Mixed nutritional flagellum and application thereof in promotion of tomato growth and prevention and control of bacterial wilt

Abstract

The invention discloses a mixed nutritional flagellum and application thereof in promotion of tomato growth and prevention and control of bacterial wilt. The preservation number of the mixed nutritional flagellates NJAU-K1 is CCTCC (China Center For Type Culture Collection) NO: V202536. Experiments show that the mixed nutritional flagellum NJAU-K1 disclosed by the invention not only shows a remarkable growth promoting effect on tomatoes, but also can effectively prevent and control tomato bacterial wilt. The invention shows that the mixed nutritional flagellate NJAU-K1, as a novel plant growth promoting and biocontrol microorganism, shows great application potential in the aspects of crop yield increase and soil-borne disease control, and provides theoretical and technical support for green development of agriculture.

Description

Technical Field

[0001] The present invention belongs to the technical fields of microbiology and biological control of plant diseases, and particularly relates to a mixotrophic flagellate and its application in promoting the growth of tomatoes and controlling bacterial wilt disease. Background Art

[0002] Tomato is a horticultural crop widely cultivated globally, with extremely high agricultural and economic value. Tomato bacterial wilt caused by Ralstonia solanacearum (abbreviated as Rs) is a soil-borne disease that seriously harms tomato production and greatly hinders the development of the tomato industry. Rs mainly invades tomato plants through root wounds or natural orifices of the plants. After the pathogen invades, it will multiply and spread in large numbers in the vascular bundles of the plants, and symptoms will begin to appear on the tomato plants. In the initial stage, the leaves at the top of the plant will wilt and droop during the day, and can return to normal when the temperature is lower in the morning and evening. As the disease progresses, the leaves at the lower part of the plant will gradually wilt, and the wilting symptoms are irreversible. At this time, brown patches will appear on the cortex at the base of the stem and gradually spread upward. At the same time, the roots of the plant will turn brown and rot, losing the ability to absorb water and nutrients. Finally, the plant wilts and dies, and the leaves still remain green.

[0003] Bacterial wilt disease causes serious losses to tomato yields every year. Currently, many methods have been adopted to control its damage, including soil fumigation and improvement, biological control, biological agents, and breeding of resistant varieties. However, some methods are not very ideal. For example, non-selectively eliminating beneficial microorganisms in the soil is likely to cause damage; soil improvement requires a large amount of manpower, material resources, and financial resources, and it takes a long time to show results; in the changing environmental conditions, breeding resistant varieties lacks excellent parents with stable resistance; although biological agents can be used to induce plant resistance, this increases the planting cost, affects the economic benefits of the industry, and various factors such as plant varieties, growth stages, environmental conditions, and the use concentration and method of the inducer will affect the control effect.

[0004] Biological control mainly relies on the mutual relationships among organisms, such as predation, parasitism, competition, etc. It uses beneficial organisms or their products to inhibit or control the population size of harmful organisms, so as to achieve the prevention and control of harmful organisms. In current research, regarding the prevention and control of Ralstonia solanacearum, many biocontrol bacteria have excellent application potential. For example, Pseudomonas, Bacillus, and Streptomyces are potential highly effective biocontrol bacterial species for controlling bacterial wilt. These biocontrol bacteria inhibit the growth of pathogenic bacteria by secreting metabolites, or reduce the colonization number of pathogenic bacteria by competing for ecological niches and nutrients. Recent research has shown that protists also play an important role in soil ecosystems including the rhizosphere. Predatory protists can regulate the structure and population size of soil microbial communities through predation and metabolic activities, enrich beneficial microorganisms for plants at the early stage of plant growth to promote the inhibition of bacterial wilt, or directly inhibit Ralstonia solanacearum through predation. Summary of the Invention

[0005] To solve the problems existing in the prior art, the present invention provides a mixotrophic protist that promotes the growth of tomatoes and controls bacterial wilt.

[0006] Another object of the present invention is to provide a culture of this protist.

[0007] Still another object of the present invention is to provide the applications of this mixotrophic protist and the culture.

[0008] The objects of the present invention can be achieved by the following technical solutions: A mixotrophic flagellate ( Poterioochromonas malhamensis ) NJAU-K1, which is deposited in the China Center for Type Culture Collection. The deposit date is May 8, 2025, and the deposit number is CCTCC NO: V202536.

[0009] The culture of the mixotrophic flagellate ( Poterioochromonas malhamensis ) NJAU-K1, and the culture is prepared by the following method: inoculate the mixotrophic flagellate NJAU-K1 into NMAS culture medium, add inactivated microorganisms as food, place it in an incubator at 18 - 20 °C, and let it stand for 40 - 50 h.

[0010] The preparation method of the culture, inoculate the mixotrophic flagellate NJAU-K1 into NMAS culture medium, add inactivated microorganisms as food, place it in an incubator at 18 - 20 °C, and let it stand for 40 - 50 h.

[0011] As a preferred embodiment of the present invention, the formula of the NMAS culture medium is as follows: sodium chloride 0.12 g / L, magnesium sulfate heptahydrate 0.004 g / L, calcium chloride hexahydrate 0.006 g / L, sodium phosphate 0.142 g / L, potassium phosphate 0.136 g / L.

[0012] As a preferred embodiment of the present invention, the inactivated microorganism is inactivated Escherichia coli.

[0013] Use of the mixotrophic flagellate ( Poterioochromonas malhamensis ) NJAU-K1 in promoting the growth of tomatoes and / or controlling tomato bacterial wilt.

[0014] Use of the mixotrophic flagellate ( Poterioochromonas malhamensis ) NJAU-K1 in the preparation of a product for promoting the growth of tomatoes and / or controlling tomato bacterial wilt.

[0015] Preferably, the product is a biological pesticide or a growth promoter.

[0016] Use of the culture in promoting the growth of tomatoes and / or controlling tomato bacterial wilt.

[0017] Use of the culture in the preparation of a product for promoting the growth of tomatoes and / or controlling tomato bacterial wilt.

[0018] Beneficial effects

[0019] The present invention isolates and screens a mixotrophic flagellate ( Poterioochromonas malhamensis ) NJAU-K1 with strong resistance to the pathogen of tomato bacterial wilt from the rhizosphere of healthy crops. Co-culture experiments with bacterial pathogens show that the mixotrophic flagellate ( Poterioochromonas malhamensis ) NJAU-K1 has a significant control effect on tomato bacterial wilt. In addition, the mixotrophic flagellate ( Poterioochromonas malhamensis ) NJAU-K1 can also significantly promote the growth of tomatoes. Therefore, the mixotrophic flagellate ( Poterioochromonas malhamensis ) NJAU-K1 of the present invention is of great significance for the control of soil-borne diseases and the promotion of plant growth. Description of the drawings

[0020] Figure 1 Photographs showing the morphological characteristics of the dormant cysts (left), autotrophic state (middle), and predatory state (right) of the mixotrophic protist NJAU-K1 of the present invention.

[0021] Figure 2 Phylogenetic tree of the 18S rDNA gene sequence of the protist NJAU-K1 of the present invention.

[0022] Figure 3For the protist NJAU-K1 in Example 3 of the present invention and Ralstonia solanacearum the bacteriostatic circle effect diagram of the protist NJAU-K1 in the co-culture of the two on CPG plates.

[0023] Figure 4 For Example 4 of the present invention, the growth conditions of the above-ground and underground parts of tomato plants in the blank control group (left) and the treatment group inoculated with the protist NJAU-K1 (right).

[0024] Figure 5 For Example 4 of the present invention, the comparison of plant height and root weight between the blank control group and the treatment group inoculated with the protist NJAU-K1.

[0025] Biological material preservation information

[0026] Mixotrophic flagellate NJAU-K1 Poterioochromonas malhamensis , was preserved in the China Center for Type Culture Collection, the preservation address is Wuhan University, Wuhan, China, the preservation date is May 8, 2025, and the preservation number is CCTCC NO: V202536. Detailed implementation manners

[0027] The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all purchased from conventional biochemical reagent companies unless otherwise specified. The tests in the following examples are all set up with three repeated experiments.

[0028] Example 1 Isolation and purification of mixotrophic flagellates The inventor of the present application collected rhizosphere soil of healthy plants from tomato-growing soil in Nanjing City, Jiangsu Province. The collection depth range was 0-20 cm, and it was sealed in a sterile self-sealing bag and taken back to the laboratory. The test target pathogen, Ralstonia solanacearum, was provided by the Soil Organic Fertilizer Team of Nanjing Agricultural University.

[0029] The soil dilution method was used to isolate mixotrophic protists. The specific method was as follows: The rhizosphere soil samples of healthy crops were mixed evenly. 1 g of soil was weighed and added into a 50 mL centrifuge tube, and 30 mL of sterile deionized water was added. Then the centrifuge tube was placed in a shaker at 250 rpm and 20 °C and shaken for 15 min to fully mix the rhizosphere soil and release the protists in the soil. After taking the centrifuge tube out of the shaker and standing for 10 min, the upper liquid was taken and added into a 96-well plate, and Escherichia coli (OD = 0.04) was added as food. Then it was cultured for 2 d under the condition of dark culture at 20 °C. The growth of protists was examined under an inverted microscope at magnifications of 100×, 200×, and 400×, and gradient dilution was carried out and continued to be cultured for 2 d at 20 °C. Finally, a single mixotrophic protist cell was picked with a capillary tube and transferred into a new 96-well plate to obtain a pure culture of protists.

[0030] Example 2 Identification of Mixotrophic Protist NJAU-K1 The screened protists were identified by 18S rDNA using a method combining morphological observation and molecular biology. PCR reaction system (50 μL system): 2 μL of each primer, 25 μL of 2× Mix, and supplemented with ddH2O to 50 μL. The primers were the most commonly used universal primers P-FLA-F / P-FLA-R, P-FLA-F: SEQ ID NO.2, P-FLA-R: SEQ ID NO.3. Reaction procedure: Pre-denaturation at 94 °C for 3 min, denaturation at 94 °C for 55 s, annealing at 50 °C for 50 s, extension at 72 °C for 1 min, extension at 72 °C for 10 min, and stored at 16 °C after 35 cycles. After the PCR products were detected, recovered by 1.5% agarose gel electrophoresis, they were sent to Beijing Tsingke Biotechnology Co., Ltd. for Sanger sequencing, and the sequencing results were shown as SEQ ID NO.1.

[0031] Using BLAST software, the obtained 18S rDNA gene sequence was aligned with the PR2 (Protist Ribosomal Reference) database and the NT (Nucleotide Sequence Database) database of the NCBI website. MEGA software was used to perform phylogenetic analysis on the isolated mixotrophic protists. The sequencing result of protist NJAU-K1 Poterioochromonas malhamensis had the highest similarity, up to 99.72%, with the model mixotrophic protist. Therefore, the isolated mixotrophic protist NJAU-K1 was identified as Poterioochromonas malhamensis . Figure 2 is the phylogenetic tree constructed based on the 18S rDNA gene sequence.

[0032] In the NMAS culture medium, the mixotrophic protist NJAU-K1 is in a dormant cyst state, which is round, in a static state, and without flagella ( Figure 1 left); its autotrophic state is round, swims relatively fast, and has a single-terminal flagellum ( Figure 1 middle); the predatory state shows an active state, with a single-terminal flagellum and swims relatively fast ( Figure 1 right). Figure 1 This is a photograph of the morphological characteristics of the mixotrophic protist NJAU-K1 of the present invention.

[0033] This strain was deposited at the China Center for Type Culture Collection (address: Culture Collection Center of Wuhan University, Wuhan, China) on May 8, 2025, and the deposit number is: CCTCC NO: V202536.

[0034] Example 3 Plate co-culture experiment of mixotrophic flagellate NJAU-K1 inhibiting Ralstonia solanacearum The tested Ralstonia solanacearum( Ralstonia solanacearum ), and the tested mixotrophic protist is the mixotrophic flagellate NJAU-K1 of Example 2, deposit number: CCTCC NO: V202536.

[0035] Preparation of mixotrophic flagellate NJAU-K1: Inoculate mixotrophic flagellate NJAU-K1 into liquid NMAS medium, add heat-inactivated Escherichia coli (OD = 0.04), place it in a constant temperature incubator at 20 °C, and incubate statically. After 48 h, aspirate 100 μL of the culture solution and count it under an inverted microscope, and dilute the number to 3×10 4 cells / mL.

[0036] Preparation of the bacterial suspension of Ralstonia solanacearum: Inoculate the preserved Ralstonia solanacearum into liquid CPG medium, place it in a constant temperature shaker, and incubate with shaking. After 2 d, wash and resuspend it with sterile water, and adjust it to OD 600 = 1.

[0037] Preparation of CPG plate: Pour CPG with an agar content of 1.5% onto a plate, and then add the Ralstonia solanacearum with OD 600 = 1 into CPG with an agar content of 0.7% at a volume ratio of 1:30, and then pour it onto CPG with an agar content of 1.5%.

[0038] Set a control group (CK): Drop 20 μL of sterile NMAS buffer on the CPG solid medium plate, with 6 replicates in total.

[0039] Set a treatment group (L1): Drop 20 μL of the solution with a concentration of 3×10 4The NJAU-K1 in the predatory state of [number] CFU / mL was dripped onto the CPG solid medium plate, with 6 replicates in total.

[0040] A total of 12 CPG solid medium plates from the above two experimental groups were placed in a bacterial incubator at 28 °C, and the growth of the inhibition zones was observed and recorded.

[0041] Figure 3 For the mixotrophic flagellate NJAU-K1 and Ralstonia solanacearum Co-culture of the two on plates, the effect diagram of the inhibition zone of the mixotrophic flagellate NJAU-K1.

[0042] As can be seen above, the mixotrophic flagellate NJAU-K1 can effectively inhibit the growth of the pathogen of tomato bacterial wilt Ralstonia solanacearum .

[0043] Example 4 Pot experiment on the promotion of tomato growth by the mixotrophic flagellate NJAU-K1 Two treatments were set up in the experiment: (1) blank control group CK; (2) treatment group inoculated with the mixotrophic flagellate NJAU-K1. Neither of the two experimental treatments was inoculated with the pathogen.

[0044] The test tomatoes were red dwarf tomato seedlings, and the test mixotrophic protist was the mixotrophic flagellate NJAU-K1 of Example 2, deposit number: CCTCC NO: V202536.

[0045] Preparation of the mixotrophic flagellate NJAU-K1: The protist NJAU-K1 was inoculated into the liquid NMAS medium, inactivated Escherichia coli (OD = 0.04) was added, and it was placed in a constant temperature incubator at 20 °C for static culture. After 48 h, 100 μL of the culture solution was taken and counted under an inverted microscope.

[0046] Cultivation of tomato plants: The test tomato seedlings were red dwarf tomato seedlings. Healthy tomato seedlings with a height of 3 cm (not infected with the pathogen) were selected and transplanted into 0.5 gallon flower pots. The potting soil was from a farmland in Jiangyan District, Taizhou City, Jiangsu Province. The soil was air-dried and sieved, and plant residues were removed before being used for the pot experiment. The soil used did not contain and was not inoculated with the pathogen. After transplantation, the potting soil of the treatment group was inoculated with NJAU-K1 cultured in the NMAS culture solution, Poterioochromonas malhamensis at a rate of 5×10 3 CFU per gram of soil; the blank control group was inoculated with an equal amount of NMAS culture solution (without mixotrophic protists). After one month of greenhouse cultivation, the growth of the tomato pots was observed.

[0047] Figure 4Growth status of the aboveground and underground parts of tomatoes in the blank control group (left) and the treatment group inoculated with the mixotrophic flagellate NJAU-K1 (right). Figure 5 It shows that in Example 4 of the present invention, inoculation with the mixotrophic flagellate NJAU-K1 significantly increased both the plant height and root weight of tomatoes. It can be seen that the mixotrophic flagellate NJAU-K1 can significantly promote the growth of tomatoes.

Claims

1. A mixotrophic flagellate Poterioochromonas malhamensis ), NJAU-K1, characterized in that It is deposited in the China Center for Type Culture Collection. The deposition date is May 8, 2025, and the deposition number is CCTCC NO: V202536.

2. The culture of the mixotrophic flagellate Poterioochromonas malhamensis ) NJAU-K1 as claimed in claim 1, characterized in that The described culture is prepared by the following method: inoculating the mixotrophic flagellate ( Poterioochromonas malhamensis ) NJAU-K1 into the NMAS culture medium, adding inactivated microorganisms as food, placing it in an incubator at 18-20 °C, and allowing it to stand for 40-50 h.

3. The preparation method of the culture according to claim 2, characterized in that, Inoculate the mixotrophic flagellate ([ Poterioochromonas malhamensis ]) NJAU-K1 described in claim 1 into NMAS culture medium, add inactivated microorganisms as food, place it in an incubator at 18 - 20 °C, and let it stand for 40 - 50 h. Poterioochromonas malhamensis ​ 4. The preparation method according to claim 3, characterized in that, The formula of the NMAS culture medium is as follows: sodium chloride 0.12 g / L, magnesium sulfate heptahydrate 0.004 g / L, calcium chloride hexahydrate 0.006 g / L, sodium phosphate 0.142 g / L, potassium phosphate 0.136 g / L.

5. The preparation method according to claim 3, wherein The inactivated microorganism is inactivated Escherichia coli.

6. Use of the mixotrophic flagellate ([ Poterioochromonas malhamensis Poterioochromonas malhamensis ) NJAU-K1 in promoting the growth of tomatoes and / or controlling tomato bacterial wilt.

7. Use of the mixotrophic flagellate Poterioochromonas malhamensis ) NJAU-K1 in the preparation of a product for promoting the growth of tomatoes and / or controlling tomato bacterial wilt.

8. The application according to claim 7, characterized in that The product is a biological pesticide or a growth promoter.

9. Use of the culture according to claim 2 in promoting the growth of tomatoes and / or controlling tomato bacterial wilt.

10. Use of the culture according to claim 2 in the preparation of a product for promoting the growth of tomatoes and / or controlling tomato bacterial wilt.

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