A method for preventing and controlling low-temperature resistance and continuous cropping obstacles of celery seedling tubes

Abstract

The application discloses a low-temperature-resistant celery seedling tube protection and continuous cropping obstacle prevention and control method, and relates to the technical field of vegetable planting.The specific steps of the method are as follows: firstly, soil detection pathogenic nucleic acid is taken to divide the continuous cropping grade and build a file; then, straw, lime and nitrogen are configured according to the grade to carry out a shed soil improvement, base fertilizer is applied after the soil re-inspection reaches the standard; after seed warm water treatment, nucleic acid screening is carried out to divide the grade, low-temperature germination is carried out according to the grade, and low-temperature seedling is trained according to the land grade gradient; during the seedling period, rhizosphere bacterial community dynamics is monitored, and the seedling is selected and planted according to the monitoring; during multiple growth periods, leaf resistance and disease resistance genes are detected to adjust field management, the file is updated after harvesting to optimize the planting scheme in the next year; the application relies on soil nucleic acid grading shed soil improvement, accurately reduces soil-borne pathogenic bacteria, and repairs soil microecology, so that the celery continuous cropping obstacle is solved; meanwhile, seed grading low-temperature germination and seedling are carried out, rhizosphere bacterial community and plant resistance gene dynamic regulation and control are monitored, and a year-by-year optimization mechanism is formed.

Description

Technical Field

[0001] This invention relates to the field of vegetable cultivation technology, specifically to a method for the management and control of low-temperature resistant celery seedlings and the prevention and control of continuous cropping obstacles. Background Technology

[0002] Celery is a mainstream vegetable in autumn and winter low-temperature greenhouse cultivation, with stable market demand and considerable planting profits, leading to the continued promotion of large-scale contiguous planting models. However, long-term repetitive planting of a single crop can easily lead to continuous cropping obstacles, manifested as the accumulation of soil-borne pathogens in the topsoil, imbalance of soil microbial community structure, decreased nutrient availability, and deterioration of physicochemical properties. Under low-temperature cultivation conditions, celery seedlings have weak stress resistance and limited tolerance to low-temperature stress, making them susceptible to soil pathogen infection during the seedling stage, resulting in problems such as seed rot, seedling death, and weak growth, which adversely affect seedling survival rate and later yield.

[0003] Currently, molecular detection technology, microbial improvement materials, and low-temperature acclimatization seedling technology have been gradually applied in vegetable cultivation. Various biological agents, straw-based greenhouse improvement methods, and supporting systems for low-temperature seedling facilities are also continuously being improved, providing a technological foundation for targeted regulation of soil microecology and enhancing crop low-temperature tolerance. There is a growing trend in the industry towards refined field management based on nucleic acid quantification and gene expression analysis, which provides a feasible technical approach to solving the overlapping problems of low-temperature seedling cultivation and continuous cropping obstacles in celery.

[0004] In current celery cultivation practices, soil disease assessment primarily relies on growers' observations of disease manifestations in the field, lacking standardized quantitative screening procedures. Therefore, disease assessment exhibits a degree of lag and subjectivity. The application rate of soil amendments and the duration of fumigation treatments are typically configured with relatively uniform parameters, failing to adequately match the differentiated needs of plots with varying pathogen levels. This may lead to incomplete eradication or excessive material input in some plots. Seed treatment often employs conventional hot water soaking, without the widespread adoption of quantitative pathogen screening and grading procedures. Infected seeds directly enter the seedling stage, increasing the risk of disease transmission during the seedling stage. Low-temperature seedling cultivation often uses fixed temperatures and hardening-off periods without tailoring to the specific cropping conditions of the plots. Furthermore, dynamic monitoring methods for seedling rhizosphere microbiota are not yet fully developed, making it difficult to optimize management measures in a timely manner when low-temperature stress and pathogen invasion overlap. Field management mainly relies on adjusting water, fertilizer, and temperature control measures based on plant appearance, making it difficult to predict changes in plant stress and disease resistance in advance. The lack of a unified mechanism for archiving and utilizing annual planting data makes it difficult to iterate and optimize planting techniques year after year. These factors combined have resulted in prominent issues such as continuous cropping obstacles and yield reduction due to low temperatures in large-scale celery production, while the problem of low seedling vigor has remained unresolved for a long time. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for the cultivation, management, and control of low-temperature resistant celery seedlings and continuous cropping obstacles. This method relies on nucleic acid quantification and gene expression detection to achieve precise control throughout the entire process. First, soil pathogens are detected to classify continuous cropping levels. This is followed by soil improvement using straw and lime nitrogen fumigation. Once the levels are met, base fertilizer is applied to restore the soil microecology. Next, seeds are screened and graded using nucleic acid, and differentiated germination treatments are applied. Gradual low-temperature hardening-off is carried out based on the disease level of the plot. During the seedling stage, the rhizosphere microbiota is dynamically monitored to adjust the environment and inoculant application. The expression of stress- and disease-resistant genes in plants is detected at different growth stages in the field, and water, fertilizer, temperature control, and microbial agent application are dynamically adjusted. After harvest, the entire cycle of monitoring data is integrated to update the plot records. The following year, the planting process is optimized based on the records. This entire solution simultaneously addresses the problems of soil-borne diseases and low-temperature stress in celery caused by continuous cropping, cultivates high-quality, robust seedlings, reduces the use of chemical agents, and achieves iterative optimization of the planting process year by year, making it suitable for large-scale, low-temperature celery cultivation in facilities.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for the management and control of low-temperature resistant celery seedlings and the prevention and control of continuous cropping obstacles, the specific steps of which are as follows: S1. Before celery seedling cultivation, topsoil samples were collected from the planting plots, total DNA was extracted from the soil samples, and the copy number of pathogenic bacteria nucleic acid was quantitatively detected using specific primers. Based on the nucleic acid load, the plots were classified into continuous cropping levels, and a plot-specific file was established. S2, retrieve the continuous cropping level data of the plot, determine the amount of straw and calcium cyanamide to be used according to the continuous cropping level, apply straw and calcium cyanamide to the plot, complete deep plowing, irrigation and sealed fumigation operations, after the fumigation is completed and ventilation is finished, retest the soil pathogen nucleic acid, apply base fertilizer to the plot with qualified nucleic acid and rotary tillage and leveling. S3. Celery seeds were selected for warm water soaking, and the seed coat oil was removed by rubbing. The seed coat extract was collected as a detection template and loop-mediated isothermal nucleic acid amplification was carried out to quantify the number of pathogen nucleic acid copies carried by the seeds. Severely infected seeds were removed, and the remaining qualified seeds were divided into two grades. S4. For qualified seeds of different grades, use germination solution corresponding to the seed grade to carry out low-temperature and light-proof germination. Determine the total time of low-temperature hardening according to the continuous cropping level of the plot. Prepare seedling substrate and fill the seedling trays. Sow the seeds into the substrate after they sprout and carry out gradient low-temperature acclimatization. S5. During the hardening-off period, collect seedling rhizosphere substrate samples, extract microbial nucleic acids and quantify the target bacterial community nucleic acid copy number, adjust hardening-off environmental parameters and bacterial agent application scheme according to preset thresholds, retest and screen qualified strong seedlings at the end of the hardening-off period, complete the transplanting and lay the drip irrigation network under the film. S6. Celery functional leaves were collected at multiple growth stages. Total RNA was extracted from the leaves and the expression abundance of endogenous stress-resistant mRNA was detected. Based on the mRNA expression status, corresponding water and fertilizer application schemes and field facility management schemes were adopted. After harvesting, the full-cycle nucleic acid test data were summarized to update the plot file and adjust the seedling and field management process parameters for the following year.

[0007] Furthermore, in S1, soil sampling employs a five-point method, taking soil layers from 0 to 20 cm in depth and sieving them through a 2 mm sieve; the detection targets are *Celtis elata* leaf spot pathogen and *Fusarium solani* root rot pathogen, and quantitative real-time PCR is used to quantify nucleic acid copy numbers; continuous cropping is classified as follows: total nucleic acid load is less than 10... 3 Copy number per gram of dry soil indicates mild severity, ranging from 10. 3 ~10 5 Copy number per gram of dry soil is considered moderate, above 10. 5 The copy number per gram of dry soil indicates severe contamination; the plot record includes the plot number, sampling date, copy numbers of the two pathogenic bacteria, and continuous cropping level.

[0008] Furthermore, in S2, for lightly continuously cropped plots, apply 1000-1400 kg of chopped straw and 25-35 kg of calcium cyanamide per mu (667 square meters), and seal the soil for 18-22 days; for moderately continuously cropped plots, apply 1250-1450 kg of chopped straw and 32-38 kg of calcium cyanamide per mu, and seal the soil for 21-25 days; for heavily continuously cropped plots, apply 1400-1600 kg of chopped straw and 37-43 kg of calcium cyanamide per mu, and seal the soil for 26-30 days. The chopped straw should be 2-3 cm long, deeply plowed to a depth of 25 cm, and the soil moisture content should be 85%-90% after irrigation. Nucleic acid retesting should be conducted 7 days after the sealing and ventilation period, and the standard for compliance is that the nucleic acid load of both pathogens is lower than 1×10⁻⁶. 3 Copy number / gram of dry soil; for qualified plots, apply 150 kg / mu of bio-organic fertilizer and 40 kg / mu of NPK compound fertilizer as base fertilizer. For plots with severe continuous cropping, apply an additional 20 kg / mu of biochar granules loaded with Bacillus subtilis. If the standard is not met, repeat the fumigation process for 5-7 days and retest until the standard is met.

[0009] Furthermore, in step S3, celery seeds are soaked in hot water at 48–50°C with stirring for 10 minutes, then soaked at room temperature for 12 hours, turning them over every 2 hours; LAMP uses dual-target primers targeting both leaf spot blight and root rot Fusarium, with real-time fluorescence quantitative interpretation; the seed nucleic acid copy number is higher than 1×10⁻⁶. 4 The number of copies / μL was considered a severe case of bacterial contamination and the bacteria were eliminated; a value below 1×10⁻⁶ was considered acceptable. 2 copies / μL represent healthy seeds, 1×10 2 ~1×10 4 copies / μL indicates slightly contaminated seeds.

[0010] Furthermore, in step S4, the germination temperature is 10℃, and the period of darkness is 6–8 days; healthy seeds are moisturized by spraying with clean water; and seeds with mild bacterial contamination are treated with a concentration of 1×10⁻⁶. 9 Soak seeds in Bacillus subtilis germination solution (CFU / mL) for 4–6 hours, then remove them and place them on a damp gauze. Spray them with fresh germination solution daily. The seedling substrate is a mixture of well-rotted organic fertilizer and garden soil at a mass ratio of 4:6, with 5% straw shreds added, and filled into 72-cell trays.

[0011] Furthermore, in S4, the hardening-off period is determined according to the continuous cropping level: 6-8 days for mild cases, 9-11 days for moderate cases, and 11-13 days for severe cases; the seedlings are sown and covered with 0.5cm of soil, and thoroughly watered; the gradient low-temperature acclimatization is as follows: the initial daytime temperature is 16-18℃ and the nighttime temperature is 8-10℃, the daytime temperature decreases by 1-2℃ and the nighttime temperature decreases by 0.5-1℃ each day, and after 3-5 days, the temperature is transitioned to 12℃ for daytime and 4℃ for nighttime and maintained until the end; the light is the same as natural sunlight; during the hardening-off period, rhizosphere substrate samples are taken and refrigerated every 1-3 days.

[0012] Furthermore, in step S5, the root surface layer (1-2 cm) of the rhizosphere substrate is sampled for testing for Bacillus subtilis, Fusarium root rot, and celery leaf spot pathogens; the beneficial bacteria threshold is not less than 5 × 10⁻⁶. 6 The copy number per gram of dry substrate should be less than 1 × 10⁻⁶. 3 Copy number per gram of dry substrate; pathogenic bacteria copy number greater than 1 × 10⁻⁶ 3 And not exceeding 1×10 4 The copy number per gram of dry matrix is ​​slightly excessive, reaching or exceeding 1×10⁻⁶. 4 The copy number per gram of dry substrate is severely excessive; when beneficial bacteria are insufficient and slightly excessive, adjust the night temperature to 5-7℃ and spray the leaves and substrate with Bacillus subtilis solution; when the level is severely excessive, suspend low-temperature acclimatization by constructing an insulated greenhouse and spray the whole plant with Trichoderma harzianum solution; at the final stage, retest, and if beneficial bacteria meet the standard and pathogenic bacteria are below 1×10⁻⁶, the level should be within acceptable limits. 3 Seedlings with a copy number per gram of dry substrate are considered strong.

[0013] Furthermore, in S5, the row spacing for planting strong seedlings is 25cm and the hole spacing is 10cm, with one seedling per hole; the drip irrigation network is laid under the mulch film, and drip irrigation is used to water the seedlings after planting.

[0014] Furthermore, in S6, the leaf sampling nodes are the end of the hardening-off period, the transplanting and seedling establishment period, the tillering period, and the canopy closure period, taking the 2nd to 3rd functional leaves from the top; detecting low-temperature stress response mRNA and disease resistance defense mRNA; low expression refers to the target mRNA expression level being downregulated by more than 50% compared to the control; when low expression of low-temperature stress mRNA occurs during the hardening-off period, drip irrigation with humic acid water-soluble fertilizer and extending the nighttime covering of the heat-insulating greenhouse; when low expression of disease resistance defense mRNA occurs during the tillering period, foliar spraying with Bacillus subtilis and drip irrigation with phosphorus and potassium water-soluble fertilizer; when both types of mRNA are lowly expressed during the canopy closure period, shading nets are activated, and the opening and closing of the heat-insulating greenhouse are adjusted according to the day-night temperature difference, compound Bacillus subtilis bacterial solution is drip-irrigated twice, and nitrogen fertilizer supply is reduced.

[0015] Furthermore, in S6, after the annual harvest, the soil pathogen nucleic acid, seed nucleic acid screening, rhizosphere microbial nucleic acid and plant endogenous mRNA expression data are summarized and stored in the plot archive; the archive is retrieved before seedling cultivation in the following year, and the amount of fumigation material, seed treatment parameters, low-temperature hardening time, water and fertilizer plan and microbial agent application time are adjusted accordingly.

[0016] Compared with existing technologies, this method for low-temperature resistant celery seedling management and continuous cropping obstacle control has the following beneficial effects: I. This invention classifies plots into continuous cropping levels by quantitative soil nucleic acid testing before seedling cultivation and establishes exclusive plot files. Combined with a graded straw and lime cyanide fumigation disinfection system, it can accurately adapt to the soil improvement needs of plots with different disease baselines, reduce the stock of target pathogens in the soil from the source, and weaken the problems of soil microbial imbalance and high incidence of soil-borne diseases caused by long-term continuous cropping of celery. Relying on the nucleic acid retesting mechanism to control the effectiveness of fumigation improvement, and combined with graded base fertilizer and functional biochar to supplement beneficial microorganisms, it can continuously optimize the soil physicochemical and micro-ecological environment, avoid the material waste or incomplete disease control caused by uniform improvement schemes. The entire soil pretreatment process relies on nucleic acid quantitative data to achieve refined management, without relying on manual experience to judge the degree of soil diseases, greatly improving the stability of continuous cropping obstacle management, and building a basic planting environment free from high pathogen stress for subsequent low-temperature seedling cultivation of celery, while simultaneously achieving soil fertility restoration and long-term inhibition of soil-borne diseases.

[0017] II. This invention combines seed nucleic acid screening and grading with differentiated low-temperature germination treatment, along with a gradient low-temperature hardening-off mode customized according to the disease level of the plot. It simultaneously links dynamic nucleic acid monitoring of seedling rhizosphere microorganisms to adjust the seedling environment and inoculant application strategies in real time. It takes into account the dual needs of celery's low-temperature tolerance cultivation and seedling disease control. Relying on the monitoring of the abundance of endogenous stress-resistance and disease-resistant mRNA expression in leaves at different growth stages of the plant, it dynamically matches water and fertilizer, temperature control, and microbial inoculant management measures. This can promptly alleviate the inhibition of celery growth by low-temperature stress, strengthen the plant's own disease resistance and metabolic capacity, and integrate full-cycle nucleic acid and gene expression data to update the plot file after harvest, forming an annual cycle optimization management system. This continuously simplifies subsequent seedling management operations, stably cultivates cold-resistant and strong seedlings, reduces the probability of field disease occurrence, reduces the input of chemical agents, and improves the germination rate and finished product yield of celery in low-temperature planting scenarios.

[0018] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination or study, or may be learned from the practice of the invention. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0020] Figure 1 A flowchart of the overall process for the cultivation, management, and control of continuous cropping obstacles in low-temperature resistant celery seedlings; Figure 2 Bar chart comparing the nucleic acid load of soil pathogens before and after fumigation in plots with different continuous cropping levels; Figure 3 A bar chart comparing celery diseases, yield, and stress resistance mRNA expression under different field management programs. Detailed Implementation

[0021] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0022] Example 1: Complete process flow for low-temperature resistant celery seedling cultivation, management, and continuous cropping obstacle control, with detailed step-by-step diagrams. Figure 1 As shown.

[0023] Contiguous celery greenhouse planting sites were selected, and soil sampling was conducted using a five-point method, with the soil depth controlled between 0 and 20 cm. Soil samples from the five points were thoroughly mixed and passed through a 2 mm sieve. Total DNA was extracted from the mixed soil samples. Celery leaf spot pathogen and Fusarium root rot pathogen were selected as detection targets, and the nucleic acid copy numbers of the two pathogens were quantified using quantitative real-time PCR.

[0024] The classification of land parcels based on nucleic acid load is as follows: parcels with a total nucleic acid load below 10 are classified as crop contiguous plots. 3 The copy number per gram of dry soil indicates a slightly continuously cropped plot, with the total nucleic acid load at 10. 3 Up to 10 5 The copy number / gram dry soil range indicates moderately continuous cropping plots, with total nucleic acid load exceeding 10. 5 The copy number per gram of dry soil indicates a severely continuously cropped plot. A complete record is kept of the plot number, soil sampling date, celery leaf spot pathogen nucleic acid copy number, Fusarium root rot pathogen nucleic acid copy number, and continuous cropping level. A unique plot file is generated and permanently stored.

[0025] Retrieve the continuous cropping level from the plot's records, determine the amount of chopped straw and calcium cyanamide to use based on the continuous cropping level, spread the chopped straw and calcium cyanamide evenly on the plot, cut the chopped straw to a length of 2 to 3 centimeters, mechanically deep-plow the soil to a depth of 25 centimeters, irrigate and adjust the soil moisture holding capacity to maintain 85% to 90%, and seal the greenhouse to complete the fumigation operation.

[0026] For lightly continuously cropped fields, apply 1200 kg of chopped straw and 30 kg of calcium cyanamide per mu (0.067 hectares), and maintain the soil conditioner for 20 days. For moderately continuously cropped fields, apply 1350 kg of chopped straw and 35 kg of calcium cyanamide per mu (0.067 hectares), and maintain the soil conditioner for 23 days. For heavily continuously cropped fields, apply 1500 kg of chopped straw and 40 kg of calcium cyanamide per mu (0.067 hectares), and maintain the soil conditioner for 28 days.

[0027] After the fumigation period ended, the greenhouse was opened for ventilation for 7 days. Soil samples were collected again for pathogen nucleic acid retesting. The nucleic acid load of both pathogens in the greenhouse was below 1×10⁻⁶. 3 Subsequent operations can be carried out by applying a copy number per gram of dry soil. For qualified plots, apply 150 kg of bio-organic fertilizer and 40 kg of NPK compound fertilizer per acre as base fertilizer. For plots with severe continuous cropping, apply an additional 20 kg of Bacillus subtilis biochar granules per acre. After fertilization, rotary till and level the plot.

[0028] If the soil nucleic acid retest value does not meet the standard, add 100 kg of chopped straw per mu, seal the greenhouse and extend the fumigation period for 5 to 7 days, and conduct nucleic acid testing again after ventilation until the soil pathogen value meets the standard.

[0029] Celery seeds were soaked in hot water at 48-50 degrees Celsius for 10 minutes with continuous stirring, then transferred to room temperature water and soaked for 12 hours, turning the seeds every 2 hours during the soaking process. After soaking, the seed coat was repeatedly rubbed to remove the seed coat oil, and all seed coat extract was collected as a template for nucleic acid detection. The copy number of pathogen nucleic acid carried by the seeds was quantified using dual-target loop-mediated isothermal nucleic acid amplification technology.

[0030] Seed nucleic acid copy number higher than 1×10 4 All seeds with copies / μL were removed, and nucleic acid copy numbers below 1×10 were removed. 2 Healthy seeds were defined as those with a nucleic acid copy number of 1×10⁻⁶ copies / μL. 2 Up to 1×10 4 Seeds in the range of copies / μL were classified as slightly contaminated seeds, and the two types of qualified seeds were stored separately for later use.

[0031] The germination environment was uniformly set at 10 degrees Celsius, with complete darkness throughout the process, and the total germination time was 7 days. Healthy seeds were only sprayed with water daily to maintain moisture, while seeds with slight bacterial contamination were treated with a concentration of 1×10. 9 Soak the seeds in a CFU / mL Bacillus subtilis germination solution for 4 to 6 hours, then remove them and spread them evenly on a damp gauze. Spray the seeds with fresh germination solution daily to keep them moist.

[0032] The seedling substrate is prepared by mixing well-rotted organic fertilizer and garden soil in a mass ratio of 4:6, with an additional 5% of straw fragments added to the total substrate mass. After thorough mixing, the mixture is filled into 72-cell seedling trays. Once the seeds show signs of sprouting, they are sown into the substrate. After sowing, the seeds are covered with a 0.5 cm thick layer of substrate and thoroughly watered.

[0033] The total duration of low-temperature hardening was determined based on the degree of continuous cropping in the plot: 7 days for plots with mild continuous cropping, 10 days for plots with moderate continuous cropping, and 12 days for plots with severe continuous cropping. The initial environment for gradient low-temperature acclimatization was a daytime temperature of 16-18 degrees Celsius and a nighttime temperature of 8-10 degrees Celsius. The daytime temperature was lowered by 1-2 degrees Celsius and the nighttime temperature by 0.5-1 degrees Celsius each day. After 3-5 days of continuous adjustment, the daytime temperature was stabilized at 12 degrees Celsius and the nighttime temperature at 4 degrees Celsius until the hardening process was completed. Light intensity inside the greenhouse followed natural sunlight without artificial adjustment.

[0034] During the hardening-off period, samples of the top 1-2 cm of rhizosphere substrate from the seedling roots were collected every 2 days and stored at low temperature. The substrate microbial nucleic acid was extracted to quantify the nucleic acid copy number of Bacillus subtilis, Fusarium root rot, and Celery leaf spot pathogen. The nucleic acid load of Bacillus subtilis was not less than 5 × 10⁻⁶. 6 Copy number per gram of dry matrix, pathogenic bacterial nucleic acid load less than 1×10 3 A copy number per gram of dry matrix is ​​within the normal range.

[0035] The pathogenic bacteria copy number is 1×10 3 Up to 1×104 When the copy number per gram of dry substrate is within the specified range, and the number of beneficial bacteria is insufficient, raise the greenhouse temperature to 6 degrees Celsius at night and spray the leaves and substrate thoroughly with Bacillus subtilis solution. 4 When the copy number per gram of dry substrate is reached, the low-temperature acclimatization should be suspended by constructing an insulated greenhouse, and the entire plant should be sprayed with Trichoderma harzianum solution. At the end of the hardening-off period, the rhizosphere microbial nucleic acid was retested; the Bacillus subtilis value was within the standard range and the nucleic acid load of pathogenic bacteria was less than 1×10⁻⁶. 3 Seedlings with a copy number per gram of dry substrate are considered strong seedlings.

[0036] After the selected strong seedlings are planted, they are planted with a row spacing of 25 cm and a hole spacing of 10 cm, with one seedling in each hole. A drip irrigation network is laid under the mulch film, and the drip irrigation is turned on to water the seedlings after planting.

[0037] Four fixed leaf sampling nodes were set up: late hardening-off stage, transplanting and seedling establishment stage, tillering stage, and canopy closure stage. The second to third functional leaves from the top of the plant were harvested each time, and total RNA was extracted from the leaves to detect the expression abundance of low-temperature stress response mRNA and disease resistance defense mRNA. A target mRNA expression level downregulated by more than 50% compared to the control was defined as a low expression state.

[0038] When low-temperature stress causes low mRNA expression during the seedling hardening stage, drip irrigation with humic acid water-soluble fertilizer and extend the nighttime covering time of the heat-insulating greenhouse. When disease resistance and defense mRNA expression is low during the tillering stage, foliar spraying with Bacillus subtilis inoculum solution and drip irrigation with phosphorus and potassium water-soluble fertilizer are performed simultaneously. When both types of mRNA show low expression during the canopy closure stage, shade netting is laid on the greenhouse, the opening and closing of the heat-insulating greenhouse are adjusted according to the diurnal temperature difference, compound Bacillus inoculum solution is drip-irrigated twice, and the supply of nitrogen fertilizer in the field is reduced simultaneously.

[0039] After the celery harvest is completed, soil pathogen nucleic acid detection data, seed nucleic acid screening data, rhizosphere microbial nucleic acid quantification data, and plant endogenous mRNA expression detection data are compiled and entered into the plot's dedicated file. The following year, before the start of celery seedling cultivation, the file records are retrieved, and the total amount of fumigation material applied, seed treatment operation parameters, total low-temperature hardening time, water and fertilizer application plan, and inoculant application time points are adjusted accordingly.

[0040] Example 2: Soil fluorescence quantitative PCR detection and verification of the effect of fumigation on plots with different continuous cropping levels.

[0041] Experimental plot grouping and soil sample pretreatment: Three celery-growing plots were selected for a parallel experiment. The three plots showed significant differences in the number of consecutive cropping years and were divided into three groups: Group 1, Group 2, and Group 3. Soil samples from the 0-20 cm topsoil layer were collected from each group using a five-point method. Three parallel sampling groups were established for each group. Each soil sample was thoroughly mixed and passed through a 2 mm sieve to remove stones and plant root residues. Total DNA was extracted from the soil for quantitative nucleic acid testing to determine the continuous cropping level of each group.

[0042] Group 1: Total nucleic acid load of soil pathogens: 2.6 × 10⁻⁶ 2 Copy number per gram of dry soil, Group 2: Total pathogenic bacteria nucleic acid load in soil 2.7 × 10⁻⁶ 4 Copy number per gram of dry soil; total nucleic acid load of pathogenic bacteria in soil of group 3: 2.1 × 10⁻⁶ 5 Copy number / gram of dry soil. The material dosage and fumigation time were matched according to the process requirements for fumigation treatment. Seven days after the fumigation ended and ventilation was completed, soil samples were collected again to test the pathogen nucleic acid load, and the changes in values ​​before and after fumigation were recorded.

[0043] Application of materials for fumigation and control of greenhouse sealing: Group 1: For plots with mild continuous cropping, apply 1200 kg of chopped straw and 30 kg of calcium cyanamide per mu (667 square meters), and seal the greenhouse for 20 days. Group 2: For plots with moderate continuous cropping, apply 1350 kg of chopped straw and 35 kg of calcium cyanamide per mu (667 square meters), and seal the greenhouse for 23 days. Group 3: For plots with severe continuous cropping, apply 1500 kg of chopped straw and 40 kg of calcium cyanamide per mu (667 square meters), and seal the greenhouse for 28 days. All plots should be tilled to a uniform depth of 25 cm. After irrigation, maintain soil moisture content at 85% to 90%. During the sealing period, the greenhouse film should be completely sealed without any ventilation gaps.

[0044] Statistical analysis of soil pathogen nucleic acid test results before and after fumigation: The total nucleic acid load of celery leaf spot pathogen and root rot Fusarium in each group of plots was measured before and after the fumigation treatment, and all data were summarized to form a comparison table 1.

[0045] Table 1

[0046] Table 1 shows a significant difference in pathogen levels among the three experimental groups before fumigation. Group 1 had the lowest initial pathogen count, which decreased slightly after fumigation. This group initially had a low pathogen load, indicating low underlying disease pressure and limited potential for improvement in fumigation effectiveness. Group 2 initially had a moderate pathogen load. After prolonged fumigation with a matched dose of chopped straw and lime cyanide, the pathogen nucleic acid load decreased by over 98%, directly reducing the soil pathogen count to below the target threshold. Group 3 had the highest initial pathogen load, representing a heavily continuously cropped plot. This group received the highest dose of straw and lime cyanide and the longest fumigation period. After fumigation, the pathogen nucleic acid load decreased significantly, ultimately falling below 1×10⁻⁶. 3The standard for copy number per gram of dry soil is as follows: Figure 2 As shown.

[0047] All three groups of plots were matched with materials and fumigation durations according to their corresponding continuous cropping levels. After fumigation and ventilation, soil pathogen levels returned to pre-planting soil indicators, demonstrating that classifying continuous cropping levels based on soil nucleic acid load and setting differentiated fumigation parameters can accurately reduce the total amount of soil pathogens and effectively alleviate contamination from accumulated continuous cropping pathogens. For severely continuously cropped plots, higher material application rates and longer sealed fumigation times can achieve rapid remediation of heavily contaminated soil. For moderately contaminated plots, moderate materials and medium fumigation cycles are sufficient for soil disinfection. For lightly contaminated plots, low-dose materials and short-cycle fumigation are sufficient to maintain a low initial level of soil pathogens. This differentiated fumigation scheme is suitable for plots with different levels of contamination, and the disinfection effect is stable and controllable.

[0048] Verification of supplementary treatment for plots of land that failed to meet the standards for fumigation: Four additional experimental plots with severe continuous cropping were added. After fumigation according to the severe standard for 28 days, the total pathogenic bacteria nucleic acid load was retested and found to be 1.2 × 10⁻⁶. 4 The copy number per gram of dry soil did not meet the soil quality standards. 100 kg of chopped straw was added per acre, and the fumigation period was extended by 7 days. After 7 days of ventilation, the total pathogenic bacteria nucleic acid load was retested and found to be 6.8 × 10⁻⁶. 2 The copy number per gram of dry soil meets the standard, verifying that extending the fumigation period can further reduce the number of soil pathogens.

[0049] Example 3: A comparative experiment on LAMP nucleic acid screening and grading of celery seeds and the germination effect of different seeds.

[0050] Seed soaking oil removal and nucleic acid template preparation: Celery seeds were collected in batches and divided into three parallel experimental groups, with each group containing 200 grams of seeds. All seeds were placed in a 49°C constant temperature water bath and stirred for 10 minutes. Then, they were transferred to room temperature water and soaked for 12 hours, turning the seeds every 2 hours. After soaking, the seed coats were rubbed to collect all the leachate, which was used as a test template. The copy number of seed pathogen nucleic acid was quantified using dual-target loop-mediated isothermal nucleic acid amplification technology, and the seeds were graded.

[0051] Nucleic acid copy number higher than 1×10 4 Severely infected seeds with copies / μL were directly discarded. The remaining healthy seeds and slightly infected seeds were divided into two groups and treated with different germination methods. They were all germinated in a 10°C dark environment for 7 days, and the final seed emergence rate was recorded.

[0052] Differentiated germination process for graded seeds: Healthy seeds were kept moist by daily spraying with water only. Seeds with mild contamination were divided into two treatment groups; the first group received 1×10⁻⁶ seeds.9 Seeds were soaked in a Bacillus subtilis germination solution at CFU / mL for 5 hours, and sprayed with fresh bacterial solution daily. The second group, with slightly contaminated seeds, was treated with only water spraying as a control. At the end of germination, the number of seeds showing white sprouts in the three groups was counted, and the sprouting rate was calculated to create a comparison table (Table 2).

[0053] Table 2

[0054] Table 2 clearly shows the impact of seed pathogen contamination and germination treatment methods on the whitening rate. Treatment group 1 consisted of healthy seeds free from pathogen contamination; simply keeping them moist with water was sufficient to achieve an extremely high whitening rate, as the seeds themselves did not suffer from pathogen infection hindering germination. Treatment group 2 consisted of slightly contaminated seeds that were soaked in Bacillus subtilis germination solution and continuously sprayed. Beneficial microorganisms inhibited the reproduction of leaf spot pathogens and Fusarium root rot bacteria carried on the seed surface, eliminating the inhibitory effect of pathogens on seed germination. Ultimately, the whitening rate was only slightly lower than that of healthy seeds. The control group, consisting of slightly contaminated seeds without any inoculant treatment, experienced continuous proliferation of pathogens carrying the seeds, damaging embryo activity and significantly reducing seed germination ability, resulting in a whitening rate decrease of over 22 percentage points.

[0055] Seed grading using LAMP nucleic acid quantitative screening, followed by treatment with Bacillus subtilis germination solution for mildly infected seeds, significantly reduces germination inhibition caused by pathogens and greatly improves the germination ability of infected seeds. Without nucleic acid grading, directly mixing heavily infected and mildly infected seeds during sowing leads to a significant increase in seedling disease incidence and a marked decrease in overall seed germination. Nucleic acid screening combined with graded germination can eliminate heavily infected seeds that have completely lost their planting value while rescuing mildly infected seeds, improving overall seed utilization efficiency and reducing the initial incidence of seedling diseases.

[0056] Unified tray sowing operation for white seeds After the three groups of seeds germinated and showed white sprouts, they were all filled into 72-cell seedling trays with a substrate of 4:6 well-rotted organic fertilizer and garden soil, with 5% straw fragments added. The seeds were sown, covered with 0.5 cm of soil, and thoroughly watered. After that, they were transferred to a low-temperature hardening-off greenhouse for gradient temperature acclimatization.

[0057] Example 4: Comparative experiment on the regulation of seedling hardening time under gradient low temperature and dynamic management of rhizosphere microorganisms.

[0058] Seedling grouping and gradient low-temperature acclimatization parameter settings: The seedling hardening-off experiment was divided into three groups based on the continuous cropping level of the plots: a mild group with a total hardening-off period of 7 days, a moderate group with a total hardening-off period of 10 days, and a severe group with a total hardening-off period of 12 days. A control group with conventional seedling raising and no gradient low temperature acclimatization was set up simultaneously. The initial daytime temperature for all groups of seedlings during acclimatization was 17 degrees Celsius and the nighttime temperature was 9 degrees Celsius. The daytime temperature was decreased by 1.5 degrees Celsius and the nighttime temperature was decreased by 0.8 degrees Celsius each day. After 4 days, the daytime temperature was stabilized at 12 degrees Celsius and the nighttime temperature was 4 degrees Celsius. Natural sunlight was used uniformly.

[0059] Rhizosphere substrate samples were collected every two days during the hardening-off period to test for microbial nucleic acids. At the end of the hardening-off period, the rhizosphere microbial community values ​​were retested uniformly, and the final seedling vigor rate of each group was calculated to form a comparison table.

[0060] Rhizosphere microbial control and seedling vigor assessment criteria: The nucleic acid load of Bacillus subtilis is not less than 5 × 10⁻⁶. 6 Copy number per gram of dry substrate, nucleic acid load of *Fusarium root rot*, the causal agent of celery leaf spot, is less than 1 × 10⁻⁶. 3 Seedlings with a copy number per gram of dry substrate were considered strong seedlings. The number of strong seedlings in the four groups of experiments was recorded to calculate the strong seedling rate.

[0061] Table 3

[0062] Table 3 shows that the four sets of data directly demonstrate the enhancing effect of gradient low-temperature hardening and dynamic regulation of rhizosphere microorganisms on the cultivation of robust seedlings. The mild hardening group had the shortest hardening time, low initial levels of soil pathogens, stable natural proliferation of beneficial rhizosphere bacteria without exceeding standards, and ultimately the highest robust seedling rate. The moderate hardening group had a longer hardening time, with slight exceedances of rhizosphere pathogens in the middle of the experiment. By increasing the nighttime greenhouse temperature and spraying Bacillus subtilis solution to adjust the microbial community structure, the microbial community values ​​eventually returned to standard, and the robust seedling rate decreased slightly but remained at a high level. The severe hardening group had the longest hardening period, with severe exceedances of pathogens during the experiment. A heated greenhouse was built to suspend low-temperature acclimatization, and Trichoderma harzianum was sprayed to control the pathogens. Ultimately, the robust seedling rate was the lowest among the three acclimatization groups, but significantly higher than the conventional control group.

[0063] The conventional control group did not undergo gradient low-temperature acclimatization, resulting in seedlings that did not complete low-temperature stress training. The number of Bacillus subtilis in the rhizosphere was far below the standard threshold, while the total number of pathogenic bacteria significantly exceeded the standard. Seedlings exhibited weak cold resistance, were prone to root diseases, and had a seedling vigor rate of less than 65%. Gradient low-temperature acclimatization can gradually improve the low-temperature tolerance of celery seedlings. Simultaneously, real-time rhizosphere nucleic acid detection dynamically adjusts greenhouse temperature and inoculant application, continuously optimizing the rhizosphere microbial community structure and inhibiting pathogenic bacteria reproduction. The hardening-off period was extended in tandem with the degree of continuous cropping contamination in the plot, and differentiated microbial community control methods were applied. Even in heavily continuously cropped plots, the proportion of vigorous seedlings produced was still far higher than in traditional seedling cultivation methods, demonstrating that gradient low-temperature hardening combined with dynamic management of rhizosphere microbial nucleic acid can simultaneously improve seedling low-temperature tolerance and disease resistance.

[0064] Transplanting of qualified and robust seedlings: The selected strong seedlings from each group were planted with a row spacing of 25 cm and a hole spacing of 10 cm. Drip irrigation network was laid under the mulch film. After planting, drip irrigation was used to water the seedlings to help them establish roots, and then the entire growth period was managed in the field.

[0065] Example 5: Comparative experiment on leaf mRNA detection and the effects of differentiated water and fertilizer management throughout the entire growth period.

[0066] Sampling of reproductive nodes and criteria for determining mRNA expression levels: Four celery plots with uniform growth were selected, each corresponding to one of four field management schemes. Total RNA was extracted from the top 2-3 functional leaves of each plot simultaneously at the late hardening-off stage, transplanting establishment stage, tillering stage, and canopy closure stage. The expression abundance of mRNAs responding to low-temperature stress and those for disease resistance was detected. Low expression was defined as a reduction of more than 50% in target mRNA expression compared to the healthy control. Corresponding water, fertilizer, and facility management measures were implemented for different low expression states. At harvest, the incidence of celery diseases and the fresh weight per plant were recorded for each group, and the data were compiled into a comparative table.

[0067] Differentiated field management plan setup: In Plot 1, water, fertilizer, and greenhouse management were dynamically adjusted based on mRNA detection results throughout the entire process, with corresponding treatment measures promptly applied to low expression levels. In Plot 2, disease control was only implemented during the tillering stage, without adjusting insulation facilities in response to low-temperature stress mRNA. In Plot 3, only water and fertilizer management related to low-temperature resistance was adjusted, without supplementing with antibacterial agents or phosphorus and potassium fertilizers. Plot 4 adopted a traditional fixed water and fertilizer application method, without leaf RNA detection or dynamic regulation.

[0068] Table 4

[0069] Table 4 shows that the four core indicators fully demonstrate the application value of dynamic management throughout the entire growth period based on leaf RNA detection. In Field 1, both low-temperature stress response and disease resistance mRNA expression states were considered simultaneously. Precise matching of humic acid water-soluble fertilizer, phosphorus and potassium fertilizer, compound Bacillus inoculum solution, shading nets, and greenhouse opening and closing measures were implemented at different growth stages. This maintained high expression levels of endogenous stress-resistance and disease-resistance genes in the plants, with an average relative expression value of 0.91 for both types of mRNA. Pathogenic bacteria in the field had difficulty infecting the plants, resulting in a disease incidence rate of only 3.8%. Plant growth was not inhibited under low-temperature conditions, and the fresh weight of a single plant reached 1.36 kg, the highest among the four groups. Figure 3 As shown.

[0070] In Plot 2, only fungicides were sprayed to address the low expression of disease-resistant mRNA during the tillering stage, without addressing the low gene expression caused by low-temperature stress. This resulted in insufficient cold tolerance and stunted growth under low-temperature conditions. Furthermore, low-temperature stress damaged the root system during the seedling establishment stage, leading to continued high disease incidence (over 12%) and a significant decrease in yield per plant. In Plot 3, only water and fertilizer related to low-temperature resistance were applied, lacking the supply of disease-resistant fungicides and phosphorus and potassium nutrients. This resulted in persistently low expression of disease-resistant genes, widespread spread of leaf spot and root rot (14.1%), stunted plant development, and low fresh weight. In Plot 4, without any RNA detection or dynamic management methods, and with fixed water and fertilizer application, plant stress-resistant and disease-resistant genes remained in a state of prolonged low expression. The combined effects of low-temperature freezing damage and soil-borne diseases led to an outbreak with a disease incidence exceeding 30%, resulting in a single plant fresh weight of only 0.83 kg and a significant reduction in planting profits.

[0071] By quantitative detection of leaf RNA at multiple growth nodes, the plant's low-temperature stress and disease stress status can be accurately identified. Corresponding water and fertilizer facility microbial agent management programs can be matched in stages to maintain high expression of endogenous stress and disease resistance genes in the plant, reduce the probability of disease occurrence in the field, and improve the growth rate and marketable yield of celery under low-temperature conditions. Neither of the two stresses can achieve ideal planting results by single management. Only comprehensive dynamic management that takes into account the expression status of the two types of mRNA can achieve high quality and high yield.

[0072] Example 6: The entire process and set of techniques have been verified for two consecutive years to ensure planting stability and the effectiveness of plot file iteration.

[0073] Two-year comparative trial group design: A single, heavily continuously cropped celery greenhouse was selected and evenly divided into experimental and control areas. The experimental area underwent a complete process including soil nucleic acid testing and grading, greenhouse fumigation, seed LAMP screening and grading, germination promotion, gradient low-temperature hardening, dynamic regulation of rhizosphere microorganisms, and RNA and water / fertilizer management throughout the entire growth cycle. All test data were compiled and updated annually after harvest, and process parameters were adjusted based on the data the following year. The control area adopted a traditional celery cultivation model throughout, without quantitative nucleic acid testing, gradient low-temperature acclimatization, or dynamic water / fertilizer regulation. A two-year planting trial was conducted, and soil pathogen counts, field disease incidence, seedling vigor, and material input costs were recorded annually at harvest. The two years' data were compiled into a comparative table.

[0074] Annual file iteration and process parameter adjustment operations: After the first year's harvest in the experimental area, all nucleic acid test data from the soil, seeds, rhizosphere, and leaves were compiled and stored in the plot archive. In the following year, the archive was retrieved, and the application rate of calcium cyanamide was reduced by 5 kg per mu, the low-temperature hardening period was shortened by 2 days, and the number of microbial sprays during the tillering stage was reduced. The planting parameters in the control area were kept completely fixed every year without any technological adjustments.

[0075] Table 5

[0076] Table 5 shows that the data from the two consecutive years of trials clearly demonstrate the long-term stability of the entire control process and the dual advantages brought about by the iterative optimization of the plot records. In the first year of the trial, the entire process was fully implemented, and the soil pathogen count decreased to 5.1 × 10⁻⁶ after fumigation and disinfection. 2 The copy number per gram of dry soil was 89.1%, the seedling vigor rate was 89.1%, and the disease incidence rate was only 4.2%. The overall material cost was higher than that of the control area. In the second year, based on the complete field records from the first year, the amount of calcium cyanamide was reduced, the hardening-off period was shortened, and the frequency of microbial agent spraying was decreased. Under the premise of reducing material input costs by 18 yuan per mu, the average soil pathogen count further decreased to 4.3×10. 2 The number of copies per gram of dry soil increased slightly, the seedling vigor rate increased to 90.3%, the incidence of diseases in the field decreased to 3.5%, the prevention and control effect improved simultaneously, and the planting cost continued to decrease.

[0077] In the control area, traditional planting methods were used for both years, without soil disinfection and grading, seed sterilization and screening, or low-temperature acclimatization. In the first year, the soil pathogen population reached as high as 1.3 × 10⁻⁶. 5 The number of copies per gram of dry soil was low, the seedling vigor rate was less than 62%, and the disease incidence rate exceeded 32%. In the second year, continuous cropping obstacles continued to accumulate in the plot, and the soil pathogen count rose to 1.8 × 10⁻⁶. 5 The number of copies per gram of dry soil indicates that the severity of root diseases in seedlings has led to a decline in the rate of robust seedlings to 58.6%, and the incidence of diseases in the field has increased to 37.4%. Although the material cost is lower, the yield reduction and losses caused by diseases in celery far exceed the amount of material savings, resulting in a serious loss of overall planting income.

[0078] This entire process relies on multi-stage quantitative nucleic acid detection to monitor pathogens and stress resistance across the entire chain of soil, seeds, seedlings, and plants. It can continuously suppress the initial number of pathogenic bacteria in the soil, mitigating the planting risks caused by continuous cropping obstacles year by year. The plot archive summarizes full-cycle testing data annually, accurately matching the changes in soil microorganisms and streamlining material inputs and seedling management time each year. This continuously improves seedling vigor and reduces disease incidence while decreasing agricultural input consumption, resulting in stable quality improvement and cost reduction for long-term large-scale planting. Traditional planting methods lack full-process quantitative detection and dynamic adjustment methods, leading to the accumulation of continuous cropping obstacles year after year, a continuous increase in diseases, and a decline in seedling quality, making long-term stable planting impossible.

[0079] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for the management and control of low-temperature resistant celery seedlings and the prevention and control of continuous cropping obstacles, characterized in that, The specific steps of this method are as follows: S1. Before celery seedling cultivation, topsoil samples were collected from the planting plots, total DNA was extracted from the soil samples, and the copy number of pathogenic bacteria nucleic acid was quantitatively detected using specific primers. Based on the nucleic acid load, the plots were classified into continuous cropping levels, and a plot-specific file was established. S2, retrieve the continuous cropping level data of the plot, determine the amount of straw and calcium cyanamide to be used according to the continuous cropping level, apply straw and calcium cyanamide to the plot, complete deep plowing, irrigation and sealed fumigation operations, after the fumigation is completed and ventilation is finished, retest the soil pathogen nucleic acid, apply base fertilizer to the plot with qualified nucleic acid and rotary tillage and leveling. S3. Celery seeds were selected for warm water soaking, and the seed coat oil was removed by rubbing. The seed coat extract was collected as a detection template and loop-mediated isothermal nucleic acid amplification was carried out to quantify the number of pathogen nucleic acid copies carried by the seeds. Severely infected seeds were removed, and the remaining qualified seeds were divided into two grades. S4. For qualified seeds of different grades, use germination solution corresponding to the seed grade to carry out low-temperature and light-proof germination. Determine the total time of low-temperature hardening according to the continuous cropping level of the plot. Prepare seedling substrate and fill the seedling trays. Sow the seeds into the substrate after they sprout and carry out gradient low-temperature acclimatization. S5. During the hardening-off period, collect seedling rhizosphere substrate samples, extract microbial nucleic acids and quantify the target bacterial community nucleic acid copy number, adjust hardening-off environmental parameters and bacterial agent application scheme according to preset thresholds, retest and screen qualified strong seedlings at the end of the hardening-off period, complete the transplanting and lay the drip irrigation network under the film. S6. Celery functional leaves were collected at multiple growth stages. Total RNA was extracted from the leaves and the expression abundance of endogenous stress-resistant mRNA was detected. Based on the mRNA expression status, corresponding water and fertilizer application schemes and field facility management schemes were adopted. After harvesting, the full-cycle nucleic acid test data were summarized to update the plot file and adjust the seedling and field management process parameters for the following year.

2. The method for low-temperature resistant celery seedling management and continuous cropping obstacle control according to claim 1, characterized in that, In S1, soil sampling was performed using a five-point method, taking soil layers from 0 to 20 cm in depth and sieving them through a 2 mm sieve. The detection targets were *Celtis elata* leaf spot pathogen and *Fusarium solani* root rot pathogen, and nucleic acid copy number was quantified using quantitative real-time PCR. Continuous cropping was classified as follows: total nucleic acid load below 10... 3 Copy number per gram of dry soil indicates mild severity, ranging from 10. 3 ~10 5 Copy number per gram of dry soil is considered moderate, above 10. 5 Copy number per gram of dry soil indicates heavy concentration.

3. The method for low-temperature resistant celery seedling management and continuous cropping obstacle control according to claim 1, characterized in that, In S2, for lightly continuously cropped plots, apply 1000-1400 kg of chopped straw and 25-35 kg of calcium cyanamide per mu (667 square meters), and seal the soil for 18-22 days; for moderately continuously cropped plots, apply 1250-1450 kg of chopped straw and 32-38 kg of calcium cyanamide per mu, and seal the soil for 21-25 days; for heavily continuously cropped plots, apply 1400-1600 kg of chopped straw and 37-43 kg of calcium cyanamide per mu, and seal the soil for 26-30 days. The chopped straw should be 2-3 cm long, deeply plowed to a depth of 25 cm, and the soil moisture content should be 85%-90% after irrigation. Nucleic acid retesting should be conducted 7 days after the sealing and ventilation period, with the standard being that the nucleic acid load of both pathogens is below 1×10⁻⁶. 3 Copy number / gram of dry soil; for qualified plots, apply 150 kg / mu of bio-organic fertilizer and 40 kg / mu of NPK compound fertilizer as base fertilizer. For plots with severe continuous cropping, apply an additional 20 kg / mu of biochar granules loaded with Bacillus subtilis. If the standard is not met, repeat the fumigation process for 5-7 days and retest until the standard is met.

4. The method for low-temperature resistant celery seedling management and continuous cropping obstacle control according to claim 1, characterized in that, In step S3, celery seeds are soaked in hot water at 48–50°C for 10 minutes with stirring, then soaked at room temperature for 12 hours, turning them over every 2 hours; LAMP is performed using dual-target primers targeting both leaf spot blight and root rot Fusarium, with real-time fluorescence quantitative analysis; the seed nucleic acid copy number is higher than 1×10⁻⁶. 4 The number of copies / μL was considered a severe case of bacterial contamination and the bacteria were eliminated; a value below 1×10⁻⁶ was considered acceptable. 2 copies / μL represent healthy seeds, 1×10 2 ~1×10 4 copies / μL indicates slightly contaminated seeds.

5. The method for low-temperature resistant celery seedling management and continuous cropping obstacle control according to claim 1, characterized in that, In step S4, the germination temperature is 10℃, and the period of darkness is 6-8 days; healthy seeds are sprayed with clean water to keep them moist; seeds with slight bacterial contamination are treated with a concentration of 1×10⁻⁶. 9 Soak seeds in Bacillus subtilis germination solution (CFU / mL) for 4–6 hours, then remove them and place them on a damp gauze. Spray them with fresh germination solution daily. The seedling substrate is a mixture of well-rotted organic fertilizer and garden soil at a mass ratio of 4:6, with 5% straw shreds added, and filled into 72-cell trays.

6. The method for low-temperature resistant celery seedling management and continuous cropping obstacle control according to claim 1, characterized in that, In S4, the hardening-off period is determined according to the continuous cropping level: 6-8 days for mild cases, 9-11 days for moderate cases, and 11-13 days for severe cases; sowing is done by covering the seedlings with 0.5cm of soil and watering thoroughly; the gradient low-temperature acclimatization is as follows: the initial daytime temperature is 16-18℃ and the nighttime temperature is 8-10℃, with the daytime temperature decreasing by 1-2℃ and the nighttime temperature decreasing by 0.5-1℃ each day, and after 3-5 days, the temperature is transitioned to 12℃ for the daytime and 4℃ for the nighttime and maintained until the end; the light is the same as natural sunlight; during the hardening-off period, rhizosphere substrate samples are taken and refrigerated every 1-3 days.

7. The method for low-temperature resistant celery seedling management and continuous cropping obstacle control according to claim 1, characterized in that, In step S5, the rhizosphere substrate is sampled from the top 1-2 cm of the root system for testing for Bacillus subtilis, Fusarium root rot, and celery leaf spot pathogens; the beneficial bacteria threshold is not less than 5 × 10⁻⁶. 6 The copy number per gram of dry substrate should be less than 1 × 10⁻⁶. 3 Copy number per gram of dry substrate; pathogenic bacteria copy number greater than 1 × 10⁻⁶ 3 And not exceeding 1×10 4 The copy number per gram of dry matrix is ​​slightly excessive, reaching or exceeding 1×10⁻⁶. 4 The copy number / gram of dry substrate is seriously excessive; when the beneficial bacteria are insufficient and slightly excessive, adjust the night temperature to 5-7℃ and spray the leaves and substrate with Bacillus subtilis solution; when the level is seriously excessive, build an insulated greenhouse to suspend low-temperature acclimatization and spray the whole plant with Trichoderma harzianum solution. Final retest showed that beneficial bacteria met the target and pathogenic bacteria were below 1×10⁻⁶. 3 Seedlings with a copy number per gram of dry substrate are considered strong.

8. The method for low-temperature resistant celery seedling management and continuous cropping obstacle control according to claim 1, characterized in that, In S5, strong seedlings are planted with a row spacing of 25cm and a hole spacing of 10cm, with one seedling per hole; drip irrigation network is laid under the mulch film, and drip irrigation is used to water the seedlings after planting.

9. The method for low-temperature resistant celery seedling management and continuous cropping obstacle control according to claim 1, characterized in that, In S6, leaf sampling points are the end of hardening-off period, the transplanting and seedling establishment period, the tillering period, and the canopy closure period, taking the 2nd to 3rd functional leaves from the top; low-temperature stress response mRNA and disease resistance defense mRNA are detected; low expression refers to the target mRNA expression level being downregulated by more than 50% compared to the control; when low expression of low-temperature stress mRNA occurs during the hardening-off period, drip irrigation with humic acid water-soluble fertilizer and extending the nighttime covering of the heat-insulating greenhouse; when low expression of disease resistance defense mRNA occurs during the tillering period, foliar spraying with Bacillus subtilis and drip irrigation with phosphorus and potassium water-soluble fertilizer; when both types of mRNA are lowly expressed during the canopy closure period, shading nets are activated, and the opening and closing of the heat-insulating greenhouse are adjusted according to the day-night temperature difference, compound Bacillus subtilis bacterial solution is drip-irrigated twice, and nitrogen fertilizer supply is reduced.

10. A method for the management and control of low-temperature resistant celery seedlings and the prevention and control of continuous cropping obstacles according to claim 1, characterized in that, In S6, after the annual harvest, the soil pathogen nucleic acid, seed nucleic acid screening, rhizosphere microbial nucleic acid and plant endogenous mRNA expression data are summarized and stored in the plot archive; the archive is retrieved before the seedling raising in the following year, and the amount of fumigation material, seed treatment parameters, low temperature hardening time, water and fertilizer plan and microbial agent application time are adjusted accordingly.

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