Compound M-1 with anti-phytopathogen activity and application thereof

Abstract

The invention relates to the technical field of natural medicinal chemistry, and discloses a compound M-1 with phytopathogen resisting activity and application thereof. The invention provides a novel compound and a fermentation liquor separated from a Ralstonia MWSTR-5 strain, and by measuring the inhibitory activity of the compound M-1 on fusarium oxysporum and alternaria solani, the compound M-1 is found to have relatively strong phytopathogen resistance activity and is a good natural bactericide.

Description

Technical Field

[0001] The present invention relates to the technical field of natural medicinal chemistry, and in particular to a compound M-1 having anti-plant pathogenic bacteria activity and an application thereof. Background Art

[0002] Plant diseases and insect pests are the main cause of global crop losses. At least 20-40% of these losses are caused by pathogenic infections, resulting in global losses of US$40 billion annually. Fusarium oxysporum belongs to the genus Fusarium and is a globally distributed soil-borne pathogenic fungus with a wide host range. This species can cause continuous cropping problems in agricultural and cash crops, and can also cause plant wilt and browning of vascular tissue. Rhizopus stolonifer is one of the pathogens that causes a variety of plant diseases. It belongs to the subdivision Synmycota and is a common root mold. It can cause a variety of stored grain molds, such as the initial white cotton-like colonies on grains and fruits, which later turn gray-black and emit a strong alcohol odor, and later produce blue-green mold. Rhizoctonia solani is one of the most important pathogens causing sheath blight in maize. Its symptoms are necrosis of the basal leaf sheaths and the formation of brown streaks. The disease is widespread in Southeast Asian countries and has also been reported in other parts of the world, resulting in yield losses of 11–40%, with some varieties experiencing up to 100% losses. Botrytis cinerea is the most extensively studied plant pathogen and the second most important plant pathogen worldwide, making it one of the most extensively studied necrotrophic fungal plant pathogens. The disease it causes, known as gray mold, affects the flowers, leaves, buds, seeds, and fruits of many crops worldwide. Sclerotinia sclerotiorum is a pathogen that causes multiple plant diseases, infecting a wide range of crops and severely impacting their yield and quality. Fusarium graminearum is a major threat to wheat, causing Fusarium foot rot (FFR) and Fusarium root rot (FRR), as well as Fusarium head blight (FHB), which are the main causes of wheat losses. Alternaria solani is a pathogen that causes a variety of plant diseases. It can infect a variety of crops such as potatoes, peppers, tobacco, and tomatoes, causing diseases such as early blight.

[0003] Currently, the main methods for controlling plant diseases include chemical pesticides and biological control. Chemical pesticides offer advantages such as high efficiency, rapid effectiveness, and ease of use, enabling rapid control of the spread of diseases. However, long-term use of chemical pesticides can lead to resistance in pests and diseases, causing plant damage and even poisoning in humans and animals, while also polluting the environment. Therefore, in line with social development, the development of new green, economical, environmentally friendly, and safe pesticide products is crucial to promote the development of modern pesticides, reduce losses from plant diseases, and ensure the safety of crop production.

[0004] Natural products have the characteristics of easy degradation, low toxicity, reduced drug resistance, health and safety, etc. They meet the requirements of green pesticides and are expected to replace chemical pesticides. They have become one of the research hotspots in the pesticide field. Summary of the Invention

[0005] The object of the present invention is to provide a compound M-1 having anti-plant pathogenic fungus activity and its application. The present application isolates an endophytic actinomycete from Pinus massonian, which is identified as a Ralstonia sp. strain by morphology and molecular biology. The activity of the strain against Fusarium oxysporum, Rhizopus stolonifer, Rhizoctonia solani, Botrytis cinerea, Sclerotinia sclerotiorum, Fusarium graminearum, and Alternaria solani was determined, and the results showed that the strain had good and broad-spectrum antibacterial activity. Further, monomeric compounds were separated from the fermentation extract of the strain to obtain compound M-1, and the activity of M-1 against Fusarium oxysporum and Alternaria solani was determined. The test results showed that compound M-1 has strong anti-Fusarium oxysporum and Alternaria solani activity and can be used as a fungicide.

[0006] In order to achieve the above-mentioned invention objectives, the technical solutions adopted in this application are:

[0007] In a first aspect, the present application provides a compound M-1 having anti-plant pathogenic fungus activity, the structural formula of the compound M-1 is as follows:

[0008]

[0009] In a second aspect, the present application provides a fermentation extract of a Ralstonia strain, wherein the fermentation extract contains the above-mentioned compound M-1.

[0010] In an optional embodiment, the fermentation extract is a fermentation extract of Ralstonia MWSTR-5 strain.

[0011] In a third aspect, the present application provides a pesticide comprising the Ralstonia MWSTR-5 strain or the above-mentioned compound M-1 or a fermentation extract.

[0012] In a fourth aspect, the present application provides a method for preparing the above-mentioned fermentation extract, comprising the following steps:

[0013] activating the Ralstonia strain and performing fermentation culture to obtain a fermentation product;

[0014] The liquid in the fermentation product is separated and extracted with ethyl acetate, and the ethyl acetate layer is collected and concentrated.

[0015] The activation method and conditions can be selected within a certain range, and any activation method that can achieve the purpose of activating the strain can be used in this application. In a particularly preferred embodiment, the activation can be specifically: the frozen strain is inoculated onto a plate of Gao's No. 1 solid medium under a sterile environment and cultured in a constant temperature incubator at 28°C for 7 days.

[0016] The fermentation method and conditions can be selected within a certain range, and any fermentation method that can produce an effective fermentation product can be used in this application. In a particularly preferred embodiment, the fermentation culture can specifically be:

[0017] First, prepare Gao's No. 1 liquid medium and dispense it into Erlenmeyer flasks, typically filling 1 / 3-1 / 2 of the flask's volume. Sterilize the medium in an autoclave at 121°C and 103.4 kPa for 20-30 minutes. Light an alcohol burner in a clean bench and sterilize the inoculating loop by burning it. Then, use the sterilized inoculating loop to pick a small amount of activated mycelium from the strain and quickly inoculate it into Gao's No. 1 liquid medium. Immediately seal and label the flasks. Place the inoculated flasks on a shaker and culture at 28°C and 150-200 rpm for 14 days. The fermentation medium contains 20.00 g soluble starch, 1.00 g potassium nitrate, 0.50 g sodium chloride, 0.50 g dipotassium hydrogen phosphate, 0.50 g magnesium sulfate heptahydrate, and 0.01 g ferric sulfate heptahydrate.

[0018] In a fifth aspect, the present application provides a method for preparing the above-mentioned compound M-1, comprising the following steps:

[0019] The fermentation extract of the Ralstonia strain was crudely fractionated using MCI medium-pressure column chromatography, and gradient elution was performed using a series of gradient concentrations of methanol-water system. The same fractions were combined to obtain the primary differential fractions.

[0020] Take the primary difference components, separate them by high performance liquid chromatography, and collect R =The peak at 21.00 min gave compound M-1.

[0021] The chromatographic conditions of the HPLC can be selected within a certain range, and any HPLC method that can achieve the separation purpose can be used in this application. In a particularly preferred embodiment, the flow rate of the HPLC is 2.0 mL / min, the mobile phase is 80% methanol, the detection wavelength is 254 nm, and the chromatographic column is a YMC-Pack ODS-A column.

[0022] In a sixth aspect, the present application provides the use of the Ralstonia MWSTR-5 strain or the above-mentioned compound M-1 or the fermentation extract or the pesticide in preventing and controlling agricultural plant diseases.

[0023] In a seventh aspect, the present application provides the use of the Ralstonia MWSTR-5 strain or the above-mentioned compound M-1 or fermentation extract or pesticide in the preparation of products for preventing and controlling agricultural plant diseases.

[0024] In an optional embodiment, the agricultural plant disease is caused by at least one of the following plant pathogens:

[0025] Fusarium oxysporum, Rhizopus stolonifer, Rhizoctonia solani, Botrytis cinerea, Sclerotinia sclerotiorum, Fusarium graminearum, and Alternaria solani.

[0026] Based on the above technical solution, this application has at least the following beneficial effects:

[0027] The present application provides a novel compound, namely compound M-1, which is isolated from the fermentation broth of the Ralstonia MWSTR-5 strain. By measuring the inhibitory activity of compound M-1 against Fusarium oxysporum and Alternaria solani, it was found that compound M-1 has strong anti-plant pathogenic fungus activity and is a good natural fungicide. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the specific implementation methods or the description of the prior art. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0029] Figure 1 This is a picture of the culture results of the MWSTR-5 strain in Example 1 of the present application;

[0030] Figure 2 is the phylogenetic tree of the MWSTR-5 strain in Example 1 of the present application;

[0031] Figure 3This is the inhibition curve of the fermentation product of MWSTR-5 strain against some plant pathogens in Example 2 of the present application;

[0032] Figure 4 This is the LC-MS spectrum of compound M-1 in Example 3 of the present application after hydrolysis with 6N HCl. DETAILED DESCRIPTION

[0033] To further illustrate the technical means and results employed by this application to achieve the intended invention objectives, the following describes in detail the specific implementation methods, technical solutions, and features of this application using preferred embodiments. The specific features, structures, or characteristics of the various embodiments described below may be combined in any suitable manner.

[0034] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0035] The present application is further described in detail below with reference to specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in this application.

[0036] Example 1 Isolation and Identification of Ralstonia sp. MWSTR-5 Strain

[0037] (1) Weigh 1.00 g of rhizosphere soil from Pinus massoniana, place it in a clean bench, and sterilize it with ultraviolet light for 10 minutes. After sterilization, transfer the rhizosphere soil to a 10 mL sterile centrifuge tube, add 5 mL of sterile water, shake and dissolve the soil thoroughly, and mark it as the soil stock solution. On this basis, the soil stock solution was diluted 10 times in a gradient to obtain 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 Solution. Take the original solution, 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 100 μL of the solution was spread onto a solid flat surface of Gao's No. 1 culture medium using a spreading rod for inoculation. After inoculation, the culture dish was sealed with sealing film, labeled, and placed in an inverted culture incubator at 28°C for 3-7 days.

[0038] A new strain was isolated from the culture results, such as Figure 1 As shown, the number is MWSTR-5.

[0039] (2) First, refer to the bacterial identification manual and perform preliminary identification of the strain by morphological methods. Then, use molecular biological methods to sequence the strain. After sequencing, enter the obtained 16S sequence into the NCBI database for Blast analysis to find the strain sequence with the highest similarity (those with a similarity of 97% or more can be considered to be of the same genus). Download and save it into MEGA11.0 software for comparison, and construct a phylogenetic tree of the strain. Identification is carried out by combining phylogenetic and evolutionary analysis, as well as colony and bacterial morphological characteristics. Finally, submit the strain 16S sequence to the NCBI database to obtain the GeneBank accession number.

[0040] (3) The 16S RNA sequence of MWSTR-5 strain was aligned with the sequence of Ralstonia sp. Strain QHR39 (MZ956308.1), and the similarity was 99.88%. The colonies were white-gray, dense, with a tight filamentous surface. The colonies were tightly connected to the culture medium, and the matrix was yellow. Combined with the sequencing results and phylogenetic tree analysis ( Figure 2 ), the MWSTR-5 strain was identified as a Ralstonia sp. GeneBank accession number: PV104007.

[0041] After the separation was completed, the obtained MWSTR-5 strain was frozen.

[0042] Example 2 Fermentation of Ralstonia sp. MWSTR-5 strain

[0043] (1) Strain activation

[0044] The frozen Ralstonia sp. MWSTR-5 strain was inoculated onto a plate of Gao's No. 1 solid culture medium in an ultra-clean workbench and cultured in a constant temperature incubator at 28°C for 7 days.

[0045] (2) Inoculation and batch fermentation of strains

[0046] The activated MWSTR-5 strain was inoculated into 38 500-mL Erlenmeyer flasks containing 300 mL of Gao's No. 1 liquid medium. The flasks were then incubated in a 28°C shaker for 14 days. The Gao's No. 1 liquid medium contained the following ingredients: 20.00 g soluble starch, 1.00 g potassium nitrate, 0.50 g sodium chloride, 0.50 g potassium hydrogen phosphate, 0.50 g magnesium sulfate heptahydrate, and 0.01 g ferric sulfate heptahydrate.

[0047] (3) Preparation of fermentation products of Ralstonia sp. MWSTR-5 strain

[0048] After the fermentation was completed, the fermentation product was filtered to obtain a yellow filtrate and yellow mycelia. The filtrate was extracted three times with equal volumes of ethyl acetate, and the ethyl acetate layers were combined and concentrated under reduced pressure to obtain the MWSTR-5 fermentation product.

[0049] (4) Determination of antibacterial activity of fermentation products

[0050] The antimicrobial activity was determined using potato dextrose agar (PDA) and complex potato culture medium (CPDA).

[0051] Prepare PDA medium as follows: Weigh 200g of potatoes, wash and peel them, cut them into small pieces, add them to 1500mL of water, boil them for 30 minutes, and filter through eight layers of gauze to obtain 1000mL of potato liquid. After cooling, add 20.00g each of glucose and agar, stirring to dissolve evenly. Aliquot the solution into 100mL Erlenmeyer flasks, seal them with parafilm, and place them in an autoclave along with the glass Petri dishes. Stir at 121°C for 30 minutes before use.

[0052] The CPDA culture medium was prepared as follows: 10.00 g potato extract, 20.00 g glucose, 3.00 g potassium hydrogen phosphate, 1.50 g magnesium sulfate heptahydrate, 0.008 g thiamine, and 15.00-20.00 g agar were weighed, added to 1000 mL of hot water, and stirred to dissolve evenly. The dissolved culture medium was dispensed into 50 mL conical flasks. After dispensing, the flasks were sealed with sealing film and placed in a high-pressure steam sterilizer together with the glass culture dishes. Sterilize at 121°C for 30 minutes and set aside.

[0053] Dissolve the MWSTR-5 fermentation product in DMSO and add it to the culture medium. Mix thoroughly to a concentration of 100 μg / mL. Use an equal concentration of DMSO as a blank control, and the marketed drug tebuconazole as a positive control. Pour the product into the plates, cool them, inoculate the culture cake, and incubate them in a 28°C incubator. Determine the antibacterial efficiency of the extract until the blank control mycelium fills the culture plate. All experiments were performed in triplicate. The antibacterial efficiency was calculated using the following formula:

[0054]

[0055] In the above formula, dc represents the colony diameter of the blank control group, d0 represents the diameter of the bacterial cake, and ds represents the colony diameter of the sample treatment group.

[0056] The results of the anti-plant pathogen activity test of the fermentation product of MWSTR-5 strain are shown in Table 1. The inhibition curves of the fermentation product against some plant pathogens are shown in Table 1. Figure 3 shown. Figure 3In the figure, (a) is the inhibition curve of the fermentation product of MWSTR-5 strain on Fusarium oxysporum; (b) is the inhibition curve of the fermentation product of MWSTR-5 strain on Rhizopus stolonifer; (c) is the inhibition curve of the fermentation product of MWSTR-5 strain on Botrytis cinerea; (d) is the inhibition curve of the fermentation product of MWSTR-5 strain on Sclerotinia sclerotiorum; (e) is the inhibition curve of the fermentation product of MWSTR-5 strain on Fusarium graminearum; (f) is the inhibition curve of the fermentation product of MWSTR-5 strain on Alternaria solani.

[0057] Table 1 Results of the determination of anti-plant pathogenic bacteria activity of MWSTR-5 strain fermentation products

[0058]

[0059] The results of the test in Table 1 show that the fermentation product of the Ralstonia sp. MWSTR-5 strain isolated above has an inhibition rate of more than 70% against various plant pathogens at a final concentration of 100 μg / mL. 50 The values ​​ranged from 4 to 54 μg / mL, showing good and broad-spectrum antibacterial activity.

[0060] (5) Separation of active ingredients from fermentation products

[0061] A. The fermentation product was crudely fractionated by MCI medium-pressure column chromatography, with gradient elution using 20%, 40%, 60%, 80%, and 100% methanol-water systems, and the same fractions were combined by TLC analysis to obtain 13 fractions, which were designated as: M1, M2, M3, M4, M5, M6, M7, M8, M9, M10, M11, M12, and M13;

[0062] B. The anti-Fusarium oxysporum activity of the above 13 components was determined, and the inhibition rates (final concentration 100 μg / mL) were 6.12%, 4.60%, 6.41%, 4.60%, 10.33%, 13.49%, 12.51%, 13.21%, 9.56%, 1.16%, 4.60%, 74.58%, and 59.55%, respectively;

[0063] C. Select the most active component M12 and use semi-preparative HPLC to separate it. Liquid chromatography conditions: wavelength 254nm, mobile phase 80% methanol-water, flow rate 2.0mL / min, collection retention time t R1 =21.00min. After the preparation, the mixture was concentrated to dryness to obtain compound M-1.

[0064] Example 3 Structure confirmation of compound M-1

[0065] (1) Physicochemical properties of compound M-1

[0066] Upon testing, compound M-1 was an orange-red powder with absorption at 254 nm UV and orange coloration with ethanolic sulfuric acid. The developing system for thin-layer chromatography was dichloromethane:methanol = 20:1.

[0067] (2) Determination of the structure of compound M-1

[0068] Its HRESIMS shows [M+H] + Peak m / z 1269.6142, indicating that its molecular weight should be 1268 and its molecular formula is C 56 H 92 N 12 O 17 S2(its [M+H] + Peak C 56 H 93 N 12 O 17 The calculated S2 value was 1269.6218). The secondary mass spectrometry showed the loss of multiple amino acid fragments, including [M+H-Leu] + The fragment peak 1156.5159, [1156.5159-Leu-Ser] + Fragment peak 956.3998, [956.3998-Val] + Fragment peak 857.3314, [857.3314-Thr-Val] + Fragment peak 657.2153, [300.1904-Pro] + Fragment peak 203.1376, [203.1376-Ala] + The fragment peak is 132.1005. The above secondary fragments indicate that compound M-1 is a peptide containing leucine (Leu), valine (Val), alanine (Ala), serine (Ser), threonine (Thr), and proline (Pro). The LC-MS spectrum of compound M-1 after hydrolysis with 6N HCl (see Figure 4) showed the presence of amino acids such as propyl, succinate, proline, valerian, threonine, and leucine, confirming the above speculation. We tried to combine 2-3 common amino acid connections for the cleavage fragments that appeared in the secondary mass spectrum with m / z from 657.2153 to 300.1904 (this section differs by 357 mass units), but none of them could explain it well. Therefore, it is speculated that this part of the structure may contain uncommon amino acids or a special structure with amino and carboxyl groups at both ends. The LC-MS spectrum of the acid hydrolysis product of M-1 found a peak with a strong abundance, with 168.0657 and its dehydration peak of 150.0547 in its MS spectrum. It is speculated that the hydrolysis product is 2-methyl-3-amino-4-hydroxybenzoic acid. The carboxyl group of this compound loses water with the amino group of an amino acid, while the 3-amino group loses water with the carboxyl group of another amino acid, thereby connecting to the M-1 structure. The other 4 hydroxyl groups may be connected to a chain structure through an ether bond. The 2-methyl-3-amino-4-oxy-benzoyl fragment is in 1 H. 13 C NMR spectrum also supports this. 1 H NMR showed δ H 2.57 (3H, s) for the proton signal of the aromatic ring methyl group; 13 C spectrum shows δ C The six aromatic ring C signals at 146.4, 141.8, 131.4, 130.6, 129.2, and 126.8, the amide C signal at 168.1, and the H-11 / C-13, H-12 / C-13, H-12 / C-14, H-16 / C-14, H-16 / C-15, and H-16 / C-13 related peaks in the HMBC spectrum also confirmed the presence of the 2-methyl-3-amino-4-oxy-benzoyl fragment. In addition, 13 δ in C spectrum C Multiple amide C signals were shown at positions 168-175, indicating that the compound was a peptide.

[0069] A peak at 132.1014 nm was detected at the low-mass end of the MS spectrum. This peak is hypothesized to be the hydrogenation peak of methionine (Met) after dehydration. This is because only when the Ala residue is followed by a Met residue, then linked to the initial cleaved Leu residue to form a cyclic peptide, can the molecular weight of 1268 be met, thus proposing a more plausible structure. However, no evidence of Met was found in the hydrolysis product of M-1, likely because Met is unstable after hydrolysis, resulting in no associated mass spectral peak.

[0070] The 4-hydroxyl group is connected to a chain substructure A through an ether bond, which is confirmed by one-dimensional and two-dimensional NMR. 1 H NMR and 13 CNMR showed a glycerol fragment with substructure A (δ H3.59(1H,dd,J=11.2,4.9Hz,H-1a),3.51(1H,dd,J=11.2,6.0Hz,,H-1a),3.70(1H,m,H-2),3.65(1H,m,H-3),δ C 64.4,71.8,73.9), 1 H- 1 The H-1 / H-2, H-2 / H-3 correlation peaks in the HCOSY spectrum and the H-1 / C-3 correlation peaks in the HMBC spectrum also indicate the presence of glycerol fragments. H 2.96 (3H, s), CH3-9 (δ H 2.87 (3H, s) and amide carbonyl (δ C 175.6, C-6) indicates that substructure A contains an N, N ’ -dimethylurea oxide. After connecting the above analyzed amino acid residues, 2-methyl-3-amino-4-oxy-benzoyl fragment and substructure A, it was found that the molecular weight was 32 less than the given molecular weight of 1268. Combined with the molecular formula C 56 H 92 N 12 O 17 S2, we speculate that compound M-1 should have another sulfur atom, which connects the glycerol fragment and urea.

[0071] In summary, the structure of M-1 is as follows:

[0072]

[0073] Table 2 Compound M-1 1 H NMR (600M) and 13 C NMR (150M) data (CD3OD, Jin Hz) a

[0074]

[0075]

[0076] Note: a Indicates based on 1 H- 1 H COSY, HMQC and HMBC spectra were assigned.

[0077] Example 4 Determination of the anti-plant pathogen activity of compound M-1 and its results

[0078] The antibacterial activity was determined using the same method as in step (4) of Example 2. The results are shown in Table 3. For demonstration purposes, only the activity of compound M-1 against Alternaria solani and Fusarium oxysporum was determined. However, the present invention is not limited to the activity of these two bacteria. Since the fermentation product has good activity against Rhizopus stolonifer, Rhizoctonia solani, Botrytis cinerea, Sclerotinia sclerotiorum, and Fusarium graminearum, it is reasonable to expect that compound M-1 also has the above activities. However, due to the limited sample size of M-1, no further determination was performed.

[0079] Table 3 Results of the anti-plant pathogen activity test of compound M-1

[0080]

[0081] The above description is merely a preferred embodiment of the present application; however, the scope of protection of the present application is not limited thereto. Any person skilled in the art who, within the technical scope disclosed in the present application, makes equivalent substitutions or modifications based on the technical solutions and improved concepts of the present application shall be covered by the scope of protection of the present application.

Claims

1. A compound M-1 having anti-plant pathogenic activity, characterized in that: The structural formula of the compound M-1 is shown below:

2. A fermentation extract of a Ralstonia strain, characterized in that The fermentation extract contains the compound M-1 according to claim 1.

3. The fermentation extract according to claim 2, characterized in that The fermentation extract is a fermentation extract of Ralstonia MWSTR-5 strain.

4. A pesticide, characterized in that The pesticide comprises the Ralstonia MWSTR-5 strain or the compound M-1 according to claim 1 or the fermentation extract according to claim 2 or 3.

5. The method for preparing the fermentation extract according to claim 2 or 3, characterized in that: The steps include: activating the Ralstonia strain and performing fermentation culture to obtain a fermentation product; The liquid in the fermentation product is separated and extracted with ethyl acetate, and the ethyl acetate layer is collected and concentrated.

6. The method for preparing compound M-1 according to claim 1, characterized in that: The steps include: The fermentation extract of the Ralstonia strain was crudely fractionated using MCI medium-pressure column chromatography, and gradient elution was performed using a series of gradient concentrations of methanol-water system. The same fractions were combined to obtain the primary differential fractions. Take the primary difference components, separate them by high performance liquid chromatography, and collect R =The peak at 21.00 min gave compound M-1.

7. Use of the Ralstonia MWSTR-5 strain, or the compound M-1 according to claim 1, or the fermentation extract according to claim 2 or 3, or the pesticide according to claim 4 in preventing and controlling agricultural plant diseases.

8. Use of the Ralstonia MWSTR-5 strain, or the compound M-1 according to claim 1, or the fermentation extract according to claim 2 or 3, or the pesticide according to claim 4 in the preparation of a product for preventing and controlling agricultural plant diseases.

9. The use according to claim 7 or 8, characterized in that The agricultural plant disease is caused by at least one of the following plant pathogens: Fusarium oxysporum, Rhizopus stolonifer, Rhizoctonia solani, Botrytis cinerea, Sclerotinia sclerotiorum, Fusarium graminearum, and Alternaria solani.

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