Application of Rice Leaf Extract in Inhibiting Phytopathogens

Abstract

The present invention discloses the application of rice leaf extract in inhibiting plant pathogens. The present invention provides the application of rice leaf extract in any one of the following: antibacterial; preparing an antibacterial agent. The rice leaf extract provided by the present invention has inhibitory effects on both fungi and bacteria, especially has a wide range of effects on fungi. The present invention is of great significance for preventing and controlling corresponding pathogens in agriculture.

Description

Technical Field

[0001] The present invention relates to the field of agriculture, and particularly to the application of rice leaf extract in inhibiting plant pathogens. Background Art

[0002] Flavonoids are a class of natural metabolites widely distributed in plants, with a C6-C3-C6 basic skeleton. According to the degree of oxidation of the central three-carbon chain, whether it forms a ring, and the position of the B-ring connection (2- or 3-position), etc., the main natural flavonoids can be divided into flavonoids, flavonols, dihydroflavonoids, dihydroflavonols, isoflavonoids, anthocyanins, etc. Most flavonoids are combined with sugars to form glycosides in plants, and some exist in the free form (aglycone). Flavonoids have very strong anti-inflammatory and anti-cancer effects and have great application prospects in the treatment of cardiovascular diseases, tumor diseases, etc.

[0003] Rice leaves contain a large number of secondary metabolites, including flavonoids. These compounds play important roles in rice's resistance to ultraviolet rays and diseases (Peng et al., 2017; Zhan et al., 2020). However, currently, it is not very clear whether rice leaf metabolites can inhibit bacteria or fungi, especially the growth of plant pathogens. Summary of the Invention

[0004] The purpose of the present invention is to provide a rice leaf extract and its application in inhibiting plant pathogens.

[0005] In the first aspect, the present invention claims the application of the rice leaf extract in any one of the following:

[0006] P1. Bacteriostasis;

[0007] P2. Preparation of a bacteriostatic agent.

[0008] In the second aspect, the present invention claims a bacteriostatic agent.

[0009] The bacteriostatic agent claimed by the present invention contains the rice leaf extract.

[0010] In the first and second aspects, the rice leaf extract is an ethanol extract of rice leaves.

[0011] Furthermore, the rice leaf extract is an extract obtained by extracting rice leaves with 75% (volume percentage) ethanol.

[0012] Among them, the rice leaves can be fresh rice leaves.

[0013] Further, the rice leaf extract can be prepared by a method including the following steps: adding rice leaves in a ratio of 1 Kg (fresh weight) to 1 L to 75% (volume percentage) ethanol, soaking for 30 min, extracting twice continuously, and obtaining the rice leaf extract from the soaked liquid.

[0014] Further, after soaking, there is also a step of rotary evaporation and concentration.

[0015] Further, the mass ratio of tricin-5-O-glucoside to isoorientin in the rice leaf extract can be (1 - 1.5):1, such as 1.35:1.

[0016] In a specific embodiment of the present invention, the total concentration of the target substances (tricin-5-O-glucoside and isoorientin) in the stock solution of the rice leaf extract obtained after rotary evaporation and concentration reaches about 2000 mg / L, such as 2040 mg / L (wherein, the concentration of tricin-5-O-glucoside is 1173 mg / L, and the concentration of isoorientin is 867 mg / L).

[0017] In a third aspect, the present invention claims to protect an antibacterial method.

[0018] The antibacterial method claimed by the present invention may include the following steps: applying the aforementioned rice leaf extract to the target bacteria; wherein the total working concentration of the target substances (tricin-5-O-glucoside and isoorientin) in the rice leaf extract is 144 μg / mL or more (such as 144 - 500 μg / mL, or 200 - 400 μg / mL or 288 - 500 μg / mL).

[0019] In the first aspect to the third aspect, the rice is indica rice.

[0020] Further, the indica rice can be any variety of indica rice such as Montakcl, J.P.5, PD 46, PATNAI 6, Shui Ya Jien, and / or YOU-I B, etc.

[0021] In a specific embodiment of the present invention, the rice leaves are obtained by mixing the leaves of Montakcl, J.P.5, PD 46, PATNAI6, Shui Ya Jien, and YOU-I B in equal mass.

[0022] In the first aspect to the third aspect, the bacteria are plant pathogenic bacteria.

[0023] Among them, the plant pathogenic bacteria can be plant pathogenic fungi or plant pathogenic bacteria.

[0024] Further, when the bacterium is a plant pathogenic fungus, the total working concentration of the target substances (tricin-5-O-glucoside and isoorientin) in the rice leaf extract in the third aspect is preferably 200-400 μg / mL. When the bacterium is a plant pathogenic bacterium, the total working concentration of the target substances (tricin-5-O-glucoside and isoorientin) in the rice leaf extract in the third aspect is preferably 288-500 μg / mL.

[0025] In a specific embodiment of the present invention, the plant pathogenic fungus is specifically selected from: Alternaria solani Sorauer, Fusarium graminearum, Phytophthora capsici Leonian, Colletotrichum fragariae, Rhizoctonia solani, Fusarium oxysporum, Fusarium graminearum, Fusarium verticillioide, Cercospora arachidicola, Sclerotinia sclerotiorum and / or Magnaporthe oryzae.

[0026] In a specific embodiment of the present invention, the plant pathogenic bacterium is specifically selected from: Xanthomonas oryzae and / or Pseudomonas syringae.

[0027] Fourthly, the present invention claims the application of a mixture composed of tricin-5-O-glucoside and isoorientin in any one of the following:

[0028] P1, inhibiting bacteria;

[0029] P2, preparing a bacteriostatic agent.

[0030] Among them, in the mixture, the mass ratio of tricin-5-O-glucoside to isoorientin can be (1-1.5):1. Such as 1.35:1.

[0031] In the application, dissolve the mixture composed of tricin-5-O-glucoside and isoorientin in ethanol (such as 75% ethanol) so that the total working concentration of tricin-5-O-glucoside and isoorientin is above 144 μg / mL (such as 144 - 500 μg / mL, or 200 - 400 μg / mL or 288 - 500 μg / mL).

[0032] In a fifth aspect, the present invention claims protection for an antibacterial agent, and its active ingredients include tricin-5-O-glucoside and isoorientin.

[0033] Among them, the mass ratio of tricin-5-O-glucoside to isoorientin can be (1 - 1.5):1. Such as 1.35:1.

[0034] In the fourth and fifth aspects, the bacteria are plant pathogenic bacteria.

[0035] Among them, the plant pathogenic bacteria can be plant pathogenic fungi or plant pathogenic bacteria.

[0036] In a specific embodiment of the present invention, the plant pathogenic fungi are specifically selected from: Alternaria solani Sorauer, Fusarium graminearum, Phytophthora capsici Leonian, Colletotrichum fragariae, Rhizoctonia solani, Fusarium oxysporum, Fusarium graminearum, Fusarium verticillioide, Cercospora arachidicola, Sclerotinia sclerotiorum and / or Magnaporthe oryzae.

[0037] In a specific embodiment of the present invention, the plant pathogenic bacteria are specifically selected from: Xanthomonas oryzae and / or Pseudomonas syringae.

[0038] In the present invention, the antibacterial effect can also be understood as controlling the corresponding plant pathogenic bacteria in agriculture.

[0039] Experiments have proved that the rice leaf extract provided by the present invention has antibacterial effects, and can inhibit both plant pathogenic fungi and plant pathogenic bacteria, especially having a wide range of effects on plant pathogenic fungi. The present invention is of great significance for preventing and controlling corresponding plant pathogenic bacteria in agriculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is the inhibitory effect on the pathogen of tomato early blight.

[0041] Figure 2 It is the inhibitory effect on the pathogen of wheat head blight.

[0042] Figure 3 It is the inhibitory effect on the pathogen of rice blast.

[0043] Figure 4 It is the inhibitory effect on the pathogen of pepper Phytophthora blight.

[0044] Figure 5 It is the inhibitory effect on the pathogen of rapeseed Sclerotinia sclerotiorum.

[0045] Figure 6 It is the inhibitory effect on the pathogen of rice sheath blight.

[0046] Figure 7 It is the inhibitory effect on Fusarium graminearum.

[0047] Figure 8 It is the inhibitory effect on Fusarium verticillioides.

[0048] Figure 9 It is the inhibitory effect on the pathogen of strawberry anthracnose.

[0049] Figure 10 It is the inhibitory effect on the pathogen of castor wilt.

[0050] Figure 11 It is the inhibitory effect on the pathogen of peanut brown spot. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0051] The present invention will be further described in detail below in conjunction with the specific embodiments. The embodiments given are only for clarifying the present invention, rather than limiting the scope of the present invention. The following embodiments provided can be used as a guide for those of ordinary skill in the art to make further improvements, and do not constitute any limitation to the present invention in any way.

[0052] Unless otherwise specified, the experimental methods in the following embodiments are all conventional methods, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials, reagents, etc. used in the following embodiments can all be obtained from commercial channels.

[0053] Example 1: Preparation of Rice Leaf Extract

[0054] 1. Collect fresh leaves of rice (indica rice) about 60 days after sowing, specifically, fresh leaves of indica rice varieties Montakcl, J.P.5, PD 46, PATNAI 6, Shui Ya Jien, and YOU-I B are mixed in equal mass (these indica rice varieties are all recorded in "Supplemental Table 1" of the article "Wang et al., The power of inbreeding: NGS based GWAS of rice reveals convergent evolution during rice domestication. 2016 Molecular Plant 9:975", which is available to the public from the applicant and can only be used for repeating the experiments of the present invention and shall not be used for other purposes). Then, according to the ratio of adding 1 L of 75% ethanol (volume percentage, with the other 25% being water) to 1 Kg of fresh leaves, soak the rice leaves in 75% ethanol for 30 min. Then, collect the soaking solution to obtain the rice leaf extract, and add an appropriate amount of 75% ethanol to the leaves again and repeat the extraction once.

[0055] 2. Collect all the obtained leaf extracts (the liquid after soaking), and then concentrate them using a rotary evaporator. During this process, continuously detect the concentration of the concentrate until the total concentration of the target substances (i.e., tricin-5-O-glucopyranoside and isoorientin) in the rice leaf extract reaches approximately 2000 mg / L. The specific detection method is as follows: Pipette 200 μL of the concentrate of the rice leaf extract into a 1.5 mL Eppendorf centrifuge tube, centrifuge at 4°C and 12,000 rpm for 15 min. Pipette 100 μL of the supernatant into an Agilent injection vial containing a liner with a volume of 200 μL. Then detect it using UPLC-MS. Mobile phase A: 0.1% formic acid aqueous solution (% represents volume percentage); Mobile phase B: acetonitrile. Elution gradient: 0 - 2 min: 5% B - 10% B, 2 - 12 min: 10% B - 25% B, 12 - 18 min: 25% B - 70% B, 18 - 23 min: 70% B - 90% B, 23 - 25 min: 90% B - 100% B, 25 - 30 min: 100% B, post-run for 5 min, % all represent volume percentages. Flow rate: 0.3 mL / min, column temperature: 40°C, injection volume: 5 μL. Use an electrospray ionization source (ESI), detect in positive ion mode, the carrier gas is high-purity nitrogen, pressure 40 psi, temperature 325°C. Finally, using the standard curve of the target substances (tricin-5-O-glucopyranoside and isoorientin) drawn, convert the peak area of the target substances in the obtained mass spectrum into the absolute content of the target substances in the concentrate. After detection, the total concentration of the target substances tricin-5-O-glucopyranoside and isoorientin in the concentrate is 2040 mg / L (the concentration of tricin-5-O-glucopyranoside is 1173 mg / L, and the concentration of isoorientin is 867 mg / L). Use the obtained rice leaf extract as the stock solution of the biological pesticide (WSE) for subsequent experiments.

[0056] The structural formula of tricin-5-O-glucopyranoside is as follows:

[0057]

[0058] The structural formula of isoorientin is as follows:

[0059]

[0060] Example 2. Verification of the antibacterial effect of biological pesticide WSE

[0061] I. Verification of the antibacterial effect against pathogenic fungi

[0062] 1. Experimental materials

[0063] Test targets: Alternaria solani Sorauer, Fusarium graminearum, Phytophthora capsici Leonian, Colletotrichum fragariae, Rhizoctonia solani, Fusarium oxysporum, Fusarium graminearum, Fusarium verticillioide, Cercospora arachidicola, Sclerotinia sclerotiorum, Magnaporthe oryzae.

[0064] Test agents: Different dilution solutions (dilution solution is water) of the biological pesticide WSE prepared in Example 1.

[0065] Instruments and equipment: Ultra-clean workbench, constant temperature incubator, sterile room, electronic balance.

[0066] 2. Experimental methods

[0067] (1) Concentration design

[0068] The following concentration gradients were set for the test agents: 400, 200, 100, 50, 25, 12.5 μg / mL, with 4 replicates for each treatment. Here, the concentration is the total concentration of the target substances (tricin-5-O-glucoside and isoorientin).

[0069] (2) Culture conditions

[0070] The culture medium was PDA medium, and the conditions of the incubator and the room were set as: temperature 25 - 26 °C.

[0071] (3) Treatment methods and investigation methods for pathogenic fungi tests

[0072] The biological pesticide WSE was formulated into a certain concentration (μg / mL), then a certain amount of PDA medium was added, shaken well, and cooled. The final liquid medicine concentration was 400, 200, 100, 50, 25, 12.5 μg / mL (here, the concentration is the total concentration of the target substances tricin-5-O-glucoside and isoorientin). After punching out circular bacterial cakes with a puncher, they were picked to the center of the culture dish with an inoculation needle, and then the culture dish was placed in sunlight during the day and cultured in an incubator at 25 °C at night.

[0073] Measure the colony diameter 3 days after the experimental treatment, and calculate the growth inhibition rate (%).

[0074] D = D 1 −D 2 (1)

[0075] In formula (1): D represents the increased diameter of the colony; D 1 represents the colony diameter; D 2 represents the diameter of the mycelial disc.

[0076]

[0077] In formula (2): I represents the mycelial growth inhibition rate; D 0 represents the increased diameter of the colony in the blank control (PDA medium without the tested WSE); D t represents the increased diameter of the colony treated with the WSE agent.

[0078] 3. Results and Analysis

[0079] The results are shown in Table 1 and Figures 1 to 11 as follows. The biopesticide WSE has excellent effects on 11 pathogenic fungi such as Alternaria solani and Gibberella zeae at concentrations of 400 and 200 μg / mL. Especially at a concentration of 400 μg / mL, for 11 pathogenic fungi, except for the inhibition rate of 96.9% against Fusarium graminearum, the other 11 are all 100%. At a concentration of 200 μg / mL, the inhibition rates against 5 pathogenic fungi such as Alternaria solani, Gibberella zeae, Phytophthora capsici, Sclerotinia sclerotiorum, and Rhizoctonia solani are all above 90%, and the bactericidal spectrum is extremely wide. However, the inhibition rate rapidly decreases at a concentration below 100 μg / mL. It is possible that this concentration is the control threshold of this biopesticide, and further experiments are needed for evaluation.

[0080] Table 1. Antibacterial activities of the biopesticide WSE against 12 pathogenic fungi

[0081]

[0082] II. Verification of the antibacterial effect against pathogenic bacteria

[0083] 1. Experimental materials

[0084] Test targets: Xanthomonas oryzae, Pseudomonas syringae.

[0085] Test agents: Different-fold dilution solutions (the dilution solution is water) of the biopesticide WSE prepared in Example 1.

[0086] Control agent: 12% Zhongshengmycin, Zhejiang Longwan Chemical Co., Ltd.

[0087] 2. Experimental Methods

[0088] (1) In vitro test: For the test agents against Xanthomonas oryzae, seven concentrations (final concentrations) of 500, 288, 144, 72, 36, 18, and 9 μg / mL were set, and for the control agents, seven concentrations (final concentrations) of 2, 1, 0.67, 0.44, 0.3, 0.2, and 0.1 μg / mL were set. For the test agents against Pseudomonas syringae, seven concentrations (final concentrations) of 500, 288, 144, 72, 36, 18, and 9 μg / mL were set, and for the control agents, eight concentrations (final concentrations) of 100, 50, 25, 12.5, 6.25, 3.125, 1.56, and 0.39 μg / mL were set. The concentration of the test agents here is the total concentration of the target substances (tricin-5-O-glucoside and isoorientin).

[0089] (2) According to the experimental design, a certain volume of the WSE stock solution was respectively pipetted into 27 mL of LB liquid medium to make the final concentration of WSE the concentration shown in (1). There were 5 replicates for each treatment. Adding an equal volume of sterile water to the LB liquid medium served as the blank control group. The formula for the LB liquid medium (1 L) is as follows: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, and 10 g of tryptone. After preparation, it needs to be autoclaved.

[0090] (3) For the prepared bacterial suspensions of Xanthomonas oryzae and Pseudomonas syringae, 0.1 mL of the bacterial suspension was added to the LB liquid medium with the test liquid in (2); before starting the culture, 3 mL was taken from one of the replicates in each treatment to measure its OD 600 value, which served as the agent control for each treatment, and it was cultured at 28 °C with constant shaking (180 rpm) for 24 h.

[0091] (4) After 24 h, the OD 600 absorbance value

[0092] (5) Calculate the control efficacy of WSE at different concentrations: The control efficacy of WSE was calculated using the following formula: I(%) = (OD 对照组 - OD 处理组 ) / OD 对照组 * 100%

[0093] 3. Experimental Results and Analysis

[0094] The results are shown in Table 2 and Table 3. It can be seen that: the test agents have good control effects against Xanthomonas oryzae pv. oryzae and Pseudomonas syringae pv. lachrymans at high concentrations. The biological pesticide WSE has an inhibitory effect of 100% on Xanthomonas oryzae pv. oryzae at 500 and 288 μg / mL, and 96.1% at 144 μg / mL. The biological pesticide WSE has an inhibitory effect of 100% on Pseudomonas syringae pv. lachrymans at 500 and 288 μg / mL, but only 25.6% at 144 μg / mL. This test is for the inhibitory effect on bacteria in vitro, and the specific actual effect requires further experiments in the field.

[0095] Table 2. Test results of the antibacterial activity of the biological pesticide WSE against Xanthomonas oryzae pv. oryzae

[0096]

[0097]

[0098] Table 3. Test results of the antibacterial activity of the biological pesticide WSE against Pseudomonas syringae pv. lachrymans

[0099]

[0100] The above has described the present invention in detail. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any changes, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application but are made by using conventional techniques known in the art. Some basic features can be applied within the scope of the following appended claims.

Claims

1. Use of rice leaf extract in any of the following: P1, antibacterial; P2, preparation of antibacterial agent; The rice leaf extract is prepared by a method comprising the following steps: adding rice leaves to 75% (v / v) ethanol at a ratio of 1 kg fresh weight to 1 L, soaking for 30 min, extracting twice continuously, and obtaining the rice leaf extract from the soaked liquid; after soaking, there is also a step of rotary evaporation and concentration; The rice is indica rice; the indica rice is a mass mixture of Montakcl, J.P. 5, PD 46, PATNAI 6, Shui Ya Jien, and YOU-I B, etc.; The bacteria are plant pathogens; The plant pathogens are plant pathogenic fungi or plant pathogenic bacteria; The plant pathogenic fungi are selected from: Alternaria solani, Gibberella zeae, Phytophthora capsici, Colletotrichum acutatum, Rhizoctonia solani, Fusarium oxysporum f. sp. ricini, Fusarium graminearum, Fusarium verticillioides, Cercospora arachidicola, Sclerotinia sclerotiorum, and / or Magnaporthe oryzae; The plant pathogenic bacteria are selected from: Xanthomonas oryzae pv. oryzae and / or Pseudomonas syringae pv. lachrymans.

2. The use according to claim 1, characterized in that: The mass ratio of tricin-5-O-glucoside to isoorientin in the rice leaf extract is (1 - 1.5):

1.

3. An antibacterial method, comprising the following steps: treating the target bacteria with the rice leaf extract described in claim 1 or 2; wherein the total working concentration of the target substances tricin-5-O-glucoside and isoorientin in the rice leaf extract is 144 μg / mL or more; The bacteria are plant pathogens; The plant pathogens are plant pathogenic fungi or plant pathogenic bacteria; The plant pathogenic fungi are selected from: Alternaria solani, Gibberella zeae, Phytophthora capsici, Colletotrichum acutatum, Rhizoctonia solani, Fusarium oxysporum f. sp. ricini, Fusarium graminearum, Fusarium verticillioides, Cercospora arachidicola, Sclerotinia sclerotiorum, and / or Magnaporthe oryzae; The plant pathogenic bacteria are selected from: Xanthomonas oryzae pv. oryzae and / or Pseudomonas syringae pv. lachrymans.

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