An ecological control method for tomato bacterial wilt based on multi-trophic level food web and its application

Abstract

The invention discloses an ecological control method for tomato bacterial wilt based on a multi-trophic level food web and its application, and belongs to the technical field of crop disease control. The ecological control method for tomato bacterial wilt provided by the invention regulates the structure of the soil food web and enhances the natural inhibitory effect of tomatoes on bacterial wilt by introducing indigenous dominant omnivorous nematodes and bacterivorous nematodes widely distributed in the soil; by improving the ecological balance of the soil and increasing the activity of beneficial microorganisms, the colonization of bacterial wilt bacteria in the rhizosphere is inhibited, and the incidence of plants is significantly reduced; thereby, it is conducive to the healthy growth and biomass accumulation of tomatoes, and significantly increases the plant height and dry weight of the aboveground part of the plants; the control method provided is simple to operate and has a wide range of applications: the method is easy to implement, does not rely on chemical drugs, reduces environmental pollution and food safety risks, and has a wide range of promotion value.

Description

Technical Field

[0001] The invention relates to the technical field of crop disease prevention and control, and in particular to an ecological prevention and control method for tomato bacterial wilt based on a multi-trophic level food web and an application thereof. Background Art

[0002] tomato( Solanum lycopersicum ) is an important fruit and vegetable, widely grown around the world. Its high nutritional value and unique flavor have led to a continuous expansion of tomato planting areas and a steady increase in production. As of 2020, China's tomato production reached 65.15 million tons, accounting for nearly 1 / 3 of the world's tomato production, making it the country with the largest tomato production in the world. However, Ralstonia solanacearum ( Ralstonia solanacearum ) is a devastating soil-borne disease that is widely distributed in major tomato-producing areas in southern China, seriously threatening the quality and yield of tomatoes. Ralstonia solanacearum is a highly pathogenic Gram-negative bacterium that can be spread through soil and water. Once it encounters a susceptible host (such as tomatoes), the pathogen can colonize in large numbers at the roots of the plant, invade through micro-wounds at the root system or the base of the stem, then invade the xylem vessels, and eventually spread rapidly through the vascular bundles to the entire above-ground part of the plant, resulting in reduced crop yields and eventual wilting and death.

[0003] Avoiding crop yield reduction due to crop diseases has become a key factor in ensuring agricultural production. At present, chemical control is still the main means of preventing and controlling tomato bacterial wilt, that is, using broad-spectrum fungicides (such as inorganic fungicides, organic sulfur fungicides and organic phosphorus fungicides) to reduce the content of pathogens in the soil. However, the effect of chemical control is not ideal, and it is easy for pathogens to develop drug resistance. At the same time, its high toxicity and high residual characteristics will also cause serious soil ecological damage and food safety problems. With the continuous advancement of agricultural sustainable development, ecological control has gradually become an important supplement to chemical control. For example, it regulates the multi-trophic level food web structure of the soil and improves the balance and stability of the food web by enhancing the interaction between different biological communities in the soil food web. Summary of the invention

[0004] The purpose of the present invention is to provide an ecological control method for tomato bacterial wilt based on a multi-trophic level food web and its application, so as to solve the problems that the chemical control effect of bacterial wilt is not ideal, the pathogenic bacteria are prone to develop drug resistance, and the high toxicity and high residual of chemical control can cause soil ecological damage and food safety.

[0005] To achieve the above object, the present invention provides an ecological control method for tomato bacterial wilt based on a multi-trophic level food web, wherein the ecological control method is to inoculate omnivorous nematodes and bacterivorous nematodes in the rhizosphere of planted tomatoes.

[0006] Preferably, the total inoculation amount of the nematodes is 5 to 15 per gram of soil, and the inoculation amount ratio of the omnivorous nematodes to the bacteriovorous nematodes is 1:1.

[0007] Preferably, the omnivorous nematode is selected from the genus Raspis P. quartusdecimus , bacteria-feeding nematodes selected from the family Rhabditidae D. veechi and Penpharyngeal nematodes Panagrolaimus sp. Any one or more of .

[0008] Preferably, the nematodes are obtained by the following method: inoculating the nematodes into Escherichia coli In the NGM medium of OP50, repeat this step every 5 days, and a large number of nematodes can be obtained at the same time after 15 days.

[0009] Preferably, the Escherichia coli The NGM medium for OP50 was prepared by the following method: Escherichia coli OP50 was inoculated into LB liquid medium and cultured in a shaking incubator at 200 r / min and 37°C until the logarithmic phase. 100 μL of Escherichia coli OP50 bacterial solution was evenly spread on the surface of NGM culture medium and cultured at 37°C for 6-8 hours.

[0010] Preferably, the inoculation of omnivorous nematodes and bacteriovorous nematodes is achieved by pouring an aqueous suspension of nematodes into the rhizosphere of tomatoes.

[0011] Preferably, the tomato bacterial wilt is caused by Ralstonia solanacearum Ralstonia solanacearum cause.

[0012] An application of the above-mentioned ecological control method for tomato bacterial wilt based on multi-trophic level food web in the control of tomato bacterial wilt.

[0013] The present invention provides a Ralstonia solanacearum ( Ralstonia solanacearum ) is an ecological control method for tomato bacterial wilt caused by tomato pests. By introducing a certain amount of omnivorous nematodes and bacteriovorous nematodes into the soil, the structure of the soil multi-trophic level food web is adjusted, the disease resistance of tomato plants is improved, thereby effectively controlling tomato bacterial wilt and promoting the healthy growth of crops.

[0014] Therefore, the present invention provides an ecological control method for tomato bacterial wilt based on a multi-trophic level food web and its application, and its specific technical effects are as follows:

[0015] (1) Regulating the structure of the soil food web: By introducing indigenous dominant omnivorous nematodes and bacterivorous nematodes that are widely distributed in the soil, the structure of the soil food web can be adjusted to enhance its natural inhibitory effect on bacterial wilt;

[0016] (2) Enhance crop disease resistance: By improving the ecological balance of the soil, increasing the activity of beneficial microorganisms, inhibiting the colonization of bacterial wilt in the rhizosphere, and significantly reducing the incidence of plant diseases;

[0017] (3) Promoting crop growth: The method provided by the present invention promotes the formation of a healthy soil microecological environment, which helps the healthy growth and biomass accumulation of tomatoes and significantly increases the plant height and aboveground dry weight;

[0018] (4) The prevention and control method provided by the present invention is simple to operate and has a wide range of applications: the method is easy to implement and does not rely on chemical drugs, reduces environmental pollution and food safety hazards, and has wide promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0020] Figure 1 This is a photo of the plant 30 days after inoculation with bacterial wilt pathogen in Example 2 of the present invention;

[0021] Figure 2 The statistical results of the plant morbidity rate 30 days after inoculation with bacterial wilt pathogen in Example 2 of the present invention;

[0022] Figure 3 is a photograph of the stem base of the plant 30 days after inoculation with bacterial wilt pathogen in Example 2 of the present invention;

[0023] Figure 4 is the statistical result of plant height 30 days after inoculation with bacterial wilt pathogen in Example 2 of the present invention;

[0024] Figure 5 This is the statistical result of the aboveground dry weight of the plants 30 days after inoculation with bacterial wilt pathogen in Example 2 of the present invention. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0026] In order to make the purpose, technical scheme and advantages of the present application clearer, more thorough and more complete, the technical scheme of the present invention is clearly and completely described below through the accompanying drawings and examples. The following detailed descriptions are all descriptions of the embodiments, and are intended to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present application belongs.

[0027] The instruments, equipment, reagents and materials used in the examples were all obtained through commercial channels; the nematodes used in the examples were isolated, identified and propagated by the inventors themselves, and the methods are shown in "Study on the main controlling factors of soil bacterial traits affecting the body size of nematodes", Master's thesis of Xie Wangliang, Nanjing Agricultural University, June 2024.

[0028] The culture medium used in the examples is a conventional biological culture medium, and the specific formula is as follows:

[0029] LB liquid culture medium: 10 g trypsin powder, 5 g yeast powder, 10 g sodium chloride, 1 L deionized water, sterilized at 121°C for 20 min.

[0030] NA liquid culture medium: 10 g glucose, 3 g beef extract, 5 g peptone, 0.5 g yeast powder, 1 L deionized water, sterilize at 115°C for 30 min.

[0031] NGM solid culture medium: 2.5 g peptone, 3.0 g sodium chloride, 17.0 g agar, 1 L deionized water, 121 ° C, 20 min high temperature and high pressure sterilization, when the temperature drops to 65 ° C, add 25 mL K3PO4 buffer (K2HPO4 3.56 g, KH2PO4 10.83 g, deionized water 100 mL), 1 mL magnesium sulfate (1 mol / L) solution, 1 mL calcium chloride (1 mol / L) solution, 1 ml cholesterol ethanol (5 mg / mL) solution, mix evenly and pour into a 60 mm culture dish.

[0032] Example 1

[0033] An ecological control method for tomato bacterial wilt based on a multi-trophic level food web is as follows:

[0034] S1. Obtain tomato plants. The specific steps are as follows:

[0035] A: Soak tomato seeds in room temperature water for 24 hours, place them on 1 / 2MS medium, and culture at 28°C until radicles grow;

[0036] B: Fill the 72-hole seedling tray evenly with the matrix (special matrix for seedling cultivation), sow the seeds in step A, and culture them in the usual way until the "two leaves and one heart" stage;

[0037] C: Transplant the tomato seedlings in step B into pots filled with natural soil taken from the field and manage them normally.

[0038] S2. Indoor cultivation and inoculation of nematodes. The specific steps are as follows:

[0039] A: Escherichia coliOP50 was inoculated into LB liquid medium and cultured in a shaking incubator at 200 r / min and 37°C until the logarithmic phase. Escherichia coli OP50 bacterial solution was evenly spread on the surface of NGM medium and cultured at 37°C until colonies were formed;

[0040] B: The genus Raspis ( P . fourteenth )、Microbacillaceae( D . old people ) nematodes and Pelpharyngeus ( Panagrolaimus sp.) nematodes were inoculated onto the surface of the culture medium in A, and a scalpel was used to cut the nematodes ( P . fourteenth )、Microbacillaceae( D . old people ) nematodes and Pelpharyngeus ( Panagrolaimus Sp.) nematode culture medium cut into pieces about 5 cm 2 Transfer the small pieces with the back side facing down to the culture medium in step A. Repeat this step every 5 days. After 15 days, a large number of nematodes can be obtained at the same time.

[0041] C: Rinse the nematodes in step B into a 50 mL centrifuge tube, wash with water three times, and prepare a nematode suspension with water;

[0042] D: Use the root irrigation method to inoculate the nematode suspension in step C into the rhizosphere of tomatoes planted in S1 for one week at a ratio of 10 nematodes / g soil, and perform routine management until the nematodes are fully adapted to the soil environment.

[0043] S3. Cultivation and inoculation of Ralstonia solanacearum. The specific steps are as follows:

[0044] The Ralstonia solanacearum was inoculated into NA liquid medium, cultured in a shaker at 200 r / min and 30°C for 24 h, transferred to a 50 mL centrifuge tube, washed with water three times, and prepared into a bacterial suspension with water. 7 The ratio of CFU / g soil was inoculated into the tomato rhizosphere where the nematodes were fully adapted to the soil environment in S2, and conventional pot management was carried out.

[0045] Example 2

[0046] The control method of Example 1 was used to conduct a tomato potted plant effect test. A total of three treatments were set up, namely, the control method of Example 1 was used, which were respectively recorded as N1, N2 and N3, and a blank control group (CK). One pot plant was one replication, and each treatment was repeated 12 times. The pot had a diameter of 7 cm, a height of 9 cm, and was filled with 100 g of soil.

[0047] N1: The omnivorous Raspberry ( P . fourteenth ) nematodes and bacteria-eating Rhabditidae ( D. old people ) nematodes were inoculated into the rhizosphere of tomatoes one week after planting at a ratio of 10 / g soil (500 nematodes of each type, for a total of 1000 nematodes inoculated). When the nematodes were fully adapted to the soil environment, Ralstonia solanacearum was inoculated at a ratio of 10 7 Inoculate the tomato rhizosphere at a ratio of CFU / g soil, water regularly, and carry out potted management.

[0048] N2: The omnivorous Raspis genus ( P . fourteenth ) nematodes and bacteria-eating Pelpharyngeus ( Panagrolaimus sp.) nematodes were inoculated into the rhizosphere of fully planted tomatoes at a ratio of 10 / g soil (500 nematodes of each type, for a total of 1000 nematodes inoculated). When the nematodes were fully adapted to the soil environment, Ralstonia solanacearum was inoculated at a ratio of 10 7 Inoculate the tomato rhizosphere at a ratio of CFU / g soil, water regularly, and manage the potted plants.

[0049] N3: The omnivorous Raspberry ( P . fourteenth ) Nematodes, Bacteriophagous Rhabditidae ( D . old people ) nematodes and Pelpharyngeus ( Panagrolaimus sp.) nematodes were inoculated into the rhizosphere of fully planted tomatoes at a ratio of 10 nematodes / g soil (500 Raspberry nematodes and 250 each of the other two nematodes, for a total of 1000 nematodes), and the nematodes were inoculated until they were fully adapted to the soil environment.

[0050] Ralstonia solanacearum Ralstonia solanacearum Add red fluorescent marker (refer to Song D, Shi B, Xue H, Li Y, Yu B, Xu Z, Liu F, Li J. Greenfluorescent protein labeling Escherichia coli TG1 confirms intestinalbacterial translocation in a rat model of chemotherapy. Curr Microbiol. 2006Jan;52(1):69-73. doi: 10.1007 / s00284-005-0177-9. Epub 2005 Dec 26. PMID:16392000. Only the GFP protein was replaced with RFP protein) to obtain Ralstonia solanacearum with red fluorescent label ( Ralstonia solanacearum -RFP labled). According to 10 7The tomato rhizosphere was inoculated with a ratio of CFU of Ralstonia solanacearum with a red fluorescent label / g soil, and the plants were watered regularly for potted management.

[0051] CK: After the tomato was fully planted, the nematode suspension was not inoculated. At the same time as the treatment groups N1, N2 and N3, Ralstonia solanacearum was inoculated at 10 7 Inoculate the tomato rhizosphere at a ratio of CFU / g soil, water regularly, and manage the potted plants.

[0052] Thirty days after inoculation with bacterial wilt, the disease severity, colonization of bacterial wilt in the stem, plant height and aboveground biomass were measured, and statistical analysis was performed on the results of N1, N2, N3 and CK. Figure 1-5 .

[0053] like Figure 1 and Figure 2 As shown, the ecological control method for tomato bacterial wilt based on the multi-trophic level food web provided by the present invention can effectively reduce the incidence of plants. Compared with the blank control group (CK), the disease severity of plants inoculated with Raspberry nematodes and Rhabditis elegans (N1), Raspberry nematodes and Pelargonia nematodes (N2), Raspberry nematodes, Pelargonia nematodes and Pelargonia nematodes (N3) was significantly reduced ( p <0.05), both decreased by 80%.

[0054] The stem bases of the treated groups N1, N2 and N3 and the control group CK were observed using a confocal microscope. Figure 3 As shown, the ecological control method for tomato bacterial wilt based on a multi-trophic level food web provided by the present invention can effectively reduce the colonization of bacterial wilt bacteria in the stems of plants. Obvious red fluorescence was observed in the stems of the control (CK) plants, while no fluorescence was observed in the stems of the plants inoculated with Raspipes and Rhabditis (N1), Raspipes and Pelargonia (N2), Raspipes, Rhabditis and Pelargonia (N3).

[0055] like Figure 4 As shown, the ecological control method for tomato bacterial wilt based on a multi-trophic level food web provided by the present invention can effectively increase the plant height. Compared with the control (CK), the plant heights of the plants inoculated with Raspberry nematodes and Rhabditis elegans (N1), Raspberry nematodes and Pelargonia nematodes (N2), Raspberry nematodes, Pelargonia nematodes and Pelargonia nematodes (N3) were significantly increased ( p <0.05), increased by 11.3%, 14.1% and 21.4% respectively.

[0056] like Figure 5As shown, the ecological control method for tomato bacterial wilt based on a multi-trophic level food web provided by the present invention can effectively increase the aboveground dry weight of the plant. Compared with the control (CK), the aboveground dry weight of the plants inoculated with Raspberry nematodes and Rhabditis elegans (N1), Raspberry nematodes and Pelargonia nematodes (N2), Raspberry nematodes, Pelargonia nematodes and Pelargonia nematodes (N3) increased significantly ( p <0.05), an increase of 14.4%, 20.7% and 30.6% respectively.

[0057] Therefore, the ecological control method for tomato bacterial wilt provided by the present invention regulates the structure of the soil food web and enhances its natural inhibitory effect on bacterial wilt by introducing indigenous dominant omnivorous nematodes and bacteriovorous nematodes widely distributed in the soil; by improving the ecological balance of the soil, increasing the activity of beneficial microorganisms, inhibiting the colonization of bacterial wilt bacteria in the rhizosphere, and significantly reducing the incidence of plants; thereby contributing to the healthy growth and biomass accumulation of tomatoes, and significantly increasing the plant height and aboveground dry weight; the control method provided is simple to operate and has a wide range of applications: the method is easy to implement, does not rely on chemical drugs, reduces environmental pollution and food safety risks, and has broad promotion value.

[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. An ecological control method for tomato bacterial wilt based on a multi-trophic level food web, characterized in that: The ecological control method comprises inoculating omnivorous nematodes and bacterivorous nematodes in the rhizosphere of planted tomatoes; the omnivorous nematodes are selected from the genus Raspis. P. quartusdecimus , bacteria-feeding nematodes selected from the family Rhabditidae D. veechi and Penpharyngeal nematodes Panagrolaimus sp. Any one or more of the following; The total inoculation amount of the nematodes is 5 to 15 per gram of soil, and the inoculation ratio of the omnivorous nematodes to the bacteriovorous nematodes is 1:1; Ralstonia solanacearum Ralstonia solanacearum cause.

2. The ecological control method for tomato bacterial wilt based on a multi-trophic level food web according to claim 1, characterized in that: The nematodes are obtained by the following method: inoculating the nematodes into Escherichia coli In the NGM medium of OP50, repeat this step every 5 days, and a large number of nematodes can be obtained at the same time after 15 days.

3. The ecological control method for tomato bacterial wilt based on a multi-trophic level food web according to claim 2, characterized in that: The said Escherichia coli The NGM medium for OP50 was prepared by the following method: Escherichia coli OP50 was inoculated into LB liquid medium and cultured in a shaking incubator at 200 r / min and 37°C until the logarithmic phase. 100 μL Escherichia coli OP50 bacterial solution was evenly spread on the surface of NGM culture medium and cultured at 37°C for 6-8 hours.

4. The ecological control method for tomato bacterial wilt based on a multi-trophic level food web according to claim 1, characterized in that: The inoculation of omnivorous nematodes and bacterivorous nematodes is achieved by pouring a water suspension of nematodes into the rhizosphere of tomatoes.

5. Use of the ecological control method for tomato bacterial wilt based on multi-trophic level food web as claimed in any one of claims 1 to 4 in the control of tomato bacterial wilt.

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