A streptomyces ydoxyferiae strain and its metabolite cis-3-indoleacrylic acid

Abstract

The application discloses a kind of streptomyces youssoufiensis strain and the application of its metabolite cis-3-indoleacrylic acid, belongs to the technical field of microbial pesticides.The preservation name of streptomyces youssoufiensis strain is streptomyces youssoufiensis YMF3.862 strain, is preserved in China Microbial Culture Collection Center;Preservation date: May 20, 2024;Preservation number: CGMCC No.30698.The application has the characteristics of low cost, good effect, no residue, safety to human and environment, and the preparation of biological pesticide by YMF3.862 strain and its metabolite cis-3-indoleacrylic acid has more than 60% of the control effect on tobacco root-knot nematode disease, tobacco bacterial wilt and the composite disease caused by the two.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial pesticides, and particularly relates to a S. youssoufiensis strain and an application of a metabolite cis-3-indoleacrylic acid of the S. youssoufiensis strain in preventing and treating root-knot nematode disease, bacterial wilt and a combined disease caused by the two. BACKGROUND

[0002] Root-knot nematodes (RKNs) are one of the main pathogens of various crops, and are distributed all over the world. There are more than 100 species of RKNs, which can parasitize more than 3000 crops, and cause economic losses of more than 157 billion US dollars in the world every year. The second instar larvae of RKNs invade the roots of crops, form root nodules at the roots, prevent crops from absorbing water and nutrients, and cause poor growth of crops, and even cause the crops to wither and die. Ralstonia solanacearum causes bacterial wilt of various crops, and is listed as one of the ten important soil-borne bacterial pathogens. The bacterium can infect more than 200 plants and grow and reproduce by using plant nutrients, causing blockage and destruction of vascular tissues, and finally causing the host to wither and die. The wounds caused by RKNs invading the roots of crops are conducive to the invasion of R. solanacearum, and the combined occurrence of root-knot nematode disease and bacterial wilt is widespread in agricultural planting (Huang et al., 2015).

[0003] At present, the main prevention and treatment measures for root-knot nematode disease, bacterial wilt and the combined disease caused by the two are to apply chemical pesticides respectively for RKNs and R. solanacearum, and the amount of pesticide used is large, the cost is high, and pesticide residues have brought many negative effects on the ecological environment safety, human and animal health and the like. The biocontrol products developed by using microorganisms and their metabolites have the advantages of environmental protection, safety and superimposed effect of prevention and treatment, and are favored by domestic and foreign people. Many microorganisms have the functions of killing nematodes and resisting bacteria, and it is of great application value to explore such microbial resources and develop biocontrol products for resisting both nematodes and R. solanacearum by using microorganisms and their active metabolites. Streptomyces is the main group of actinomycetes, and is widely distributed in soil, and can produce various active metabolites for antagonizing RKNs and R. solanacearum. S. youssoufiensis is a new species of actinomycetes reported in 2011 (Hamdali et al., 2011).

[0004] The Actinomycete metabolites reported so far mainly include reedsmycins (Che et al., 2015; Yao et al., 2018), unsaturated fatty acid amides (Hou et al., 2019), polyene acids (youssoufenes A1, B1-B4) (Li et al., 2020), dimeric cinnamoyl lipids (youssoufenes A2, A3) (Liu et al., 2022), benzoic nigericin, epinigericin, abierixin, grisorixin methyl ester (Leulmi et al., 2019), glucosylpiericidins (Liu et al., 2021). The biological activities of these compounds involve antifungal, antibacterial and cancer cell toxicity. The present inventors screened an Actinomycete YMF3.862 from cave soil, which was identified as S. youssoufiensis based on morphological and molecular biological characteristics. The fermentation broth of YMF3.862 strain and its metabolite cis-3-indoleacrylic acid have good antagonistic activity against second instar larvae and eggs of root-knot nematode and Pseudomonas solanacearum. Through literature retrieval, the biocontrol activity of S. youssoufiensis and cis-3-indoleacrylic acid against root-knot nematode and P. solanacearum has not been reported, which highlights the innovation and potential application value of the present application. SUMMARY

[0005] The present application aims to overcome the shortcomings of the prior art and provide a Streptomyces youssoufiensis strain and its metabolite cis-3-indoleacrylic acid that are resistant to root-knot nematode and plant pathogenic bacteria.

[0006] To achieve the above application purposes, the technical solutions adopted by the present application are as follows:

[0007] In a first aspect, the present application provides a Streptomyces youssoufiensis strain.

[0008] In a second aspect, the present application provides a Streptomyces youssoufiensis microbial agent.

[0009] In a third aspect, the present application provides a method for preparing the fermentation culture of Streptomyces youssoufiensis.

[0010] In a fourth aspect, the present application provides a compound cis-3-indoleacrylic acid.

[0011] Fifthly, this application provides a method for preparing the compound cis-3-indoleacrylic acid.

[0012] Sixthly, this application provides a biological agent.

[0013] Seventhly, this application provides the application of a biological agent in the prevention and control of tobacco root-knot nematode disease, tobacco bacterial wilt, and the combined diseases caused by the two.

[0014] This invention discloses a Streptomyces youssoufiensis strain, whose preservation name is Streptomyces youssoufiensis YMF3.862, deposited at the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; deposit date: May 20, 2024; accession number: CGMCC No. 30698.

[0015] The present invention relates to a Streptomyces eugenol strain prepared from Streptomyces eugenol.

[0016] Furthermore, its active ingredient is at least one of the following (a), (b), and (c):

[0017] (a) Fermentation culture of Streptomyces eustachiana;

[0018] (b) The supernatant of the ultrasonic lysis of the obtained Streptomyces eugenolae cells;

[0019] (c) Ultrasonic lysis and precipitation of the obtained Streptomyces eugenolae cells.

[0020] The method for preparing the fermentation culture of *Streptomyces eugenolus* of the present invention includes the following steps:

[0021] (1) Inoculate the strain of Streptomyces youssoufiensis YMF3.862 onto the slant of solid culture medium in test tubes. The culture medium used is ISP4 medium. Incubate at 28-36℃ for 4-7 days to obtain the slant strain.

[0022] (2) Dispense ISP4 liquid culture medium into 500 ml Erlenmeyer flasks, with a liquid volume of 300 ml / flask. After sterilization according to conventional microbial methods, use an inoculation needle to pick up YMF3.862 cells from the slant of the test tube in a clean bench and inoculate them into the culture medium. After culturing at 25-30℃ and 130-200 rpm for 10-15 days, the fermentation culture of strain YMF3.862 is obtained.

[0023] The compound cis-3-indoleacrylic acid of this invention was obtained by extraction with ethyl acetate, column chromatography, and purification by silica gel column chromatography using petroleum ether / methanol as the eluent from a culture of *Streptomyces youssoufiensis* strain YMF3.862. The structural formula of cis-3-indoleacrylic acid is shown in formula (I).

[0024] .

[0025] The preparation method of the compound cis-3-indoleacrylic acid of the present invention includes the following steps: extracting the culture of strain YMF3.862 with ethyl acetate, combining the obtained organic phases and concentrating them at 40°C using a rotary evaporator, eluing the concentrate with methanol and then performing Sephadex LH-20 column chromatography to obtain a component active against southern root-knot nematodes and tobacco wilt pathogens, and further purifying the active component with petroleum ether / methanol using silica gel column chromatography to obtain the compound cis-3-indoleacrylic acid.

[0026] Furthermore, the biological agent is obtained by dissolving the compound cis-3-indoleacrylic acid in methanol, mixing it with water, and then adding Tween-20, wherein the content of cis-3-indoleacrylic acid is ≥10%, the content of methanol is ≤5%, and the content of Tween-20 is ≤0.3%.

[0027] The application of the biological agents of the present invention in the prevention and control of tobacco root-knot nematode disease, tobacco bacterial wilt, and the combined diseases caused by the two.

[0028] Furthermore, cis-3-indoleacrylic acid in the biological agent can inhibit the activity of tobacco root-knot nematodes and bacterial wilt pathogens.

[0029] Beneficial effects: This invention features low cost, good efficacy, no residue, and safety for humans, animals, and the environment. The biopesticide prepared using YMF3.862 strain and its metabolite cis-3-indoleacrylic acid has an efficacy of over 60% against tobacco root-knot nematode disease, tobacco bacterial wilt, and the combined diseases caused by the two.

[0030] Compared with the prior art, the present invention has the following advantages:

[0031] (1) The YMF3.862 strain of the present invention is easy to ferment and culture. Its culture has both nematicidal and antibacterial activities and can be used as a nematicide and fungicide to prevent and control root-knot nematode disease, bacterial wilt and the combined diseases caused by the two.

[0032] (2) The compound Z-IAA of the present invention is easy to obtain. The compound has both nematicidal and antibacterial activity and can be used as a nematicide and fungicide to prevent and control root-knot nematode disease, bacterial wilt and the combined diseases caused by the two.

[0033] (3) The YMF3.862 strain and its compound Z-IAA of the present invention have the advantages of high efficiency, low toxicity and low residue in the prevention and control of root-knot nematode disease, bacterial wilt and the combined diseases caused by the two. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1 This diagram illustrates the morphological characteristics and phylogenetic analysis of the *Streptomyces youssoufiensis* strain YMF3.862, as presented in this invention. Figure 1 A: Colony morphology of strain YMF3.862 after 14 days of incubation at 28°C on ISP4 medium; Figure 1 Morphology of spores and spore chains of strain B:YMF3.862; Figure 1 C: Phylogenetic analysis based on the 16S rDNA sequence showed that strain YMF3.862 is S. youssoufiensis.

[0036] Figure 2 This is a diagram showing the inhibition of the YMF3.862 strain culture and Z-IAA against the bacterial wilt of tobacco in this invention. Figure 2 A: Comparison; Figure 2 B: Culture of strain YMF3.862; Figure 2 C: 500 times dilution of 10% Z-IAA. Detailed Implementation

[0037] To make the technical problems, technical solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0038] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0039] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0040] The first aspect of this application provides a Streptomyces youssoufiensis strain, whose preservation name is Streptomyces youssoufiensis YMF3.862, deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; deposit date: May 20, 2024; accession number: CGMCC No. 30698.

[0041] The second aspect of this application provides a Streptomyces eugenol strain prepared from Streptomyces eugenol.

[0042] In some embodiments, the active ingredient is a fermentation culture of Streptomyces eustoniae.

[0043] In some embodiments, the active ingredient is the ultrasonic lysis supernatant of the obtained Streptomyces eugenolae cells;

[0044] In some embodiments, the active ingredient is the ultrasonic lysis and precipitation of the obtained Streptomyces eugenolans cells.

[0045] The third aspect of this application provides a method for preparing a fermentation culture of *Streptomyces eustachiana*, comprising the following steps:

[0046] (1) Inoculate Streptomyces youssoufiensis YMF3.862 strain onto the slant of solid culture medium in test tubes. The culture medium used is ISP4 medium. Incubate at 28-36℃ for 4-7 days to obtain slant strains.

[0047] (2) Dispense ISP4 liquid culture medium into 500 ml Erlenmeyer flasks, with a liquid volume of 300 ml / flask. After sterilization according to conventional microbial methods, use an inoculation needle to pick up YMF3.862 cells from the slant of the test tube in a clean bench and inoculate them into the culture medium. After culturing at 25-30℃ and 130-200 rpm for 10-15 days, the fermentation culture of strain YMF3.862 is obtained.

[0048] In some embodiments, (1) Streptomyces youssoufiensis YMF3.862 strain is inoculated onto a solid culture medium slant in a test tube. The culture medium used is ISP4 medium, and the culture is carried out at 28°C for 4 days to obtain the slant strain.

[0049] In step (2), the ISP4 liquid culture medium is dispensed into 500 ml Erlenmeyer flasks, with a liquid volume of 300 ml / flask. After sterilization according to conventional microbial methods, YMF3.862 cells are picked from the slant of the test tube and inoculated into the culture medium using an inoculation needle in a clean bench. After culturing at 25°C and 200 rpm for 10 days, the fermentation culture of strain YMF3.862 is obtained.

[0050] In some embodiments, (1) Streptomyces youssoufiensis YMF3.862 strain is inoculated onto a solid culture medium slant in a test tube. The culture medium used is ISP4 medium, and the culture is carried out at 36°C for 6 days to obtain the slant strain.

[0051] In step (2), the ISP4 liquid culture medium is dispensed into 500 ml Erlenmeyer flasks, with a liquid volume of 300 ml / flask. After sterilization according to conventional microbial methods, YMF3.862 cells are picked from the slant of the test tube and inoculated into the culture medium using an inoculation needle in a clean bench. After culturing at 28°C and 130 rpm for 15 days, the fermentation culture of strain YMF3.862 is obtained.

[0052] In some embodiments, (1) Streptomyces youssoufiensis YMF3.862 strain is inoculated onto a solid culture medium slant in a test tube. The culture medium used is ISP4 medium, and the culture is carried out at 32°C for 6 days to obtain the slant strain.

[0053] In step (2), the ISP4 liquid culture medium is dispensed into 500 ml Erlenmeyer flasks, with a liquid volume of 300 ml / flask. After sterilization according to conventional microbial methods, YMF3.862 cells are picked from the slant of the test tube and inoculated into the culture medium using an inoculation needle in an ultra-clean workbench. After culturing at 30℃ and 170 rpm for 12 days, the fermentation culture of strain YMF3.862 is obtained.

[0054] The fourth aspect of this application provides a compound cis-3-indoleacrylic acid, which is obtained by ethyl acetate extraction, column chromatography, and then purification by silica gel column chromatography using petroleum ether / methanol as the eluent from a culture of *Streptomyces youssoufiensis* strain YMF3.862. The structural formula of cis-3-indoleacrylic acid is shown in formula (I).

[0055] .

[0056] The fifth aspect of this application provides a method for preparing the compound cis-3-indoleacrylic acid. A culture of strain YMF3.862 is extracted with ethyl acetate. The resulting organic phases are combined and concentrated using a rotary evaporator at 40°C. The concentrate is then subjected to Sephadex LH-20 column chromatography using methanol as the eluent to obtain a component active against southern root-knot nematodes and tobacco wilt pathogens. This active component is further purified by silica gel column chromatography using petroleum ether / methanol (100:1, 80:1, 60:1, 40:1, 20:1, v / v) as the eluent to obtain the compound cis-3-indoleacrylic acid.

[0057] The sixth aspect of this application provides a biological agent, a solution containing the compound cis-3-indoleacrylicacid.

[0058] In some embodiments, the biological agent is obtained by dissolving the compound cis-3-indoleacrylic acid in methanol, mixing it with water, and then adding Tween-20, wherein the content of cis-3-indoleacrylic acid is ≥10%, the content of methanol is ≤5%, and the content of Tween-20 is ≤0.3%.

[0059] The seventh aspect of this application provides the application of biological agents in the prevention and control of tobacco root-knot nematode disease, tobacco bacterial wilt, and the combined diseases caused by the two.

[0060] In some embodiments, cis-3-indoleacrylic acid in biological agents can inhibit the activity of tobacco root-knot nematodes and bacterial wilt pathogens.

[0061] Example 1

[0062] The actinomycete of this invention is *Streptomyces youssoufiensis* (YMF3.862). Strain YMF3.862 was deposited on May 20, 2024, at the China General Microbiological Culture Collection Center (CGMCC); address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; accession number: CGMCC No. 30698.

[0063] When strain YMF3.862 was cultured on ISP4 medium (soluble starch 10 g / L, peptone 1 g / L, yeast extract 0.5 g / L, (NH4)2SO4 2 g / L, MgSO4 1 g / L, K2HPO4 1 g / L, NaCl 1 g / L, CaCO3 2 g / L, agar 18 g / L, pH 7.2) at 28°C for 14 days, the colonies were grayish-white and dry, exhibiting typical actinomycete colony characteristics. Figure 1 A); aerial hyphae are white, with columnar spores that grow in chains on the hyphae ( Figure 1 B); The 16S rDNA sequence of this strain has been submitted to the GeneBank public database, with the accession number PP858108. This sequence is the main basis for the molecular identification of strain YMF3.862. Phylogenetic analysis combining the morphology and 16S rDNA sequence of strain YMF3.862 ( Figure 1 C) indicates that the strain is *Streptomyces youssoufiensis*.

[0064] Figure 1 This diagram illustrates the morphological characteristics and phylogenetic analysis of the *Streptomyces youssoufiensis* strain YMF3.862, as presented in this invention. Figure 1 A shows the colony morphology of the YMF3.862 strain of the present invention after being cultured at 28°C for 14 days on ISP4 medium; Figure 1 B represents the morphology of spores and spore chains of the YMF3.862 strain of the present invention; Figure 1 C indicates that the phylogenetic analysis based on the 16S rDNA sequence of this invention shows that strain YMF3.862 is S. youssoufiensis.

[0065] This invention utilizes ISP4 liquid medium to culture strain YMF3.862 using conventional microbial fermentation methods at 28°C and 180 rpm for 14 days. The resulting culture exhibits good antagonistic activity against southern root-knot nematode (Meloidogyne incognita) and tobacco bacterial wilt fungus (R. solanacearum). The application of the YMF3.862 strain culture of this invention in the preparation of nematicidal biopesticides and bacterial wilt-resistant biological agents is also discussed.

[0066] The culture of strain YMF3.862 was extracted with ethyl acetate. The resulting organic phases were combined and concentrated using a rotary evaporator. The concentrate was subjected to Sephadex LH-20 column chromatography, and the active component was purified by silica gel column chromatography using petroleum ether / methanol (100:1, 80:1, 60:1, 40:1, 20:1, v / v) as the eluent to obtain the compound of the present invention.

[0067] Based on multispectral analysis, the chemical characteristics of the compounds in this invention are as follows: 1.2 (c = 0.10, methanol); UV (methanol)λ max (logε)nm: 223 (3.93), 282 (3.39), 315 (3.57); based on high-resolution mass spectrometry HR-ESI-MS ([M+H] + The molecular formula is determined to be C (m / z 188.0702). 11 H9NO2; 13 C-NMR and DEPT spectra revealed four quaternary carbons (δC 110.7, 128.3, 137.0 and 168.7) and seven methylene groups (δC 127.8, 118.8, 123.6, 121.4, 112.7, 125.9 and 104.3), indicating that the compound is an indole derivative. 1Combined with H-NMR and COSY, the data from the COSY spectrum revealed key correlation points for this compound (H-4 / H-5 / H-6 / H-7; H-9 / H-10), from which it can be deduced that the compound has two segments: -C-4-C-5–C-6-C-7- and –C-9-C-10-. The fine structure of the compound was determined by 2D-NMR. According to data from HMBC, H-1 (δH 8.04) correlates with C-2 (δC 110.7), C-3 (δC 128.3), and C-8 (δC 137.0); H-4 (δH 7.68) correlates with C-5 (δC 123.6) and C-8 (δC 137.0); and H-5 (δH 7.20) correlates with C-4 (δC 118.8). The relative configurations of the compound were determined by NOESY. The NOESY data showed that the coupling constant at the double bond -C-9-C-10- was 8.8 Hz, revealing that the double bond at C-9 was in the cis configuration. Therefore, the structure of the compound of the present invention was determined to be cis-3-indoleacrylic acid (hereinafter referred to as Z-IAA for ease of description). The structural formula of cis-3-indoleacrylic acid is shown in formula (I):

[0068] .

[0069] Experiments showed that Z-IAA has significant activity against southern root-knot nematodes, and the median lethal concentration (LC50) for southern root-knot nematodes is [not specified]. 50 The concentration of Z-IAA was 16.31 μg / ml; Z-IAA also exhibited significant antagonistic activity against *Ralstonia solanacearum*, with an IC50 concentration of 16.31 μg / ml. 50 The concentration was 3.1 μg / ml.

[0070] The compound Z-IAA of the present invention is first dissolved in methanol, then in water, and finally Tween-20 is added as an emulsifier to prepare a solution containing 10% Z-IAA, wherein the final concentration of methanol does not exceed 5% and the final concentration of Tween-20 does not exceed 0.3%. This solution is used in the preparation of nematicides and fungicides.

[0071] Example 2

[0072] 1. Fermentation production method of strain YMF3.862

[0073] (1) The Streptomyces youssoufiensis YMF3.862 strain was inoculated onto the slant of solid culture medium in test tubes. The culture medium used was ISP4 medium. The culture was carried out at 28℃ for 4 days to obtain the slant strain.

[0074] (2) Dispense ISP4 liquid culture medium into 500 ml Erlenmeyer flasks, with a liquid volume of 300 ml / flask. After sterilization according to conventional microbial methods, use an inoculation needle to pick up YMF3.862 cells from the slant of the test tube in a clean bench and inoculate them into the culture medium. After culturing at 25-30℃ and 130-200 rpm for 10-15 days, the fermentation culture of strain YMF3.862 is obtained.

[0075] 2. Preparation method of compound Z-IAA

[0076] The culture of strain YMF3.862 was extracted with ethyl acetate. The resulting organic phases were combined and concentrated using a rotary evaporator at 40°C. The concentrate was then subjected to Sephadex LH-20 column chromatography using methanol as the eluent to obtain the fraction active against southern root-knot nematodes and tobacco wilt pathogens. The active fraction was further purified by silica gel column chromatography using petroleum ether / methanol (100:1, 80:1, 60:1, 40:1, 20:1, v / v) to obtain the compound of this invention. The structural formula of cis-3-indoleacrylicacid is shown in formula (I):

[0077]

[0078] The difference between Example 3 and Example 2 is as follows:

[0079] In step (1), the Streptomyces youssoufiensis YMF3.862 strain was inoculated onto the slant of solid culture medium in a test tube. The culture medium used was ISP4 medium, and the culture was carried out at 36°C for 6 days to obtain the slant strain.

[0080] In step (2), the ISP4 liquid culture medium is dispensed into 500 ml Erlenmeyer flasks, with a liquid volume of 300 ml / flask. After sterilization according to conventional microbial methods, YMF3.862 cells are picked from the slant of the test tube and inoculated into the culture medium using an inoculation needle in a clean bench. After culturing at 28°C and 130 rpm for 15 days, the fermentation culture of strain YMF3.862 is obtained.

[0081] The difference between Example 4 and Example 2 is as follows:

[0082] In step (1), the Streptomyces youssoufiensis YMF3.862 strain was inoculated onto the slant of solid culture medium in a test tube. The culture medium used was ISP4 medium, and the culture was carried out at 32°C for 6 days to obtain the slant strain.

[0083] In step (2), the ISP4 liquid culture medium is dispensed into 500 ml Erlenmeyer flasks, with a liquid volume of 300 ml / flask. After sterilization according to conventional microbial methods, YMF3.862 cells are picked from the slant of the test tube and inoculated into the culture medium using an inoculation needle in an ultra-clean workbench. After culturing at 30℃ and 170 rpm for 12 days, the fermentation culture of strain YMF3.862 is obtained.

[0084] Example 5

[0085] Application methods of YMF3.862 strain culture and compound Z-IAA for the control of nematodes and bacterial diseases.

[0086] When using the culture of strain YMF3.862 to control diseases, the liquid obtained by adding Tween-20 to the culture at a final concentration of 0.3% can be used to control root-knot nematode disease, tobacco bacterial wilt, and combined diseases caused by the two; the dosage is 200 ml / plant, and the method of application is root irrigation.

[0087] When using compound Z-IAA to control diseases, Z-IAA is first dissolved in methanol, then in water, and finally Tween-20 is added as an emulsifier. The resulting solution contains Z-IAA ≥ 10%, methanol ≤ 5%, and Tween-20 ≤ 0.3%. When using, dilute with water 500-1000 times, use 200 ml per plant, and apply by root drenching.

[0088] Experimental Example 1

[0089] Methods for detecting the activity of YMF3.862 strain culture and compound Z-IAA against root-knot nematodes and bacterial wilt pathogens.

[0090] 1) Preparation of second-instar larvae (J2) of root-knot nematodes and the pathogen of tobacco bacterial wilt

[0091] Preparation of J2: The tomato roots infected by the southern root-knot nematode were washed with sterile water. The oocysts of the root-knot nematode were picked from the root nodules with a dissecting needle and placed in a 5ml centrifuge tube. The oocysts were lysed with 0.1% NaClO solution for 5 minutes. The NaClO solution was removed by centrifugation and the oocysts were washed 3 times with sterile water. The obtained eggs were placed in sterile water and incubated at 25℃ for 4-6 days. J2 was obtained by centrifugation.

[0092] Preparation of *Ralstonia solanacearum* causal agent: *Ralstonia solanacearum* causal agent was inoculated into NA medium and cultured at 37℃ and 150 rpm for 3 days to obtain the *Ralstonia solanacearum* culture medium. The NA formulation (g / L) was: peptone 10, beef extract 3, sodium chloride 5, pH 7.2.

[0093] Experimental Example 2

[0094] Methods for detecting the toxic activity of YMF3.862 strain culture and Z-IAA against root-knot nematodes.

[0095] Add 1.9 ml of culture or different dilutions of 10% Z-IAA to a sterile culture dish with a diameter of 3.5 cm. Then add 0.1 ml of nematode suspension containing more than 200 J2 worms, mix well, and let stand at room temperature. Use ISP4 liquid medium containing 0.3% Tween-20 as a control for YMF3.862 strain culture, and use an equivalent dilution containing 0.3% Tween-20 and 5% methanol as a Z-IAA control. Each treatment is repeated in triplicate. After 24 h of standing, count the number of dead J2 worms under a dissecting microscope, and calculate the J2 mortality rate and corrected mortality rate using the following formula.

[0096] Mortality rate (%) = (Number of surviving J2s / Total number of J2s counted) × 100

[0097] Corrected mortality rate (%) = (treatment group mortality rate - control mortality rate) / (1 - control mortality rate) × 100 (Example 3)

[0098] YMF3.862 strain culture and its antibacterial activity against Ralstonia solanacearum were detected by a method.

[0099] Prepare NA solid medium (add 18g agar to the NA medium formulation), sterilize routinely, and cool to 45℃. Add 1ml of *Ralstonia solanacearum* culture solution to every 100ml of medium, mix well, and pour into plates. After cooling, punch 6 wells evenly on the plates using a 1cm diameter punch. Remove the medium from the wells and add 100μl of *YMF3.862* strain culture or different dilutions of 10% Z-IAA to each well. Use NA liquid medium containing 0.3% Tween-20 as the control for *YMF3.862* strain culture, and use an equivalent dilution of 0.3% Tween-20 and 5% methanol as the Z-IAA control. Each treatment is repeated three times. After incubating the plates at 37℃ for 48h, measure the inhibition zone for each treatment. The size of the inhibition zone represents the antibacterial activity of *YMF3.862* strain culture and 10% Z-IAA.

[0100] Test Example 4

[0101] Methods for determining the efficacy of YMF3.862 strain culture and Z-IAA against root-knot nematode and tobacco bacterial wilt pathogens.

[0102] The experiment was conducted using a greenhouse pot experiment. Specifically, 2 kg of sterilized soil was placed in each pot, and one tobacco seedling (variety: Honghua Dajinyuan) was transplanted into each pot. Immediately after transplanting, the roots were drenched with 200 ml of YMF3.862 strain culture or different dilutions of 10% Z-IAA. Three days after transplanting, each seedling was inoculated with 1000 J2 strains or 100 ml of tobacco wilt bacteria fermentation broth. ISP4 liquid medium containing 0.3% Tween-20 served as the control for the YMF3.862 strain culture, and a Z-IAA control was prepared using a dilution of 0.3% Tween-20 and 5% methanol. Each treatment was replicated 15 times and randomly arranged. After 60 days of cultivation in the greenhouse, the disease index and control efficacy for each treatment were investigated using the following formula.

[0103] Disease index = (n1×1+n2×2+n3×3+n4×4+n5×5) / (S×5)×100, where n1-n5 represent the total number of plants at levels 1-5 respectively; and S represents the total number of plants surveyed.

[0104] Efficacy (%) = (1 - Treatment disease severity / Control disease severity) × 100

[0105] 5) Method for determining the control efficacy of YMF3.862 strain culture and Z-IAA against root-knot nematodes and bacterial wilt combined disease.

[0106] The experiment was conducted using a greenhouse pot experiment. Specifically, 2 kg of sterilized soil was placed in each pot, and one tobacco seedling (variety: Honghua Dajinyuan) was transplanted into each pot. Immediately after transplanting, the roots were drenched with 200 ml of YMF3.862 strain culture or a 10% Z-IAA dilution. Three days after transplanting, each seedling was simultaneously inoculated with 1000 J2 strains and 100 ml of tobacco wilt bacteria fermentation broth. ISP4 liquid medium containing 0.3% Tween-20 served as the control for the YMF3.862 strain culture, and an equivalent dilution of 0.3% Tween-20 and 5% methanol served as the Z-IAA control. Each treatment had 15 replicates, randomly arranged. After 60 days of cultivation in the greenhouse, the disease index and control efficacy for each treatment were investigated using the following formula.

[0107] Disease index = (n1×1+n2×2+n3×3+n4×4+n5×5) / (S×5)×100, where n1-n5 represent the total number of plants at levels 1-5 respectively; and S represents the total number of plants surveyed.

[0108] Efficacy (%) = (1 - Treatment disease severity / Control disease severity) × 100

[0109] Experimental Example 5

[0110] Activity of YMF3.862 strain culture and compound Z-IAA against root-knot nematodes and bacterial wilt pathogens.

[0111] The culture of strain YMF3.862 and a 10% Z-IAA solution were prepared according to the above method, and their activity was determined using the above plate assay method. Specifically, the stock solution of strain YMF3.862 was used for testing, and a 500-fold dilution of 10% Z-IAA was used. Nematode mortality was measured 24 hours after treatment, and the inhibition zone was measured 48 hours after treatment. The results (Table 1) showed that the lethality of strain YMF3.862 and the 500-fold dilution of 10% Z-IAA against *Rhizoctonia solani* J2 at 24 hours were 67.36% and 98.45%, respectively, demonstrating good nematicidal activity. The inhibition zones of strain YMF3.862 and the 500-fold dilution of 10% Z-IAA against *Rhizoctonia solani* at 48 hours were 20.68 mm and 49.44 mm, respectively. Figure 2 The culture of YMF3.862 and Z-IAA showed good antibacterial activity. The activities of the culture and Z-IAA against southern root-knot nematodes and bacterial wilt pathogens are shown in Table 1.

[0112] Table 1

[0113]

[0114] Figure 2 This diagram shows the inhibition of *Ralstonia solanacearum* by the YMF3.862 strain culture and Z-IAA. A: Control; B: YMF3.862 strain culture; C: 500-fold dilution of 10% Z-IAA.

[0115] Experimental Example 6

[0116] The efficacy of YMF3.862 strain culture and compound Z-IAA against southern root-knot nematode disease.

[0117] The YMF3.862 strain culture and 10% Z-IAA compound solution were prepared according to the above method, and the control efficacy was determined using the above greenhouse pot test method. Specifically, the YMF3.862 strain culture was tested with 200 ml of the stock solution per plant, and Z-IAA was tested with 500, 1000, and 1500 times dilutions of 10% Z-IAA, with a dosage of 200 ml per plant. A sterile hydroponic crop was used as a control, and the results were investigated after 60 hours of pot cultivation.

[0118] The results (Table 2) showed that the control efficacy of the YMF3.862 strain culture against southern root-knot nematode disease in tobacco was 61.43%; the control efficacies of 500, 1000, and 1500 times dilutions of 10% Z-IAA against southern root-knot nematode disease in tobacco were 87.25%, 73.18%, and 66.94%, respectively, all showing good control efficacy. The control efficacy of the YMF3.862 strain culture and 10% Z-IAA against southern root-knot nematode disease in tobacco is shown in Table 2.

[0119] Table 2

[0120]

[0121] Experimental Example 7

[0122] The efficacy of YMF3.862 strain culture and compound Z-IAA against tobacco bacterial wilt.

[0123] The YMF3.862 strain culture and 10% Z-IAA compound solution were prepared according to the above method, and the control efficacy was determined using the above greenhouse pot test method. Specifically, the YMF3.862 strain culture was tested with 200 ml of the stock solution per plant, and Z-IAA was tested with 500, 1000, and 1500 times dilutions of 10% Z-IAA, with a dosage of 200 ml per plant. A sterile hydroponic crop was used as a control, and the results were investigated after 60 hours of pot cultivation.

[0124] The results (Table 3) showed that the control efficacy of the YMF3.862 strain culture against tobacco bacterial wilt was 65.74%; the control efficacies of 500, 1000, and 1500 times dilutions of 10% Z-IAA against tobacco bacterial wilt were 73.86%, 68.45%, and 67.92%, respectively, all demonstrating good control efficacy. The control efficacy of the YMF3.862 strain culture and 10% Z-IAA against tobacco bacterial wilt is shown in Table 3.

[0125] Table 3

[0126]

[0127] Experimental Example 8

[0128] The efficacy of YMF3.862 strain culture and compound Z-IAA against tobacco root-knot nematodes and bacterial wilt combined disease.

[0129] The YMF3.862 strain culture and 10% Z-IAA compound solution were prepared according to the above method, and the control efficacy was determined using the above greenhouse pot test method. Specifically, the YMF3.862 strain culture was tested with 200 ml of the stock solution per plant, and Z-IAA was tested with 500, 1000, and 1500 times dilutions of 10% Z-IAA, with a dosage of 200 ml per plant. A sterile hydroponic crop was used as a control, and the results were investigated after 60 hours of pot cultivation.

[0130] The results (Table 3) showed that the YMF3.862 strain culture had a control efficacy of 61.47% against the combined disease of tobacco root-knot nematode and bacterial wilt; the control efficacies of 500, 1000, and 1500 times dilutions of 10% Z-IAA against the combined disease were 68.62%, 65.36%, and 62.85%, respectively, all showing good control efficacy. The control efficacy of the YMF3.862 strain culture and 10% Z-IAA against the combined disease of tobacco root-knot nematode and bacterial wilt is shown in Table 4.

[0131] Table 4

[0132]

[0133] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope. The scope of protection of the present invention is defined by the appended claims, specification, and their equivalents.

Claims

1. A Streptomyces eustachiana strain, characterized in that: The preservation name of the *Streptomyces eusfieldii* strain is *Streptomyces eusfieldii*. Streptomyces youssoufiensis Strain YMF3.862 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; deposit date: May 20, 2024; accession number: CGMCC No. 30698.

2. The *Streptomyces eugenolus* inoculum prepared by the *Streptomyces eugenolus* strain according to claim 1.

3. The Streptomyces eugenolactone inoculant according to claim 2, wherein the active ingredient is at least one of the following (a), (b), and (c): (a) Fermentation culture of Streptomyces eustachiana as claimed in claim 1; (b) The supernatant of ultrasonic lysis of *Streptomyces eugenolans* cells obtained according to claim 1; (c) The ultrasonic lysis and precipitation of Streptomyces eugenolae cells obtained according to claim 1.

4. The method for preparing the fermentation culture of *Streptomyces eustachiana* as described in claim 3, characterized in that... Includes the following steps: (1) Sterilella eustachiana Streptomyces youssoufiensis YMF3.862 strain was inoculated onto the slant of solid culture medium in test tubes using ISP4 medium and cultured at 28-36℃ for 4-7 days to obtain the slant strain; (2) Dispense ISP4 liquid culture medium into 500 ml Erlenmeyer flasks, with a liquid volume of 300 ml / flask. After sterilization according to conventional microbial methods, use an inoculation needle to pick up YMF3.862 cells from the slant of the test tube in a clean bench and inoculate them into the culture medium. After culturing at 25-30℃ and 130-200 rpm for 10-15 days, the fermentation culture of strain YMF3.862 is obtained.

5. A method for preparing the compound cis-3-indoleacrylic acid, characterized in that: The culture of strain YMF3.862 described in claim 1 was extracted with ethyl acetate. The resulting organic phases were combined and concentrated using a rotary evaporator at 40°C. The concentrate was then subjected to Sephadex LH-20 column chromatography with methanol as the eluent to obtain a component active against southern root-knot nematode and tobacco wilt pathogen. The active component was further purified by silica gel column chromatography with petroleum ether / methanol as the eluent to obtain the compound cis-3-indoleacrylic acid. cis The structural formula of 3-indoleacrylic acid is shown in formula (I): （I） 。 6. A biological agent, characterized in that: A solution containing the compound cis-3-indoleacrylic acid as described in claim 5.

7. The biological agent according to claim 6, characterized in that: The biological agent is obtained by dissolving the compound cis-3-indoleacrylic acid in methanol, mixing it with water, and then adding Tween-20, wherein the content of cis-3-indoleacrylic acid is ≥10%, the content of methanol is ≤5%, and the content of Tween-20 is ≤0.3%.

8. The application of the biological agent according to claim 6 in the prevention and control of tobacco root-knot nematode disease, tobacco bacterial wilt, and the combined diseases caused by the two.

9. The application according to claim 8, characterized in that: The cis-3-indoleacrylicacid in the biological agent can inhibit the activity of tobacco root-knot nematodes and bacterial wilt fungus.

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