Application of cucumber-marigold intercropping mode in prevention and treatment of cucumber root knot nematode disease

Abstract

The invention relates to the technical field of green prevention and control of plant diseases, in particular to application of a cucumber-marigold intercropping mode to prevention and control of cucumber root knot nematode diseases. Cucumbers and marigold are intercropped and cultivated, and cucumber root-knot nematode infection and / or reproduction are / is inhibited by the allelopathy of the marigold; wherein marigold plants are planted in soil and / or a culture medium, so that stable root systems are formed before cucumber planting; 7-21 days after field planting of the marigold, cucumber seedlings are planted in the same cultivation container or the adjacent rhizosphere space, the root system of the cucumber and the root system of the marigold are located in the same cultivation environment but not directly twined, and the marigold keeps surviving and growing in the early stage and the middle stage of cucumber growth. The method solves the problem that an existing intercropping mode lacks a system method with a cultivation structure and a planting time sequence as the core, and has the advantages that operation is easy and convenient, applicability is high, popularization is easy, compared with cucumber single cropping, the root knot index of cucumbers under an intercropping system is remarkably reduced, and the requirement for green agriculture development is met.

Description

Technical Field

[0001] This invention relates to the field of green prevention and control technology for plant diseases, specifically to the application of cucumber-marigold intercropping in the control of cucumber root-knot nematode disease. Background Technology

[0002] Cucumber root-knot nematode disease is a soil-borne disease caused by parasitic nematodes such as the southern root-knot nematode, and is prevalent in cucumber-growing areas worldwide. Root-knot nematodes primarily infect cucumber roots, forming root knots of varying sizes, damaging the root's vascular system, and hindering the absorption and transport of water and nutrients. Infected cucumber plants exhibit slow growth, yellowing leaves, and decreased resistance; in severe cases, root rot leads to plant death. This disease causes a significant reduction in cucumber yield by 30%–80%, and deteriorates fruit quality. Furthermore, the wounds created by nematodes are susceptible to secondary infections by soil-borne fungi and bacteria, exacerbating disease losses. Root-knot nematodes are characterized by their high reproductive capacity and resilience; under suitable conditions of 25℃–30℃ and soil moisture of 40%–70%, they can have 5–10 generations per year, with widespread generation overlap. Even in soil without host plants, nematodes can survive for 2–3 years, making complete eradication difficult and posing a significant challenge to control efforts.

[0003] Currently, the main control methods for cucumber root-knot nematode disease include chemical control, biological control, breeding of disease-resistant varieties, and intercropping. However, all of these methods have significant limitations: First, chemical control mainly relies on chemical nematicides such as abamectin and thiazophos. Long-term use can easily lead to increased nematode resistance, resulting in a gradual decline in control effectiveness. Furthermore, chemical agents are difficult to degrade in the soil, leaving large residues that can damage the beneficial microbial community, leading to decreased soil fertility and potentially posing safety risks to agricultural products, even causing root damage and seedling burn. Second, biological control utilizes microbial preparations such as Bacillus amyloliquefaciens and Bacillus brevis. While environmentally friendly, it suffers from unstable effectiveness, is highly dependent on soil conditions, and has high control costs, making large-scale application in production difficult. Third, regarding the breeding of disease-resistant varieties, the number of cucumber varieties resistant to root-knot nematodes is limited, and resistance often targets specific nematode physiological races, making it difficult to cope with the infection pressure brought about by the rapid evolution of nematodes. Additionally, disease-resistant varieties may have defects such as poor yield or quality. Fourthly, intercropping for control: Intercropping cucumbers with allelopathic plants utilizes allelochemicals to inhibit cucumber root-knot nematode infection and / or reproduction, which is an important sustainable control method. Studies have shown that marigolds, as a member of the Asteraceae family, release various active substances with inhibitory effects on root-knot nematodes during their root system growth, demonstrating certain nematode control potential in some crop systems. However, existing intercropping models suffer from insufficient synergistic optimization of control efficacy and yield, and unclear technical parameters.

[0004] Key evaluation indicators for the control of cucumber root-knot nematode disease include nematode population density, root-knot index, disease index, control effect, crop growth indicators, and yield indicators. The core is the synergistic assessment of nematode suppression, disease severity, and crop growth and yield. Auxiliary evaluation indicators include nematode mortality rate, nematode tropism, and soil microbial community, etc.

[0005] The comparative literature retrieved by the applicant includes:

[0006] The study, "The Effects of Marigold Intercropping Density on the Control Efficacy of Cucumber Root-Knot Nematodes and on Cucumber Production and Yield," investigated the control effects of different marigold intercropping densities on cucumber root-knot nematodes and studied the effects of marigold intercropping density on cucumber growth, physiology, and yield.

[0007] The study on the control efficacy of calcium cyanamide and marigold against southern root-knot nematodes in cucumber under organic substrate cultivation showed that intercropping with marigold significantly reduced the number of second-instar larvae in the cultivation substrate and the disease index of cucumber, alleviating the damage caused by southern root-knot nematodes to cucumber and improving cucumber root vitality. Direct sowing of a certain proportion of pre-germinated marigold seeds significantly reduced the number of second-instar larvae in the substrate and the disease index of cucumber, alleviating the damage caused by southern root-knot nematodes to cucumber and improving cucumber root vitality. Sowing marigold at the time of cucumber transplanting has the advantages of simple operation and no impact on the normal growth of cucumber, but its control effect on southern root-knot nematodes is somewhat inferior to that of calcium cyanamide.

[0008] Patent document CN201210453057 discloses a method for controlling nematodes through crop rotation or intercropping with plants such as marigolds. Intercropping marigolds with cucumbers ensures that cucumbers are protected from root-knot nematodes throughout the entire growing season, resulting in low control costs and significant effectiveness. The planting techniques include: a method of simultaneous seedling cultivation and transplanting of cucumbers and marigolds, and a method of direct sowing of cucumbers followed by seedling cultivation and transplanting of marigolds. In the simultaneous seedling cultivation and transplanting method, cucumbers are sown 20-25 days earlier than marigolds; cucumbers are transplanted when they have 3-5 true leaves, and the transplanting time for marigolds should be the same as or 1-4 days later than that for cucumbers. In the direct sowing method, marigolds are sown 15-20 days earlier for seedling cultivation, and the transplanting time for marigolds is the same as that for cucumbers.

[0009] The main problem with the existing technologies mentioned above is that they are mostly focused on marigold rotation, the application of preparations or extracts. There is still a lack of technical solutions for the stable control of cucumber root-knot nematode disease by rationally constructing an intercropping cultivation model of marigold and cucumber, especially a lack of systematic methods centered on cultivation structure and planting sequence. Summary of the Invention

[0010] The purpose of this invention is to address the shortcomings of the prior art by providing an application of a cucumber-marigold intercropping model in the prevention and control of cucumber root-knot nematode disease. By regulating the cultivation sequence of marigold and cucumber and their rhizosphere symbiotic relationship, the degree of cucumber root-knot nematode infection can be effectively reduced without the application of chemical nematicides or soil disinfection, while not affecting the growth and development of cucumber.

[0011] The overall technical concept of this invention is:

[0012] The application of cucumber-marigold intercropping in the control of cucumber root-knot nematode disease involves intercropping cucumbers with marigolds, utilizing the allelopathic effects of marigolds to inhibit cucumber root-knot nematode infection and / or reproduction; this includes the following steps:

[0013] A. Plant marigold plants in soil and / or cultivation substrate to allow the marigolds to develop a stable root system before the cucumbers are planted.

[0014] B. 7–21 days after the marigolds are planted, transplant cucumber seedlings into the same cultivation container or adjacent root space, so that the cucumber roots and marigold roots are in the same cultivation environment but do not directly intertwine, and the marigolds can survive and grow in the early and middle stages of cucumber growth.

[0015] In existing technologies, intercropping cucumbers and marigolds for nematode control often involves simultaneous planting or increasing marigold density to enhance efficacy. This relies primarily on marigolds releasing nematode-inhibiting substances during their growth. However, these methods often fail to cover the early stages when cucumber roots are most vulnerable to infection, and in some cases, competition between the two crops can negatively impact early cucumber growth.

[0016] Based on this, the present invention shifts the focus of prevention and control from "simple spatial parallel intercropping" to "temporal regulation of the rhizosphere formation stage". By allowing marigolds to grow preferentially before cucumbers are planted, a rhizosphere environment that inhibits the infestation of second-instar larvae of root-knot nematodes is established before the cucumber roots are formed, thereby moving the prevention and control window forward.

[0017] Studies have shown that marigolds enter a stable growth period approximately 10–15 days after planting, with significantly enhanced root activity. This root system continues to influence the rhizosphere soil environment for the following 30–60 days, interfering with the survival, migration, and infection behavior of root-knot nematodes. This invention utilizes this growth characteristic of marigolds to quantitatively control their introduction time.

[0018] The applicant further demonstrated through experiments that when marigolds were planted 7–21 days before cucumber transplanting and continued to grow symbiotically with cucumbers during the early and middle stages of cucumber growth, the inhibitory effect on cucumber root-knot nematode disease was significantly better than the treatment where marigolds and cucumbers were planted simultaneously. This result indicates that preferential planting of marigolds and the establishment of a stable rhizosphere environment are key technical conditions for exerting the inhibitory effect.

[0019] The specific technical concept of this invention also includes:

[0020] To avoid competition for light or nutrients due to excessively high intercropping density, the preferred technical approach is to plant marigolds to cucumbers in a ratio of 1:1 to 3.

[0021] To balance disease control effectiveness with normal cucumber growth, and to ensure that marigold and cucumber plants are within the same rhizosphere without significant root entanglement, allowing for quantitative control of their rhizosphere spatial relationship, the preferred technique is to plant adjacent marigold and cucumber plants at a horizontal spacing of 5–10 cm.

[0022] To suppress the development of nematode resistance to improve control efficacy, reduce the residual amount of chemical agents in the soil, maintain the structure of beneficial soil microbial communities and soil fertility, and reduce potential risks to agricultural product quality and safety, the preferred technical means is to avoid applying chemical nematicides and / or soil disinfectants during cultivation.

[0023] To simplify the unified management of intercropping crops, the preferred technical means are to ensure that the water holding capacity of the soil and / or cultivation substrate is 60% to 70% during the cultivation process.

[0024] To facilitate observation of root-knot nematode disease and promote its widespread application in production, the preferred technical approach is to select the cucumber variety "Zhongnong 26," which is widely used in production, highly susceptible to root-knot nematodes, and highly representative in the field.

[0025] The essential features and significant technical advancements achieved by this invention are as follows:

[0026] This invention is based on conventional cultivation practices and does not involve complex formulation preparation or additional treatment processes. Firstly, it can be implemented simply by rationally arranging the introduction and cultivation sequence of marigolds. This invention shifts the focus of control from "simple spatial intercropping" to "temporal regulation of the rhizosphere formation stage." By prioritizing the growth of marigolds before cucumber transplanting, a rhizosphere environment that inhibits root-knot nematodes is established before the cucumber root system forms, thus shifting the control window forward. It is simple to operate and highly applicable. Secondly, while controlling cucumber root-knot nematode disease, it does not affect the normal growth and development of cucumbers. It is suitable for various application scenarios such as pot experiments and facility cultivation, providing a safe, sustainable, and easily promoted technical solution for the green control of cucumber root-knot nematode disease. Thirdly, according to the applicant's experiments, compared with cucumber monoculture, the average number of root knots per cucumber plant is reduced by approximately 30% to 55%, and the root knot index is significantly decreased. Fourth, the cultivation process does not use chemical nematicides and / or soil disinfectants, which inhibits the development of nematode resistance and improves the control effect, reduces the residual amount of chemical agents in the soil, maintains the structure of beneficial soil microbial communities and soil fertility, reduces potential risks to the quality and safety of agricultural products, and meets the requirements of green agricultural development. Attached Figure Description

[0027] The accompanying drawings of this invention are as follows:

[0028] Figure 1 This is a diagram illustrating the cultivation of cucumbers in a monoculture manner.

[0029] Figure 2 This is a schematic diagram of a cucumber-marigold intercropping system.

[0030] Figure 3 Phenotypic diagram of marigold roots after inoculation with root-knot nematodes.

[0031] Figure 4 This shows the growth of cucumber plants and roots under a monoculture system.

[0032] Figure 5 This shows the growth of cucumber plants and roots under a cucumber-marigold intercropping system.

[0033] Figure 6 The growth of cucumber plants and roots 35 days after inoculation with Southern Root-Knot Nematode in a monoculture cucumber crop.

[0034] Figure 7 The growth of cucumber plants and roots 35 days after inoculation with Southern Root-Knot Nematode in a cucumber-marigold intercropping system.

[0035] Figure 8 Analysis of the number of root knots per cucumber plant 35 days after inoculation with Southern root-knot nematodes in cucumber monoculture and intercropping.

[0036] Figure 9Analysis of the number of egg masses per cucumber plant 35 days after inoculation with Southern root-knot nematodes in cucumber monoculture and intercropping.

[0037] Figure 10 Analysis of the number of root knots per gram of cucumber roots 35 days after inoculation with Southern root-knot nematodes in cucumber monoculture and intercropping.

[0038] Figure 11 Analysis of the number of root egg masses per gram of cucumber 35 days after inoculation with Southern root-knot nematodes in cucumber monoculture and intercropping. Detailed Implementation

[0039] The present invention will be further described below with reference to embodiments, but this is not intended to limit the present invention. The scope of protection of the present invention shall be determined by the contents of the claims. Any equivalent means substitution made in accordance with the description of the present invention shall not depart from the scope of protection of the present invention.

[0040] Example 1

[0041] The application of cucumber-marigold intercropping in the control of cucumber root-knot nematode disease involves intercropping cucumbers with marigolds, utilizing the allelopathic effects of marigolds to inhibit cucumber root-knot nematode infection and / or reproduction; this includes the following steps:

[0042] A. Plant marigold plants in soil and / or cultivation substrate to allow the marigolds to develop a stable root system before the cucumbers are planted.

[0043] B. 7–21 days after the marigolds are planted, transplant cucumber seedlings into the same cultivation container or adjacent root space, so that the cucumber roots and marigold roots are in the same cultivation environment but do not directly intertwine, and the marigolds can survive and grow in the early and middle stages of cucumber growth.

[0044] The ratio of marigolds to cucumbers planted is 1:1 to 3.

[0045] The horizontal spacing between adjacent marigold and cucumber plants should be 5-10 cm.

[0046] No chemical nematicides and / or soil disinfectants are used during cultivation, and the water holding capacity of the soil and / or cultivation substrate is 60% to 70% during cultivation.

[0047] The cucumber variety selected was "Zhongnong 26", which is widely used in production, highly susceptible to root-knot nematodes, and has strong field representativeness.

[0048] Example 2

[0049] Comparison of the chemotactic and lethality effects of cucumber and marigold root exudates on second-instar larvae (J2) of the southern root-knot nematode.

[0050] To further verify the mechanism of intercropping cucumber and marigold in controlling cucumber root-knot nematode disease, this example compares and analyzes the chemotaxis and lethality of root exudates from cucumber and marigold to J2. Both cucumber and marigold plants were selected based on uniform growth, lack of mechanical damage, and seedling age. Before the experiment, the roots were rinsed with clean water and allowed to acclimatize briefly under nutrient-free conditions to reduce interference from soil residues on root exudates.

[0051] The preparation and inoculation of J2 were carried out according to the following steps: Fresh egg masses were collected from the roots of tomato plants infested with southern root-knot nematodes and rinsed in clean water to remove dirt; the egg masses were placed in a 0.5%–1.0% sodium hypochlorite solution and gently shaken for 2–3 minutes to separate the eggs, followed by repeated rinsing with sterile water until no chlorine odor remained; the eggs were transferred to incubation dishes and incubated in the dark at 25°C. The hatched J2 cells were collected every 24 hours and counted using a stereomicroscope. To ensure inoculation consistency, J2 cells with high viability within 24 hours of hatching were selected as experimental materials; high viability was characterized by active movement and a transparent, intact body surface.

[0052] The preferred method for collecting root exudates is hydroponics: Plant roots are placed in sterile deionized water or a 0.5 mmol / L calcium chloride solution, ensuring the roots are submerged and slightly aerated. Root exudates are collected for 12–24 hours at 25°C. After collection, suspended solids are removed by coarse filtration through gauze, followed by sterilization through a 0.22 μm filter membrane to obtain crude extracts of cucumber and marigold root exudates. Root exudates are stored for a short period at 4°C in the dark. J2 was prepared according to the "egg mass separation-hatching-collection" process to ensure that the J2 individuals used for bioassays were highly viable individuals within 24 hours of hatching.

[0053] The lethality rate was verified using a microplate or petri dish immersion method: 100 insects per well were added to each well / dish with an equal volume of treatment solution and an equal volume of J2. Marigold secretion group, cucumber secretion group, and sterile water blank control group were set up, with at least 9 biological replicates in each group. The activity status of J2 was observed at 6, 12, and 24 hours. Survival was determined using a light touch stimulation method; no response and rigidity were considered death. The number of live and dead insects were counted. The mortality rate was calculated as "number of deaths / total number × 100%".

[0054] Chemotaxis verification is preferably performed using the two-choice agar plate method: equal volumes of marigold secretion and control solution (or cucumber secretion) are spotted at opposite ends of a 1.5% water agar plate. One hundred J2 nematodes are then added to the center of the plate. After incubation at 28°C for 6 hours, the number of J2 nematodes aggregated at both ends is counted. The chemotaxis index CI is calculated as: CI = (number of treated nematodes - number of control nematodes) / (number of treated nematodes + number of control nematodes). CI > 0 indicates strong chemotaxis, and CI < 0 indicates weak chemotaxis.

[0055] Table 1. Effects of marigold and cucumber root exudates on mortality and tidal activity of root-knot nematodes.

[0056] deal with 24-hour mortality rate (%) 48-hour mortality rate (%) directional Marigold root secretions 53.27±7.34 90.68±11.31 ＜0 Cucumber root secretions 24.39±7.71 39.84±9.67 ＞0 Sterile water control 4.41±3.11 11.0±2.16

[0057] The results, as shown in Table 1, indicated that marigold root exudates had a stronger lethal effect on J2 compared to cucumber root exudates, with a significantly increased corrected mortality rate 24–48 hours after treatment. Furthermore, in the chemotaxis test, J2 exhibited a clear migration inhibition phenomenon in response to marigold exudates. These phenomena may indicate that marigold exudates have a toxic effect on J2, or they may indicate that marigold exudates have a repellent / interference effect on J2 migration.

[0058] Example 3

[0059] For potted plants, marigolds should be planted first and intercropped with cucumbers to control cucumber root-knot nematode disease.

[0060] 30cm x 70cm rectangular plastic flowerpots were used as cultivation containers. Drainage holes were installed at the bottom of the flowerpots, and a filter screen was laid to prevent substrate loss. Healthy, pathogen-free cultivation soil was used as the substrate, a mixture of horticultural soil and river sand in a 3:1 volume ratio. The soil was thoroughly mixed after passing through a 2mm sieve, and each pot was filled with uniform weight and watered to 60%–70% of field capacity. The experiment included an intercropping treatment of cucumber and marigold and a single-cropping control of cucumber, with 10–15 replicates for each treatment.

[0061] The cucumber variety used in the test was "Zhongnong 26", which is widely used in production, highly susceptible to root-knot nematodes, and highly representative in the field. The seedlings were raised in plug trays in a uniform manner. When the seedlings reached 2-3 true leaves, were of uniform age, and grew uniformly, they were transplanted to reduce the interference of seedling age differences on the disease.

[0062] The experimental setup and treatments are as follows: Figure 1 The cucumber shown is grown alone, as shown Figure 2 The cucumber-marigold intercropping is shown. Each treatment has 15 replicates, with two cucumber plants planted per pot, and the pots are randomly arranged. The plants are rotated periodically during cultivation to eliminate marginal effects. In the intercropping treatment, two marigold seedlings of the same age with 2-3 true leaves are planted in each pot first. After transplanting, the marigolds are managed with water and fertilizer as usual to promote their preferential growth. Preferably, the marigolds are allowed to grow for 14 days. Subsequently, cucumber seedlings are transplanted into the same pot, controlling the horizontal distance between the roots of the cucumber and marigold at 5-10 cm, preferably 6-8 cm, and ensuring consistent ventilation and light conditions within the pot to avoid shading bias caused by intercropping competition.

[0063] Inoculate 14 days after cucumber transplanting using the rhizosphere inoculation method: make 2-4 small holes 2-3 cm around the main root of each cucumber plant, inject an equal volume of J2 suspension into the rhizosphere soil, cover with soil and compact, ensuring that the number of nematodes inoculated on each cucumber plant is uniform. Immediately after inoculation, water should be added to encourage the nematodes to migrate to the rhizosphere. Afterward, maintain stable substrate moisture content, keeping the field water holding capacity at 60%-70%. Do not apply any chemical nematicides or soil disinfectants during the growing season.

[0064] Disease surveys were conducted about 35 days after cucumbers began growing: the plants were removed along with their roots, the roots were gently rinsed with running water, and plant phenotypes including plant height, number of leaves, growth vigor, etc. were recorded, and the number of root knots per plant was counted.

[0065] The results showed that the marigold root system was normal, and no root-knot nematode disease was found. See details below. Figure 3 In a cucumber monoculture system, inoculation with root-knot nematodes resulted in cucumber plants that were stunted and had paler leaves, as shown in the specific results below. Figure 4 and Figure 6 As shown; Figure 5 and Figure 7 As shown, under the "marigold priority planting and intercropping" treatment of this invention, although no significant differences were found in the number of root knots per plant, the number of oocytes per plant, and the number of oocytes per unit, the specific results are shown in [the original text]. Figure 8 , 9 However, the average number of root knots per cucumber plant decreased by about 30% to 55%, and the number of root knots per unit decreased significantly. See the details below. Figure 10 Meanwhile, the results in Table 2 show that the plant height, number of leaves, and overall growth vigor of cucumber plants under intercropping treatment did not decrease significantly compared with monoculture treatment, indicating that this intercropping pattern effectively inhibits root-knot nematode disease without affecting the normal growth of cucumbers.

[0066] Table 2. Statistics on growth phenotypes of cucumber-marigold intercropping 35 days after transplanting.

[0067]

Claims

1. Application of cucumber-marigold intercropping in the control of cucumber root-knot nematode disease: Cucumber and marigold are intercropped, utilizing the allelopathic effect of marigolds to inhibit cucumber root-knot nematode infection and / or reproduction; characterized by… Includes the following steps: A. Plant marigold plants in soil and / or cultivation substrate to allow the marigolds to develop a stable root system before the cucumbers are planted. B. 7–21 days after the marigolds are planted, transplant cucumber seedlings into the same cultivation container or adjacent root space, so that the cucumber roots and marigold roots are in the same cultivation environment but do not directly intertwine, and the marigolds can survive and grow in the early and middle stages of cucumber growth.

2. The application according to claim 1, characterized in that... The ratio of marigolds to cucumbers planted is 1:1 to 3.

3. The application according to claim 1, characterized in that... The horizontal spacing between adjacent marigold and cucumber plants should be 5-10 cm.

4. The application according to claim 1, characterized in that... No chemical nematicides and / or soil disinfectants are used during cultivation.

5. The application according to claim 1, characterized in that... During cultivation, the water holding capacity of the soil and / or cultivation substrate should be 60% to 70%.

6. The application according to claim 1, characterized in that... The cucumber variety selected is "Zhongnong 26".

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