Bacillus inoculant and technology for preventing and treating tomato root knot nematode disease by using bacillus inoculant

Abstract

The invention discloses a foliar spraying bacillus inoculant and a technology for preventing and treating tomato root knot nematode disease, and belongs to the technical field of application of microorganisms to prevention and treatment of plant diseases. Bacillus used in the technology is bacillus amyloliquefaciens and bacillus safensis, the two kinds of bacillus are cultured in an LB liquid culture medium in a shaking mode at the temperature of 28 DEG C and the speed of 200 rpm for 24 h, the two kinds of bacillus are equivalently mixed, then a surfactant fatty amine polyoxyethylene ether is added till the final concentration is 1%, the two kinds of bacillus are evenly mixed, and a microbial inoculum is prepared. According to the spraying technology, the fungicide is evenly sprayed to the surfaces of stems and leaves of tomato seedlings about 2 weeks through a spraying can, and 1 mL of fungicide is sprayed to each tomato plant. The microbial agent and the foliar spraying technology can effectively reduce invasion of root-knot nematodes and formation of root knots, relieve the destructive effect of the nematodes on root tissues and improve the fresh weight of roots, and compared with traditional methods such as root irrigation, seed dressing and seed coating, the microbial agent and the foliar spraying technology are easy and fast to apply, do not affect soil micro-ecology and have wide application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of application of microorganisms to prevent and control plant diseases, and is specifically about using Bacillus to prevent and control tomato root knot nematode disease. Background Art

[0002] Tomato root-knot nematode disease is an important disease in tomato production, caused by root-knot nematodes (mostly southern root-knot nematodes). Root-knot nematodes in the soil invade from the roots of tomatoes, feed and develop inside the roots, and swell to form root knots. Root-knot nematode disease affects the physiological functions of the root system and causes serious yield losses. The wounds caused by the invasion of nematodes also facilitate the invasion of other pathogens in the soil, further aggravating the disease. Because it is a soil-borne disease, the prevention and control of root-knot nematode disease relies on chemical pesticides for root irrigation, seed coating, seed mixing, etc. Long-term use of chemical pesticides may cause nematodes to develop drug resistance, and pesticide residues in the soil are also a problem that cannot be ignored. Therefore, the development of green prevention and control technologies for root-knot nematode disease will help sustainable development.

[0003] As a common biocontrol bacteria, Bacillus has been widely studied in the prevention and treatment of root-knot nematode disease. Some Bacillus have been developed into microbial agents and sold on the market, such as Bacillus subtilis and Bacillus laterosporus. However, both scientific and commercial Bacillus are currently applied directly to the soil (such as root irrigation) or seeds are treated in advance (such as coating) before entering the soil when preventing and treating root-knot nematode disease. This is not conducive to trying to reduce the amount of microbial agents to reduce the pressure on the soil, reduce interference with the ecological environment, and thus maintain sustainable development. In addition, microbial agents directly applied to the soil will be diluted by the soil, and often require a larger amount to ensure the prevention effect.

[0004] In order to reduce the amount of biocontrol bacteria used, alleviate the impact on the soil environment, and improve the sustainability of agricultural production, the present invention sprays the Bacillus liquid on the surface of the stems and leaves of tomato seedlings, inducing the plants to produce resistance to reduce the invasion of root-knot nematodes, and has broad application prospects. Summary of the invention

[0005] In view of the above problems, the first object of the present invention is to provide a Bacillus agent for preventing and controlling tomato root-knot nematode diseases. The second object of the present invention is to provide a biological control technology for tomato root-knot nematode diseases which is easy to apply, requires a small application amount, and has little impact on the soil microecology.

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

[0007] The bacillus agent provided by the invention is a highly effective biocontrol agent, which can prevent and control tomato root-knot nematode disease safely and conveniently.

[0008] The main active ingredients of the Bacillus inoculant are Bacillus amyloliquefaciens and Bacillus safensis, which are placed in LB liquid culture medium and cultured at 28°C and 200 rpm for 24 hours until the OD 600 =1.2, and then mix them in equal amounts. In order to improve the permeability of the bacterial solution, fatty amine polyoxyethylene ether is added to the mixture to a final concentration of 1%.

[0009] Furthermore, the culture medium for culturing Bacillus can be beef extract peptone medium, LB medium or other complete medium. The culture conditions are 28°C 200rpm, shaking culture for 24h. The Bacillus used for spraying is a bacterial suspension containing bacterial cells and metabolites, and the bacterial suspension is cultured to a concentration of OD 600 =1.2.

[0010] The bacillus described in the present invention is Bacillus amyloliquefaciens and Bacillus safensis separated from soil and stored in the laboratory of this study. These two types of bacillus are widely distributed in nature, and those skilled in the art can separate them from environmental soil by themselves, or purchase them from various strain collection centers.

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

[0012] The Bacillus inoculum (OD 600 =1.2) after shaking for 24 hours, and then sprayed evenly on the stem and leaf surfaces of tomatoes about 2 weeks old, with an average of about 1 mL of bacterial solution sprayed on each tomato plant. The sprayed tomato seedlings were cultured at 25°C, 16 h of light, and 8 h of darkness. After 5 days, the second-instar larvae of southern root-knot nematodes were inoculated around the roots. After 10 days, 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), and a small amount is caused by the northern root-knot nematode (M. hapla). Those skilled in the art can isolate the root-knot nematodes from the soil or the diseased host.

[0014] Furthermore, the tomato is a susceptible variety (susceptible to root-knot nematodes), such as Zhongza 201, Zhongza 9 (Zhongshu Seed Industry), etc. The soil for planting tomatoes is sterilized sandy loam, and the tomatoes are cultured in a greenhouse at 25°C. The tomatoes about 2 weeks old are used for spraying the bacterial solution. The bacterial solution is evenly sprayed on the surface of leaves and stems, and then the second-instar larvae of the root-knot nematode are inoculated in the root-surrounding soil, and 200-500 second-instar larvae are inoculated per tomato.

[0015] Furthermore, the control effect of tomato root-knot nematode disease was 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 the present invention are:

[0017] (1) The bacillus agent provided by the present invention is a biocontrol agent to which fatty amine polyoxyethylene ether is added. Fatty amine polyoxyethylene ether is a nonionic surfactant that exhibits wetting, penetration and adhesion properties and is commonly used as a leveling agent for reducing acid dyes, a lubricant, an emulsifier and a dispersant in the pesticide, textile, papermaking and leather industries. Adding fatty amine polyoxyethylene ether to a water-soluble bacillus bacterial solution helps promote the penetration, spreading and adsorption of the active ingredients, thereby reducing the dosage, significantly reducing the number of nematodes invading the roots, reducing the root knot index, increasing the fresh weight of the roots, and improving the control effect.

[0018] (2) The method for controlling tomato root knot disease of the present invention adopts spraying control technology. Compared with the traditional method of directly applying the biocontrol agent to the soil (such as root irrigation), the amount of biocontrol agent used is reduced, the properties of the soil are not affected, and the normal rhizosphere microbial ecology is not disturbed. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the root knot condition of tomatoes after spraying with Bacillus amyloliquefaciens and Bacillus safovici. DETAILED DESCRIPTION

[0020] In order to illustrate the content of the present invention in detail, the following examples will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be pointed out that the examples are preferred embodiments of the present invention, which are intended to illustrate the implementation conditions that can be used to implement the present invention, rather than to limit the experimental conditions. Those skilled in the art can still obtain similar beneficial effects by changing some of the conditions such as bacterial liquid concentration, tomato variety, etc.

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

[0022] The Bacillus amyloliquefaciens and Bacillus safensis strains stored in the laboratory of the present invention were streaked on LB medium plates for activation and placed in a 28°C incubator for 24 hours. A single colony was picked and inoculated into LB liquid medium and placed in a 28°C shaker for 24 hours with shaking at 200 rpm. At this time, the OD 600 About 1.2.

[0023] After germination, Zhongza 201 tomato seeds were sown in sterilized sandy loam and cultured in a 25°C 16-hour light / 8-hour dark environment. After 2 weeks, the bacterial solution prepared in the previous step was sprayed on the surface of the tomato stems and leaves with a small spray pot, with each tomato sprayed with about 1 mL of bacterial solution. Five days later, a suspension of freshly hatched second-instar larvae of southern root-knot nematodes was dripped into the root-surrounding soil, and each tomato was inoculated with about 250 nematodes.

[0024] Take tomato seedlings 10 days after inoculation with nematodes, wash the root tissue with running water, and stain the infected nematodes in the roots using the following method: soak the roots in 1% sodium hypochlorite solution for 5 minutes, wash with running water, soak in distilled water for 15 minutes, transfer to about 0.01% acid fuchsin (containing glacial acetic acid) solution and boil for 30 seconds, rinse with running water after cooling, transfer to acid glycerol (add a few drops of hydrochloric acid to glycerol), boil in boiling water for 30 seconds, and observe and count the number of nematodes in the roots under a microscope.

[0025] The obtained data were analyzed by variance analysis and multiple comparison using SPSS29 software, and different letters indicate significant differences.

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

[0027] Take tomato seedlings about 6-7 weeks after nematode inoculation, wash the root tissue, take photos and calculate the root knot index. The root knot grading standard is as follows:

[0028] Level 0: No root knot

[0029] Level 1: Few small knots

[0030] Level 2: Small root knots, taproot without root knots

[0031] Level 3: Some large knots, no knots on the taproot

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

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

[0034] Level 6: The taproot has a large number of knots

[0035] Level 7: Most taproots have knots

[0036] Level 8: All taproots have knots, and few roots are knotless

[0037] Level 9: All roots have large knots and the plant is dying

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

[0039] The formula for calculating the root knot index is: (∑root knot level×number of tomato plants at that level) / highest root knot level×total number of tomato plants.

[0040] The excess water on the root surface was sucked dry and the root fresh weight was weighed. The results are shown in Table 1 and Figure 1 As shown, Bacillus amyloliquefaciens and Bacillus safovich inhibited root nodule formation to a certain extent and significantly increased root weight, which helped the root system to function normally.

[0041] Table 1 The number of infected nematodes, root knot index and root fresh weight of tomatoes sprayed with Bacillus amyloliquefaciens and Bacillus saffron

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

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

[0044] The cultivation of Bacillus and the planting of tomato seedlings were the same as in Example 1. The cultured Bacillus amyloliquefaciens and Bacillus safensis were mixed in a ratio of 1:1, and then fatty amine polyoxyethylene ether (AC1810, Haian Petrochemical) was added to the Bacillus bacterial solution to a final concentration of 1% and mixed. The prepared mixed solution was evenly sprayed on the tomato leaves, and the spraying method, nematode inoculation method, staining method and data analysis method were the same as in Example 1. The results are shown in Table 2. The addition of fatty amine polyoxyethylene ether further reduced the nematode infection amount and root knot index, and increased the root fresh weight.

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

[0046] deal with Number of infected second instar larvae Root knot index Root fresh 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 saffron 52.2+13.4b 3.2 1.9+0.7b Bacillus safovici + AC1810 27.3+10.1c 2.6 2.8+0.6a Bacillus amyloliquefaciens + Bacillus saffron 46.6+9.2b 3.2 2.0+0.4b Bacillus amyloliquefaciens + Bacillus sabdariffa + 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 summary, spraying treatment with Bacillus amyloliquefaciens and Bacillus safensis can effectively inhibit the infection of southern root-knot nematodes to tomatoes, reduce root knot formation to a certain extent, and increase root fresh weight. Adding fatty amine polyoxyethylene ether can further promote this control effect. Since this method is easy to apply and does not affect soil ecology, it can be used as a potential biocontrol bacteria application technology.

[0048] The above embodiment is an implementation of the present invention and is a method optimized by the inventor. Those skilled in the art may improve and replace various technical parameters in the embodiment, and all of them are within the protection scope of the present invention without exceeding the scope of the core content of the present invention.

Claims

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

2. A technology for preventing and controlling tomato root knot nematode disease, characterized in that: The technology is to spray the Bacillus agent described in claim 1 on the stem and leaf surfaces of tomato seedlings of about 2 weeks old, with a spraying amount of about 1 mL per tomato plant.

3. The technology for preventing and controlling tomato root knot nematode disease according to claim 2, characterized in that: The root-knot nematode disease is caused by the southern root-knot nematode.

4. The technology for controlling tomato root knot nematode disease according to any one of claims 2 to 3, characterized in that: 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.

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