阅读说明：本技术 两种蒽醌类化合物在防治根结线虫病方面的应用 (Application of two anthraquinone compounds in preventing and treating root knot nematode disease ) 是由 黄衍章 李彩斌 李章海 毛振坤 杨云飞 丁涛 于 2021-10-12 设计创作，主要内容包括：本发明公开了两种蒽醌类化合物在防治根结线虫病方面的应用,涉及生物农药应用领域。本发明所述两种蒽醌类化合物源于掌叶大黄根茎提取物,分别为大黄素(1,3,8-三羟基-6-甲基蒽醌)和芦荟大黄素(1,8-二羟基-3-羟甲基蒽醌),其对南方根结线虫等有较高的致死率,可用于开发源于中药草的杀线剂；可用于制备防治根结线虫病的水乳剂、可湿性粉剂、可溶性粉剂、可溶粒剂、水分散粒剂、悬浮剂、油悬浮剂和片剂。本发明难以使线虫产生抗药性,也不存在药剂残留问题,符合有害生物绿色防控技术的基本要求；本发明用于防治植物根结线虫病,有利于保护农田生态环境和生物多样性,具有良好的生态效益。(The invention discloses application of two anthraquinone compounds in preventing and treating root knot nematode diseases, and relates to the field of application of biopesticides. The two anthraquinone compounds are derived from Rheum palmatum rhizome extracts, are emodin (1,3, 8-trihydroxy-6-methylanthraquinone) and aloe-emodin (1, 8-dihydroxy-3-hydroxymethyl anthraquinone), have high lethality to Meloidogyne incognita and the like, and can be used for developing a nematicide derived from traditional Chinese medicinal herbs; can be used for preparing emulsion in water, wettable powder, soluble granules, water dispersible granules, suspending agents, oil suspending agents and tablets for preventing and treating root knot nematode diseases. The invention is difficult to cause the nematode to generate drug resistance, has no problem of drug residue, and meets the basic requirements of the green prevention and control technology of pests; the invention is used for preventing and controlling plant root knot nematode disease, is beneficial to protecting the ecological environment and biological diversity of farmlands, and has good ecological benefit.)

1. The application of two anthraquinone compounds in the aspect of preventing and treating root knot nematode diseases is characterized in that:

the two anthraquinone compounds are derived from Rheum palmatum rhizome extract, and are emodin (1,3, 8-trihydroxy-6-methylanthraquinone) and aloe-emodin (1, 8-dihydroxy-3-hydroxymethylanthraquinone), and the chemical structural formulas of the two anthraquinone compounds are respectively shown in figure 1 and figure 2;

② the two anthraquinone compounds show stronger poisoning activity to Meloidogyne incognita, the using concentration of emodin is 1-10 mg/mL, and the lethality to Meloidogyne incognita is higher than 50% when treated for 72 h; the using concentration of the aloe-emodin is 2-20 mg/mL, and the lethality to meloidogyne incognita is higher than 50% when the aloe-emodin is treated for 72 hours;

③ the concentration proportion of the mixture of the two anthraquinone compounds emodin and the aloe-emodin is 2: 1-3: 1, the using concentration is 1-10 mg/mL, and the lethality to Meloidogyne incognita is higher than 60% when the Meloidogyne incognita is treated for 72 hours;

fourthly, the anthraquinone compound is a plant source extract which is screened from the traditional Chinese herbal medicine and has the poisoning effect on the meloidogyne incognita, and can be used for developing a nematicide from the traditional Chinese herbal medicine;

fifthly, the using concentration of the Rheum palmatum rhizome extract for preventing and treating root-knot nematode is 1-4 mg/mL, the filling amount is 100-500 mL, and the potted plant prevention and treatment effect on tomato root-knot nematode is higher than 60%;

sixthly, the Rheum palmatum rhizome extract can be used for preparing emulsion in water, wettable powder, soluble granules, water dispersible granules, suspending agent, oil suspending agent and tablets for preventing and treating root knot nematode diseases.

2. The use of two anthraquinones according to claim 1 for controlling root knot nematode disease, wherein:

example 1: the ethanol extract of the rhizome of the rhubarb palmate leaves has the bioactivity on the meloidogyne incognita.

3. The use of two anthraquinones according to claim 1 for controlling root knot nematode disease, wherein:

example 2: the anthraquinone compound extracted from the rhizome of Rheum palmatum has poisoning activity on Meloidogyne incognita.

4. The use of two anthraquinones according to claim 1 for controlling root knot nematode disease, wherein:

example 3: the Rheum palmatum rhizome extract has effect in preventing and treating tomato root knot nematode disease in potted plants.

Technical Field

The invention relates to the field of application of biopesticides, in particular to application of two anthraquinone compounds in preventing and treating root knot nematode diseases.

Background

Root knot nematode disease is one of the important bottlenecks restricting the sustainable production of agriculture, and serious economic loss is easily caused by improper control. The root knot nematode disease belongs to typical soil-borne diseases, has hidden pathogenic habits and strong stress resistance, is easy to be confused with fertilizer deficiency symptoms in production to cause misjudgment, and is more difficult to control compared with diseases in propagation ways of air, water, seedlings and the like. The root-knot nematode can not only directly damage the plants to cause wilting and yellowing, growth retardation and even death of the whole plants, but also induce other soil-borne diseases such as bacterial wilt, black shank, root black rot and the like.

In order to effectively control the root knot nematode disease, related researches are carried out at home and abroad in the aspects of chemical control, physical control, biological control and the like. In contrast, chemical fumigation of soil is the most thorough and effective method, but due to high cost and serious damage to the soil micro-ecological environment, fumigants such as dazomet, metham and the like are difficult to popularize and apply in a large area in a tobacco field. With the gradual elimination or limited use of high-pollution, high-residue and high-toxicity chemical nematicides (such as aldicarb, carbofuran and the like), the development of green and efficient nematicides is particularly important, wherein botanical pesticides are an important research direction for green prevention and control of root-knot nematodes.

The botanical nematicide is natural and has the advantages of low residue, less pollution, fast decomposition, low toxicity and the like. The effective active part or plant extract of the nematocide (inhibiting) plant or the mixed breeding of the nematocide plant and the host plant of the nematode can be used for controlling the harm of the nematode. At present, marigold (Tagetes), Euphorbia (Euphorbia), Ricinus (Ricinus), albizia (Acacia), Eucalyptus (Acacia), basidium (Ocimum), Allium (Allium), Cannabis (Cannabis) and other plants are mainly discovered through research. In the aspect of preventing and treating the tobacco root-knot nematode, the lightyellow Sophora root (Sophora flavescens), the Chinese feverfew (Crotalaria pallida), the maidenhair (Tagetes patula), the marigold (Tagetes erecta) and the like have better preventing and treating effects. In addition, Horseradish (Horseradish individual) and pepper (Zanthoxylum bungeanum) also have good control effect on tomato root-knot nematode of solanaceae crops. Bamboo vinegar, Nerium indicum (Nerium indicum), Melia azedarach (Melia azedarach), Lantana camara (L.) have good biological activity against Meloidogyne incognita.

Rheum palmatum L is a perennial herb, belongs to Polygonaceae (Polygonaceae) Rheum genus (Rheum), and is widely distributed in northwest and southwest of China. So far, the research of the rhubarb palmate on the aspect of pest control mainly focuses on the aspect of sterilization, and few reports on the aspect of pest and nematode control are available. The resources of China are rich, the development of plant-source pesticides is scientifically and reasonably carried out from the nematode prevention and control efficacy of the rhubarb palmate, and the market prospect is wide. At present, no research report about the processing of the palmate rhubarb pesticide dosage form is available at home and abroad.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide the application of two anthraquinone compounds in the aspect of preventing and treating root knot nematode diseases.

In particular, aiming at the current situation, the plant source green nematicide can be applied in the field, is safe and friendly to the environment and has better control effect on the plant root knot nematode disease.

The purpose of the invention is realized by the following steps of:

the two anthraquinone compounds are derived from Rheum palmatum rhizome extract, and are emodin (1,3, 8-trihydroxy-6-methylanthraquinone) and aloe-emodin (1, 8-dihydroxy-3-hydroxymethylanthraquinone), and the chemical structural formulas of the two anthraquinone compounds are respectively shown in figure 1 and figure 2;

② the two anthraquinone compounds show stronger poisoning activity to Meloidogyne incognita, the using concentration of emodin is 1-10 mg/mL, and the lethality to Meloidogyne incognita is higher than 50% when treated for 72 h; the using concentration of the aloe-emodin is 2-20 mg/mL, and the lethality to meloidogyne incognita is higher than 50% when the aloe-emodin is treated for 72 hours;

③ the concentration ratio of the mixture of the two anthraquinone compounds of emodin and aloe-emodin is 2: 1-3: 1, the use concentration is 1-10 mg/mL, and the lethality to meloidogyne incognita is higher than 60% when the mixture is treated for 72 hours;

fourthly, the anthraquinone compound is a plant source extract which is screened from the traditional Chinese herbal medicine and has the poisoning effect on the meloidogyne incognita, and can be used for developing a nematicide from the traditional Chinese herbal medicine;

fifthly, the using concentration of the Rheum palmatum rhizome extract for preventing and treating root-knot nematode is 1-4 mg/mL, the filling amount is 100-500 mL, and the potted plant prevention and treatment effect on tomato root-knot nematode is higher than 60%;

sixthly, the Rheum palmatum rhizome extract can be used for preparing emulsion in water, wettable powder, soluble granules, water dispersible granules, suspending agent, oil suspending agent and tablets for preventing and treating root knot nematode diseases.

The invention has the following advantages and positive effects:

firstly, natural Chinese herbal medicine plant rhubarb palmate leaf is adopted as a material, two anthraquinone compounds with good poisoning activity on root-knot nematode larvae are separated from a root extract of the natural Chinese herbal medicine plant rhubarb palmate leaf, and the anthraquinone compounds are natural in origin, safe and friendly to the environment and relatively wide in source.

Secondly, the prevention and control technology is difficult to cause the pests to have drug resistance, and the problem of drug residue does not exist, thereby meeting the basic requirements of the green prevention and control technology of the pests.

The invention is used for preventing and controlling plant root knot nematode disease, is beneficial to protecting the ecological environment and biological diversity of farmlands, and has good ecological benefit.

Drawings

FIG. 1 is a chemical structure diagram of emodin, a thread-killing active compound, isolated from the rhizome of Rheum palmatum;

FIG. 2 is a chemical structure diagram of the line-killing active compound aloe-emodin separated from the rhizome of Rheum palmatum;

table 1 shows the effect of ethanol extract of Rheum palmatum rhizome on Meloidogyne incognita J₂The poisoning activity of (a);

table 2 shows the effect of ethanol extract of Rheum palmatum rhizome on Meloidogyne incognita J₂Analyzing the toxicity;

table 3 shows the nematicidal activity of different fractions of Rheum palmatum rhizome extract;

table 4 shows the virulence analysis of meloidogyne incognita for two anthraquinone compounds;

table 5 shows the combined virulence of emodin and aloe-emodin against Meloidogyne incognita;

table 6 shows the pot control effect of Rheum palmatum rhizome extract on tomato root knot nematode disease.

Detailed description of the preferred embodiments

The present invention will be described in detail with reference to examples.

1. Example 1: biological activity of ethanol extract of palmate rhubarb rhizome on meloidogyne incognita

1) Root-knot nematode feeding

Carrying out propagation and breeding on potted tomatoes; after the tomato seedlings are transplanted and survive, a nematode suspension is accessed at a position 1-2 cm away from the tomato roots by using a liquid moving machine, water and fertilizer management is carried out regularly, the root systems are dug out within about 40 days, and oocysts are picked; putting the oocysts into a glass culture dish, adding a small amount of clear water, placing and incubating for 2-3 days at the temperature of 22-24 ℃ in a dark place, and performing microscopic examination on nematode liquid to enable the concentration of nematodes to be about 200 heads/mL; the experiment selects the J with vigorous activity₂The larval instar is tested for its poisoning activity.

2) Preparation of Rheum palmatum rhizome extract

Weighing 200g of rheum palmatum rhizome dry powder, putting into a brown wide-mouth bottle, adding 800mL of absolute ethyl alcohol, soaking at 24-27 ℃ at room temperature, and stirring once by using a glass rod in the morning and at night every day; soaking for 7 days, filtering, concentrating the filtrate under reduced pressure in a rotary evaporator under 40 deg.C water bath to obtain viscous paste extract, and storing in 4 deg.C refrigerator in dark place.

3) Method for testing thread killing activity

The pest-soaking method is adopted to test the poisoning effect of the extract on the meloidogyne incognita; weighing 0.2g of extract, adding 9.8mL of clear water at room temperature, and diluting to 10mL in advance by 50 times; fully and uniformly mixing 3mL of extract diluent with the 3mL of nematode liquid to obtain 100 times of extract liquid, namely preparing a mixed liquid of 10mg/mL of nematodes and the extract; adding the mixed solution into a 48-hole cell culture plate by using a pipettor, wherein 1.5mL of the mixed solution is added into each hole; sterile water is set as a control; 4 wells were added for each treatment, i.e. repeated 4 times; placing the cover on a sterile and dark condition at 25 ℃ after covering, observing under an optical microscope at 40 times after 24h, 48h and 72h, investigating the death number of the nematodes, and calculating the corrected mortality.

The investigation method comprises the following steps: 0.6mL (6 times multiplied by 0.1 mL/time) is investigated per well, 0.1mL is pipetted per time and counted on a glass slide, and the total number of worms is 200-240 per treatment and investigation.

Corrected mortality (%) - (treatment mortality-control mortality)/(100-control mortality) × 100

Nematode death criteria: when the nematodes are observed under a microscope, if the nematodes move or are in an S shape, the curly shape can be judged as live nematodes, the nematodes are dead or in a J shape and are judged as inactivated and dead, the picking needles are used for lightly touching the nematodes when the nematodes are difficult to judge, and the nematodes are judged as dead if the nematodes do not have any reaction.

Diluting the plant extract with sterile water to 4.00, 2.00, 1.00, 0.67, 0.50mg/mL respectively; after 24h, 48h and 72h of treatment at 25 ℃ in a dark place, the number of nematode deaths is investigated according to the method, and the corrected mortality, the virulence regression equation and the semi-lethal concentration LC are calculated₅₀And a correlation coefficient.

4) Test results

TABLE 1 poisoning Activity of ethanol extract of Rheum palmatum rhizome on Meloidogyne incognita (10mg/mL)

Control mortality was less than 10%.

As shown in Table 1, after the rheum palmatum L extract is used for treating the test insects for 24 hours, 48 hours and 72 hours at the concentration of 10mg/mL, the corrected mortality rates of the test insects are 88.41%, 91.30% and 95.22%, respectively, which shows that the rheum palmatum L rhizome ethanol extract has higher poisoning activity on Meloidogyne incognita.

TABLE 2 toxicity analysis of Rheum palmatum rhizome ethanol extract on Meloidogyne incognita

As can be seen from Table 2, LC was obtained after treating the test insects with Rheum palmatum extract for 24h, 48h and 72h₅₀The toxicity regression equation correlation coefficients of the ethanol extract of the rheum palmatum rhizome and the rhizomes of the rheum palmatum are respectively 0.9455mg/mL, 0.7040mg/mL and 0.5310mg/mL, and the toxicity regression equation correlation coefficients under different treatment times are all larger than 0.9500, so that the tested toxicity test result is reliable, and the test further shows that the rheum palmatum rhizome and the rhizomes ethanol extract have higher toxicity activity on Meloidogyne incognita.

Example 2: poisoning activity of anthraquinone compound extracted from Rheum palmatum rhizome on Meloidogyne incognita

1) Root-knot nematode feeding

The same as in example 1.

2) Separation and preparation of anthraquinone compound extracted from Rheum palmatum rhizome

Weighing 5g of the rheum palmatum ethanol extract concentrate in example 1, separating by silica gel column chromatography, and packing by wet column method; the eluent is petroleum ether and ethyl acetate eluent is 4:1(V/V), the dosage of the eluent is about 15 times of the retention volume, the eluent is collected according to 50 mL/bottle, and the elution flow rate is about 3 mL/min; performing primary identification on each group of elution fractions by adopting Thin Layer Chromatography (TLC), and merging and collecting elution fractions with similar TLC patterns; after all elution was completed, concentration under reduced pressure yielded a total of 3 fractions: identifying component A as mixed component by thin layer chromatography (further separating to obtain single components A1 and A2), and B and C as single components; the chemical structure identification shows that A1 and A2 are chrysophanol and physcion, and B and C are emodin (figure 1) and aloe-emodin (figure 2).

3) Method for testing thread killing activity

The same as in example 1.

4) Test results

TABLE 3 nematicidal Activity of different fractions of Rheum palmatum rhizome extract (2mg/mL)

The same letter in each column indicates no significant level of difference (P >0.05), the different letter indicates significant level of difference (P < 0.05); the control mortality was less than 10%.

As can be seen from Table 3, after the root-knot nematodes are treated by the components at 2mg/mL for 24 hours, the corrected mortality rate of the test nematodes is lower than 30%, and the nematicidal activity is weak. After 48 hours of treatment, the corrected mortality rates of the emodin and the aloe-emodin are both higher than 30%; the corrected mortality rate for emodin after 72h treatment was 76.68% and aloe-emodin was 56.47%. The components of the rhubarb are emodin and aloe-emodin with stronger activity to Meloidogyne incognita, but the activity is lower than that of the crude extract of the rhizome of the rhubarb.

TABLE 4 virulence analysis of Meloidogyne incognita by two anthraquinones

As can be seen from the toxicity test results (Table 4) of emodin and aloe-emodin on root-knot nematodes, when the emodin is treated for 24h, 48h and 72h, the indoor toxicity of the emodin on the root-knot nematodes is superior to that of the aloe-emodin, and better indoor poisoning effects are shown, and LC is treated for 24h, 48h and 72h₅₀Values of 4.7568, 1.8783 and 0.8818mg/mL, respectively, were found to increase virulence effects significantly for 48h and 72h compared to 24 h.

TABLE 5 Combined virulence of emodin and Aloe-emodin against Meloidogyne incognita (72h)

As can be seen from Table 5, after 72 hours of drug treatment, 5 different ratios of emodin and aloe-emodin against root-knot nematode LC₅₀1.0009mg/mL, 0.6935mg/mL, 0.7197mg/mL, 1.3190mg/mL and 1.7110 respectivelymg/mL. From table 5, it can be seen that the co-toxicity coefficients CTC values are 101, 135, 126, 83 and 67, respectively; when the emodin and the aloe-emodin are mixed according to the ratio of 2:1 and 3:1, the CTC value is more than 120, which shows that the emodin and the aloe-emodin have certain coordination and synergy effects in the two ratios, and the ratio of the emodin to the aloe-emodin is 2: the CTC value is 135 at 1, and the synergistic effect is most obvious.

Example 3: potted plant control effect of Rheum palmatum rhizome extract on tomato root knot nematode disease

The Rheum palmatum rhizome was extracted as in example 1 and concentrated to obtain the rhizome extract. Diluting the rhizome extract with clear water to obtain 0.1%, 0.2% and 0.4% dilutions for root irrigation.

And (3) irrigating roots during tomato transplanting: before application, the clean nutrient soil and the disease-infecting soil of the root-knot nematode are mixed in a proportion of 2: 1(w/w), fully and uniformly mixing, putting into a plastic flowerpot with the diameter of 15cm and the height of 20cm, and putting 2Kg of diseased soil mixed sample into each pot; transplanting the healthy tomato seedlings into the pretreated flowerpots after third true leaves grow out; after transplanting, 100mL of the diluent is poured into each pot, and 3 times of the dilution are set, wherein 20 pots are repeated. The control was filled with 100mL of clear water. Placing the plastic film in a plastic greenhouse, and uniformly managing water and fertilizer; and after 60 days, the tomato roots are pulled out slightly, the incidence of diseases is investigated according to root knot conditions of the roots, and the potted plant control effect on the root knot nematode disease is counted.

The root knots were graded according to the method of Barker, with the following criteria:

level 0: the root system is healthy and complete, and root knots are not generated; level 1: a small amount of root knots are slightly formed, and the root knots are small; and 2, stage: the lateral root has clearly visible small root knots, and the main root is normal; and 3, level: the lateral root has a large root knot, and the main root is normal; 4, level: the lateral root has more knots, and the main root is normal; and 5, stage: the main root begins to have sporadic root knots; and 6, level: obvious root knots appear on part of the main roots; and 7, stage: most of the main roots have root knots; and 8, stage: all the main roots have root knots; and 9, stage: all root systems have root knots; 10 level: all root systems have serious root knots and are seriously infected.

Calculating disease index and prevention and treatment effect according to the grading standard, wherein the calculation formulas are respectively as follows:

disease index ∑ (number of each disease-grade plant × number of grade)/(total number of investigated plants × number of most serious disease-grade) × 100

The prevention and treatment effect is (contrast disease index-treatment disease index)/contrast disease index multiplied by 100 percent

TABLE 6 potted plant control effect of Rheum palmatum rhizome extract on tomato root knot nematode disease

Treatment of	Index of disease condition	Control effect (%)
			0.1％	20.4	62.2±5.0b
0.2％	15.4	71.5±3.6b
			0.4％	8.9	83.5±1.4a
Control	53.9	-

From table 6, after the tomatoes are planted for 60 days, the root knot number of the root systems of the tomatoes treated by the concentrations of the 3 extracts is reduced compared with that of the tomatoes treated by the control group, the disease indexes of the tomatoes treated by the 0.1%, 0.2% and 0.4% extracts are respectively 20.4, 15.4 and 8.9, the control effects are respectively 62.2%, 71.5% and 83.5%, wherein the control effect of the pot culture after the 0.4% rheum palmatum extract is treated is higher than 80%, and the control effect is obviously better than that of the 0.1% rheum palmatum extract and the 0.2% rheum palmatum extract.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.