A green control method for Fusarium wilt based on banana extracellular DNA and its application

Abstract

This invention discloses a green control method and application for banana wilt disease based on banana extracellular DNA, belonging to the field of plant disease biological control technology. The method includes: extracting total DNA from healthy banana tissue, obtaining esDNA fragments of 100-500 bp size through physical shearing; preparing the esDNA into a working solution, and applying it to banana plants via root irrigation. This invention utilizes banana-derived esDNA as an immune elicitor, which not only directly inhibits the mycelial growth of *Fusarium oxysporum* Cuban-specific serotypes (especially Foc TR4), but also systematically activates the immune response of banana plants, enhances their defensive enzyme activity, and reduces the accumulation of reactive oxygen species, thereby significantly reducing rhizosphere colonization of pathogens, alleviating yellowing and wilting symptoms, and improving plant survival rate and biomass. This method is environmentally friendly, highly safe, and easy to operate, providing a new green solution for the sustainable management of banana wilt disease.

Description

Technical Field

[0001] This invention belongs to the field of plant disease biological control technology, specifically relating to a method for inducing plant systemic resistance and controlling soil-borne diseases by using banana extracellular DNA (esDNA), which is particularly suitable for the control of banana wilt caused by Fusarium oxysporum f. sp. cubense. Background Technology

[0002] Bananas (Musa spp.) are an important global food and economic crop, but their production has long been severely threatened by banana wilt disease (Panama disease), with the disease caused by Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc TR4) being particularly serious. This pathogen is highly virulent, widely spread, and has a long soil residue time. There are currently no highly effective chemical agents, and breeding for disease resistance is a long and challenging process. Physical and chemical methods such as soil fumigation are costly, cause environmental pollution, and damage soil ecology.

[0003] Recent studies have revealed that plant extracellular self-DNA (esDNA), as an endogenous damage-associated molecular pattern (DAMP), is released when plants are damaged or attacked by pathogens. It can be recognized by plants and trigger a strong pattern-triggered immune response, thereby inducing systemic resistance. This provides new insights for developing environmentally friendly, novel biocontrol strategies based on plant self-immunity mechanisms.

[0004] While some studies have shown that exogenous application of plant esDNA may induce resistance, there is still a lack of systematic, operable, and effective implementation plans for transforming this into a stable, effective, and green control technology applicable to field production, especially for a devastating disease like banana wilt. Therefore, developing a safe, efficient, and easy-to-operate method for controlling wilt based on banana's own esDNA has significant theoretical value and practical necessity. Summary of the Invention

[0005] This invention aims to overcome the shortcomings of existing banana wilt control technologies and provide a new method that is environmentally friendly, biosafe, and can effectively utilize the banana's own immune mechanism to enhance disease resistance.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: An application of esDNA in the control of banana wilt disease, wherein the banana esDNA is an extracellular DNA fragment of the banana plant, which is obtained by processing the total DNA extracted from healthy banana leaves into DNA fragments with a size of 100-500 bp through physical shearing.

[0007] Furthermore, the banana wilt disease is caused by Fusarium oxysporum, specifically the Cuban variant.

[0008] Furthermore, the *Fusarium oxysporum* Cuban variant is tropical race 4.

[0009] Furthermore, the application includes: formulating the banana esDNA into a working solution and applying it to banana plants by root irrigation.

[0010] Furthermore, the concentration of the working solution is 30 ng / μL.

[0011] Furthermore, the physical shearing method is an ultrasonic crushing method using glass beads with a diameter of 0.5 mm, the ultrasonic treatment time is 4 minutes, and it is carried out under water bath conditions of 5-10℃; wherein, the filling volume of the glass beads accounts for 1 / 3 of the solution volume.

[0012] Furthermore, the root irrigation application begins 24 hours after inoculation with the Foc spore suspension, is applied once a week, and 30 mL of the working solution is applied per plant each time, for a continuous period of 3 weeks.

[0013] Furthermore, the banana esDNA can directly inhibit the mycelial growth of Foc.

[0014] Furthermore, the application can induce systemic resistance in banana plants, which is manifested by: increasing the activity of phenylalanine ammonia-lyase, peroxidase, and catalase in the plant leaves, and reducing the content of hydrogen peroxide.

[0015] Furthermore, the application can reduce the colonization of Foc in banana roots and alleviate symptoms of yellowing and wilting in the plants. Beneficial effects

[0016] (1) The banana esDNA provided by the present invention has a direct in vitro inhibitory effect on the pathogen Foc TR4 and can effectively activate the systemic immune response in banana plants, including significantly increasing the activity of key defense enzymes such as phenylalanine ammonia-lyase (PAL), peroxidase (POD), and catalase (CAT), regulating the metabolic homeostasis of reactive oxygen species (such as H2O2), and enhancing the plant's own disease resistance.

[0017] (2) By applying the root irrigation, the present invention can significantly reduce the number of Foc TR4 colonizing the banana root system, effectively alleviate the symptoms of yellowing and wilting caused by the disease, improve the survival rate of infected plants, promote biomass accumulation, and improve root development.

[0018] (3) The banana esDNA provided by this invention is derived from the plant itself, has high biocompatibility and environmental compatibility, and does not have the risk of chemical residues or pathogen resistance. It provides an important technical support for the green and sustainable development of the banana industry. Attached Figure Description

[0019] Figure 1 This is a photograph showing the direct inhibitory effect of banana esDNA on the mycelial growth of Fusarium oxysporum f. sp. Cuban (Foc) in Example 2.

[0020] Figure 2 These are comparative photographs of the appearance of banana potted plants in different treatment groups 60 days after inoculation with the pathogen in Example 3.

[0021] Figure 3 This is a bar chart showing the colonization abundance (gene copy number) of the Cuban specific type (Foc) of Fusarium oxysporum in banana roots under different treatments, as determined by real-time quantitative PCR (qPCR) in Example 3. Detailed Implementation

[0022] To enable those skilled in the art to better understand the technical solutions of this invention, the present application will be further described in detail below with reference to embodiments.

[0023] Example 1: Preparation of banana esDNA 1. Material Collection: Select vigorous "Brazilian banana" (Musa AAACavendish cv. 'Brazilian') plants from healthy banana plantations with no history of wilt disease. Collect tender, healthy leaves, flash-freeze them in liquid nitrogen, and store them at -80℃ for later use.

[0024] 2. Total DNA Extraction: Weigh 0.1g of frozen tissue and grind it thoroughly into powder in liquid nitrogen. Use the Plant Genomic DNA Extraction Kit (DP304) from Tiangen Biotech Co., Ltd. to extract total DNA strictly according to the instructions.

[0025] 3. DNA Quality and Concentration Detection: The concentration (ng / μL) and purity of the obtained DNA solution were determined using a NanoDrop 2000 spectrophotometer. DNA integrity was then assessed by 1% agarose gel electrophoresis to ensure the main band was clear and free of significant degradation.

[0026] 4. esDNA Fragmentation Preparation: Place the extracted high-purity intact genomic DNA solution (concentration approximately 100-200 ng / μL) into a 2 mL centrifuge tube. Add an appropriate amount of acid-washed glass beads with a diameter of 0.5 mm (filling the tube to approximately 1 / 3 of the solution volume). Use a Jiemeng JP-1005 ultrasonic cleaner to sonicate in a water bath (5-10℃) for 4 minutes to physically cleave the large DNA fragments into smaller fragments of 100-500 bp.

[0027] 5. Fragment Validation and Working Solution Preparation: The cleaved DNA product was subjected to 1% agarose gel electrophoresis to verify that the fragment size was mainly distributed in the range of 100-500 bp. The cleaved esDNA solution was quantitatively diluted to 20 ng / μL (for antibacterial experiments) or 30 ng / μL (for pot experiment) with sterile, nuclease-free deionized water to prepare the standard working stock solution. After aliquoting, it was stored at -20℃ protected from light, avoiding repeated freeze-thaw cycles.

[0028] Example 2: Determination of the direct inhibitory effect of banana esDNA on Foc TR4 1. Pathogen activation and culture medium preparation: Prepare potato dextrose agar (PDA), autoclave at 121℃ for 20 minutes, then pour it into petri dishes in a sterile laminar flow hood and allow it to solidify. Inoculate the preserved Fusarium oxysporum Cuban race Tropical 4 (Foc TR4) into the center of the PDA plate and incubate in the dark at 28℃ for 5-7 days.

[0029] 2. Preparation of drug-containing plates: Under aseptic conditions, different volumes of the banana esDNA working stock solution (20 ng / μL) prepared in Example 1 were taken and evenly spread onto the surface of the solidified PDA plates using a sterile spreader. Plates coated with an equal volume of sterile deionized water were used as blank controls (CK).

[0030] 3. Mycelial disc inoculation and culture: Using a sterile punch, cut a mycelial disc with a diameter of about 6 mm from the edge of the activated Foc TR4 colony. Using a sterile inoculation needle, inoculate the mycelial disc (mycelial side down) into the center of the PDA plates containing different concentrations of esDNA and the control.

[0031] 4. Data Observation and Recording: The inoculated plates were placed in a 28℃ incubator for dark incubation. Mycelial growth was observed daily. When the control group colonies covered approximately two-thirds of the plate area, the colony diameter of each treatment group was measured using the cross-sectional method.

[0032] 5. Results Analysis and Judgment: By comparing the differences in colony growth between the 100 μL 20 ng / μL and 200 μL 20 ng / μL esDNA treatment groups and the control group, the inhibition rate was calculated (inhibition rate (%) = [(control group colony diameter - treatment group colony diameter) / control group colony diameter) × 100%]), and the direct antibacterial activity of banana esDNA against Foc TR4 was systematically evaluated, and its effective concentration range was determined.

[0033] The results show that ( Figure 1 The addition of esDNA to the plates directly inhibited FOC. On day four of inoculation, the FOC colony diameter treated with esDNA was significantly smaller than that in the control (CK) plate (with added sterile water). By day seven, the FOC in the CK plate had completely covered the plate, while the inhibition rates of esDNA treatment were 31.3% (200 μL 20 ng / μL) and 27.7% (100 μL 20 ng / μL).

[0034] Example 3: Greenhouse pot experiment to verify the esDNA-induced disease resistance of bananas 1. Preparation of test materials (1) Banana seedlings tested: Selected “Brazilian banana” tissue culture bottle seedlings with uniform growth and free from pests and diseases, transplanted into nutrient pots containing sterilized substrate, and cultivated in a greenhouse (day / night temperature of 28±2℃ / 25±2℃, relative humidity of 70%-80%, natural light supplemented by LED plant growth lamps) for 4 weeks to allow the seedlings to recover.

[0035] (2) Test soil: Soil from the 0-20 cm topsoil layer of banana plantations affected by banana wilt disease was collected. After removing impurities and air-drying, the soil was sieved through a 2 mm sieve and then exposed to sunlight and fumigated to serve as a uniform base soil.

[0036] (3) Preparation of pathogen spore suspension: Activated Foc TR4 strain was inoculated into potato dextrose liquid medium (PDB) and cultured at 28℃ and 150 rpm / min for 48 hours with shaking. Mycelia were collected, washed with sterile water, filtered, and the spore concentration was adjusted to 1.28 × 10⁻⁶ using a hemocytometer. 8 CFU / mL available for use.

[0037] (4) esDNA working solution: The esDNA from healthy banana roots prepared by the method in Example 1 was diluted with sterile water to 30 ng / μL.

[0038] 2. Experimental Design and Processing A greenhouse pot experiment was conducted, with four treatment groups, each with six replicates, arranged in a randomized block design.

[0039] CK (blank control): No pathogen inoculation, weekly root irrigation with an equal volume of sterile water.

[0040] Foc (pathogen control): Inoculate with Foc spore suspension and drench the roots with an equal volume of sterile water weekly.

[0041] Foc+esDNA (treatment group): 24 hours after inoculation with Foc spore suspension, drench the roots weekly with esDNA working solution (30 mL / plant) for the first three weeks, and then irrigate weekly with an equal volume of sterile water.

[0042] esDNA (DNA control): No pathogens were inoculated. For the first three weeks, the roots were irrigated with an equal volume of esDNA working solution each week, followed by irrigation with an equal volume of sterile water.

[0043] After the banana seedlings had recovered from inoculation, they were inoculated with the pathogen. A root drenching method was used, with each plant individually irrigated with 30 mL of the prepared Foc TR4 spore suspension (Foc group and Foc+esDNA group). The CK group and esDNA group were irrigated with an equal volume of sterile water. esDNA treatment began 24 hours after pathogen inoculation and was repeated weekly for three consecutive weeks.

[0044] 3. Indicator Measurement and Methods (1) Plant growth observation: After inoculation, the plant height, vertical height from the base of the stem to the base of the petiole of the newly unfolded leaf, stem diameter, pseudostem base diameter and leaf yellowing index were measured and evaluated according to the proportion of yellowing area of ​​the leaves and photographed and recorded.

[0045] (2) Root morphology analysis: After sampling, the roots were washed and placed in a transparent plastic root plate. The lateral roots were gently dispersed by tweezers. The cover of the scanner was removed, and the roots were scanned with an Epson scanner (Perfection V800). The root length, root diameter, root surface area and other parameters were analyzed using the WinRHIZO root analysis system.

[0046] (3) Determination of disease resistance-related physiological and biochemical indicators: Leaf tissues from each treatment group were collected, flash-frozen in liquid nitrogen, and stored at -80℃. Crude enzyme solution was extracted by cold grinding, and the activities of enzymes such as phenylalanine ammonia-lyase (PAL) and peroxidase (POD) were determined using the corresponding kits.

[0047] (4) Quantitative analysis of Foc TR4 in roots: On day 30 after treatment, root samples of banana plants from each treatment were collected and ground in liquid nitrogen. Total DNA was extracted using a plant genomic DNA extraction kit. Amplification was performed on a real-time quantitative PCR instrument using specific primer pairs for Foc TR4 (FOF1, 5′-ACATACCACTTGTTGCCTC-3′, SEQ ID NO.1; FOR1, 5′-CGCCAATCAATTTGAGGAACG-3′, SEQ ID NO.2) with SYBR Green as the fluorescent dye. Reaction system (20 μL): 10 μL of 2×SYBR Green premix, 0.5 μL each of forward and reverse primers (10 μM), 1 μL of template DNA, and 8 μL of ddH2O. Reaction program: 95℃ pre-denaturation for 5 minutes; 95℃ denaturation for 30 seconds, 60℃ annealing for 30 seconds, and 72℃ extension for 30 seconds, for a total of 40 cycles. The number of Foc TR4 gene copies per gram of fresh root weight was calculated using a standard curve containing plasmids with known copy numbers.

[0048] 4. Data statistics: SPSS or R software was used to perform one-way ANOVA on the experimental data. Duncan's new multiple range test was used to test the significance of differences (p<0.05). Origin or Excel software was used to draw charts and display the results.

[0049] 5. Test Results (1) Disease control and plant growth: The results showed that ( Figure 2 The Foc-treated group of bananas began to yellow 18 days after inoculation and all died after 30 days. Although the Foc+esDNA-treated group showed mild yellowing, they maintained growth after 60 days and retained a significant number of green leaves. Furthermore, the growth of bananas treated with esDNA alone was basically the same as the CK control, indicating that esDNA itself has no toxic effect on bananas.

[0050] According to Table 1, each treatment had a significant impact on banana growth characteristics (p<0.05). Compared with the control (CK), the FOC treatment significantly inhibited banana plant growth, with plant height, stem diameter, and aboveground and underground dry weight all significantly reduced, reaching the lowest levels overall. Compared with the FOC treatment, the application of esDNA under FOC stress improved all growth indicators, with plant height and aboveground and underground dry weight significantly higher than the FOC treatment, but still lower than the CK level; stem diameter showed some changes, but the difference from the FOC treatment was not significant. Under pathogen-free conditions, esDNA treatment showed a certain promoting effect on plant growth, with plant height significantly higher than the CK, aboveground dry weight not significantly different from the CK, underground dry weight slightly lower than the CK but still significantly higher than the FOC treatment, while stem diameter was lower than other treatments. Overall, FOC stress significantly inhibited banana growth, while the application of esDNA under FOC conditions improved plant growth indicators to varying degrees compared with the FOC treatment, especially in terms of biomass recovery.

[0051] Table 1. Banana growth characteristics under different treatments

[0052] (2) Root growth: As shown in Table 2, the root length and surface area of ​​the treatment with pathogens alone were significantly lower than those of the CK and esDNA treatments, while the root length and surface area of ​​the FOC+esDNA treatment were significantly higher than those of the FOC treatment, indicating that esDNA alleviated the inhibition of root growth by FOC.

[0053] Table 2. Root morphology parameters of bananas under different treatments

[0054] (3) Physiological resistance response: According to the results in Table 3, each treatment had a significant impact on the physiological indicators of banana leaf resistance. Regarding osmotic regulators, the FOC treatment significantly increased proline (Pro) content, significantly higher than the control (CK), indicating that FOC stress induced a stress response in the plant. While the Pro content in the FOC+esDNA treatment was slightly lower than in FOC, it remained at a high level, indicating that esDNA maintained a certain osmotic regulatory capacity while alleviating stress. In terms of reactive oxygen species (ROS) metabolism, the FOC treatment significantly inhibited superoxide dismutase (SOD) activity and significantly increased H2O2 content, indicating that pathogen infection led to ROS accumulation and exacerbated oxidative stress. In contrast, the FOC+esDNA treatment further reduced SOD activity but significantly reduced H2O2 content. In the defense enzyme system, phenylalanine ammonia-lyase (PAL) activity reached its highest level in the FOC+esDNA treatment, significantly higher than in both FOC and CK, indicating that esDNA significantly activated secondary metabolic pathways related to disease resistance. Furthermore, the activities of catalase (CAT) and peroxidase (POD) in the FOC+esDNA treatment reached 21.86 and 6566.5, respectively, both significantly higher than other treatments. In particular, POD activity increased by approximately 2.5 times compared to the FOC treatment, indicating that esDNA significantly enhanced the plant's ability to scavenge reactive oxygen species. Notably, even without pathogen inoculation, application of esDNA alone could also increase CAT and POD activities to some extent while maintaining low H2O2 levels, suggesting that esDNA itself has the function of inducing a basal immune response in plants. In summary, esDNA enhances the resistance of banana plants to FOC infection by significantly increasing the activities of key defense enzymes such as PAL, CAT, and POD, and effectively regulating reactive oxygen species homeostasis.

[0055] Table 3. Banana leaf resistance enzyme activities under different treatments

[0056] (4) Colonization of pathogens in the roots: Quantitative analysis results of Fusarium rhizosphere are as follows Figure 3 As shown, the pathogen gene abundance in the FOC-only inoculation group was significantly higher than that in the CK control group (P<0.05), indicating that the pathogen successfully colonized and proliferated in the roots. Compared with FOC alone, esDNA pretreatment significantly inhibited pathogen enrichment, and the gene abundance in the FOC+esDNA treatment group was significantly reduced. Furthermore, there was no significant difference between the esDNA-only treatment group and the CK control group; both maintained a low baseline level of pathogens, significantly lower than the FOC-only infection group, indicating that esDNA itself does not promote pathogen growth but can effectively reduce the rhizosphere colonization pressure of FOC.

[0057] In summary, this invention, through exogenous application of fragmented autologous esDNA extracted from healthy banana tissue via root irrigation, not only directly inhibits the mycelial growth of Foc TR4 in vitro, but also significantly enhances the banana plant's resistance to Fusarium wilt, reduces the root colonization of the pathogen, and ultimately alleviates disease symptoms and improves plant survival rate and biomass by activating the banana plant's systemic immune response in vivo (including increasing the activity of key defense enzymes, regulating reactive oxygen species metabolism, and promoting root growth). This method provides a novel, environmentally friendly, and effective solution for the green control of banana Fusarium wilt.

Claims

1. The application of esDNA in the control of banana wilt disease, characterized in that, The banana esDNA is an extracellular DNA fragment of the banana plant, which is obtained by processing the total DNA extracted from healthy banana leaves into DNA fragments with a size of 100-500 bp through physical shearing. The banana wilt disease is caused by Fusarium oxysporum, specifically tropical race 4, also known as Foc TR4.

2. The application according to claim 1, characterized in that, The application includes: preparing the banana esDNA into a working solution and applying it to banana plants by root irrigation.

3. The application according to claim 2, characterized in that, The concentration of the working solution is 30 ng / μL.

4. The application according to claim 1, characterized in that, The physical shearing method is an ultrasonic crushing method using glass beads with a diameter of 0.5 mm. The ultrasonic treatment lasts for 4 minutes and is carried out in a water bath at 5-10℃. The glass beads fill 1 / 3 of the solution volume.

5. The application according to claim 2, characterized in that, The root irrigation application begins 24 hours after inoculation with Foc TR4 spore suspension, and is applied once a week, with 30 mL of the working solution applied per plant each time, for 3 consecutive weeks.

6. The application according to claim 1, characterized in that, The banana esDNA can directly inhibit the mycelial growth of Foc TR4.

7. The application according to claim 1, characterized in that, The application can induce systemic resistance in banana plants, which is manifested by increasing the activity of phenylalanine ammonia-lyase, peroxidase, and catalase in the plant leaves, and reducing the content of hydrogen peroxide.

8. The application according to claim 1, characterized in that, The application can reduce the colonization of Foc TR4 in banana roots and alleviate symptoms of yellowing and wilting in the plants.

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