Brevundimonas sp. Fermentation medium and application thereof in prevention and treatment of plant root-knot nematodes

Abstract

The invention discloses a brevundimonas sp. Fermentation medium and application thereof in prevention and treatment of plant root-knot nematodes. Each liter of the brevundimonas chinensis fermentation medium comprises the following components by mass or volume: 6-10 g / L of yeast powder, 16-18 g / L of peptone, 31-45 ml / L of nematode lysis buffer, 0.8-2.5 g / L of chitin and 5.6-6 g / L of yeast extract, and the pH value of the brevundimonas chinensis fermentation medium is 6.5-6.8. The application of the fermentation broth culture medium refers to the application of the obtained fermentation broth in prevention and treatment of plant root-knot nematodes. According to the present invention, with the fermentation medium, the fatality rate of the obtained fermentation broth on the plant root-knot nematode exceeds 95%, the action time does not exceed 12 h, the nematode can be rapidly killed before the larva enters the plant, and the good application prospect is provided in the plant root-knot nematode prevention and control.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and particularly relates to a Brevundimonas sinensis fermentation culture medium and application thereof in preventing and controlling plant root-knot nematodes. Background Art

[0002] Brevundimonas sinensis is a new member of the Brevundimonas genus that has been extensively studied in recent years. It possesses unique metabolic properties and demonstrates significant potential for applications in environmental remediation, biocatalysis, and biosynthesis. Studies on Brevundimonas sinensis cultivation, combined with culture conditions, have revealed a wide range of applications, with the same strain exhibiting significant variations in function and efficiency under different culture conditions. Currently, fermentation cultures of Brevundimonas sinensis typically utilize general bacterial culture media (e.g., LB, nutrient broth) or formulations based on related strains (e.g., Brevundimonas diminuta). However, specialized liquid culture media suitable for fermenting Brevundimonas sinensis to produce fermentation broths with high plant root knot inhibition activity have not been reported.

[0003] The present invention aims to provide a fermentation medium with an inhibitory effect on plant root-knot nematodes, so as to improve the function and control effect of Brevundimonas sinensis fermentation liquid in preventing and controlling plant root-knot nematodes. Summary of the Invention

[0004] The first object of the present invention is to provide a fermentation medium for Brevundimonas sinensis, and the second object of the present invention is to provide an application of the fermentation medium.

[0005] The first object of the present invention is achieved by providing a fermentation medium for Brevundimonas sinensis, wherein each liter of the fermentation medium comprises the following components by mass or volume: 6-10 g / L yeast powder, 16-18 g / L peptone, 31-45 ml / L nematode lysate, 0.8-2.5 g / L chitin, 5.6-6 g / L yeast extract, and a pH value of 6.5-6.8.

[0006] The second object of the present invention is achieved in that the application of the fermentation liquid culture medium is the application of the fermentation liquid obtained therefrom in preventing and controlling plant root-knot nematodes.

[0007] The invention adds chitin as a substrate and uses nematode lysate as an induction factor, thereby promoting the directional production of anti-nematode components by Brevundimonas sinensis, thereby improving the ability of Brevundimonas sinensis to produce effective components for preventing and treating plant root-knot nematodes by fermentation.

[0008] The beneficial effects of the present invention are as follows: the fermentation liquid obtained by fermenting the Chinese root-knot nematode 1.547-1 with the fermentation medium provided by the present invention has a mortality rate of more than 95% against southern root-knot nematodes, an action time of no more than 12 hours, and can quickly kill nematodes before the larvae enter the plant, thus having good application prospects in preventing and controlling plant root-knot nematodes. DETAILED DESCRIPTION

[0009] The present invention will be further described in detail below with reference to the embodiments, but the present invention is not limited in any way. Any changes or improvements made based on the teachings of the present invention fall within the scope of protection of the present invention.

[0010] The present invention provides a Brevundimonas huaxiensis fermentation medium. The fermentation medium comprises, per liter, the following components by mass or volume: 6-10 g / L yeast powder, 16-18 g / L peptone, 31-45 ml / L nematode lysate, 0.8-2.5 g / L chitin, 5.6-6 g / L yeast extract, and a pH value of 6.5-6.8.

[0011] The preparation method of the nematode lysate is as follows: place root-knot nematodes in a container, add lysate and shake well, act for 10-20 minutes, add water to 100 ml, and adjust the pH to 6.5; the mass volume ratio of root-knot nematodes to lysate is 1:1 g / ml.

[0012] The lysate consists of the following components: 20 mL of 5M sodium hydroxide, 10 mL of 10% sodium hypochlorite solution, and 80 mL of water.

[0013] The Chinese shortwave mononas is Chinese shortwave mononas 1.5471, which is deposited in the Guangdong Provincial Microbial Culture Collection Center with the number GDMCC No: 65867, the preservation date is January 16, 2025, and the preservation address is No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province.

[0014] The present invention further provides a fermentation broth prepared based on the fermentation medium, and the preparation method thereof is as follows: inoculating the fermentation seed liquid of Brevundimonas sinensis into the fermentation medium, and culturing the fermentation broth on a shaker at 35-37° C. and 150-200 r / min for 2-3 days.

[0015] The present invention further provides application of the fermentation liquid in preventing and controlling plant root-knot nematodes.

[0016] The present invention also provides a bacterial agent for preventing and controlling plant root-knot nematodes based on the fermentation liquid, wherein the bacterial agent uses the fermentation liquid as a main active ingredient or one of the active ingredients.

[0017] Examples 1-3 1. Seed culture: Transfer pure strains of Brevundimonas huaxiensis 1.5471 into multiple 250mL triangular flasks, There is 100 mL of seed culture medium (LB culture medium), and then culture it at 37℃±1℃ on a reciprocating shaker at a speed of 200 r / min for 20 hours to obtain the fermentation seed liquid.

[0018] Fermentation culture: 9 ml of fermentation seed liquid was inoculated into a 250 mL Erlenmeyer flask containing 150 mL of fermentation medium, and cultured at 25°C ± 1°C and 150 r / min for 4 days to obtain fermentation liquid.

[0019] Nematode lysis solution: Place 1g of root-knot nematodes or Caenorhabditis elegans in a container, add 1ml of lysis solution, shake well, and incubate for 10-20 minutes. Observe the solution until turbid and free of visible solid particles. Add water to 100ml and adjust the pH to 6.5 with acid. Lysis solution recipe: 20ml 5M sodium hydroxide, 10ml 10% sodium hypochlorite solution, 80ml water. Mix well and set aside.

[0020] The components of the fermentation medium of Examples 1-3 are shown in Table 1.

[0021] Table 1 Components and ratios of fermentation medium in Examples 1-3 Comparative Examples 1-15 The preparation method of the fermentation broth of Comparative Example 1-15 is the same as that of Example 1-3, except that the proportions of the fermentation medium components are different, as shown in Table 2.

[0022] Table 2 Comparative Examples 1-15 Fermentation Medium Components and Ratios Experimental Example 1 Nematicidal test was performed on the fermentation broth prepared in Examples 1-3 Test Method: 700 µL of each fermentation broth prepared in Examples 1-3 and Comparative Examples 1-15 was added to a cell culture dish. A suspension of 2nd-instar southern root-knot nematodes (20 µL: 80-100 southern root-knot nematodes) was then added and mixed thoroughly. The test cell culture dish containing the bacterial culture and southern root-knot nematodes was placed in a 22°C incubator and incubated. The number of live and dead nematodes was counted under a stereomicroscope every 30 minutes. Nematodes were considered dead if they showed stiffness and did not respond to needle prodding. Blank culture medium and Escherichia coli OP50 were used as controls. 12-hour nematode mortality and adjusted mortality were calculated according to Zhang et al. (2016). The experiment was repeated three times.

[0023] The test results are shown in Table 3.

[0024] Table 3 12-hour lethality of fermented liquids prepared in Examples 1-3 and Comparative Examples 1-15 against nematodes As shown in Table 3, the fermentation liquid prepared in Examples 1-3 has a 12-hour lethality rate of over 95% against nematodes, which can quickly kill nematodes before the larvae enter the plants. It has good application prospects in controlling plant root-knot nematodes.

Claims

1. A fermentation medium for Brevundimonas sinensis, characterized in that: Each liter of fermentation medium includes the following components by mass or volume: yeast powder 6-10 g / L, peptone 16-18 g / L, nematode lysate 31-45 ml / L, chitin 0.8-2.5 g / L, yeast extract 5.6 -6 g / L, and pH 6.5-6.

8.

2. The fermentation medium of Brevundimonas sinensis according to claim 1, characterized in that: The preparation method of the nematode lysate is as follows: place root-knot nematodes in a container, add lysate and shake well, act for 10-20 minutes, add water to 100 ml, and adjust the pH to 6.5; the mass volume ratio of root-knot nematodes to lysate is 1:1 g / ml.

3. The fermentation medium of Brevundimonas sinensis according to claim 2, characterized in that: The lysate consists of the following components: 20 mL of 5M sodium hydroxide, 10 mL of 10% sodium hypochlorite solution, and 80 mL of water.

4. The fermentation medium of Brevundimonas sinensis according to claim 1, characterized in that: The Brevundimonas sinensis is Brevundimonas sinensis 1.547-1, which is deposited in Guangdong Provincial Microbiological Culture Collection Center with the number GDMCC No: 65867 and the deposit address is No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province.

5. A fermentation broth prepared based on the fermentation medium according to claim 1, characterized in that: The preparation method of the fermentation liquid is as follows: the fermentation seed liquid of Brevundimonas huaxiensis is inoculated into the fermentation medium, and cultured on a shaker at 35-37° C. and 150-200 r / min for 2-3 days.

6. Use of the fermentation liquid according to claim 5 in preventing and controlling plant root-knot nematodes.

7. A bacterial agent for controlling plant root-knot nematodes based on the fermentation liquid of claim 5, characterized in that: The bacterial agent uses the fermentation broth as a main active ingredient or one of the active ingredients.

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