A biocontrol bacterium for controlling root-knot nematodes, Cossackia coli, and its application

Abstract

The present invention belongs to the technical field of agricultural production, and specifically relates to a biocontrol bacterium, Cossackia coli, for controlling root-knot nematodes and its application. The present invention uses fermentation liquid of the biocontrol bacterium, Cossackia coli, to treat second-instar larvae of root-knot nematodes, thereby achieving a certain contact killing activity and inhibiting egg hatching. Seeds are soaked and coated to promote seed germination without inhibiting plant growth. In an indoor potted plant experiment, root irrigation treatment of tomato seedlings can effectively control southern root-knot nematode disease. Cossackia coli has an anti-nematode effect and can be effectively used for biological control of plant parasitic nematodes.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural production, in particular to a biocontrol bacterium Cossackia coli for preventing and controlling root-knot nematodes and its application. Background Art

[0002] Globally, the occurrence and damage of plant parasitic nematodes have become increasingly serious with the development of agriculture and forestry and changes in cultivation and farming systems. According to statistics, by 1990, 207 genera and 4,832 species of plant parasitic nematodes had been reported worldwide. It is estimated that plant parasitic nematodes cause an annual loss of 12.8% of the world's major crops, exceeding US$110 billion. However, because the damage caused by many nematodes is more hidden than that of other organisms and there are no obvious visible field symptoms or specialized characteristics, the actual losses have not attracted attention, resulting in far more than estimated losses. In my country, there are mainly Soybean cyst nematode, root knot nematode, pine wood nematode, sweet potato stem nematode, etc.; among them, root knot nematode (Meloidogyne) disease is an important type of plant parasitic nematode disease. Once it occurs, it is difficult to prevent and eradicate. It has a wide range of hosts and can parasitize on more than 3,000 plant species. Widely cultivated facility vegetables can be its good hosts, thus causing huge economic losses to agricultural production; in my country, especially in protected facility vegetable cultivation, the disease rate in greenhouses generally reaches 20%-30%, and the losses caused by severe disease can reach 60%-70%, or even total loss of production.

[0003] Due to the limited availability of highly effective, low-toxic, and low-residue chemical nematicides and the rapid development of resistance, biological control methods for plant nematode diseases have become a research hotspot. Currently, biocontrol bacteria are abundant, environmentally friendly, and generally safe for humans and livestock. Using organisms or their metabolites to control root-knot nematode disease is highly consistent with the sustainable development strategy of modern agriculture. Biocontrol bacteria can prevent and control plant diseases without causing significant environmental damage, making them a safe and effective means of controlling root-knot nematode disease and a hot topic of research and development.

[0004] Based on the above theory, the present invention targets plant root-knot nematodes and screens Kosakonia cowanii with high anti-nematode activity from the soil, intending to provide new resources for the research and development of new and effective biocontrol agents against root-knot nematodes. Summary of the Invention

[0005] In order to make up for the deficiencies of the prior art and solve the technical problems existing in the background technology, the present invention proposes a biocontrol bacterium Cossackia coli for controlling root-knot nematodes and its application.

[0006] The present invention is achieved through the following technical scheme: a biocontrol bacterium Kosakonia cowanii for controlling root-knot nematodes, the Kosakonia cowanii Sneb2002; deposited at the General Microbiology Center of China Culture Collection Administration; address: Zhongguancun, Beijing, China; deposit date: 20230523; deposit registration number: CGMCC No. 27415; the Kosakonia cowanii is isolated and purified from soybean field soil by a plate dilution method, and the soil is sourced from Xinglong Primary School in Boli County, Qitaihe City, Heilongjiang Province (longitude 130°533856, latitude 45°943791).

[0007] Preferably, the fermentation culture method of Cossackia coli comprises the following steps:

[0008] S1: Activate the Cossackia coli stored in the cryopreservation tube in NA solid medium;

[0009] S2: Pick a single colony with good growth in S1 and ferment it in 150 ml of liquid culture medium. The liquid culture medium is placed on a shaker for shaking culture.

[0010] S3: directly or indirectly applying the fermentation liquid formed in S2 or the product of the fermentation liquid to the plants damaged by the root-knot nematodes.

[0011] Preferably, the NA solid medium in S1 comprises:

[0012] Beef extract peptone agar medium (NA), ingredients (g / L): beef extract 3-5g; peptone 10g; sodium chloride 5g; agar 15-20g; distilled water 1000mL; pH: 7.4-7.6.

[0013] Preferably, the liquid culture medium in S2 comprises:

[0014] LB broth, ingredients (g / L); tryptone 10.0 g; yeast extract powder 5.0 g; sodium chloride 10.0 g; pH: 6.9-7.1.

[0015] Preferably, the activation temperature in S1 is 28° C., and the activation culture is performed for 2 days.

[0016] Preferably, the fermentation temperature in S2 is 28° C., the fermentation time is 2 days, and the shaking speed is 200 r / min.

[0017] The use of the biocontrol bacterium Cossackia coli for controlling root-knot nematodes as described above is characterized in that the Cossackia coli directly or indirectly treats tomato seeds.

[0018] The use of the biocontrol bacterium Cossackia coli for controlling root-knot nematodes as described above is characterized in that the Cossackia coli is directly irrigated into the roots of seedlings.

[0019] The beneficial effects of the present invention are:

[0020] The present invention uses fermentation liquid of the biocontrol bacterium Cossackia coli to treat second-instar larvae of root-knot nematodes, thereby achieving certain contact killing activity and inhibiting egg hatching; soaking and coating seeds can promote seed germination without inhibiting plant growth; and in indoor potted plant experiments, root irrigation treatment of tomato seedlings can effectively prevent and control southern root-knot nematode disease. Cossackia coli has an anti-nematode effect and can be effectively used for biological control of plant parasitic nematodes. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a flow chart of the fermentation culture method of the present invention; DETAILED DESCRIPTION

[0022] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0023] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. The reagents or raw materials used in the present invention can be purchased through conventional channels. Unless otherwise specified, the reagents or raw materials used in the present invention are used in a conventional manner in the art or in accordance with the product instructions. In addition, any method and material similar to or equivalent to the described content can be applied to the method of the present invention. The present invention is further described with reference to the accompanying drawings and specific embodiments. The preferred embodiments and materials described in the present invention are for demonstration purposes only.

[0024] like Figure 1 As shown:

[0025] Example 1: Cultivation of Kosakonia cowanii Sneb2002 strain

[0026] Kosakonia cowanii (Sneb 2002); deposited at the General Microbiology Center of China Culture Collection Administration; Zhongguancun, Beijing, China; date of deposit: May 23, 2023; registration number: CGMCC No. 27415. This bacterium was isolated and purified from soybean field soil by the plate dilution method. The soil was sourced from Xinglong Primary School, Boli County, Qitaihe City, Heilongjiang Province (longitude 130°533856, latitude 45°943791).

[0027] The fermentation culture method of Cossackia coli comprises the following steps:

[0028] S1: Activate the Cossackia coli stored in cryopreserved tubes in NA solid medium; activate the cells from cryopreserved tubes at -80°C onto plates;

[0029] Beef extract peptone agar medium (NA), composition (g / L): beef extract 3-5g; peptone 10g; sodium chloride 5g; agar 15-20g; distilled water 1000mL; pH: 7.4-7.6;

[0030] The activation temperature in S1 was 28°C, and the activation culture was carried out for 2 days.

[0031] S2: Pick a single colony with good growth in S1 and ferment it in 150 ml of liquid culture medium. The liquid culture medium is placed on a shaker for shaking culture.

[0032] LB broth, composition (g / L): tryptone 10.0 g; yeast extract powder 5.0 g; sodium chloride 10.0 g; pH: 6.9-7.1;

[0033] The fermentation temperature in S2 was 28° C., the fermentation time was 2 days, and the shaking speed was 200 r / min.

[0034] S3: directly or indirectly applying the fermentation liquid formed in S2 or the product of the fermentation liquid to the plants damaged by the root-knot nematodes.

[0035] The formed fermentation liquid or the fermentation liquid product can be used to directly or indirectly treat tomato seeds and directly irrigate seedlings; the treated tomato seeds and irrigated seedlings can reduce the damage caused by root knot nematode diseases.

[0036] Example 2: Contact killing test of Kosakonia cowanii Sneb2002 strain against root-knot nematodes

[0037] The fermentation broth of Kosakonia cowanii prepared by the aforementioned liquid method was set aside. The fermentation broth was centrifuged at 8000 rpm for 5 minutes to obtain a fermentation filtrate for use in a toxicity test against second-instar larvae of the southern root-knot nematode.

[0038] Oocysts of the southern root-knot nematode (Meloidogyne incognita) were obtained by propagating tomato roots. J2 nematodes were hatched using the shallow tray method and prepared into a nematode suspension at 250 nematodes / mL.

[0039] Take 800 μL of the fermentation filtrate and add 200 μL of the nematode suspension (approximately 50 nematodes). Stand for 12 h and 24 h, and observe the number of dead nematodes to calculate the mortality rate. Mortality rate (%) = number of dead nematodes / total number of nematodes × 100.

[0040] The results of the experiment showed that the mortality rate of the fermentation filtrate of the Sneb2002 strain on the second-instar larvae of the southern root-knot nematode could reach 86.53% in 12 hours and 92.07% in 24 hours.

[0041] Example 3: Seed coating germination test of Kosakonia cowanii Sneb2002 strain

[0042] Prepare the fermentation broth of the Sneb2002 strain according to the above method and set aside.

[0043] Tomato variety L-402; the seeds were first surface-sterilized with 3% sodium hypochlorite solution, then rinsed repeatedly with sterile water, and the seeds were soaked in the strain fermentation liquid for 3 minutes. The seeds were dried and then cultured.

[0044] Instruments and appliances: steam sterilizer, electric constant temperature blower drying oven, electric constant temperature incubator, clean workbench, disposable culture dish, shaking incubator, seedling tray.

[0045] Sow the coated seeds in a sterile Petri dish (d = 9 cm) lined with filter paper. Each treatment consisted of 20 seeds, replicated three times. Sterile water (CK) served as a control. Culture was performed in a 25°C constant temperature and humidity incubator. Germinated seeds were counted daily after 24 hours. After 7 days of incubation, the germination rate was calculated and root length was measured. Germination rate (%) = number of seeds germinated on the specified day / total number of seeds tested × 100.

[0046] Table 1: Tomato seeds coated with Kosakonia cowanii

[0047]

[0048] Note: The data in the table are mean ± standard deviation; different letters in the same column indicate significant differences at the P < 0.05 level tested by Duncan's method.

[0049] The results of the experiment showed that the germination rate and root length of tomato seeds treated with Sneb2002 fermentation products were significantly higher than those of the control.

[0050] Example 4: Potted experiment of Kosakonia cowanii

[0051] Prepare the Kosakonia cowanii fermentation broth as described above and set aside. Prepare the nematode suspension as described above and set aside. Tomato (L-402) seeds should be surface-sterilized with a 3% sodium hypochlorite solution, then rinsed repeatedly with sterile water. After drying, the seeds should be cultured until they reach the four-leaf stage.

[0052] The experiment involved the following treatments: a control (CK) using sterile water for root irrigation, a root irrigation using sterile water and inoculation with J2 (CK+J2), and a root irrigation treatment using a fermented liquid from the strain and inoculation with J2. The fermented liquid concentration was approximately 1 × 108 cfu / mL, with 10 mL of liquid applied to each seedling. A suspension of second-instar larvae (approximately 2,000 J2) was also added to each plant. A second 10 mL irrigation was performed at mid-growth. Five seedlings were randomly arranged in each treatment. After 45 days of incubation, plant height, root length, root fresh weight, root dry weight, plant fresh weight, and plant dry weight were measured. The number of root knots and oocysts was also measured, and the root knot reduction rate and oocyst reduction rate were calculated. The root knot reduction rate was used to represent relative control efficacy. Control effect (%) = (root knot index of control group - root knot index of treatment group) / root knot index of control group × 100; Oocyst reduction rate (%) = (number of oocysts in control group - number of oocysts in treatment group) / number of oocysts in control group × 100;

[0053] Table 2: Effects of Kosakonia cowanii fermentation broth treatment on the number of root nodules and oocysts in pot experiments

[0054]

[0055] Table 3 Effects of Kosakonia cowanii fermentation broth treatment on the growth and development of tomato plants in pot experiments

[0056]

[0057] Note: The data in the table are mean ± standard deviation. Different letters in the same column indicate significant differences at the P < 0.05 level as tested by Duncan's method.

[0058] The above are only preferred embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structures or equivalent process changes made using the contents of the present invention's description and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A biocontrol bacterium for controlling root-knot nematodes, Cossackia coli, characterized in that: The Cossackia coli ( Kosakonia cowanii )Sneb2002; depositor: General Microbiology Center of China Culture Collection Administration of Microorganisms; deposit date: 20230523; deposit registration number: CGMCC No.27415.

2. The biocontrol bacterium for controlling root-knot nematodes according to claim 1, characterized in that: The fermentation culture method of Cossackia coli comprises the following steps: S1: Activate the Cossackia coli stored in the cryopreservation tube in NA solid medium; S2: Pick a single colony with good growth in S1 and ferment it in 150 ml of liquid culture medium. The liquid culture medium is placed on a shaker for shaking culture. S3: directly or indirectly applying the fermentation liquid formed in S2 or the product of the fermentation liquid to the plants damaged by the root-knot nematodes.

3. The biocontrol bacterium for controlling root-knot nematodes according to claim 2, characterized in that: The NA solid medium described in S1 includes: The components of the beef extract peptone agar medium are 3-5 g / L beef extract; 10 g / L peptone; 5 g / L sodium chloride; 15-20 g / L agar; and 1000 mL distilled water; pH: 7.4-7.

6.

4. The biocontrol bacterium for controlling root-knot nematodes according to claim 2, characterized in that: The liquid culture medium in S2 comprises: The composition of LB broth is tryptone 10.0 g / L; yeast extract 5.0 g / L; and sodium chloride 10.0 g / L; pH: 6.9-7.

1.

5. The biocontrol bacterium for controlling root-knot nematodes according to claim 2, characterized in that: The activation temperature in S1 was 28°C, and the activation culture was carried out for 2 days.

6. The biocontrol bacterium for controlling root-knot nematodes according to claim 2, characterized in that: The fermentation temperature in S2 was 28° C., the fermentation time was 2 days, and the shaking speed was 200 r / min.

7. Use of the biocontrol bacterium Cossackia coli for controlling root-knot nematodes according to any one of claims 1 to 6, characterized in that: The Cossackia coli directly or indirectly treats tomato seeds.

8. Use of the biocontrol bacterium Cossackia coli for controlling root-knot nematodes according to any one of claims 1 to 6, characterized in that: The Cossackia coli bacteria are directly injected into the roots of tomato seedlings.

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