A mixotrophic flagellate and its application in promoting tomato growth and controlling bacterial wilt

Abstract

The present invention discloses a mixotrophic flagellate and its application in promoting tomato growth and preventing and controlling bacterial wilt. The mixotrophic flagellate NJAU‑K1 has a deposit number of CCTCC NO: V202536. Experiments have shown that the mixotrophic flagellate NJAU‑K1 involved in the present invention not only exhibits a significant growth-promoting effect on tomatoes, but also can effectively prevent and control tomato bacterial wilt. The present invention shows that the mixotrophic flagellate NJAU‑K1, as a new type of plant growth-promoting and biocontrol microorganism, has great application potential in increasing crop yields and preventing and controlling soil-borne diseases, providing theoretical and technical support for the green development of agriculture.

Description

Technical Field

[0001] The invention belongs to the technical field of microbiology and plant disease biocontrol, and particularly relates to a mixotrophic flagellate and application thereof in promoting tomato growth and preventing and controlling bacterial wilt. Background Art

[0002] Tomatoes are a widely cultivated horticultural crop with high agricultural and economic value. Tomato bacterial wilt, caused by the bacterium Ralstonia solanacearum (Rs) in the Solanaceae family, is a soil-borne disease that severely harms tomato production and significantly hinders the development of the tomato industry. Rs primarily invades tomato plants through root wounds or natural openings. Once inside, the pathogen rapidly multiplies and spreads within the plant's vascular bundles, causing the tomato plants to begin showing symptoms. Initially, the top leaves of the plant wilt and droop during the day, but return to normal during cooler morning and evening temperatures. As the disease progresses, the lower leaves of the plant also gradually wilt, and the wilting symptoms are irreversible. Brown patches appear on the cortex at the base of the stem, gradually spreading upward. Simultaneously, the roots turn brown and rot, losing their ability to absorb water and nutrients. Ultimately, the plant withers and dies, while the leaves remain green.

[0003] Bacterial wilt causes significant annual yield losses in tomatoes. Numerous methods are currently used to control its damage, including soil fumigation and improvement, biological control, biological agents, and breeding of resistant varieties. However, some methods are less than ideal. For example, non-selective elimination can easily damage beneficial microorganisms in the soil; soil improvement requires significant human, material, and financial resources, and takes a long time to be effective; and, under constantly changing environmental conditions, breeding resistant varieties lacks excellent parents with stable resistance. While biological agents can be used to induce plant resistance, this increases cultivation costs, impacting the industry's economic benefits. Furthermore, multiple factors, including plant variety, growth stage specificity, environmental conditions, and the concentration and method of inducer application, all affect control effectiveness.

[0004] Biological control primarily relies on interactions between organisms, such as predation, parasitism, and competition, using beneficial organisms or their products to suppress or control pest populations, thereby achieving pest control. Currently, many biocontrol bacteria have shown great potential for controlling bacterial wilt. For example, Pseudomonas, Bacillus, and Streptomyces are potentially highly effective biocontrol agents for bacterial wilt. These bacteria inhibit pathogen growth by secreting metabolites or reduce pathogen colonization through competition for niches and nutrients. Recent research has demonstrated that protists also play an important role in soil ecosystems, including the rhizosphere. Predatory protists can regulate soil microbial community structure and population abundance through predation and metabolic activities, enriching beneficial plant microorganisms during the early stages of plant growth to promote bacterial wilt suppression, or directly inhibiting bacterial wilt through predation. Summary of the Invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a mixotrophic protist that promotes tomato growth and prevents and controls bacterial wilt.

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

[0007] Another object of the present invention is to provide applications of the mixed nutritional protists and cultures.

[0008] The purpose of the present invention can be achieved through the following technical solutions:

[0009] A mixotrophic flagellate ( Poterioochromonas malhamensis ) NJAU-K1, deposited in China Center for Type Culture Collection, the deposit date is May 8, 2025, and the deposit number is CCTCC NO: V202536.

[0010] The mixed nutritional flagellate ( Poterioochromonas malhamensis ) NJAU-K1 culture, the culture is prepared by the following method: inoculating the mixotrophic flagellate NJAU-K1 into NMAS culture medium, adding inactivated microorganisms as food, placing in an incubator at 18-20° C., and standing for 40-50 hours.

[0011] The culture preparation method comprises the following steps: inoculating the mixotrophic flagellate NJAU-K1 into NMAS culture medium, adding inactivated microorganisms as food, placing the culture medium in an incubator at 18-20° C., and allowing the culture medium to stand for 40-50 hours.

[0012] As a preferred embodiment of the present invention, the formula of the NMAS culture medium is 0.12 g / L of sodium chloride, 0.004 g / L of magnesium sulfate heptahydrate, 0.006 g / L of calcium chloride hexahydrate, 0.142 g / L of sodium phosphate, and 0.136 g / L of potassium phosphate.

[0013] As a preferred embodiment of the present invention, the inactivated microorganism is inactivated Escherichia coli.

[0014] The mixed nutritional flagellate ( Poterioochromonas malhamensis )Application of NJAU-K1 in promoting tomato growth and / or preventing and controlling tomato bacterial wilt.

[0015] The mixed nutritional flagellate ( Poterioochromonas malhamensis )Application of NJAU-K1 in the preparation of products for promoting tomato growth and / or preventing and controlling tomato bacterial wilt.

[0016] The product is preferably a biological pesticide or a growth promoter.

[0017] The culture is used in promoting tomato growth and / or preventing and controlling tomato bacterial wilt.

[0018] The culture is used in preparing products for promoting tomato growth and / or preventing and treating tomato bacterial wilt.

[0019] Beneficial effects

[0020] The present invention isolates and screens a mixotrophic flagellate with strong resistance to tomato bacterial wilt pathogens from the rhizosphere of healthy crops ( Poterioochromonas malhamensis )NJAU-K1, bacterial pathogen co-culture experiments showed that the mixed nutritional flagellate ( Poterioochromonas malhamensis )NJAU-K1 has a significant control effect on tomato bacterial wilt. In addition, the mixed nutritional flagellate ( Poterioochromonas malhamensis )NJAU-K1 can also significantly promote the growth of tomatoes. Therefore, the mixed nutritional flagellate of the present invention ( Poterioochromonas malhamensis )NJAU-K1 It is of great significance to the prevention and control of soil-borne diseases and the promotion of plant growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 These are photographs of the morphological characteristics of the mixotrophic protist NJAU-K1 in the present invention in its dormant cyst (left), autotrophic state (middle), and predatory state (right).

[0022] Figure 2 This is a phylogenetic tree of the 18S rDNA gene sequence of the protist NJAU-K1 of the present invention.

[0023] Figure 3For the protists NJAU-K1 and Ralstonia solanacearum The two were co-cultured on CPG plates, and the inhibition zone effect of the protist NJAU-K1 was shown.

[0024] Figure 4 The figures show the growth conditions of the aboveground and underground parts of tomato plants in the blank control group (left) and the treatment group inoculated with the protist NJAU-K1 (right) in Example 4 of the present invention.

[0025] Figure 5 This is a comparison of plant height and root weight between the blank control group and the treatment group inoculated with the protist NJAU-K1 in Example 4 of the present invention.

[0026] Biomaterial deposit information

[0027] Mixotrophic flagellate NJAU-K1 Poterioochromonas malhamensis , deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, China, the deposit date is May 8, 2025, and the deposit number is CCTCC NO: V202536. DETAILED DESCRIPTION

[0028] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent companies. The experiments in the following examples were all performed in triplicate.

[0029] Example 1 Isolation and purification of mixotrophic flagellates

[0030] The inventors collected rhizosphere soil from healthy tomato plants in Nanjing, Jiangsu Province, at a depth of 0-20 cm. The soil was sealed in sterile ziplock bags and brought back to the laboratory. The target pathogen, Ralstonia solanacearum, was provided by the Soil and Organic Fertilizer Team at Nanjing Agricultural University.

[0031] Mixotrophic protists were isolated using the soil dilution method. The following steps were performed: Rhizosphere soil samples from healthy crops were mixed thoroughly. 1 g of soil was weighed and placed in a 50 mL centrifuge tube, followed by 30 mL of sterile deionized water. The centrifuge tube was then shaken at 250 rpm and 20°C for 15 minutes to thoroughly mix the rhizosphere soil and release the protists. The centrifuge tube was removed from the shaker and allowed to stand for 10 minutes. The supernatant was then transferred to a 96-well plate and supplemented with Escherichia coli (OD = 0.04) as food. The plate was then incubated at 20°C in the dark for 2 days. Protist growth was examined under an inverted microscope at 100×, 200×, and 400× magnifications. Serial dilutions were then performed and incubated at 20°C for another 2 days. Finally, a single mixotrophic protist cell was picked using a capillary pipette and transferred to a new 96-well plate to obtain a pure culture of the protist.

[0032] Example 2 Identification of the Mixotrophic Protist NJAU-K1

[0033] The 18S rDNA of the screened protists was identified using a combination of morphological observation and molecular biology. The PCR reaction system (50 μL system) consisted of 2 μL of each primer, 25 μL of 2× Mix, and ddH2O to a final volume of 50 μL. The most commonly used universal primers were P-FLA-F / P-FLA-R, with P-FLA-F (SEQ ID NO. 2) and P-FLA-R (SEQ ID NO. 3). The reaction procedure included 35 cycles of initial denaturation at 94°C for 3 minutes, denaturation at 94°C for 55 seconds, annealing at 50°C for 50 seconds, extension at 72°C for 1 minute, and extension at 72°C for 10 minutes. After 35 cycles, the product was stored at 16°C. The PCR product was recovered by electrophoresis on a 1.5% agarose gel and sent to Beijing Qingke Biotechnology Co., Ltd. for Sanger sequencing. The sequencing result is shown in SEQ ID NO. 1.

[0034] The obtained 18s rDNA gene sequences were compared with the PR2 (Protist Ribosomal Reference) database and the NT (Nucleotide Sequence Database) database of the NCBI website using BLAST software, and the phylogenetic analysis of the isolated mixotrophic protists was performed using MEGA software. Poterioochromonas malhamensis The similarity was the highest, reaching 99.72%, so the isolated mixotrophic protist NJAU-K1 was identified as Poterioochromonas malhamensis . Figure 2 This is a phylogenetic tree constructed based on 18S rDNA gene sequences.

[0035] In NMAS culture medium, the dormant cysts of the mixotrophic protist NJAU-K1 are round, static, and without flagella ( Figure 1 Left); its autotrophic state is round, swims faster, and has a single flagellum ( Figure 1 In the predatory state, it is active, has a single flagellum, and swims fast ( Figure 1 right). Figure 1 This is a photograph of the morphological characteristics of the mixotrophic protist NJAU-K1 of the present invention.

[0036] The strain was deposited in the China Center for Type Culture Collection (address: China, Wuhan, Wuhan University Collection Center), on May 8, 2025, with the deposit number: CCTCC NO: V202536.

[0037] Example 3 Co-culture experiment of the mixotrophic flagellate NJAU-K1 against Ralstonia solanacearum

[0038] For the test tomato solanacearum ( Ralstonia solanacearum ), the mixotrophic protist used was the mixotrophic flagellate NJAU-K1 of Example 2, with a deposit number of CCTCC NO: V202536.

[0039] Preparation of Mixotrophic Flagellate NJAU-K1: Inoculate mixotrophic flagellate NJAU-K1 into liquid NMAS medium, add inactivated Escherichia coli (OD = 0.04), and incubate in a constant temperature incubator at 20°C. After 48 hours, aspirate 100 μL of the culture medium and count under an inverted microscope. Dilute to 3 × 10 4 pieces / mL.

[0040] Preparation of tomato bacterial wilt pathogen suspension: inoculate the preserved tomato bacterial wilt pathogen Ralstonia solanacearum into liquid CPG culture medium, place in a constant temperature shaker, and shake culture. After 2 days, wash and resuspend with sterile water and adjust to OD 600 =1.

[0041] Preparation of CPG plates: Pour 1.5% CPG agar onto the plates, and then 600 =1 Ralstonia solanacearum was added to CPG with an agar content of 0.7% at a volume ratio of 1:30, and then poured onto CPG with an agar content of 1.5%.

[0042] Set up a control group (CK): add 20 μL of sterile NMAS buffer dropwise onto the CPG solid culture medium plate, with a total of 6 replicates.

[0043] Set up a treatment group (L1): add 20 μL of 3×10 4The preyed NJAU-K1 at a concentration of 100 / mL was dropped onto a CPG solid culture medium plate, with a total of 6 replicates.

[0044] A total of 12 CPG solid culture medium plates from the above two experimental groups were placed in a 28°C bacterial incubator, and the growth of the inhibition zones was observed and recorded.

[0045] Figure 3 Mixotrophic flagellates NJAU-K1 and Ralstonia solanacearum The two were co-cultured on a plate, and the inhibition zone effect of the mixed nutritional flagellate NJAU-K1 was shown.

[0046] As can be seen from the above, the mixed nutritional flagellate NJAU-K1 can effectively inhibit the pathogen of tomato bacterial wilt Ralstonia solanacearum growth.

[0047] Example 4 Potted experiment on promoting tomato growth by mixotrophic flagellate NJAU-K1

[0048] Two treatments were set up in the experiment: (1) blank control group CK; (2) treatment group inoculated with mixotrophic flagellate NJAU-K1. No pathogens were inoculated in either treatment.

[0049] The tested tomatoes were red dwarf tomato seedlings, and the tested mixotrophic protists were the mixotrophic flagellate NJAU-K1 of Example 2, with a deposit number of CCTCC NO: V202536.

[0050] Preparation of mixotrophic flagellate NJAU-K1: Inoculate the protist NJAU-K1 into liquid NMAS medium, add inactivated Escherichia coli (OD = 0.04), and incubate in a 20°C incubator for 48 hours. Aspirate 100 μL of the culture medium and count under an inverted microscope.

[0051] Tomato plant cultivation: The test tomato seedlings were red dwarf tomato seedlings. Healthy tomato seedlings (not infected with pathogens) with a height of 3 cm were selected and transplanted into 0.5 gallon pots. The potting soil was obtained from farmland in Jiangyan District, Taizhou City, Jiangsu Province. The soil was air-dried and sieved to remove plant debris before use in potted experiments. The soil used did not contain and was not inoculated with pathogens. After transplanting, the soil in the treatment group pots was inoculated with NMAS culture medium. Poterioochromonas malhamensis NJAU-K1, 5×10 3 indivual The blank control group was inoculated with an equal amount of NMAS culture medium (without mixotrophic protists). The growth of the tomato potted plants was observed after one month of greenhouse cultivation.

[0052] Figure 4The growth 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 The results show that in Example 4 of the present invention, inoculation with the mixotrophic flagellate NJAU-K1 significantly increased both plant height and root weight of tomatoes. This indicates that the mixotrophic flagellate NJAU-K1 can significantly promote the growth of tomatoes.

Claims

1. A mixotrophic flagellate Poterioochromonas malhamensis NJAU-K1, characterized by It was deposited in the China Center for Type Culture Collection on May 8, 2025, and the deposit number is CCTCC NO: V202536.

2. The mixed nutritional flagellate according to claim 1 Poterioochromonas malhamensis The culture of NJAU-K1 is characterized in that The culture is prepared by the following method: Poterioochromonas malhamensis NJAU-K1 was inoculated into NMAS culture medium, inactivated microorganisms were added as food, and the culture medium was placed in an incubator at 18-20°C for 40-50 h.

3. The method for preparing the culture according to claim 2, characterized in that: The mixotrophic flagellate according to claim 1 Poterioochromonas malhamensis NJAU-K1 was inoculated into NMAS culture medium, inactivated microorganisms were added as food, and the culture medium was placed in an incubator at 18-20°C for 40-50 h.

4. The preparation method according to claim 3, characterized in that The formula of the NMAS culture medium is as follows: 0.12 g / L sodium chloride, 0.004 g / L magnesium sulfate heptahydrate, 0.006 g / L calcium chloride hexahydrate, 0.142 g / L sodium phosphate, and 0.136 g / L potassium phosphate.

5. The preparation method according to claim 3, characterized in that The inactivated microorganism is inactivated Escherichia coli.

6. The mixed nutritional flagellate according to claim 1 Poterioochromonas malhamensis Application of NJAU-K1 in promoting tomato growth and / or preventing and controlling tomato bacterial wilt.

7. The mixed nutritional flagellate according to claim 1 Poterioochromonas malhamensis Application of NJAU-K1 in preparing products for promoting tomato growth and / or preventing and controlling tomato bacterial wilt.

8. The use 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 tomato growth and / or preventing tomato bacterial wilt.

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

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