Use of endogenous hormone zeatin in banana tissue in combating banana fusarium wilt

Abstract

The application provides a use of a banana tissue endogenous hormone zeatin in resisting banana wilt, and belongs to the technical field of plant protection. The banana tissue endogenous hormone is zeatin, and experiments prove that in vitro tests (tissue culture bottles and greenhouses) of the zeatin or lutein show that the zeatin can effectively inhibit the growth of TR4 mycelium, exhibits good anti-banana wilt fungus activity, has a significant prevention and control effect on the banana wilt fungus (TR4), and does not affect the growth of seedlings. The application provides a new idea and method for preventing and controlling TR4, and lays a foundation for in-depth exploration of the prevention and control of the banana wilt disease TR4.

Description

Technical Field

[0001] This invention relates to the field of plant protection technology, and more specifically, to the use of the endogenous hormone zeatin in banana tissues in the fight against banana wilt disease. Background Technology

[0002] Plant hormones are trace organic compounds synthesized in specific parts of a plant and transported from their site of synthesis to other parts of the plant, playing a significant regulatory role in the plant's overall life activities. They are also known as natural plant hormones or endogenous plant hormones. Plant hormones typically exert significant regulatory effects on plant growth and development even at low concentrations.

[0003] Plant hormones, also known as natural plant hormones or endogenous plant hormones, are trace organic compounds produced within plants that regulate (promote or inhibit) their own physiological processes. There are six known classes of plant hormones: auxins, gibberellins, cytokinins, abscisic acid, ethylene, and brassinosteroids. These are all simple small-molecule organic compounds, but their physiological effects are extremely complex and diverse, ranging from influencing cell division, elongation, and differentiation to affecting germination, rooting, flowering, fruiting, sex determination, dormancy, and abscission. Therefore, plant hormones play a crucial regulatory role in plant growth and development. Auxins, gibberellins, and cytokinins promote plant growth and development, while abscisic acid and ethylene inhibit plant growth and promote maturation and senescence. These hormones, in addition to their unique functions at different stages of plant growth and development, can also mutually promote or inhibit each other, fully exerting their regulatory effects on plant growth and development. Some mineral nutrients, such as nitrogen, phosphorus, and potassium, and soil stress can affect the content and distribution of hormones in plant roots, thereby regulating root growth.

[0004] Zeatin is an organic compound with the molecular formula C2. 10 H 13 N5O appears as white crystals or powder, sparingly soluble in water, soluble in alcohol and DMF, and is non-toxic. It is a naturally occurring cytokinin found in plants. It was the first natural cytokinin extracted and crystallized from the kernels of sweet corn during the grain-filling stage, and it can now be synthesized artificially. The exogenous zeatin used in production is a bio-based plant growth regulator.

[0005] Lutein, also known as plant lutein, is a carotenoid with the chemical formula C6H2O. 40 H 56O2 is widely present in vegetables, fruits, flowers, and other plants. It is slightly soluble in oils and n-hexane, soluble in acetone, dichloromethane, and ethanol, and readily soluble in ethyl acetate, tetrahydrofuran, and chloroform. Lutein has poor stability and is mainly affected by factors such as oxygen, light, heat, metal ions, and pH. However, lutein is highly safe, non-toxic, and harmless. It is an important natural pigment and natural health product, and a green health food ingredient.

[0006] Fusarium oxysporum f.sp.cubense, a Cuban-specific variant 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. This pathogen spreads rapidly, and the disease has now spread from parts of Southeast Asia to more than 20 banana-growing countries, including Jordan, Oman, and Peru. 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 are often ineffective in controlling its spread. Due to its rapid spread, difficulty in control, and the lack of systematic control methods, numerous banana plantations have been destroyed and abandoned because of TR4. Therefore, developing effective and green methods for controlling TR4 is crucial for the sustainable development of the banana industry.

[0007] A search revealed that Bu Jiajia, in her master's thesis "A Study on the Relationship between Endogenous Hormones in Banana Tissues and Resistance to Fusarium Wilt," clearly stated: "Agrophytes may participate in the resistance response to Fusarium wilt in bananas. Changes in zeatin content showed no significant correlation with banana resistance, suggesting that zeatin may play a minor role in banana resistance to Fusarium wilt, while abscisic acid may play a role." No existing research has found any use for zeatin or lutein in inhibiting plant pathogens, particularly in resistance to banana Fusarium wilt. Summary of the Invention

[0008] To address the above technical problems, this invention provides the use of endogenous hormones in banana tissues in combating banana wilt disease.

[0009] This invention protects the application of endogenous hormones in banana tissues in combating banana wilt disease, wherein the endogenous hormone is zeatin.

[0010] Furthermore, the banana wilt disease is caused by Fusarium oxysporum f.sp.cubense, physiological race 4, tropical type.

[0011] Furthermore, the application of the zeatin in inhibiting the growth of banana wilt pathogen.

[0012] Furthermore, the role of zeatin in preventing banana wilt pathogen infection.

[0013] The research team of this invention discovered through KEGG pathway enrichment analysis that the disease-resistant treatment group showed a significant enrichment of 19 KEGG pathways compared to the control group. Among these, 10 pathways were individually induced, including Carotenoid biosynthesis (map00906) and Zeatin biosynthesis (map00908). This differential gene enrichment may be an important response leading to banana disease resistance. Therefore, the inventors hypothesized that substances synthesized in the later stages of these two pathways (map00906 and map00908) may play an important role in the growth of the pathogen TR4. Consequently, the inventors attempted to conduct experiments on resistance to banana wilt disease TR4 using zeatin or lutein. The results showed that zeatin or lutein effectively inhibited the growth of TR4 mycelia, exhibiting good anti-bacterial activity against banana wilt pathogens (TR4) and demonstrating significant control effects.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] 1) This invention protects zeatin as the endogenous hormone in banana tissue. Experiments have shown that both in vitro and in-tissue culture experiments demonstrate that zeatin or lutein can effectively inhibit the growth of TR4 mycelia. Treatment of rooting medium with zeatin or lutein can improve the resistance of bananas to Fusarium wilt. However, greenhouse hardening-off experiments show that seedlings treated with zeatin and inoculated with TR4 exhibit consistent survival rates compared to the uninoculated control. In the greenhouse, zeatin also improved the resistance of banana seedlings to TR4 without affecting seedling growth, while the survival rate of lutein in the greenhouse was basically the same as that of the TR4-inoculated control. This indicates that although lutein improves the resistance of banana seedlings to TR4, it also affects seedling growth. Therefore, this invention protects the activity of zeatin against Fusarium wilt of banana, and zeatin has a significant control effect on Fusarium wilt of banana (TR4).

[0016] 2) This invention provides new ideas and methods for the control of TR4, laying the foundation for further exploration of the control of TR4, a disease of banana wilt. Plant hormones originate from the host plant. Gene expression screening revealed that genes of these two types of hormone signaling pathways were enriched, and appropriate doses would not have adverse effects on the plant. Attached Figure Description

[0017] Figure 1 The effect of in vitro hormone treatment on the change in TR4 colony diameter;

[0018] Figure 2 Effects of in vitro hormone treatment on TR4 growth;

[0019] Figure 3 The effect of hormone treatment on TR4 resistance in banana tissue culture seedlings;

[0020] Figure 4 The effect of hormone treatment on the survival rate of banana tissue culture rooted seedlings inoculated with TR4;

[0021] Figure 5 Effects of hormone treatment on the plant height of banana tissue culture seedlings;

[0022] Figure 6 The effect of hormone treatment on the fresh weight of banana tissue culture seedlings;

[0023] Figure 7 The effect of hormone treatment on the root growth of banana tissue culture seedlings. Detailed Implementation

[0024] Example 1: Screening of anti-TR4 substances in bananas

[0025] 1. Experimental processing and sample collection

[0026] Banana rooted seedling culture: Select the best-growing seedlings from the propagation medium of the banana TR4 variety - Brazilian banana and place them into tissue culture bottles containing rooting medium, 15 seedlings per bottle. Culture them for 15-20 days under the conditions of light intensity of 1600-2000 lux and light time of 8 hours / day to obtain rooted seedlings that can be used for hardening.

[0027] Hardening off and seedling cultivation: After washing the culture medium, the rooted tissue culture seedlings of Brazilian banana are cultivated in seedling pots with substrate and coconut coir (1:1) in the greenhouse for about 3 weeks, and then transferred to pots with raw soil / banana substrate (1:1) for cultivation, 1 plant per pot. When they grow to 10cm, they are artificially inoculated with TR4 (15-1), and the leaves are sprayed with 0.05% urea solution every week.

[0028] Treatment of seedlings by inoculation with TR4 and sample collection: Previous studies have found that the selenium form used can effectively improve the resistance of Brazilian banana seedlings to TR4 in greenhouses (Liu Lina et al., 2023: Application of sodium selenite in the control of banana wilt pathogen, patent number CN202210010596.8). Therefore, this invention selects this selenium form to induce disease resistance in banana seedlings in order to screen for induced disease resistance-related substances. Ten days before inoculation with TR4, the Brazilian banana seedlings were divided into four groups: blank control (Control), TR4 control (TR4), selenium form treatment (Se80: 80 mg / L selenium form liquid, 50 mL / pot), and selenium form treatment + TR4 (Se80 + TR4). The root-damage inoculation method was used for inoculation (Liu Lina et al., 2021). After removing banana seedlings cultured in coconut coir, the seedlings to be inoculated were arranged in an orderly manner according to germplasm resources. The roots of the banana seedlings were trimmed to a length of about 5 cm with scissors, and then the roots were immersed in the spore suspension, with the suspension stirred every 5-10 minutes. After 30 minutes, the soaked banana seedlings were removed and transplanted into pots with mixed substrate soil. On the 7th day after TR4 inoculation, banana bulb samples were collected, with 3 bulbs pooled into 1 sample, and 5 samples / treatment were used for transcriptome sequencing. The remaining approximately 20 plants from each group were used for disease incidence survey 42 days after TR4 inoculation.

[0029] 2. Transcriptome sequencing and data analysis

[0030] RNA extraction, library construction, and sequencing: Total RNA was extracted from plant tissues using the TRIzol method, and genomic DNA was removed using DNase I. RNA integrity was assessed using 1.0% agarose gel electrophoresis, RNA quality was determined using Bioanalyser 2100, and quantification was performed using ND-2000. High-quality RNA samples (OD260 / 280 = 1.8–2.2, OD260 / 230 ≥ 2.0, RIN ≥ 8.0, 28S:18S ≥ 1.0, > 1 μg) were selected to construct sequencing libraries. RNA purification, reverse transcription, library construction, and sequencing were performed by Shanghai Meiji Biotechnology Co., Ltd., following Illumina's operating instructions. After passing the TBS380 test, the bidirectional RNA sequencing libraries were sequenced using an Illumina NovaSeq 6000 sequencer.

[0031] Gene expression analysis: Raw data was cleaned and quality-controlled using FASTP (https: / / github.com / OpenGene / fastp) (Chen et al., 2018) to obtain Clean Reads. Then, HISAT2 software (http: / / ccb.jhu.edu / software / hisat2 / index.shtml) (Kim et al., 2015) was used to align the Clean Reads to the banana reference genome (https: / / banana-genome-hub.southgreen.fr / ) (Belser et al., 2021) in a directional mode. Each sample of Reads was assembled into the reference genome using StringTie (https: / / ccb.jhu.edu / software / stringtie / ) (Pertea et al., 2015). The expression levels of each gene were calculated using the TPM method, and RSEM (http: / / deweylab.biostat.wisc.edu / rsem / ) (Li et al., 2011) was used to quantify gene abundance.

[0032] Functional enrichment analysis: Differential expression analysis was performed using a combination of DESeq2 (Love et al., 2014) / DEGseq (Wang et al., 2009) / EdgeR (Robinson et al., 2010). Genes with |log2 FC| ≥ 1 and P-adjust ≤ 0.05 were defined as differentially expressed genes (DEGs). DEGs were then subjected to GO functional gene enrichment analysis (Gene Ontology, http: / / www.geneontology.org) and KEGG pathway analysis (Kyoto Encyclopedia of Genes and Genomes, http: / / www.genome.jp / kegg / ).

[0033] 3. Experimental Results

[0034] The disease index results of the four treatment groups 42 days after TR4 inoculation were as follows: the disease indices for Control, TR4, Se80, and Se80+TR4 were 0.0, 63.0, 0.0, and 42.0, respectively. The control effect of Se80 reached 33.3%, achieving the treatment effect. The incidence rates of Control and Se80 were 0, proving that the experiment was uncontaminated and the treatment results were reliable. Therefore, the collected bulb samples can be used for transcriptome sequencing research.

[0035] Transcriptome analysis of Se80+TR4 and TR4 revealed a total of 5709 differentially expressed genes. Compared to the TR4 group, Se80+TR4 showed an increase of 2674 genes and a decrease of 3035 genes. GO enrichment analysis of the differentially expressed genes identified 19 biological processes specifically enriched in Se80+TR4 (Table 1), including hormone metabolism (GO:0042445), phenylpropanoid metabolism (GO:0009698), lignin metabolism (GO:0009808), and secondary metabolism (GO:0019748). KEGG enrichment analysis showed significant enrichment in 10 pathways, including diterpenoid phytoalexin biosynthesis (map00904), carotenoid biosynthesis (map00906), and zeatin biosynthesis (map00908). Based on the results of GO enrichment, KEGG pathway enrichment and gene expression levels, lutein from the carotenoid biosynthesis pathway and zeatin from the zeatin biosynthesis pathway were selected for further functional studies on improving the resistance of banana plants to TR4.

[0036] Table 1. GO enrichment results of differentially expressed genes

[0037]

[0038] Table 2. Results of differentially expressed KEGG pathway enrichment.

[0039]

[0040] Example 2: The in vitro inhibitory effect of zeatin or lutein on TR4 growth.

[0041] 1. Experimental Materials

[0042] Fusarium wilt pathogen strain: TR4 (a highly pathogenic wild-type strain 15-1 isolated from Xishuangbanna, Yunnan).

[0043] 2. Experimental Methods

[0044] Culture of TR4(15-1) mycelium: Peel 200g of potatoes, cut them into small pieces, boil them in 1000mL of water for 15min, filter out the potato residue with gauze, add 20g of white sugar and 20g of agar powder, add water to make up to 1000mL, and autoclave at 121℃ for 20min. Inoculate TR4(15-1) stored at -80℃ onto PDA plates and incubate in the dark at 28℃ for 5 days.

[0045] TR4 cultured in hormone-treated medium: Purchased lutein from the carotenoid synthesis pathway (Soluble, SL8650, C) for screening. 40 H 56 O2), zeatin in the zeatin biosynthesis pathway (Soleb, X8040, C) 10H 13 N 50 10 mM stock solutions were prepared using DMSO: 5 mg of zeatin was dissolved in 2.281 mL of DMSO, and 20 mg was dissolved in 3.516 mL of DMSO. 1 mL of Mixture I (80 μL stock solution + 920 μL DMSO) was added to 100 mL of PDA medium to obtain PDA medium with a final concentration of 8 μM zeatin or lutein. Correspondingly, 1 mL of Mixture II (640 μL stock solution + 360 μL DMSO) was added to 100 mL of PDA medium to obtain PDA medium with a final concentration of 64 μM zeatin or lutein. PDA medium with 1 mL of dd H2O or DMSO was used as a control. TR4 mycelial discs with a diameter of 5 mm were punched at the edge of the colony using a puncher. These mycelial discs were inoculated onto PDA medium with different concentrations and hormone treatments. After culturing for 5 days, the colony diameter was measured. Six biological replicates were set up, and the experiment was repeated three times.

[0046] Data analysis: T-test was used to analyze the significance of diameter differences between different treatments.

[0047] 3. Experimental Results

[0048] The results showed that the effects of hormones with significantly enriched differentially expressed genes (zeatin and lutein) on TR4 mycelial growth were determined on PDA plates treated in vitro. (See attached figures.) Figure 1 and Figure 2 .

[0049] As shown in the figure, the inhibitory effects of different concentrations of zeatin or lutein on TR4 mycelial growth were significantly higher than those on the control and the control containing DMSO, indicating that high concentrations had a greater inhibitory effect than low concentrations. The TR4 colony diameters on PDA medium treated with the control, DMSO, 8 μM zeatin, 64 μM zeatin, 8 μM lutein, and 64 μM lutein were 65.25 ± 2.83 cm, 63.17 ± 2.03 cm, 59.17 ± 1.14 cm, 57.83 ± 1.95 cm, 58.75 ± 1.64 cm, and 57.08 ± 1.89 cm, respectively. The inhibition rates of the hormones (lutein or zeatin) on TR4 mycelial growth ranged from 9.3% to 12.5%. Zeatin treatment significantly inhibited TR4 growth, with higher concentrations showing better inhibitory effects, but there was no significant difference between different concentrations. Lutein treatment also significantly inhibited TR4 growth, and the inhibitory effect of the 64 μM treatment was significantly higher than that of the 8 μM treatment. The results showed that plant hormones (zeatin or lutein) could effectively inhibit the growth of TR4 mycelia in vitro.

[0050] Example 3: Effects of zeatin or lutein on TR4 resistance in banana seedlings in tissue culture flasks.

[0051] 1. Experimental Materials

[0052] Banana variety: Brazilian banana.

[0053] Fusarium wilt pathogen strain: TR4 (a highly pathogenic wild-type strain 15-1 isolated from Xishuangbanna, Yunnan).

[0054] 2. Experimental Methods

[0055] Culture of TR4(15-1) mycelia: Conidia on PDA plates were washed off with sterile water and injected into Erlenmeyer flasks containing 300 mL of PDB liquid medium. The flasks were incubated at 25°C and 150 rpm for 3 days. Mycelia were removed by filtration through two layers of gauze. The spore concentration was adjusted to 1 × 10⁻⁶ using a hemocytometer under a microscope. 6 Spore count / mL. PDB liquid medium is PDA medium without agar powder.

[0056] Culture of rooted seedlings of different treatments of Brazilian banana: Brazilian banana seedlings with a height of 3cm or more and uniformity were selected and cut. They were inoculated into tissue culture flasks containing rooting medium (MS medium + NAA 0.5mg / L + sucrose 10-15g / L + agar 5.0g / L, pH 5.6-5.8). 100mL of medium was added to each flask, and 1mL of zeatin or lutein mixed solution I (80μL 10uM stock solution + 920μL DMSO) was added respectively. The final concentration of zeatin or lutein in the rooting medium was 8uM. Five flasks were used for each treatment, with five Brazilian banana seedlings cut in each flask. The seedlings were cultured for 20-25 days under a light intensity of 1600-2000 lux and a light duration of 8 hours / day to obtain rooted seedlings of different treatments. The experiment was repeated twice.

[0057] Leaf inoculation with TR4: Leaves of rooted seedlings under different treatments were inoculated with 1×10⁻⁶ under aseptic conditions. 6 The disease resistance assessment based on spore count / mLTR4 was conducted according to the patent (Liu Lina et al., 2022: A method for identifying the resistance of different banana varieties to Fusarium wilt pathotropic type 4 using tissue culture seedlings, patent number CN202110658725.X). To increase the contact time of the spore solution on the leaves, Tween20 was added to the bacterial solution at a final concentration of approximately 0.2% for adhesion.

[0058] Hardening-off cultivation of rooted seedlings after TR4 inoculation: Brazilian banana rooted seedlings treated with zeatin and lutein, control Brazilian bananas inoculated with TR4, and Brazilian bananas not inoculated with TR4 were divided into 4 groups, with 24 seedlings in each group. After 7 days of inoculation, the seedlings were hardened off in a greenhouse using a medium with a coconut coir: substrate ratio of 1:1 for about 40 days. The survival rate of the plants was counted and then transferred to a soil-containing substrate for planting (soil mass: substrate mass = 1:1).

[0059] 3. Experimental Results

[0060] Disease incidence was investigated on day 7 after inoculation with TR4 at different concentrations of lutein or zeatin-treated Brazilian banana seedlings grown in rooting medium for 20 days. Results are shown below. Figure 3 Data showed that the leaves of banana seedlings in the control or DMSO treatments exhibited varying degrees of susceptibility to TR4 infection, while leaves treated with lutein or zeaxanthin did not show lesion expansion upon TR4 infection. This indicates that hormone (zeaxanthin or lutein) treatment of the rooting medium can improve the resistance of bananas to Fusarium wilt. The survival rate of greenhouse seedlings after TR4 inoculation with different treatments is shown in the figure. Figure 4 As shown in the figure, after 40 days of hardening-off, the survival rates of the 24 rooted seedlings in the control group (without TR4), the control group (inoculated with TR4), the control group (inoculated with zeatin TR4), and the control group (inoculated with lutein TR4) were 21, 15, 21, and 13, respectively. Seven days after being transferred to pots with soil, two plants in the TR4-inoculated control group died, while the rest grew well. The experimental results indicate that zeatin TR4 and the uninoculated TR4 control showed consistent survival rates. In the greenhouse, zeatin also improved the resistance of banana seedlings to TR4, while lutein did not significantly improve the resistance, and the survival rate was basically the same as the TR4 control. This may be because the effective dose of lutein inhibiting TR4 affected the plant growth of the banana seedlings.

[0061] Example 4: Effects of zeatin or lutein on the growth of banana plants in tissue culture bottles. 1. Experimental methods

[0062] After 20 days of rooting of Brazilian banana seedlings under different treatments, five bottles were prepared for each treatment, with five seedlings per bottle, to determine the growth indicators of the banana plants. The culture medium was removed from the seedlings, they were washed, and excess water was absorbed with absorbent paper. The plant height (height from the first fibrous root to the top of the plant) was measured, and the total weight of the plant was weighed. Root growth activity was observed. A T-test was used to analyze the significance of differences in diameter between different treatments.

[0063] 2. Experimental Results

[0064] The effects of zeatin or lutein on the growth of Brazilian banana rooted seedlings compared to the control plant height are shown in [link to relevant documentation]. Figure 5 (Plant height) Figure 6 (Fresh weight) Figure 7(Root Vigor). The plant height of Brazilian banana seedlings treated with zeatin or lutein was 10.7±1.7cm and 11.0±0.8cm, respectively, consistent with the control or DMSO-treated seedling height (10.9±3.7cm and 11.2±2.4cm). Compared with the control (2.89±1.17g and 2.50±0.82g), the fresh weight of Brazilian banana seedlings treated with zeatin or lutein was 2.21±0.72g and 2.06±0.81g, respectively, both showing a decrease, with the lutein treatment showing a significant decrease in fresh weight. Root vigor indicated no significant changes in root number or growth. The data show that within the range of concentrations that significantly increased TR4 resistance in Brazilian banana seedlings, zeatin had no effect on seedling growth, while lutein significantly reduced the fresh weight of Brazilian banana seedlings.

Claims

1. The application of zeatin, an endogenous hormone in banana tissues, in the resistance to banana wilt disease, characterized by: The aforementioned banana wilt disease is caused by Fusarium oxysporum cucumeriforme (Cuba spp.). Fusarium oxysporum f.sp. cubense The concentration of zeatin in the 4th physiological race, tropical type, is 8 μM or 64 μM.

2. The application according to claim 1, characterized in that: The application of zeatin in inhibiting the growth of banana wilt pathogen.

3. The application according to claim 1, characterized in that: The role of zeatin in preventing banana wilt pathogen infection.

4. The application according to any one of claims 1-3, characterized in that: The zeatin mentioned above, while resisting banana wilt disease, does not affect the growth of banana plant height, fresh weight, or root vitality.

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