Moraxella roselle NRS-133, fungicide and application of Mraxella roselle NRS-133 in inhibition of cotton verticillium wilt

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to Moraxella marina NRS-133, a fungicide and application of the Mraxella marina NRS-133 in inhibition of cotton verticillium wilt. The seawater Moraxella roselle NRS-133 disclosed by the invention is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.35930. The Moraxella roselle NRS-133 provided by the invention has a remarkable inhibition effect on verticillium dahliae (verticillium dahliae), and can be used for preventing and treating cotton verticillium wilt.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microorganisms, and particularly relates to a seawater Moraxella rossius NRS-133, a bacterial agent and application of the bacterial agent in inhibiting cotton verticillium wilt. BACKGROUND

[0002] Cotton verticillium wilt is an important soil-borne disease of cotton caused by the fungal pathogen Verticillium dahliae, which widely exists in global cotton planting areas and causes serious threats to cotton production. The disease has strong pathogenicity, and the pathogen can survive in the soil for a long time and infect plants through the roots, leading to symptoms such as wilting, yellowing and growth restriction of cotton plants. Cotton verticillium wilt causes huge economic losses to agricultural production every year. At present, the main method for preventing and treating cotton verticillium wilt at home and abroad relies on chemical pesticides such as bromomethane. However, long-term reliance on chemical agents not only leads to drug resistance of the pathogen, thereby reducing the prevention and treatment effect, but also may cause environmental pollution and pesticide residues. In addition, due to the weak host plant specificity of the cotton verticillium wilt pathogen, the difference in disease resistance between crop varieties is very small, and the prevention and treatment work faces great challenges. In order to reduce the negative impact of chemical pesticides on the environment, in recent years, biological control as a green and environmentally friendly control method has attracted widespread attention. In particular, the use of microorganisms with antagonistic effects to inhibit pathogens has become an effective and sustainable solution. At present, the strains with strong effects on cotton verticillium wilt reported mainly include Bacillus, such as Bacillus subtilis NCD-2, Bacillus sp. SHZ-24, SHT-15, SMT-24 and BHZ-29. Trichoderma, Chaetomium globosum, Streptomyces TD3-2-1 and Streptomyces albus KF-43-1 have certain antagonistic effects on cotton verticillium wilt. However, most of the biocontrol strains are still in the laboratory research stage. Therefore, it is of great significance to develop a functional strain that can effectively inhibit cotton verticillium wilt for promoting agricultural production. Therefore, it is necessary to provide an improved technical solution to solve or improve the problems in the prior art that chemical agents are usually used to prevent and treat cotton verticillium wilt, which is prone to drug resistance and reduces the prevention and treatment effect. SUMMARY

[0003] The application aims to provide a seawater Moraxella rossius NRS-133, a bacterial agent and application of the bacterial agent in inhibiting cotton verticillium wilt, so as to help solve or improve the problem in the prior art that chemical agents are usually used to prevent and treat cotton verticillium wilt, which is prone to drug resistance and reduces the prevention and treatment effect.

[0004] In order to achieve the above-mentioned purpose, the present application provides the following technical scheme: a Rossellomora aquimaris NRS-133, which is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 35930.

[0005] The present application also provides a microbial agent, which adopts the following technical scheme: a microbial agent containing the Rossellomora aquimaris NRS-133 as described above.

[0006] Preferably, the concentration of the Rossellomora aquimaris NRS-133 in the microbial agent is 1×10 7 cfu / mL.

[0007] The present application also provides a preparation method of a microbial agent, which adopts the following technical scheme: the preparation method of the microbial agent as described above, comprising the following steps: culturing the Rossellomora aquimaris NRS-133 by using a beef extract tryptone agar medium, adjusting the concentration of the Rossellomora aquimaris NRS-133 in the culture solution, and obtaining the microbial solution.

[0008] The present application also provides the application of the Rossellomora aquimaris NRS-133 or the microbial agent as described above, which adopts the following technical scheme: the application of the Rossellomora aquimaris NRS-133 or the microbial agent as described above in inhibiting cotton verticillium wilt.

[0009] Preferably, the Rossellomora aquimaris NRS-133 or the microbial agent is used for inhibiting L. theoma.

[0010] Preferably, the following step is included: inoculating the Rossellomora aquimaris NRS-133 or the microbial agent when the cotton seedlings grow to two true leaves.

[0011] Beneficial effects: The Rossellomora aquimaris NRS-133 of the present application is preserved in the China General Microbiological Culture Collection Center (address: No. 3, Beichen West Road, Chaoyang District, Beijing), and the preservation date is September 16, 2025, and the preservation number is CGMCC No. 35930. The Rossellomora aquimaris NRS-133 of the present application has a significant inhibitory effect on cotton verticillium wilt (L. theoma), and can be used for the prevention and treatment of cotton verticillium wilt. BRIEF DESCRIPTION OF DRAWINGS

[0012] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the application and together with the description, serve to explain the application. In the drawings, Figure 1 Colony morphology of Rossellomorea aquimaris NRS-133 of the application; Figure 2 Phylogenetic tree of Rossellomorea aquimaris NRS-133 and related strains of the application based on 16S rRNA sequence; Figure 3 Antagonistic effect of Rossellomorea aquimaris NRS-133 of the application on Verticillium dahliae; Figure 4 Cotton seedling Verticillium wilt; Figure 5 Agarose gel electrophoresis detection result of 16S rDNA sequence PCR amplification of Rossellomorea aquimaris NRS-133 of the application. DETAILED DESCRIPTION

[0013] The technical solutions in the embodiments of the application will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments of the application. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art belong to the protection scope of the application.

[0014] The application will be described in detail below with reference to the embodiments. It should be noted that the embodiments in the application and the features in the embodiments can be combined with each other without conflict.

[0015] The application provides Rossellomorea aquimaris NRS-133 to solve the problem that the cotton Verticillium wilt is usually prevented and treated by chemical agents in the prior art, and the resistance is easy to be generated to reduce the prevention and treatment effect.

[0016] Rossellomorea aquimaris NRS-133 of the embodiments of the application is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 35930.

[0017] Rossellomorea sp. is a new genus divided from Bacillus, which is generally gram-negative, forms endospores, and moves by unipolar flagella or peritrichous flagella. The genus currently includes four validly published species (Rossellomorea aquimaris, R. enclensis, R. marisflavi and R. vietnamensis). In recent years, more and more Rossellomorea sp. are discovered, and it is proved that Rossellomorea sp. has significant potential in improving saline-alkali soil and promoting plant growth. However, there is no report on the inhibition of cotton verticillium wilt.

[0018] The seawater Rossellomorea sp. NRS-133 of the present application has a significant inhibitory effect on cotton verticillium wilt (L. theicola), and can be used for the prevention and treatment of cotton verticillium wilt. The seawater Rossellomorea sp. NRS-133 of the present application helps to solve the problem that the use of chemical agents for the prevention and treatment of cotton verticillium wilt is prone to drug resistance, thereby reducing the prevention and treatment effect.

[0019] The present application also provides a bacterial agent, and the bacterial agent of the present application contains the seawater Rossellomorea sp. NRS-133 as described above.

[0020] In a preferred embodiment of the bacterial agent of the present application, the concentration of the seawater Rossellomorea sp. NRS-133 in the bacterial agent is 1x10 7 cfu / mL.

[0021] The present application also provides a preparation method of the bacterial agent as described above, and the preparation method of the bacterial agent of the present application comprises the following steps: culturing the seawater Rossellomorea sp. NRS-133 by using beef extract tryptone agar medium, then inoculating and culturing in beef extract tryptone liquid medium, adjusting the concentration of the seawater Rossellomorea sp. NRS-133, and obtaining the bacterial agent.

[0022] The present application also provides an application of the seawater Rossellomorea sp. NRS-133 or the bacterial agent as described above, and the seawater Rossellomorea sp. NRS-133 or the bacterial agent as described above is used for inhibiting cotton verticillium wilt.

[0023] In a preferred embodiment of the application of the present application, the seawater Rossellomorea sp. NRS-133 or the bacterial agent as described above is used for inhibiting L. theicola.

[0024] In a preferred embodiment of the application of the present application, the following steps are included: inoculating the seawater Rossellomorea sp. NRS-133 or the bacterial agent as described above when the cotton seedlings grow to two true leaves.

[0025] The application will be described in detail below by specific examples.

[0026] The sources of the main raw materials used in the following examples are as follows: PDA, PDB medium, beef extract tryptone medium and agar are all Coolaber brand. The strain identification company is Shanghai Generay.

[0027] Example 1 Isolation, screening and identification of seawater Moraxella rossius NRS-133: In 2025, soil samples were collected from the rhizosphere of cotton in Weixian County, Hebei Province. 10 g of freshly collected soil was poured into a triangular flask containing 90 mL of sterile water and shaken for 10 min to form a 1 / 10 concentration suspension. The room temperature was continued to be shaken for 30 min. 1 mL of supernatant was taken and prepared into a 10 -4 or 10 -5 concentration diluent for standby.

[0028] The melted medium (beef extract tryptone agar medium: beef extract 10 g, tryptone 5 g, sodium chloride 5 g, agar 18 g, distilled water 1000 mL, pH 7.0-7.2) was sterilized and poured into a sterilized culture dish. After solidification, 20 μL of 10 -4 concentration diluent was dropped on the surface of the solidified medium. A sterile coating rod was used to evenly coat the culture dish, which was inverted in a 28℃ constant temperature incubator for 1-2 d. After the colonies grew, the single colonies with round shape, milky white color, flat, regular edge, slightly convex center, moist and smooth surface without pigment were streaked on beef extract tryptone agar medium and repeated isolation and purification for 3 times. Finally, the single colony was named NRS-133 and subjected to physiological and biochemical identification and 16S rDNA sequencing.

[0029] The results of physiological and biochemical identification are shown in Table 1. Table 1. Results of physiological and biochemical identification

[0030] 16S rDNA gene identification: Identification method: the cultured seawater Moraxella rossius NRS-133 plate is sent to Shanghai Shenguo, the Ezup column type bacterial genome DNA extraction kit is used to extract DNA, and 16S rDNA sequence is amplified by using 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') primers. The PCR reaction system and conditions are carried out according to table 2 to table 3 (the corresponding experiment is entrusted to Shanghai Shenguo). After PCR amplification, the product is subjected to 1% agarose gel electrophoresis (1X TAE electrophoresis buffer) test, and there is obvious characteristic band (such as Figure 5 indicated).

[0031] Table 2 PCR reaction system for 16S rRNA amplification

[0032] Table 3 PCR reaction conditions

[0033] The sequence length of 16S rDNA of the seawater Moraxella rossius NRS-133 of the application is 1488bp, and the sequence is as follows:

[0034] The 16S rDNA sequencing results were compared and analyzed with the sequences in GenBank, and the results showed that the strain NRS-133 had the highest similarity with the Bacillus aquimaris strain M2 16S ribosomal RNA gen gene sequence, and the homology was 100%. Bacillus aquimaris was reclassified as Rossellomorea aquimaris. Based on the above identification results of the strain NRS-133, it was determined that the strain NRS-133 belonged to Rossellomorea aquimaris, and the strain was named as Rossellomorea aquimaris NRS-133. The phylogenetic tree of Rossellomorea aquimaris NRS-133 and related strains based on 16S rRNA sequence is shown in Figure 2 .

[0035] Example 2 Application of Rossellomorea aquimaris NRS-133 in inhibiting cotton verticillium wilt: Two treatments were set in the test. The control group (CK) was to place a 6mm-diameter Verticillium dahliae disc in the center of a culture dish containing PDA medium (as shown in the left part of Figure 3 the middle of the figure), and the treatment group was to place a 6mm-diameter Verticillium dahliae disc, and then use a loop dipped with Rossellomorea aquimaris NRS-133 strain to draw a circle (as shown in the right part of Figure 3 the middle of the figure) at a distance of 2.5cm above, below, left and right of the disc, and then cultivate at 25℃ for 7d, and then measure the colony diameter of Verticillium dahliae.

[0036] The inhibition rate (%) = colony diameter (CK) - colony diameter (treatment group) / colony diameter (CK). Among them, "colony diameter (CK)" refers to the colony diameter of Verticillium dahliae after the control group is cultivated at 25℃ for 7d; "colony diameter (treatment group)" refers to the colony diameter of Verticillium dahliae after the Rossellomorea aquimaris NRS-133 strain is inoculated according to the above method and cultivated at 25℃ for 7d.

[0037] The test results showed that the strain Rossellomorea aquimaris NRS-133 could strongly inhibit the growth of cotton verticillium wilt (Verticillium dahliae), and the inhibition rate reached 83.1% ( Figure 3 ). The results showed that the Rossellomorea aquimaris NRS-133 of the application had the effect of inhibiting Verticillium dahliae.

[0038] Example 3 The bacterial agent of the present embodiment contains seawater Roseburia NRS-133, which is prepared by a method comprising the following steps: The seawater Roseburia NRS-133 was transferred to fresh beef extract tryptone agar solid medium and incubated at 28°C for 1 day, then inoculated into a 500 mL triangular flask containing 100 mL of fresh beef extract tryptone liquid medium, and cultured on a rotary shaker at 28°C and 180 r / min for 24-48 h to obtain a bacterial suspension. The suspension was diluted with sterile water, and 2-3 spores were observed under a microscope in the smallest grid of a hemocytometer, which was 1.0 x 10 7 cfu / mL, and prepared immediately before use.

[0039] The application of the bacterial agent of the present embodiment is to explore the suitable amount of application of the bacterial agent.

[0040] The 8 cm x 8 cm x 6 cm flowerpots were washed with clean water, then wiped with 70% alcohol and dried; 100 g of nutrient soil was placed in each flowerpot, and 6 cotton seeds were sown in each pot with a planting depth of 2-3 cm; the nutrient pots were placed in 28 cm x 44 cm plastic trays, 15 flowerpots were placed in each tray, and an equal amount of 1 L distilled water was added to each tray to allow the soil to naturally absorb the water; the plastic trays were placed in an artificial climate chamber for cultivation, and the cultivation conditions were as follows: 16 h of light, 24°C, 50% humidity, and 60% light intensity during the day; 8 h of darkness, 20°C, 70% humidity, and 0% light intensity at night; and the seedlings were watered with an equal amount of distilled water every 4 days to facilitate germination, and cotyledons emerged after about 3 days; after the cotyledons unfolded, 3 well-grown cotton seedlings were left in each pot, and the other seedlings were manually removed. The cotton seedlings grew to two true leaves after about 15 days, and the bacterial agent of the present embodiment was applied according to different treatments. The cotton seedlings grew to three true leaves after about 20 days, and were inoculated with Lecanicillium fungicidum using the wounded root method, with a concentration of 1.0 x 10 7 cfu / mL and a dosage of 20 mL per pot. After 20 days of inoculation with L. fungicidum, the cotton seedlings were observed for yellow wilt disease.

[0041] The test was set up with a blank control (CK), a negative control (V), inoculation of the bacterial agent at 2 mL / pot (T1), 10 mL / pot (T2), 20 mL / pot (T3), 40 mL / pot (T4), and 200 mL / pot (T5), wherein: the CK group was inoculated with sterile water only (i.e., 20 mL of sterile water per pot when the cotton seedlings grew to two true leaves and three true leaves); and the V group was inoculated with L. fungicidum only (i.e., 20 mL of L. fungicidum solution per pot when the cotton seedlings grew to two true leaves and three true leaves). The total number of plants in each group was 45.

[0042] The cotton Verticillium wilt liquid (Lagerstroemia ferrea liquid) is prepared by the following method: the Verticillium wilt (Lagerstroemia ferrea) existing at 4°C is transferred to fresh PDA medium, sealed with a sealing film, and then the culture dish is inverted and cultured in a dark incubator at 25°C for 7 days; then the grown colonies are divided into about 1 cm blocks with an inoculation needle, and the blocks are placed in PDB culture solution, and cultured in a constant temperature shaker (25°C, 130 rpm / min); after the spore suspension is obtained by filtering the mycelium with gauze, the suspension is diluted with corresponding sterile water, and 2-3 spores are observed in the smallest grid of a hemocytometer under a microscope, preferably 1.0 x 10 7 cfu / mL, and it is prepared immediately before use.

[0043] The prevention and treatment effect of the microbial agent of the present embodiment on cotton Verticillium wilt is shown in the following table: Table 4

[0044] Compared with the negative control group (V), the experimental groups were inoculated with different amounts of microbial agents containing seawater Moraxella rossius NRS-133 of the present embodiment; among them, the cotton Verticillium wilt incidence of 2 mL and 10 mL inoculation concentration was higher than that of 20 mL, 40 mL and 200 mL, and the cotton seedling Lagerstroemia ferrea incidence of 20 mL, 40 mL and 200 mL inoculation was the same (incidence = number of diseased plants / total number of plants; the number of diseased plants in the V group was 30, the number of diseased plants in the T1 group was 29, the number of diseased plants in the T2 group was 19, and the number of diseased plants in the T3, T4 and T5 groups was 6; the total number of plants was 45). This result shows that the optimal application amount of the microbial agent containing NRS-133 is 20 mL / pot.

[0045] Example 4 The microbial agent of the present embodiment contains seawater Moraxella rossius NRS-133, which is prepared by the following method: The seawater Moraxella rossius NRS-133 is transferred to fresh beef extract tryptone agar solid medium, and after 1 day of static culture at 28°C, it is inoculated into a 500 mL triangular flask containing 100 mL of fresh beef extract tryptone liquid medium, and then it is cultured in a rotary shaker at 28°C and a rotation speed of 180 r / min for 24-48 h to obtain a bacterial liquid. The suspension is diluted with corresponding sterile water, and 2-3 spores are observed in the smallest grid of a hemocytometer under a microscope, preferably 1.0 x 10 7 cfu / mL, and it is prepared immediately before use.

[0046] The application of the microbial agent of the present embodiment to the prevention and treatment of cotton Verticillium wilt (N+V) includes the following steps: 8cm x 8cm x 6cm flower pots are washed with clean water, then wiped with 70% alcohol, and dried; 100g of nutrient soil is loaded into each flower pot, 6 cotton seeds are sown in each pot, and the planting depth is 2cm-3cm; the nutrient pots are placed in 28cm x 44cm plastic trays, 15 flower pots are placed in each tray, and an equal amount of 1L distilled water is added to each tray to allow the soil to naturally absorb the water; the plastic trays are placed in an artificial climate chamber for cultivation, and the cultivation conditions are: 16h of daylight, 24℃, 50% humidity, and 60% light; 8h of night, 20℃, 70% humidity, and 0% light; the cotton seedlings are watered with an equal amount of distilled water every 4d during the seedling period to facilitate germination, and cotyledons grow out after about 3d, 3 well-grown cotton seedlings are left in each pot after the cotyledons unfold, and the other seedlings are manually removed. The cotton seedlings grow to two true leaves after about 15d, the inoculum of the present embodiment (the concentration of seawater Roseburia inositorum NRS-133 is 1.0 x 10 7 cfu / mL) is irrigated, and 20mL is inoculated per pot. The cotton seedlings grow to three true leaves after about 20d, and Verticillium dahliae is inoculated using the wounded root method, the concentration is 1.0 x 10 7 cfu / mL, and the amount is 20mL per pot. After 20d of Verticillium dahliae inoculation, the cotton seedling yellow wilt disease is observed, and the disease index is investigated.

[0047] During the test, a blank control group (CK) and a negative control group (V) are set, wherein: the CK group is only inoculated with sterile water (i.e., 20mL of sterile water is inoculated per pot when the cotton seedlings grow to two true leaves and three true leaves); and the V group is only inoculated with Verticillium dahliae (i.e., 20mL of Verticillium dahliae liquid is inoculated per pot when the cotton seedlings grow to two true leaves and three true leaves).

[0048] The cotton Verticillium wilt fungus liquid (Verticillium dahliae liquid) is prepared by the following method: the Verticillium dahliae (Verticillium dahliae) present at 4℃ is transferred to fresh PDA culture medium, sealed with a sealing film, and then inverted in a 25℃ constant temperature incubator for dark culture for 7d; then the grown colonies are divided into about 1cm blocks with a inoculation needle, the blocks are placed in PDB culture solution, and a constant temperature shaker (25℃, 130rpm / min) is used; after the fungus liquid is filtered with gauze to remove the mycelium, a spore suspension is obtained, the suspension is diluted with sterile water, and 2-3 spores in the smallest grid of a hemocytometer under a microscope are appropriate, i.e., 1.0 x 10 7 cfu / mL, and it is prepared as needed.

[0049] The disease index investigation method is as follows: 0 level: healthy plants, well-developed stems and leaves without disease; 1 level: 1-2 cotyledons of the plant turn yellow, a small number of true leaves turn yellow and dry, and curl; Grade 2: 1 true leaf disease, yellowing and wilting, leaf edge dry, curled or brown mottling; Grade 3: more than 2 true leaf disease, yellowing and wilting, leaf edge dry, curled or leaf abscission only heart leaf; Grade 4: all leaf abscission or plant death; Calculation formula: Disease index = Σ (number of plants at each level x corresponding disease level) / total number of plants surveyed x highest disease level.

[0050] The prevention and treatment effect of the bacterial agent of the present embodiment on cotton Verticillium wilt is as shown in the following table and the following figure: Figure 4 Figure 4 In the figure, from left to right, the test results of the CK group, the V group and the N+V group are shown in order. Table 5

[0051] Compared with the negative control group (V), the disease index of cotton seedlings L. theochromogenes was reduced by 52.87% after inoculation with the bacterial agent containing seawater Moraxella NRS-133 of the present application. This result shows that NRS-133 has the ability to reduce the occurrence of cotton Verticillium wilt and can be used to inhibit cotton Verticillium wilt.

[0052] The above description is only the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.​

Claims

1. A marine Rossellomorea aquimaris NRS-133, characterized in that, The *Rossellomorea aquimaris* NRS-133 strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35930.

2. A microbial agent, characterized in that, The bacterial agent contains *Moraxella rosenbergii* NRS-133 as described in claim 1.

3. The microbial agent as described in claim 2, characterized in that, The concentration of *Moraxella rosenbergii* NRS-133 in the bacterial agent was 1 × 10⁻⁶. 7 cfu / mL.

4. The method for preparing the microbial agent as described in claim 2, characterized in that, The process includes the following steps: culturing *Moraxella rosenbergii* NRS-133 in beef extract tryptone agar medium, then inoculating it into beef extract tryptone liquid medium, adjusting the concentration of *Moraxella rosenbergii* NRS-133 to obtain the bacterial agent.

5. The application of *Moraxella rosenbergii* NRS-133 as described in claim 1 or the bacterial agent as described in claim 2 in the suppression of cotton Verticillium wilt.

6. The application as described in claim 5, characterized in that, The *Roseolario Moraxella salina* NRS-133 or the bacterial agent of claim 2 is used to inhibit *Verticillium dahliae*.

7. The application as described in claim 5, characterized in that, The procedure includes the following steps: when cotton seedlings have grown to two true leaves, inoculate them with *Moravir roselliae* NRS-133 or the aforementioned inoculum.

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