Brevundimonas sp. For preventing and treating root-knot nematode and application of brevundimonas sp.

Abstract

The invention discloses brevundimonas chinensis for preventing and treating root-knot nematode and application of the brevundimonas chinensis. The brevundimonas sp. Is preserved in the Guangdong Microbial Culture Collection Center, the number is GDMCC No: 65867, and the preservation address is No.100, Xianlie Middle Road, Xiaolian District, Guangzhou City, Guangdong Province. The fermentation liquor of the brevundimonas sp. Provided by the invention not only can attract nematodes, but also can kill the nematodes, can actively attract the nematodes to a pesticide action area and intensively kill the nematodes, improves the control effect, can avoid waste caused by large-scale spraying of traditional pesticides, and can reduce the pesticide consumption, the cost and the environmental pollution. According to the present invention, the mortality rate of the fermentation broth of the brevundimonas chinensis on the root-knot nematode exceeds 89%, the mortality rate of the fermentation broth diluted by 50 times on the root-knot nematode exceeds 80%, the action time does not exceed 12 h, nematodes can be rapidly killed before larvae enter plants, and the brevundimonas chinensis strain has good application prospects in plant root-knot nematode prevention and control.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to Brevundimonas sinensis for preventing and treating root-knot nematodes and an application thereof. Background Art

[0002] Root-knot nematodes are sessile, obligate endoparasitic nematodes that obtain nutrients from their hosts through feeding sites. These enlarged feeding sites cause root swelling, forming knots, which impair the plant's ability to absorb nutrients and water. Symptoms include weakened plant growth, leaf wilting, yellowing, root deformities, low yields, and poor fruit quality. Root-knot nematodes have a wide host range, encompassing vegetables such as cucumbers, tomatoes, peppers, and beans, as well as cash crops such as corn, potatoes, and soybeans, including grains and oilseeds. They are found throughout much of southern my country and some warmer regions in northern China, and are found on almost all landmasses worldwide except Antarctica. Parasitic nematodes undoubtedly cause significant crop losses in temperate, subtropical, and tropical regions. Globally, they cause a total annual economic loss of $358.24 billion to 37 important crops, with an estimated annual loss of $215.77 billion for 20 essential food crops and $142.47 billion for 17 important cash crops. In addition, the proportion of important crop planting areas in the world that are infected by plant nematodes is also at a high level. For example, 90% of the world's banana planting areas have plant nematode diseases.

[0003] During the crop planting process, root knot nematodes and other diseases often occur together. In this process, root knot nematodes play an important role as pioneers. Root knot nematodes cause a large number of wounds on the roots, allowing pathogens to invade the host through the wounds caused by nematode infection. The combined infection of multiple pathogens will show multiple symptoms on the host, and the result of 1+1>2 will be formed, which greatly aggravates the damage. For example, the pathogen of tobacco bacterial wilt, Ralstonia solanacearum Ralstoma solanacearum Interaction with the southern root-knot nematode. With global warming, the increase in protected areas, long-term continuous cropping, and the complexity of the soil environment, complex root diseases have become the norm, seriously restricting agricultural production in my country.

[0004] Brevundimonas sinensis is a Gram-negative bacterium belonging to the genus Brevundimonas and family Caulobacteraceae. It was first described by Segers et al. There are 33 species in the genus Brevundimonas, which can be found in diverse environments, such as soil, water, activated sludge, and plant roots. Brevundimonas has broad potential applications, such as cadmium biosorption, soil bioremediation, water pollutant treatment, and promoting plant growth in sustainable agriculture in arid regions. However, Brevundimonas is an opportunistic pathogen in humans, causing a range of hospital-acquired infections, including bacteremia, eye infections, peritonitis, urinary tract infections, and skin and soft tissue infections. However, there are limited reports on the control of nematodes by Brevundimonas.

[0005] The present invention aims to provide a novel Brevundimonas sinensis strain with excellent nematode trapping and killing effect. Summary of the Invention

[0006] The first object of the present invention is to provide a Brevundimonas sinensis ( Brevundimonas huaxiensis ) 1.547-1, the second object of the present invention is to provide the said Shortwave Mononas sinoprocta ( Brevundimonas huaxiensis ) Application of 1.547-1.

[0007] The first object of the present invention is achieved by: a strain of Brevundimonas sinensis ( Brevundimonas huaxiensis ) 1.547-1 ， It is deposited in Guangdong Provincial Microbiological Culture Collection with the number GDMCC No: 65867, the deposit date is January 16, 2025, and the deposit address is No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province.

[0008] The second object of the present invention is achieved in that the application of Brevundimonas sinensis 1.547-1 is used in preventing and controlling plant root-knot nematodes.

[0009] The beneficial effects of the present invention are: 1. The present invention provides Brevundimonas sinensis ( Brevundimonas huaxiensis )1.547-1 strain is derived from the nematode-feeding fungus Arthrosporum oligosporum ( Arthrobotrys oligospora )1.5470, which is an endophytic bacterium of the fungal strain. The two form a symbiotic relationship, which enables the strain to assist the nematode-feeding fungus in inhibiting the existence of nematodes to a certain extent, improving the environmental control ability of the nematode-feeding fungus Arthrospora oligosporida or the strain as a biocontrol agent, thereby improving the control efficiency of the nematode-feeding fungus strain and the strain as a biocontrol agent against nematodes.

[0010] 2. The fermentation liquid of Brevundimonas sinensis provided by the present invention can not only attract nematodes but also kill nematodes. It can actively attract nematodes to the action area of the agent and eliminate them in a concentrated manner, thereby improving the prevention and control effect. It can also avoid the waste of large-scale spraying of traditional pesticides, reduce the amount of pesticides used, reduce costs, and reduce environmental pollution.

[0011] 3. The fermentation liquid of Brevundimonas sinensis provided by the present invention has a mortality rate of over 89% against root-knot nematodes, and the mortality rate of the fermentation liquid diluted 50 times against root-knot nematodes exceeds 80%. The action time does not exceed 12 hours, and it can quickly kill nematodes before the larvae enter the plant. It has good application prospects in the prevention and control of plant root-knot nematodes. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The endoparasitic micrograph (Figure A) and fluorescence morphology (Figures CD) of Brevundimonas huaxiensis 1.547-1 of the present invention are shown; Figure 2 This is the phylogenetic tree of Brevundimonas huaxiensis 1.547-1 of the present invention; Figure 3 These are pictures of the culture solution of Brevundimonas sinensis 1.547-1 of the present invention attracting nematodes, where Figure a is a bisected plate, and Figure b is an enlarged picture of the nematodes crossing the septum of the plate and crawling towards the strain growth area; the left side of the bisected plate is the strain growth area, and the right side is the nematode living area. DETAILED DESCRIPTION

[0013] The present invention will be further described in detail below with reference to the accompanying drawings and 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.

[0014] The present invention provides a strain of Brevundimonas sinensis ( Brevundimonas huaxiensis ) 1.547-1 ， The strain 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.

[0015] The present invention also provides the use of the Brevundimonas sinensis 1.547-1 in preventing and controlling plant root-knot nematodes.

[0016] The application is to kill root-knot nematodes by culturing metabolites produced by Brevundimonas huaxiensis 1.547-1.

[0017] The application is to control plant root-knot nematodes by culturing metabolites produced by Brevundimonas huaxiensis 1.547-1.

[0018] The application is to prepare a preparation by using Brevundimonas sinensis 1.547-1 or to prepare a preparation in combination with the nematode-feeding fungus Arthrospora oligosporida to prevent and control plant root-knot nematodes.

[0019] The present invention further provides a bacterial agent prepared based on the Brevundimonas sinensis 1.547-1.

[0020] The preparation method of the bacterial agent is as follows: 1) Inoculate Brevundimonas huaxiensis 1.547-1 into a seed culture medium, and then culture on a shaker at 36-38°C and 200 rpm for 20 h to obtain a fermentation seed solution; 2) The fermentation seed liquid was inoculated into the fermentation medium and cultured on a shaker at 37°C and 185 r / min for 40 h. The resulting fermentation liquid was the target bacterial agent.

[0021] In step 1), the seed culture medium is LB culture medium.

[0022] In step 2), the fermentation medium is LB medium.

[0023] Example 1: Isolation, purification and identification of strains The present invention is a strain of Brevundimonas huaxiensis isolated from the nematode-feeding fungus Arthrosporum oligosporum ( Arthrobotrys oligospora ), the fungal strain is collected in the "Southwest Germplasm Resources-Microbial Bank" of Yunnan University (the bank is managed and operated by the State Key Laboratory of Bioresource Conservation and Utilization of Yunnan University), the original strain number is 1.5470, and the preservation number is YMF1.05470.

[0024] Strain isolation method: Fungal colonies were inoculated into CMB liquid medium and cultured in a constant temperature shaker (28°C, 180 rpm / min) for 10 days. Mycelium was then extracted from the shake flask using a vacuum pump and transferred to a 2 mL sterile centrifuge tube. Sterile deionized water and sterile steel balls were added. The collected mycelium was disrupted using a high-throughput tissue disruptor to obtain a fungal tissue homogenate. Appropriate amounts were then added dropwise to three solid media: LB, TWYE, and YEM. After spreading evenly with a spreader, the plates were sealed with Parafilm and incubated in a 30°C constant temperature incubator for 3-4 days. Colony growth was observed and recorded. Single colonies were then picked based on their morphological characteristics and transferred to LB solid medium. This procedure was repeated to purify and culture the strain, obtaining Brevundimonas huaxiensis 1.547-1. Molecular biological identification confirmed that strain 1.547-1 was a new species of Brevundimonas huaxiensis. Its ITS sequence is shown in SEQ ID No. 1.

[0025] The culture medium components used for strain isolation and culture are as follows: 1. Fungal culture medium: CMB liquid medium: 20 g crushed corn, dilute to 1 L with water. Sterilize by high-pressure steam at 121°C for 20 min.

[0026] 2. Bacterial culture media: LB, TWYE and YEM LB medium: 5 g yeast powder, 10 g tryptone, 10 g sodium chloride, add water to 1 L. Sterilize with high-pressure steam at 121°C for 20 min.

[0027] TWYE medium: 0.25 g yeast powder, 0.5 g potassium hydrogen phosphate, dilute to 1 L with water. Autoclave at 121°C for 20 min.

[0028] YEM medium: 0.5 g yeast extract, 5 g mannitol, 0.5 g potassium hydrogen phosphate (KHPO), 0.2 g magnesium sulfate heptahydrate, 0.1 g sodium chloride. Adjust pH to 7.0 and add water to 1 L. Autoclave at 121°C for 20 min.

[0029] NGM solid medium: 2.5 g tryptone, 3 g sodium chloride, 15 g agar, dilute to 1 L with deionized water. After sterilization at 121°C for 20 min, wait until the temperature drops to approximately 55°C. Add 1 mL each of 1 M phosphate buffer filtered through a 0.22 µm microporous membrane, 1 M magnesium sulfate solution, and 1 mL of 5 mg / mL cholesterol (dissolved in anhydrous ethanol) to the medium. Shake evenly and pour onto a plate.

[0030] Example 2 Preparation of fermentation broth 1. Seed Culture: Transfer the pure strain 1.547-1 from the plate of Example 1 to multiple 250 mL Erlenmeyer flasks containing 100 mL of seed culture medium (LB medium). The flasks were then incubated at 37°C ± 1°C on a reciprocating shaker at 200 r / min for 20 h to obtain fermentation seed liquid.

[0031] 2. Fermentation culture: inoculate the prepared fermentation seed liquid into the fermentation medium (LB medium) in a 250 mL triangular flask. The liquid volume was 150 mL, the inoculation amount was 5%, and the culture was carried out at 37°C and a rotation speed of 185 r / min for 40 hours to obtain the fermentation liquid.

[0032] Experimental Example 1 The fermentation liquid prepared in Example 2 was subjected to a nematode attraction test. Experimental method: Sterilized and melted NGM culture medium was injected into a 9 cm diameter two-divided culture dish in advance and used after solidification. 100-150 2nd-instar southern root-knot nematodes were added to the center of one compartment; 20 μL of bacterial solution (OD 600 = 1.0) and evenly spread with a coating rod. Once the liquid stops flowing, immediately seal all test plates with Parafilm and place in a 22°C incubator. After 2, 4, and 8 hours, observe under a stereomicroscope the number of nematodes that cross the grid and reach the end of the bacterial solution. Calculate the attraction index (AR) and record the results. E. coli OP50 and blank LB culture medium were used as controls. The experiment was repeated five times.

[0033] The larger the AR value, the stronger the attraction of endosymbiotic bacteria to nematodes.

[0034] Table 1 Results of the nematode attraction test of the fermentation liquid prepared in Example 2 from Figure 3 It can be seen that, attracted by the fermentation liquid, the nematodes move to the left area and even crawl across the middle partition.

[0035] The results in Table 1 show that the fermentation liquid prepared in Example 2 has an AR value of >0.3 after 4 hours and an AR value of >0.5 after 8 hours, indicating that the fermentation liquid has a significant attraction to nematodes. The AR value of the control is zero, indicating that the control has no attraction.

[0036] Experimental Example 2: Test on the lethality of the fermentation liquid prepared in Example 2 to nematodes Test Method: 700 µL of fermentation broth was added to a cell culture dish. A suspension of 80–100 second-instar M. incognita (20 µL) was then added. The mixture was thoroughly mixed. The test cell culture dish containing the bacterial culture and nematodes was placed in a 22°C incubator. The number of live and dead nematodes was counted under a stereomicroscope every 30 minutes. Nematodes were considered dead if they became rigid and showed no response to needle prodding. Blank culture medium and E. coli OP50 were used as controls. Nematode mortality and adjusted mortality were calculated according to Zhang et al. (2016). The experiment was repeated five times. The results are shown in Table 2.

[0037] Table 2 Results of the lethality test of the fermentation broth prepared in Example 2 against plant root-knot nematodes As shown in Table 2, the fermentation liquid prepared in Example 2 had a 12-hour lethality rate against incognita root-knot nematodes exceeding 89%.

[0038] The fermentation liquid prepared in Example 2 was diluted 50 times and then subjected to the nematode lethality test according to the above method. The test results were As shown in Table 3.

[0039] Table 3 Results of the lethality test of the fermentation broth dilution (×50) prepared in Example 2 against plant root-knot nematodes As shown in Table 3, the fermentation liquid prepared in Example 2, after being diluted 50 times, had a lethality rate of over 80% against the second-instar larvae of the southern root-knot nematode after 12 hours of action.

[0040] Experimental Example 3 Field Test 1. Test agent: fermentation broth prepared in Example 2 Control group: 2% avermectin suspension (Jiangsu Shuangning Chemical) 2. Experimental treatment Treatment 1: 10 ml / plant of fermentation broth prepared in Example 2 Treatment 2: 20 ml / plant of fermentation broth prepared in Example 2 Treatment 3: 30 ml / plant of fermentation broth prepared in Example 2 Treatment 4: 40 ml / plant of fermentation broth prepared in Example 2 Treatment 5: 50 ml / plant of fermentation broth prepared in Example 2 Positive control: 2% avermectin suspension 20 ml / plant Blank control: 50 ml water / plant 3. Experimental methods The test crop was tomato, infected with southern root-knot nematode ( Meloidogyne incongnita ), yellow loam soil. The experimental plots were 100 square meters in size, with 50 seedlings per treatment, spaced 50 cm apart, and randomly arranged. The pesticide was applied to the rhizosphere of the tomato seedlings at transplanting. Conventional management was followed during the experimental period.

[0041] 4. Survey Methods After all tomatoes were harvested, the incidence rate, disease index and relative control effect were investigated.

[0042] Table 4 Disease classification standards 5. Experimental results Table 5 Control effect of the fermentation liquid prepared in Example 1 on root-knot nematodes in the field The results in Table 5 show that, compared with the control, the relative control effects of the five concentrations of the fermentation broth prepared in Example 2 on field nematodes are better than the control effects of avermectin (the main drug for field nematode control) during the same period.

Claims

1. A strain of Brevundimonas sinensis ( Brevundimonas huaxiensis ) 1.547-1 ， It is deposited in Guangdong Provincial Microbiological Culture Collection with the number GDMCC No: 65867, and the deposit address is No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province.

2. Use of Brevundimonas sinensis 1.547-1 according to claim 1 in preventing and controlling plant root-knot nematodes.

3. The application according to claim 2, characterized in that: The application is to kill plant root-knot nematodes by culturing metabolites produced by Brevundimonas huaxiensis 1.547-1.

4. The application according to claim 2, characterized in that The application is to prepare a preparation by using Brevundimonas sinensis 1.547-1 or to prepare a preparation in combination with the nematode-feeding fungus Arthrospora oligosporida to prevent and control plant root-knot nematodes.

5. A bacterial agent prepared based on the Brevundimonas sinensis 1.547-1 described in claim 1.

6. The bacterial agent prepared by Brevundimonas sinensis 1.547-1 according to claim 5, characterized in that: The preparation method of the bacterial agent is as follows: 1) Transfer the strain of Brevundimonas huaxiensis 1.547-1 to the seed culture medium and then shake it at 35-37℃ at 150-200℃. r / min for 20 h to obtain the fermentation seed liquid; 2) Inoculate the fermentation seed liquid into the fermentation medium and culture it on a shaker at 35-37°C and 150-200 r / min for 40-72 hours. The resulting fermentation liquid is the target bacterial agent.

7. The bacterial agent prepared by Brevundimonas sinensis 1.547-1 according to claim 6, characterized in that: In step 1), the seed culture medium is LB culture medium.

8. The bacterial agent prepared by Brevundimonas sinensis 1.547-1 according to claim 6, characterized in that: In step 2), the fermentation medium is LB medium.

9. Use of the bacterial agent according to claim 4 in controlling root-knot nematodes in plants.

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