阅读说明：本技术 月桂酸在防治疫霉菌中的应用 (Application of lauric acid in prevention and treatment of phytophthora ) 是由 王群青 胥倩 梁畅汇 汪勇 吉亚泰 于 2020-12-09 设计创作，主要内容包括：本发明公开了月桂酸在防治疫霉菌中的应用,植物源杀菌剂技术领域。本发明研究发现,月桂酸对大豆疫霉菌菌丝径向生长、对大豆疫霉菌游动孢子囊产生及游动孢子的产生、萌发均具有明显的抑制作用。本发明通过月桂酸对土壤进行处理,能够对土壤中的大豆疫霉菌起到有效的防治作用,对防治大豆疫病有重要意义。(The invention discloses application of lauric acid in prevention and treatment of phytophthora and belongs to the technical field of plant-derived bactericides. The research of the invention finds that the lauric acid has obvious inhibiting effect on the radial growth of the phytophthora sojae hyphae, the generation of the zoosporangium of the phytophthora sojae, and the generation and germination of the zoospores. According to the invention, the lauric acid is used for treating the soil, so that the effective prevention and treatment effect on phytophthora sojae in the soil can be achieved, and the method has an important significance for preventing and treating phytophthora sojae.)

1. Application of lauric acid in preventing and treating phytophthora is disclosed.

2. Use according to claim 1, wherein the phytophthora is phytophthora sojae.

3. The use according to claim 2, wherein lauric acid controls Phytophthora sojae by at least one of the following (1) to (3):

(1) inhibiting the radial growth of phytophthora sojae hyphae;

(2) inhibiting phytophthora sojae zoosporangium and zoospore production;

(3) inhibiting the germination of phytophthora sojae zoospores.

4. A method for preventing and treating phytophthora by using lauric acid is characterized by comprising the step of treating soil by using the lauric acid.

5. The method of claim 4, wherein: the treatment of the lauric acid on the soil is to uniformly mix the lauric acid with the soil according to the dosage of 25-35 kg/mu in a fallow period so as to prevent and treat phytophthora in the soil.

6. The method of claim 4, wherein: the treatment of the lauric acid on the soil is that the lauric acid is evenly mixed with the soil according to the dosage of 25-35 kg/mu when the soil is turned over 3-5 days before sowing.

7. The method of claim 4, wherein: the phytophthora is phytophthora sojae.

8. Application of lauric acid in preparation of phytophthora bactericide.

9. The phytophthora fungicide is characterized by taking effective amount of lauric acid as an active ingredient.

Technical Field

The invention relates to the technical field of plant-derived bactericides, and particularly relates to application of lauric acid in prevention and control of phytophthora.

Background

Phytophthora sojae belongs to the phylum Oomycetes, the order Peronospora, the genus Phytophthora, is an Oomyces pathogenic agent with extremely strong pathogenicity, has a narrow host range, and can only infect soybeans and lupins under natural conditions. Phytophthora sojae can damage the seed and seedling rot in the whole soybean growing period, seeding period and seedling period, root and stem rot in the adult plant period, pod damage in the pod setting period, even no grain harvest in severe occurrence, and is a destructive disease in soybean production. Because the phytophthora sojae is high in infection speed, long in infection duration, rapid in morbidity, extremely short in incubation period, insensitive to a plurality of fungicides and rich in genetic diversity, the phytophthora sojae is the most serious in loss in soybean production and is one of the diseases which are difficult to control.

Phytophthora sojae sexual reproduction produces oospores with thick walls, which germinate to produce germ tubes, with the appropriate conditions, followed by the formation of hyphae or zoosporangia, which are the primary source of the initial infection. The phytophthora sojae asexual reproduction produces zoospores which become a re-infection source, and the zoospores are dormant after swimming for a period of time to form resting spores which can swim again. When it encounters the appropriate host tissue, the resting spores germinate to produce germ tubes that invade the epidermis of the host.

When zoospores contact the surface of a host plant, the zoospores stop swimming, flagella disappear, and resting spores are formed. When the conditions are proper, the germination produces a sucker, and the sucker continuously invades new cells through intercellular spaces to suck nutrients, promote the further colonization of the new cells and cause the necrosis of the host.

As an important precursor source of green bactericides, plant-derived antibacterial substances widely exist in the nature. Plants generate a plurality of secondary metabolites in vivo, and the secondary metabolites have strong antibacterial activity, wherein the secondary metabolites comprise a plurality of different types such as organic acids, phenolic compounds, flavonoids, alkaloids, proteins and the like, in the previous report, 1389 plants are known, and the plants are likely to become bactericides later. At present, people carry out very deep and detailed research on the biological activity and the chemical structure of main active ingredients in antibacterial plants, and research a plurality of high-efficiency and low-toxicity pesticides which play an extremely important role in important diseases of crops, such as quasi-ginsengs, propylene formates and the like. In the future, the application of the plant source bactericide will be very wide, and the plant source bactericide will play an increasingly important role in protecting the ecological environment.

At present, the medicines for preventing and treating soybean epidemic disease are mainly bactericides such as metalaxyl, azoxystrobin, dimethomorph, flumorph, cymoxanil, propamocarb, boscalid, propiconazole and the like. However, these drugs have been found to have a problem of decreasing the drug efficacy in recent years. On the one hand, it may be misuse by the user and, on the other hand, it may be the evolution of the species into drug-resistant species.

If a new compound with medicinal effect is found, the compound has great significance for preventing and treating soybean epidemic disease, and no relevant report about using lauric acid for preventing and treating phytophthora is provided at present.

Disclosure of Invention

Aiming at the prior art, the invention aims to provide the application of lauric acid in prevention and treatment of phytophthora, and researches of the invention find that the lauric acid has obvious inhibition effects on hypha radial growth of phytophthora sojae, phytophthora sojae zoosporangium and zoospore production, zoospore and phytophthora sojae zoospore germination, and has great significance on prevention and treatment of phytophthora sojae.

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

in a first aspect of the invention, there is provided the use of lauric acid for the control of phytophthora.

Preferably, the phytophthora is phytophthora sojae.

Preferably, lauric acid controls phytophthora sojae by at least one of the following routes (1) to (3):

(1) inhibiting the radial growth of phytophthora sojae hyphae;

(2) inhibiting phytophthora sojae zoosporangium and zoospore production;

(3) inhibiting the germination of phytophthora sojae zoospores.

Preferably, the lauric acid has a chemical structural formula as follows:

in a second aspect of the invention, there is provided a method of controlling phytophthora using lauric acid, comprising the step of treating soil with lauric acid.

Preferably, the lauric acid is uniformly mixed with the soil according to the dosage of 25-35 kg/mu in the fallow period to prevent and treat phytophthora in the soil.

Preferably, the lauric acid is used for treating the soil 3-5 days before sowing, and when the soil is turned, the lauric acid is uniformly mixed with the soil according to the dosage of 25-35 kg/mu.

Preferably, the phytophthora is phytophthora sojae.

In a third aspect of the invention, there is provided the use of lauric acid in the preparation of a phytophthora fungicide.

In a fourth aspect of the present invention, there is provided a phytophthora fungicide containing lauric acid.

The invention has the beneficial effects that:

1. the lauric acid has obvious inhibiting effect on the radial growth of phytophthora sojae hyphae, the generation of phytophthora sojae zoosporangium and the generation and germination of zoospores, can play an effective prevention and treatment effect on the phytophthora sojae in soil, and has great significance on the prevention and treatment of phytophthora sojae.

2. The lauric acid has extremely low production raw material cost, simple production process and short production period, is beneficial to industrial production and transportation, and provides a new method for developing and applying a new bactericide preparation.

3. Lauric acid is a natural plant extract, is used in the field of food, is environment-friendly, is not easy to generate resistance, can effectively inhibit the occurrence of soil-borne diseases, and simultaneously can not cause the 3R phenomenon (residual quantity, drug resistance and rampant recurrence) of the traditional control method.

Drawings

FIG. 1 is a mass chromatogram of lauric acid in the gaseous volatiles of susceptible soybeans of the present invention.

FIG. 2 is a mass chromatogram of lauric acid in the antiviral soybean volatile gaseous compounds of the present invention.

FIG. 3 is a graph showing the inhibition of radial growth of phytophthora sojae hyphae by lauric acid in accordance with the present invention.

FIG. 4 is a graph showing the effect of lauric acid on the morphology of Phytophthora sojae hyphae according to the present invention.

FIG. 5 is a graph showing the inhibition of germination of phytophthora sojae zoospore by lauric acid in accordance with the present invention.

FIG. 6 is a control diagram of the control effect of lauric acid on the soybean epidemic disease control potted plant experiment of the invention.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

As introduced in the background section, the current medicines for preventing and treating soybean blight are mainly bactericides such as metalaxyl, azoxystrobin, dimethomorph, flumorph, cymoxanil, propamocarb, boscalid, and propiconazole. However, these drugs have been found to have a problem of decreasing the drug efficacy in recent years. On the one hand, it may be misuse by the user and, on the other hand, it may be the evolution of the species into drug-resistant species.

It was found experimentally that lauric acid was detected in disease and infection resistant soy volatiles (as shown in figures 1 and 2). The invention further discovers that lauric acid has obvious inhibiting effect on radial growth of phytophthora sojae hyphae, phytophthora sojae zoosporangium production and zoospore production and germination, and has great significance on prevention and treatment of phytophthora sojae.

In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.

The test materials used in the examples of the present invention are all conventional in the art and commercially available. Wherein: lauric acid was purchased from 143-07-7 of Shandong Youso chemical science and technology, Inc.; phytophthora sojae strain R2(P6497), Phytophthora sojae strain R6(P7063), Phytophthora sojae strain R17(P7074), and Phytophthora sojae strain R19(P7076) are all from Nanjing university of agriculture.

Example 1: effect of lauric acid on radial growth of Phytophthora sojae hyphae

Activating strains: cutting and placing the low-temperature preserved phytophthora sojae R2, R6, R17 and R19 blocks on a V8 solid medium plate for culturing for 5 days at 25 ℃, cutting the phytophthora sojae R2, R6, R17 and R19 colonies into small blocks on a V8 solid or liquid medium plate, and culturing for 3 days at 25 ℃.

Solid V8 medium formulation: v8 beverage 100.0mL, agar 20.0g, water 900.0 mL.

Liquid V8 medium formula: v8 beverage 100.0mL, water 900.0 mL.

Adding a certain amount of lauric acid into 10% solid V8 culture medium to prepare a drug-containing culture medium with final concentration of lauric acid of 0mg/L, 100mg/L, 200mg/L, 300mg/L, 400mg/L and 500mg/L, beating a plurality of cakes of phytophthora sojae R2 after strain activation on the culture medium, and placing the cakes in an incubator at 25 ℃ for dark culture by taking the culture medium with final concentration of lauric acid of 0mg/L as a control, as shown in figure 3. The colony diameter for each treatment in the experiment was measured using the cross method. The half-maximal effect concentration (EC50), 95% maximal effect concentration (EC95) and 99% maximal effect concentration (EC99) of each concentration of lauric acid to phytophthora sojae R2, R6, R17 and R19 were calculated and are shown in Table 1.

Phytophthora hyphae growth inhibition (%) was [ (] control colony diameter-cake diameter) - (treated colony diameter-cake diameter) ]/(] control colony diameter-cake diameter) × 100%

Table 1: lauric acid for hypha radial growth EC50, EC95 and EC99

	EC50(mg/L)	EC95(mg/L)	EC99(mg/L)
				R2	237.8416	457.2265	594.5051
R6	427.4813	674.6127	840.4823
				R17	347.1482	548.3617	719.2173
R19	407.5918	607.4972	791.2056

Example 2: influence of lauric acid on Phytophthora sojae R2 zoosporangia and zoospore production

Taking liquid culture with the same mycelium amount after activation, rinsing with sterile tap water for 3-4 times, finally adding lauric acid to the final concentration of 0mg/L, 2mg/L, 4mg/L, 8mg/L, 16mg/L, 32mg/L, 64mg/L, 125mg/L and 250mg/L respectively, repeating the treatment for 3 times, taking V8 culture medium with the lauric acid concentration of 0mg/L as a control, culturing for 6h at 25 ℃, taking 5 visual fields randomly under a microscope, observing and recording the number of zoosporangia. The inhibition rate of lauric acid on the formation of zoosporangium of phytophthora sojae R2 was calculated.

The inhibition rate (%) of phytophthora zoosporangium formation was ═ number of zoosporangia in control group-number of zoosporangia in treated group)/number of zoosporangia in control group × 100%

Taking liquid culture with the same amount of activated mycelium, rinsing with sterile tap water for 3-4 times, finally adding equal volume of sterile tap water, repeating 3 times of treatment, culturing for 3 hours at 25 ℃, adding lauric acid until the final concentration is 0mg/L, 2mg/L, 4mg/L, 8mg/L, 16mg/L, 32mg/L, 64mg/L, 125mg/L and 250mg/L, standing for 3 hours, taking 5ul zoospore suspension in each treatment, observing and recording the number of zoospores under a microscope, and calculating the inhibition rate of the lauric acid on the phytophthora sojae R2 zoospores, wherein the inhibition rate is shown in Table 2.

The phytophthora zoospore production inhibition ratio (%) was (control group zoospore number-treatment group zoospore number)/control group zoospore number × 100%.

Table 2: inhibitory rate of lauric acid on zoosporangium and zoospore production

Example 3: effect of lauric acid on the form of the mycelium of Phytophthora sojae R2

Taking two parts of activated phytophthora sojae R2 liquid culture mycelia, adding lauric acid into one part of the mycelia, standing for 3h, taking the other part of the mycelia as a control without any treatment, and observing the hypha form under a microscope.

Compared with the hyphae of the control group, the hyphae of the control group treated by lauric acid naturally spread, have no twisting, bending and increase of terminal branches, have uniform and consistent hypha growth points, are far away from the top in the formation of branches, and have more zoosporangia. The lauric acid-treated hyphae grew disorderly, the terminal branches increased significantly, and the formation of zoosporangia was inhibited, as shown in fig. 4.

Example 4: effect of lauric acid on Phytophthora sojae R2 zoospore germination

Preparation of zoospore suspension: taking activated phytophthora sojae R2 for liquid culture, sucking out all liquid V8 culture medium, discarding, adding sterilized tap water into a culture dish, immersing mycelium blocks, washing with sterilized tap water every 10min, after washing for 3 times, changing water for the last time, wherein the water does not need to be added too much, hyphae can be just immersed, and placing the plate in the dark at 25 ℃ for 6 h.

The zoospore suspension concentration is controlled to 4 x 10 by counting with a blood counting plate³Per mL, prepared in such a way thatThe final concentrations of lauric acid were 0mg/L, 2mg/L, 4mg/L, 8mg/L, 16mg/L, 32mg/L, 64mg/L, 125mg/L, 250mg/L, and 500mg/L of the drug-containing medium, 1mL of zoospore suspension was added, and uniformly spread with a spreader, and spore germination was observed for all the treated media, as shown in FIG. 5. As can be seen from FIG. 5, when the lauric acid concentration reached 8mg/L, the germination of zoospores was significantly inhibited.

Table 3: effect of lauric acid on the Germination of zoospores

Concentration (mg/L)	500	250	125	64	32	16	8	4	2	CK
											Lauric acid	-	-	-	-	-	-	-	+	+	+

Note: "+" there was zoospore germination; "-" No zoospore germination

Example 5: pot culture experiment of lauric acid for preventing soybean epidemic disease

Simulating the diseased soil: and (3) uniformly mixing the activated liquid culture phytophthora sojae R2 hyphae with soil, and subpackaging into a plurality of small pots.

Soil treatment: adding lauric acid powder with different concentrations into small basin filled with simulated disease soil, mixing, and making into 0.25g/dm²、0.5g/dm²、1g/dm²Respectively marked as 1, 2 and 3, and treating the soil in the pot for 3-5 days;

the small pot with the simulated disease soil was recorded as CK0 without lauric acid treatment.

A small pot of ordinary soil without P.sojae R2 was designated CK1 as a control.

Planting soybeans: after the dormancy state of soybean was broken, the soybean seeds were put in the treated soil, and after 5d, the growth of soybean was observed as shown in fig. 6.

In conclusion, lauric acid has obvious inhibition effect on phytophthora sojae R2, R6, R17 and R19, and can be widely applied to prevention and control of phytophthora sojae.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.