Liuan nematode-based mollusk pest biological control method

Abstract

The invention relates to a method for biologically preventing and controlling mollusc pests, in particular to a method for biologically preventing and controlling the mollusc pests by utilizing a new nematode species of Phamarabis, namely, Phamarabis lanensis. The method comprises the following steps: separating and purifying the Liuan nematode from a natural environment, and carrying out species identification through morphological and molecular biological technologies. The invention further relates to a culture technology of the Lu'an nematode. The culture technology comprises selection and optimization of solid and liquid culture media. An infection experiment proves that the Lu'an nematode has high-efficiency killing capability on mollusk pests such as slugs and snails. The biological control method aims to reduce the use of chemical pesticides, reduce chemical residues, protect human health and improve the economic value of crops. The invention provides a new biological control strategy for a sustainable agricultural ecological environment.

Description

Technical Field

[0001] The invention relates to the technical field of plant protection and biological control, in particular to a biological control method for agricultural pests, namely, mollusks (such as slugs and snails). Background Art

[0002] In ecological farms and Chinese herbal medicine plantations, mollusks (such as slugs and snails) often cling to plant leaves and shoots at night to feed, resulting in reduced edible value of crops and economic losses. In addition, mollusks may carry viruses and parasites, posing a threat to human health.

[0003] Although traditional chemical pesticides such as metaldehyde and iron phosphate compounds can effectively control mollusks, they pose great ecological risks and are not suitable for use in ecological farms and Chinese herbal medicine plantations that pursue ecological and sustainable development.

[0004] Currently, biological control has become an effective way to reduce dependence on chemical pesticides and protect the ecological environment. In this field, Phasmarhabditis nematodes have attracted attention due to their lethal effects on mollusks. Since their discovery in 1990, biological control products based on Phasmarhabditis nematodes have been promoted in the European market, providing environmentally friendly mollusc management solutions for local agricultural production. However, the promotion of these products worldwide is limited, mainly due to the lack of nematode strains suitable for various regions, especially in regions such as the United States and China.

[0005] In China, the research on the biological control of mollusks using nematodes is still in its infancy and there is a gap with the international advanced level. Molluscs pose a serious threat to my country's ecological farms and Chinese herbal medicine plantations. At the same time, with the increasing public concern about food safety, it has become particularly urgent to develop biological control products suitable for my country's local ecological environment. Therefore, the present invention aims to screen out nematode species that have a high efficiency in killing specific mollusks and are friendly to the local ecological environment through in-depth research, and develop corresponding biological control products to solve the problem of mollusc control in my country's ecological agriculture, ensure crop yield and quality, and at the same time enhance my country's competitiveness in the field of global biological control technology to ensure people's food safety.

[0006] The invention aims to fill the gap in the field of mollusk biological control technology in my country and promote the sustainable and healthy development of my country's ecological agriculture. Summary of the invention

[0007] The present invention relates to a Phasmarhabditis nematode product adapted to my country's ecological environment and a biological control method for controlling molluscs (such as slugs and snails), aiming to fill the gap in the field of biological control of mollusc pests in my country. The core objective of the present invention is to provide a Phasmarhabditis nematode product adapted to my country's ecological environment to solve the technical problem and commercial demand of the current lack of special, efficient and environmentally friendly mollusc biological killing means.

[0008] The technical solution of the present invention is as follows:

[0009] The Phasmarhabditis nematode involved in the present invention is isolated from a mollusk in a natural environment. The nematode is widely distributed in organic habitats such as piles of rotten leaves in nature. When a mollusk passes by, the nematode can keenly sense its presence and invade the mollusk through the pores. After successful parasitism, the infected mollusk will gradually show pathological symptoms such as swelling of the mantle and die in a short time. Based on the above phenomenon, the present invention adopts the following strategy to collect nematodes: after catching the mollusk, the excrement in its breeding box can be checked and the nematodes can be preliminarily collected by taking advantage of the characteristic that the nematodes are discharged with the excrement; or, the mollusk can be directly dissected to obtain the Phasmarhabditis nematode from its body. After the collected nematodes are properly treated and cultured, they can be used as active ingredients of biological pesticides for biological control of mollusks.

[0010] The nematode culture method of the present invention is flexible and diverse, and solid or liquid culture medium can be used. When selecting the culture medium, either a standard culture medium with precisely controlled chemical composition or a crude culture medium with relatively extensive composition can be used. The key is to ensure that the culture medium can provide the protein, sterol, vitamin, mineral and carbohydrate required for the growth and development of the nematodes.

[0011] From the perspective of cost-effectiveness and production convenience, commercial production tends to use crude culture media such as egg yolk salt solution medium (including 0.3% to 1.5% NaCl, 0.5% to 3% yeast extract, 0.5% to 3% tryptone, 1% to 3% olive oil, 3% to 6% egg yolk salt solution) to reduce costs and simplify operating procedures. In order to improve the efficiency of nematode collection, liquid culture medium has become the preferred option because it is more suitable for large-scale culture and harvesting. In the laboratory stage, researchers usually use conical flasks for liquid culture experiments, while in the commercial production stage, large fermentation tanks are used as the main equipment. Combined with an efficient stirring system and aeration device, the healthy growth of nematodes in a liquid environment can be ensured.

[0012] After the nematode culture is completed, the nematodes in the dauer stage (dormant stage) can be efficiently collected by separation techniques such as centrifugation or sedimentation. Nematodes in this stage are more stable during storage and transportation, while retaining excellent biological control activity.

[0013] In the present invention, nematodes exhibit a wide range of feeding habits. Experiments have shown that nematodes can not only grow normally in a laboratory environment by feeding on common OP50 Escherichia coli, but can also use a variety of standardized bacteria including Top10 Escherichia coli as a source of nutrition to maintain their healthy growth. In addition, a variety of symbiotic bacteria isolated from slugs have also been shown to effectively support the growth and development of nematodes.

[0014] In the commercial production stage, the relationship between bacterial species and nematode growth rate, as well as whether certain bacteria can secrete toxins that kill mollusks, can be considered comprehensively to select the most suitable bacterial species as a nutrient source for nematodes. This will not only optimize the production efficiency of nematodes, but also improve their biological control effect.

[0015] Since only Phasmarhabditis nematodes have been proven to have the ability to kill mollusks in current scientific research, it is particularly important to accurately identify the species of the collected nematodes. The present invention combines two complementary methods, epigenetic identification and molecular identification, to ensure that the identification results are accurate and reliable.

[0016] In terms of phenotypic identification, we made a preliminary judgment by comparing the descriptive features of Phasmarhabditis nematodes in the literature, conducting morphological classification. Molecular identification further verified the species identity by analyzing conservative genetic markers such as 18SrDNA, 28S rDNA and ITS sequences of the collected nematodes and comparing them with known Phasmarhabditis nematode sequences. Using these two methods, our research team successfully identified and named a new species: Phasmarhabditis luanensis.

[0017] In subsequent culture experiments, we selected female nematodes carrying eggs in their bodies for solid culture. The experimental results showed that not only female nematodes appeared in the culture medium, but also male nematodes were observed, and the male-female ratio was about 0.85. This discovery provides valuable clues for a deeper understanding of the reproductive strategy and population dynamics of this nematode.

[0018] In order to meet the long-term storage requirements of commercially available nematodes at room temperature, the present invention uses a variety of carrier materials such as 0.5% to 5% agarose gel, 3% to 15% polyacrylamide gel or diatomaceous earth to embed the nematodes. The nematodes used for embedding are preferably in the dauer stage (dormant stage), because the nematodes at this stage not only have the ability to actively infect mollusks, but also have a significantly extended life cycle of up to 4 months, far exceeding the normal growth cycle of nematodes of only about 3 weeks.

[0019] Through this method, the stability and activity of nematode products in market circulation are effectively improved, ensuring that they continue to exert efficient biological control effects during use.

[0020] The present invention has the following beneficial effects:

[0021] The present invention proposes a nematode product that can efficiently kill mollusks, which is particularly suitable for places such as ecological farms and Chinese herbal medicine plantations that pursue green ecology and strictly limit the use of chemical pesticides. This technology can significantly reduce the harm of mollusks to crops and Chinese herbal medicines, and effectively ensure the quality and ecological safety of agricultural products.

[0022] Foreign literature records show that at high doses, Phasmarhabditis hermaphrodita, Phasmarhabditis papillosa and Phasmarhabditis californica can cause 86% to 97% mortality in molluscs within 14 days. Experimental verification found that the killing effect of P. luanensis nematodes on molluscs is better than the results in foreign literature.

[0023] The core of the invention is to break through the bottleneck of my country's mollusc biological control technology and fill the technical gap in this field. This not only reflects a high sense of responsibility for environmental protection, but also provides a new idea and method for sustainable agricultural development and ecological balance maintenance.

[0024] The promotion and implementation of this technology will significantly promote the development of ecological agriculture, reduce the negative impact of chemical pesticides on soil, water sources and biodiversity, and enhance the market competitiveness of agricultural products to meet consumers' growing demand for green and healthy products. In addition, the successful application of this invention will drive the research and development and promotion of related biological control technologies, and make important contributions to agricultural biosafety and sustainable development in my country and even the world. DETAILED DESCRIPTION

[0025] The technical content of the present invention is further described in detail below in combination with laboratory specific test data and examples. It should be noted that the provided examples are intended to clearly demonstrate the practical application of the present invention and are not intended to limit the wide range covered by the present invention. Unless otherwise specified, the methods used in the examples all follow the conventional operating specifications of those skilled in the art; all reagents and materials, if their sources are not specifically stated, are considered to be commercially available standard products.

[0026] Step 1: Isolate nematodes

[0027] (1) The present invention collects slugs and snails from Huoshan County, Lu'an City, Anhui Province, rinses them with tap water and distilled water, and then places them in a plastic incubator for cultivation using lettuce and dog food;

[0028] (2) Spray a small amount of water every day to maintain the humidity of the breeding box, and collect the excrement in the breeding box to pick out the nematodes under a microscope.

[0029] Step 2: Cultivate nematodes

[0030] (1) preparing a solid culture medium in the proportion of 3% agar, 0.6% NaCl, 2.5% yeast extract, and 0.5% tryptone, and sterilizing it in an autoclave;

[0031] (2) preparing an egg yolk salt solution in a ratio of 1:1 between egg yolk and 0.9% NaCl solution;

[0032] (3) Cool the solid culture medium in step 2.1 to about 60° C., add the egg yolk salt solution prepared in step 2.2 and 3% olive oil at a rate of 5% of the total amount of the culture medium, and shake well;

[0033] (4) After the plate is cooled and solidified, Top10 Escherichia coli is smeared and cultured at 25°C for 48 h;

[0034] (5) Inoculate the nematodes picked in step 1.2 onto the solid culture medium in step 2.4 and culture for 4 days;

[0035] (6) Pick a single nematode from step 2.5 and inoculate it on the solid culture medium in step 2.4, and continue to culture for 7 days;

[0036] (7) preparing a liquid culture medium in the ratio of 0.6% NaCl, 2.5% yeast extract, and 0.5% tryptone, and sterilizing it in an autoclave;

[0037] (8) After the liquid culture medium in step 2.7 is cooled to room temperature, the egg yolk salt solution prepared in step 2.2 and 3% olive oil are added at a rate of 5% of the total amount of the culture medium and shaken thoroughly;

[0038] (9) Add Top10 Escherichia coli to the liquid egg yolk medium and culture in a shaking incubator at 28°C for 32 h;

[0039] (10) Rinse the nematodes in step 2.6 with distilled water and add them to sterilized liquid culture medium. Culture in a shaking incubator at 25°C for 7 days.

[0040] (11) The nematode density was calculated using the hemocytometer method.

[0041] Step 3: Identify the nematodes

[0042] (1) Use distilled water to wash off the nematodes in step 1.2 and step 2.6 for species identification. Figure 1 ;

[0043] (2) Rinse the nematodes cultured in step 2.6 with distilled water, extract the nematode DNA, and amplify the 28S rDNA sequence using universal primers (28S forward primer: 5'-AGCGGAGGAAAAGAAACTAA-3', 28S reverse primer: 5'-TCGGAAGGAACCAGCTACTA-3') for species molecular identification.

[0044] Step 4: Nematode vector

[0045] (1) Prepare 1.1% agarose solution;

[0046] (2) When the temperature reaches 30°C, add the dauer stage nematodes collected by centrifugation in step 2 and stir.

[0047] Step 5: Two-line mucophagous slug infection experiment

[0048] (1) Prepare 6 feeding boxes, each with a bottom area of ​​100 cm 2 Pure cotton non-woven fabric as the base;

[0049] (2) 30 two-lined mucophagous slugs were selected, with an individual weight of 1.07 g ± 0.22 g (variation range 20%). The slugs were divided into 6 groups, with 5 slugs in each group, including 3 experimental groups (infected groups) and 3 control groups (such as Figure 2 );

[0050] (3) In the experimental group, 120,000 P. luanensis nematodes were added to each feeding box, which was equivalent to adding 1200 nematodes per square centimeter of the bottom fabric; in the control group, 1 mL of Top10 Escherichia coli for feeding nematodes was added to each box, and distilled water was added to ensure that the total amount of liquid was consistent with that of the experimental group;

[0051] (4) Lettuce leaves and dog food were used as feed for mollusks. The temperature was controlled at 25°C. The substrate was kept moist to facilitate the survival and activity of nematodes, but no water accumulation was formed. The observation was continued for 5 days, and the survival rate of the mollusks (e.g. Figure 3 ).

[0052] Step 6: Homotype snail infection experiment

[0053] (1) Prepare 6 feeding boxes, each with a bottom area of ​​100 cm 2 Pure cotton non-woven fabric as the base.

[0054] (2) Thirty-six snails of the same type, with an individual diameter of 8 mm ± 1 mm, were selected and divided into six groups, with six snails in each group. There were three experimental groups (infection groups) and three control groups.

[0055] (3) In the experimental group, 90,000 P. luanensis nematodes were added to each feeding box, which was equivalent to adding 900 nematodes per square centimeter of the bottom fabric; in the control group, 1 mL of Top10 Escherichia coli for feeding nematodes was added to each box, and distilled water was added to make the total amount of liquid consistent with that of the experimental group.

[0056] (4) Lettuce leaves were used as feed for mollusks. The temperature was controlled at 25°C. The substrate was kept moist to facilitate the survival and activity of nematodes, but no water accumulation was formed. The survival rate of the mollusks was calculated for 10 consecutive days (e.g. Figure 4 )

[0057] Finally, it should be noted that the above are only specific embodiments of the present invention. The present invention is not limited to these embodiments, and various modifications or adjustments can be made. All modifications and improvements directly derived or associated with the disclosure of the present invention by ordinary technicians in this field should be deemed to belong to the protection scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] The specific implementation is further described in detail below with reference to the accompanying drawings.

[0059] Figure 1 , Physical characteristics of male and female P. luanensis nematodes

[0060] Note: The figure shows two sexes of nematodes. The upper left is a male individual, whose tail is umbrella-shaped, typically associated with male reproductive structures; the lower is a female individual, whose tail is needle-shaped, consistent with the morphological characteristics of female individuals.

[0061] Figure 2 , Phylogenetic tree of P. luanensis and other Phasmarhabditis nematodes constructed by 28S rDNA

[0062] Note: The phylogenetic tree was constructed using the neighbor-joining method (NJ method) and 1000 Bootstrap tests were performed. Only support values ​​with a confidence level of ≥50% are displayed. The analysis results showed that P. luanensis nematodes belong to the genus Phasmarhabditis.

[0063] Figure 3 Multiple alignment of 28S rDNA sequences of P. luanensis and other Phasmarhabditis nematodes

[0064] Note: The results of multiple sequence alignment cover some Phasmarhabditis nematodes found at home and abroad. The analysis shows that P. luanensis and other Phasmarhabditis nematodes belong to different species.

[0065] Figure 4 , Experiment on P. luanensis nematodes infecting two-lined mucoid slugs

[0066] Note: There are 3 experimental groups and 3 control groups in the experiment. The area of ​​each feeding box is 100cm 2 The pure cotton non-woven fabric was used as the substrate, and 5 double-line mucus-loving slugs were placed, with an individual weight of 1.07g±0.22g (variation range 20%). 1200 P. luanensis nematodes were added per square centimeter in the experimental group; 1mL of Top10 Escherichia coli for feeding nematodes was added to the control group, and distilled water was added to make the total amount of liquid the same as that of the experimental group ( Figure 4 a, 4b). The experimental results showed that no slugs in the control group died, while all slugs in the experimental group died on the 5th day. Figure 4 c shows some of the individuals that died in the experimental group.

[0067] Figure 5 , Survival rate of two-lined mucophilous slugs infected with P. luanensis

[0068] Note: The figure shows the survival rate of two-lined mucophilous slugs exposed to 1200 P. luanensis nematodes per square centimeter, as well as the survival rate of two-lined mucophilous slugs in the control group (no nematode infestation). The experimental results showed that in the nematode treatment group, the survival rate of slugs gradually decreased over time and dropped to 0% on the 5th day; while the survival rate of slugs in the control group remained 100% within 5 days.

[0069] Figure 6 , survival rate of the same type of snails infected with P. luanensis

[0070] Note: The figure shows the survival rate of the same type of snails exposed to 900 P. luanensis nematodes per square centimeter, as well as the survival rate of the same type of snails in the control group (without nematode infection). The experimental results showed that in the nematode treatment group, the survival rate of snails gradually decreased over time and dropped to 0% on the 10th day; while the survival rate of the snails in the control group remained 100% within 10 days.

Claims

1. A nematode-based pest control agent, comprising a culture medium and a biological pesticide carrier.

2. The nematode-based pest control agent according to claim 1, wherein the nematode comprises Phasmarhabditis luanensis.

3. A nematode-based pest control agent according to claim 1 or 2, used in combination with a suitable biological insecticide carrier, such as NGM culture medium or egg yolk culture medium.

4. A nematode-utilizing pesticide according to any one of claims 1 to 3, for controlling Camaenidae molluscs, wherein the Camaenidae molluscs include Bradybaenasimilaris, Cathaica fasciola, Acusta ravida, and Acusta redfieldi.

5. A nematode-utilizing pesticide according to any one of claims 1 to 3, for controlling Philomycidae molluscs, wherein the Philomycidae molluscs include Meghimatium bilineatum.

6. A nematode-utilizing pesticide according to any one of claims 1 to 3, for controlling Limacidae, which includes Limacus flavus and Ambigolimaxvalentianus.

7. A nematode-utilizing pesticide according to any one of claims 1 to 3, for controlling Agriolimacidae, including Deroceras laeve.

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