Paenibacillus polymyxa strain FB 9-3 and application thereof

Abstract

The invention discloses a paenibacillus polymyxa strain FB 9-3 and application thereof. The preservation number of the paenibacillus polymyxa strain FB 9-3 is CCTCC (China Center for Type Culture Collection) NO: M 20241788. The paenibacillus polymyxa strain FB 9-3 belongs to bacillus, is used as a biocontrol bacterium for preventing and controlling root-knot nematodes and a growth-promoting bacterium for promoting plant growth, can form spores with extremely strong stress resistance, is suitable for long-time transportation and preservation, and has a great application prospect in the aspect of preparing root-knot nematode biocontrol or microbial fertilizer products.

Description

Technical Field

[0001] The invention relates to the technical field of microbial pesticides, and in particular to a Paenibacillus polymyxa strain FB 9-3 and an application thereof. Background Art

[0002] There are more than 5,000 species of plant parasitic nematodes, which can parasitize more than 3,000 plants, including various food crops, cash crops and ornamental trees. It is reported that they can cause 12.6% losses to the global agricultural economy each year, equivalent to about US$216 billion per year. Meloidogyne spp. is one of the most damaging plant parasitic nematodes and has the greatest impact on economic benefits. Root-knot nematode is an obligate plant parasitic nematode that is widely distributed throughout the world. Root-knot nematode can parasitize almost all vascular plants. As of 2019, 109 species have been reported worldwide, and about 58 species have been found in my country. Among the root-knot nematodes, southern root-knot nematode is the most widely distributed in my country, which can cause serious damage to plants in the Solanaceae, Cruciferae and Cucurbitaceae families. In recent years, with the promotion and expansion of cultivation facilities such as solar greenhouses in my country and changes in overall environmental conditions, the damage area of ​​southern root-knot nematodes has gradually spread to the north of my country, and there is a trend of widespread infestation across the country.

[0003] The second-instar larvae (J2) of the root-knot nematode invade the roots of plants by physical damage caused by piercing with their needles and by secreting cellulolytic enzymes and pectin-degrading enzymes to destroy the cell walls. The roots of the host plants infected by the root-knot nematodes will gradually swell to form nodules of varying sizes and irregular shapes. These nodules are composed of multiple giant cells and are called root knots. Typical symptoms caused by root-knot nematodes include root deformities, poor plant growth and development, wilting, leaf discoloration, and small and immature fruits. The wounds caused by the invasion of the root-knot nematodes on the host roots will also promote the invasion of other pathogens, especially fungal diseases, which will cause greater secondary damage to the host plants. The most common is the combined infection of root-knot nematodes and the pathogenic fungus Fusarium, which causes crops such as tomatoes and cucumbers to suffer from stem base rot and neck root rot or wilt, resulting in large-scale yield reduction. Combined infection can deepen the degree of disease damage and sometimes lead to a significant advance in the onset of the disease.

[0004] Root-knot nematodes reproduce quickly, have strong colonization ability, and have a wide host range. It is very difficult to completely eradicate root-knot nematode diseases. There are agricultural control, chemical control, physical control, and biological control methods for root-knot nematodes. Among them, the application of chemical nematicides is widely used to control root-knot nematodes because of its simplicity and high efficiency. The main agricultural, physical, and chemical control methods currently used have problems such as environmental unfriendliness, high toxicity, and high economic costs, which are not conducive to the sustainable development of agriculture. In contrast, biological control has the advantages of being green and environmentally friendly, highly safe, highly specific, and extremely rich potential resources available, which makes people increasingly optimistic about the prospects of biological control. However, due to the scarcity of biocontrol strain resources, there are relatively few types of biocontrol preparations available at this stage, and there is an urgent need to continuously explore and discover new root-knot nematode biocontrol strains.

[0005] At present, many strains have been identified as biocontrol strains for root-knot nematodes. However, many strains have poor adaptability to the environment and weak survival ability, making it difficult to make commercial preparations that can be stored for a long time. Paenibacillus polymyxa belongs to the genus Bacillus, which is currently widely used in the control of root-knot nematodes. Because it can form spores with strong stress resistance, it is suitable for long-term transportation and storage, and has great application prospects in the preparation of root-knot nematode biocontrol products. Summary of the invention

[0006] The purpose of the present invention is to effectively prevent and control root-knot nematodes and reduce the harm caused by them. The present invention provides a polymyxa bacillus strain FB 9-3 and an application thereof. The present invention screens and obtains a polymyxa bacillus FB 9-3 from soil. The polymyxa bacillus belongs to the genus Bacillus. The fermentation liquid of the strain can significantly reduce the number of root knots at the roots of plants, increase the dry weight of the aboveground part, promote the growth and development of plants, and has good application prospects.

[0007] To achieve the above purpose, the technical solution designed by the present invention is as follows:

[0008] The invention provides a Paenibacillus polymyxa strain FB 9-3, which is named as Paenibacillus polymyxa FB 9-3 and has a preservation number of CCTCC NO: M20241788.

[0009] The present invention also provides an application of the above-mentioned Paenibacillus polymyxa strain FB 9-3 in preventing and treating root-knot nematode disease.

[0010] The present invention also provides a use of the above-mentioned Paenibacillus polymyxa strain FB 9-3 in promoting plant growth and development.

[0011] The present invention also provides a use of the above-mentioned Paenibacillus polymyxa strain FB 9-3 in preparing a fermentation liquid for preventing and treating root-knot nematode disease.

[0012] The present invention also provides a method for preparing a fermentation broth of the Paenibacillus polymyxa strain FB 9-3, comprising the following steps:

[0013] 1) activating the Paenibacillus polymyxa strain FB 9-3;

[0014] 2) The activated Paenibacillus polymyxa strain FB 9-3 is inoculated into LB liquid culture medium and cultured to obtain a fermentation liquid of the Paenibacillus polymyxa strain FB 9-3.

[0015] The present invention also provides an application of the above-mentioned Paenibacillus polymyxa strain FB 9-3 or the fermentation liquid of the Paenibacillus polymyxa strain FB 9-3 prepared by the above-mentioned method in preparing a biological agent for preventing and controlling root-knot nematode disease and promoting plant growth and development.

[0016] The present invention also provides a biological nematicide for preventing and treating root-knot nematode disease, wherein the biological nematicide contains the above-mentioned Paenibacillus polymyxa strain FB 9-3 or contains the fermentation liquid of Paenibacillus polymyxa strain FB 9-3 prepared by the above-mentioned method; wherein the number of live bacteria of Paenibacillus polymyxa strain FB 9-3 in the insecticide is 1 to 2 billion / ml.

[0017] Furthermore, in the insecticide, the number of viable bacteria of the Bacillus polymyxa strain FB 9-3 is 1 billion / ml.

[0018] Beneficial effects of the present invention:

[0019] 1. The raw materials of the nematicide of the present invention are derived from soil, and have no adverse effects on the environment and all organisms such as plants, humans, and animals. In addition, the production cost is low, the production process is simple, and the environment is not polluted.

[0020] 2. The present invention is of great significance for the biological control of root-knot nematodes, the development and application of microbial nematicides and microbial fertilizers, and the pollution-free production of agricultural products, and is suitable for popularization and application.

[0021] 3. The biological agent of the present invention is a pure biological agent and can be used for biological control of root-knot nematodes. The number of root knots caused by root-knot nematodes in potted tomatoes treated with fermentation liquid was reduced by 58.8% compared with the treatment group.

[0022] 4. The present invention has a significant promoting effect on the growth and development of crops, and can increase the accumulation of fresh biomass of the above-ground part of crops by 35.7% and the accumulation of dry matter by 31.1%.

[0023] In summary: Paenibacillus polymyxa strain FB 9-3 belongs to the genus Bacillus. It is a biocontrol bacterium for root-knot nematode control and a growth-promoting bacterium for promoting plant growth. Because it can form spores with extremely strong stress resistance, it is suitable for long-term transportation and storage. It has great application prospects in the preparation of root-knot nematode biocontrol or microbial fertilizer products. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a schematic diagram of the Paenibacillus polymyxa colony;

[0025] Figure 2 This is a schematic diagram of the growth of the aboveground part of tomato plants under different treatments after 25 days of normal water and fertilizer management;

[0026] Figure 3 This is a schematic diagram showing the comparison of the aboveground dry weight of tomato plants under different treatments after 25 days of normal water and fertilizer management;

[0027] Figure 4 This is a schematic diagram of the root growth of tomato plants after 25 days of normal water and fertilizer management under different treatments;

[0028] Figure 5 This is a schematic diagram of the number of root knots caused by root-knot nematode infection in different treatments after 25 days of normal water and fertilizer management;

[0029] Figure 6 This is a comparison picture of lettuce plants after 20 days of treatment;

[0030] Figure 7 This is a comparison chart of the fresh weight and dry weight of the aerial part of lettuce after 20 days of treatment. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand.

[0032] Example 1

[0033] Isolation and identification of Paenibacillus polymyxa strain FB 9-3

[0034] 1. Sample Collection

[0035] The soil samples were from cucumber rhizosphere soil in Xiaogan City, Hubei Province

[0036] 2. Isolation of Paenibacillus polymyxa strains

[0037] (1) Weigh 1 g of powdered soil sample and add it to a 250 mL Erlenmeyer flask containing 20 mL of LB+NaAC liquid culture medium (the NaAc content in 1 L of culture medium is 34.02 g); while weighing the soil sample, measure the pH value of the soil sample itself; wherein, the formula of LB culture medium (1 L) is: peptone 10 g; yeast powder 5 g; NaCl 10 g;

[0038] (2) Then, incubate the flask in a shaking incubator at 30°C / 220 rpm for 4 h. After incubation, place the flask in an 80°C water bath for about 180 s.

[0039] (3) After the water bath treatment, wait for the liquid in the flask to cool to room temperature, then shake the culture medium and then take 100 μL of it to apply to the LA plate. Apply 3 plates for each soil sample, and then place the plate in a 30°C incubator and invert it for 18-24 hours until the colony morphology can be determined; the LA medium formula is 1L LB medium with 15g of agar powder added.

[0040] 3. Morphological identification of Paenibacillus polymyxa strains

[0041] Observe the LA plate and pick out the suspected Bacillus polymyxa colonies. To maximize the number of Bacillus polymyxa strains obtained, pick out as many suspected strains as possible.

[0042] General characteristics of Bacillus polymyxa colonies: The colonies are mostly light yellow or white and sticky, with a moist and smooth surface; they can move using peritrichous flagella and produce oval spores in the expanded sporangium ( Figure 1 ).

[0043] 4. Molecular Biology Analysis

[0044] (1) The results of 16S rRNA gene sequence determination are as follows:

[0045]

[0046] After identification, the Paenibacillus polymyxa strain was Paenibacillus polymyxa FB 9-3. On August 14, 2024, the Paenibacillus polymyxa strain FB 9-3 was deposited in the China Center for Type Culture Collection, with the preservation address being Wuhan University, Wuhan City, Hubei Province, and its preservation number is: CCTCC NO: M 20241788.

[0047] Example 2

[0048] A method for preparing a fermentation broth of Paenibacillus polymyxa strain FB 9-3 comprises the following steps:

[0049] 1) activating the Paenibacillus polymyxa strain FB 9-3;

[0050] 2) The activated Paenibacillus polymyxa strain FB 9-3 was inoculated into LB liquid culture medium and cultured to obtain 1 billion / ml of Paenibacillus polymyxa strain FB 9-3 fermentation liquid.

[0051] Example 3

[0052] A biological nematicide for preventing and controlling root-knot nematode disease is prepared by diluting the fermentation liquid of Paenibacillus polymyxa strain FB 9-3 prepared by the method of Example 2 by 100 times; wherein the number of live bacteria of Paenibacillus polymyxa strain FB 9-3 in the insecticide is 1 billion / ml.

[0053] Example 4: Effect of biological nematicides on the control of root-knot nematodes in potted plants

[0054] After germinating the tomato seeds for 2 days, the tomato seedlings were sown and raised for 14 days and then planted in 10 cm flower pots. The substrate was peat soil and coarse river sand in a ratio of 1:2. The seedlings were allowed to grow slowly for 2 to 3 days. About 1,500 second-instar root-knot nematodes were added to each pot. After 1 day, different treatments were performed on the tomato seedlings in different pots:

[0055] a. Experimental group: add 50mL of biological nematicide,

[0056] b. Control group: add 50mL deionized water,

[0057] c. Blank group: tomato seedlings without the addition of second-instar root-knot nematodes.

[0058] After 25 days of treatment, the weights were measured and weighed.

[0059] like Figures 2 to 5As shown: The growth status of tomatoes in the experimental group and the blank control group is basically the same, and the tomato plants in the control group are slightly thinner and weaker. By weighing the aboveground parts of the dried tomatoes, there is no significant difference in the accumulation of dry matter among the three groups, but the dry weight of the experimental group is higher than that of the control group. Statistical analysis of the number of root knots in the roots of the three groups of treated tomatoes shows that the number of root knots in the experimental group is reduced by 58.8% compared with the control group.

[0060] Combining the growth conditions of the aboveground and underground parts, Bacillus polymyxa 9-3 can significantly alleviate the growth inhibition of potted tomatoes caused by root-knot nematode infection.

[0061] Example 5 Effect of biological nematicides on promoting plant growth in potted plants

[0062] Sow the seeds directly for 2-3 days, select lettuces with the same growth status as the experimental objects, and pour the fermentation liquid evenly on the root zone of the lettuce according to the treatment:

[0063] a. Experimental group: add 1mL of biological nematicide,

[0064] b. Control group: add 50mL deionized water,

[0065] After 20 days of unified water and fertilizer management, the fresh weight and dry weight of the aboveground part of the lettuce were measured respectively.

[0066] like Figures 6-7 As shown: 20 days after treatment, the lettuce plants in the experimental group were significantly larger than those in the control group. The fresh weight of the aboveground part of the lettuce in the experimental group was 35.7% higher than that in the control group. The dry weight of the aboveground part of the lettuce after drying was 31.1% higher than that in the control group. The growth rate and the accumulation of nutrients in the potted lettuce treated with Paenibacillus polymyxa FB 9-3 were significantly higher than those in the control group. Paenibacillus polymyxa FB 9-3 had a significant growth-promoting effect on plants in potted plants. It can be used in combination with microbial agents in microbial fertilizers or used alone to promote plant growth and development and achieve an increase in yield, and has a high application potential.

[0067] Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all belong to the protection scope of the present invention.

Claims

1. A polymyxa bacillus strain FB 9-3, characterized in that: The strain is named as Paenibacillus polymyxaFB 9-3, and its deposit number is CCTCC NO: M20241788.

2. Use of the Paenibacillus polymyxa strain FB 9-3 according to claim 1 in preventing and controlling root-knot nematode disease.

3. Use of the Paenibacillus polymyxa strain FB 9-3 according to claim 1 in promoting plant growth and development.

4. Use of the Paenibacillus polymyxa strain FB 9-3 according to claim 1 in preparing a fermentation liquid for preventing and treating root-knot nematode disease.

5. A method for preparing a fermentation broth of Paenibacillus polymyxa strain FB 9-3, characterized in that: The following steps are involved: 1) activating the Paenibacillus polymyxa strain FB 9-3; 2) The activated Paenibacillus polymyxa strain FB 9-3 is inoculated into LB liquid culture medium and cultured to obtain a fermentation liquid of the Paenibacillus polymyxa strain FB 9-3, wherein the number of live bacteria of the Paenibacillus polymyxa strain FB 9-3 in the fermentation liquid of the Paenibacillus polymyxa strain FB 9-3 is 1 to 2 billion / ml.

6. A fermentation broth of Paenibacillus polymyxa strain FB 9-3 prepared by the method according to claim 5, characterized in that: In the fermentation liquid of the Paenibacillus polymyxa strain FB 9-3, the number of live bacteria of the Paenibacillus polymyxa strain FB 9-3 is 1 to 2 billion / ml.

7. Use of the Paenibacillus polymyxa strain FB 9-3 according to claim 1 or the fermentation liquid of the Paenibacillus polymyxa strain FB 9-3 prepared by the method according to claim 4 in preparing a biological agent for preventing and controlling root-knot nematode disease and promoting plant growth and development.

8. A biological nematicide for preventing and treating root-knot nematode disease, characterized in that: The biological nematicide contains the Bacillus polymyxa strain FB 9-3 described in claim 1 or contains the fermentation liquid of the Bacillus polymyxa strain FB 9-3 prepared by the method described in claim 5; wherein the number of live bacteria of the Bacillus polymyxa strain FB 9-3 in the insecticide is 1 to 2 billion / ml.

9. The biological nematicide according to claim 8, characterized in that: In the insecticide, the number of viable bacteria of the Paenibacillus polymyxa strain FB 9-3 is 1 billion / ml.

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