Use of isocitric acid in preventing and treating plant bacterial wilt

Abstract

This invention discloses the application of isocitric acid in the control of bacterial wilt in plants, wherein the isocitric acid is derived from tomato root exudates. The inhibitory effect of isocitric acid on *Ralstonia solanacearum* was evaluated through shake-flask culture experiments and plate inhibition zone experiments, and the control effect of isocitric acid on bacterial wilt was determined using greenhouse pot experiments. The results showed that within the concentration range of 10–200 μM, isocitric acid significantly inhibited the growth of *Ralstonia solanacearum*, forming a distinct inhibition zone. Under greenhouse pot conditions, applying 0.2–0.5 mg / kg of isocitric acid to the soil effectively reduced the incidence of bacterial wilt in tomatoes, with a disease control rate of 21.43%–52.38%. Furthermore, isocitric acid can indirectly inhibit the occurrence of bacterial wilt by influencing the soil bacterial community. The isocitric acid used in this invention is derived from natural plant root exudates, possessing advantages such as environmental friendliness and high safety, and can be used as a novel inhibitor of tomato bacterial wilt, showing promising application prospects in the field of green control of plant diseases.

Description

Technical Field

[0001] This invention belongs to the field of plant disease control technology, specifically relating to the application of isocitric acid in inhibiting bacterial wilt in plants. Background Technology

[0002] Bacterial wilt is a disease caused by Rhesus spp. (Solanaceae family) Ralstonia solanacearum Bacterial wilt, a plant disease caused by pathogens, poses a significant threat to the production of economic crops such as tomatoes. This pathogen can invade the vascular bundles through the plant roots and rapidly multiply within the xylem, causing blockage of the xylem vessels and hindering water transport, ultimately leading to typical bacterial wilt symptoms. In the early stages, the leaves turn pale, gradually wilt, then yellow and wither. The newest leaves at the top wilt and droop first, followed by the lower leaves, and in severe cases, the entire plant dies rapidly. Existing control measures mainly include chemical pesticides, breeding resistant varieties, agricultural cultivation practices, and biological control. However, long-term use of chemical pesticides poses risks such as environmental pollution and the development of pathogen resistance; breeding resistant varieties has a long cycle; and biological control is unstable and easily affected by the environment. Therefore, developing safe, efficient, and environmentally friendly stable control measures for bacterial wilt is of great importance.

[0003] In recent years, the role of plant root exudates in plant-microbe interactions has received widespread attention. Plant roots can release a variety of low-molecular-weight compounds into the rhizosphere, including organic acids, amino acids, sugars, phenols, and secondary metabolites. These substances not only participate in plant nutrient absorption and signal transduction but also regulate the structure of the rhizosphere microbial community, thereby affecting the plant's resistance to pathogens. Isocitric acid is a naturally occurring intermediate metabolite of the tricarboxylic acid cycle (TCA cycle), widely found in plant tissues, microbial cells, and various natural metabolic systems. Studies have shown that isocitric acid participates in energy metabolism, carbon metabolism, and various physiological regulatory processes in plant metabolism, and is also an important component of plant root exudates. Due to its natural source, low toxicity, and easy degradation, isocitric acid has broad application prospects in the food, pharmaceutical, and biotechnology fields. However, current research on the use of isocitric acid in plant disease control is still limited, particularly regarding its role in inhibiting the growth of Ralstonia solanacearum and controlling bacterial wilt of tomato. This invention analyzes the metabolites of tomato root exudates and finds that they contain isocitric acid. Furthermore, it verifies that isocitric acid has a significant inhibitory effect on Ralstonia solanacearum, thus providing a new technical approach for controlling tomato bacterial wilt using natural plant metabolites. Summary of the Invention

[0004] The purpose of this invention is to provide the application of isocitric acid in inhibiting the growth of Ralstonia solanacearum and controlling bacterial wilt of tomato, so as to overcome the shortcomings of existing control technologies.

[0005] Specifically, this application identified plant root exudates using gas chromatography-mass spectrometry (GC-MS) and found that isocitric acid in them has the application of preventing and controlling bacterial wilt in plants.

[0006] Specifically, in application, an isocitric acid aqueous solution is first prepared, and then the isocitric acid aqueous solution is applied by root irrigation.

[0007] When applying, add the solution to the roots 5-9 days after transplanting the seedlings.

[0008] The amount of isocitric acid added is 0.2~0.45 mg / kg soil, preferably 0.215~0.430 mg / kg soil. Preferably, the concentration of the isocitric acid aqueous solution is 100~200 μM.

[0009] The plant in question is a member of the Solanaceae family, preferably a tomato. This application has discovered that isocitrate has an effect on Ralstonia solanacearum (…). Ralstonia solanacearum It has an inhibitory effect.

[0010] The present invention also proposes the application of isocitric acid in the preparation of formulations for inhibiting Ralstonia solanacearum in tomatoes.

[0011] Furthermore, this invention proposes a method for controlling bacterial wilt in plants, specifically, using isocitric acid solution to drench the roots of the plants.

[0012] The plant in question is a member of the Solanaceae family, and preferably, the plant is a tomato.

[0013] When applying this solution, add isocitric acid solution to the root irrigation 5-9 days after the tomatoes are transplanted.

[0014] When applying, the amount of isocitric acid added is 0.2~0.45 mg / kg soil, preferably 0.215~0.430 mg / kg soil. Preferably, the concentration of the isocitric acid aqueous solution is 10~200 μM, more preferably 100~200 μM.

[0015] The isocitric acid of the present invention has the following characteristics: (1) Isocitric acid can significantly inhibit the growth of Ralstonia solanacearum in the culture system, and the inhibitory effect increases with increasing concentration.

[0016] (2) Isocitric acid formed a clear inhibition zone against Ralstonia solanacearum on solid culture medium, indicating that it has good antibacterial activity.

[0017] (3) Applying isocitric acid under greenhouse pot conditions can significantly reduce the incidence of bacterial wilt in tomatoes.

[0018] (4) Isocitric acid can indirectly reduce the incidence of bacterial wilt in tomatoes by inducing changes in the composition of soil microbial communities. Attached Figure Description

[0019] Figure 1 shows the composition of tomato root exudates analyzed by GC-MS; Figure 2 shows the inhibitory effect of isocitric acid on the growth of Ralstonia solanacearum in a 96-well plate; Figure 3 shows the antagonistic zone of isocitrate against Ralstonia solanacearum on solid culture medium; Figure 4 shows the control effect of isocitrate on bacterial wilt of tomato; Figure 5 shows the effect of isocitrate-induced soil microbial community on the incidence of bacterial wilt; Figure 6. Effects of transplanting isocitrate-induced soil microbial communities on the rhizosphere bacterial community of tomato (Principal coordinate axis analysis PCoA plot). Figure 7 shows the effect of transplanting isocitrate-induced soil microbial communities on the composition of the rhizosphere bacterial community in tomatoes. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0021] Existing research on bacterial wilt control mainly focuses on chemical pesticides or antagonistic microorganisms. This invention is the first to discover that isocitric acid, as a natural organic acid, has a significant inhibitory effect on bacterial wilt fungus of tomato and shows good control effect on bacterial wilt under pot conditions.

[0022] Example 1: Isolation and identification of isocitric acid in root exudates of disease-resistant tomato plants.

[0023] The composition of tomato root exudates was analyzed using gas chromatography-mass spectrometry (GC-MS). Pretreatment was performed first: 50 mg of root exudate was dissolved in 1 mL of 75% methanol solution, 20 μL of L-2-chlorophenylalanine was added, and the mixture was vortexed for 30 s. Glass beads were then added, and the mixture was homogenized in a 35 Hz grinder and sonicated at 4°C for 5 min. The sample was then placed in a 2 mL centrifuge tube and centrifuged at 12000 rpm at 4°C for 20 min. 200 μL of the supernatant was transferred to a new 2 mL centrifuge tube. 50 μL of each sample was taken and mixed to prepare a quality control (QC) sample. The sample was dried using a vacuum desiccator, and 60 μL of methoxyamine salt reagent was added. After mixing, the sample was placed in an 80°C oven for 30 min. Each sample was treated with 79.2 μL of trifluoroacetamide (BSTFA) and 0.8 μL of trimethyl thionyl chloride (TMCS), incubated at 70 °C for 1.5 h, cooled to room temperature, and then 5 μL of saturated fatty acid methyl esters (FAMEs) dissolved in chloroform was added. The samples were analyzed using an Agilent 7890 gas chromatography-time-of-flight mass spectrometry (GC-TOF-MS) instrument equipped with an Agilent DB-5MS capillary column (30 m long, 250 μm inner diameter, 0.25 μm film thickness, J&W Scientific, USA).

[0024] Mass spectrometry data analysis was performed using ChromaTOF software (Version 4.3x), including peak extraction, baseline correction, and deconvolution. Qualitative compound analysis was based on the LECO-Fiehn Rtx5 database, employing retention time matching and mass spectrometry matching. In the quality control process, characteristic peaks with an in-sample detection rate of less than 50% or a coefficient of variation greater than 30% were removed.

[0025] The results are as follows Figure 1 As shown, isocitric acid components were isolated and identified from root exudates of tomato plants.

[0026] Example 2: Shake-flask antibacterial experiment of isocitric acid against Ralstonia solanacearum.

[0027] Remove the Ralstonia solanacearum stored at -80℃. Ralstonia solanacearum(CGMCC No. 9487, courtesy of Nanjing Agricultural University) The culture was streaked onto NA agar plates and incubated at 37°C for 1-2 days until single colonies appeared. Single colonies of *Ralstonia solanacearum* were picked and transferred to 5 mL of NA liquid medium and incubated at 200 rpm and 30°C for 16 hours. The culture was centrifuged at 12000 rpm for 3 minutes, the supernatant was discarded, and the bacterial cells were retained. The cells were resuspended in sterile water, and this process was repeated 2-3 times to thoroughly wash away the culture medium. The absorbance of the bacterial suspension at 600 oz was measured using a microplate reader. The solution was then diluted or concentrated to OD100 based on the absorbance reading. 600 =0.5 for later use; effective viable count of diluted or concentrated solutions ≥1×10⁻⁵ 7 CFU / mL. The tested strain was grown in 96-well plates, each well containing 188 μL of 20% NB medium and 2 μL of bacterial suspension (uniform OD). 600 =0.5, approximately 10 7 CFU mL -1 10 μL of different root exudate fractions (sterilized by filtration through a 0.22 μm filter membrane) were used as wells. The final concentrations of the root exudate fractions were: 0 (control), 10, 20, 40, 60, 80, 100, 150, and 200 μM. The 96-well plates were incubated at 200 rpm and 30 °C for 26 h, with each concentration treatment repeated three times. OD was measured using a SpectraMax M5 (Molecular Devices, CA, USA). 600 To determine bacterial growth status, calculate the percentage of growth inhibition or promotion using the following formula: Growth promotion / inhibition percentage = [(Isocitric acid treatment group OD]] 600 Value - Control Group OD 600 (value) / control group OD 600 The value was multiplied by 100%, and a curve showing the relationship between isocitric acid concentration and the growth inhibition rate of Ralstonia solanacearum was plotted. The experimental results showed that, compared with the control group, the growth of Ralstonia solanacearum in the tomato treatment group with added isocitric acid was significantly inhibited, and the inhibitory effect increased with the increase of isocitric acid concentration, indicating that isocitric acid has a significant growth inhibitory effect on Ralstonia solanacearum.

[0028] Example 3: Determination of the antagonistic ability of isocitrate against Ralstonia solanacearum.

[0029] The antibacterial activity of isocitric acid against Ralstonia solanacearum was determined using the plate antagonism method.

[0030] Pre-preparation of Ralstonia solanacearum bacterial suspension for isocitric acid antibacterial activity testing: After culturing Ralstonia solanacearum on NA medium plates for 2 days, the bacterial cells were collected, suspended in deionized water, and OD was prepared. 600 =0.5 (1.0×10 7A bacterial suspension (CFU / mL) was prepared. A 200 μM isocitrate solution was prepared, and 10 μL was spotted into the center of a solid culture medium. After the plate dried, the prepared Ralstonia solanacearum suspension was evenly sprayed onto the solid culture medium. The petri dishes were incubated at 30℃ for 48 h, and the results were observed and recorded. The results are as follows: Figure 3 As shown, isocitric acid has a significant inhibitory effect on the growth of Ralstonia solanacearum.

[0031] Example 4: Greenhouse pot experiment to verify the efficacy of isocitric acid in preventing bacterial wilt of tomato.

[0032] Tomato seeds (variety "Hezuo 906") were soaked in 75% ethanol for 30 seconds, then surface-sterilized with 2.5% sodium hypochlorite solution for 5 minutes, followed by rinsing with deionized water 5–8 times. The seeds were evenly placed on sterile, moistened filter paper and germinated at 30°C for 2 days. 3–5 seeds with relatively uniform germination were selected and sown into the holes of a seedling tray containing 50 g of seedling substrate. After one week of growth, the seedlings were thinned so that there was only one tomato plant in each seedling hole. The seedlings with uniform growth were transplanted into pots (each pot contained 600 g of soil and 4 tomato seedlings, with water added at 60% of the maximum soil moisture content). After one week of growth, each pot was inoculated with 5 mL of isocitric acid solution of different concentrations.

[0033] A total of 3 processing groups were set up: 1) CK: Apply 5 mL of deionized water as a root drench. 2) Treatment group ①: 5 mL of a solution containing 0.129 mg isocitrate (i.e., 0.215 mg / kg soil) was applied by root irrigation. 2) Treatment group ②: 5 mL of a solution containing 0.258 mg isocitrate (i.e., 0.430 mg / kg soil) was applied by root irrigation. Four tomato plants in each pot were considered as one replicate, with three replicates per treatment group. The tissue culture bottles were placed in an artificial climate chamber with a 16-hour light exposure, day and night temperatures of 26 °C and 16 °C respectively, and an air humidity of 65%–70%. Water was added twice a week to maintain consistent soil moisture during the incubation period. The pots were randomly placed and changed weekly. One week after the tomato seedlings began to grow, OD200 was added to the pots. 600 =0.5 (2.0×10 7 A suspension of Ralstonia solanacearum (CFU / mL) was cultured for 4 weeks, and the disease incidence in tomatoes was statistically analyzed.

[0034] The disease severity grading standards for tomato bacterial wilt are as follows (NY / T 1858.4-2010): Grade 0: No symptoms; Grade 1: One leaf wilts; Grade 2: Two to three leaves wilt; Grade 3: Except for the top two to three leaves, all other leaves wilt; Grade 4: All leaves on the entire plant wilt. The formulas for calculating the disease index and control efficacy are as follows: Incidence rate (DI) = ×100%; Biosecurity rate = ×100% Table 1. Biocontrol effect of isocitric acid on bacterial wilt of tomato in greenhouse

[0035] The results showed that applying isocitric acid under greenhouse pot conditions could significantly reduce the incidence of bacterial wilt in tomatoes.

[0036] Example 5: The effect of isocitric acid-induced soil microbial community on the incidence of bacterial wilt.

[0037] The effect of exogenous addition of isocitric acid on the regulation of soil microbial community structure and its influence on the occurrence of bacterial wilt in tomato was evaluated. The tested soil samples were thoroughly mixed and dispensed into tissue culture bottles, with 700 g of soil added to each bottle. The culture conditions were set at 25±2 ℃ and relative humidity of 65%–70%. 0.301 mg of isocitric acid was dissolved in 5 mL of deionized water to prepare an isocitric acid solution. This solution was added to the soil, with a total application of 0.301 mg (0.430 mg / kg soil) of isocitric acid per pot for the treatment group. The control group received 5 mL of deionized water. Soil moisture content was maintained at 60% of its maximum moisture content, with three replicates for both the control and treatment groups. Soil samples were collected after 5 weeks. Soil suspensions were prepared using a soil:deionized water ratio of 1:2 (v:v). Next, add the prepared soil suspension to a pot containing 700 g of sterile soil (sterilized twice at 121℃ for 60 min) at 60% of the maximum soil moisture content. After standing for 1 week to allow the microbial community to stabilize, transplant tomato seedlings with 2-3 true leaves of uniform growth. Three weeks later, inoculate the soil with bacterial wilt pathogen and measure the incidence rate. The incidence rate is as follows: Figure 5 As shown, the incidence rate in the control group was 83.33%, while the incidence rate decreased significantly by 43.75% after the addition of isocitric acid.

[0038] Example 6: Effects of isocitric acid on soil microbial communities.

[0039] In Example 5, after the tomato disease incidence stabilized, rhizosphere soil (the soil layer tightly attached to the plant roots; during collection, the complete plant roots must be carefully removed, and the soil shaken off; the soil attached to the root surface at this point is the rhizosphere soil) was collected. Soil DNA was extracted using a soil DNA extraction kit (TIANamp Soil DNA Kit DP336-02) according to the instructions, and the concentration and quality of the extracted DNA were detected using Nano Drop (Thermo Scientific).

[0040] PCR amplification of the V4-V5 region of 16S rRNA was performed using primers 515F (5'-GTGCCAGCMGCCGCGGTAA-3') and 907R (5'-CCGTCAATTCMTTTRAGTTT-3'). The PCR amplification system (25 μL) contained 5 μL 5× reaction buffer, 5 μL 5× GC buffer, 2 μL 2.5 mM dNTP, 1 μL 10 μM pre-primer, 1 μL 10 μM post-primer, 2 μL DNA template, 8.75 μL ddH2O, and 0.25 μL Q5 DNase. The amplification parameters were: 98℃ pre-denaturation for 2 min, 98℃ denaturation for 15 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, and 72℃ final extension for 5 min, for 25-30 cycles. PCR products were analyzed by agarose gel electrophoresis and Nano Drop micro-nucleic acid quantification, and then sent to Shanghai Paisenuo Biotechnology Co., Ltd. for bidirectional Miseq sequencing. The effects of isocitrate-induced soil microbial communities on the rhizosphere bacterial community structure of tomato were investigated. Figure 6 and 7 As shown, principal component analysis (PCoA) revealed that isocitrate-induced soil microbial community significantly influenced the composition of the tomato rhizosphere bacterial community, with contribution rates of 45.3% and 19.9% ​​for the two principal axes, respectively. Sequencing results identified 24 bacterial phyla, including Proteobacteria (…). Pseudomonadota ), Actinobacteria ( Actinomycetota Bacteroidetes ( Bacteroidota ), Acidobacteria ( Acidobacteriota ), Bacillus phylum ( Gemmatimonadota ) and Myxococci ( Myxococcota Bacteroidetes, accounting for 92.7% of the total sequence count, were the dominant phylum. Independent samples t-tests showed that the relative abundance of Bacteroidetes, Acidobacteria, and Bacillus in the isocitrate-treated group was significantly increased by 4%, 3%, and 5%, respectively, compared to the control group.

[0041] This invention provides the application of isocitric acid in inhibiting the growth of Ralstonia solanacearum and controlling bacterial wilt of tomato. Various methods and approaches can be used to implement this technical solution, and the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications to the application method, concentration, timing, and combination with other biocontrol agents or microbial preparations of isocitric acid without departing from the technical principles of this invention. All such improvements and modifications should be considered within the scope of protection of this invention. All technical features and components not explicitly defined in this embodiment can be implemented using conventional techniques known in the art.

Claims

1. Application of isocitric acid in the prevention and control of bacterial wilt in plants.

2. The application according to claim 1, characterized in that, The bacterial wilt disease is caused by the bacterial wilt bacterium (Ralstonia solanacearum). Ralstonia solanacearum )cause.

3. The application according to claim 1, characterized in that, When applying, isocitric acid is prepared into an aqueous solution and applied to the rhizosphere soil of plants by root irrigation.

4. The application according to claim 1, characterized in that, Apply the solution to the roots 5–9 days after transplanting the seedlings.

5. The application according to claim 1, characterized in that, The application rate of isocitric acid in soil is 0.2–0.5 mg / kg soil.

6. The application according to claim 1, characterized in that, The plant is a member of the Solanaceae family, and preferably, the plant is a tomato.

7. A method for controlling bacterial wilt of plants, characterized in that, After isocitric acid is prepared into an aqueous solution, it is used for root irrigation treatment of plants.

8. The method according to claim 7, characterized in that, When applying this solution, add isocitric acid solution to the root irrigation 5-9 days after the tomatoes are transplanted.

9. According to the method of claim 7, the application rate of isocitric acid in the soil is 0.2~0.5 mg / kg soil.

10. The method according to claim 7, characterized in that, The plant is a member of the Solanaceae family, and preferably, the plant is a tomato.

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