CN112602709B - Oomycete inhibitor - Google Patents

Abstract

The invention provides a novel oomycete inhibitor, the active ingredient of which is lovastatin hydrolysate. The invention also provides application of lovastatin in the aspect of preventing and treating plant oomycete diseases. By adding lovastatin into the culture medium, the growth rate of phytophthora sojae hyphae is obviously reduced, the sporulation ability and the pathogenic ability are inhibited, and the culture medium has similar growth inhibiting effect on other plant pathogenic oomycetes. Lovastatin can be used as a potential inhibitor for preventing and controlling phytophthora sojae and other oomycetes.

Description

Oomycete inhibitor

Technical Field

The invention relates to the field of plant disease control, and particularly relates to an oomycete inhibitor.

Background

Oomycetes (oomycetes) are a large group of eukaryotes, whose morphology is similar to that of fungi, growing in filaments, with undivided hyphae. Oomycetes have long been classified as fungi, belonging to the subdivision flagellates. But are greatly different from fungi in terms of major components of cell walls, chromosome multiples of trophozoites, types of asexual spores, sizes of genomes, and the like. Phylogenetic analysis at molecular level finds that the relationship between oomycetes and plants such as diatom is very close, but the relationship between the oomycetes and fungi is very far, and the new classification system classifies the oomycetes into the kingdom of the animal testes and penis (Stramnopila) and the phylum of oomycetes (oomycota). The plant pathogenic oomycetes are important pathogenic bacteria groups, and can infect hundreds of plants, so that serious economic loss of agriculture and forestry is caused. Phytophthora infestans (p.infestans) can cause late blight of potatoes, a devastating disease in potato production. Phytophthora sojae (p.sojae) can cause soybean root rot, causing economic losses in the world of over 20 billion dollars each year. At present, the plant is classified as an imported plant epidemic-resistant pest in China, and the difficulty in disease prevention and control is continuously increased. Phytophthora capsici (p. capsicii) and Pythium species (Pythium species) cause epidemic diseases of vegetables such as pepper, cucumber, pumpkin and the like, and the loss is very serious. Because of the differences between the metabolic pathways of oomycetes and fungi, the quantity of bactericides effective on the oomycetes is small at present. Triazole pesticides targeting CYP51 in the sterol synthesis pathway are not effective against oomycetes, for example, due to the inability to synthesize sterols by themselves. After the specific bactericide metalaxyl is used for a long time of nearly 30 years, the drug resistance risk is continuously increased, and an effective medicament and an action target for preventing and controlling plant oomycetes diseases are developed, so that the specific bactericide metalaxyl has important significance for formulating a prevention and control strategy of plant pathogenic oomycetes.

In 1976, the Japanese biochemist Akira Endo isolated lovastatin from Penicillium citrinum (Penicillium citrinum) was marketed in the 80 th century as the first choice for the treatment of hyperlipidemia, arteriosclerosis, coronary heart disease and cerebrovascular disease. Lovastatin acts on the liver after administration, and acts on acetyl-CoA reductase (HMGR), a rate-limiting enzyme in cholesterol synthesis after being metabolized into an active form. Lovastatin competitively binds to HMGR, changing its conformation and preventing normal substrate binding, resulting in decreased cholesterol synthesis. At present, no report related to the application of lovastatin to oomycetes and the control of plant diseases caused by the oomycetes is found.

Disclosure of Invention

The invention aims to provide a novel oomycete inhibitor.

Another purpose of the invention is to provide a new application of lovastatin, in particular to the application of lovastatin in the aspect of plant oomycete disease control.

In order to achieve the object, in a first aspect, the present invention provides an oomycete inhibitor, the effective component of which is lovastatin hydrolysate, and the structure of which is shown in formula I):

the chemical name of the compound of the formula I) is (3R,5R) -7- [ (1S,2S,6R,8S,8aR) -2,6-dimethyl-8- [ (2S) -2-methylbutanoyl ] oxy-1,2,6,7,8,8 a-hexahydrophthalalen-1-yl ] -3, 5-dihydroheptaneoic acid. See Guo, M.R., et al (2016), Simultaneous determination of lovastatin and its metabolism lovastatin acid in a tissue using UPLC-MS/MS with positive/negative ion-switching electronics, Application to a pharmacological test of lovastatin negative selectivity, journal of Chromatography B-Analytical Technologies in the biological and Life Sciences 1023:55-61.

The preparation method of lovastatin hydrolysate comprises: adding lovastatin into alkaline ethanol solution, and heating until lovastatin is completely dissolved.

In the preparation method, the alkaline ethanol solution is NaOH aqueous solution containing 10-20% v/v ethanol, wherein the concentration of NaOH is 0.1-0.3% w/v. Preferably, the alkaline ethanol solution is an aqueous NaOH solution containing 15% v/v ethanol, wherein the concentration of NaOH is 0.25% w/v.

In the aforementioned preparation method, lovastatin is completely dissolved by heating to 50-70 deg.C (preferably 60 deg.C).

In a second aspect, the present invention provides the use of lovastatin hydrolysate for inhibiting oomycetes and plant diseases caused thereby.

In a third aspect, the invention provides the use of lovastatin hydrolysate in the preparation of oomycete inhibitors and medicaments for controlling plant oomycete diseases.

The oomycetes can be selected from the group consisting of Phytophthora sojae (Phytophthora sojae), Phytophthora infestans (Phytophthora infestans), Phytophthora capsici (Phytophthora capsici), Pythium ultimum (Pythium ultimum) and the like.

Further, the application includes:

1) the lovastatin hydrolysate reduces the hyphal growth speed of the phytophthora sojae;

2) lovastatin hydrolysate inhibits the production of phytophthora sojae sporangia;

3) the lovastatin hydrolysate reduces the yield of phytophthora sojae oospores;

4) the lovastatin hydrolysate inhibits the pathogenic ability of phytophthora sojae zoospores.

In the invention, lovastatin is converted into an active form through hydrolysis in vitro, and the growth speed of phytophthora sojae hyphae can be obviously reduced after lovastatin hydrolysis products with different concentrations are added into a culture medium. The sporangium and the zoospore are main propagation modes of phytophthora sojae in fields, play an important role in disease propagation and infection, inhibit the generation of the sporangium by using lovastatin treatment, and improve the resting rate of the zoospore. The oospore is a dormant structure formed in the later period of disease occurrence, and can cause primary infection in the next year after overwintering. Treatment with lovastatin significantly reduced oospore production. The pathogenic capability of zoospores is reduced by using lovastatin treatment. The addition of lovastatin hydrolysate at different concentrations to the culture medium can also significantly reduce the growth of phytophthora infestans (p.infestans), phytophthora capsici (p.capsici) and pythium ultimum (py.ultimum) hyphae. The results show that the lovastatin has the capability of inhibiting the growth, sporulation and pathogenicity of the phytophthora sojae hyphae, has a certain broad-spectrum bacteriostatic action on part of oomycetes, and can be used as a potential inhibitor for preventing and controlling the phytophthora sojae and other oomycetes.

Drawings

FIG. 1 is a graph showing the colonies and growth rates of Phytophthora sojae under various concentrations of lovastatin treatment in a preferred embodiment of the present invention.

FIG. 2 is a graph showing the sporangium morphology and yield of Phytophthora sojae under treatment with different concentrations of lovastatin in a preferred embodiment of the present invention.

FIG. 3 is a graph showing the sporulation morphology and yield of Phytophthora sojae at various concentrations of lovastatin in the preferred embodiment of the present invention.

FIG. 4 is a graph showing the resting rate of Phytophthora sojae zoospores treated with different concentrations of lovastatin in a preferred embodiment of the present invention.

FIG. 5 is a graph showing the pathogenic ability of Phytophthora sojae zoospores treated with different concentrations of lovastatin in a preferred embodiment of the present invention.

FIG. 6 shows the growth rates of Phytophthora infestans (A), Phytophthora capsici (B) and Pythium ultimum (C) under various concentrations of lovastatin treatment in a preferred embodiment of the present invention.

FIG. 7 is a graph showing that lovastatin hydrolysate reduces the incidence of soybean root rot caused by Phytophthora sojae in a preferred embodiment of the present invention. Wherein denotes P < 0.05.

In the figure, different lower case letters indicate significant differences.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.

The percent in the present invention means mass percent unless otherwise specified; but the percent of the solution, unless otherwise specified, refers to the grams of solute contained in 100mL of the solution.

Lovastatin used in the following examples was purchased from Beijing Sorleibao technologies, Inc.

Phytophthora sojae is a (genome sequencing) standard strain P6497, provided by Nanjing university of agriculture. See Tyler, B., et al (2006), "photophthora genome sequences and mechanisms of pathogenesis"Science 313(5791):1261.

Phytophthora infestans is (genome sequencing) the standard strain T30-4, provided by Nanjing university of agriculture. See Haas, B.J., et al (2009), "Genome sequence and analysis of the Irish potato gene pathogen Phosphohthora armans"Nature 461(7262):393-398.

Phytophthora capsici is (genome sequencing) standard strain LT263, supplied by Nanjing university of agriculture. See Cui, C., et al (2019). "Draft Assembly of photophtora capsacici from Long-Read Sequencing Uncevers compatibility"Mol Plant Microbe Interact 32(12):1559-1563.

Pythium ultimum is (genome sequencing) the standard strain DAOM BR144, provided by the university of agriculture of tokyo. See Levesque, C.A., et al (2010), "Genome sequence of the transgenic plant pathogen pathology tissue and effector microorganism pathogenesis"Genome Biol 11(7):R73.

EXAMPLE 1 preparation of lovastatin hydrolysate

Adding lovastatin 400mg into 20mL alkaline ethanol (15% v/v ethanol, 0.25% w/v NaOH), heating in water bath at 60 deg.C until lovastatin is completely dissolved to obtain active form, filtering, sterilizing, and storing at-20 deg.C.

EXAMPLE 2 inhibition of lovastatin hydrolysate

1. Inhibiting the growth of phytophthora sojae hyphae

The lovastatin hydrolysates were added to V8 medium at final concentrations of 20, 40, and 80. mu.g/mL, respectively, and phytophthora sojae cakes having a diameter of 5mm were inoculated into the center of the medium and cultured in the dark at 25 ℃ for 3 days. The growth rate of phytophthora sojae hyphae on the control plate was calculated to be 4.85mm/d, 4.07mm/d and 3.30mm/d under the treatment of 20, 40 and 80. mu.g/mL lovastatin hydrolysate. The addition of lovastatin hydrolysate at different concentrations in the culture medium significantly reduced the hyphal growth rate of phytophthora sojae (fig. 1).

2. Inhibition of phytophthora sojae sporangium production

Dissolving lovastatin hydrolysate in water, and respectively preparing into solution with concentration of 20, 40, 80 μ g/mL, and stimulating phytophthora sojae hyphae to produce sporangium. The results showed that when pure water was used to induce phytophthora sojae to produce sporangia, an average of 130 sporangias were produced; inducing by using 20 mu g/mL lovastatin hydrolysate solution to generate 42 sporangia on average; inducing by using 40 mu g/mL lovastatin hydrolysate solution to generate 9 sporangia on average; while induction with 80. mu.g/mL lovastatin hydrolysate solution failed to produce sporangia at all. There was no significant change in the morphology of the sporangia (FIG. 2).

3. Reducing the yield of phytophthora sojae oospores

Adding lovastatin hydrolysate into V8 culture medium at final concentration of 20, 40 and 80 μ g/mL, inoculating Phytophthora sojae cake with diameter of 5mm in the center of the culture medium, culturing in dark at 25 deg.C for 30d, and counting the output of oospore. The control group (no lovastatin hydrolysate added to the medium) phytophthora sojae produced an average number of oospores of 800; the average number of oospores produced was 500 in V8 medium containing 20 μ g/mL lovastatin hydrolysate; the average number of oospores produced was 440 in V8 medium containing 40 μ g/mL lovastatin hydrolysate; in V8 medium containing 80. mu.g/mL lovastatin hydrolysate, the number of produced average oospores was 280 (FIG. 3).

4. Inhibiting pathogenic capability of phytophthora sojae zoospores

Preparing a fresh phytophthora sojae zoospore suspension, adding lovastatin hydrolysate with final concentration of 20, 40 and 80 mu g/mL into the zoospore suspension respectively, and standing for 1h to count the resting rate of the zoospores. And inoculating 5 mu L of zoospore suspension treated by lovastatin hydrolysate to hypocotyls of soybean etiolated seedlings, culturing at 25 ℃ in the dark for 48h, and counting the length of lesion spots. The results showed that the resting rates after addition of lovastatin hydrolysate to zoospore suspension were 5.9%, 6.6% and 10.4% at concentrations of 0, 20 and 40 μ g/mL, respectively, with no significant difference (fig. 4). The resting rate increased to 49% with 80. mu.g/mL lovastatin hydrolysate treatment. Under the treatment of concentrations of 0, 20 and 40 mu g/mL, the pathogenic capability of zoospores is not obviously changed, and under the treatment of lovastatin hydrolysate of 80 mu g/mL, the pathogenic capability is reduced, and the length of lesion spots is reduced by 25 percent (figure 5).

5. Inhibiting other oomycetes

Adding lovastatin hydrolysate with final concentration of 20, 40 and 80 μ g/mL into V8 culture medium respectively, inoculating Phytophthora infestans, Phytophthora capsici and Pythium ultimum cake with diameter of 5mm in the center of the culture medium, and performing dark culture at 25 ℃ to count the growth rate of hyphae. The results show that the growth rates of phytophthora infestans are 3.63mm/d, 3.65mm/d, 3.37mm/d and 2.39mm/d respectively under the treatment of lovastatin hydrolysate with the concentration of 0, 20, 40 and 80 mu g/mL; the growth rates of the phytophthora capsici are 7.83mm/d, 7.04mm/d, 6.96mm/d and 5.21mm/d respectively; the growth rates of Pythium ultimum were 27mm/d, 26.83mm/d, 24.92mm/d and 22.67mm/d, respectively. The above results show that the hyphal growth of three important oomycetes, Phytophthora infestans, Phytophthora capsici and Pythium ultimum is significantly inhibited by 80. mu.g/mL lovastatin hydrolysate (FIG. 6).

The experimental results show that the growth rate of phytophthora sojae hyphae is remarkably reduced by adding the lovastatin through an external source, the sporulation capacity and the pathogenic capacity are inhibited, the lovastatin has similar growth inhibition effects on other three plant pathogenic oomycetes, and the lovastatin can be used as a potential inhibitor for prevention and control of the phytophthora sojae and other oomycetes.

Example 3 lovastatin hydrolysate reduces the incidence of soybean root rot caused by Phytophthora sojae

And selecting 1-2 concentrations to perform a disease control experiment according to the indoor flat plate bacteriostatic experiment result. Sowing susceptible soybean varieties, obtaining a Hefeng variety, growing for 2 weeks under the conditions of 25 ℃, 16h of illumination/8 h of darkness until the first true leaves are developed. After the phytophthora sojae hyphae is inoculated, water solution containing lovastatin hydrolysate (with the concentration of 0, 40 and 80 mu g/mL respectively) is uniformly sprayed on soybean seedlings, and the incidence is counted after 3 days of culture. The results show that the soybeans treated by the control (with the concentration of 0 mug/mL) are all attacked and lodged, and the incidence rate is 100 percent; after the lovastatin hydrolysate with the final concentration of 40 mug/mL is used for treatment, the morbidity is reduced to 80 percent; particularly, after the lovastatin hydrolysate with the concentration of 80 mu g/mL is treated, the morbidity is reduced to 70 percent, and the control effect can reach 30 percent or more (figure 7).

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (3)

1. The application of lovastatin hydrolysate in inhibiting oomycetes and plant diseases caused by the oomycetes, wherein the oomycetes are selected from Phytophthora sojae (Phytophthora sojae), Phytophthora infestans (Phytophthora infestans), Phytophthora capsici (Phytophthora capsaici), and Pythium ultimum;

the preparation method of the lovastatin hydrolysate comprises the following steps: adding lovastatin 400mg into 20mL of alkaline ethanol, and heating in water bath at 60 ℃ until the lovastatin is completely dissolved to obtain the lovastatin-containing water solution; wherein the alkaline ethanol is 15% v/v ethanol and 0.25% w/v NaOH.

2. Use of lovastatin hydrolysate in the preparation of an oomycete inhibitor and a medicament for controlling plant oomycete diseases, wherein the oomycete is selected from the group consisting of Phytophthora sojae (Phytophthora sojae), Phytophthora infestans (Phytophthora infestans), Phytophthora capsici (Phytophthora capsici), Pythium ultimum (Pythium ultimum);

the preparation method of the lovastatin hydrolysate comprises the following steps: adding lovastatin 400mg into 20mL of alkaline ethanol, and heating in water bath at 60 ℃ until the lovastatin is completely dissolved to obtain the lovastatin-containing water solution; wherein the alkaline ethanol is 15% v/v ethanol and 0.25% w/v NaOH.

3. The application according to claim 1 or 2, characterized in that it comprises:

1) the lovastatin hydrolysate reduces the hyphal growth speed of the phytophthora sojae;

2) lovastatin hydrolysate inhibits the production of phytophthora sojae sporangia;

3) the lovastatin hydrolysate reduces the yield of phytophthora sojae oospores;

4) the lovastatin hydrolysate inhibits the pathogenic ability of phytophthora sojae zoospores.

Images