Disease prevention and seedling raising method for grapefruits

Abstract

The invention relates to the technical field of plant disease prevention and control, and particularly discloses a grapefruit disease prevention and seedling raising method, specifically, according to the prevention and control method provided by the invention, a bactericide with an effective component of an ethanol extract of branches and leaves of kukuhua catalpa is used, and the bactericide is sprayed on grapefruit tree seedlings. The Yunnan catalpa bungei extract can effectively prevent and control pomelo tree canker caused by infection of pathogenic bacteria Xanthomonas citri subsp.citri (Xcc), when the effective concentration is 500 mg / L, the relative control effect reaches up to 83.52%, a green and sustainable way is provided for prevention and control of pomelo tree canker, and occurrence of the pomelo tree canker is expected to be relieved.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural biological control, and particularly relates to a method for preventing and controlling canker disease caused by Xcc during the pomelo seedling stage by utilizing a natural antibacterial agent extracted from a plant. Background Art

[0002] Pomelo (Citrus maxima) is a plant of the genus Citrus in the Rutaceae family and is a major economic crop. Canker is a major disease that threatens the pomelo industry. It is mainly caused by pathogenic variants of Xanthomonas spp. Xanthomonas citri subsp. citri (Xcc) It has occurred in all major planting areas of my country. The pathogen can be spread through rain splash, irrigation water, and agricultural operations, infecting multiple parts of pomelo trees, including leaves, branches, and fruit. Long-distance transmission is mainly through diseased seedlings, scions, and fruit. If these infected cultivation materials are not quarantined or treated, they may carry the pathogen to new planting areas. During the seedling stage, once the seedlings are infected with the pathogen, they may cause water-soaked lesions on the leaves and die back of branches and branches at the very least, or even the death of the entire plant at the worst, seriously affecting the quality and survival rate of the seedlings. According to surveys, in nurseries with severe disease, ulcer disease can cause a seedling mortality rate of up to 30% to 50%, resulting in huge economic losses for growers.

[0003] Currently, the prevention and control of grapefruit canker mainly relies on chemical agents, such as Bordeaux mixture and copper preparations like copper hydroxide. However, the long-term use of these chemicals alone can not only easily lead to the development of resistance in pathogens, but can also cause copper accumulation in the soil, disrupting ecological balance, and causing phytotoxicity to seedlings. Furthermore, as demand for agricultural product quality and safety continues to rise, the use of chemical pesticides is subject to increasingly stringent restrictions.

[0004] Botanical fungicides are gaining increasing attention as an environmentally friendly alternative control method. These fungicides, primarily derived from plant secondary metabolites such as alkaloids, phenols, and terpenes, possess a broad antimicrobial spectrum, diverse mechanisms of action, and good environmental compatibility. Importantly, botanical fungicides are less likely to induce resistance in pathogens and are safe against non-target organisms, meeting the requirements of green development in modern agriculture. However, research on botanical fungicides for grapefruit seedling canker is relatively underdeveloped, and the efficacy and stability of existing products remain to be improved. Therefore, the development of effective and safe botanical fungicides and the establishment of supporting application techniques are crucial for the sustainable development of the grapefruit industry. Summary of the Invention

[0005] The main purpose of the present invention is to provide a method for preventing and controlling pomelo seedling canker disease, so as to solve the problems existing in the background technology.

[0006] In order to achieve the above objectives, the present invention provides the following technical solutions: The invention provides a pomelo disease prevention and seedling raising method, which comprises spraying a fungicide containing a Chinese catalpa ovata extract on the pomelo seedlings to prevent and treat ulcer disease. The Chinese catalpa ovata extract is an ethanol extract of Chinese catalpa ovata branches and leaves.

[0007] Furthermore, the concentration of the ethanol extract of Catalpa yunnanensis branches and leaves is not less than 500 mg / L.

[0008] The present invention also provides an ethanol extract of catalpa ovata branches and leaves for preventing and treating pathogenic bacteria. Xanthomonas citri subsp. citri (Xcc) Application in plant diseases caused by.

[0009] Furthermore, the plant is a citrus plant.

[0010] Furthermore, the plant is pomelo.

[0011] As an embodiment, the present invention also provides a fungicide for preventing and treating pathogens. Xanthomonas citri subsp. citri (Xcc) for use in infection, wherein the active ingredient of the fungicide is an ethanol extract of Catalpa yunnanensis branches and leaves. In some embodiments, the fungicide further comprises an adjuvant, a carrier, a preservative, a pH adjuster, or a buffer, and in some embodiments, the adjuvant comprises a surfactant, an emulsifier, a dispersant, or a stabilizer.

[0012] As an embodiment, the present invention also provides a bactericidal composition for preventing and treating pathogens. Xanthomonas citri subsp. citri (Xcc) For use in infection, the fungicidal composition comprises the ethanol extract of Catalpa ovata branches and leaves. In some embodiments, the fungicidal composition comprises any other fungicidal active ingredient that can synergize with the ethanol extract of Catalpa ovata branches and leaves.

[0013] The technical effects achieved by the present invention are: The results of indoor fungicide toxicity test show that the four plant-derived antibacterial agents, namely, Rauwolfia yunnanensis extract, Oroxylum indica extract and Catalpa yunnanensis extract, are effective against the pathogen of grapefruit canker. Xcc All had a certain inhibitory effect, with the order of strength being: Catalpa yunnanensis extract > Tiezai extract > Rauwolfia yunnanensis extract > Oroxylum indica extract. Seedling efficacy tests showed that Catalpa yunnanensis extract and Rauwolfia yunnanensis extract at varying concentrations were effective in preventing and treating pomelo canker. Catalpa yunnanensis extract showed the greatest efficacy at a concentration of 500 mg / L, with a relative efficacy of 83.52%. Furthermore, in vivo seedling experiments showed that Catalpa yunnanensis extract did not cause phytotoxicity to pomelo seedlings at varying concentrations, allowing them to grow normally. DETAILED DESCRIPTION

[0014] The present invention is further described below by way of examples. It should be emphasized that these examples are merely examples for illustrating the principles and effects of the present invention and do not constitute a limitation on the scope of protection of the present invention. In the following examples, the experimental methods mentioned are, unless otherwise specified, conventional methods commonly used in the art. Similarly, the materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained through conventional commercial channels.

[0015] Table 1 Medicinal plants tested

[0016] The term "branches and leaves" refers to the terminal branches of a plant, which are formed by leaves and stems. Leaves consist of two parts: blades and petioles. Therefore, the structural components on the branches are divided into three parts: blades, petioles and stems.

[0017] Example 1 Drug sensitivity test of the test medicinal plant extracts against grapefruit canker pathogen Xcc Test materials Test pathogen: The test bacteria was Xanthomonas citri subsp. citri (Xcc), a common pathogen of grapefruit canker, provided by Yunnan Academy of Agricultural Sciences and inoculated on LB solid medium.

[0018] LB solid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 15 g / L agar.

[0019] Preparation method of medicinal plant extracts: Weigh 20 g of dried and crushed plant powder, place them in 500 mL conical flasks respectively, add 100 mL of 90% ethanol, and extract with ultrasound for 30 minutes. Then add 100 mL of 90% ethanol to the filter residue and repeat the extraction once. Combine the two filtrates and concentrate them using a rotary evaporator (40°C, 100 r / min). After drying, add anhydrous ethanol to dissolve it and then add distilled water to make up the volume to prepare a medicinal plant extract mother solution with a mass concentration of 10 mg / mL. Store it in a refrigerator at 4°C for later use.

[0020] Pathogen activation Inoculate pathogen Xcc onto freshly prepared LB solid medium plates using the streak method, controlling the force of the streak to ensure a single colony is obtained. Incubate the inoculated plate upside down in a 28°C incubator in the dark for 72 hours, regularly monitoring colony growth. Once a typical, uniform colony appears on the plate, select a representative colony for subsequent experiments or store it at 4°C for short-term use.

[0021] Bacterial suspension preparation First, a representative single colony was picked from a 72-hour LB solid medium plate and inoculated into 20 mL of sterile LB liquid medium (composition: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride). The inoculated culture flask was placed in a 28°C incubator and shaken at 200 rpm for 18–24 hours until the logarithmic growth phase. An appropriate amount of the bacterial solution was measured using a spectrophotometer to determine the OD value. 600 value, and the bacterial solution OD 600 The value was adjusted to 1.0. The bacterial suspension was centrifuged at 4000 rpm for 5 minutes to collect the cells. The supernatant was discarded and the pellet was resuspended in sterile saline (0.85% NaCl). The bacterial suspension concentration was determined by plate count method. The bacterial suspension was diluted 10-fold and then spread on LB solid medium. The suspension was incubated at 28°C for 18 hours and then counted. Finally, based on the count results, the bacterial suspension concentration was accurately adjusted to 1×10 using sterile saline. 8 cfu / mL and stored at 4°C for future use. All operations were performed under sterile conditions.

[0022] Indoor antibacterial effect test The concentration of bacterial suspension was determined by spectrophotometry to evaluate the effect of plant extracts on Xcc The four test agents were prepared into 10 mg / mL stock solutions, and five concentration gradients of drug-containing LB liquid culture medium were prepared by two-fold dilution method. At the same time, LB liquid culture medium without drug was set as blank control (CK), and three replicates were set for each concentration. The treated culture medium was placed in a constant temperature incubator at 28°C for 16 hours and the OD was measured. 600 Antibacterial rate (%) = 1- [(OD after drug treatment 600 ) / (control group OD 600 )]×100%.

[0023] In order to further quantify the effects of the four botanical fungicides on Xcc The toxicity effect of each agent was analyzed by converting the five concentration gradients (mg / L) of each agent into logarithmic values ​​(x). At the same time, the average value of the colony growth inhibition rate obtained by three repeated experiments was converted into a probability value (y) to establish a dose-response relationship data set. Subsequently, statistical software was used to perform regression analysis on these data and fit the four botanical fungicides to the Xcc Finally, the EC of each drug was obtained by solving the equation 50 EC value, that is, the concentration of drug required to achieve 50% growth inhibition rate. 50 The smaller the value, the better the Xcc The stronger the virulence, the more significant the antibacterial effect.

[0024] Indoor antibacterial test results 4 kinds of plant extracts Xcc The results of growth inhibition are shown in Table 2. As the concentration of fungicide increases, the antibacterial effect becomes more obvious, among which the extract of Catalpa yunnanensis has a Xcc The best inhibitory effect was achieved when the concentration reached 400 mg / L, with an inhibition rate of 86.98%. The second was the iron extract, which had an inhibition rate of 67.78% when the concentration reached 400 mg / L. Xcc The inhibitory effect of the four agents was the worst. When the concentration reached 400 mg / L, the inhibition rate was only 42.89%. XCC EC 50 The toxicity of catalpa asiatica extracts is different from that of XCC The most virulent, EC 50 The value was 129.10 mg / L, followed by the iron extract, EC 50 The toxicity of the extracts of Rauwolfia yunnanensis and Oroxylum indica decreased in turn, and the EC 50 They are 442.41 and 573.17 mg / L respectively.

[0025] Table 2 Inhibitory effect of botanical fungicides on Xcc and toxicity analysis

[0026] Example 2 Determination of the efficacy of botanical fungicides against grapefruit seedling canker Potted pomelo seedlings with healthy growth and no pests and diseases were selected as experimental materials. The seedling height was controlled at 30±2cm and the number of leaves was 8-10. Three new leaves in the rapid growth period (before reaching the maximum leaf area) were selected from each seedling as treatment objects. Before inoculation, the leaf surface was disinfected with 75% alcohol. After the alcohol evaporated, 10 evenly distributed puncture points (spacing 1 cm×1 cm) were made on the back of the leaf with a sterile injection needle. The depth of the wound should just penetrate the epidermis. Dip a sterile cotton swab in Xcc Bacterial suspension (concentration 1×10 6 CFU / mL) was evenly applied to the wound and the leaf surface area within 1 cm around it, and a treatment with sterile water was set as a negative control.

[0027] During the incubation period, two antibacterial agents, Tiezai extract and Catalpa yunnanensis extract, were prepared into working solutions at three concentration gradients: 300 mg / L, 400 mg / L, and 500 mg / L, respectively. Foliar spraying was performed using a sprayer, maintaining the nozzle 30 cm from the leaves to ensure even coverage of the entire leaf surface (front and back). Approximately 20 mL of solution was applied per leaf at a time (equivalent to 60 L per mu). The first application was made 24 hours after inoculation, followed by applications every three days for a total of four applications. All treated seedlings were cultured in a rainproof canopy under controlled environmental conditions: day / night temperatures of 28 ± 2°C / 22 ± 2°C, relative humidity of 70%–80%, and natural light. Disease investigations were conducted seven days after the final application.

[0028] Disease index statistics and grading Incidence rate (%) = (number of lesions at the wound site on the leaf ÷ total number of lesion sites on the leaf) × 100; disease grading standard: Level 0 means no lesions at the wound site; Level 1 means 1 to 3 lesions at the wound site; Level 2 means 4 to 6 lesions at the wound site; Level 3 means 7 or more lesions at the wound site; disease index (%) = (0 × n0 + 0.25 × n1 + 0.5 × n2 + 0.75 × n3) ÷ n × 100, where n0 represents the number of leaves at level 0, n1, n2, n3, and so on; and n is the total number of leaves at each level.

[0029] Relative prevention effect (%) = (disease index of control group - disease index of treatment group) / disease index of control group × 100.

[0030] Prevention results The results of the comparative test on the efficacy of pesticides showed that the treatment with 500 mg / L Chinese catalpa ovata extract had the best control effect, with an incidence rate of only 20.16%, a disease index of 13.98, and a relative control effect of 83.52%; the treatment with 500 mg / L iron pine extract had a lower control effect, with an incidence rate of 49.96%, a disease index of 38.96, and a relative control effect of 54.07%.

[0031] Table 4 Field efficacy test results of the tested agents on young leaves of pomelo trees.

[0032]

[0033] The test showed that 500 mg / L of Catalpa yunnanensis extract was the most effective in preventing pomelo seedlings from the pathogenic bacteria causing the disease, with a relative protection rate of 83.52%. Therefore, the Catalpa yunnanensis extract in the present invention can be used as a recommended agent for preventing pomelo seedling canker.

[0034] By pathogens ( Xcc) is a major disease that seriously restricts the healthy development of my country's pomelo industry, not only causing yield losses but also directly affecting the commercial value of the fruit. Currently, there is a lack of effective tools for green and sustainable control in the prevention and treatment of pomelo canker. In the present invention, the results of the indoor drug sensitivity test showed that the four botanical fungicides, Tiezai extract, Yunnan Rauwolfia extract, Oroxylum indica extract and Yunnan Catalpa asiatica extract, were effective against the pathogen of pomelo canker. Xcc All had a certain inhibitory effect, with the order of strength being Catalpa yunnanensis extract > Tiezai extract > Rauwolfia yunnanensis extract > Oroxylum indica extract. Pomelo control efficacy tests showed that Catalpa yunnanensis extract and Tiezai extract at different concentrations were effective in preventing and treating pomelo canker. Catalpa yunnanensis extract had the best control effect at a concentration of 500 mg / L, with a relative efficacy of 83.52%.

[0035] Through a series of rigorous drug sensitivity tests and crop efficacy verification, this study successfully selected agents with excellent control effects against grapefruit canker. This discovery not only opens up new avenues for grapefruit disease prevention and control, providing strong support for reducing disease occurrence, but also demonstrates that the selected antibacterial agents are all naturally derived and environmentally friendly.

[0036] The above description is only a preferred embodiment of the present invention and does not limit the patent scope of the present invention. All equivalent transformations made using the contents of the present invention specification under the concept of the present invention, or direct / indirect applications in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. A pomelo disease prevention seedling raising method, characterized in that: A fungicide containing a Chinese catalpa ovata extract is sprayed on pomelo seedlings to prevent and treat ulcer disease. The Chinese catalpa ovata extract is an ethanol extract of Chinese catalpa ovata branches and leaves.

2. The method according to claim 1, characterized in that The usage concentration of the ethanol extract of Catalpa yunnanensis branches and leaves is not less than 500 mg / L.

3. Application of ethanol extract of Catalpa yunnanensis branches and leaves in preventing and controlling plant diseases caused by the pathogen Xanthomonas citri subsp. citri (Xcc).

4. The use according to claim 3, characterized in that The plant is a citrus plant.

5. The use according to claim 4, characterized in that The plant is pomelo.

6. A fungicide in preventing and controlling pathogens Xanthomonas citri subsp. citri (Xcc) Application in infection, characterized in that The active ingredient of the fungicide is the ethanol extract of Catalpa yunnanensis branches and leaves.

7. A bactericidal composition for preventing and treating pathogens Xanthomonas citri subsp. citri (Xcc) application in infection, characterized in that The bactericidal composition contains the ethanol extract of Catalpa yunnanensis branches and leaves.

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