Green prevention and control method for virus diseases of passion fruits

Abstract

The invention discloses a green prevention and control method for viral diseases of passion fruits, which comprises the following steps: purifying seedlings: breeding the seedlings: selecting passion fruit cutting seedlings or grafted seedlings with the plant height of 15-20cm in a 4-6 leaf stage, and screening healthy seedlings without yellow leaves and without spots on stalks; in the 2-6 leaf stage, 0.2% monopotassium phosphate leaf fertilizer is sprayed every 7 days, potassium and phosphorus nutrition is supplemented, the stress resistance of plants is improved by 40% or above, the virus infection probability is reduced by 60%, diseased plants and soil with the depth of 30-40 cm within the range of 1 m around the diseased plants are dug out with a garden shovel disinfected with 75% alcohol, and the diseased plants are moved to special pits with the depth of 50 m to be buried; the disease hole is subjected to quick lime and high-temperature soil covering treatment, the soil virus Ct value is larger than 38 (negative), residual viruses are thoroughly inactivated, the morbidity of plants around the disease hole is reduced to 0, and secondary infection is avoided.

Description

Technical Field

[0001] This invention relates to the field of green prevention and control technology for passion fruit viruses, and in particular to a green prevention and control method for passion fruit viral diseases. Background Technology

[0002] Passion fruit, a specialty economic crop in tropical and subtropical regions, is rich in vitamin C, amino acids, flavonoids, and dietary fiber. It has dual value for both fresh consumption and processing (juice, jam). Its cultivation scale has been expanding year by year in production areas such as Guangxi, Fujian, and Yunnan in my country, making it an important economic crop driving farmers' income growth. However, the viral diseases encountered in passion fruit production have become a core bottleneck restricting the high-quality development of the industry. Among them, TeMV (night-blooming jasmine mosaic virus), PWV (passion fruit wooding virus), and CMV (cucumber mosaic virus) are the three most harmful pathogenic viruses. They can spread through multiple pathways, including infected seedlings, aphids / thrips, and residual viruses in the soil, disrupting the entire growth cycle of passion fruit.

[0003] Passion fruit plants infected with the virus will exhibit typical symptoms: yellowing, wrinkling, and mottling of leaves; abnormal lignification of stems (hardening and cracking); and a sharp drop in photosynthetic efficiency (photosynthetic rate can drop to 11 μmol / m³). 2 (Below s), ultimately leading to a 30%-60% reduction in fruit yield, and a drop in soluble solids content from over 16% to below 14%, resulting in a sour and astringent taste and loss of marketability. Current prevention and control technologies have many key shortcomings, making it difficult to overcome the bottleneck in viral disease control. Specific problems are as follows:

[0004] 1. Failure of source control and high rate of seedling infection: In current planting, most farmers use ordinary cuttings or grafted seedlings that have not undergone strict virus testing, or even directly use seedlings propagated from infected mother plants; some virus elimination techniques rely only on single heat treatment or simple shoot tip culture, lacking accurate RT-PCR detection, resulting in a generally low virus elimination rate, which leads to the virus entering the field from the initial stage of planting, creating a "congenital virus-carrying" risk.

[0005] 2. Inefficient vector control leads to significant environmental pollution and pesticide resistance. Traditional control methods rely on chemical insecticides such as imidacloprid and acetamiprid to control aphids and thrips. Long-term use not only leads to increased pesticide resistance in vectors but also causes pesticide residues in the soil and contamination of fruits, failing to meet green food standards. At the same time, the lack of physical barriers and precise monitoring systems makes it impossible to provide timely warnings of vector density.

[0006] 3. Lack of immune support, resulting in weak plant disease resistance. Current technologies focus on "treating disease after it occurs" while neglecting "preventing disease before it occurs": no immune programs have been developed for periods of high viral incidence, nor have disease-resistant nutrients been supplemented in conjunction with the plant's growth stages; plants are in a state of "passive disease resistance" for a long time, with chlorophyll SPAD values ​​often below 40, resulting in poor stress resistance and rapid disease development once infected by a virus.

[0007] 4. Incomplete treatment of diseased plants poses a high risk of secondary infection. After discovering diseased plants, they are simply pulled out without disinfecting the roots and the soil within 1 meter around them. The virus remaining in the soil can spread through cultivation and watering, leading to secondary infection of healthy plants in the surrounding area.

[0008] Therefore, developing a comprehensive green prevention and control method that is controllable at the source, preventable by vectors, strengthens immunity, and treats diseased plants has become an urgent need to solve the problem of passion fruit viral diseases and ensure the economic benefits of growers. Summary of the Invention

[0009] In order to overcome the shortcomings of the existing technology, one of the objectives of this invention is to provide a green prevention and control method for passion fruit viral diseases.

[0010] One of the objectives of this invention is achieved through the following technical solution:

[0011] A green method for controlling passion fruit viral diseases includes the following steps:

[0012] Seedling purification:

[0013] ① Seedling selection: Select passion fruit cuttings or grafted seedlings with 4-6 leaves and a plant height of 15-20cm, and select healthy seedlings without yellow leaves and without spots on the stems;

[0014] RT-PCR was used to detect night-blooming jasmine mosaic virus (TeMV), passion fruit woodiness virus (PWV), and cucumber mosaic virus (CMV). Healthy seedlings with a Ct value > 35 were used as treatment subjects.

[0015] Strengthening the mother plant: Healthy seedlings that pass the test are treated to strengthen them by foliar spraying with 0.3% amino acid foliar fertilizer (total amino acid ≥10%) + 5% amino oligosaccharide soluble concentrate at a dilution of 2000 times, once every 7 days for 2 consecutive times; at the same time, maintain a daytime temperature of 25-28℃ and a nighttime temperature of 18-20℃. After cultivating strong mother plants, they enter a staged heat treatment.

[0016] ② Staged heat treatment: Place healthy seedlings in an intelligent constant temperature incubator. Maintain 38℃±0.5℃ and relative humidity of 65%-70% for the first 7 days, and raise the temperature to 40℃±0.5℃ and relative humidity of 60%-65% for the next 8 days. The light intensity is 2000-2500 lux and the light duration is 12h / d throughout the process.

[0017] ③ Shoot tip culture: After heat treatment, 0.2-0.3 mm shoot tips (with 1-2 leaf primordia) are taken from the terminal buds and inoculated into MS medium containing 0.5 mg / L 6-BA and 0.1 mg / L NAA. The culture is carried out at 25℃±1℃ and 14 h / d light for 30-35 days to obtain tissue culture seedlings.

[0018] ④ Transplanting guidelines: Harden tissue culture seedlings for 7-10 days:

[0019] First 3 days: Daytime temperature 25-28℃, nighttime temperature 18-20℃, shading rate 50%;

[0020] Days 4-7: Daytime temperature transitions to 28-30℃, nighttime temperature to 20-22℃, and shading rate decreases to 30%;

[0021] Last 3 days (or 10 days if hardening off): Full sun, daytime temperature 30-32℃, nighttime temperature 22-24℃;

[0022] During the hardening-off period: Spray the leaves with 0.2% potassium dihydrogen phosphate foliar fertilizer (K2O≥34%, P2O5≥52%) every 3 days, with a spraying amount of 80-100mL / plant;

[0023] Soil disinfection before planting: 7 days before planting, spray the planting area evenly with a mixture of 50% carbendazim wettable powder (500 times dilution) and 0.5% matrine aqueous solution (800 times dilution), at a rate of 200-250 mL / m², to kill residual viruses and pathogens in the soil.

[0024] Media control:

[0025] ① Physical isolation: A protective fence with a height of 2.8-3.0m is built around the planting area. High-density polyethylene insect-proof net (60 mesh high-density polyethylene insect-proof net, mesh deviation ≤0.08mm, breaking strength ≥550N) is fixed inside the protective fence. The bottom of the insect-proof net is buried in the soil for 30-40cm and compacted.

[0026] ② Monitoring system: Hang one double-sided yellow sticky trap (20cm×30cm, coated with 0.1% aphid pheromone attractant) for every 10㎡ in the field. The yellow sticky trap should be 20-30cm away from the top of the plant. Count the number of aphids every day from 9:00 to 10:00. When the aphid density per plant is ≥5 aphids / plant, start emergency control.

[0027] ③ Emergency treatment: For emergency prevention and control, use a 6000-fold dilution of 5% Isatis indigotica extract soluble solution (chlorogenic acid content ≥5%), spray it on the leaves between 9-11 am (temperature 20-25℃, wind speed ≤3m / s), and evenly cover the front and back of the leaves with the solution. Apply twice consecutively, with an interval of 7 days.

[0028] Immune activation:

[0029] ①Basic protection: Every 15 days after transplanting, spray the leaves with a 1800-fold dilution of 5% amino oligosaccharide soluble agent at 8-10 am or 4-6 pm (avoiding temperatures above 30℃), at a rate of 120-150 mL / m².

[0030] ② Enhanced protection during peak season: During the spring and summer peak season for viruses (average daily temperature ≥25℃, relative humidity ≥70%), first spray with a 0.5g / L rhamnolipin solution (fermentation product of Pseudomonas aeruginosa, pH 6.5-7.5, purity ≥98%), and 24 hours later spray with a passion fruit attenuated mutant compound preparation (containing attenuated mutant CMV-m1 of cucumber mosaic virus and attenuated mutant PWV-m2 of passion fruit lignification virus, both at a concentration of 1×10⁻⁶). 6 (Copies / mL, store at 4℃ protected from light, shelf life 6 months);

[0031] ③ Enhanced resistance: During the seedling stage (2-6 leaf stage), spray 0.2% potassium dihydrogen phosphate foliar fertilizer (K2O≥34%, P2O5≥52%) every 7 days, with each spraying amount of 80-100mL / plant, for a total of 3-4 times;

[0032] Treatment of diseased plants:

[0033] ① Diseased plant identification: Diseased plants are identified and marked by observation of symptoms (yellowing, wrinkling, and mottling of leaves, and abnormal lignification of stems) combined with rapid RT-PCR detection;

[0034] ② Mechanical removal: Use a sterilized horticultural shovel (wipe with 75% alcohol for disinfection, disinfect once for each plant) to dig up the diseased plant, removing it along with the roots and the surrounding soil within 1m (30-40cm deep). Move it to a special pit (1.5m deep) more than 50m away from the planting area, cover it with quicklime and then fill it in.

[0035] ③ Treatment of diseased holes: Evenly spread 100-200 mesh quicklime (0.5 kg / hole) in the diseased hole, mix it into the soil to a depth of 20-25 cm, water it with 200-300 mL to activate it, and then cover it with a transparent plastic film (thickness ≥ 0.08 mm) for high-temperature soil treatment; use natural light to raise the temperature during the day and keep the temperature inside the film ≥ 50℃ for 5 days; after removing the plastic film, leave it idle for 2 days, and then replant healthy seedlings. (High-temperature soil treatment combined with quicklime can completely inactivate residual viruses in the soil and avoid secondary infection.)

[0036] As a further improvement to the above technical solution:

[0037] In step (1)③, the MS medium was further supplemented with 30 g / L sucrose and 7 g / L agar, and the pH was adjusted to 5.8-6.0. When the tissue culture seedlings reached the 3-4 leaf stage, RT-PCR was used to detect viruses. The detection targets included night-blooming jasmine mosaic virus (TeMV), passion fruit woodiness virus (PWV), and cucumber mosaic virus (CMV). The primers for TeMV detection were F: 5'-GGTGGTGACGGTGGTGGT-3' and R: 5'-CCTGCTGCTGCTGCTGCT-3'. The PCR amplification conditions were 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 45 s, for a total of 35 cycles, and 72℃ final extension for 10 min. Seedlings with a Ct value ≤35 were considered virus-free.

[0038] The planting area mentioned in step (2)① should be at least 500m away from tobacco and solanaceous crops (tomato, pepper, eggplant), and one insect monitoring point should be set up for every 200㎡. The monitoring point should be equipped with an insect-attracting lamp (wavelength 365nm, power 20W), which should be turned on from 19:00 to 6:00 the next day. The number of insect vectors (aphids, thrips) captured should be counted weekly.

[0039] The rhamnolipin solution described in step (3) ② needs to be filtered through a 0.22μm filter membrane for sterilization before use. If it rains within 24 hours after spraying, it needs to be sprayed again. When spraying the attenuated mutant R181K preparation of night-blooming jasmine mosaic virus, it is necessary to avoid mixing it with alkaline pesticides, and maintain a relative humidity of 60%-70% in the field within 48 hours after spraying.

[0040] Step (1) Before transplanting, the seedling substrate should be prepared with a formula of garden soil: peat moss: fine river sand = 4:1:1. The substrate should be disinfected by spraying with 300-500 times diluted 50% carbendazim wettable powder (200L of solution per cubic meter of substrate). After disinfection, cover with plastic film and seal for 7 days. After ventilating for 3 days, test the substrate colony count. If it is ≤100CFU / g, it can be used. The substrate EC value should be controlled at 1.2-1.8mS / cm and the pH value should be controlled at 5.5-6.5.

[0041] The rapid RT-PCR detection described in step (4)① uses a virus detection kit (detection limit ≤ 10). 3 The sampling site was the new leaves of the diseased plant (the 2nd-3rd leaf from the top), with a sample size of 0.1g. After grinding, total RNA was extracted using the TRIzol method, and cDNA was synthesized by reverse transcription. The cDNA was then amplified by PCR. The amplified products were detected by 1.5% agarose gel electrophoresis. The presence of the target bands (TeMV 580bp, PWV 420bp, CMV 350bp) indicated that the plant was infected.

[0042] After spraying the 5% amino oligosaccharide soluble agent mentioned in step (3)①, it is necessary to regularly monitor the chlorophyll content (SPAD value ≥ 45) and photosynthetic rate (≥ 15 μmol / m²) of the plant leaves. 2 •s), when SPAD value < 40 or photosynthetic rate < 12 μmol / m 2 At 10:00 pm, apply an additional 0.3% amino acid foliar fertilizer (total amino acid content ≥10%).

[0043] It also includes soil conditioning steps: 15 days before transplanting, apply 2000-3000 kg / mu of well-rotted organic fertilizer (chicken manure: sheep manure = 3:1, decomposition period ≥ 60 days) to the soil, combined with bio-fertilizer (effective live bacteria count ≥ 2 × 10⁻⁶). 8 Apply 50 kg / mu (CFU / g) of fertilizer and tillage to a depth of 30-35 cm to adjust the soil organic matter content to ≥2.5% and available nitrogen content to 80-100 mg / kg, thus providing a foundation for disease resistance in plant growth.

[0044] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0045] 1. Select healthy seedlings with 4-6 leaves and a height of 15-20cm. RT-PCR testing (Ct value > 35) ensures the absence of TeMV, PWV, and CMV. Combined with a staged heat treatment of 38℃±0.5℃ for the first 7 days and 40℃±0.5℃ for the next 8 days, and cultured in MS medium (with 0.5mg / L 6-BA + 0.1mg / L NAA) with 0.2-0.3mm shoot tips (with 1-2 leaf primordia), the virus elimination rate of seedlings reached 98.5%, significantly higher than that of traditional single virus elimination techniques.

[0046] Before transplanting, the soil is disinfected with 50% carbendazim + 0.5% matrine, and the seedling substrate is strictly controlled (colony count ≤100 CFU / g, EC value 1.2-1.8 mS / cm) to thoroughly remove residual viruses in the soil and substrate, thus blocking the spread of viruses from the source of planting.

[0047] 2. Construct a 2.8-3.0m protective fence + 60-mesh high-density insect-proof net (bury the bottom in soil 30-40cm) around the planting area to form a physical barrier and reduce the vector invasion rate by more than 90%; hang pheromone yellow sticky traps every 10㎡ and set up 365nm insect-attracting lamps every 200㎡ to achieve real-time monitoring of aphid and thrips density;

[0048] Emergency control measures use a 5% Isatis root extract soluble solution to replace chemical insecticides. After spraying, the aphid mortality rate exceeds 85%, and the amount of pesticide used is reduced by more than 90%, avoiding soil and fruit pollution and solving the problems of resistance and residues in traditional chemical control.

[0049] 3. Basic protection: Spray with 5% amino oligosaccharide soluble concentrate (1800 times dilution) every 15 days after transplanting to ensure that the leaf SPAD value is ≥45 and the photosynthetic rate is ≥15μmol / m². 2 •s, to maintain the plant's basic disease resistance;

[0050] Enhanced protection during peak season: In spring and summer, spray with 0.5 g / L rhamnolipin solution first, and then spray with passion fruit attenuated mutant compound preparation 24 hours later. This can reduce the viral load of wild type virus and form cross protection.

[0051] Enhanced nutrition during the seedling stage: Spray 0.2% potassium dihydrogen phosphate foliar fertilizer every 7 days (3-4 times) during the 2-6 leaf stage to supplement potassium and phosphorus nutrition, improve plant resistance by more than 40%, and reduce the probability of virus infection by 60%.

[0052] 4. Use a gardening shovel disinfected with 75% alcohol to dig up the diseased plant and the surrounding soil within 1m and 30-40cm deep, and move it to a special pit 50m away for burial; treat the diseased hole with quicklime and high-temperature soil saturation, the soil virus Ct value >38 (negative), completely inactivating the residual virus, reducing the incidence of disease in plants around the diseased hole to 0, and avoiding secondary infection.

[0053] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0054] Figure 1 This is a flowchart of the method in this embodiment. Detailed Implementation

[0055] The present invention will now be further described in conjunction with the accompanying drawings and specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0056] It should be noted that when a component is described as "fixed to" another component, it can be directly on the other component or may have a component in between. When a component is considered "connected to" another component, it can be directly connected to the other component or may have a component in between. When a component is considered "set on" another component, it can be directly set on the other component or may have a component in between. The terms "vertical," "horizontal," "left," "right," and similar expressions used in this document are for illustrative purposes only.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items. I. Specific Implementation Methods

[0059] I. Experimental Location and Basic Conditions

[0060] Location: Passion fruit planting base in Nanning, Guangxi (22.82°N, 108.37°E), with an average annual temperature of 21.6°C. The average daily temperature in spring and summer (April-September) is 25-32°C, and the relative humidity is 70%-85%. The soil type is red soil with an initial organic matter content of 1.8% and available nitrogen of 72 mg / kg. The previous crop was tomato (there is a risk of CMV residue). The planting area is 550m away from surrounding solanaceous crops such as tobacco, tomatoes, and peppers, which meets the prevention and control requirement of "interval not less than 500m".

[0061] Variety: Tainong No. 1 passion fruit (susceptible to TeMV, PWV, CMV), seedling age 4-leaf stage, initially tested by RT-PCR (detection limit ≤10). 3 The Ct values ​​of TeMV, PWV, and CMV were all >35, indicating that the vaccine was virus-free.

[0062] Seedling substrate: The formula of garden soil: peat moss: fine river sand = 4:1:1 is used. The substrate is disinfected by spraying with 50% carbendazim wettable powder at a dilution of 400 times (200L of solution per cubic meter of substrate). After disinfection, the substrate is covered with plastic film and sealed for 7 days, then the film is removed and the substrate is ventilated for 3 days. The substrate colony count is 85 CFU / g (≤100 CFU / g), EC value is 1.5 mS / cm (1.2-1.8 mS / cm), and pH value is 6.0 (5.5-6.5), which meets the standards for seedling substrate before transplanting.

[0063] II. Implementation Steps

[0064] (I) Soil conditioning (15 days before transplanting)

[0065] Apply 2500 kg / mu of well-rotted organic fertilizer (chicken manure: sheep manure = 3:1, decomposition period 65 days, ≥60 days) to the soil, combined with bio-fertilizer (effective live bacteria count 2.5×10⁻⁶). 8 CFU / g, ≥2×10 8 CFU / g) 50kg / mu;

[0066] Mechanical tillage was used, with a tillage depth of 32cm (30-35cm), and the soil pH was adjusted to 6.0. After 15 days, the soil organic matter content was tested to be 2.6% (≥2.5%) and available nitrogen was 88mg / kg (80-100mg / kg), which met the conditions of "providing a disease-resistant foundation for plant growth".

[0067] (II) Seedling purification and transplanting

[0068] 1. Seedling selection and parent plant strengthening: Passion fruit cuttings with 4 leaves and a height of 18cm (15-20cm) were selected, and healthy seedlings without yellow leaves and stem spots were screened; RT-PCR testing was performed (detection limit ≤10). 3 The Ct values ​​of TeMV, PWV, and CMV were all >35, and these were identified as treatment targets. For qualified healthy seedlings, the mother plant was strengthened by foliar spraying with a 0.3% amino acid foliar fertilizer (total amino acids ≥10%) + a 2000-fold dilution of 5% amino oligosaccharide soluble concentrate, once every 7 days for two consecutive times; simultaneously, a daytime temperature of 26℃ (25-28℃) and a nighttime temperature of 19℃ (18-20℃) were maintained to cultivate robust mother plants.

[0069] 2. Staged heat treatment: Place healthy seedlings in an intelligent constant temperature incubator. Maintain a temperature of 38℃±0.5℃ and a relative humidity of 68% (65%-70%) for the first 7 days. Increase the temperature to 40℃±0.5℃ and the relative humidity to 62% (60%-65%) for the next 8 days. The light intensity is 2200 lux (2000-2500 lux) and the light duration is 12h / d throughout the process.

[0070] 3. Shoot tip culture: After heat treatment, 0.25 mm (0.2-0.3 mm) shoot tips (with 2 leaf primordia) of the terminal bud are taken and inoculated into MS medium (6-BA 0.5 mg / L + NAA 0.1 mg / L, 30 g / L sucrose and 7 g / L agar added, pH adjusted to 5.9 (5.8-6.0)). The tissue culture seedlings are obtained under the conditions of 25℃±1℃ and 14 h / d light.

[0071] 4. Virus Detection in Tissue Culture Seedlings: Tissue culture seedlings were tested for viruses using RT-PCR at the 3-leaf stage (3-4 leaf stage). The targets for detection were TeMV, PWV, and CMV. The primers for TeMV detection were F: 5'-GGTGGTGACGGTGGTGGT-3' and R: 5'-CCTGCTGCTGCTGCTGCT-3'. PCR amplification conditions were: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 58℃ annealing for 30 s, and 72℃ extension for 45 s, for a total of 35 cycles, with a final extension at 72℃ for 10 min. The results showed that the Ct values ​​for all three viruses were >35, indicating that the seedlings were virus-free.

[0072] 5. Planting specifications:

[0073] Hardening off seedlings: Harden tissue culture seedlings for 8 days (7-10 days). For the first 3 days: daytime temperature 26℃ (25-28℃), nighttime temperature 19℃ (18-20℃), shading rate 50%; for days 4-7: daytime temperature 29℃ (28-30℃), nighttime temperature 21℃ (20-22℃), shading rate reduced to 30%; for the last day (because hardening off takes 8 days, the "last 3 days" full light step is omitted; if hardening off takes 10 days, then "full light, daytime temperature 30-32℃, nighttime temperature 22-24℃"). During the hardening off period, spray the leaves with 0.2% potassium dihydrogen phosphate foliar fertilizer (K2O3 4.5% ≥ 34%, P2O5 52.3% ≥ 52%) every 3 days, at a rate of 90mL / plant (80-100mL / plant).

[0074] Soil disinfection: 7 days before planting, spray the planting plot evenly with a mixture of 50% carbendazim wettable powder (500 times dilution) and 0.5% matrine aqueous solution (800 times dilution), at a rate of 220 mL / m² (200-250 mL / m²), to kill residual viruses and pathogens in the soil.

[0075] Transplanting procedure: After hardening off the seedlings, soak the roots of the tissue culture seedlings in a 2000-fold dilution of 5% amino oligosaccharide soluble solution for 15 minutes, and transplant them with a row spacing of 2.8m and a plant spacing of 1.8m, for a total of 1000 seedlings.

[0076] (III) Media Control

[0077] 1. Physical isolation: Construct a protective fence with a height of 2.9m (2.8-3.0m) around the planting area. Fix a 60-mesh high-density polyethylene insect-proof net (mesh size deviation 0.07mm≤0.08mm, breaking strength 560N≥550N) to the inside of the protective fence. Bury the bottom of the insect-proof net 35cm (30-40cm) in the soil and compact it to form a physical barrier to block the virus transmission medium.

[0078] 2. Monitoring System:

[0079] Hang one double-sided yellow sticky trap (20cm×30cm, coated with 0.1% aphid pheromone attractant) for every 10 square meters in the field, with the yellow sticky trap 25cm (20-30cm) away from the top of the plant;

[0080] The number of aphids is counted every day from 9:00 to 10:00. At the same time, one insect monitoring point is set up for every 200 square meters in the planting area. Each monitoring point is equipped with an insect-attracting lamp with a wavelength of 365nm and a power of 20W. The lamp is turned on from 19:00 to 6:00 the next day. The number of aphids, thrips and other insect vectors that are trapped is counted every week.

[0081] 3. Emergency Response: On June 5th, the aphid density was measured at 6.2 aphids / plant (≥5 aphids / plant), triggering emergency control measures. At 10:00 AM that day (temperature 23℃-20-25℃, wind speed 2.5m / s≤3m / s), a 6000-fold dilution of 5% Isatis indigotica extract soluble concentrate (chlorogenic acid content 5.2% ≥5%) was sprayed on the leaves, ensuring even coverage of both sides of the leaves. A second spray was applied 7 days later, for a total of 2 applications.

[0082] (iv) Immune activation

[0083] 1. Basic Protection: Every 15 days after transplanting, spray the leaves with a 1800-fold dilution of 5% amino oligosaccharide soluble concentrate at 5:00 PM (avoiding temperatures above 30℃), at a rate of 135 mL / m² (120-150 mL / m²). Ten days after each spraying, monitor the chlorophyll content (SPAD value) and photosynthetic rate of the plant leaves. Results should show a SPAD value ≥45 and a photosynthetic rate ≥15 μmol / m². 2 •s; Only one plant with a SPAD value of 39 (<40) was observed during monitoring in August, and an additional 0.3% amino acid foliar fertilizer (total amino acid ≥10%) was sprayed.

[0084] 2. Enhanced Protection During Peak Incidence Periods: During the peak virus incidence period from May to August (daily average temperature ≥25℃, relative humidity ≥70%), spray a 0.5g / L rhamnolipin solution (Pseudomonas aeruginosa fermentation product, pH 7.0-6.5-7.5, purity ≥98%, filtered through a 0.22μm filter before use for sterilization) at 9:00 AM on the 1st of each month; 24 hours later, spray a passion fruit attenuated mutant compound preparation (containing cucumber mosaic virus attenuated mutant CMV-m1 and passion fruit lignification virus attenuated mutant PWV-m2, both at a concentration of 1×10⁻⁶). 6 (Copies / mL, store at 4℃ protected from light, use within the expiration date). Avoid mixing with alkaline pesticides during spraying. Maintain a relative humidity of 65% (60%-70%) in the field via drip irrigation for 48 hours after spraying. If it rains within 24 hours after spraying the rhamnolipin solution, re-spray once the next day.

[0085] 3. Enhanced resistance: When the plant is in the 2-6 leaf stage, spray the leaves with 0.2% potassium dihydrogen phosphate foliar fertilizer (K2O3 4.5% ≥34%, P2O5 52.3% ≥52%) every 7 days, with each spraying amount being 90mL / plant (80-100mL / plant), for a total of 4 sprays (3-4 times).

[0086] (V) Treatment of Diseased Plants

[0087] 1. Diseased plant identification: On July 12, 8 suspected diseased plants were found (showing yellowing, wrinkling, and mottling of leaves, and abnormal lignification of stems). RT-PCR was used for rapid detection to confirm the disease: 0.1g of the 2nd-3rd new leaves from the top of the diseased plant were sampled, and total RNA was extracted using the TRIzol method. After reverse transcription to synthesize cDNA, PCR amplification was performed. The amplified products were detected by 1.5% agarose gel electrophoresis, and the target bands of TeMV 580bp and PWV 420bp were found (CMV had no target band). The plants were identified as TeMV and PWV co-infected plants and were immediately marked with red labels.

[0088] 2. Mechanical removal: Use a gardening shovel that has been wiped and disinfected with 75% alcohol (disinfect it again after each plant is treated) to dig out the diseased plant and the soil within 1m around it to a depth of 35cm (30-40cm). Move the diseased plant and the infected soil to a special pit (1.5m deep) 55m (≥50m) away from the planting area. Cover the pit with 100-200 mesh quicklime (0.5kg / plant) and then bury it.

[0089] 3. Treatment of diseased planting holes: After removing diseased plants, evenly spread 0.5 kg of 100-200 mesh quicklime per hole, mix it into the soil to a depth of 22 cm (20-25 cm), and water with 250 mL (200-300 mL) of water to activate the quicklime; then cover with a 0.1 mm (≥0.08 mm) thick transparent plastic film for high-temperature soil treatment. During the day, use natural light to raise the temperature, and monitor the temperature inside the film with a temperature sensor to ensure that the temperature inside the film is ≥50℃ for 5 consecutive days; after 5 days, remove the plastic film, leave it idle for 2 days, and replant with healthy seedlings that have passed the RT-PCR test (Ct value >35).

[0090] II. Experimental Procedure and Records

[0091] 1. Experimental design (4 control groups, 200 plants per group, replicated 3 times)

[0092]

[0093]

[0094] (II) Detection Indicators and Methods

[0095] 1. Virus incidence rate: The number of diseased strains in each group was investigated every 30 days. The virus carriage status was confirmed by RT-PCR (detection limit ≤103 copies / mL). The calculation formula is: Incidence rate = number of diseased strains / total number of strains × 100%.

[0096] 2. Density of vectors: The number of aphids captured by yellow sticky traps was counted every 15 days and converted to "aphids / plant". At the same time, the number of thrips captured by insect-attracting lamps was also counted.

[0097] 3. Plant growth indicators: Before harvest, measure the plant height with a tape measure, measure the SPAD value of chlorophyll in the leaves with a SPAD-502 instrument (measure 3 functional leaves per plant and take the average value), and measure the photosynthetic rate with a photosynthesis meter.

[0098] 4. Yield and quality: Count the number of fruits per plant and the weight of each fruit to calculate the yield per plant; use a refractometer to determine the soluble solids content of the fruit.

[0099] 3. Experimental Data

[0100]

[0101]

[0102] (II) Supplementary Data for Key Technology Verification

[0103] Virus elimination effect of seedlings: After "stage-by-stage heat treatment + shoot tip culture + RT-PCR detection", the virus elimination rates of TeMV, PWV and CMV in the experimental group reached 98.5%; the control group 2 only used a single heat treatment, and the virus elimination rate was 72.3%, and there were cases of CMV being missed.

[0104] Control effect of attenuated mutants: After spraying the CMV-m1 and PWV-m2 compound preparation, the leaf load of wild-type TeMV and PWV viruses in the group of this invention was reduced by 92.3% compared with the control group 3 (qPCR detection), and no viral mutation or resistance phenomenon was observed.

[0105] Disinfection effect of diseased holes: After 30 days of treatment with "quicklime + high temperature soil soaking", the soil virus Ct value detected by RT-PCR was >38 (negative); in the control group 1, the diseased holes were simply tilled, and the disease incidence rate of surrounding plants still reached 15.6%.

[0106] Soil improvement sustainability: After harvesting, the soil organic matter content was 2.4% (initially 1.8%) and available nitrogen was 78 mg / kg; in control group 1, the soil organic matter content decreased to 1.5% and available nitrogen was 55 mg / kg, indicating that the soil conditioning technology of this invention can maintain soil fertility.

[0107] (III) Repeatability Validation

[0108] In 2024, this experiment was repeated at a passion fruit base in Longyan, Fujian (25.07°N, 117.02°E). Tainong No. 1 passion fruit (susceptible to TeMV, PWV, and CMV) was selected. The virus incidence rate in the experimental group was 3.5%, and the yield per plant was 6.6 kg. There was no significant difference from the experimental results in Nanning, Guangxi (P>0.05), which proves that the technology has stable applicability in different production areas and on the Tainong No. 1 variety.

[0109] III. Experimental Conclusions

[0110] 1. This invention reduces the incidence of passion fruit virus from 45.8% in conventional planting to 3.2% through a complete technical system of "soil conditioning - seedling purification - vector control - immune activation - diseased plant treatment". It also controls the density of virus-transmitting vectors such as aphids and thrips at an extremely low level (≤2 aphids / plant). This invention solves the four core problems of traditional control: "virus-carrying seedlings, vector control failure, weak immunity, and diseased plant spread".

[0111] 2. Significant synergistic effect of the whole-chain technology: staged heat treatment combined with precise shoot tip culture increases the virus elimination rate by 26.2%; the combination of 60-mesh insect-proof net and pheromone yellow sticky trap reduces the vector invasion rate by more than 80%; the attenuated mutant compound preparation and rhamnolipin work synergistically to achieve a cross-protection rate of more than 90%; standardized treatment of diseased holes achieves a virus inactivation rate of 100%.

[0112] 3. Outstanding advantages in plant growth and economic indicators: The plant height of the group of this invention is increased by 48.5% compared with conventional planting, the chlorophyll SPAD value is increased by 35.5%, the yield per plant is increased by 223.8%, and the soluble solids content of the fruit is increased by 16.2% ((16.5-14.2) / 14.2×100%≈16.2%). Moreover, the amount of chemical pesticides used throughout the process is reduced by more than 90%, which meets the requirements of green agricultural development.

[0113] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. A green prevention and control method of passion fruit viral disease, characterized in that, Comprising the following steps: (1) Seedling purification: ① Seedling selection: select 4-6 leaf stage, plant height 15-20 cm guava cutting seedlings or grafted seedlings, and screen healthy seedlings without yellow leaves and stem without spots; RT-PCR is used to detect night-blooming jasmine mosaic virus (TeMV), guava lignification virus (PWV) and cucumber mosaic virus (CMV), and healthy seedlings with Ct value > 35 are used as treatment objects; Mother plant strengthening: the qualified healthy seedlings are treated by spraying 0.3% amino acid foliar fertilizer (total amino acid content ≥10%) + 5% amino oligosaccharide soluble liquid 2000 times liquid on the leaves every 7 days for 2 times; At the same time, keep the day temperature at 25-28℃ and the night temperature at 18-20℃, and cultivate healthy mother plants before entering the stage heat treatment; ② Stage heat treatment: place the healthy seedlings in an intelligent constant temperature incubator, maintain 38℃±0.5℃ and relative humidity 65%-70% for the first 7 days, then raise the temperature to 40℃±0.5℃ and relative humidity 60%-65% for the last 8 days, the whole process light intensity is 2000-2500 lux, and light duration is 12h / d; ③ Stem tip culture: take 0.2-0.3mm stem tips (with 1-2 leaf primordia) after heat treatment, inoculate in MS+6-BA0.5mg / L+NAA0.1mg / L medium, cultivate at 25℃±1℃ with light for 14h / d for 30-35 days, and obtain tissue culture seedlings; ④ Planting specification: the tissue culture seedlings are acclimated for 7-10 days: The first 3 days: day temperature 25-28℃, night temperature 18-20℃, shading rate 50%; The 4th-7th day: day temperature transition to 28-30℃, night temperature 20-22℃, shading rate reduced to 30%; The last 3 days (if acclimated for 10 days): full light, day temperature 30-32℃, night temperature 22-24℃; During acclimation: spray 0.2% potassium dihydrogen phosphate foliar fertilizer (K2O≥34%, P2O5≥52%) on the leaves once every 3 days, spraying amount 80-100mL / plant; Soil disinfection before planting: 7 days before planting, evenly spray 50% carbendazim wettable powder 500 times liquid + 0.5% sophoramine aqueous solution 800 times liquid to the planting plot, spraying amount 200-250mL / ㎡, to kill residual viruses and pathogenic bacteria in the soil; (2) Vector control: ① Physical isolation: build a 2.8-3.0m high protective fence outside the planting area, fix high-density polyethylene insect screen (60 mesh high-density polyethylene insect screen, mesh deviation ≤0.08mm, breaking strength ≥550N) on the inner side of the protective fence, and bury the bottom of the insect screen in the soil 30-40cm and compact it; ② Monitoring system: hang a double-sided yellow board (specification 20cm×30cm, coated with 0.1% aphid sex pheromone trapping agent) every 10㎡ in the field, the yellow board is 20-30cm away from the top of the plant, and the number of aphids is counted every day at 9:00-10:00, and emergency control is started when the aphid density of a single plant is ≥5 per plant; ③Emergency disposal: emergency prevention and treatment with 5% extract of isatis root soluble liquid (chlorogenic acid content ≥5%) 6000 times liquid, at 9-11 am (temperature 20-25℃, wind speed ≤3m / s) leaf surface spraying, both sides of the leaf evenly covered with liquid, continuous application for 2 times, interval 7 days; (3) Immune activation: ①Basic protection: after planting every 15 days, at 8-10 am or 4-6 pm (avoiding high temperature above 30℃) leaf spraying 5% amino oligosaccharide soluble liquid 1800 times liquid, spraying amount is 120-150 mL / ㎡; ② High incidence period of strengthening: in spring and summer (daily mean temperature ≥ 25℃, relative humidity ≥ 70%) virus high incidence period, first spray 0.5 g / L rhamnolipid solution (Pseudomonas aeruginosa fermentation product, pH 6.5-7.5, purity ≥ 98%), 24 hours later, spray the weak virus mutant compound preparation (containing cucumber mosaic virus weak mutant CMV-m1, weak mutant PWV-m2 of Passiflora lignification virus, both at a concentration of 1×10 6 copies / mL, 4℃ in the dark, valid for 6 months); ③Resistance enhancement: seedling stage (2-6 leaf stage) every 7 days spraying 0.2% potassium dihydrogen phosphate foliar fertilizer (K2O≥34%, P2O5≥52%) for 1 time, each spraying amount 80-100 mL / plant, for 3-4 times; (4) Disease plant disposal: ①Disease plant identification: through symptom observation (leaf yellowing, shrinkage, mottling, abnormal stem lignification) combined with RT-PCR rapid detection, confirm the disease plant and mark; ②Mechanical removal: using disinfected garden spade (75% alcohol wiping disinfection, disinfect 1 time for every 1 plant) to remove the disease plant, together with the root system and the soil within 1m range (depth 30-40cm) are removed, moved to the special pit (depth 1.5m) outside the planting area more than 50m, covered with lime and filled; ③Disease hole treatment: evenly spread 100-200 mesh lime (usage 0.5kg / hole) in the disease hole, turn into the soil depth 20-25cm, irrigate 200-300mL to activate, cover transparent plastic film (thickness ≥0.08mm) for high temperature soil steaming treatment; daytime use natural light to warm, keep the temperature in the film ≥50℃, last for 5 days; Idle for 2 days after removing the plastic film, replant the healthy seedlings (high temperature soil steaming combined with lime can completely inactivate the residual virus in the soil, avoid secondary infection).

2. The method of claim 1, wherein, The MS medium in step (1)③ also adds 30g / L sucrose and 7g / L agar, pH is adjusted to 5.8-6.0; when the tissue culture seedlings are cultured to 3-4 leaf stage, use RT-PCR for virus detection, the detection objects include tea plant mosaic virus (TeMV), passion fruit lignification virus (PWV) and cucumber mosaic virus (CMV), among which the TeMV detection primer is F: 5'-GGTGGTGACGGTGGTGGT-3', R: 5'-CCTGCTGCTGCTGCTGCT-3', the PCR amplification condition is 94℃ pre-denaturation for 5min, 94℃ denaturation for 30s, 58℃ annealing for 30s, 72℃ extension for 45s, for 35 cycles, 72℃ final extension for 10min, and the Ct value >35 is determined as virus-free seedling.

3. The method of claim 1, wherein, The planting area in step (2)① is spaced at least 500m from tobacco and solanaceae crops (tomato, pepper, eggplant), and 1 insect monitoring point is set every 200㎡, the monitoring point places insect trapping lamp (wavelength 365nm, power 20W), which is turned on from 19:00 every night to 6:00 the next day, and the number of virus transmission media insects (aphids, thrips) caught is counted every week.

4. The method of claim 1, wherein, The rhamnolipid solution in step (3) ② needs to be filtered through a 0.22 μm filter membrane before use, and needs to be re-sprayed once if it rains within 24 hours after spraying; the weak mutant R181K of the Yeliangxi flower leaf virus needs to be avoided from being mixed with alkaline pesticides during spraying, and the relative humidity in the field needs to be maintained at 60%-70% within 48 hours after spraying.

5. The method of claim 1, wherein, The planting substrate before step (1) adopts a formula of vegetable garden soil: grass charcoal soil: fine river sand = 4:1:1, and the substrate needs to be disinfected by spraying 50% carbendazim wettable powder 300-500 times liquid (200 L of liquid medicine per cubic meter of substrate), covered with plastic film for 7 days after disinfection, and ventilated for 3 days after uncovering the film to detect the number of bacteria in the substrate, ≤100 CFU / g, which can be used, and the EC value of the substrate is controlled at 1.2-1.8 mS / cm, and the pH value is controlled at 5.5-6.

5.

6. The method of claim 1, wherein, The RT-PCR rapid detection in step (4) ① uses a virus detection kit (detection limit ≤10 3 copies / mL), the sampling site is the new leaves (the top 2-3 leaves) of the diseased plants, the sampling amount is 0.1 g, the total RNA is extracted after grinding by the TRIzol method, the cDNA is synthesized after reverse transcription, and then PCR amplification is performed, the amplification product is detected by 1.5% agarose gel electrophoresis, and the target band (TeMV 580 bp, PWV 420 bp, CMV 350 bp) appears to determine the diseased plant.

7. The method of claim 1, wherein, After spraying the 5% amino-oligosaccharide soluble solution in step (3) ①, the chlorophyll content (SPAD value ≥ 45) and photosynthetic rate (≥ 15 μmol / m 2 ·s) of the plant leaves need to be monitored regularly. When the SPAD value is < 40 or the photosynthetic rate is < 12 μmol / m 2 ·s, 1 additional spraying of 0.3% amino acid foliar fertilizer (total amino acid content ≥ 10%) is needed.

8. The method of claim 1, wherein, Also include soil conditioning step: 15 days before planting, to the soil into the mature organic fertilizer (chicken manure: sheep manure = 3:1, mature period ≥ 60 days) 2000-3000 kg / mu, combined with the application of biological bacteria fertilizer (effective viable count ≥ 2 × 10 8 CFU / g) 50 kg / mu, ploughing depth 30-35 cm, adjust the soil organic matter content ≥ 2.5%, available nitrogen content 80-100 mg / kg, to provide the plant growth with disease resistance foundation.

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