A bacillus bacterial agent and a method for preventing and treating tomato root knot nematode disease

Abstract

The application discloses a kind of foliage spray bacillus agent and method for preventing and treating tomato root-knot nematode disease, belong to the application technical field of plant disease prevention and cure of microorganism.The method uses bacillus for solubilizing starch bacillus and shafu bacillus, two kinds of bacillus are in LB liquid culture medium 28 o C 200 rpm shaking culture 24 h, after mixing in equal amounts, surfactant fatty amine polyoxyethylene ether is added to final concentration 1%, mix uniformly to make agent.Spraying method is that agent is evenly sprayed in the stem and leaf surface of tomato seedling growth 2 with spray pot, and each tomato is sprayed 1 mL.The agent and foliage spraying method proposed in the application can effectively reduce the invasion and root knot formation of root-knot nematode, alleviate the damage of nematode to root tissue, increase root fresh weight, compared with traditional irrigation root, mix seed, seed coating and other methods, simple, fast, do not affect soil microecology, and has wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial application technology for the control of plant diseases. Specifically, it relates to Bacillus subtilis for the control of root-knot nematode disease in tomatoes. Background Technology

[0002] Root-knot nematode disease is a significant pest in tomato production, caused by root-knot nematodes (most commonly the southern root-knot nematode). Soil-borne nematodes invade the tomato roots, feeding and developing within them, forming root knots. Root-knot nematode disease impairs root physiological functions, causing severe yield losses. The wounds created by nematodes also facilitate the invasion of other pathogens from the soil, further exacerbating the disease. As it is a soil-borne disease, control methods for root-knot nematode disease rely on chemical pesticide drenching, seed coating, and seed treatment. However, long-term use of chemical pesticides may lead to nematode resistance, and pesticide residues in the soil are also a significant concern. Therefore, developing green control technologies for root-knot nematode disease is crucial for sustainable development.

[0003] Bacillus, as a common biocontrol bacterium, has been extensively studied in the control of root-knot nematode disease, and some Bacillus species, such as Bacillus subtilis and Bacillus laterosporus, have been developed into inoculants and sold on the market. However, currently, both research and commercial Bacillus species are applied directly to the soil (e.g., through root irrigation) or pre-treated seeds (e.g., seed coating) before being introduced into the soil to control root-knot nematodes. This approach is detrimental to efforts to reduce the amount of inoculant used, thereby lessening soil stress and environmental impact, and ultimately maintaining sustainable development. Furthermore, inoculants applied directly to the soil are diluted by the soil, often requiring larger doses to ensure efficacy.

[0004] To reduce the amount of biocontrol bacteria used, mitigate the impact on the soil environment, and improve the sustainability of agricultural production, this invention sprays Bacillus bacterial solution onto the surface of tomato seedling stems and leaves, inducing plant resistance to reduce the invasion of root-knot nematodes, and has broad application prospects. Summary of the Invention

[0005] To address the aforementioned problems, the first objective of this invention is to provide a Bacillus agent for the prevention and control of tomato root-knot nematode disease. The second objective of this invention is to provide a biological control method for tomato root-knot nematode disease that is convenient to apply, requires a small amount of application, and has minimal impact on the soil microecology.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] The Bacillus agent provided by this invention is a highly efficient biocontrol agent that can safely and conveniently control root-knot nematode disease in tomatoes.

[0008] The main active ingredients of the Bacillus inoculant are Bacillus amyloliquefaciens and Bacillus safensis, which are placed separately in LB liquid medium at 28°C. o Incubate at 200 rpm for 24 h with shaking until OD is reached. 600 =1.2, then mix the equal amounts evenly. To improve the permeability of the bacterial solution, add fatty amine polyoxyethylene ether to the mixture until the final concentration is 1%.

[0009] Furthermore, the culture medium used for culturing Bacillus can be beef extract peptone medium, LB medium, or other complete media. The culture conditions are 28°C. o C. Incubate at 200 rpm with shaking for 24 h. The Bacillus used for spraying is a bacterial suspension containing bacterial cells and metabolites, cultured until the bacterial concentration reaches OD0.05. 600 =1.2.

[0010] The Bacillus species described in this invention are *Bacillus amyloliquefaciens* and *B. safensis*, isolated from soil and preserved in our research laboratory. These two Bacillus species are widely distributed in nature and can be isolated from environmental soil by those skilled in the art, or purchased from various bacterial culture collection centers.

[0011] This invention provides a biological control method for tomato root-knot nematode disease.

[0012] The Bacillus agent (OD) 600 =1.2) Shake and incubate for 24 hours, then spray directly and evenly onto the surface of the stems and leaves of tomatoes that have been growing for 2 weeks, with an average of 1 mL of bacterial solution sprayed per tomato plant. After spraying, place the tomato seedlings at 25°C. o C. Cultured under 16 h light and 8 h darkness conditions, 5 days later, second-instar larvae of Southern Root-knot Nematodes were inoculated around the roots. 10 days later, the number of nematodes infecting the roots was counted by acid fuchsin staining.

[0013] Furthermore, the root-knot nematode disease is mainly caused by the southern root-knot nematode (Meloidogyne incognita), with a small number caused by the northern root-knot nematode (M. hapla). Those skilled in the art can isolate root-knot nematodes from soil or diseased hosts.

[0014] Furthermore, the tomatoes mentioned are susceptible varieties (susceptible to root-knot nematodes), such as Zhongza 201 and Zhongza 9 (Zhongshu Seed Industry), etc., and the soil for planting the tomatoes is sterilized sandy loam, and the tomatoes are cultivated at 25°C. oIn greenhouse C, tomatoes that have been growing for two weeks are sprayed with bacterial solution. The bacterial solution is sprayed evenly on the surface of the leaves and stems, and then second-instar larvae of root-knot nematodes are inoculated into the soil around the roots, with 200-500 second-instar larvae inoculated into each tomato plant.

[0015] Furthermore, the control effect of tomato root-knot nematode disease is defined by the number of nematodes invading the roots, the root knot index, or the fresh weight of the roots.

[0016] The beneficial effects of this invention are as follows:

[0017] (1) The Bacillus inoculant provided by this invention is a biocontrol agent with added fatty amine polyoxyethylene ether. Fatty amine polyoxyethylene ether is a nonionic surfactant that exhibits wetting, penetrating and adhesive properties. It is commonly used as a leveling agent for vat acid dyes and as a lubricant, emulsifier and dispersant in the pesticide, textile, paper and leather industries. Adding fatty amine polyoxyethylene ether to water-soluble Bacillus inoculant solution helps to promote the penetration, spreading and adsorption of active ingredients, thereby reducing the amount of pesticide used, significantly reducing the number of nematodes invading the roots, reducing the root knot index, increasing the fresh weight of roots, and improving the control efficacy.

[0018] (2) The method for preventing root-knot disease of tomatoes in this invention adopts the spraying method. Compared with the traditional method of applying biocontrol agents directly to the soil (such as root irrigation), it reduces the amount of biocontrol agents used, has no impact on soil properties, and does not interfere with the normal rhizosphere microbial ecology. Attached Figure Description

[0019] Figure 1 This study investigated the root knot formation in tomato roots after treatment with Bacillus amyloliquefaciens and Bacillus saffron bacterial solutions. Detailed Implementation

[0020] To illustrate the invention in detail, the following embodiments will help those skilled in the art to further understand the invention, but do not limit the invention in any way. It should be noted that the embodiments are preferred embodiments of the invention and are intended to illustrate the conditions that can be used to implement the invention, rather than limiting the experimental conditions. Those skilled in the art can obtain similar beneficial effects by changing some of the conditions, such as the bacterial concentration or the tomato variety.

[0021] Example 1: Effects of Bacillus spraying on tomato root-knot nematode disease

[0022] Bacillus amyloliquefaciens and Bacillus safensis strains preserved in the laboratory of this invention were streaked onto LB agar plates for activation and placed at 28°C. o Incubate in a C24 incubator for 24 hours. Pick a single colony and inoculate it into LB liquid medium, then incubate at 28°C. oIncubate in a shaker at 200 rpm for 24 h, at which point the OD... 600 It is 1.2.

[0023] After germination, Zhongza 201 tomato seeds were sown in sterilized sandy loam soil and placed at 25°C. o Cultivate in a 16-hour light / 8-hour dark environment for 2 weeks. After 2 weeks, spray the prepared bacterial solution onto the surface of the tomato stems and leaves using a small spray bottle, with 1 mL of bacterial solution sprayed onto each tomato plant. 5 days later, add a suspension of freshly hatched second-instar larvae of the southern root-knot nematode to the soil around the roots, with 250 nematodes inoculated onto each tomato plant.

[0024] Take tomato seedlings inoculated with nematodes 10 days ago, wash the root tissue with running water, and stain the nematodes in the roots as follows: soak the roots in 1% sodium hypochlorite solution for 5 minutes, wash with running water, soak in distilled water for 15 minutes, transfer to 0.01% acidic fuchsin (containing glacial acetic acid) solution and boil for 30 seconds, cool and rinse with running water, transfer to acidic glycerol (with a few drops of hydrochloric acid added), boil in water for 30 seconds, and observe and count the number of nematodes in the roots under a microscope.

[0025] The obtained data were analyzed using SPSS 29 software for ANOVA and multiple comparisons, with different letters indicating significant differences.

[0026] The results are shown in Table 1. Compared with the negative control LB medium, Bacillus amyloliquefaciens and B. safensis significantly reduced the number of second-instar larvae infecting the roots.

[0027] Tomato seedlings inoculated with nematodes were collected 6-7 weeks prior. The root tissue was washed, photographed, and the root knot index was calculated. The root knot grading criteria are as follows:

[0028] Level 0: No root nodes

[0029] Level 1: Very few small root knots

[0030] Grade 2: Small root knots, no root knots on the main root.

[0031] Level 3: Some large root knots, no root knots on the main root.

[0032] Level 4: Large root knots are the majority, and the main root has no root knots.

[0033] Level 5: 50% of roots have root knots, and some taproots have root knots.

[0034] Level 6: Numerous root knots distributed on the taproot

[0035] Level 7: Most taproots have root knots.

[0036] Level 8: All taproots have root knots; very few roots lack root knots.

[0037] Level 9: All roots have large root knots, and the plant gradually dies.

[0038] Level 10: All roots have large root knots, root tissue collapses, and the plant usually dies.

[0039] The formula for calculating the root knot index is: / Root knot highest level × Total number of tomato plants.

[0040] After drying the root surface to remove excess moisture, the fresh weight of the roots was measured. The results are shown in Table 1 and 2. Figure 1 As shown, Bacillus amyloliquefaciens and Bacillus safranin inhibited root knot formation to some extent and significantly increased root weight, which helped the root system to perform normal functions.

[0041] Table 1. Number of nematodes, root knot index, and root fresh weight in tomatoes treated with Bacillus amyloliquefaciens and Bacillus saffron.

[0042] deal with Number of second instar larvae infected Root knot index Fresh root weight (g) Bacillus amyloliquefaciens 47.1+21.9b 3.3 2.1+0.7a Bacillus sarfusae 53.3+24.1b 3.2 1.9+0.7a LB 101.6+31.1a 4.3 0.7+0.5b

[0043] Example 2: The effect of fatty amine polyoxyethylene ether on promoting the spraying of Bacillus to control root-knot nematode disease

[0044] The cultivation of Bacillus and the planting of tomato seedlings were the same as in Example 1. Cultured Bacillus amyloliquefaciens and Bacillus safensis were mixed 1:1. Fatty amine polyoxyethylene ether (Hai'an Petrochemical, AC1810) was added to the Bacillus culture to a final concentration of 1% and mixed thoroughly. The prepared mixture was evenly sprayed onto the tomato leaves using the same spraying, nematode inoculation, staining, and data analysis methods as in Example 1. The results are shown in Table 2. The addition of fatty amine polyoxyethylene ether further reduced nematode infestation and root knot index, and increased root fresh weight.

[0045] Table 2. The promoting effect of adding fatty amine polyoxyethylene ether on the control of Bacillus.

[0046] deal with Number of second instar larvae infected Root knot index Fresh root weight (g) Bacillus amyloliquefaciens 49.3+11.6b 3.3 2.1+0.7b Bacillus amyloliquefaciens + AC1810 29.1+10.9c 2.9 2.9+0.5a Bacillus sarfusae 52.2+13.4b 3.2 1.9+0.7b Bacillus sarcodactylis + AC1810 27.3+10.1c 2.6 2.8+0.6a Bacillus amyloliquefaciens + Bacillus safranin 46.6+9.2b 3.2 2.0+0.4b Bacillus amyloliquefaciens + Bacillus safranin + AC1810 26.4+9.5c 2.7 3.1+0.7a LB 106.9+25.2a 4.3 0.7+0.5c LB+AC1810 105.5+25.6a 4.4 0.8+0.5c

[0047] In conclusion, spraying with *Bacillus amyloliquefaciens* and *B. safensis* effectively inhibited the infection of tomatoes by southern root-knot nematodes, reduced root knot formation to some extent, and increased root fresh weight. The addition of fatty amine polyoxyethylene ether further enhanced this control effect. Because this method is convenient to apply and does not affect the soil ecology, it can be considered a promising method for applying biocontrol agents.

[0048] The above embodiments are one implementation of the present invention and belong to the inventor's optimized method. Those skilled in the art can improve or substitute various technical parameters in the embodiments, and all such improvements or substitutions are within the protection scope of the present invention without exceeding the core content scope of the present invention.

Claims

1. A Bacillus bacterial inoculant, characterized in that: The active ingredient of the bacillus bacterial agent is an equal mixture of liquid shake culture products of Bacillus amyloliquefaciens and B. safensis with OD 600 = 1.2, and addition of fatty amine polyoxyethylene ether to a final concentration of 1%.

2. A method of controlling root-knot nematode disease of tomato, characterized by: The method is to spray the bacillus agent of claim 1 on the stem and leaf surface of the tomato seedling grown for 2 weeks, and the spraying amount is 1 mL per tomato.

3. The method of controlling tomato nematode according to claim 2, wherein: The root-knot nematode disease is caused by southern root-knot nematode.

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