Method for identifying seedling resistance of salvia miltiorrhiza to root-knot nematode and application thereof

Abstract

The application discloses a seedling stage resistance identification method for salvia miltiorrhiza root knot nematode disease and application thereof. After seedling of salvia miltiorrhiza seeds to the 5-6 leaf stage, 2mL of a 2-stage young nematode (J2) suspension with a concentration of 200 pieces / mL is inoculated, and the root knot condition of salvia miltiorrhiza and the influence of different infection durations on the growth condition of salvia miltiorrhiza are investigated after inoculation. The number of root knots of the to-be-tested variety is investigated on the 9th day after inoculation of the nematodes, if the number of root knots is less than 15, the to-be-tested variety enters the next stage of identification, that is, comprehensive trait investigation is carried out on the to-be-tested variety on the 21st day after inoculation of the nematodes, if there is no significant difference between the root fresh weight, the height of the aboveground part and the leaf fresh weight of the to-be-tested variety and those of the non-inoculation group, the to-be-tested variety is preliminarily determined as a resistant variety. The application provides a rapid, simple and efficient indoor identification method for seedling stage resistance identification of salvia miltiorrhiza root knot nematode disease.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agricultural disease prevention and treatment, in particular to a seedling stage resistance identification method for root-knot nematode disease of Salvia miltiorrhiza and application thereof. BACKGROUND

[0002] Salvia miltiorrhiza Bunge is a perennial herb of Labiatae, which is a medicinal plant widely used for treating coronary heart disease and cerebrovascular disease. In recent years, with the high incidence of cardiovascular diseases, wild Salvia miltiorrhiza has been unable to meet the demand for medicinal materials, and the area of artificially planted Salvia miltiorrhiza has gradually increased. Compared with wild Salvia miltiorrhiza, artificially planted Salvia miltiorrhiza has single variety, reduced biodiversity, serious diseases and insect pests, and the continuous cropping obstacles in most areas make the root-knot nematode more serious, resulting in the decline of the quality and quantity of medicinal materials, and causing huge economic losses to the farmers.

[0003] Salvia miltiorrhiza is used as a medicine, so the quality of Salvia miltiorrhiza root is relatively high. However, after being damaged by nematodes, the symptoms are that the branches and young shoots are yellow and withered first, the plant is dwarfed and poorly developed, and then the whole plant is dead. The root is small, the fibrous root is increased, and many size different nodular or nodular objects grow on the root, and the diseased plant cannot be used as medicine, causing great economic losses. The early stage of root-knot nematode infection is often difficult to find, and it can be clearly seen in the later stage. SUMMARY

[0004] At present, there is a lack of deep understanding and systematic report on Salvia miltiorrhiza varieties resistant to root-knot nematodes, and there is no good seedling stage resistance identification method for root-knot nematode disease of Salvia miltiorrhiza. Therefore, the present application preliminarily establishes a simple and efficient method for identifying the resistance of Salvia miltiorrhiza to nematodes by observing the root knot condition of Salvia miltiorrhiza after inoculation of root-knot nematodes and the growth condition of Salvia miltiorrhiza, and the influence of different root-knot nematode infection times on the growth of Salvia miltiorrhiza.

[0005] To solve the above technical problems, the present application provides a seedling stage resistance identification method for root-knot nematode disease of Salvia miltiorrhiza and application thereof.

[0006] The present application is realized by the following technical scheme:

[0007] The present application provides a seedling stage resistance identification method for root-knot nematode disease of Salvia miltiorrhiza, comprising the following steps:

[0008] ST1, preparing a nematode suspension; selecting Salvia miltiorrhiza varieties for seedling culture, and inoculating root-knot nematodes 10 days after transplanting seedlings;

[0009] ST2, roots are pulled out respectively within 3-21 days after inoculation, and the soil is washed, the root knot situation of Salvia miltiorrhiza after inoculation of root-knot nematode and the influence of root-knot nematode disease on the growth of Salvia miltiorrhiza are investigated; the fresh weight of aboveground and underground parts of the plant is weighed, the height of aboveground and underground parts of the plant is measured, the root knot number is counted, and the root knot index is calculated;

[0010] ST3, the root knot number of the tested variety is investigated on the 9th day after inoculation, if the root knot number is less than 15, the variety is determined as a resistant variety, that is, the comprehensive trait investigation is carried out on the 21st day after inoculation of the nematode, if there is no significant difference between the fresh weight of root, the height of aboveground part, the fresh weight of leaf and the non-inoculation group, the variety is preliminarily determined as a resistant variety.

[0011] Preferably, in the step ST1, the nematode suspension is prepared by placing the nematode eggs on a 30 μm nylon screen cloth, then placing the nylon cloth in an inverted culture dish, adding appropriate deionized water to the bottom, and sealing the culture dish with a plastic wrap to prevent water evaporation; when collecting the second instar larvae, only the suspension at the bottom of the culture dish needs to be collected.

[0012] Preferably, in the step ST1, the inoculation concentration of root-knot nematode is 100, 200 or 500 pieces / mL.

[0013] More preferably, in the step ST1, the inoculation concentration of root-knot nematode is 200 pieces / mL.

[0014] Preferably, in the step ST2, the roots are pulled out respectively on the 3rd, 6th, 9th, 12th, 15th, 18th, 21st day after inoculation, the soil is washed, the root knot situation of Salvia miltiorrhiza after inoculation of root-knot nematode and the influence of root-knot nematode disease on the growth of Salvia miltiorrhiza are investigated.

[0015] Preferably, in the step ST2, the roots are pulled out respectively on the 15th, 18th, 21st day after inoculation, the soil is washed, the root knot situation of Salvia miltiorrhiza after inoculation of root-knot nematode and the influence of root-knot nematode disease on the growth of Salvia miltiorrhiza are investigated.

[0016] Preferably, in the step ST3, if the fresh weight of leaf of the treatment group is significantly lower than that of the control group after inoculation for 21 days, the variety is a weakly resistant variety.

[0017] Preferably, in the step ST3, if the height of aboveground part of the treatment group is significantly lower than that of the control group after inoculation for 21 days, the variety is a weakly resistant variety.

[0018] Preferably, in the step ST3, if the fresh weight of root of the treatment group is significantly lower than that of the control group after inoculation for 18 days, the variety is a weakly resistant variety.

[0019] Preferably, in the step ST3, if the fresh weight of root of the treatment group is significantly lower than that of the control group after inoculation for 21 days, the variety is a weakly resistant variety.

[0020] The beneficial effects of the present application are: the present application inoculates 2mL of 2-stage young nematode (J2) suspension with a concentration of 200 strips / mL after seedling of Salvia miltiorrhiza seeds to 5-6 leaf stage, investigates the number of root nodules on the test variety on the 9th day after inoculation, if the number of root nodules is less than 15, it enters the next stage of identification, that is, comprehensive trait investigation is carried out on the 21st day after inoculation of nematodes, if there is no significant difference between the root fresh weight, the height of the aboveground part and the leaf fresh weight and the non-inoculated group, it is preliminarily determined as a resistant variety. The present application provides a rapid, simple and efficient indoor identification method for seedling stage identification of root-knot nematode disease of Salvia miltiorrhiza. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 It is the condition of the root knot of Salvia miltiorrhiza when inoculated with different concentrations of root-knot nematodes for 21 days. Among them, the left Salvia miltiorrhiza root system is inoculated with a concentration of 100 strips / mL, the middle Salvia miltiorrhiza root system is inoculated with a concentration of 200 strips / mL, and the right Salvia miltiorrhiza root system is inoculated with a concentration of 500 strips / mL.

[0022] Figure 2 It is a comparison diagram of Salvia miltiorrhiza plants in the treatment group inoculated with nematodes for 3-21 days and the control group

[0023] Figure 3 It is a diagram of root fresh weight of Salvia miltiorrhiza in the treatment group and the control group inoculated with nematodes for 6-21 days. Among them, * indicates that the difference between the control group and the treatment group is significant at the 5% significant level. ** indicates that the difference between the control group and the treatment group is extremely significant at the 5% significant level. DETAILED DESCRIPTION

[0024] Example Seedling stage resistance identification method of root-knot nematode disease of Salvia miltiorrhiza is established

[0025] 1. MATERIALS AND METHODS

[0026] 1.1 Experimental materials

[0027] 1.1.1 Test material

[0028] Salvia miltiorrhiza seeds were collected from local planting of purple flower Salvia miltiorrhiza local varieties in Shandong in 2020, and were planted in the first crop land of the medicinal plant garden of Shandong Agricultural University.

[0029] 1.1.2 Test insect source

[0030] Disease roots of Salvia miltiorrhiza with obvious disease and a large number of root nodules were collected from areas with serious root-knot nematode disease or plots with continuous cropping for more than three years, and the collected diseased plants were stored in a 4℃ refrigerator for extraction of nematode eggs and preparation of root-knot nematode egg suspension.

[0031] 1.2 Experimental method

[0032] 1.2.1 Preparation of nematode suspension

[0033] (1) Collect the roots of Salvia miltiorrhiza Bunge from the area where the disease is very serious, and the roots have obvious disease and a large number of nodules

[0034] (2) Wash the roots, cut them into pieces, put them into a grinder, add appropriate amount of water, and grind them into homogenate;

[0035] (3) Pass the root pieces and the solution through the screens with the mesh size of 60, 200 and 500 in sequence, and wash the root pieces and the homogenate on the screen with the mesh size of 200 with distilled water so as to collect the nematode eggs on the screen with the mesh size of 500 as much as possible;

[0036] (4) Collect the root-knot nematode eggs obtained on the screen with the mesh size of 500 in a 250 mL beaker, and make the volume to 100 mL;

[0037] (5) Take 0.5 mL of the egg suspension with a pipette and move it into a small beaker containing 4.5 mL of distilled water, and shake it thoroughly;

[0038] (6) Immediately take 1 mL of the diluted egg suspension in a counting dish, and make the eggs randomly fall on the bottom of the counting dish;

[0039] (7) Count the number of the eggs on the bottom of the counting dish under a Wild Makroskop M410 stereomicroscope;

[0040] (8) Dilute the egg suspension to 1000 eggs / mL, and store it at 4°C for standby, and perform the inoculation treatment within 24 h.

[0041] 1.2.2 Inoculation of nematodes

[0042] Select different varieties of Salvia miltiorrhiza Bunge to grow seedlings, inoculate the root-knot nematodes to the seedlings after 10 days of transplanting, repeat 10 times for each variety of Salvia miltiorrhiza Bunge, inoculate 100, 200 and 500 eggs in one group to determine the optimal inoculation concentration and perform the following inoculation experiment. Before inoculation, pour water into the pots; when inoculating, make two holes in the rhizosphere soil of the seedlings, about 1-2 cm deep, and uniformly inject the nematode egg suspension into the holes, 2 mL per pot, 1 mL in each hole; after inoculation, normally manage, and adjust the conditions in the plant incubator to be: temperature 22±0.5°C, humidity 60%-70%, and constant illumination.

[0043] 1.2.3 Observation of the infection of root-knot nematodes

[0044] The roots were pulled out at 3, 6, 9, 12, 15, 18, 21 days after inoculation (dpi), washed clean, and the root knot situation after inoculation of root-knot nematodes and the effect of root-knot nematode disease on the growth of Salvia miltiorrhiza was investigated. The fresh weight of the aboveground and underground parts of the plants was weighed, the height of the aboveground and underground parts of the plants was measured, and the number of root knots was counted to calculate the root knot index. Each identification day was a treatment, with one plant per pot, 20 plants per group, a total of 6 groups, 4 groups inoculated with root-knot nematode suspension, and the other 2 groups as controls. Root knot index (GI) = number of root knots per plant / fresh weight of root per plant.

[0045] 2 Results and analysis

[0046] 2.1 Improvement of hatching method of root-knot nematodes

[0047] In this experiment, the hatching method of root-knot nematodes was improved. The nematode eggs were placed on a 30 μm nylon screen cloth, then the nylon cloth was placed in an inverted culture dish, a suitable amount of deionized water was added to the bottom, and the culture dish was sealed with plastic wrap to prevent water evaporation. Since the size of the eggs is 101.8-81.8 μm x 32.2-45.8 μm, the body length of the second instar larvae is about 359.3-426.7 μm, and the body width is about 12.5-18.1 μm (Dang Jinhuan, 2021), the 30 μm screen cloth can keep the nematode eggs above the screen cloth. The second instar larvae, due to their water-seeking nature, will move towards the water at the bottom of the culture dish, and the width of the second instar larvae is smaller, so they can pass through the pores of the screen cloth. Therefore, this method can effectively separate the nematode eggs from the hatched second instar larvae, and the second instar larvae can be collected by collecting the suspension at the bottom of the culture dish.

[0048] 2.2 Selection of inoculation concentration

[0049] Using second instar larvae as the inoculation stage, the inoculation effect of three concentrations, 100, 200 and 500 worms / mL, on the roots of Salvia miltiorrhiza was investigated. From Figure 1 It can be seen that after 21 days of inoculation of root-knot nematodes, compared with 100 worms / mL, the root knots inoculated with 200 worms / mL of second instar larvae suspension were more obvious after 3 weeks of inoculation and easier to observe. Compared with 500 worms / mL, the inoculation effect of 200 worms / mL of second instar larvae suspension was not much different, but it was easier to cultivate, so 200 worms / mL was preliminarily determined as the best inoculation concentration.

[0050] 2.3 Investigation of root knots of Salvia miltiorrhiza after inoculation of root-knot nematodes

[0051] 2.3.1 Observation of root knot morphology

[0052] The root knot conditions of 3-21 days after inoculation of root-knot nematode were observed, including root knot size, shape, number, color, formation position, etc. Root knots were observed 6 days after inoculation of nematodes, at which time the root knots were oval and slightly thicker than other positions of the roots, and were not easy to observe. With the increase of inoculation time, the volume of root knot gradually increased, and the boundaries of two root knots close to each other gradually connected after 15 days of inoculation of nematodes, and part of the root knots began to turn red, and after 21 days of inoculation of nematodes, multiple root knots began to appear, and the specific root knot conditions are shown in Table 1.

[0053] Table 1 Change of root knot of Salvia miltiorrhiza Bunge after inoculation of nematodes for 3-21 days

[0054]

[0055]

[0056] After 21 days of inoculation of root-knot nematodes, the root knot was gradually grown from a small volume and a color similar to the root system oval root knot to a large volume near-circular root knot. If two small root knots were close to each other in the early stage, then with the increase of volume, the two root knots would gradually approach, and the edges would be unclear, and would be fused into one root knot (multiple root knots), and at the same time, the color of part of the root knots would deepen.

[0057] 2.3.2 Investigation of root knot index

[0058] The results of investigation of the number of root knots and root knot index of Salvia miltiorrhiza Bunge after 6-21 days of inoculation of root-knot nematodes are shown in Table 2. The overall trend of the change of the number of root knots is to increase with the increase of time. The root knot is obvious after 9 days of inoculation of nematodes, and the number of root knots is basically stable, which indicates that most of the second instar larvae need about 9 days from entering the soil to establishing the feeding site to form giant cells, and no large-scale re-infection occurs after 9-21 days.

[0059] The root knot index of Salvia miltiorrhiza Bunge after infection of root-knot nematodes shows a trend of first increasing and then decreasing, which indicates that about 9 days after inoculation of nematodes is the period when the root-knot nematodes have the greatest impact on the root system. After 12-21 days of inoculation of nematodes, the root knot index is continuously decreasing, which may be due to the fact that after 9 days of inoculation of nematodes, with the growth of the root system of Salvia miltiorrhiza Bunge, the fresh weight of the root system is continuously increasing, while most of the root-knot nematodes have completed the infection of the root system and the induction of the plant to produce root knots, and have not yet reproduced to produce the second generation of root-knot nematodes. Therefore, the growth rate of the plant root system exceeds the formation rate of the root knot after 9 days of inoculation of nematodes, so that the peak value of the root knot index is 9 days, and the root knot index shows a downward trend after 9 days.

[0060] Table 2 Number of root knots of Salvia miltiorrhiza Bunge after 6-21 days of inoculation of nematodes

[0061] Inoculation time (d) Nodule number (piece) Nodule index (piece / g) 6 5.880±5.617b 89.330±57.185c 9 15.000 ± 9.230 ab 182.101±59.259a 12 13.670 ± 8.128 ab 160.961 ± 65.112 ab 15 18.750±12.159a 130.603 ± 110.266 abc 18 20.800±13.343a 100.540 ± 75.085 bc 21 20.380±14.264a 70.277±33.056e

[0062] 2.4 Effect of root-knot nematodes on Salvia miltiorrhiza Bunge seedlings under different inoculation times

[0063] 2.4.1 Leaf Fresh Weight

[0064] The overall trend of leaf fresh weight of the treatment group and the control group increased over time. During the 6-21 days of inoculation with root-knot nematodes, the leaf fresh weight of the treatment group was lower than that of the control group. By the 21st day, there was a very significant difference between the treatment group and the control group, indicating that 21 days after the root-knot nematode infected the salvia seedlings, it had a significant impact on the leaf fresh weight.

[0065] According to the results Figure 2 It can be seen that during the 3-18 days of root-knot nematode infection of salvia plants, there was no significant difference in plant growth between the treatment group and the control group. At 18 days, leaf wilting occurred, and at 21 days, leaf wilting worsened, and the growth of the treatment group was significantly weaker than that of the control group.

[0066] 2..4.2 Height of Aboveground Part

[0067] The overall trend of the height of the aboveground part of salvia (treatment group) and the control group increased over time during the 6-21 days of inoculation with root-knot nematodes. Except for 12 days, the height of the aboveground part of the treatment group was lower than that of the control group, but there was no significant difference. Until 21 days after inoculation with nematodes, the height of the aboveground part of the treatment group was lower than that of the control group, and the difference was extremely significant. Combining the investigation results, it can be found that after 18 days of root-knot nematode infection of salvia seedlings, a small amount of leaf wilting symptoms appeared in the aboveground part, and the height of the aboveground part and the leaf fresh weight of the treatment group were significantly lower than those of the control group after 21 days of inoculation with nematodes, indicating that root-knot nematode infection had some impact on the aboveground part of salvia, but this impact was not obvious in the early stage of infection.

[0068] 2.4.3 Root Length

[0069] After 6-21 days of inoculation with root-knot nematodes, the overall trend of the root length of salvia increased over time, but the infection of root-knot nematodes had no significant effect on the root length of salvia plants.

[0070] 2.4.4 Root Fresh Weight

[0071] The investigation results of the root fresh weight of salvia inoculated with root-knot nematodes for 6-21 days are shown in Figure 3 The overall trend of the root fresh weight of the treatment group and the control group increased over time. The root fresh weight of the treatment group was lower than that of the control group at each period. After 18 days of root-knot nematode infection, there was a significant difference in root fresh weight between the treatment group and the control group. After 21 days of root-knot nematode infection of salvia, the root fresh weight of the treatment group was significantly lower than that of the control group. This indicates that the root system of salvia was severely affected by root-knot nematodes, and changes occurred early. The longer the time of root-knot nematode infection, the more serious the loss of root yield of salvia.

[0072] From the above results, compared with the aboveground part, the root of Salvia miltiorrhiza seedling is easier to observe after inoculation of nematode, and changes earlier. The number of root knot and root knot index change obviously after 9 days of inoculation of nematode. Therefore, when screening Salvia miltiorrhiza varieties resistant to root-knot nematode, Salvia miltiorrhiza can be comprehensively evaluated through multiple conditions. According to Table 2, after 9 days of inoculation of nematode, the average number of root knot formed after Salvia miltiorrhiza is infected by nematode is 15. Through this value, preliminary screening can be carried out. After 9 days of inoculation of nematode, the number of root knot of the test variety is counted. If the average number of root knot of the test material is greater than 15, it means that the material cannot resist the invasion of the second instar nematode well. If the average number of root knot of the test material is less than 15, it is preliminarily determined that the material has certain resistance to the second instar nematode. Then it enters the next stage of identification, that is, after 21 days of inoculation of nematode, the fresh weight of root, fresh weight of leaf and height of aboveground part are counted, and whether these indexes are significantly different from the indexes of the control group is calculated. If the three indexes are not significantly different from the control group, it can be determined as an excellent resistant variety.

[0073] The above only describes the preferred embodiments of the patent, and it should be noted that for ordinary skilled persons in the technical field, several improvements and replacements can be made without departing from the technical principles of the patent, and these improvements and replacements should also be considered as the protection scope of the patent.

Claims

1. A method for identifying seedling resistance to root-knot nematode disease of Salvia miltiorrhiza, characterized in that The method comprises the following steps: ST1, preparing a southern root-knot nematode second instar larva suspension with a concentration of 200 pieces / mL; selecting a salvia miltiorrhiza variety to grow seedlings, and performing artificial inoculation 10 days after the seedlings grow to the 5-6 leaf stage, with 2 mL of the suspension inoculated per plant; ST2, performing root knot number investigation on the salvia miltiorrhiza variety to be tested on the 9th day after inoculation, and if the number of root knots per plant is less than 15, the next stage of identification is entered; ST3, on the 21st day after inoculation, measuring the root fresh weight, aboveground height and leaf fresh weight of the treatment group and the non-inoculated control group, and if the root fresh weight, aboveground height and leaf fresh weight of the treatment group have no significant difference with those of the control group, the salvia miltiorrhiza variety is determined to be a resistant variety.

2. The method of claim 1, wherein: In step ST2, the root knot number investigation is used to calculate the root knot index, and the root knot index GI is calculated as the number of root knots per plant / the root fresh weight.

3. The method of claim 1, wherein: In step ST3, the no significant difference is determined by independent sample t test, and the significance level is α=0.

05.

4. The method of claim 1, wherein: In step ST1, the preparation method of the suspension comprises the following steps: placing root-knot nematode eggs on a 30-μm nylon screen cloth, inverting the screen cloth in a culture dish, adding deionized water to the bottom, and collecting the second instar larva suspension after hatching.

5. The method of claim 1, wherein: In step ST3, if the leaf fresh weight of the treatment group is significantly lower than that of the control group 21 days after inoculation, the salvia miltiorrhiza variety is determined to be a susceptible variety.

6. The method of claim 1, wherein: In step ST3, if the aboveground height of the treatment group is significantly lower than that of the control group 21 days after inoculation, the salvia miltiorrhiza variety is determined to be a susceptible variety.

7. The method of claim 1, wherein: If the root fresh weight of the treatment group is significantly lower than that of the control group 18 days after inoculation, the salvia miltiorrhiza variety is determined to be a susceptible variety.

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