Application of farnesene in prevention and treatment of southern root-knot nematode

Abstract

The application provides application of farnesene in prevention and treatment of southern root-knot nematode, belongs to the technical field of prevention and treatment of root-knot nematode, and the concentration of farnesene in prevention and treatment of southern root-knot nematode is 92-118 ng / h per plant. The application finds that farnesene exposure treatment can effectively inhibit the invasion and reproduction of southern root-knot nematode and significantly reduce the feeding preference of southern root-knot nematode to corn by using farnesene to expose and treat corn plants, and farnesene can improve the resistance of plants by regulating and improving the content of defense hormone jasmonic acid isoleucine complex in corn. In conclusion, the application provides a new idea for green prevention and control of southern root-knot nematode.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of root-knot nematode control, and particularly relates to application of farnesene in prevention and treatment of southern root-knot nematode. BACKGROUND

[0002] Root-knot nematodes (Meloidogyne spp.) are a class of the most important plant parasitic nematodes, which have a wide host range and can harm almost all crops, and cause the largest economic losses among all plant parasitic nematodes. Among them, M. incognita is the most destructive species in the genus of root-knot nematodes, has a wide host range, a high reproductive rate, and is easy to form complex diseases with other soil-borne pathogens, thereby causing serious harm to agricultural production. The life history of M. incognita includes three stages of egg, juvenile and adult. Under suitable temperature (20-30℃), the life history is generally 20-28 days. The first instar juvenile hatches in the egg, then develops into the second instar juvenile and escapes from the egg shell, invades the root from the root tip of the nutrient root, establishes a feeding site, forms multinucleate and swollen giant cells that are the source of nematode nutrients, and further develops into the third instar juvenile, the fourth instar juvenile and adult after colonization, thereby forming root knot on the root system after root damage; the female adult lays eggs in an egg sac, usually 300-500 eggs in each egg sac, and the eggs further hatch into second instar juveniles for re-invasion, finally causing serious damage to the root system, leading to a decrease in the ability of the root system to absorb nutrients and water, and thereby causing plant malnutrition, slow growth, and even death of the whole plant.

[0003] Maize is one of the most widely planted crops in the world and plays an important role in global food security. Due to various factors such as continuous cropping of crops, nematode diseases have become a common disease of maize, and M. incognita is an important pathogen of maize. After infecting maize, M. incognita can inhibit the growth and development of the root system of maize, and even cause the death of maize, resulting in complete loss of maize. Traditional measures for preventing and treating M. incognita of maize mainly include agricultural prevention and chemical pesticide prevention. However, traditional chemical nematicides mostly have the disadvantages of strong sterilization, pesticide residues, great influence on ecological environment, and easy development of drug resistance. Therefore, it is of great significance to find excellent plant-derived nematicides for the prevention and treatment of root-knot nematodes.

[0004] Farnesene is a volatile sesquiterpene compound that is specifically released after plants are fed by phytophagous animals; it is also the main or even the only component of the alarm pheromone of most aphid species, which is secreted from the abdominal tube when aphids encounter threats such as natural enemies. Studies have shown that farnesene, as a plant volatile, is involved in the direct and indirect defense responses of plants to pests, affects the habitat, feeding or oviposition of pests, and attracts natural enemies of pests. At present, there is no report on the use of farnesene to prevent and treat plant nematodes. SUMMARY

[0005] Therefore, the application provides the application of farnesene in the prevention and treatment of southern root-knot nematode, which is safe, non-toxic, economical and environmentally friendly, and does not bring negative effects to the growth of crops after exposure treatment.

[0006] To achieve the above-mentioned object, the application provides the following technical solutions.

[0007] The application provides the application of farnesene in the prevention and treatment of southern root-knot nematode, and the concentration of farnesene in the prevention and treatment of southern root-knot nematode is 92-118 ng / h per plant.

[0008] The application also provides a method for preventing and treating southern root-knot nematode, which comprises the following steps: exposing and treating crops with farnesene.

[0009] Preferably, the concentration of farnesene in the exposure treatment of crops is 92-118 ng / h per plant.

[0010] Preferably, the exposure treatment is performed at the 14th-18th day after the sowing of crops.

[0011] Preferably, the exposure treatment is performed once or twice per day.

[0012] Preferably, the exposure treatment is performed for 1-2 hours each time.

[0013] Preferably, the crops comprise corn.

[0014] The application also provides the application of farnesene in the improvement of the content of jasmonic acid-isoleucine complex in corn.

[0015] The application also provides the application of farnesene in the preparation of a medicament for preventing and treating southern root-knot nematode.

[0016] Preferably, the dosage form of the medicament comprises powder, spray, suspension and sustained-release preparation.

[0017] Compared with the prior art, the application has the following beneficial effects:

[0018] The application exposes and treats corn plants with farnesene, and it is found that the exposure treatment of farnesene can effectively inhibit the invasion and reproduction of southern root-knot nematode and significantly reduce the feeding preference of southern root-knot nematode to corn; farnesene has a certain repellent effect on nematodes, and the prevention and treatment effect is good. Research shows that farnesene can improve the content of defense hormone jasmonic acid-isoleucine complex in corn by regulation to improve plant resistance, thereby inhibiting the invasion and reproduction of southern root-knot nematode, mainly including the invasion number, reproduction amount and feeding preference of second instar larvae of southern root-knot nematode, and the application has no adverse effects on plant growth and ecological environment. In addition, the application can provide a new idea for the green prevention and control of southern root-knot nematode. Attached Figure Description

[0019] Figure 1 A schematic diagram of maize plants exposed to farnesene;

[0020] Figure 2 The number of second-instar larvae of the southern root-knot nematode in maize roots was measured 3 days after inoculation.

[0021] Figure 3 The number of root knots in maize roots was measured 10 days after inoculation with southern root-knot nematodes.

[0022] Figure 4 A schematic diagram of an apparatus for determining the feeding preferences of the southern root-knot nematode.

[0023] Figure 5 The number of southern root-knot nematodes around the maize root system was measured in Example 5.

[0024] Figure 6 The content of jasmonic acid-isoleucine in maize roots was measured under different exposure treatment times;

[0025] Figure 7 The dry weight of the aboveground parts of the maize plant after farnesene exposure treatment, as measured in Example 7;

[0026] Figure 8 The dry weight of the underground part of the maize plant after farnesene exposure treatment, as measured in Example 7;

[0027] Figure 9 The height of the maize plant after farnesene exposure treatment was measured in Example 7. Detailed Implementation

[0028] This invention provides the application of farnesene in the control of southern root-knot nematodes. The concentration of farnesene for controlling southern root-knot nematodes is 92-118 ng / h per plant, preferably 96-114 ng / h per plant, and more preferably 100 ng / h.

[0029] The present invention also provides a method for controlling southern root-knot nematodes, comprising the following steps: treating crops with farnesene exposure.

[0030] In the present application, the concentration of the farnesene exposure treatment of the crop is 92-118 ng / h per plant, preferably 96-114 ng / h per plant, and further preferably 100 ng / h per plant; the period of the exposure treatment is preferably 14-18 d after the sowing of the crop, further preferably 15-17 d, and more preferably 16 d; the frequency of the exposure treatment is preferably 1-2 times per day; the time of the exposure treatment is preferably 1-2 h per time, further preferably 1.2-1.8 h per time, more preferably 1.4-1.6 h per time, and further preferably 1.5 h per time; and the crop preferably includes corn.

[0031] The present application also provides the use of farnesene in increasing the content of jasmonic acid-isoleucine complex in corn.

[0032] The present application also provides the use of farnesene in preparing a medicament for preventing and treating southern root-knot nematode.

[0033] In the present application, the medicament preferably includes a powder, a spray, a suspension and a slow-release agent.

[0034] The technical solutions provided by the present application are described in detail below in conjunction with examples, but they should not be understood as limiting the scope of protection of the present application.

[0035] Example 1: Preparation of a farnesene slow-release device

[0036] Glass wool (CAS: 65997-17-3, purchased from Shanghai Aldrich Biochemical Technology Co., Ltd.) was wound and placed in a blue 9 mm open thread cap (VEAP-5397-09B-100, purchased from Shanghai Anpu Experimental Technology Co., Ltd.), and 400 μL of farnesene pure solution (purity 98%, purchased from Shanghai Maikelin Biochemical Technology Co., Ltd.) was added to make the release concentration of farnesene reach 105 ng / h.

[0037] Example 2: Planting corn

[0038] Corn B73 seeds were sown in cultivation pots, the size of the cultivation pots was 10 cm in length, 10 cm in width and 9 cm in height, 250 g of soil was placed in each pot, the temperature of the cultivation greenhouse was 26.5℃, the humidity was 55%, the carbon dioxide concentration was 456 mL·L -1 , the light duration was 14 h (8:00-22:00), and watering was performed during the period, 100 mL of water was poured into each pot each time, and watering was performed twice a day to ensure the normal growth of the corn.

[0039] After 14 d, the farnesene slow-release device prepared in Example 1 was placed beside the plant, and it was placed in a 2 L glass cover together with the corn cultivation pot, and the corn plant was exposed to treatment (such as Figure 1The roots were treated with the exposure concentration of 105 ng / h per plant for 1.5 h, and the exposure treatment was performed once. Fifteen replicates were set for each treatment, and the treatment group and the control group (furon exposure treatment) were placed alternately. Then, the nematodes were inoculated immediately.

[0040] Example 3 Preparation of southern root-knot nematode second instar larva suspension

[0041] The southern root-knot nematodes were isolated by the shallow plate method. Specifically, the shallow plate isolation device mainly consisted of two plastic shallow plates, one of which had a coarse mesh screen at the bottom and was placed on the other shallow plate. Two layers of gauze were wetted and placed on the screen plate, and the tomato root systems infected with the southern root-knot nematodes (tomatoes were specially planted in large quantities to breed nematodes) were picked and cut into 5 mm in size, and then placed on the gauze of the screen plate. Clean water was slowly injected to immerse the sample. After 2 days of culture, the liquid in the shallow plate was collected in a 50 mL centrifuge tube, 25 mL of solution was collected in each centrifuge tube, and the centrifuge tube was placed in a centrifuge, centrifuged at 6000 rpm for 5 min, the supernatant was removed, 3 mL of the lower liquid was retained and enriched, and the enriched nematode suspension was obtained.

[0042] After the suspension was stirred uniformly, the number of southern root-knot nematodes in each 100 μL of liquid was observed under a microscope, and then the total number of nematodes in the liquid was calculated.

[0043] Example 4 Nematode infection experiment

[0044] In example 2, 5 positions were selected on the roots of the corn, the sand was loosened down 2 cm, and the southern root-knot nematode second instar larva suspension prepared in example 3 was inoculated on the corn roots in 5 equal portions by using a pipette gun, 1000 second instar larvae were inoculated on each pot. Then the sand was filled in the corn roots. The control group was also inoculated with nematodes.

[0045] After 3 days of inoculation, the sample was collected, the roots were washed with running tap water until there was no sand on the roots, and the root surface was wiped dry. The root system was frozen in a refrigerator at -20°C for 24 h, and then cut into 1 cm in size after thawing. According to the proportion of 10 mL of tap water added to 1 g of corn roots, tap water was added, stirred thoroughly, and then passed through a 100 mesh and a 500 mesh nested screen. The residue on the 500 mesh screen was washed into a 50 mL centrifuge tube, and the number of invading nematodes was counted under a microscope (OLYMPUS CKX53-HOUN). The number of invasions was used as an index of the infection of the southern root-knot nematodes. The results are shown in Figure 2 .

[0046] After 10 days of inoculation, the sample was collected, and the number of root knots on the roots was counted, which was used as an index of the reproduction of the nematodes. The results are shown in Figure 3 .

[0047] The results show that the invasion rate is reduced by 48.36% and the number of root nodules on the corn root is reduced by 9.52% compared with the control group, indicating that farnesene exposure treatment can effectively inhibit the invasion and reproduction of southern root-knot nematodes; at the same time, farnesene has a repellent effect on nematodes, and after farnesene exposure treatment of corn plants, the resistance of corn root system to southern root-knot nematodes can be improved.

[0048] Experimental Example 5: Feeding preference test experiment

[0049] According to the method in Example 2, corn was planted, and a 14 cm long and 1 cm diameter black straw was inserted in the middle of the corn cultivation pots in the control group and the treatment group, 2000 pieces of southern root-knot second instar nematode suspension prepared in Example 3 were inoculated in the straw (the schematic diagram of the treatment is shown in Figure 4 After 10 h, the sand in the cultivation pots in the treatment group and the control group was collected respectively, and observed and counted under a microscope to obtain the feeding preference index of root-knot nematodes after farnesene treatment. Each group of the experiment was set with 15 replicates. The results are shown in Figure 5

[0050] The results show that the feeding preference of southern root-knot nematodes on corn is reduced by 49.67% compared with the control group; it is shown that farnesene exposure treatment has obvious repellent effect on southern root-knot nematodes.

[0051] Experimental Example 6: Determination of plant hormones

[0052] According to the method in Example 2, corn was planted, and corn roots treated with farnesene for 0 h, 0.5 h and 1.5 h were collected respectively, and were quickly frozen in liquid nitrogen for 30 min, and sample grinding treatment was performed to obtain corn root powder. The internal standard solution was prepared in advance, and the internal standard solution contained deuterated jasmonic acid-D5 (d5-JA) and carbon 13 labeled jasmonic acid-isoleucine (13C-JA-ILE) as solvents. The final internal standard solution was prepared by adding 0.5 mg / L d5-JA and 0.5 mg / L 13C-JA-ILE into 100% methanol. 13 ​C6-JA-Ile), deuterated salicylic acid-D4 (d4-SA), deuterated abscisic acid-D6 (d6-ABA) and deuterated indole acetic acid-D5 (d5-IAA) were each 100 ng / mL. Then the mixed standard solution was prepared, 990 μL of ethyl acetate as extraction solution and 10 μL of internal standard solution were contained in each 1 mL of the mixed standard solution. 0.1 g of corn root powder was weighed, 1 mL of the mixed standard solution was added to each 0.1 g of corn root powder, vortexed on a vortexer for 10 min, centrifuged at 12000 rpm for 20 min at 4°C, and the supernatant was then pipetted into a new 2 mL centrifuge tube, and concentrated by rotary evaporation for 35 min; finally, 200 μL of 70% methanol was added to each of the concentrated centrifuge tubes, vortexed for 5 min, centrifuged at 13000 rpm for 10 min at 4°C, and 100 μL of the supernatant was pipetted into a sample bottle (2 mL) with an inserted pipette (250 μL) to extract jasmonic acid, jasmonic acid isoleucine complex and abscisic acid from the corn root powder, and then analyzed by UHPLC-MS / MS to obtain the hormone content in the corn at different time periods before and after farnesene treatment. There were 8 replicates for each treatment. The results are shown in Table 1. Figure 6

[0053] The results show that farnesene exposure treatment can promote the synthesis of defense-related hormones in corn roots; and compared with the control group, the content of jasmonic acid isoleucine complex in the corn root system treated with farnesene for 0.5-1.5 h increased significantly from 13.63 ng / g.FW (Fresh Weight) to 16.23 ng / g.FW, proving that farnesene can inhibit the invasion of southern root-knot nematodes by increasing the hormone content in corn roots.

[0054] Example 7 Determination of corn growth indicators

[0055] According to the method in Example 2, the corn plant height after farnesene exposure treatment for 1.5 h was determined; the aboveground and underground parts of the control group and the treatment group were collected, dried at 60°C for 48 h, and weighed to obtain the corn growth indicators after farnesene treatment. The results are shown in Table 2. Figures 7 to 9

[0056] The results show that after farnesene treatment, the dry weight of the aboveground and underground parts of the corn plant and the plant height are not affected compared with the untreated; indicating that farnesene treatment will not bring negative effects to the growth of crops.

[0057] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered within the protection scope of the present application.​​

Claims

1. The use of farnesene in the control of Meloidogyne incognita, characterized in that, The concentration of farnesene for preventing and treating southern root-knot nematode is 92-118 ng / h per plant.

2. A method of controlling southern root-knot nematodes, characterized by, The method comprises the following steps: exposing the crops to farnesene.

3. The method of claim 2, wherein, The concentration of farnesene for exposing the crops is 92-118 ng / h per plant.

4. The method of claim 2, wherein, The exposing time is 14-18 days after the crops are sowed.

5. The method of claim 2, wherein, The exposing frequency is 1-2 times per day.

6. The method of claim 2, wherein, The exposing time is 1-2 hours per time.

7. The method according to any one of claims 2 to 6, characterized in that, The crops include corn.

8. Application of farnesene in increasing the content of jasmonic acid-isoleucine complex in corn.

9. Application of farnesene in preparing a medicine for preventing and treating southern root-knot nematode.

10. Use according to claim 9, characterized in that, The dosage form of the medicine includes powder, spray, suspension and slow-release agent.

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