Banana root exudate 10-hydroxydecanoic acid as well as preparation method and application thereof

Abstract

The invention provides application of banana root exudates or an ethanol extract of the banana root exudates in preparation of 10-hydroxydecanoic acid. The new streptomyces is used for inducing the change of banana root exudates, the key root exudates 10-hydroxydecanoic acid can be obtained through separation, gibberellic disease and the like can be effectively antagonized, but no inhibition effect is achieved on banana fusarium oxysporum fourth physiological race and the like; however, the 10-hydroxydecanoic acid can effectively inhibit the fourth physiological race of the banana wilt pathogen in the banana rhizosphere soil, effectively prevent and treat the banana wilt caused by the fourth physiological race of the banana wilt pathogen, improve the abundance of Sphingomonas bacteria, Bacillus bacteria, Pseudolaabrys bacteria and the like in the banana rhizosphere soil, and improve the yield of the banana wilt disease caused by the fourth physiological race of the banana wilt pathogen and the banana wilt disease caused by the fourth physiological race of the banana wilt pathogen. The invention provides a new method and idea for preventing and controlling fusarium wilt and preventing and controlling wheat gibberellic disease, strawberry colletotrichum gloeosporioides, banana colletotrichum gloeosporioides, litchi colletotrichum gloeosporioides, mango colletotrichum gloeosporioides, grape botrytis

Description

Technical Field

[0001] The invention relates to banana root exudate, in particular to banana root exudate 10-hydroxydecanoic acid and a preparation method and application thereof. Background Art

[0002] banana( Musa spp.) is a plant of the genus Musa in the family Musaceae. It is the most important fruit and grain crop in the world, the second largest fruit and the fourth largest grain crop in the world (Zhang et al., 2023). However, banana wilt disease seriously threatens the banana industry. There are no resistant strains or effective chemical agents. The banana industry was once facing the situation of extinction. In order to solve the industry problem, it took 20 years for the national banana industry technology system to basically achieve the prevention and control of banana wilt disease through a series of measures. To achieve long-term and effective prevention and control of banana wilt disease in the field, cultivating resistant (tolerant) strains and screening antagonistic biocontrol bacteria are two important measures.

[0003] Banana wilt is caused by the Cuban-specific pathogen Fusarium oxysporum ( Fusarium oxysporum f. sp. Cubense , Foc TR4) is a devastating soil-borne disease caused by bananas. Its prevention and control is a global problem. When the disease is severe, it will lead to a sharp decline in the banana planting area. Breeding disease-resistant varieties is the most effective and fundamental way to prevent and control it. However, most of the current edible bananas are triploid and sterile, and parthenocarpy, which limits the effective recombination and exchange of genetic material during the evolution process, resulting in a relatively simple genetic background, which is difficult to improve through sexual hybridization. Therefore, the progress of breeding resistant strains is relatively slow.

[0004] Microecological control based on antagonistic microorganisms is one of the green and effective control measures for Fusarium wilt, and has become a current research hotspot. The project team has been conducting long-term research on banana root exudates. Through the interaction between root exudates and rhizosphere microbial communities, the bottleneck problems of single control measures for Fusarium wilt and poor control effects have been solved. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a banana root exudate 10-hydroxydecanoic acid and a preparation method and application thereof.

[0006] The first aspect of the present invention is to provide Streptomyces Streptomyces Yongxingensis sp. nov., or banana root exudates or banana root exudates ethanol extract in the preparation of 10-hydroxydecanoic acid.

[0007] The banana root exudates described in the context of the present invention are: placing part of the root system of the same banana plant in a nutrient solution inoculated with Streptomyces Yongxingensis sp. nov. and banana wilt pathogen race 4, and placing part of the root system in a sterile nutrient solution, culturing for 1-3 days, then replacing the sterile nutrient solution with sterile water, continuing to culture for 2-4 days, and collecting the liquid corresponding to the sterile water, which is the banana root exudates.

[0008] The second aspect of the present invention is to provide a method for preparing 10-hydroxydecanoic acid, which is separated from banana root exudates.

[0009] Preferably, the specific steps of the preparation method are: (1) concentrating banana root secretions into an extract; (2) dissolving the extract with methanol and then adsorbing it through Diaion HP20 macroporous adsorption resin with MeOH / H 2 O (1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, MeOH) gradient elution was performed, and after detection and comparison with the 10-hydroxydecanoic acid standard, the effective component Fr.A9 containing 10-hydroxydecanoic acid was obtained; (3) The effective component Fr.A9 was passed through an ODS reverse silica gel column and eluted with MeOH / H 2 O (5:5, 6:4,7:3, 8:2, 9:1, MeOH) was used as the mobile phase for elution. After combined detection and comparison with the 10-hydroxydecanoic acid standard, the effective component Fr.B6 containing 10-hydroxydecanoic acid was obtained; (4) The effective component Fr.B6 was subjected to Sephadex LH-20 gel column chromatography and purified by CH 2 Cl 2 / MeOH (2:1) eluent, combined with detection and comparison with 10-hydroxydecanoic acid standard, the effective component Fr.C5 containing 10-hydroxydecanoic acid was obtained; (5) Fr.C5 was passed through an ODS-C18 reverse silica gel column with MeOH / H 2 After elution with O (80:20) as the mobile phase, combined detection and comparison with the 10-hydroxydecanoic acid standard were performed to obtain the effective component Fr.D4-3 containing 10-hydroxydecanoic acid; (6) The component Fr.D4-3 was repeatedly separated and purified by RP-HPLC (RID A:Refractive Index Signal; λ=230) to obtain a high-purity monomer compound H5.

[0010] The third aspect of the present invention is to provide 10-hydroxydecanoic acid prepared by the preparation method described in the second aspect of the present invention.

[0011] A fourth aspect of the present invention is to provide Streptomyces Streptomyces Yongxingensissp. nov., or banana root exudates, or banana root exudate ethanol extract, or 10-hydroxydecanoic acid in the preparation of preparations for antagonizing wheat fusarium sphaerocephala, and / or strawberry anthracnose, and / or banana anthracnose, and / or litchi anthracnose, and / or mango anthracnose, and / or grape gray mold.

[0012] The fifth aspect of the present invention is to provide the use of Streptomyces Yongxingensis sp. nov., or banana root exudates, or banana root exudate ethanol extract, or 10-hydroxydecanoic acid in the preparation of a preparation that antagonizes wheat fusarium sphaeroides, and / or strawberry anthracnose, and / or banana anthracnose, and / or litchi anthracnose, and / or mango anthracnose, and / or grape gray mold but does not antagonize banana wilt pathogenic species I and / or banana wilt pathogenic species IV.

[0013] The sixth aspect of the present invention is to provide the use of Streptomyces Yongxingensis sp. nov., or banana root exudates, or banana root exudate ethanol extract, or 10-hydroxydecanoic acid in the preparation of a preparation for preventing and controlling diseases caused by wheat fusarium, and / or strawberry anthracnose, and / or banana anthracnose, and / or litchi anthracnose, and / or mango anthracnose, and / or grape gray mold.

[0014] The seventh aspect of the present invention is to provide Streptomyces Streptomyces Yongxingensis sp. nov., or banana root exudates, or banana root exudate ethanol extract, or 10-hydroxydecanoic acid in the preparation of a preparation for inhibiting banana wilt pathogenic fungus No. 4 physiological subspecies in banana rhizosphere soil.

[0015] The eighth aspect of the present invention is to provide Streptomyces Streptomyces Yongxingensis sp. nov., or banana root exudates, or banana root exudate ethanol extract, or 10-hydroxydecanoic acid in the preparation of a preparation for preventing and controlling banana wilt disease caused by banana wilt pathogenic fungus race 4.

[0016] The ninth aspect of this method maze is to provide Streptomyces Streptomyces Yongxingensis sp. nov., or banana root exudates, or banana root exudate ethanol extract, or 10-hydroxydecanoic acid in the preparation of banana rhizosphere soil to improve Sphingomonas Bacteria, and / or Bacills Bacteria, and / or Pseudolabrys Application of preparations with high abundance of bacterial genus.

[0017] Beneficial effects of the present invention: The invention uses a new species of Streptomyces Yongxingensis sp. nov. to induce changes in banana root secretions, and can separate and obtain the key root secretion 10-hydroxydecanoic acid. The 10-hydroxydecanoic acid can effectively antagonize wheat fusarium, strawberry anthracnose, banana anthracnose, litchi anthracnose, mango anthracnose, grape gray mold, etc., but has no inhibitory effect on banana wilt pathogenic species I and banana wilt pathogenic species IV. However, the 10-hydroxydecanoic acid can effectively inhibit banana wilt pathogenic species IV in banana rhizosphere soil, effectively prevent and control banana wilt caused by banana wilt pathogenic species IV, and improve the banana rhizosphere soil. Sphingomonas Bacteria, Bacills Bacteria, Pseudolabrys The abundance of fungi, etc., the present invention provides a new method and idea for the prevention and control of wilt disease, and also provides a new method and idea for the prevention and control of wheat fusarium wilt, strawberry anthracnose, banana anthracnose, litchi anthracnose, mango anthracnose, grape gray mold, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Flowchart for banana root exudate collection.

[0019] Figure 2 Dig in for critical root exudates.

[0020] Figure 3 It is the structure of the compound 10-hydroxydecanoic acid.

[0021] Figure 4 This is the hydrogen spectrum of the compound 10-hydroxydecanoic acid.

[0022] Figure 5 This is the carbon spectrum of the compound 10-hydroxydecanoic acid.

[0023] Figure 6 This is the result of the inhibitory effect of 10-hydroxydecanoic acid on plant pathogens.

[0024] Figure 7 The results show that 10-hydroxydecanoic acid has a preventive and therapeutic effect on banana wilt disease in potted banana seedlings.

[0025] Figure 8 Effect of 10-hydroxydecanoic acid on banana rhizosphere soil Foc Effects of TR4 colonization.

[0026] Fig. 9 Effects of 10-hydroxydecanoic acid on the rhizosphere microbiome of banana. DETAILED DESCRIPTION

[0027] The present invention will be further described below with reference to the accompanying drawings in conjunction with specific embodiments to better understand the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. Where the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be obtained commercially.

[0028] 1 Test materials

[0029] 1.1 Test pathogens Fusarium wilt race 4 Fusarium oxysporum f. sp. Cubense tropicalRace 4 ( Foc TR4, ATCC 76255), Fusarium wilt race 1 F. oxysporum f. sp. cubense Race 1 (ACCC 31271) Foc 1); Wheat fusarium wilt pathogen Fusarium graminearum Schwabe (ATCC MYA-4620); Colletotrichum schwabe Colletotrichum fragariae Brooks (ATCC58718); banana anthracnose Colletotrichum musae (ACCC 96167), Colletotrichum gloeosporioides (Penz) Saec (ATCC MYA-456), Colletotrichum gloeosporioides (Penz) Saec (ATCC MYA-456) Colletotrichum musae (ATCC 96167); Botrytis cinerea Botrytis cinerea Persoon (ATCC11542). The above pathogens were all strains preserved in our laboratory.

[0030] 1.2 Main Reagents The main reagents used in this experiment are shown in Table 1.

[0031] Table 1 Main reagents and sources Reagent name source Bacterial Genomic DNA Rapid Extraction Kit Beijing Biotech Biotechnology Co., Ltd. 2×Es Taq Master Mix (Dye) Biomed Biopharm DNA marker (Maker D2000) Beijing Kangwei Century Biotechnology Co., Ltd. Nucleic acid stains GoldView 99.8% Potassium dichromate Guangzhou Chemical Reagent Factory Nystatin Shanghai Jinsui Biotechnology Co., Ltd. Streptomycin sulfate Solarbio Rifampicin 95% Solarbio

[0032] 1.3 Test instruments and equipment The main instruments and equipment required for this experiment are shown in Table 2.

[0033] Table 2 Instruments and equipment name model Manufacturer Biochemical incubator SPX-150 Beijing Hengrui Tianchuang Mechanical and Electrical Equipment Co., Ltd. pH meter Delta 320 Mettler-Toledo Instruments Constant temperature water bath HHS-11-2 Hangzhou Huier Instrument Co., Ltd. High Pressure Steam Sterilizer HVE-2510 HIRAYAMA,Japan PCR Amplifier TAdvanced 96 SG Biometra, Germany Gel imaging analysis system UVP EC3 UVP, USA Horizontal electrophoresis apparatus HR / 022 Beijing Hengrui Tianchuang Mechanical and Electrical Equipment Co., Ltd. Scanning electron microscopy Sigma VP Zeiss, Germany Electric heating blast drying oven DHG-9140A Shanghai Yiheng Scientific Instrument Co., Ltd. Single-person single-sided horizontal clean workbench SW-CF-1F Suzhou Su Clean Equipment Co., Ltd. Desktop Refrigerated Centrifuge ST16R Thermo Company of Germany Laser confocal fluorescence microscopy ZEISS Axio Scope A1 Carl Zeiss AG

[0034] 1.4 Main culture medium The main culture media tested in this study are shown in Table 3.

[0035] Table 3 Main culture media and formulations Culture medium name Medium formulation Potato Dextrose Agar (PDB) Potato 200.0g, glucose 20.0g, water 1000mL. LB medium Yeast powder 5g, tryptone 10g, NaCl 10g, agar 20g, water 1000mL, pH 7.2~7.5

[0036] 1.5 Banana seedlings for testing The banana tissue culture seedlings all have relatively uniform growth and are healthy Brazilian banana cup seedlings in the 5-6 leaf stage, provided by the Danzhou Tissue Culture Center of the Chinese Academy of Tropical Agricultural Sciences.

[0037] 1.6 Test soil The soil used in the experiment was red soil from a banana plantation in Danzhou City, Hainan Province (109°54'66"E, 19°44′63"N).

[0038] 2 Test methods and results

[0039] 2.1 Experimental design, root exudate collection, and metabolomics (LC-MS) analysis Previous studies have found that Streptomyces Streptomyces Yongxingensis sp. nov. (deposited in China Center for Type Culture Collection (CCTCC) on March 30, 2021, with the deposit number CCTCC NO: M 2021303, and its characteristics refer to Chinese patent CN202111205925.6) can induce changes in banana root secretions and produce new substances that resist banana wilt. Therefore, this study intends to use a hydroponic root splitting system to collect banana root secretions under controlled conditions ( Figure 1 The root system is divided into two independent culture chambers, the left chamber is used for inoculation of microorganisms (the spore liquid of the strain can be directly added to the culture liquid), and the right chamber is used for collecting root secretions. The roots of a seedling are divided into two parts, and placed in the left and right chambers respectively. Hoagland's nutrient solution is used for initial culture. After the left chamber is inoculated with microorganisms ( Foc The inoculum concentration of TR4 was 1.0×10 5 cfu / mL, the inoculation concentration of Streptomyces Sy was 1.0×10 7 cfu / mL), cultured at 28°C for 48 h with a day / night ratio of 16 / 8; the nutrient solution in the right chamber was replaced with sterile water, cultured for 72 h and the liquid in the right chamber was collected as root exudates, freeze-dried and then subjected to metabolomics (LC-MS) determination and analysis. There were two treatments in the experiment: inoculation Foc TR4 blank control and simultaneous inoculation Foc TR4+Streptomyces Streptomyces Yongxingensis sp. nov. treatment group. Each treatment group had 6 biological replicates, and 10 banana seedlings were collected from each replicate to collect root exudates.

[0040] A liquid chromatography-mass spectrometry system (LC-MS) consisting of a Waters UPLC Acquity I-Class PLUS ultra-high performance liquid phase coupled with a Waters UPLC XevoG2-XS QTOF high-resolution mass spectrometer was selected as the instrument platform for metabolomics analysis. Mass spectrometry data were collected under the control of the acquisition software (MassLynx V4.2, Waters). The collected raw data were imported into the processing software Progenesis QI for baseline filtering, peak identification, integration, retention time correction, peak alignment, etc., and finally a data matrix of retention time, mass-to-charge ratio and peak intensity was obtained. The software was then used to search the library for characteristic peaks, match the mass spectrometry information with the metabolic database, and set the mass spectrometry mass error to less than 10 ppm. At the same time, metabolites were identified based on the secondary mass spectrometry matching score, and the relative concentration of each metabolite was calculated using the internal standard normalization method. All qualitative data were imported into the SIMCA 14.1 software for Pareto-scaling normalization, combined with univariate statistical analysis (Student's t-test, P < 0.01) and fold change (FC > 2) were used to screen for differential metabolites or root exudates.

[0041] use Figure 1 Flowchart method, root exudates were collected and metabolome sequencing was performed, a total of 542 root exudates were identified, and the differential metabolites produced between the two treatments were compared and analyzed, and 198 differential metabolites were screened, of which 98 were down-regulated and 100 were up-regulated in the treatment ( Figure 2 To further screen the key secretory metabolites, we filtered out the metabolites with low abundance (peak < 1e-5), and combined with the parameters (|log2FC| ≥ 2 & VIP ≥ 1 & p-value ≤ 0.05), we identified a total of 20 candidate metabolites ( Figure 2 b). Based on the content and functional analysis of key metabolites, a candidate key root exudate 10-hydroxycapric acid was located. 10-hydroxycapric acid is a white powder with a molecular formula of C 10 H 20 O 3 , its planar structure, such as Figure 3 10-Hydroxydecanoic acid standard (purity ≥ 99.9%) was purchased from Dingxiangtong for subsequent experiments.

[0042] 2.2 Isolation and purification of key root exudate 10-hydroxydecanoic acid The root exudates of the right chamber were collected based on the method in 2.1 to obtain 60 L of root exudate solution, which was concentrated under reduced pressure at 45°C to an extract. The extract was dissolved in methanol and passed through Diaion HP20 macroporous adsorption resin with MeOH / H 2 O (1:9, 2:8,3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, MeOH) gradient elution, 10 components (Fr.A1 ~Fr.A10) were obtained. After TLC detection and comparison with the TLC of 10-hydroxydecanoic acid standard, the effective component Fr.A9 (6.3221 g) containing 10-hydroxydecanoic acid was obtained. The effective component Fr.A9 was passed through an ODS reverse silica gel column and eluted with MeOH / H 2 After elution with O (5:5, 6:4, 7:3,8:2, 9:1, MeOH) as the mobile phase, 65 tubes of fractions were collected, and the fractions containing 10-hydroxydecanoic acid were detected by TLC. The fractions were combined to obtain 1.8657 g of the effective component Fr.B6. Fr.B6 was purified by Sephadex LH-20 gel column chromatography and purified by CH 2 Cl 2 / MeOH (2:1) eluent, a total of 42 tubes of fractions were collected, and the fractions containing 10-hydroxydecanoic acid were determined by TLC and HPLC detection, and the effective component Fr.C5 (1.1343 g) was obtained by merging. Fr.C5 was passed through an ODS-C18 reverse silica gel column with MeOH / H 2 After elution with O (80:20) as the mobile phase, the fractions containing 10-hydroxydecanoic acid were determined by TLC and HPLC and combined to obtain the effective component Fr.D4-3 (0.5108), which was then purified by RP-HPLC in the later stage.

[0043] Component Fr.D4-3 was repeatedly separated and purified by RP-HPLC (RID A:Refractive Index Signal; λ=230) to obtain high-purity monomer compound H5 (63.85 mg, MeOH:H 2 O =80:20, 2 mL / min, tR =36 min), use CD 2 After OD dissolution, the sample was sent to 600M NMR for spectrum scanning and structural identification.

[0044] Compound H5 is a white powder with a molecular formula of C 10 H 20 O 3 According to the NMR spectrum of the compound ( Figure 4-5), and compared with the literature, it was found that the spectral data of compound H5 was consistent with the literature report, and compound H5 was determined to be 10-hydroxycapric acid. The planar structure of the compound was analyzed by 2D-NMR spectrum, such as Figure 3 shown.

[0045] 2.3 Broad-spectrum antifungal activity of 10-hydroxydecanoic acid In order to evaluate the broad-spectrum antifungal activity of 10-hydroxydecanoic acid, the growth rate method was used to determine the broad-spectrum antifungal activity of 8 plant pathogens. 10-Hydroxydecanoic acid dissolved in DMSO (20.0 mg / ml) was added to PDA medium at 45-50°C to prepare a PDA plate with a final concentration of 10 mg / ml, and an equal amount of DMSO was added as a control. Foc TR4, Foc The fungal cakes (Φ=5 mm) of TR1, wheat fusarium sphaeroides, strawberry anthracnose, banana anthracnose, litchi anthracnose, mango anthracnose, and grape gray mold were inoculated in the center of different plates and cultured at 28 ± 2 °C until the control mycelium reached the edge of the plate. The average vertical diameter of each colony was determined. Each treatment was repeated 3 times. The formula for calculating the mycelium growth inhibition rate is as follows (Nimaichand et al., 2015):

[0046] Where: C is the average diameter of the colonies in the control group, and T is the average diameter of the colonies in the treatment group.

[0047] The results are as follows Figure 6 As shown in the results, there were significant differences in the inhibitory effects of 10-hydroxydecanoic acid on different pathogenic fungi. The inhibition rate of 10-hydroxydecanoic acid on litchi anthracnose was 53.94 ± 0.72%, which was significantly higher than that of the other 7 pathogenic fungi. However, it had no antibacterial activity against banana wilt species 1 and 4; the inhibitory activity against mango anthracnose was also low, with an inhibition rate of 44.48 ± 0.68%, which was not significantly different from the inhibitory effects of wheat fusarium head blight, strawberry anthracnose, banana anthracnose, litchi anthracnose and grape gray mold. These results show that 10-hydroxydecanoic acid has antagonistic effects on plant pathogenic fungi such as wheat fusarium head blight, strawberry anthracnose, banana anthracnose, litchi anthracnose, mango anthracnose and grape gray mold, but has no inhibitory effect on banana wilt.

[0048] 2.4 Control effect of root exudate 10-hydroxydecanoic acid on banana wilt

[0049] 2.4.1 Potted experiment The pot experiment was conducted at the Sanya Research Institute of the Chinese Academy of Tropical Agricultural Sciences from April to June 2024. The greenhouse conditions were 28°C, 70% humidity, and natural light. Healthy soil from banana gardens was collected from Danzhou City, Hainan Province, and sieved through 20 mesh. Banana seedlings with uniform growth and 3-4 leaves were selected, rinsed with sterile water, and planted in plastic pots containing 1000 g of soil, with 30 plants in each treatment. The experiment set up 3 treatment groups: Control (uninoculated Foc TR4, sterile water; Treatment (inoculation Foc TR4, Streptomyces Yongxingensis sp. nov. 2-11, deposit number: CCTCC NO: M 2021303, positive control); 10-hydroxycapric acid (inoculation Foc TR4, 1 mM 10-hydroxydecanoic acid). Each treatment was repeated three times.

[0050] 2.4.2 Disease Index Statistics According to Himaman et al., (2016), 30 banana seedlings were selected from each group to evaluate the disease index (DI) 49 days after inoculation. It is divided into 5 levels, calculated by the proportion of yellowing leaves in a plant, level 0: healthy plant, level 1: 1-25% yellowing leaves, level 2: 26-50% yellowing leaves, level 3: 51-75% yellowing leaves, level 4: more than 75% yellowing leaves. The calculation formula of the disease index (DI) of banana wilt is as follows:

[0051] Effect of 10-hydroxydecanoic acid on the control of banana wilt disease in potted banana seedlings Figure 7 The control group had a morbidity rate of 78.33% after 49 days, with severe disease and obvious wilt of the seedlings. However, after the banana seedlings were treated with strain 2-11 and 10-hydroxydecanoic acid, the disease symptoms were significantly suppressed and the seedlings were healthy and disease-free ( Figure 7 a in the figure). Compared with the control group (contrlo), the disease index of strain 2-11 treatment (Treatment) and 10-hydroxydecanoic acid (10-hydroxydecanoic acid) was reduced to 19.16% and 19.88% respectively after 49 days of treatment, and there was no significant difference between the two ( Figure 7 b). This indicates that 10-hydroxydecanoic acid has a good control effect on banana wilt and can inhibit the infection of banana wilt pathogen.

[0052] 2.4.3 In soil Foc TR4 quantitative detection The rhizosphere soil samples of potted plants were collected for 49 days for the quantitative detection of banana wilt pathogens. The rhizosphere soil suspension was serially diluted on plates containing modified Komada selective medium to determine the banana wilt pathogens ( Foc TR4) Colony forming units (cfu / g). Colony forming units (cfu / g) = average number of colonies per plate × dilution factor × 5 / soil weight.

[0053] The results showed that the banana rhizosphere soil under different treatments Foc The colonization of TR4 is as follows Figure 8 The results showed that in the Control group Foc TR4 had the largest number of pathogens, reaching 5.58×10 4 CFU / g, while after treatment with strain 2-11 and 10-hydroxydecanoic acid, the number of banana rhizosphere pathogens was significantly reduced to 1150 CFU / g and 950 CFU / g, respectively. The results show that 10-hydroxydecanoic acid can inhibit Foc The colonization of TR4 in the banana rhizosphere has a good preventive effect against banana wilt disease.

[0054] 2.5 Effects of root exudate 10-hydroxydecanoic acid on the banana rhizosphere microbiome

[0055] 2.5.1 Soil DNA extraction and PCR amplification According to EZNA ® Soil DNA Kit Protocol (Omega Bio-tek, Norcross, GA, US) was used to extract soil microbial total DNA, and the total DNA concentration was measured using a NanoDrop 2000 UV-vis spectrophotometer (Thermo Scientific, Wilmington, USA). DNA integrity was tested by 1% agarose gel electrophoresis. The extracted soil total DNA was used as a template and a thermal cycle PCR system (GeneAmp 9700, ABI, USA) was used to amplify the bacterial 16S rRNA gene V3-V4 region using bacterial universal primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') / 806R (5'-GGACTACHVGGGTWTCTAAT-3'). The bacterial 16S rRNA polymerase chain reaction was performed with the following program: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 45 s, 27 cycles, and a final extension at 72°C for 10 min.

[0056] 2.5.2 High-throughput (Illumina MiSeq) sequencing Purified DNA fragments were pooled in equimolar amounts and subjected to paired-end sequencing (2 × 300) using the Illumina MiSeq platform (Illumina, San Diego, USA) according to the standard method of Majorbio Bio Pharm Technology Co. Ltd. (Shanghai, China).

[0057] 2.5.3 Sequence processing and bioinformatics analysis Raw fastq files were decomposed, quality filtered by Trimmomatic, and merged by FLASH with the following criteria: (i) reads were truncated at any position that received an average quality score <20 over a 50 bp sliding window; (ii) primers were perfectly matched, 2-nucleotide mismatches were allowed, and reads containing ambiguous bases were removed; and (iii) sequences with overlap lengths greater than 10 bp were merged based on overlapping sequences. Operational taxonomic units (OTUs) were clustered using UPARSE (version 7.1 http: / / drive5.com / UPARSE / ) at 97% similarity, and chimeric sequences were identified and removed using UCHIME. Classification analysis of each 16srRNA gene sequence and sequences were analyzed against the Silva (SSU123) 16srRNA database, and fungal ITS sequences were analyzed using the RDP classifier algorithm (http: / / RDP.cme.msu.edu / ) with a confidence threshold of 70%.

[0058] In order to study the effect of 10-hydroxydecanoic acid on the banana rhizosphere microbiome, high-throughput sequencing was performed on the banana rhizosphere soil. The results showed that the microbiome analysis found that there was no significant difference in the α diversity index between the 10-hydroxydecanoic acid and strain 2-11 treatments, but it was significantly different from the control group ( Fig. 9 a in ). β The same is true for diversity, with the 10-hydroxydecanoic acid and strain 2-11 treatment groups located on the right side of PCoA1 (48.01%), while the control group was on the left ( Fig. 9 b), which suggests that the 10-hydroxydecanoic acid and strain 2-11 inoculant treatments have more similar microbial compositions. Sphingomonas Genus (Sphingomonas), Bacills Genus (Bacillus) and Pseudolabrys Genus (Pseudomonas), and in the 10-hydroxydecanoic acid and strain 2-11 groups, the levels were significantly higher than those in the control group ( Fig. 9c in the figure). This also shows that 10-hydroxydecanoic acid can induce directional assembly of microorganisms, and the assembly direction is similar to that of strain 2-11 treated with bacterial agents. By comparison, it was found that Bacillus was more abundant in the 10-hydroxydecanoic acid treatment group and was also the genus with the largest change fold difference ( Fig. 9 in de).

[0059] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. Use of banana root exudates or banana root exudates ethanol extract in the preparation of 10-hydroxydecanoic acid, wherein: The banana root exudates are prepared by placing part of the root system of the same banana plant in a nutrient solution inoculated with Streptomyces Yongxingensis sp. nov. and banana wilt pathogenic bacteria No. 4 physiological species, and placing part of the root system in a sterile nutrient solution, culturing for 1-3 days, then replacing the sterile nutrient solution with sterile water, continuing to culture for 2-4 days, and collecting the liquid corresponding to the sterile water, which is the banana root exudates.

2. A method for preparing 10-hydroxydecanoic acid, characterized in that: The method is separated from banana root secretions. The banana root secretions are obtained by placing part of the root system of the same banana plant in a nutrient solution inoculated with Streptomyces Yongxingensissp. nov. and banana wilt pathogenic bacteria No. 4 physiological species, and placing part of the root system in a sterile nutrient solution, culturing for 1-3 days, then replacing the sterile nutrient solution with sterile water, continuing to culture for 2-4 days, and collecting the liquid corresponding to the sterile water, which is the banana root secretions.

3. The preparation method according to claim 2, characterized in that: The specific steps are: (1) Banana root exudates are concentrated into extracts; (2) The extract was dissolved in methanol and then eluted with Diaion HP20 macroporous adsorption resin using MeOH / H2O (1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, MeOH) gradient. After detection and comparison with the 10-hydroxydecanoic acid standard, the effective component Fr.A9 containing 10-hydroxydecanoic acid was obtained; (3) The effective component Fr.A9 was eluted by ODS reverse silica gel column with MeOH / H2O (5:5, 6:4, 7:3, 8:2, 9:1, MeOH) as the mobile phase. After combined detection and comparison with 10-hydroxydecanoic acid standard, the effective component Fr.B6 containing 10-hydroxydecanoic acid was obtained; (4) The effective component Fr.B6 was eluted by Sephadex LH-20 gel column chromatography with CH2Cl2 / MeOH (2:1) eluent, and after combined detection and comparison with the 10-hydroxydecanoic acid standard, the effective component Fr.C5 containing 10-hydroxydecanoic acid was obtained; (5) Fr.C5 was eluted by ODS-C18 reversed phase silica gel column with MeOH / H2O (80:20) as the mobile phase, and then detected and compared with the 10-hydroxydecanoic acid standard to obtain the effective component Fr.D4-3 containing 10-hydroxydecanoic acid; (6) Component Fr.D4-3 was repeatedly separated and purified by RP-HPLC (RID A:Refractive Index Signal; λ=230) to obtain a high-purity monomer compound H5.

4. 10-hydroxydecanoic acid obtained by the preparation method according to claim 2 or 3.

5. Use of banana root exudates, or banana root exudate ethanol extract, or 10-hydroxydecanoic acid in the preparation of a preparation for antagonizing wheat fusarium, and / or strawberry anthracnose, and / or banana anthracnose, and / or litchi anthracnose, and / or mango anthracnose, and / or grape gray mold; wherein, The banana root exudates are prepared by placing part of the root system of the same banana plant in a nutrient solution inoculated with Streptomyces Yongxingensis sp. nov. and banana wilt pathogenic bacteria No. 4 physiological species, and placing part of the root system in a sterile nutrient solution, culturing for 1-3 days, then replacing the sterile nutrient solution with sterile water, continuing to culture for 2-4 days, and collecting the liquid corresponding to the sterile water, which is the banana root exudates.

6. Use of banana root exudates, or banana root exudate ethanol extract, or 10-hydroxydecanoic acid in the preparation of a preparation that is antagonistic to wheat fusarium head blight, and / or strawberry anthracnose, and / or banana anthracnose, and / or litchi anthracnose, and / or mango anthracnose, and / or grape gray mold, but not antagonistic to banana wilt pathogen race 1 and / or banana wilt pathogen race 4; wherein, The banana root exudates are prepared by placing part of the root system of the same banana plant in a nutrient solution inoculated with Streptomyces Yongxingensis sp. nov. and banana wilt pathogenic bacteria No. 4 physiological species, and placing part of the root system in a sterile nutrient solution, culturing for 1-3 days, then replacing the sterile nutrient solution with sterile water, continuing to culture for 2-4 days, and collecting the liquid corresponding to the sterile water, which is the banana root exudates.

7. Use of banana root exudates, or banana root exudate ethanol extract, or 10-hydroxydecanoic acid in the preparation of a preparation for preventing and controlling diseases caused by wheat fusarium, and / or strawberry anthracnose, and / or banana anthracnose, and / or litchi anthracnose, and / or mango anthracnose, and / or grape gray mold; wherein: The banana root exudates are prepared by placing part of the root system of the same banana plant in a nutrient solution inoculated with Streptomyces Yongxingensis sp. nov. and banana wilt pathogenic bacteria No. 4 physiological species, and placing part of the root system in a sterile nutrient solution, culturing for 1-3 days, then replacing the sterile nutrient solution with sterile water, continuing to culture for 2-4 days, and collecting the liquid corresponding to the sterile water, which is the banana root exudates.

8. Use of banana root exudates, or banana root exudate ethanol extract, or 10-hydroxydecanoic acid in the preparation of a preparation for inhibiting banana wilt pathogenic bacteria No. 4 in banana rhizosphere soil; wherein, The banana root exudates are prepared by placing part of the root system of the same banana plant in a nutrient solution inoculated with Streptomyces Yongxingensis sp. nov. and banana wilt pathogenic bacteria No. 4 physiological species, and placing part of the root system in a sterile nutrient solution, culturing for 1-3 days, then replacing the sterile nutrient solution with sterile water, continuing to culture for 2-4 days, and collecting the liquid corresponding to the sterile water, which is the banana root exudates.

9. Use of banana root exudates, or banana root exudate ethanol extract, or 10-hydroxydecanoic acid in the preparation of a preparation for preventing and treating banana wilt disease caused by banana wilt pathogen race 4; wherein: The banana root exudates are prepared by placing part of the root system of the same banana plant in a nutrient solution inoculated with Streptomyces Yongxingensis sp. nov. and banana wilt pathogenic bacteria No. 4 physiological species, and placing part of the root system in a sterile nutrient solution, culturing for 1-3 days, then replacing the sterile nutrient solution with sterile water, continuing to culture for 2-4 days, and collecting the liquid corresponding to the sterile water, which is the banana root exudates.

10. Streptomyces Yongxingensis sp. nov., or banana root exudates, or banana root exudate ethanol extract, or 10-hydroxydecanoic acid in the preparation of banana rhizosphere soil to improve Sphingomonas Bacteria, and / or Bacills Bacteria, and / or Pseudolabrys Application in preparations with high abundance of bacteria; wherein, The banana root exudates are prepared by placing part of the root system of the same banana plant in a nutrient solution inoculated with Streptomyces Yongxingensis sp.nov. and banana wilt pathogenic bacteria No. 4 physiological species, and placing part of the root system in a sterile nutrient solution, culturing for 1-3 days, then replacing the sterile nutrient solution with sterile water, continuing to culture for 2-4 days, and collecting the liquid corresponding to the sterile water, which is the banana root exudates.

Images