Use of lipid-related metabolites of banana tissue in combating banana fusarium wilt

Abstract

The application relates to the technical field of plant protection, and discloses the use of lipid-related metabolites of banana tissues in resisting banana wilt. The application finds the role of lipid-related metabolites generated in banana corm tissues in resisting Fusarium oxysporum f. sp. Cubense Tropical Race 4 (TR4), and for the first time reports the function of lipid-related metabolites 16-hydroxyhexadecanoic acid, erucic acid, D-sphingosine and glycerophosphates in inhibiting the activity of TR4 in plants in vivo and in vitro. The application shows that the lipid-related metabolites generated by plants have a significant prevention and control effect on TR4, and provides a green method and reference for the prevention and control of TR4.

Description

Technical Field

[0001] This invention relates to the field of plant protection technology, and more specifically, to the use of lipid-related metabolites in banana tissues in resistance to banana wilt disease. Background Technology

[0002] Fusarium oxysporum f.sp.cubense, a specialized strain of the fungus *Fusarium wilt*, severely restricts the sustainable development of the banana industry. The most severe case is physiological race 4 (TR4), which infects banana roots, corms, pseudostems, leaves, xylem, and rachis (Bai et al., 2020). TR4 is extremely difficult to control. Once it spreads to a region, the application of fungicides, soil fumigants, and improved cultivation practices such as crop rotation and soil amendments fail to control its spread (Heslop-Harrison and Schwarzacher, 2007; Zheng et al., 2018). Due to its rapid spread, difficulty in control, and lack of systematic control methods, numerous banana plantations have been destroyed and abandoned because of TR4 (Ploetz RC, 2015; Li et al., 2019). Developing effective and green methods for controlling TR4 is crucial for the sustainable development of the banana industry.

[0003] Plant lipids are mainly composed of molecules such as fatty acids, glycerol, and phosphate. Their synthesis involves two steps: the synthesis of fatty acids and the synthesis of glycerophosphate esters, including a variety of compounds such as fatty acids, oils, glycerophospholipids, galactolipids, sphingolipids, sterols, and neutral lipids (such as triacylglycerols, sterol esters, cuticles, waxes, and lignin) (Ischebeck, 2016). As primary biomolecules, lipids play four main roles in most organisms, including as structural components, sustainable carbon and energy storage molecules, active signal sensors, and surface coverings (LiBeisson et al., 2016). In plants, cuticle waxes, cuticles, and lignin are generally defined as lipids covering the surface (Kunstand Samuels, 2009), and a relatively complete biosynthetic pathway for waxes and cuticles has been constructed in the vegetative epidermis (Yeats and Rose, 2013).

[0004] Plant lipids are major components of cell membranes and participate in various physiological processes, including plant growth, development, and adaptation to stress. These processes primarily involve biosynthesis and transport, playing a crucial role in plant reproductive development, particularly in the development of the anther cuticle and pollen wall, anther dehiscence, pollen maturation, and pollen hydration (Ariizumi and Toriyama, 2011; Shi et al., 2015; Wan et al., 2020). The two lipid layers in plants (anther cuticle and pollen exine) are essential for male reproductive development; disruption of these lipids often leads to microspore abortion and male genetic sterility (Zhang et al., 2016). Lipids participate in tissue growth processes, including the loosening of the epidermal wall (Xin et al., 2021) and the synthesis of cuticle wax components (Bian et al., 2022). They not only affect the physicochemical properties of cell membranes but also participate in plant hormone signal transduction (Ling et al., 2022), regulating plant responses to stresses such as drought, hypoxia, and pathogenic stress (Shao et al., 2022; Liu et al., 2021). A search revealed no research on the function of plant lipids in resistance to banana wilt disease. Summary of the Invention

[0005] In summary, in view of the above technical problems, the present invention provides the use of lipid-related metabolites in banana tissue in the fight against banana wilt disease.

[0006] Preferably, the lipid-related metabolite is any one of 16-hydroxyhexadecanoic acid, erucic acid, D-sphingosine, and glycerophosphate.

[0007] Furthermore, the banana wilt disease is caused by Fusarium oxysporum f.sp.cubense, physiological race 4, tropical type.

[0008] This invention also protects the use of the lipid-related metabolites in inhibiting the growth of banana wilt pathogen.

[0009] The present invention also protects the role of the lipid-related metabolites in preventing infection by Fusarium wilt pathogens in bananas.

[0010] This invention also protects a method for improving resistance to banana wilt disease, wherein any one of the above-mentioned lipid-related metabolites, 16-hydroxyhexadecanoic acid, erucic acid, D-sphingosine, and glycerophosphate, is added to a rooting medium to culture banana tissue culture seedlings, and the resulting banana tissue culture seedlings have resistance to the banana wilt pathogen TR4.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] 1) The inventors attempted to screen for disease-resistant metabolites using banana tissues treated with disease resistance and control treatments. The selected lipid-related metabolites (16-hydroxyhexadecanoic acid, erucic acid, D-sphingosine, and glycerophosphate) were validated against Fusarium wilt pathogen TR4. Results showed that in vitro tests of 16-hydroxyhexadecanoic acid, erucic acid, D-sphingosine, and glycerophosphate effectively inhibited the growth of Fusarium wilt pathogen TR4 mycelia, exhibiting good anti-Fusarium wilt activity. In vivo test results were consistent with in vitro results. When lipid-related metabolites were added to rooting medium to cultivate banana tissue culture seedlings, inoculation with TR4 significantly improved the disease resistance of the seedlings. This indicates that the lipid-related metabolites produced by the plant itself have a significant control effect on Fusarium wilt pathogen (TR4), providing a green method for TR4 control.

[0013] 2) This invention discovers the role of lipid-related metabolites produced in banana bulb tissue in resisting banana wilt fungus (TR4), and reports for the first time the function of lipid-related metabolites (16-hydroxyhexadecanoic acid, erucic acid, D-sphingosine, glycerophosphate) in inhibiting the activity of wilt fungus TR4 in plants and in vitro. Attached Figure Description

[0014] Figure 1 Graph showing the difference in response of bananas to Fusarium wilt pathogen TR4 between disease-resistant and control treatments.

[0015] Figure 2 Graph showing the changes in metabolites and functional types of bananas treated with disease resistance and those under control

[0016] Figure 3 Cluster analysis diagram of different metabolites of bananas

[0017] Figure 4 Figure showing the effect of plant lipid metabolites on the hyphal diameter of TR4 fungus, the causal agent of banana wilt, in vitro.

[0018] Figure 5 Figure 1. Effects of plant lipid metabolites on the mycelial growth of TR4 fungus, the causal agent of banana wilt, in vitro.

[0019] Figure 6 Effects of plant lipid metabolites on plant height and fresh weight of banana tissue culture seedlings

[0020] Figure 7 Figure 1: Plant height and root growth of banana seedlings in tissue culture flasks after treatment with plant lipid metabolites

[0021] Figure 8 Phenotypic changes in banana leaves resistant to TR4 wilt pathogen after treatment with plant lipid metabolites Detailed Implementation

[0022] Example 1: Screening of lipid-related metabolites

[0023] 1.1 Greenhouse cultivation of banana seedlings, disease resistance identification and sample collection

[0024] Brazilian banana seedlings rooted in tissue culture bottles were transferred to coconut coir for hardening off. After survival (approximately 2 weeks), the seedlings were transferred to plastic pots containing soil (1:1 ratio of native soil and banana substrate) for cultivation (approximately 6 weeks). Once they reached 20 cm in height, they were used for disease resistance treatment and artificial root injury inoculation with TR4 (a highly pathogenic wild-type strain 15-1 isolated from Xishuangbanna, Yunnan), following the inoculation method (Liu Lina et al., 2021). Soil was treated as follows: Control, Treatment, with 16 biological replicates for each treatment. On day 7, banana seedlings in the Disease Resistance and Control treatments were inoculated with TR4 at a final concentration of 2 × 10⁶ spores / g soil, labeled Control_TR4 and Treatment_TR4, respectively. After 42 days of cultivation following TR4 inoculation, disease severity was assessed, and the disease index was calculated. Disease severity was graded from 0 to 4, following the method described in (Liu Yidao et al., 2008). Disease index = Σ(number of diseased plants at each level × corresponding level value) / (total number of plants investigated × highest level value) × 100, and the T-test was used to analyze the significance of the difference. Control_TR4 and Treatment_TR4 were used for non-targeted metabolomics determination by collecting bulb samples on day 7 after inoculation (BXJ_TR4 and BXJ_S8R).

[0025] 1.2 Non-target metabolomics assay of banana bulbs by LC-MS

[0026] Add 400 μL of extraction buffer (acetonitrile:methanol = 1:1) to 50 mg solid sample, mix well, and extract by low-temperature ultrasonication for 30 min (5℃, 40 kHz). Place the sample at -20℃ for 30 min, centrifuge at 13000g for 15 min, transfer the supernatant, dry under nitrogen, reconstitute with 120 μL of reconstitution solution (acetonitrile:water = 1:1), extract by low-temperature ultrasonication for 5 min, centrifuge at 13000g for 10 min, and transfer the supernatant to a vial with an inner tube for analysis. The instrument platform for LC-MS analysis is Thermo Fisher Scientific UHPLC-Q Exactive HF-X system.

[0027] 1.3 Database Search and Data Preprocessing

[0028] Raw data were imported into the metabolomics processing software Progenesis QI (Waters Corporation, Milford, USA) for analysis to obtain a data matrix containing information such as retention time, mass-to-charge ratio, and peak intensity. The software was used for characteristic peak search and identification, matching MS and MS / MS mass spectrometry information with metabolic databases, and identifying metabolites based on the secondary mass spectrometry matching scores. The main databases used were the public databases http: / / www.hmdb.ca / and https: / / metlin.scripps.edu / , as well as a self-built database. Preprocessed data were uploaded to the Meiji Bio Cloud Platform (https: / / cloud.majorbio.com) for data analysis. A normalized data matrix was obtained, variables with a relative standard deviation >30% for QC samples were removed, and log10 normalization was performed to obtain the final data matrix used for subsequent analysis. The R package Robls (Version 1.6.2) was used for orthogonal partial least squares discriminant analysis (OPLS-DA) with variable weights (VIP), Student's T test, and fold change analysis. Metabolites were screened, with those possessing VIP>1 and p<0.05 considered significantly differentially expressed. Metabolic pathway annotation using the KEGG database identified the pathways involved by these differentially expressed metabolites. Pathway enrichment analysis was performed using the Python package Scipy.stats, and Fisher's exact test was used to identify the biological pathways most relevant to the experimental treatment.

[0029] 1.4 Screening of Fusarium wilt-related metabolites in banana tissues

[0030] In a greenhouse, banana seedlings treated with disease resistance and then inoculated with TR4 (Treatment_TR4) and a control group inoculated with TR4 (Control_TR4) showed changes in aboveground leaf phenotype, underground root phenotype, and disease index. Figure 1 .

[0031] from Figure 1 The results showed that the symptoms of Fusarium wilt in both the aboveground and underground parts of banana seedlings were reduced after the disease-resistant treatment. The average disease index was 32.54±1.12, which was significantly lower than that of the control (76.42±2.98), and the control efficacy reached 57.4%. The results showed that there was a significant difference between the disease-resistant treatment and the control. Samples collected on the 7th day after TR4 inoculation can be used for further metabolite determination and analysis.

[0032] Non-target metabolites were detected by LC-MS in seedlings treated with two methods (Control_TR4 and Treatment_TR4). A total of 12 samples were analyzed, labeled BXJ_TR4 and BXJ_S8R respectively. 653 metabolites were identified in cation mode and 396 metabolites in anion mode. PLS-DA score analysis of metabolites obtained in both cation and anion modes for Treatment_TR4 vs. Control_TR4 revealed complete separation between the two treatments, indicating a significant interaction between influencing factors. Treatment_TR4 vs. Control_TR4 yielded 77 metabolites (…). Figure 2 A). Differential metabolites mainly participate in the biosynthesis of keratin and waxes (map00073), sphingolipid metabolism (map00600), fatty acid biosynthesis (map00061), and unsaturated fatty acid biosynthesis (map01040), playing roles in lipid metabolism, secondary metabolite biosynthesis, and signal transduction. Figure 2 B).

[0033] Based on the correlation expression of all metabolites, four expression patterns M1-M4 were identified. Figure 3The expression pattern M3, consisting of 11 metabolites including N,N-Dimethyldodecylamine-N-oxide, 1-Pentanol, (+ / -)-Hydroxycitronellol, Lauryldiethanolamine, Myristicacid, 16-Hydroxyhexadecanoicacid, 1-Hexanol, PA (18:1(11Z) / 18:3(6Z,9Z,12Z)), Sphinganine, Erucicicine, and Psilocine, showed a strong correlation. Analysis revealed significant differences in VIP values ​​between the two groups for metabolites in this pattern. These metabolites included PA, which is only present in the specific KEGG pathway (map00561), and 16-HA, which is present in map00073. Sphinganine (map00600), Psilocine (map00901), Myristicacid (map00061), and Erucic acid (map01040) were highly expressed in Treatment_TR4. Figure 3 Data indicate that metabolites accumulate in the M3 mode under disease-resistant treatment, thereby enhancing host resistance. Based on the fold change in VIP expression and the function of the KEGG pathway, this invention selects lipid-related metabolites in this mode for testing against TR4 in Fusarium wilt pathogens, including 16-hydroxyhexadecanoic acid, erucic acid, D-sphingosine, and glycerophosphate (PA(18:1(11Z) / 18:3(6Z,9Z,12Z))).

[0034] Example 2. In vitro antibacterial activity of purified lipid-related metabolites

[0035] Purified lipid-related metabolites, including 16-HA (16-Hydroxyhexadecanoic acid, Solarbio), erucic acid (Solarbio), D-sphingosine (Maikelin), and PA (PA(18:1(11Z) / 18:3(6Z,9Z,12Z)), glycerophosphate (Maikelin), were purchased and diluted with DMSO. 1 mL of diluent was added to every 100 mL of PDA medium to obtain PDA medium with final concentrations of 8 μM and 64 μM. PDA medium with 1 mL of ddH2O or DMSO was used as a control. TR4 mycelial discs with a diameter of 5 mm were punched at the edge of the colony and inoculated onto PDA medium. After 5 days of incubation, the colony diameter was measured. Six biological replicates were performed, with two experimental replicates. The significance of diameter differences between different treatments was analyzed using the T-test.

[0036] The results showed that the effects of highly functionally relevant lipid-related metabolites PA, 16-HA, Sphinganine, and Erucic acid on TR4 mycelial growth were determined on isolated PDA plates. (See attached figures.) Figure 4 and Figure 5 .

[0037] As shown in the figure, the mycelial growth rate in the control medium containing DMSO was lower than that in the blank control, but the difference was not significant. The four metabolites with the same accumulation pattern and their different concentrations all had significantly higher inhibitory effects on TR4 mycelial growth than the control and the DMSO-containing control, indicating that higher concentrations had a greater inhibitory effect than lower concentrations. The metabolite with the strongest inhibitory effect on mycelial growth was PA, with the highest inhibition rate reaching 16.2%. There was no significant difference in the inhibitory effects of the two treatments with different concentrations of Sphinganine (9.2% and 9.6%), but both were significantly higher than the inhibition rate of DMSO (3.3%). The results indicate that the accumulation of candidate lipid-related metabolites can effectively inhibit the growth of TR4 mycelia in vitro.

[0038] Example 3. Lipid-related metabolites enhance banana resistance to Fusarium wilt.

[0039] Purchasing and screening purified lipid-related metabolites 16-HA (16-hydroxyhexadecanoic acid), erucic acid, D-sphingosine, and PA (glycerophosphate) were used. Each was diluted with DMSO solution, and 1 mL of the diluent was added to every 100 mL of rooting medium to obtain a tissue culture medium with a final concentration of 8 μM. Medium containing 1 mL of ddH2O was used as a control. Brazilian banana seedlings over 3 cm in height were cut and inoculated into tissue culture bottles containing rooting medium. The seedlings were cultured for 10-20 days under a light intensity of 1600–2000 lx and a light duration of 8 hours / day. Rooted seedlings with different treatments were obtained for verification of leaf inoculation with TR4 (refer to patent, Liu Lina et al., 2022: A method for identifying the resistance of different banana varieties to tropical race 4 of Fusarium wilt using tissue culture seedlings). The rooting medium used was: MS medium + NAA 0.5 mg / L + sucrose 10-15 g / L + agar 5.0 g / L, pH 5.6–5.8. After 3 weeks of cultivation, growth indicators (plant height and fresh weight) of the Brazilian banana seedlings were measured. Changes in plant height, root growth, and fresh weight are shown in the figure. Figure 6 and Figure 7 .

[0040] Data showed that compared with the control plant height (10.9±3.7cm and 11.2±2.4cm), the plant height of Brazilian banana seedlings treated with different lipid-related metabolites increased, but the differences were not significant, at 12.8±2.4cm; 12.1±2.9cm; 12.8±2.7cm; and 12.3±2.1cm, respectively. Compared with the control fresh weight (2.89±1.17g and 2.50±0.82g), there was no significant difference in the fresh weight of Brazilian banana seedlings treated with different lipid-related metabolites, at 2.43±0.89g; 2.81±1.32g; 2.71±1.04g; and 2.90±0.84g, respectively. There was no significant difference in root growth among the treatments. These data indicate that the different lipid-related metabolites screened did not affect the growth of banana seedlings.

[0041] Disease incidence was investigated on day 7 after inoculation with TR4 in Brazilian banana seedlings grown for 20 days in different rooting media. Results are shown below. Figure 8 Data showed that the leaves of rooted banana seedlings in the control group were susceptible to TR4 infection, while the leaves of banana plants treated with 16-hydroxyhexadecanoic acid, erucic acid, D-sphingosine, and glycerophosphate all showed increased resistance to TR4 infection. This indicates that treatment of the rooting medium with lipid-related metabolites (16-hydroxyhexadecanoic acid, erucic acid, D-sphingosine, and glycerophosphate) can improve the resistance of bananas to Fusarium wilt.

[0042] In summary, the inventors attempted to screen for disease-related metabolites in banana tissues treated with disease resistance and those treated with control. The selected lipid-related metabolites (16-hydroxyhexadecanoic acid, erucic acid, D-sphingosine, and glycerophosphate) were then used to verify their resistance to Fusarium wilt pathogen TR4. The results showed that in vitro tests of 16-hydroxyhexadecanoic acid, erucic acid, D-sphingosine, and glycerophosphate effectively inhibited the growth of Fusarium wilt pathogen TR4 mycelia, exhibiting good anti-Fusarium wilt activity. In vivo test results were consistent with in vitro results. When lipid-related metabolites were added to rooting medium to cultivate banana tissue culture seedlings, inoculation with TR4 significantly improved the disease resistance of the seedlings. This indicates that the lipid-related metabolites produced by the plant itself have a significant control effect on Fusarium wilt pathogen TR4, providing a green method for TR4 control.

Claims

1. The use of lipid-related metabolites in banana tissue in resistance to banana wilt disease, characterized by: The lipid-related metabolites of the banana tissue are any one of 16-hydroxyhexadecanoic acid, erucic acid, D-sphingosine, and glycerophosphates. The pathogen of banana wilt disease is Fusarium oxysporum caudata. Fusarium oxysporum f. sp.c ubense ) 4th physiological race, tropical type (TR4).

2. The use of the lipid-related metabolites of banana tissue according to claim 1 in resistance to banana wilt disease, characterized in that: The stated use is the application of lipid-related metabolites from banana tissue in inhibiting the growth of Fusarium wilt pathogens in bananas.

3. The use of the lipid-related metabolites of banana tissue according to claim 1 in resistance to banana wilt disease, characterized in that: The stated use is the application of lipid-related metabolites from banana tissue in the prevention of banana wilt pathogen infection.

4. A method for improving resistance to banana wilt disease, characterized in that: The lipid-related metabolites of the banana tissue described in claim 1 were added to the rooting medium to culture banana tissue culture seedlings, and the resulting banana tissue culture seedlings were resistant to Fusarium oxysporum Cuban serotype (…). Fusarium oxysporum f. sp. cubense Banana wilt caused by physiological race 4 (TR4) is resistant.

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