Use of compound NSC-32 in the prevention and treatment of pyricularia oryzae

Abstract

The present application relates to the field of plant filamentous fungal disease prevention and treatment, in particular to the application of compound NSC-32 (N-[(5Z)-4-oxo-5-(phenylmethylidene)-2-sulfanylidene-1,3-thiazolidin-3-yl]pyridine-4-carboxamide) in the prevention and treatment of Magnaporthe oryzae. The present application takes MoPex19 protein as a target, screens compound NSC-32, and proves that the compound can inhibit the growth of Magnaporthe oryzae and reduce the pathogenicity of the latter. In addition, the present application also explores the applicable object, use method, target specificity and action mechanism of compound NSC-32, and provides a new green pesticide and new idea for the prevention and treatment of Magnaporthe oryzae.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant filamentous fungal disease prevention and treatment, in particular to the application of compound NSC-32 in the prevention and treatment of Magnaporthe oryzae. BACKGROUND

[0002] Rice is the main food source for more than half of the world's population, and is the core crop in the food security system. The stable production of rice is related to the global food security. However, rice is threatened by a variety of biological diseases during its growth, among which the rice blast caused by Magnaporthe oryzae is particularly serious. The pathogen can systemically destroy rice leaves (leaf blast), stems (stem blast), panicle neck (panicle neck blast) and grains (grain blast), leading to a serious reduction in rice yield, and even to the death of the plant, causing serious losses to the global economy.

[0003] The field infection of rice blast is mainly mediated by conidia of Magnaporthe oryzae. These pathogens spread through multiple pathways such as air flow, raindrops, water flow and insect carrying. When the conidia successfully attach to the rice leaves, they will initiate the germination process to form a unique infection organ, the appressorium, under suitable temperature and humidity conditions. During the development process, the appressorium can accumulate osmotic adjustment substances such as glycerol to generate a turgor pressure of up to 8.0 MPa, which drives the formation of a long and thin infection peg at the base to break through the plant epidermis defense. The pathogenic potential of Magnaporthe oryzae is derived from its three biological characteristics: wide geographical distribution, strong environmental adaptability and rapid genetic variation rate. These characteristics make Magnaporthe oryzae have strong ecological plasticity, which further leads to serious challenges in the prevention and control system. Therefore, the development and implementation of a comprehensive management scheme based on multi-dimensional prevention and control strategy has important strategic value for ensuring the stability of rice production system and maintaining the international food security pattern.

[0004] The traditional rice blast prevention and control system consists of six core strategies: creation of disease-resistant high-yield germplasm, pathogen bank elimination, chemical control, optimization of agronomic management, precise regulation of water and fertilizer, and harmless treatment of diseased plants. It is worth noting that chemical control has always occupied a key position in plant protection strategies due to its rapid control ability during the epidemic period. However, the overuse of broad-spectrum fungicides has caused serious ecological crisis in the past three decades, and ecological problems such as soil degradation and imbalance of microbial community structure have continued to worsen. Moreover, the abuse of chemical pesticides has led to an increasing proportion of drug-resistant strains in the Magnaporthe oryzae population, which undoubtedly increases the difficulty of rice blast prevention and control. In summary, there is an urgent need for new compounds that can prevent and control Magnaporthe oryzae. SUMMARY

[0005] In view of the above-mentioned needs, the present application focuses on the pathogenic molecular mechanism analysis of pathogenic bacteria, and provides a new green compound NSC-32 (2-(dimethylamino)ethyl 3-[(5Z)-5-(2H-1,3-benzodioxol-5-ylmethylidene)-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]propanoate) for preventing and treating rice blast fungus by accurately positioning the key effector protein and signal hub in the infection process. The compound NSC-32 is a lead compound obtained by screening a key protein MoPex19 (SEQ ID NO: 2) for peroxisome membrane protein transport as a target, and its structural formula is as follows:

[0006] (Formula 1)

[0007] To achieve the above-mentioned purposes, the technical solutions adopted by the present application are as follows:

[0008] In a first aspect, the present application provides the use of the compound NSC-32 and its derivatives in the preparation of an agent for preventing and treating plant pathogenic bacteria, wherein the structure of the compound NSC-32 is as follows: .

[0009] Further, the plant pathogenic bacteria can express a protein represented by SEQ ID NO: 2 or a protein homologous to the sequence represented by SEQ ID NO: 2 by 50% and above.

[0010] It should be understood that the compound NSC-32 is obtained by screening small molecule compounds combined with MoPex19 protein (SEQ ID NO: 2). Further prediction results show that the benzene ring structure of the compound NSC-32 is combined with the SER-277 and GLN-273 sites of MoPex19 protein, so that the compound NSC-32 can be combined with MoPex19 protein, thereby affecting the normal physiological function of the latter, and then affecting the pathogenicity of rice blast fungus, thereby achieving the purpose of preventing and treating rice blast fungus. At the same time, by comparing the Pex19 homologous proteins in a plurality of plant pathogenic bacteria, it is found that the homology of Pex19 proteins in different plant pathogenic bacteria is as high as 50% and above, and the 3' end space structure is highly similar to MoPex19 protein, especially the existence of the NSC-32 binding site GLN-273, so it can be reasonably inferred that NSC-32 can be used for preventing and treating plant pathogenic bacteria expressing proteins homologous to MoPex19 protein by 50% and above.

[0011] In some embodiments, the plant pathogen is any one of Botrytis, Fusarium, Colletotrichum, Alternaria, Phoma, and Magnaporthe.

[0012] In some more specific embodiments, the pathogen is Magnaporthe grisea, which is capable of expressing a protein as set forth in SEQ ID NO: 2.

[0013] In some more specific embodiments, the pathogen is Botrytis cinerea, which is capable of expressing a protein as set forth in SEQ ID NO: 3, i.e., BcPex19 protein.

[0014] In some more specific embodiments, the pathogen is Fusarium fujikuroi, which is capable of expressing a protein as set forth in SEQ ID NO: 4, i.e., FfPex19 protein.

[0015] In some more specific embodiments, the pathogen is Colletotrichum orbiculare, which is capable of expressing a protein as set forth in SEQ ID NO: 5, i.e., CoPex19 protein.

[0016] In some more specific embodiments, the pathogen is Alternaria alternata, which is capable of expressing a protein as set forth in SEQ ID NO: 6, i.e., AaPex19 protein.

[0017] In some more specific embodiments, the pathogen is Fusarium oxysporum, which is capable of expressing a protein as set forth in SEQ ID NO: 7, i.e., FoPex19 protein.

[0018] In some more specific embodiments, the pathogen is Colletotrichum acutatum, which is capable of expressing a protein as set forth in SEQ ID NO: 8, i.e., CaPex19 protein.

[0019] Similarly, according to the way of the NSC-32 binding to the MoPex19 protein, it can be inferred that the derivatives with the same or similar mother nucleus structure of the compound NSC-32 can target the MoPex19 protein, and then achieve the purpose of preventing and treating the plant pathogenic fungi; specifically, on the basis of the mother nucleus structure of the compound NSC-32 unchanged, alkyl, halogen, nitro, cyano, halogenated alkyl and other groups are introduced, the introduction of the groups will not affect the binding of the derivatives and the MoPex19 protein, or even can improve the affinity between the two, or can make the binding between the two more firm, without hindering the binding between the two.

[0020] In a second aspect, the present application provides the use of the compound NSC-32 and its derivatives in the preparation of an agent for inhibiting the hypha growth of the plant pathogenic fungi and / or inhibiting the spore germination of the plant pathogenic fungi and / or inhibiting the disease spot formation of the plant pathogenic fungi and / or reducing the pathogenicity of the plant pathogenic fungi, the structure of the compound NSC-32 is .

[0021] In some specific embodiments, the plant pathogenic fungi is Magnaporthe oryzae.

[0022] The present application has explored the mechanism of the compound NSC-32 affecting the growth of Magnaporthe oryzae, and found that the compound may achieve the effect of preventing and treating Magnaporthe oryzae by promoting peroxisome autophagy.

[0023] In a third aspect, the present application provides the use of the compound NSC-32 and its derivatives in the preparation of an agent for targeting Pex19 protein, the structure of the compound NSC-32 is ; the Pex19 protein comprises the amino acid residue sequence shown in SEQ ID NO: 2 or an amino acid residue sequence homologous to the sequence shown in SEQ ID NO: 2 by 50% and above.

[0024] In a fourth aspect, the present application provides an agent for preventing and treating plant pathogens, the agent comprises the compound NSC-32; specifically, the compound is an active ingredient in the agent.

[0025] In some specific embodiments, the plant pathogenic fungi is Magnaporthe oryzae.

[0026] Further, the agent further comprises an adjuvant, such as gelatin, DMSO.

[0027] In a fifth aspect, the present application provides a method for preventing and controlling the plant pathogenic fungi and / or the diseases caused by the plant pathogens, that is, applying the compound NSC-32 or / and the agent as described above; the structure of the compound NSC-32 is .

[0028] In some embodiments, the plant pathogen is any one or more of Magnaporthe grisea, Botrytis cinerea, Fusarium fujikuroi, Guignardia bidwellii, Alternaria alternata, Fusarium oxysporum, and Erysiphe necator.

[0029] In some embodiments, the disease is any one or more of Magnaporthe grisea, Botrytis cinerea, Fusarium fujikuroi, Guignardia bidwellii, Alternaria alternata, Fusarium oxysporum, and Erysiphe necator.

[0030] In some embodiments, the disease is any one or more of Magnaporthe grisea, Botrytis cinerea, Fusarium fujikuroi, Guignardia bidwellii, Alternaria alternata, Fusarium oxysporum, and Erysiphe necator.

[0031] Further, the concentration of the compound NSC-32 is 10 ~ 50 ppm.

[0032] Preferably, the concentration of the compound NSC-32 is 20 ~ 50 ppm.

[0033] When the use object is rice, the applicable concentration of the compound NSC-32 is 20 ~ 50 ppm; when the use object is barley, the applicable concentration of the compound NSC-32 is 30 ~ 50 ppm.

[0034] Further, the plant is a plant of the family Poaceae. The plant of the family Poaceae includes but is not limited to rice and barley, and preferably rice.

[0035] Further, the compound NSC-32 and / or the agent are applied within 24 hours before and after the plant is infected by Magnaporthe grisea, and preferably the compound is applied within 24 hours after the plant is infected by Magnaporthe grisea.

[0036] The beneficial effects of the present application include:

[0037] 1. The present application discloses a new use of the compound NSC-32, which molecule can simultaneously inhibit the growth of multiple plant pathogens of different genera.

[0038] 2. The compound NSC-32 provided by the present application is a targeted preparation, which has double ecological benefits: it can specifically interfere with the development of appressorium of the pathogen, and it can maintain the dynamic balance of soil microbial community through degradable molecular design, which provides a new path for breaking through the marginal effect of traditional chemical control and realizing stable yield and quality of rice.

[0039] 3. The compound NSC-32 provided by the present application can inhibit the growth and development of Magnaporthe oryzae and inhibit the pathogenicity of Magnaporthe oryzae spores, thereby achieving the purpose of preventing and treating rice blast; and has a wide range of applications and can significantly reduce the lesion area formed by Magnaporthe oryzae in rice and barley, and the use effect of rice is better.

[0040] 4. The application concentration of the compound NSC-32 is explored, that is, 10 ~ 50 ppm, and more preferably 20 ~ 50 ppm. In particular, when the concentration is 50 ppm, NSC-32 can completely prevent and control rice blast, and has less effect on the growth and development of rice, thereby laying a foundation for the development of a new green pesticide.

[0041] 5. The use method of the compound NSC-32 is explored, that is, the NSC-32 compound is used within 24 hours before and after the plant is infected with Magnaporthe oryzae, and more preferably within 24 hours after the plant is infected with Magnaporthe oryzae.

[0042] 6. The action mechanism of the compound NSC-32 is preliminarily explored.

[0043] 7. A new target for preventing and controlling Magnaporthe oryzae, MoPex19 gene / protein, is provided. BRIEF DESCRIPTION OF DRAWINGS

[0044] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0045] Figure 1 It is a three-dimensional structure diagram of MoPex19 protein, wherein the red boxed area is a potential small molecule inhibitor binding area in MoPex19 protein.

[0046] Figure 2 It is the disease condition of detached barley leaf (n = 3) treated by 5 different compounds with a concentration of 50 ppm, wherein the ruler = 0.5 cm.

[0047] Figure 3 It is a prediction diagram of the binding site of MoPex19 and NSC-32 compound.

[0048] Figure 4 It is the inhibition of plate growth of Magnaporthe oryzae by different concentrations of NSC-32 compound.

[0049] Figure 5Figure 2 is a comparison chart of the diameters of the colony growth of Magnaporthe grisea under treatment with different concentrations of compound NSC-32, wherein different letters a, b, c, d, e or f represent significant differences in the inhibition rates of different concentrations of compound NSC-32; p < 0.05; the data in the figure were obtained by one-way ANOVA.

[0050] Figure 6 Figure 3 is a comparison chart of the inhibition rates of the mycelial growth of Magnaporthe grisea under treatment with different concentrations of compound NSC-32, wherein different letters a, b, c, d or e represent significant differences in the inhibition rates of different concentrations of compound NSC-32; p < 0.05; the data in the figure were obtained by one-way ANOVA.

[0051] Figure 7 Figure 4 is a chart showing the inhibition of the growth of different plant pathogenic fungi by 50 ppm of compound NSC-32, wherein B. cinerea: Botrytis cinerea; F. fujikuroi: Fusarium fujikuroi; C. orbiculare: Colletotrichum orbiculare; A. alternata: Alternaria alternata; F. oxysporum: Fusarium oxysporum; C. acutatum: Colletotrichum acutatum.

[0052] Figure 8 Figure 5 is a chart showing the diameters of the colony growth of different plant pathogenic fungi under treatment with 50 ppm of compound NSC-32, wherein the black columnar chart is the diameter of the colony growth of the pathogenic fungi under normal growth without addition of NSC-32 (control), the white columnar chart is the diameter of the colony growth of the pathogenic fungi under treatment with NSC-32, n = 3, * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001; wherein B. cinerea: Botrytis cinerea; F. fujikuroi: Fusarium fujikuroi; C. orbiculare: Colletotrichum orbiculare; A. alternata: Alternaria alternata; F. oxysporum: Fusarium oxysporum; C. acutatum: Colletotrichum acutatum.

[0053] Figure 9Figure 6 is a sequence alignment of Pex19 homologous proteins in different plant pathogens, wherein the red box corresponds to the site predicted to bind with NSC-32 molecules; MoPex19 protein corresponds to SEQ ID NO: 2, BcPex19 protein corresponds to SEQ ID NO: 3, FfPex19 protein corresponds to SEQ ID NO: 4, CoPex19 protein corresponds to SEQ ID NO: 5, AaPex19 protein corresponds to SEQ ID NO: 6, FoPex19 protein corresponds to SEQ ID NO: 7, and CaPex19 protein corresponds to SEQ ID NO: 8.

[0054] Figure 10 Figure 7 is the disease incidence of barley leaves treated with different concentrations of compound NSC-32 in vitro, wherein the scale = 1 cm.

[0055] Figure 11 Figure 8 is the disease incidence of rice leaves treated with different concentrations of compound NSC-32 in vitro, wherein the scale = 0.5 cm.

[0056] Figure 12 Figure 9 is the disease incidence of live rice leaves after spraying 50 ppm of compound NSC-32 at different times after inoculation with Magnaporthe oryzae spore solution, wherein the scale = 1 cm.

[0057] Figure 13 Figure 10 is a bar chart of the relative lesion area of live rice leaves after spraying 50 ppm of compound NSC-32 at different times after inoculation with Magnaporthe oryzae spore solution, wherein *** indicates p < 0.001.

[0058] Figure 14 Figure 11 is the effect of different concentrations of compound NSC-32 on the growth of rice, wherein the scale = 2 cm.

[0059] Figure 15 Figure 12 is the effect of different concentrations of compound NSC-32 on the seed germination of rice, wherein the scale = 2 cm.

[0060] Figure 16 Figure 13 is the formation of Magnaporthe oryzae appressorium at different time points after treatment with 50 ppm of compound NSC-32, wherein the scale = 20 μm.

[0061] Figure 17 Figure 14 is the formation rate of Magnaporthe oryzae appressorium at different time points after treatment with 50 ppm of compound NSC-32, wherein cc, *** indicates p < 0.001.

[0062] Figure 18The collapse of Magnaporthe grisea appressorium under different glycerol concentrations after treatment with 50 ppm of compound NSC-32; the scale = 20 μm.

[0063] Figure 19 The collapse rate of Magnaporthe grisea appressorium under different glycerol concentrations after treatment with 50 ppm of compound NSC-32, ** indicates p < 0.01, *** indicates p < 0.001.

[0064] Figure 20 The effect of NSC-32 compound on the peroxisome autophagy process of Magnaporthe grisea. DETAILED DESCRIPTION

[0065] The present application will be further described below in conjunction with the accompanying drawings and specific examples, which are only used to explain the present application and are not intended to limit the scope of the present application; all other examples obtained by those of ordinary skill in the art without making creative efforts based on the examples in the present application fall within the scope of the present application.

[0066] The test methods used in the following examples are conventional methods, all with three biological replicates, unless otherwise specified; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials available through commercial channels.

[0067] The plant pathogenic fungi used in the present application are shown in Table 1, of which Magnaporthe grisea is a wild type strain Guy11 (American ATCC strain center, https: / / www.atcc.org / products / 201236) preserved in the laboratory, and the culture medium for culturing the seven plant pathogenic fungi is complete culture medium (abbreviated as CM). The formula of CM culture medium is shown in Table 2, and the formula of 20×Nitrate salt, 1000×Vitamin solution and 1000×Trace elements solution is shown in Table 3.

[0068] Table 1 Plant pathogenic fungi

[0069]

[0070] Table 2 Formula of complete culture medium (CM)

[0071]

[0072] Table 3 Formula of 20×N salt, vitamin and trace element solution

[0073]

[0074] The preparation steps of CM medium are as follows: according to the contents of Table 1, the required reagents are weighed, and then diluted with pure water to 1 L. The corresponding reagents can be proportionally enlarged according to the volume of the required medium. A magnetic stirrer is used for complete dissolution, and the pH is adjusted to 6.5 with 5 M NaOH. It is divided into 500 mL glass bottles. In the solid medium, agar powder is added at a proportion of 18 g / L, and sterilized at 121°C for 15 minutes.

[0075] NSC-32, full name N-[(5Z)-4-oxo-5-(phenylmethylidene)-2-sulfanylidene-1,3- thiazolidin-3-yl]pyridine-4-carboxamide, is purchased from Shanghai Taoshu Biological Technology Co., Ltd., with catalog number HIT ID: HIT104123361. The molecular structure formula of compound NSC-32 is as follows: .

[0076] The mother liquor preparation method and storage conditions of compound NSC-32 are as follows: 10 mg of compound NSC-32 is diluted in 4 mL of DMSO to make the final concentration of the drug 2,500 ppm. The drug is stored at -20°C for short-term storage and at -80°C for long-term storage.

[0077] Example 1: Screening of rice blast fungus target

[0078] Peroxisome is an important organelle, which is involved in a variety of core metabolic processes in fungi, including fatty acid beta-oxidation, reactive oxygen species (ROS) degradation, glyoxylate cycle, etc., and is called the “metabolic center” of the cell. The proteins required for peroxisome formation are collectively referred to as Peroxin, which are encoded by PEX genes. Currently, 37 Peroxins have been identified in fungi, plants and animals. MoPex19 is a soluble chaperone, and its core function is to recognize newly synthesized peroxisomal membrane proteins (PMPs), bind to them, and transport them to the peroxisomal membrane, thereby completing the morphogenesis of peroxisomal membrane proteins. After the deletion of MoPex19 protein, most PMPs cannot be correctly targeted and integrated into the peroxisomal membrane, resulting in the inability of peroxisomes to form normally and complete loss of function, so it is a “upstream” and “hub” key target. The research group of the present inventors found in the previous study that MoPex19 gene (MGG_00971, SEQ ID NO: 1) is essential for the pathogenicity of Magnaporthe oryzae, and the deletion of the gene leads to the complete loss of pathogenicity of Magnaporthe oryzae, proving the necessity of the target. At the same time, the three-dimensional structure of MoPex19 protein (XP_003717979, SEQ ID NO: 2) was analyzed, and it was found that MoPex19 protein contains a site with a certain depth and volume (257 Å 3 ) which has the potential to bind small molecule inhibitors ( Figure 1 ).

[0079] In summary, the present application takes MoPex19 protein as a target to screen compounds capable of preventing and treating Magnaporthe oryzae.

[0080] Example 2: Screening of target-type bacteriostatic preparations

[0081] Based on the research results of Example 1, the present example takes MoPex19 protein as a target to screen compounds inhibiting the growth of Magnaporthe oryzae. Specifically, the protein molecule (G4ND53) is subjected to molecular docking with the Pesticide Library (43,417 compound molecules) by standard precision (SP) using the Glide software of the Schrödinger software package, the binding free energy of the top 200 molecules is calculated by prime MM-GBSA, the binding mode is analyzed, and finally 5 compound molecules are selected, which are NFD-22 (N-(3,4-dimethylphenyl)-1-[(3-fluorophenyl)methyl]-2-oxo-1,2-dihydropyridine-3-carboxamide), the structural formula is: ), BTH-75 ((6E)-6-{2-[4,6-bis(piperidin-1-yl)-1,3,5-triazin-2-yl]hydrazin-1-ylidene}hexane-1,2,3,4,5-pentol, structural formula: ), BMD-45 (2-(4-bromophenyl)-2-oxoethyl 4-(5-methyl-1,3-dioxo-2,3,3a,4,7,7a-hexahydro-1H-isoindol-2-yl)benzoate, structural formula: ), DDP-26 (3-chloro-1-[2-(3,4-dimethoxyphenyl)ethyl]-4-{[2-(3,4-dimethoxyphenyl)ethyl]amino}-2,5-dihydro-1H-pyrrole-2,5-dione, ), and NSC-32 (N-[(5Z)-4-oxo-5-(phenylmethylidene)-2-sulfanylidene-1,3-thiazolidin-3-yl]pyridine-4-carboxamide, structural formula: ).

[0082] Further, the effect on pathogenicity of barley was determined by treating the conidia of Magnaporthe oryzae wild type Guy11 with the five compounds (50 ppm) respectively, and the specific experimental procedures were as follows:

[0083] Magnaporthe oryzae strain Guy11 was inoculated on a 7 cm diameter CM solid medium plate containing 15 mL, and cultured in an incubator for 7 days (28°C, light for 16 h / dark for 8 h); under sterile conditions, 3-4 mL sterile water was added to the surface of the Guy11 plate, and the conidia were scraped off with a sterile spreader, filtered through three layers of sterile filter paper, and collected in a 1.5 mL centrifuge tube. The NSC-32 compound was mixed with the spore solution to prepare a spore solution containing 50 ppm of the compound, and the final concentration of the spore solution was 5 x 10 4spores / mL, and DMSO without the compound was used as a control; two layers of water-absorbing paper and a filter paper were placed in a square culture dish of 10 cm x 10 cm, and an appropriate amount of water was added to moisten it. 5-7 cm long barley (ZJ-8) leaves grown for 8-10 days were cut in order and placed in the square dish, 3 drops (about 20 μL in volume) of spore solution were spotted on each leaf, and each treatment was repeated 3 times; the square dish containing the leaves was carefully placed in a constant temperature incubator at 25°C (light and dark cycle 16 h: 8 h), and after 4 days of culture, the incidence of disease was observed and photographed.

[0084] Figure 2 It is shown that the virulence of Magnaporthe oryzae is significantly reduced after treatment with NSC-32 compound, while the effects of the other four small molecule compounds on the virulence of Magnaporthe oryzae are weak or have no significant effect, so NSC-32 is preferably selected to reduce the virulence of Magnaporthe oryzae.

[0085] Next, in order to further verify the targeting of NSC-32 molecules, this embodiment uses AlphaFold3 (https: / / alphafoldserver.com / ) to predict the binding site of NSC-32 molecules and MoPex19 protein, and the prediction result is as shown in Figure 3 The prediction result shows that the binding site of NSC-32 compound in MoPex19 protein is SER-277 and GLN-273.

[0086] Example 3: Virulence determination and results of compound NSC-32 on plant pathogens

[0087] In this embodiment, the virulence of compound NSC-32 is determined by mycelial growth rate method, and the specific experimental steps are as follows: Magnaporthe oryzae Guy11 is inoculated in the middle of a CM plate with a diameter of 7 cm, and is cultured at 25°C with light and dark for 16 h / 8 h for 7 days; different doses of compound NSC-32 are added to the solid CM medium (temperature is 45-55°C) to prepare drug-containing plates, wherein the final concentration of compound NSC-32 is 0, 10, 20, 30, 40, 50 ppm, respectively; under the sterile environment of a clean bench, 3-4 mL of sterile water is added to the plate of Guy11 cultured for 7 days, a disposable sterile coating rod is used to gently scrape and wash the conidia, and the conidia are filtered into a 1.5 mL centrifuge tube with 3 layers of filter paper, and the conidia are diluted to 1×10 5spores / mL; 5 μL of diluted conidial suspension was spotted in the center of the drug-containing plate; each experimental group was repeated 3 times; the CM medium containing the same concentration of DMSO was used as a control; the treated plates were inverted and cultured in an incubator (25°C, light and dark 16h / 8h) for 3 days, and the growth of the Magnaporthe oryzae mycelium in the experimental group was observed. The mycelial diameter was measured by the cross method, and the mycelial growth rate method was used to calculate the mycelial growth inhibition rate of Magnaporthe oryzae under different concentrations of compound NSC-32: mycelial growth inhibition rate (%) = (average diameter of control group colony - average diameter of experimental group colony) / average diameter of control group colony x 100. DPS statistical software was used for analysis, and a virulence regression equation was established to calculate the EC 50 of compound NSC-32 against Magnaporthe oryzae, and the virulence level of compound NSC-32 against Magnaporthe oryzae was evaluated.

[0088] Table 4 In vitro virulence of compound NSC-32 against Magnaporthe oryzae

[0089]

[0090] As shown in Table 4, the NSC-32 treatment group had different degrees of inhibition on the growth of Magnaporthe oryzae mycelium at 5 concentration gradients. Figure 4 The average diameter of the colony gradually decreased ( Figure 5 ) as the concentration of compound NSC-32 increased from 10 ppm to 50 ppm; correspondingly, the mycelial growth inhibition rate gradually increased ( Figure 6 ). Further, within the concentration range of 10 ppm to 50 ppm, the inhibition rate of compound NSC-32 against Magnaporthe oryzae was 4.5%~72%; more specifically, when the concentration of compound NSC-32 was 50 ppm, the inhibition effect on Magnaporthe oryzae was the most significant, with a relative inhibition rate of 71.77%. As can be seen from Table 4, the R 2 of the virulence regression equation was 0.9848, close to 1, indicating that the experimental data was accurate and reliable; the EC 50 of compound NSC-32 against Magnaporthe oryzae mycelial growth was 36.64394 ppm. In summary, the preferred concentration of NSC-32 is 10~50 ppm; more preferably 50 ppm.

[0091] In addition to Magnaporthe oryzae, this embodiment also detected the toxic effects of compound NSC-32 on other plant pathogens (Botrytis cinerea, Fusarium fujikuroi, Guazatine, Alternaria alternata, Fusarium oxysporum, and Colletotrichum acutatum), and the experimental steps were the same as described above. The strains were from Table 1, and the experimental results are shown in Figure 7~Figure 8 .

[0092] From the results, it can be seen that the compounds NSC-32 can inhibit the mycelial growth of the above-mentioned 6 different genera of plant pathogens to different degrees; in terms of the 7 kinds of pathogens, the inhibition effect from strong to weak is: Magnaporthe oryzae > Alternaria alternata > Colletotrichum orbiculare. These results prove the broad-spectrum bacteriostatic effect of NSC-32 molecules, which may be because NSC-32 molecules specifically target Pex19 protein, and the Pex19 protein in different pathogens has high homology, so NSC-32 molecules can bind to Pex19 homologous proteins in different pathogens, and then achieve significant bacteriostatic effect.

[0093] In order to verify the above guess, the Pex19 homologous protein sequences (SEQ ID NO: 2~ SEQ ID NO: 8) in the above-mentioned 7 pathogens were compared in this embodiment, and it was found that the homology of Pex19 protein in different pathogens was more than 50%, and the 3' end region was highly conserved, and this region was the interval combined with NSC-32 molecules; further, the Pex19 homologous proteins all contain the "GLN-273" site (SEQ ID NO: 1) described in embodiment 2. Figure 9 Therefore, combined with the bacteriostatic experiment ( Figure 4~Figure 8 ) and the results of homologous protein sequence comparison ( Figure 9 ), it can be reasonably inferred that NSC-32 molecules can inhibit plant pathogens that can express Pex19 homologous proteins, which refers to proteins with 50% and above homology with MoPex19 protein (SEQ ID NO: 2), more preferably containing "GLN-273" binding site.

[0094] Example 4: Pathogenicity determination and results of compound NSC-32 on Magnaporthe oryzae

[0095] Example 3 proves that the effect of NSC-32 molecules on inhibiting Magnaporthe oryzae is the most obvious, so in the follow-up, Magnaporthe oryzae is taken as the research object to explore the function, use method and safety of compound NSC-32. In this embodiment, the pathogenicity of compound NSC-32 is determined by ex vivo inoculation method, and the specific experimental steps are as follows:

[0096] 4.1 Ex vivo barley inoculation

[0097] Rice strain Guy 11 was inoculated on a 7 cm diameter plate containing 15 mL of CM solid medium, and incubated in an incubator for 7 days (28°C, 16 h light / 8 h dark); under sterile conditions, 3-4 mL sterile water was added to the surface of the Guy 11 plate, and spores were scraped off with a sterile spreader, filtered through 3 layers of sterile filter paper, and collected in a 1.5 mL centrifuge tube. Compound NSC-32 was mixed with the spore solution to prepare spore solutions with different concentrations (0, 10, 20, 30, 40, 50 ppm) of the compound, and the final concentration of the spore solution was 5 x 10 4 spores / mL. The dose difference of compound NSC-32 in different treatment groups was supplemented with DMSO to make the final concentration of DMSO consistent. Two layers of absorbent paper and one filter paper were placed in a 10 cm x 10 cm square dish, and the paper was wetted with an appropriate amount of water. 5-7 cm long barley (ZJ-8) leaves that had grown for 8-10 days were placed in the square dish in order, and 3 drops (about 20 μL) of spore solution were applied to each leaf, with 3 replicates for each treatment. The control group contained the same concentration of DMSO. The square dish containing the leaves was carefully placed in a 25°C incubator (16 h light: 8 h dark), and the leaves were observed for disease symptoms and photographed after 4 days of incubation.

[0098] From Figure 10 It can be seen that when the concentration of compound NSC-32 is in the range of 10-50 ppm, the size of the lesions on the detached barley leaves infected by the rice blast fungus gradually decreases as the concentration increases, indicating that the inhibitory effect of compound NSC-32 on the rice blast fungus also increases with increasing concentration. When the concentration is 30-50 ppm, the inhibitory effect on the detached barley leaves is similar, i.e., the optimal concentration of NSC-32 is 30 ppm, and further increasing the concentration of the compound cannot achieve better antibacterial effect.

[0099] 4.2 In vitro rice inoculation: The healthy second leaf of a three-leaf stage rice seedling (variety Oryza sativa CO39 obtained from the China Rice Institute) was cut and treated with the medicated spore solution using the same procedure as described in "4.1 In vitro barley inoculation".

[0100] As Figure 11As shown, compound NSC-32 has inhibitory effect on rice blast in vitro at a concentration range of 10 ~ 50 ppm, and to some extent, the lesion area decreases with the increase of concentration. More specifically, when the concentration of compound NSC-32 is increased to 20 ppm, the bacteriostatic effect is optimal, i.e. the in vitro rice leaves are basically not diseased; when the concentration of the compound is > 20 ppm, the inhibition rate of the compound on in vitro rice leaves is equivalent to that at a concentration of 20 ppm, so 20 ppm is preferred.

[0101] In summary, when the control object is barley, the use concentration of compound NSC-32 is preferably 30 ~ 50 ppm, more preferably 30 ppm; when the control object is rice, the use concentration of compound NSC-32 is preferably 20 ~ 50 ppm, more preferably 20 ppm.

[0102] On the other hand, considering the above results as a whole, Figure 10 and Figure 11 At the same concentration, the control effect of compound NSC-32 on rice blast in vitro is significantly better than that on barley in vitro; and compared with in vitro barley, a lower concentration of compound NSC-32 can achieve a better control effect on rice blast in vitro. The better effect of compound NSC-32 on rice may not be due to the difference in the inhibitory effect of the compound itself on the pathogen, but due to the excellent "synergistic effect" of the compound with the strong basic disease resistance of rice. Specifically, as a natural host of rice blast fungus, rice has a highly vigilant immune system, i.e. once the pathogen is recognized, a large amount of reactive oxygen species (ROS) will be produced, the disease resistance genes will be activated, and the plant will synthesize antifungal substances such as plant hormones; barley is not a natural host of rice blast fungus, and its specific defense response to rice blast fungus may not be strong and rapid enough.

[0103] In addition, different application times of compound NSC-32 may affect the control effect on rice blast in vivo, so in order to explore the optimal application time of compound NSC-32, this example improves the in vivo rice leaf spray inoculation scheme.

[0104] The experimental procedure of the non-optimized live rice leaf spray inoculation method is described as follows: rice seeds (Oryza sativa CO39) were placed in a humidity box and incubated at 37°C for 48 h; then they were sown in culture pots (30 seeds per pot) and incubated outdoors for 14 d under sufficient sunlight; Magnaporthe oryzae strain Guy11 was inoculated on a 9 cm diameter CM medium plate and incubated at 25°C in a light-dark cycle of 16 h:8 h for 7 d, and spore solution was obtained according to the above procedure; compound NSC-32, spore solution, and gelatin solution (to allow spores to adhere to the leaves) were mixed to prepare a spore solution containing the compound and gelatin (the final concentration of compound NSC-32 was 50 ppm, the final concentration of gelatin was 0.2%, and the final concentration of spores was 5 x 10 4 spores / mL), and the dose difference of compound NSC-32 was made up with DMSO to make the final concentration of DMSO consistent; 2 mL of the mixed spore solution containing the compound and gelatin was evenly sprayed on the rice leaves, and 3 pots of rice were inoculated in each treatment group; spore solution containing the same concentration of DMSO and gelatin, and gelatin solution containing 50 ppm of compound NSC-32 but no spores were sprayed as toxicity test controls; the inoculated live rice was incubated in a 22°C incubator in the dark for 48 h, and then incubated at 25°C in a light-dark cycle of 16 h / 8 h for 3-4 d; the incidence of disease on the rice leaves was observed and photographed, and the lesion area was calculated using Image J software, and the lesion rate was calculated as follows: lesion rate (%) = lesion area / leaf area x 100.

[0105] The specific optimization experiment procedure is as follows: the concentration of compound NSC-32 for inhibiting the incidence of rice blast was selected as 50 ppm, and 50 ppm of compound NSC-32 was applied at different time points of inoculation of Magnaporthe oryzae spore solution, and three groups of experiments were set up: (1) -24 h: compound NSC-32 was sprayed 24 h before inoculation of Magnaporthe oryzae spore solution; (2) 0 h: compound NSC-32 was applied at the same time as inoculation of Magnaporthe oryzae spore solution, i.e., compound NSC-32 was mixed with Magnaporthe oryzae spore solution and sprayed for inoculation; (3) +24 h: compound NSC-32 was sprayed 24 h after inoculation of Magnaporthe oryzae spore solution. Three pots of rice were used as replicates for each treatment, and blank and DMSO solvent controls were set up.

[0106] On the one hand, the rice in the control group sprayed with only 50 ppm of compound NSC-32 and gelatin solution grew well and had no obvious adverse effects, indicating that the compound did not affect the growth of rice and was relatively safe (P < 0.05). Figure 12 On the other hand, compared with the control group inoculated with spore solution and DMSO, the lesion area of rice was significantly reduced whether compound NSC-32 was sprayed at -24 h, 0 h, or +24 h of pathogen inoculation (P < 0.05).Figure 12 ~ Figure 13 ), indicating that the compound NSC-32 has a practical prevention and control effect on rice blast, that is, the NSC-32 can not only prevent the occurrence of rice blast, but also significantly reduce the adverse effects of Magnaporthe oryzae on plants after the rice is infected. Further, spraying the compound NSC-32 at the same time of inoculating the pathogen can maximize the efficacy of the NSC-32 and most reduce the lesion area on the rice leaves, which may be because the compound NSC-32 can quickly act on the pathogen at the key period of Magnaporthe oryzae invasion, thereby most inhibiting the invasion of the mycelium and blocking the earliest link of disease occurrence, realizing effective interference with the life cycle of the pathogen; the effect of treating the plants with the compound NSC-32 24 hours after inoculating the pathogen is the second. In summary, the compound NSC-32 can achieve an outstanding effect of preventing and controlling the pathogen within 24 hours before and after the inoculation of Magnaporthe oryzae. In order to achieve the best prevention and control effect, it is preferred to apply the NSC-32 within 24 hours after the plant is infected with Magnaporthe oryzae. In addition, these experimental results also fully prove that the compound has practical significance in preventing and controlling rice blast.

[0107] It can be understood that the NSC-32 can inhibit the pathogenicity of Magnaporthe oryzae and the plant disease (rice blast) caused thereby; similarly, the NSC-32 can also inhibit the pathogenicity of Botryosphaeria dothidea, Fusarium fujikuroi, Guignardia bidwellii, Alternaria alternata, Fusarium oxysporum, and Colletotrichum acutatum and the related diseases caused thereby (see Table 1 for details).

[0108] Example 5: Safety of the compound NSC-32

[0109] The compound NSC-32 is screened based on the target MoPex19 protein, but it is not yet clear whether it will affect the normal growth and development of rice, so this embodiment explores it.

[0110] Firstly, this embodiment compares the sequences of the MoPex19 protein of Magnaporthe oryzae and the OsPex19 protein in rice, and finds that the homology of the overall sequences of the two proteins is low. Specifically, the sequence alignment shows that only about one-third of the MoPex19 protein sequence can be clearly aligned with the corresponding region of the OsPex19 protein, which indicates that there is a large difference between the two in sequence length and domain composition; in other words, the homology of the MoPex19 protein and the OsPex19 protein is less than 30%; and in the about one-third of the conserved matching region, the sequence identity is only 41.7%, so it is preliminarily speculated that the binding force between the compound NSC-32 and the OsPex19 protein is weak or cannot be combined, and then the compound NSC-32 has little effect on the growth of rice or does not affect its growth.

[0111] Further, the present embodiment carries out experimental verification on the influence of NSC-32 on the growth of rice plants and seed germination, and the rice leaf spraying inoculation method is the same as above, and the specific optimization experiment steps are as follows: 25, 50 and 100 ppm of three different concentrations of NSC-32 compound are inoculated on the rice plants after 14 days of cultivation, wherein the inoculation volume is 2 mL, and the inoculation site is the rice leaf; after 14 days, the growth of the rice is observed. At the same time, the rice seeds are treated with three different concentrations of NSC-32 compound, and the treated germinated rice seeds are planted, and the germination rate of the rice seeds and the growth of the rice seeds after planting are observed. Each treatment uses 3 pots of rice as a repeat, and blank control (ddH2O) and DMSO solvent control are set.

[0112] As shown in Figure 14 and Figure 15 , the compound NSC-32 has no obvious difference compared with the control on the growth of rice, the germination rate of rice seeds and the growth of rice seedlings after treatment, and does not show obvious negative effects on rice, which shows that NSC-32 is safe for rice while effectively preventing rice blast.

[0113] On the other hand, the alignment results of protein sequences and the safety test results further prove that the toxic effect of NSC-32 depends largely on the homology of Pex19 protein in the application object and MoPex19 protein. When the homology of Pex protein in the application object and MoPex19 protein is higher than 50%, NSC-32 can inhibit the growth, development, pathogenicity, etc. of the application object; and when the homology is lower than 30%, NSC-32 has little or no effect on the physiological process of the application object.

[0114] The present application also carries out toxicity determination, pathogenicity determination and safety test of the other four compounds (NFD-22, BTH-75, BMD-45, DDP-26) as described in embodiment 2, and the specific steps are described in embodiments 3-5. Due to the length of the paper, the results are not shown one by one. The overall effect of the four compounds is not as good as NSC-32, so NSC-32 is preferred.

[0115] Embodiment 6: Exploration of the mechanism of compound NSC-32

[0116] The above embodiments fully prove that the compound NSC-32 can inhibit the growth of rice blast fungus and reduce the area of rice blast fungus lesion on the plant leaf, but the mechanism of action of the compound NSC-32 is not clear, so this embodiment carries out the exploration.

[0117] Firstly, the present embodiment determines the formation rate of Magnaporthe oryzae, and the specific experimental steps are as follows: Magnaporthe oryzae Guy11 is inoculated on a CM plate with a diameter of 7 cm; after being cultured at 28°C under light and dark alternation for 16 h / 8 h for 7 days, 3 mL of sterile water is added to the plate, and the conidia are scraped and washed with a disposable sterilized coating rod, filtered through 3 layers of filter paper to obtain a spore solution, and the spore solution is diluted to a final concentration of 5 x 10 4 spores / mL spore solution; the compound NSC-32 and the spore solution are mixed to prepare a drug-containing spore solution (the final concentration of spores is 5 x 10 4 spores / mL, and the final concentration of the compound is 50 ppm), the dose difference of the compound NSC-32 is supplemented with DMSO to make the final concentration of DMSO consistent, and the same concentration of DMSO is used as a control; the artificial hydrophobic membrane is soaked in 75% ethanol for 5 min and wiped clean with filter paper, a rectangular glass slide is prepared, the artificial hydrophobic membrane is placed on the glass slide (3 membranes per slide), 20 μL of the drug-containing spore solution is added to each hydrophobic membrane, and the glass slide is placed in a humidification box (with appropriate amount of sterile water added), and then the glass slide is placed in the dark at 22°C for induction. The formation of Magnaporthe oryzae appressorium at different time points (4 h, 8 h, 16 h and 24 h) is recorded by microscope photography, and the number of Magnaporthe oryzae appressorium formed at each time point is counted and the formation rate is calculated. The formation rate (%) = the number of Magnaporthe oryzae appressorium formed / the total number of Magnaporthe oryzae spores x 100%; each group of experiments is repeated 3 times.

[0118] From Figure 16~Figure 17 it can be seen that the compound NSC-32 has no significant inhibitory effect on the formation of Magnaporthe oryzae appressorium at the four different time points set in the experiment, indicating that the compound NSC-32 does not affect the formation of Magnaporthe oryzae appressorium.

[0119] Then, the present embodiment also determines the turgor pressure of Magnaporthe oryzae appressorium, and the specific experimental steps are as follows: the process of inducing appressorium is consistent with that described in the appressorium formation determination experiment; glycerol solutions with concentrations of 1 M and 2 M are prepared, after 24 h of induction, the hydrophobic membrane is taken out, the surface water is gently absorbed with a pipette or water-absorbing paper, 20 μL of the glycerol solution is quickly added, and after 5 min of room temperature standing, the number of collapsed appressorium is quickly counted, and the proportion of collapsed appressorium in the total appressorium is calculated. Each strain is repeated 3 times under each glycerol concentration.

[0120] The experimental results show that Figure 18 and Figure 19 under the same glycerol concentration, the collapse rate of Magnaporthe oryzae appressorium treated with the compound NSC-32 has no significant difference compared with the control, indicating that the compound NSC-32 (concentration of 50 ppm) has no significant effect on the turgor pressure of appressorium.

[0121] Further, the present embodiment detects peroxisome autophagy of Magnaporthe oryzae, and the specific experimental steps are as follows: the peroxisome autophagy marker protein GFP-MoPex14 is transferred into wild type Guy11 to obtain Guy11-GFP-MoPex14 strain, the mycelium of the strain is crushed and placed in 180 mL liquid CM medium, and then transferred to a 25°C, 150 rpm / min shaker for 2 days. 1 / 3 of the mycelium in the liquid is filtered with 3 layers of filter paper on a clean bench, frozen with liquid nitrogen and temporarily stored at -80°C, and the remaining 2 / 3 of the mycelium is washed with sterilized ddH2O for 2-3 times, and then slightly pressed dry with a water absorption paper. The mycelium block is divided into two parts, one part is transferred to 60 mL of CM medium added with NSC-32 compound (final concentration of 50 ppm), and the other part is transferred to 60 mL of CM medium added with DMSO (2%). After 4 hours of continuous culture, the mycelium is collected, and the total protein of the three parts of mycelium is extracted, and the degradation of GFP-MoPex14 is detected by Western blotting.

[0122] The experimental results show that (Fig. 3) Figure 20 ), compared with the untreated and the same concentration of DMSO treatment, the GFP-MoPex14 band is weaker after NSC-32 compound treatment, and the free GFP band is stronger, indicating that the degradation rate of GFP-MoPex14 after NSC-32 compound treatment is significantly faster than that of the control group, which means that after NSC-32 compound treatment, the peroxisome autophagy in Magnaporthe oryzae is enhanced, which in turn leads to the weakening of the pathogenicity of Magnaporthe oryzae.

[0123] In summary of all the embodiments, the present application takes MoPex19 protein as a target, and screens the compound NSC-32, and proves that the compound can inhibit the growth of Magnaporthe oryzae and reduce the pathogenicity of the latter. In addition, the present application also explores the applicable object, use method (including use concentration and time point of applying reagent), safety and mechanism of action of the compound NSC-32, and provides a new green pesticide and new idea for preventing and controlling Magnaporthe oryzae.

[0124] The above shows and describes the basic principles and main features of the present application and the advantages of the present application, and it is obvious for those skilled in the art that the present application is not limited to the details of the above exemplary embodiments, and the present application can be realized in other specific forms without departing from the spirit or basic characteristics of the present application; therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting, the scope of the present application is defined by the appended claims rather than the above description, and therefore all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present application, and any figure reference in the claims should not be regarded as limiting the involved claims.

Claims

1. Use of the compound NSC-32 for the preparation of an agent for the control of phytopathogenic fungi, characterized in that, The structure of the compound NSC-32 is , and the plant pathogenic fungus is any one or more of Magnaporthe grisea, Botrytis cinerea, Fusarium fujikuroi, Guignardia bidwellii, Alternaria alternata, Fusarium oxysporum, and Guignardia bidwellii.

2. Use according to claim 1, wherein The plant pathogenic fungus is capable of expressing a protein represented by SEQ ID NO:

2.

3. Use of the compound NSC-32 for the preparation of an agent for inhibiting hyphal growth of a phytopathogenic fungus and / or for inhibiting spore germination of a phytopathogenic fungus and / or for inhibiting the formation of lesions of a phytopathogenic fungus and / or for reducing the pathogenicity of a phytopathogenic fungus, characterized in that The structure of the compound NSC-32 is , and the plant pathogenic fungus is any one or more of Magnaporthe grisea, Botrytis cinerea, Fusarium fujikuroi, Guignardia bidwellii, Alternaria alternata, Fusarium oxysporum, and Erysiphe cichoracearum.

4. Use of the compound NSC-32 for the preparation of a reagent targeting the Pex19 protein, characterized in that, The structure of the compound NSC-32 is ; the Pex19 protein has a sequence shown in any one of SEQ ID NO: 2 ~ SEQ ID NO:

8.

5. A method for controlling a plant pathogenic fungus and / or a disease caused by a plant pathogenic fungus, characterized by, applying a compound NSC-32; the structure of the compound NSC-32 is , the plant pathogenic fungi being any one or more of Magnaporthe grisea, Botrytis cinerea, Fusarium fujikuroi, Guignardia bidwellii, Alternaria alternata, Fusarium oxysporum, and Erysiphe necator.

6. The method of claim 5, wherein, The concentration of the compound NSC-32 is 10 ~ 50 ppm.

7. The method of claim 5, wherein, When the plant pathogenic fungus is Magnaporthe grisea, the compound NSC-32 is applied to the plant or Magnaporthe grisea.

8. The method of claim 7, wherein, The compound NSC-32 is applied within 24 hours before and after the plant is infected by Magnaporthe grisea.

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