Microbacterium luteolum C10-2 antagonizing cucumber downy mildew and application thereof

Abstract

The application provides a strain of antagonistic cucumber downy mildew Microbacterium luteum C10-2 and application thereof, and belongs to the technical field of biocontrol bacteria. The Microbacterium luteum C10-2 is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 20253025. The strain can effectively inhibit cucumber downy mildew. Indoor test shows that the spore germination inhibition rate of the strain on Pseudoperonospora cubensis is 86.54%, the leaf disc prevention efficiency is 84.23%, and the strain is superior to the chemical agent dimethomorph; the fermentation liquor of the strain is sprayed in a field at a concentration of 1x10 8 cfu / mL, and the prevention efficiency reaches 80.50%. The strain can stably colonize in cucumber, inhibit pathogenic bacteria infection, and is environment-friendly, free of drug hazards and not prone to drug resistance. The application provides a new strain resource for green prevention and control of cucumber downy mildew, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biocontrol technology, and in particular to a strain of Rhizobium bacillus C10-2 that antagonizes cucumber downy mildew and its application. Background Technology

[0002] Cucumber is a major economic vegetable crop in my country, cultivated both in greenhouses and in open fields. Its widespread planting area and large market demand make it a core crop for ensuring vegetable supply and increasing farmers' income. Throughout the entire growth cycle of cucumbers, frequent pests and diseases consistently hinder the development of high-quality and high-yield cucumber production. Downy mildew is one of the most serious and devastating epidemic diseases caused by oomycetes. The pathogen is *Pseudoperonospora cubensis*, an obligate parasite characterized by rapid spread, wide disease range, strong outbreaks, and severe damage. It can spread rapidly through various means, including airflow, rainwater, and agricultural operations. Low temperature, high humidity, and poor ventilation in the growing environment greatly increase the risk of large-scale epidemics.

[0003] Downy mildew of cucumbers primarily affects the leaves. In the early stages, small, pale yellow, water-soaked spots appear on the leaves. As the disease progresses, these spots enlarge and merge, causing the leaves to wither, curl, and fall off. This severely impacts photosynthesis, leading to weakened plant growth, reduced fruit set, and deformed fruits, significantly decreasing cucumber yield and market quality. Statistical data shows that this disease can cause 20% to 40% yield loss annually. In rainy, humid years with continuous cropping, the disease incidence rate can reach over 90%, resulting in yield reductions exceeding 70%. In severe cases, it can cause complete crop failure, causing enormous economic losses to the vegetable farming industry. It is a core disease problem that urgently needs to be solved in the large-scale, standardized cultivation of cucumbers.

[0004] Biological control has become the mainstream development direction for crop disease control due to its advantages such as safety and non-toxicity, environmental friendliness, low likelihood of developing drug resistance, and ability to improve the crop micro-ecological environment. However, in the existing research on biocontrol strains, common biocontrol bacteria such as Bacillus and Streptomyces have defects such as weak field adaptability, poor colonization ability, and unstable control efficacy, making it difficult to adapt to complex field planting environments and limiting the actual promotion and application effects.

[0005] Plant endophytic bacteria are bacteria that colonize within plants. They not only do not harm the plant but also inhibit pathogen growth and reproduction and block disease infection through various mechanisms, such as secreting antibacterial metabolites and inducing systemic resistance in plants. Simultaneously, they can promote crop growth to a certain extent, possessing both disease prevention and growth-promoting effects, making them a highly promising biocontrol resource. Therefore, developing an endophytic bacterium capable of controlling cucumber downy mildew can provide technical support for the sustainable control of this disease, and has significant practical and application value for promoting the development of my country's agricultural biological control industry and assisting in the upgrading of the green vegetable industry. Summary of the Invention

[0006] In view of this, the present invention provides a strain of *Rhizobium bacillus* C10-2 that antagonizes cucumber downy mildew and its application. This strain is a plant endophytic strain that can stably colonize cucumber plants and has a strong inhibitory effect on the pathogen of cucumber downy mildew. It can effectively inhibit the mycelial growth, spore germination and infection spread of the pathogen, and has a stable and excellent disease control effect in the field. At the same time, it is safe and non-toxic to cucumber plants and has good environmental compatibility and biosafety.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a strain of Microbacterium testaceum C10-2, deposited at the China Center for Type Culture Collection (CCTCCNO: M 20253025), on December 29, 2025, at Wuhan University.

[0009] The present invention also provides a microbial inoculant containing the aforementioned *Rhizobium bacillus* C10-2.

[0010] Preferably, the viable bacterial concentration of *Rhizobium bacillus* C10-2 in the microbial agent is ≥1×10⁻⁶. 8 cfu / mL.

[0011] The present invention also provides a method for preparing the microbial agent, wherein Bacillus rubrum C10-2 is inoculated into a fermentation medium for fermentation culture.

[0012] Preferably, the fermentation culture temperature is 28~32℃ and the rotation speed is 160~200 r / min.

[0013] Preferably, the fermentation culture time is 48~72 h.

[0014] The present invention also provides the application of the aforementioned *Rhizobium bacillus* C10-2 in any one or more of the following:

[0015] (1) Application in inhibiting *Pseudomonas columbarium*;

[0016] (2) Application in the prevention and control of cucumber downy mildew;

[0017] (3) Application in the preparation of agents to inhibit *Peronospora cucumeroides*;

[0018] (4) Application in the preparation of fungal agents for the prevention and control of cucumber downy mildew.

[0019] The present invention also provides a method for preventing and controlling downy mildew in cucumbers by spraying the aforementioned microbial agent onto cucumber plants.

[0020] By adopting the above technical solution, the present invention has the following beneficial effects: The *Microbacterium tumefaciens* C10-2 of the present invention is deposited at the China Center for Type Culture Collection (CCTCCNO: M 20253025). Indoor experiments show that its inhibition rate against *Persona cucurbita* spore germination is 86.54%, and its leaf disc control efficacy is 84.23%, which is superior to the chemical agent dimethomorph. Field spraying at 1×10⁻⁶... 8 The fermentation broth of *Rhizobium bacillus* C10-2 (cfu / mL) achieved a control efficacy of 80.50%. This strain can stably colonize cucumbers, inhibit pathogen infection, and is environmentally friendly, phytotoxic, and unlikely to induce drug resistance. This invention provides a novel strain resource for the green control of cucumber downy mildew, with broad application prospects. Attached Figure Description

[0021] Figure 1 The image shows the control effect of strain C10-2 on cucumber downy mildew as determined by the leaf disc method.

[0022] Figure 2 Phylogenetic tree of 16S rDNA for strain C10-2.

[0023] Figure 3 This image shows the field control effect of strain C10-2 on cucumber downy mildew.

[0024] Biological Preservation Instructions

[0025] The taxonomic name of Microbacterium testaceum C10-2 of this invention is Microbacterium testaceum. It is deposited at the China Center for Type Culture Collection (CCTCCNO: M20253025) on December 29, 2025, at Wuhan University. Detailed Implementation

[0026] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0027] Example 1. Isolation, purification and preservation of endophytic bacteria in plants

[0028] Fifteen healthy cucumber plants from severely affected cucumber downy mildew-infected plots in Xinping Village and Zhongbao Village, Beiwan Town, Baiyin City, Gansu Province, were collected and placed in sterile self-sealing bags for storage at -4℃. The roots, stems, and leaves of the plants were chopped, soaked in 75% ethanol for 1 min, then in 0.1% HgCl2 for 60 s, and finally in 75% ethanol for 30 s. The tissues were then removed and rinsed four times with sterile water. A homogenate was formed by grinding the homogenate with 10 mL of sterile water and a small amount of sterile quartz sand in a sterilized mortar. After standing for 30 min, 0.1 mL of the homogenate was evenly spread onto beef extract peptone agar plates and incubated at 28℃ for 48 h. Different colonies were selected and purified on beef extract peptone agar plates. The purified bacterial strains were preserved on beef extract peptone agar slants and stored at 4℃ for later use.

[0029] The preserved bacterial strain was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of LB liquid medium and incubated on a shaker for 48 h (170 r·min). -1 The fermentation broth obtained at 28 ℃ was diluted with an appropriate amount of sterile water, and the original solution was prepared into a solution with a concentration of 10 by plate count method. 8 cfu·mL -1 The fermentation broth. All the above experiments were conducted under aseptic conditions.

[0030] Based on differences in colony size, color, and morphology, a total of 105 endophytic bacteria strains were isolated and purified from cucumber roots, stems, and leaves.

[0031] (1) Initial screening test

[0032] The initial screening test used the spore germination inhibition method. Sterile beef extract agar plates were cut into 0.5 cm diameter discs using a sterile punch, and then immersed in pre-prepared fermentation broths of each bacterial strain for 4 h. The control consisted of discs immersed in sterile water. 20 μL of a suspension of *Pseudoperonospora cubensis* pathogenic spores (10...) was added to the discs. 7 pcs·mL -1 The mycelium was randomly placed into sterile petri dishes, with 5 mycelium cakes per dish. The dishes were then covered and placed in a light incubator (25 °C, 16 h·d). -1 The spores were cultured in a medium (80% relative humidity) for 48 h, with each treatment repeated three times. The spore germination inhibition rate was examined under a microscope and calculated, as shown in Table 1.

[0033] Table 1. Inhibitory effects of different antagonistic bacteria on the germination of conidia of cucumber downy mildew.

[0034] C10-2 86.54±1.73 a C15-7 39.53±0.74 ij C3-4 75.66±1.88 b C14-3 38.54±0.65 j C4-5 66.35±2.33 c C13-6 37.30±1.03 jk C8-4 61.43±1.54 cd C12-6 37.22±0.89 jk C11-4 58.24±2.15 de C2-1 36.67±0.54 jk C2-5 55.68±1.53 ef C3-7 35.27±0..68 k C1-3 52.74±1.60 f C5-1 34.23±0.72 kl C2-7 49.12±1.42 fg C11-3 33.87±1.22 kl C5-4 47.23±1.33 g C10-1 32.22±0.93 l C6-2 47.29±1.45 gh C1-5 30.67±0.42 lm C4-6 46.65±0.89 gh C4-2 28.28±0.58 lm C7-1 45.90±0.77 gh C8-2 27.09±0.73 m C9-3 44.20±0.23 h C13-3 26.15±0.82 mn C8-5 44.01±1.08 hi C13-1 23.34±0.94 mn C10-4 43.82±0.72 hi C10-7 21.89±0.77 n C11-2 41.17±0.83 i Dimethomorph 74.33±1.82 b C12-3 40.90±1.12 ij CK -

[0035] Note: Different lowercase letters are marked after the data in the same row and column to indicate that the two groups of data are statistically significant (P < 0.05).

[0036] Table 1 shows that the endophytic bacteria C10-2, C3-4, C4-5, C8-4, C11-4, C2-5, and C1-3 all inhibited the germination of cucumber downy mildew conidia by more than 50%, with inhibition rates of 88.54%, 75.66%, 66.35%, 61.43%, 58.24%, 55.68%, and 52.74%, respectively. Among them, C10-2 had the highest inhibition rate.

[0037] (2) Secondary screening test

[0038] Seven endophytic bacteria strains (C10-2, C3-4, C4-5, C8-4, C11-4, C2-5, and C1-3) selected from the above screening experiments were used to test their leaf disc control efficacy against cucumber downy mildew. Leaf discs were cut from healthy young cucumber leaves using a 1 cm diameter punch and placed in an endophytic bacterial fermentation broth (10... 8 cfu·mL -1 After soaking in sterile water and air-drying, the control was a leaf disc soaked in sterile water, placed face down on the surface of filter paper moistened with sterile water, and 50 μL of pathogen spore suspension (10) was added to the leaf disc. 7 ·mL -1 Cover the petri dish and place it in a light incubator (22 °C, 16 h·d light). -1 (Relative humidity 80%). Four leaf discs were randomly selected as one treatment, with three replicates. The lesion area was investigated after 7-10 days, and the disease index and control effect were calculated according to the method in NY / T 1857.1-2010, as shown in Table 2.

[0039] Grading standards: Grade 0: No symptoms; Grade 1: Lesions cover less than 1 / 10 of the total leaf area; Grade 3: Lesions cover 1 / 10 to 1 / 4 of the total leaf area; Grade 5: Lesions cover 1 / 4 to 1 / 2 of the total leaf area; Grade 7: Lesions cover 1 / 2 to 3 / 4 of the total leaf area; Grade 9: Lesions cover more than 3 / 4 of the total leaf area.

[0040] Disease index = ∑ (number of disease-grade plants × number of representative grades) / (total number of plants surveyed × highest grade value) × 100;

[0041] Prevention and control effect / % = (control disease index - treatment disease index) / control disease index × 100.

[0042] Table 2. Control efficacy of 7 endophytic bacteria in cucumber downy mildew leaf disc test.

[0043] C10-2 <![CDATA[1×10 8 ]]> 12.54±1.78 e 84.23±1.62 a C3-4 <![CDATA[1×10 8 ]]> 22.94±2.90 d 71.15±1.74 b C4-5 <![CDATA[1×10 8 ]]> 30.17±2.54 c 62.07±2.18 c C8-4 <![CDATA[1×10 8 ]]> 35.12±1.66 bc 55.85±1.80 cd C11-4 <![CDATA[1×10 8 ]]> 38.45±2.75 b 51.66±2.01 de C2-5 <![CDATA[1×10 8 ]]> 40.27±1.53 b 49.37±1.91 e C1-3 <![CDATA[1×10 8 ]]> 40.35±1.22 b 49.27±1.75 e Dimethomorph 1000× 22.03±1.26 d 72.30±1.60 b Water control (CK) 79.54±1.80 a

[0044] Table 2 shows that all seven strains exhibited certain control effects against cucumber downy mildew, with significant differences compared to the control. Among them, C10-2 showed the best control effect, with significantly higher efficacy than the pesticide dimethomorph, achieving a disease index of only 12.54 and a control efficacy of 84.23%. Figure 1 It can be seen that the leaf disc lesion area treated with C10-2 fermentation broth was significantly smaller than that of the control.

[0045] Example 2. Identification of strain C10-2

[0046] (1) Morphological and physiological-biochemical characteristics

[0047] Morphological and physiological-biochemical characteristics of the antagonistic strain C10-2 were observed and determined. The C10-2 cells were short rod-shaped, with round, raised colonies, neat edges, light milky orange color, slightly glossy, and opaque. The strain was Gram-positive and aerobic. It could utilize mannitol and glucose, but not glycerol and citrate. It was catalase positive, and the gelatin test, H2S test, and indole test were all negative.

[0048] Table 3. Morphological and physiological-biochemical characteristics of strain C10-2

[0049] Bacterial shape short rod-shaped glucose utilization + Colony shape Round, raised, with neat edges glycerol utilization - Colony color Light cream orange Citrate utilization - Colony surface Slightly glossy, opaque catalase + Gram staining + Gelatin test - Anaerobic test aerobic <![CDATA[H2S Test]]> - Mannitol utilization + Indole test -

[0050] Note: + indicates positive; - indicates negative.

[0051] (2) Molecular biological identification

[0052] Single colonies were picked and placed in 1.5 mL centrifuge tubes containing 1 mL of LB medium. The cultures were incubated at 30 °C and 200 rpm for 24 h with shaking. Using this bacterial culture as a template, the 16S rRNA sequence was amplified using universal primers 27F / 1492R. The amplified products were separated and identified by 1% agarose gel electrophoresis. The PCR products were sent to Gansu Kemeiyi Biotechnology Co., Ltd. for bidirectional sequencing. The 16S rDNA sequence of strain C10-2 is 1415 bp, as shown in SEQ ID No. 1.

[0053]

[0054] The 16S rDNA sequence of the antagonistic endophytic bacterial strain C10-2 was compared on the Ezbiocloud website, and a phylogenetic tree was constructed. Figure 2 The results showed that strain C10-2 had high homology with *Microbacterium testaceum* (accession number NR026163.1), with a similarity of 99%. Based on morphological and physiological-biochemical characteristics, strain C10-2 was identified as *Microbacterium testaceum* and named *Microbacterium testaceum* C10-2.

[0055] Example 3. Control effect in field trials

[0056] The selected endophytic bacteria were tested in a field to assess their control efficacy. The field trial was conducted in June 2025 in a solar greenhouse in Xinping Village, Beiwan Town, Jingyuan County, Baiyin City, Gansu Province. This plot of land was a natural site for cucumber downy mildew.

[0057] Preparation method of fermentation broth of *Microbacterium tumefaciens* C10-2: *Microbacterium tumefaciens* C10-2 strain was inoculated into LB liquid medium and cultured at 28°C and 170 rpm with shaking for 48 h. The fermentation broth was then diluted with sterile water to an effective viable count of 1 × 10⁻⁶ cells / mL. 8 CFU / mL was used to obtain a bacterial culture of Bacillus rubrum C10-2.

[0058] Experimental Methods: Plump cucumber seeds were selected and sown in 60-cell trays when they germinated until they showed white sprouts. When the seeds had two true leaves, they were transplanted into a greenhouse. Under normal cultivation management, during the peak period of cucumber downy mildew, a bacterial suspension of *Microbacillus rubrum* C10-2 was evenly sprayed onto both sides of the cucumber leaves using a backpack electric sprayer. Chemical treatments, including a 2000-fold dilution of 40% dimethomorph suspension and water, were applied to the same *Microbacillus rubrum* C10-2 bacterial suspension. Each treatment consisted of 100 cucumber plants, replicated four times, randomly arranged, with isolation zones between treatment groups. Disease incidence was assessed 7 days after spraying, and the disease index and control efficacy were calculated.

[0059] Table 4. Field control efficacy of strain C10-2 against cucumber downy mildew.

[0060] C10-2 <![CDATA[1×10 8 ]]> 33.42±3.23 a 15.41±1.22 e 80.50±2.67 a Dimethomorph 1000× 31.19±2.47 a 19.56±1.27 d 73.49±2.03 b Water control (CK) -- 32.26±3.01 a 76.32±3.67 a --

[0061] Experimental results showed that strain C10-2 had significant field control efficacy against downy mildew in mature cucumber plants, and its control efficacy was significantly higher than that of the pesticide dimethomorph, with a disease index of only 15.41 and a relative control effect of 80.50%. Figure 3 It can be seen that the lesion area of ​​cucumber leaves treated with C10-2 fermentation broth was significantly smaller than that of the control.

[0062] Example 4. Field control effect of fermentation broth of strain C10-2 at different dilutions on cucumber downy mildew.

[0063] The original solution of fermentation broth C10-2, which has a significant control effect on cucumber downy mildew, and its diluted solutions of 10 times and 100 times were sprayed on the leaves of naturally infected cucumber plants at the mature stage to investigate the field control effect of fermentation broth of strain C10-2 at different dilutions on cucumber downy mildew. The results are shown in Table 5.

[0064] Table 5. Field control effect of fermentation broth of strain C10-2 at different dilutions on cucumber downy mildew.

[0065] C10-2 <![CDATA[1×10 8 ]]> 33.45±2.45 a 15.27±1.54 c 79.45±2.35 a C10-2 <![CDATA[1×10 7 ]]> 32.14±3.33 a 21.13±0.98 bc 70.43±1.38 b C10-2 <![CDATA[1×10 6 ]]> 32.60±2.16 a 26.42±1.65 b 63.55±3.60 c Dimethomorph 1000× 31.82±1.87 a 21.83±1.33 bc 69.14±2.62 b Water control (CK) 32.76±2.23 a 72.84±2.45 a

[0066] Table 5 shows that the fermentation broth of strain C10-2 at different dilutions all had a certain control effect on cucumber downy mildew, but the control effect gradually decreased with increasing dilution. Spraying with the undiluted fermentation broth of C10-2 showed the best control effect, reaching 79.45%; the disease index of cucumber downy mildew was the lowest, at only 15.27.

[0067] As can be seen from the above embodiments, the present invention provides a strain of *Rhizobium bacillus* C10-2 that antagonizes cucumber downy mildew and its application. This strain can effectively inhibit cucumber downy mildew.

[0068] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A strain of *Rhizobium brickii* ( Microbacterium testaceum C10-2, characterized in that, It is deposited at the China Center for Type Culture Collection, accession number CCTCCNO: M 20253025, on December 29, 2025, at Wuhan University.

2. A microbial inoculant, characterized in that, It contains the Brick-Rhodiola C10-2 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The viable bacterial concentration of *Rhizobium bacillus* C10-2 in the microbial agent is ≥1×10⁻⁶. 8 cfu / mL.

4. The method for preparing the microbial inoculant according to claim 2, characterized in that, Brick-red Microbacterium C10-2 was inoculated into fermentation medium for fermentation culture.

5. The preparation method according to claim 4, characterized in that, The fermentation culture temperature is 28~32℃ and the rotation speed is 160~200 r / min.

6. The preparation method according to claim 4, characterized in that, The fermentation culture time is 48~72 h.

7. The use of the *Rhizobium bacillus* C10-2 according to claim 1 in any one or more of the following: (1) Application in inhibiting *Pseudomonas columbarium*; (2) Application in the prevention and control of cucumber downy mildew; (3) Application in the preparation of agents to inhibit *Peronospora cucumeroides*; (4) Application in the preparation of fungal agents for the prevention and control of cucumber downy mildew.

8. A method for controlling downy mildew in cucumbers, characterized in that, Spray cucumber plants with the microbial agent described in claim 2 or 3.

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