阅读说明：本技术 藁本内酯及含其的植物精油在防治花生白绢病方面的应用 (Application of ligustilide and plant essential oil containing ligustilide in prevention and treatment of peanut southern blight ) 是由 周琳 何磊鸣 杜鹏强 崔凯娣 赵特 高飞 汪梅子 何亚 韩志远 于 2021-08-25 设计创作，主要内容包括：本发明涉及藁本内酯在防治花生白绢病方面的应用,尤其是含藁本内酯的植物精油(如川芎精油或当归精油等)在防治花生白绢病方面的应用。本发明研究了藁本内酯及含其的植物精油对花生白绢病菌菌丝生长、菌核萌发的毒力以及花生白绢病的盆栽防治效果,结果发现：藁本内酯及含其的植物精油对花生白绢病菌的菌丝生长、菌核萌发具有良好的抑制活性,随着藁本内酯浓度的升高,其抑制活性增强,EC-(50)值为7.0 mg/L,抑制活性显著优于对照药剂多抗霉素。同时,盆栽防效试验与室内毒力试验结果一致。此外,与空白对照和对照药剂多抗霉素相比,藁本内酯也能够显著降低死棵率,表明藁本内酯以及含其的植物精油在防治白绢病上有很大的应用前景。(The invention relates to application of ligustilide in preventing and treating peanut southern blight, in particular to application of ligustilide-containing plant essential oil (such as ligusticum wallichii essential oil or angelica sinensis essential oil and the like) in preventing and treating peanut southern blight. The invention researches the toxicity of ligustilide and plant essential oil containing ligustilide on the growth and sclerotium germination of sclerotium of peanut and the pot culture control effect of sclerotium of peanut, and finds that: ligustilide and plant essential oil containing the same have good inhibitory activity on hypha growth and sclerotium germination of southern blight fungus, and with the increase of ligustilide concentration, the inhibitory activity is enhanced, and EC is increased 50 The value is 7.0 mg/L, and the inhibitory activity is obviously superior to that of a control medicament polyoxin. Meanwhile, the results of the potted plant control effect test are consistent with the results of the indoor toxicity test. In addition, polyoxins were compared to placebo and control agentsCompared with the traditional Chinese medicine, the ligustilide can also obviously reduce the death rate, and shows that the ligustilide and the plant essential oil containing the ligustilide have great application prospect in preventing and treating southern blight.)

1. Application of ligustilide in preventing and treating southern blight of peanut is provided.

2. The use as claimed in claim 1, wherein the ligustilide-containing plant essential oil is used for controlling southern blight of peanuts.

3. The use of claim 2, wherein the plant essential oil is essential oil of chuanxiong rhizome or essential oil of angelicae gigantis radix.

4. The use as claimed in claim 1, wherein ligustilide is formulated with art-recognized adjuvants to make a mixed medicament.

5. The use according to claim 4, wherein the mixed medicament is a suspension, a water dispersible granule, a wettable powder, a granule, an emulsion in water, an emulsifiable concentrate or a microcapsule.

Technical Field

The invention belongs to the technical field of plant disease and insect control, and particularly relates to application of ligustilide and plant essential oil containing the ligustilide in the aspect of controlling peanut southern blight.

Background

Ligustilide is naturally present in traditional Chinese medicine Umbelliferae plants such as rhizoma Ligustici Chuanxiong and radix Angelicae sinensis, and is also main active ingredient of rhizoma Ligustici Chuanxiong essential oil and radix Angelicae sinensis essential oil. Researches on the Toujingye and the like find that the ligustilide has the effects of relieving asthma and relieving spasm. Later, pharmacological studies on ligustilide are also increasing in the fields of traditional Chinese medicine, food, and sports health. The study of the Liumeng shows that the supplementation of the ligustilide can promote the fatigue recovery and has positive influence on the physical quality. The reviews of lychee and gentlenan and the like show that the ligustilide can play a role in resisting senile dementia by resisting nerve oxidation injury, resisting neuritis, promoting cerebroprotein synthesis and the like. The review of Yangxiangjun et al shows that the pharmacological action of ligustilide in the cardiovascular system is mainly shown in four aspects of myocardial ischemia resistance, vasodilation, atherosclerosis resistance and microcirculation improvement. At present, no report on the biological activity of ligustilide on plant pathogenic fungi is found.

Southern blight of peanut is caused by sclerotium rolfsii (Sclerotium rolfsii) Fungal soil-borne diseases caused by infection. The pathogenic bacteria have wide hosts and can infect more than 500 kinds of plants such as cereals, potatoes, beans and the like. At present, southern blight has become an important factor for restricting the yield and quality of peanuts in China. The southern blight of the peanuts mostly occurs in the adult-plant stage of the peanuts, germs mainly invade from the stem bases of the plants, hypha can expand upwards and downwards along stems, and finally stems, fruit needles, roots and pods of the plants can be damaged. Leaf wilting at early stage of injury, with white silk-like bacteria covering the ground and stem baseSilk, white hyphae are also attached to the root and the pod after being pulled out; in the later period, the whole plant dies, the root part and the pod become brown and rot, and even the kernel also changes color and becomes wet and rotten. Hyphae begin to gather to form sclerotia in the later period, the hypha is white small particles in the initial period, then gradually increases to become yellow brown, finally becomes rapeseed-shaped black brown or dark brown hard sclerotia, and then the sclerotia can be spread along with irrigation, rainwater or some farming operations and other modes, so that the condition is further increased. At present, outbreak and disaster formation of peanut southern blight are reported in peanut planting areas of Henan, Shandong, Anhui, Liaoning, Jiangxi province and the like in China, the morbidity is generally 10% -40%, 20% -30% of yield loss of peanuts can be caused, and the yield can be reduced by more than 50% in serious cases. In 2020, southern Henan province agricultural rural hall lists the southern peanut southern blight as a category II pest and disease damage directory in southern Henan province. The research on the southern blight of the peanuts at home and abroad is less, and the prevention and control effects are limited due to the wide hosts and the long survival time of sclerotia of the peanuts because of the lack of disease-resistant varieties and agricultural measures such as deep ploughing, reasonable rotation and the like. At present, the control of the peanut southern blight mainly depends on chemical bactericides. However, the varieties of chemical bactericides registered on Chinese pesticide information network for preventing and controlling the peanut southern blight are few, and the long-term use of the chemical bactericides can not only cause the pests to generate drug resistance, but also cause the problems of pesticide residue exceeding standard, environmental pollution, harm to human health and the like. Therefore, a new safe, efficient and pollution-free medicament is urgently needed to be found.

Under the background of long creation period, high cost and the like of new pesticides, people begin to search high-activity compounds from natural products, and more efficient pesticide varieties can be developed by using the natural products as lead compounds for structural optimization and modification, for example, polyoxin, a metabolite generated by streptomyces aureofaciens, has a good prevention and treatment effect on southern blight, and pesticide registration is carried out. In addition, eugenol, allicin, matrine and other bactericidal substances derived from plant bodies have been registered to prevent and treat various plant diseases. The research finds that the ligustilide and the plant essential oil containing the ligustilide have strong inhibitory activity on the southern blight.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides application of ligustilide and plant essential oil containing ligustilide in the aspect of preventing and treating peanut southern blight. The test proves that: ligustilide has excellent prevention and treatment effect on southern blight of peanut.

In order to achieve the purpose, the invention adopts the following technical scheme:

application of ligustilide in preventing and treating southern blight of peanut is provided.

Further, the invention provides application of the ligustilide-containing plant essential oil in the aspect of preventing and treating peanut southern blight.

In the above application, preferably, the plant essential oil is ligusticum wallichii essential oil or angelica sinensis essential oil, and can be directly purchased from common commercial products or extracted by adopting a conventional method in the field.

The invention also provides a mixed medicament prepared by compounding ligustilide and conventional auxiliary materials in the field.

Further, the mixed medicament can be a suspending agent, a water dispersible granule, a wettable powder, a granule, an emulsion in water, missible oil or a microcapsule agent and the like.

The rhizoma Ligustici Chuanxiong essential oil is prepared from Umbelliferae plant rhizoma Ligustici Chuanxiong (rhizoma Ligustici Chuanxiong)Ligusticum sinensecv. Chuanxiong) and extracting the volatile oil from the rhizome of Chuanxiong). The essential oil of rhizoma Ligustici Chuanxiong has effects of promoting blood circulation, removing blood stasis, caring skin, whitening skin, preventing alopecia and canities, etc., and has been used in bathing agent in Japan. Muuiulin studies indicate that: the essential oil of rhizoma Ligustici Chuanxiong has certain fresh-keeping effect on fruit, and also has certain inhibiting effect on bacteria in antiseptic aspect. However, there are only few reports on the research on the prevention and treatment of plant pathogenic fungal diseases by ligusticum wallichii essential oil at present.

Compared with the prior art, the invention has the following beneficial effects:

1) the invention discovers the following effects by researching the toxicity of ligustilide on the growth of the hyphae of the peanut southern blight fungus and the pot culture control effect of the peanut southern blight: ligustilide has good inhibitory activity on hypha growth of southern blight fungus, and with the increase of ligustilide concentration, its inhibitory activity is enhanced, and EC₅₀The value is 7.0 mg/L, and the inhibitory activity is obviously better than that of a control medicament polyoxin (EC)₅₀The value was 204.7 mg/L). Potted plant control effect test and indoor toxicity test resultConsistently, the control effect of ligustilide is significantly higher than that of the registered drug polyoxin, and compared with the blank control and the control drug polyoxin, the death rate can also be significantly reduced. The ligustilide has great application prospect in preventing and treating southern blight;

2) the invention discovers the following by researching the toxicity of the ligusticum wallichii essential oil on the growth of the sclerotium rolfsii mycelium, the inhibition effect on the growth of the sclerotium germination of the sclerotium and the pot culture control of the sclerotium rolfsii mycelium: under the treatment concentrations of 1000 mg/L and 500 mg/L, the inhibition rates of the ligusticum wallichii essential oil on the growth of the hyphae of the southern blight fungus are 96.0% and 89.6% respectively, and the inhibition rates are obviously superior to those of a control medicament polyoxin (64.9% and 61.7%). The essential oil of rhizoma Ligustici Chuanxiong has good inhibitory activity on hypha growth of southern blight, and the inhibitory activity is enhanced with the increase of essential oil concentration, and EC₅₀The value is 104.2 mg/L, and the inhibitory activity is obviously better than that of a control medicament polyoxin (EC)₅₀The value was 204.7 mg/L). The ligusticum wallichii essential oil also has a good inhibition effect on sclerotium germination of southern blight bacteria, and the inhibition rate of the ligusticum wallichii essential oil on sclerotium germination is 80% under the treatment concentration of 400 mg/L; when the concentration of the essential oil is increased to 500 mg/L, the inhibition rate of the essential oil on sclerotium germination is as high as 90%. The ligusticum wallichii essential oil has good prevention and treatment effect on the southern blight of peanuts, the prevention and treatment effect of 1000 mg/L ligusticum wallichii essential oil on the southern blight of peanuts is 53.51%, and the prevention and treatment effect is not obviously different from that of a control medicament 1000 mg/L polyoxin (44.82%). .

Detailed Description

The technical solution of the present invention is further described in detail with reference to the following examples, but the scope of the present invention is not limited thereto.

In the following examples, ligustilide is a common commercially available product, and the essential oil of chuanxiong rhizome is chuanxiong rhizome oil which is purchased from natural medicinal oil of cedar from Jiangxi, and has light brown oily liquid appearance, warm nature, pungent taste and strong medicinal herb fragrance, and the content of ligustilide is more than or equal to 50%.

Test methods and results

1. Toxicity of ligustilide on growth of hyphae of florigen southern blight bacterium

The growth rate of hypha is measured by a method for inhibiting the growth rate of hypha. Dissolving ligustilide 0.02 g in DMSO 0.4 mL and Tween-80, adding sterile water 19.58 mL to constant volume of 20mL, and preparing into mother liquor of 1000 mg/L, wherein the volume ratio of DMSO to Tween-80 is 1%; then the mixture is diluted into 5 liquid medicines with the concentration of 500, 100, 50, 10, 5 mg/L by sterile water in a gradient way. Adding 1 mL of medicinal liquid with different concentrations into 9 mL of PDA culture medium, and making into medicated culture medium with final concentration of 100, 50, 10, 5, 1, 0.5 mg/L. The final concentrations of the control drug, polyoxin, were 6000, 1000, 500, 100, 50, 10 mg/L, compared to drug-free medium containing 0.1% DMSO and 0.1% Tween-80. Each concentration was replicated three times. Culturing in dark at 25 deg.C in light incubator, and measuring colony diameter by cross method after 3 d.

According to the inhibition rate of hypha growth = [ (control colony diameter-fungus cake diameter) - (treatment colony diameter-fungus cake diameter)]V (contrast colony diameter-cake diameter) x 100, calculating the inhibition rate of ligustilide and polyoxin on the growth of hyphae of southern blight, and obtaining toxicity regression equation and effective medium concentration (EC) according to a probability value analysis method₅₀) And a concentration (EC) at which the inhibition rate is 90%₉₀). The results are shown in Table 1.

TABLE 1 toxicity of ligustilide on hyphae of southern blight bacteria

As shown in Table 1, ligustilide has good inhibitory activity on hypha growth of southern blight fungus, and with the increase of ligustilide concentration, its inhibitory activity is enhanced, EC₅₀The value is 7.0 mg/L, and the inhibitory activity is obviously better than that of a control medicament polyoxin (EC)₅₀The value was 204.7 mg/L).

Potted plant control effect of ligustilide on peanut southern blight

A spraying method is adopted to test the potted plant control effect of ligustilide on the peanut southern blight.

The test peanut variety is Yuhua 9326, peanut seeds with full grains and consistent sizes are selected and placed in a basin, one third of the seeds are submerged by adding sterile water, and the peanut seeds are placed in an incubator for half a day. Then mixing the matrix soil and vermiculite according to the volume ratio of 3:1, adding sterile water to adjust the water holding capacity of the soil to 70%, placing the soil in a pot, and sowing the germinated seeds with radicles facing downwards.

Artificially inoculating germs: after one week of sowing, selecting healthy, growing and consistent peanut plants, inoculating 1 fungus cake on the stem base of each peanut plant, and burying with soil. When white hypha appears on peanut plants and soil, spraying 50 mg/L ligustilide, 100 mg/L ligustilide and 500 mg/L polyoxin liquid medicine on the roots and stems of the plants and the surrounding soil respectively, and spraying 10 mL of liquid medicine on each plant. Sterile water containing 0.1% DMSO and 0.1% Tween-80 was used as a control. Each treatment was repeated 3 times for 15 strains. After 3 days of administration, the disease condition was identified according to the following grading criteria:

level 0: no disease spots;

level 1: the lesion is smaller than 1/3 pedicles;

and 3, level: the disease spots are larger than 2/3 stems, and a small amount of hypha is on the ground;

and 5, stage: the disease spots are larger than 2/3 stems, and a large amount of hypha is on the ground;

and 7, stage: the disease spot is larger than 2/3 stems, the ground has a large amount of hyphae, and the overground part of the disease spot has wilting;

and 9, stage: the plants died.

According to the disease index = (each stage diseased plant x relative stage value)/(total plant number x 9) × 100,

The prevention and treatment effect (%) = (contrast treatment disease index-medicament treatment disease index)/contrast treatment disease index x 100, the disease index and prevention effect of different treatments are calculated, and the death rate of different treatments is counted. The results are shown in Table 2.

TABLE 2 potted plant control of southern blight by ligustilide

Note: data in the table are mean ± sem. The different letter representations in the same column are tested by the least significant difference methodP <The difference at the 0.05 level was significant.

From table 2, the potted plant control effect test is consistent with the indoor toxicity test result, the control effects of ligustilide at the doses of 50 mg/L and 100 mg/L are respectively 34.98% and 60.07%, which are obviously higher than the control effect (23.1%) of polyoxin of 500 mg/L of the registered medicament, and the ligustilide can also obviously reduce the death rate compared with the blank control medicament and the control medicament. This shows that ligustilide has a great application prospect in preventing and treating southern blight.

Inhibition effect of ligusticum wallichii essential oil on growth of hypha of peanut southern blight bacteria

The inhibitory activity of the ligusticum wallichii essential oil on sclerotium rolfsii is measured by adopting a method for inhibiting the hypha growth rate. Dissolving 0.1 g of ligusticum wallichii essential oil in 0.2 mL of DMSO and Tween-80, adding 9.7 mL of sterile water, and fixing the volume to 10 mL to prepare 10000 mg/L of mother solution, wherein the volume ratio of the DMSO to the Tween-80 is 1%. Then diluting with sterile water to obtain medicinal liquid of 5000 mg/L. 1 mL of the two kinds of liquid medicines with two concentrations are respectively added into 9 mL of PDA culture medium to prepare drug-containing culture medium with final concentration of 1000 mg/L and 500 mg/L, the final concentration of the control drug polyoxin is also 1000 mg/L and 500 mg/L, drug-free culture medium containing 0.1% DMSO and 0.1% Tween-80 is used as a control, and each concentration is repeated three times. The cells were cultured in a dark atmosphere at 25 ℃ in a light incubator for 3 days, and then the colony diameter was measured by the cross method, and the inhibition ratio of each treatment was calculated.

The hypha growth inhibition rate calculation formula is as follows: hypha growth inhibition = [ (control colony diameter-cake diameter) - (treatment colony diameter-cake diameter) ]/(control colony diameter-cake diameter) × 100. The results are shown in Table 3.

TABLE 3 inhibition ratio (%)% of different concentrations of essential oil of Ligusticum chuanxiong Hort on growth of hypha of southern blight fungus

As can be seen from Table 3, the inhibition rates of the essential oil of Ligusticum wallichii on the growth of hyphae of southern blight fungus are 96.0% and 89.6% respectively at the treatment concentrations of 1000 mg/L and 500 mg/L, which are obviously higher than the inhibition rates (64.9% and 61.7%) of the control medicament polyoxin.

Toxicity of ligusticum wallichii essential oil on growth of hypha of peanut southern blight bacteria

The growth rate of hypha is measured by a method for inhibiting the growth rate of hypha. Dissolving 0.1 g of ligusticum wallichii essential oil in 0.4 mL of DMSO and Tween-80, adding 19.5 mL of sterile water to fix the volume to 20mL, and preparing into mother solution of 5000 mg/L, wherein the volume ratio of the DMSO to the Tween-80 is 1%. Then the mixture is diluted into 6 liquid medicines with the concentration series of 2500, 1250, 625, 312.5, 156.25 and 78.125 mg/L by sterile water in a gradient way. 1 mL of the liquid medicine with different concentrations is respectively added into 9 mL of PDA culture medium to prepare 7 kinds of drug-containing culture medium with final concentrations of 500, 250, 125, 62.5, 31.25, 15.625 and 7.8125 mg/L, the final concentrations of the control drug polyoxin are 6000, 1000, 500, 100, 50 and 10 mg/L, and drug-free culture medium containing 0.1% DMSO and 0.1% Tween-80 is used as a control, and each concentration is repeated three times. Culturing in dark at 25 deg.C in light incubator, and measuring colony diameter by cross method after 3 d. Calculating the inhibition rate of the rhizoma Ligustici Chuanxiong essential oil on the growth of hyphae of southern blight bacteria, and calculating toxicity regression equation and effective intermediate concentration (EC) according to a probability value analysis method₅₀) And a concentration (EC) of up to 90% inhibition₉₀). The results are shown in Table 4.

TABLE 4 toxicity of essential oil of Ligusticum wallichii to hyphae of southern blight bacterium

As can be seen from Table 4, the essential oil of Ligusticum wallichii has good inhibitory activity on the hypha growth of southern blight, and the inhibitory activity is enhanced with the increase of the concentration of the essential oil, and EC is₅₀The value is 104.2 mg/L, and the inhibitory activity is obviously better than that of a control medicament polyoxin (EC)₅₀The value was 204.7 mg/L).

Inhibition effect of ligusticum wallichii essential oil on germination of sclerotium of sclerotium of sclerotium of sclerotium of sclerotium of sclerotium of material of sclerotium of sclerotium of material of sclerotium of material of sclerotium of ingredient of sclerotium of sclerotium of sclerotium of ingredient of material of sclerotium of sclerotium of material of sclerotium of material of the material of sclerotium of material of a of material of a of growing on flower of growing

Dissolving 0.1 g of ligusticum wallichii essential oil in 1 mL of DMSO and Tween-80, adding 18.9 mL of sterile water, and diluting to 20mL of mother liquor prepared into 5000 mg/L, wherein the volume fractions of the DMSO and the Tween-80 are 3% and 2% respectively. Then the medicine liquid is diluted into 5 medicine liquids with the concentration series of 4000, 3000, 2000, 1000 and 500 mg/L. Adding 1 mL of the medicinal liquid into 9 mL of PDA culture medium, and making into medicated culture medium with final concentration of 500, 400, 300, 200, 100, and 50 mg/L. The sclerotium with the same shape and size is selected respectively and placed on the drug-containing culture medium, 20 sclerotium are placed in each treatment, drug-free culture medium containing 0.3% DMSO and 0.2% Tween-80 is used as a control, and each concentration is repeated three times. And after 2 d of culture on a culture medium containing medicine, observing the germination condition of sclerotium, and when the hypha generated by the sclerotium is more than or equal to 2 mm, determining the sclerotium to germinate. Sclerotium germination inhibition rate = (control sclerotium germination number-treated sclerotium germination number)/(control sclerotium germination number) × 100%. The results are shown in Table 5.

TABLE 5 inhibition of sclerotium germination in southern blight flowers by essential oil of Ligusticum wallichii (2 d)

Table 5 shows that the essential oil of chuanxiong rhizome also has a good inhibition effect on sclerotium germination of southern blight bacteria. Under the treatment concentration of 400 mg/L, the inhibition rate of the ligusticum wallichii essential oil on sclerotium germination is 80%; when the concentration of the essential oil is increased to 500 mg/L, the inhibition rate of the essential oil on sclerotium germination is as high as 90%.

Potted plant control effect of ligusticum wallichii essential oil on peanut southern blight

A spraying method is adopted to test the potted plant control effect of the ligusticum wallichii essential oil on the southern blight of the peanuts.

The test peanut variety is Yuhua 9326, peanut seeds with full grains and consistent sizes are selected and placed in a basin, one third of the seeds are submerged by adding sterile water, and the peanut seeds are placed in an incubator for half a day. Then mixing the matrix soil and vermiculite according to the volume ratio of 3:1, adding sterile water to adjust the soil to 70% humidity, placing the soil in a pot, and sowing the germinated seeds with radicles facing downwards.

Artificially inoculating germs: after one week of sowing, selecting healthy, growing and consistent peanut plants, inoculating 1 fungus cake on the stem base of each peanut plant, and burying with soil. When white hypha appears on peanut plants and soil, spraying the root stems and the surrounding soil of the peanut plants with 500 mg/L of ligusticum wallichii essential oil and 1000 mg/L of polyoxin liquid medicine respectively, wherein 10 mL of liquid medicine is sprayed on each plant. Sterile water containing 0.1% DMSO and 0.1% Tween-80 was used as a blank. Each treatment of 15 plants was repeated three times. After 3 days of administration, the disease was identified according to the following grading criteria:

level 0: no disease spots;

level 1: the lesion is smaller than 1/3 pedicles;

and 3, level: the disease spots are larger than 2/3 stems, and a small amount of hypha is on the ground;

and 5, stage: the disease spots are larger than 2/3 stems, and a large amount of hypha is on the ground;

and 7, stage: the disease spot is larger than 2/3 stems, the ground has a large amount of hyphae, and the overground part of the disease spot has wilting;

and 9, stage: the plants died.

According to the disease index = (each stage diseased plant x relative stage value)/(total plant number x 9) × 100,

The prevention and treatment effect (%) = (contrast treatment disease index-medicament treatment disease index)/contrast treatment disease index x 100, the disease index and prevention effect of different treatments are calculated, and the death rate of different treatments is counted. The pot test results are shown in Table 6.

TABLE 6 prevention and treatment effect of essential oil of Ligusticum wallichii on southern blight of peanut

Note: data in the table are mean ± sem. The different letter representations in the same column are tested by the least significant difference methodP <The difference at the 0.05 level was significant.

As can be seen from Table 6, the essential oil of Ligusticum wallichii has good control effect on southern blight of peanuts. The prevention and treatment effect of 1000 mg/L of ligusticum wallichii essential oil on the southern blight of peanuts is 53.51%, and the prevention and treatment effect is not obviously different from that of a control medicament 1000 mg/L of polyoxin (44.82%).