Method for artificially inoculating ditylenchus destructor on sweet potato tubers and resistance identification method

Abstract

The invention belongs to the technical field of agricultural plant protection, and particularly relates to a method for artificially inoculating ditylenchus destructor on sweet potato tubers and a resistance identification method. The method for artificially inoculating the ditylenchus destructor onto the sweet potato tubers comprises the following steps: sealing the bottoms of the shaded surfaces of the sweet potato tubers by using paraffin, uniformly dropwise adding a ditylenchus destructor suspension along one week above the paraffin, vertically inserting the shaded surfaces of the sweet potato tubers into moisturized flower arrangement mud downwards, and carrying out dark culture at 26-30 DEG C for 38-42 days, by means of the method, the requirement of susceptible control can be met after inoculation is conducted for 30 days, the invention further provides a method for identifying the invasion resistance of the sweet potatoes to the ditylenchus destructor, the method comprises the method for artificially inoculating the ditylenchus destructor to the sweet potato blocks, and the method is accurate in result, shorter in identification time and high in identification efficiency. The method makes a contribution to establishment of rapid and environment-friendly prevention and control measures for the ditylenchus destructor.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural plant protection, and in particular relates to a method for artificially inoculating sweet potato tubers with stem nematodes and a resistance identification method. Background Art

[0002] Sweet potato stem nematode disease is one of the most serious diseases that harm sweet potatoes. The pathogen is the rot stem nematode (Ditylenchus destructor Thorne), which can cause a 10%-20% reduction in sweet potato yield, and in severe cases, up to 50%-60%, or even a total loss. The disease can cause damage throughout the sweet potato growth period, causing bed rot during the seedling stage and sweet potato rot during storage.

[0003] Breeding resistant varieties has always been the most economical and effective means of integrated crop disease prevention and control, and the primary task of resistance breeding is to screen resistance resources. The currently widely used methods for identifying sweet potato resistance to stem nematode disease are mainly field natural induction method and indoor punch inoculation method. The former is easily affected by environmental factors, so the identification results are unstable and the experimental cycle is long; the latter has destroyed the sweet potato epidermis structure during punch inoculation. Although it can accurately evaluate the resistance of sweet potato varieties to the expansion of stem nematodes, it cannot evaluate the resistance to invasiveness.

[0004] Lin Maosong et al. (Lin Maosong, He Limin, Wen Ling, Fang Zhongda, Song Bofu. Resistance mechanism of sweet potato morphology to stem nematode disease. Chinese Agricultural Science, 1996. 29 (3): 8-12) used a method of directly dripping a suspension containing 2000 nematodes onto the tuber surface and then adsorbing it with absorbent cotton to evaluate the resistance of sweet potatoes to the rot stem nematode. The main drawbacks of this method are that, on the one hand, the buds generated during the storage of the inoculated tubers will form micro-wounds on the surface, which is not conducive to the evaluation of resistance to invasion. On the other hand, the fluidity of the nematode solution will lead to a large loss of inoculated nematodes and the inability to fix the infection site of the nematodes, reducing the reliability of the subsequent resistance evaluation results. Xu Zhen et al. (Xu Zhen, Sun Houjun, Yang Dongjing, Zhang Chengling, Zhao Yongqiang, Xie Yiping. Anhui Agricultural Science, 2017.45(2):164-166,173) used a method of adding 200 nematode suspension drops onto the hemostatic cotton of a band-aid, and then applying the band-aid with nematodes to the middle surface of the tuber to evaluate the resistance of sweet potato to the rot stem nematode by adsorption. This method can fix the infection site of the nematode and better avoid the disadvantage of losing the inoculated nematodes. However, this method requires the purchase of additional band-aids, and the disease index survey method is relatively cumbersome. At the same time, since it takes a certain amount of time for the nematodes to invade the epidermis of the sweet potato tuber, the band-aid contains less cotton, and it is difficult to provide a moist and suitable infection environment for the nematodes for a long time. There is a situation where the inoculation effect is not ideal, and the ideal disease level cannot be achieved in a short time. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for artificially inoculating sweet potato tubers with Ditylenchus destructor and a method for identifying resistance.

[0006] The method for artificially inoculating sweet potato tubers with stem nematodes of the present invention comprises the following steps: sealing the bottom of the dark side of the sweet potato tubers with paraffin, dripping a suspension of stem nematodes evenly around the paraffin, inserting the tubers with the dark side facing downward into moisturizing flower-cutting mud, and culturing in the dark at 26-30°C for 38-42 days.

[0007] In the present invention, the shady side of the sweet potatoes dug out from the field is often damaged, and the paraffin wax is used for sealing. On the one hand, the wound of the potato tuber can be sealed, and on the other hand, the paraffin wax sleeve formed at one end of the shady side of the sweet potato can fix or hold a circle of nematode suspension that is dripped to prevent it from falling.

[0008] Since the buds of sweet potatoes are almost all concentrated on one end of the sunny side of the sweet potatoes, the present invention inserts the shady side of the potato tuber into the flower-inserting mud downwards, and inoculates nematodes on the shady side, which can avoid the interference of micro-wounds caused by the buds of the sweet potatoes after being placed for a long time, and the interference of anti-invasive evaluation can be avoided, and a moist and suitable infection environment for the nematodes can be provided, so that the potato tuber can reach the ideal disease degree in a short time.

[0009] Furthermore, the bottom of the dark side of the sweet potato tuber is sealed with paraffin wax and then subjected to high temperature healing to eliminate the minute wounds on the tuber surface. The high temperature healing condition is 33-37° C. for 22-26 hours in an environment of 85-90% humidity.

[0010] Furthermore, the concentration of the Ditylenchus destructans suspension is 5500 to 6500 heads / ml.

[0011] The present invention also provides a method for identifying the resistance of sweet potatoes to Ditylenchus destructans, which uses the method for artificially inoculating sweet potato tubers with Ditylenchus destructans to artificially inoculate sweet potato tubers of a test variety with Ditylenchus destructans, and sets a disease-susceptible control, and also includes:

[0012] After dark culture, the tubers were cut longitudinally along the middle of the tubers n times, and the tuber length L and the longitudinal length h of the epidermal diseased tissue were measured. The total longitudinal length H of the epidermal diseased tissue was calculated, and the diseased-healthy length ratio P was calculated by the tuber length L and the total longitudinal length H of the epidermal diseased tissue. The control effect was calculated by the diseased-healthy length ratio of the test variety and the diseased-healthy length ratio P of the susceptible control. The test sweet potato varieties were graded according to the average control effect to evaluate their resistance and susceptibility. The calculation formula is as follows:

[0013] P = H / (n + 2);

[0014] Prevention effect = (P 感病对照 -P 测试品种 ) / P 感病对照 ×100%;

[0015] P 感病对照 is the ratio of length of diseased to healthy controls, P 测试品种 The ratio of healthy length to disease length of the tested species

[0016] Furthermore, the susceptible control sweet potato variety is Longshu 9, and the test variety is any one of Lizixiang, Xu 781, Yanshu 25, and Xushu 29.

[0017] Furthermore, n is 1-3.

[0018] Furthermore, the grading standards are:

[0019] High resistance: average protection efficiency ≥ 80%;

[0020] Disease resistance: 60%≤average prevention effect<80%;

[0021] Medium resistance: 40% ≤ average protection effect < 60%;

[0022] Susceptible: 20%≤average protection effect<40%;

[0023] High sensitivity: average protection effect <20%.

[0024] The beneficial effects of the present invention are:

[0025] The method for artificially inoculating sweet potato tubers with Ditylenchus destructans of the present invention can provide a continuous moist and suitable infection environment for the nematodes, thereby making the infection result more accurate.

[0026] The identification method of the present invention is scientific and effective, the investigation method is simple, and the situation of sweet potato being infected by the destructive nematode in the field can be restored well, and the invasion resistance of sweet potato varieties to the destructive nematode can be accurately shown. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The disease severity of different varieties of sweet potato tubers inoculated with Ditylenchus destructans in Example 4 of the present invention is shown, wherein: A: Li Zixiang; B: Xu 781; C: Yanshu 25; D: Xushu 29; E: Longshu 9; F: Longshu 9 tubers to be tested 40 days after inoculation. DETAILED DESCRIPTION

[0028] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0029] Example 1: A method for artificially inoculating sweet potato tubers with Ditylenchus destructans

[0030] 1. Isolation and disinfection of Ditylenchus destructans: Collect diseased tubers in the field, separate and obtain a suspension of Ditylenchus destructans by using the shallow plate method, concentrate the suspension of Ditylenchus destructans by centrifugation at 8000 rpm for 1 min, discard the supernatant, add a disinfectant containing 0.1% streptomycin sulfate and 0.001% sodium hypochlorite to disinfect the surface of Ditylenchus destructans for 1 min, wash with sterile water for 3 times, prepare a nematode suspension with a concentration of 5500 heads / ml, and store at 4°C for later use;

[0031] 2 Preparation of sweet potato varieties: Select intact Longshu No. 9 tubers, clean them and spray the surface with 75% alcohol for disinfection. After disinfection, place the tubers to be identified in a high temperature environment of 35℃ and 85% humidity for 24 hours to eliminate minor wounds on the surface of the tubers. After high temperature healing, place the tubers in a sterilized plastic box for storage;

[0032] 3 Inoculation identification: Seal the bottom of the shady side of the sweet potato (the side facing downward in the ridge) with paraffin wax, use a pipette to draw up 100 μl of nematode suspension and drip the suspension evenly along the paraffin wax, insert the potato tubers upright with the shady side down into the flower inserting mud, and keep the flower inserting mud completely moist for the first 7 days (place the flower inserting mud in a long plastic frame, add clean water into the frame, and the color of the flower inserting mud will become darker after it completely absorbs water), culture in the dark at 28°C for 20-40 days, and conduct investigation and evaluation.

[0033] 4 Investigation and evaluation: Take the tubers out of the nutrient pots, cut them twice longitudinally along the middle of the tubers, measure the length of the tubers (L), and measure the longitudinal length of the diseased tissue (h1, h2, h3, h4).

[0034] 5. Calculation method of prevention effect: The disease severity of tubers is evaluated based on the diseased-healthy length ratio of tubers P, that is, the ratio of the longitudinal length of the diseased tissue to the length of the tuber. According to the measurement data of step 4, the calculation method of the diseased-healthy length ratio is P=(h1+h2+h3+h4) / 4 / L.

[0035] Example 2: A method for artificially inoculating sweet potato tubers with Ditylenchus destructans

[0036] 1. Isolation and disinfection of Ditylenchus destructans: Collect diseased tubers in the field, separate and obtain a suspension of Ditylenchus destructans using the shallow plate method, concentrate the suspension by centrifugation at 8000 rpm for 1 min, discard the supernatant, add a disinfectant containing 0.1% streptomycin sulfate and 0.001% sodium hypochlorite to disinfect the surface of Ditylenchus destructans for 1 min, wash with sterile water for 3 times, prepare a nematode suspension with a concentration of 6000 heads / ml, and store at 4°C for later use.

[0037] 2 Preparation of sweet potato varieties for testing: Select intact Longshu No. 9 potato tubers, clean them and spray the surface with 75% alcohol for disinfection. After disinfection, place the tubers to be identified in an environment of 35℃ and 85% humidity for 24 hours to eliminate minor wounds on the surface of the tubers. Place the tubers after high-temperature healing in a sterilized plastic box for storage.

[0038] 3 Inoculation identification: Seal the bottom of the shady side of the sweet potato (the side facing downward in the ridge) with paraffin wax, use a pipette to draw up 100 μl of nematode suspension and drip the suspension evenly along the paraffin wax, insert the potato tubers upright with the shady side down into the flower inserting mud, and keep the flower inserting mud completely moist for the first 7 days (place the flower inserting mud in a long plastic frame, add clean water into the frame, and the color of the flower inserting mud will become darker after it completely absorbs water), culture in the dark at 28°C for 20-40 days, and conduct investigation and evaluation.

[0039] 4 Investigation and evaluation: Take the tubers out of the nutrient pots, cut them twice longitudinally along the middle of the tubers, measure the length of the tubers (L), and measure the longitudinal length of the diseased tissue (h1, h2, h3, h4).

[0040] 5. Calculation method of prevention effect: The disease severity of tubers is evaluated based on the diseased-healthy length ratio of tubers P, that is, the ratio of the longitudinal length of the diseased tissue to the length of the tuber. According to the measurement data of step 4, the calculation method of the diseased-healthy length ratio is P=(h1+h2+h3+h4) / 4 / L.

[0041] Example 3: A method for artificially inoculating sweet potato tubers with Ditylenchus destructans

[0042] 1. Isolation and disinfection of Ditylenchus destructans: Collect diseased tubers in the field, separate and obtain a suspension of Ditylenchus destructans using the shallow plate method, concentrate the suspension by centrifugation at 8000 rpm for 1 min, discard the supernatant, add a disinfectant containing 0.1% streptomycin sulfate and 0.001% sodium hypochlorite to disinfect the surface of Ditylenchus destructans for 1 min, wash with sterile water for 3 times, prepare a nematode suspension with a concentration of 6500 heads / ml, and store at 4°C for later use.

[0043] 2 Preparation of sweet potato varieties for testing: Select intact Longshu No. 9 potato tubers, clean them and spray the surface with 75% alcohol for disinfection. After disinfection, place the tubers to be identified in an environment of 35℃ and 85% humidity for 24 hours to eliminate minor wounds on the surface of the tubers. Place the tubers after high-temperature healing in a sterilized plastic box for storage.

[0044] 3 Inoculation identification: Seal the bottom of the shady side of the sweet potato (the side facing downward in the ridge) with paraffin wax, use a pipette to draw 100 μl of nematode suspension and drip the suspension evenly along the paraffin wax, insert the potato tubers upright with the shady side down into the flower inserting mud, and keep the flower inserting mud completely moist for the first 7 days (place the flower inserting mud in a long plastic frame, add clean water to the frame until the flower inserting mud is completely absorbed, the color of the flower inserting mud will become darker after it is completely absorbed), culture in the dark at 28°C for 20-40 days, and conduct investigation and evaluation.

[0045] 4 Investigation and evaluation: Take the tubers out of the nutrient pots, cut them twice longitudinally along the middle of the tubers, measure the length of the tubers (L), and measure the longitudinal length of the diseased tissue (h1, h2, h3, h4).

[0046] 5. Calculation method of prevention effect: The disease severity of tubers is evaluated based on the diseased-healthy length ratio of tubers P, that is, the ratio of the longitudinal length of the diseased tissue to the length of the tuber. According to the measurement data of step 4, the calculation method of the diseased-healthy length ratio is P=(h1+h2+h3+h4) / 4 / L.

[0047] Comparative Example 1: A method for artificially inoculating sweet potato tubers with stem nematodes

[0048] The method is basically the same as Example 2, except that no moisturizing treatment is performed 7 days after inoculation.

[0049] Comparative Example 2: A method for artificially inoculating sweet potato tubers with stem nematodes

[0050] The method is basically the same as Example 2, except that soil is used to replace the flower arrangement mud.

[0051] Comparative Example 3: A method for artificially inoculating sweet potato tubers with stem nematodes

[0052] The inoculation was carried out using the method of the invention patent with application number 201610225321.0.

[0053] 12 potato tubers were selected for experiments in Examples 1-3 and Comparative Examples 1-3, respectively. The average diseased and healthy length ratios are shown in Table 1.

[0054] Table 1 Average ratio of diseased and healthy length of potato tubers under different treatment methods

[0055]

[0056]

[0057] It can be seen from Table 1 that the average diseased-healthy length ratio of potato tubers treated by the methods of Comparative Examples 1-3 is significantly lower than that of potato tubers treated by the methods of Examples 1-3, that is, the treatment methods of Comparative Examples 1-3 cannot make the potato tubers reach the ideal disease degree in a short period of time, while the potato tubers treated by the methods of Examples 1-3 for 30 days meet the requirements of being used as a susceptible control. Therefore, the inoculation method of Example 2 was selected for subsequent experiments.

[0058] Example 4: A method for identifying sweet potato resistance to Ditylenchus destructor

[0059] 1. Isolation and disinfection of Ditylenchus destructans: Collect diseased tubers in the field, separate and obtain a suspension of Ditylenchus destructans by using the shallow plate method, concentrate the suspension of Ditylenchus destructans by centrifugation at 8000 rpm for 1 min, discard the supernatant, add a disinfectant containing 0.1% streptomycin sulfate and 0.001% sodium hypochlorite to disinfect the surface of Ditylenchus destructans for 1 min, wash with sterile water for 3 times, prepare a nematode suspension with a concentration of 6000 heads / ml, and store at 4°C for later use;

[0060] 2 Preparation of sweet potato varieties for testing: Select Lizixiang, Xu 781, Yanshu 25, and Xushu 29 with intact skin as seed potatoes, clean them and spray the surface with 75% alcohol for disinfection. After disinfection, the tubers to be identified are placed in a high temperature environment of 35℃ and 85% humidity for 24 hours to eliminate minor wounds on the surface of the tubers. The tubers that have been cured by high temperature are placed in a sterilized plastic box for storage. The intact Longshu No. 9 tubers are selected for the same treatment as the disease-susceptible control;

[0061] 3 Inoculation identification: Use paraffin to seal the bottom of the shady side of the sweet potato (the side facing downward in the ridge), use a pipette to draw 100 μl of nematode suspension and drip the suspension evenly along the paraffin, insert the potato tuber with the shady side facing downward into the flower-inserting mud, and keep the flower-inserting mud completely moist for the first 7 days (place the flower-inserting mud in a long plastic frame, add clean water into the frame until the flower-inserting mud is completely absorbed, and the color of the flower-inserting mud will become darker after it is completely absorbed), and culture in the dark at 28°C for 40 days, and conduct investigation and evaluation;

[0062] 4 Investigation and evaluation: Take the tubers out of the nutrient pots, cut them twice longitudinally along the middle of the tubers, measure the length of the tubers (L), and measure the longitudinal length of the diseased tissue (h1, h2, h3, h4).

[0063] 5. Calculation method of control effect: The disease degree of tubers is evaluated based on the ratio of diseased to healthy length of tubers P, that is, the ratio of the longitudinal length of diseased tissue to the length of tubers. According to the measurement data of step 4, the calculation method of diseased to healthy length ratio is P = (h1 + h2 + h3 + h4) / 4 / L. The control effect is calculated by dividing the difference between the diseased to healthy length ratio of the control and the diseased to healthy length ratio of the test variety by the diseased to healthy length ratio of the control. The specific calculation formula is as follows:

[0064]

[0065] 6. Grading standards: The resistance level of sweet potato varieties is evaluated by the range of the prevention effect value. The specific grading standards are:

[0066] [1] High resistance: average protection efficiency ≥ 80%;

[0067] [2] Disease resistance: 60% ≤ average prevention effect < 80%;

[0068] [3] Moderate resistance: 40% ≤ Average protection effect < 60%;

[0069] [4] Susceptibility: 20% ≤ Average protection effect < 40%;

[0070] [5] High sensitivity: average protection effect <20%.

[0071] The results of this experiment are shown in Table 2. Figure 1 .

[0072] Table 2 Evaluation of invasion resistance of different sweet potato varieties

[0073]

[0074] It is known that Xu 781 variety is highly resistant and Li Zixiang is a highly susceptible variety. It can be seen that the identification method of the present invention is scientific and effective.

[0075] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0076] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0077] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for artificially inoculating sweet potato tubers with Ditylenchus destructans, characterized in that: Seal the bottom of the dark side of the sweet potato tuber with paraffin wax, drip the suspension of stem nematodes evenly around the paraffin wax, insert the tuber upright with the dark side facing down into the moist flower-cutting mud, and culture in the dark at 26-30℃ for 38-42 days.

2. The method for artificially inoculating sweet potato tubers with Ditylenchus destructans according to claim 1, characterized in that: Before sealing the bottom of the dark side of the sweet potato tuber with paraffin, high temperature healing is performed to eliminate the slight wounds on the surface of the tuber. The high temperature healing conditions are 33-37° C. for 22-26 hours in an environment of 85-90% humidity.

3. The method for artificially inoculating sweet potato tubers with Ditylenchus destructans according to claim 1, characterized in that: The concentration of the Ditylenchus destructans suspension is 5500-6500 heads / ml.

4. A method for identifying the resistance of sweet potato to Ditylenchus destructor, characterized in that: The method according to any one of claims 1 to 3 is used to artificially inoculate sweet potato tubers of a test variety with Ditylenchus destructor, and a disease-susceptible control is set, further comprising: After dark culture, the tubers were cut longitudinally along the middle of the tubers n times, and the tuber length L and the longitudinal length h of the epidermal diseased tissue were measured. The total longitudinal length H of the epidermal diseased tissue was calculated, and the diseased-healthy length ratio P was calculated by the tuber length L and the total longitudinal length H of the epidermal diseased tissue. The control effect was calculated by the diseased-healthy length ratio of the test variety and the diseased-healthy length ratio P of the susceptible control. The test sweet potato varieties were graded according to the average control effect to evaluate their resistance and susceptibility. The calculation formula is as follows: P = H / (n + 2); Prevention effect = (P 感病对照 -P 测试品种 ) / P 感病对照 ×100%; P 感病对照 is the ratio of length of diseased to healthy controls, P 测试品种 is the healthy-to-sick length ratio of the tested species.

5. The method for identifying the resistance of sweet potato to Ditylenchus destructor according to claim 4, characterized in that: The susceptible control sweet potato variety was Longshu No.

9.

6. The method for identifying the resistance of sweet potato to Ditylenchus destructor according to claim 4, characterized in that: The tested varieties are any one of Lizixiang, Xu781, Yanshu25 and Xushu29.

7. The method for identifying the resistance of sweet potato to Ditylenchus destructor according to claim 4, characterized in that: n is 1 to 3.

8. The method for identifying the resistance of sweet potato to Ditylenchus destructor according to claim 7, characterized in that: The grading standards are: High resistance: average protection efficiency ≥ 80%; Disease resistance: 60%≤average prevention effect<80%; Medium resistance: 40% ≤ average protection effect < 60%; Susceptible: 20%≤average protection effect<40%; High sensitivity: average protection effect <20%.

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