne on the roots of Swiss chard; 3) Allowing the Meloidogyne to infect the roots of Swiss chard at the infection temperature, allowing the Meloidogyne to develop at the development temperature, and then allowing the nematodes to reproduce at the reproduction temperature; the infection temperature is 20 to 30 °C, the development temperature is 25 to 28 °C, and the reproduction temperature is 28 to 30 °C; 4) Harvesting the Meloidogyne.

2. The method according to claim 1, characterized in that, The Meloidogyne is Meloidogyne incognita.

3. The method according to claim 1 or 2, characterized in that, In step 1), the mass ratio of the added asparagine to the added carotene is 1:

10.

4. The method according to claim 1 or 2, characterized in that, In step 2), the inoculation amount of the Meloidogyne is 500 nematodes per plant to 2000 nematodes per plant.

5. The method according to claim 1 or 2, characterized in that, In step 2), the inoculation amount of the Meloidogyne is 1000 nematodes per plant to 2000 nematodes per plant.

6. The method according to claim 1 or 2, characterized in that In step 2), the inoculated Meloidogyne is second-stage larvae.

7. The method according to claim 1 or 2, characterized in that In step 3), the infection time is 6 to 9 d.

8. The method according to claim 1 or 2, characterized in that, In step 3), the development time is 6 to 12 d.

9. The method according to claim 1 or 2, characterized in that In step 3), the reproduction time is 19 to 35 d.

10. The method according to claim 1 or 2, characterized in that, in In step 3), first infect at an infection temperature of 25 °C for 6 d, then transfer to a development temperature of 28 °C for 9 d, and then transfer to a reproduction temperature of 30 °C for 25 d for harvesting.

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