Cunningham echinosporium and application thereof
By using a spore suspension of Cunninghamella echinulata JSAFC 2240 to irrigate the roots of rosemary, the problems of chemical pesticide resistance and environmental pollution in the prevention and control of rosemary anthracnose are solved, and the inhibition of gloeosporial anthracnose and the promotion of rosemary growth are achieved.
Patent Information
- Application Number
- CN202511031083.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-25
- Publication Date
- 2025-10-17
AI Technical Summary
Existing chemical pesticides have the problem of drug resistance when preventing and controlling rosemary anthracnose, and long-term use causes serious environmental pollution and affects the ecological balance.
Cunninghamella echinulata JSAFC 2240 was used as a biocontrol strain. The spore suspension was applied to the roots of rosemary to inhibit the growth of Colletotrichum gloeosporioides and promote the growth of rosemary.
It effectively inhibits the growth of Colletotrichum gloeosporioides, increases the plant height and leaf number of rosemary, enhances root growth, and provides an environmentally friendly disease prevention and control method.
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Figure CN120796085A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of microorganisms and relates to a Cunninghamella echinulata and application thereof. BACKGROUND
[0002] Rosmarinus officinalis is a kind of evergreen aromatic shrubs, the stems are lignified, the leaves are linear or needle-shaped, the flowers are small, labiate, light blue, purple or white, and the plant is suitable for sunlight, planting, cluster planting and group planting. The leaves of the plant have strong pine aroma and slightly bitter and pungent taste, and the plant is widely used for western cooking. Meanwhile, the plant is rich in antioxidant ingredients such as rosmarinic acid and salvia acid, can enhance blood circulation in the brain and improve concentration. The plant also has ornamental value and is often used for courtyards, flower beds, potted plants or hedges, and can be shaped into various shapes after pruning. Anthracnose is a common disease in the growth process of the plant, and severe infection can cause the plant to grow slowly, a large number of leaves to fall off, and eventually the whole plant to die. The anthracnose caused by Colletotrichum gloeosporioides mainly damages the leaves and branches of the plant, causes the leaves to appear lesions, and the lesions are brown, the branches gradually die after infection, and orange-red conidia piles can be seen on the lesions under humid conditions.
[0003] At present, chemical pesticides are commonly used for plant disease control, but long-term use can make many pathogenic bacteria resistant, reduce the disease control effect, frequent and high-concentration use can pollute the environment, and excessive residues can also threaten human health. There are many beneficial microorganisms in the environment around the plant, and these microorganisms not only do not cause plant diseases, but also can induce plants to produce resistance and improve the survival ability of plants under biological or non-biological stress. In addition, these microorganisms are derived from the natural environment and do not pollute the environment, and the use of these microorganisms to control plant diseases is conducive to maintaining the ecological balance. SUMMARY
[0004] The purpose of the application is to provide a Cunninghamella echinulata and application thereof in inhibiting the growth of Colletotrichum gloeosporioides and promoting the growth of Rosmarinus officinalis.
[0005] Technical scheme: The application provides a Cunninghamella echinulata, and the strain number of the Cunninghamella echinulata is JSAFC 2240, which was preserved in the China General Microbiological Culture Collection Center on May 6, 2025, and the preservation number is CGMCC No. 41984.
[0006] Furthermore, the nucleotide sequence of the ITS gene of Cunninghamella echinops is shown in SEQ ID NO.1.
[0007] The present invention also provides a biocontrol fungus agent, wherein the active ingredient of the biocontrol fungus agent is the above-mentioned Cunninghamella echinulata.
[0008] The present invention also provides the use of the Cunninghamella echinulata and the biocontrol fungus agent in inhibiting the growth of Colletotrichum gloeosporioides.
[0009] The present invention also provides the use of the Cunninghamella echinulata and the biocontrol fungus agent in promoting the growth of rosemary.
[0010] Furthermore, the application is specifically to promote the plant height growth, leaf increase and root growth of rosemary by irrigating the rosemary roots with a spore suspension of Cunninghamella echinulata JSAFC 2240.
[0011] Furthermore, the preparation method of the spore suspension is as follows: (1) taking the biocontrol fungus Cunninghamella spp. JSAFC2240, under sterile conditions, picking up mycelium with an inoculation needle, inoculating it into a sterilized PDA culture medium, and culturing it for 5-7 days; (2) under sterile conditions, scraping the mycelium, washing the mycelium with sterile water and filtering it with gauze; (3) under sterile conditions, adjusting the spore concentration with sterile water.
[0012] Furthermore, the culture conditions in step (1) are 25° C., 65% humidity, and dark culture.
[0013] Furthermore, the concentration of the spore suspension is 10 5 / ml.
[0014] Furthermore, the spore suspension is applied at an amount of 5 mL / time / 2 days for 30 days.
[0015] Beneficial Effects: Compared with existing technologies, the present invention has the following significant advantages: A strain of Cunninghamella echinulata was isolated and purified from soil surrounding rosemary. This strain can be used as a biocontrol agent to inhibit Colletotrichum gloeosporioides, with an inhibition rate of 73.91% in plate-based culture. Furthermore, this strain can promote rosemary development and regulate plant growth, which is of great significance for the future development of biocontrol agents for anthracnose. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 : Phylogenetic tree of Cunninghamella echinulata JSAFC 2240.
[0017] Figure 2 :Confrontation culture of Cunninghamella echinulata JSAFC 2240 and Colletotrichum gloeosporioides.
[0018] Figure 3 : Effects of Cunninghamella echinulata JSAFC 2240 on plant height of Rosemary.
[0019] Figure 4 : Effects of Cunninghamella echinulata JSAFC 2240 on the roots of Rosemary. DETAILED DESCRIPTION
[0020] The technical solution of the present invention will be further described below with reference to the accompanying drawings.
[0021] As described in the present invention, the term "biocontrol bacteria" refers to beneficial microorganisms that can prevent and control plant diseases, mainly bacteria, fungi, and actinomycetes.
[0022] Example 1: Isolation and Identification of Cunninghamella echinulata JSAFC 2240
[0023] 1. Isolation of Cunninghamella echinulata JSAFC 2240
[0024] JSAFC 2240 was isolated from soil in Fuzhou, Fujian, where rosemary grew well. The bacterial colony characteristics were as follows: when cultured on PDA plate medium, the colony grew rapidly and was sparse and flocculent.
[0025] 2. Molecular biological identification of Cunninghamella echinulata JSAFC 2240
[0026] (1) The genomic DNA of strain JSAFC 2240 was extracted using a kit from TIANGEN.
[0027] (2) Amplification of the ITS gene of genomic DNA. DNA amplification was performed in a reaction volume of 30 μL, containing 15 μL of 2x EasyTaq PCR SuperMix (+dye), 1 μL of each primer pair ITS1 (SEQ ID NO. 2: 5'-CTTGGTCATTTAGAGGAAGTAA-3') & ITS4 (SEQ ID NO. 3: 5'-TCCTCCGCTTATTGATATGC-3') (10 μM), 2 μL of template DNA, and 11 μL of ddH2O. The PCR amplification program conditions were as follows: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 54°C annealing for 45 s, 72°C extension for 60 s, for a total of 35 cycles; and finally 72°C extension for 10 min.
[0028] (3) After amplification of the obtained PCR product was subjected to 1% agarose gel electrophoresis, the target band was observed under ultraviolet light, and the PCR product was sent to Nanjing Qikexi Biological Technology Co., Ltd. for sequencing. The sequencing result was that the ITS gene sequence of strain JSAFC 2240 was SEQ ID NO. 1
[0029]
[0030]
[0031] (4) The sequence of SEQ ID NO. 1 was aligned on the NCBI database website and a phylogenetic tree was established, see Figure 1 The alignment results are shown in Table 1. According to the results in Table 1, it can be inferred that the biocontrol strain JSAFC 2240 of the present application is Cunninghamella echinulata, named Cunninghamella echinulata JSAFC 2240, and the strain was preserved in the China General Microbiological Culture Collection Center on May 6, 2025, with the preservation number of CGMCC No. 41984, the classification and naming of Cunninghamella echinulata JSAFC 2240, and the preservation address of No. 3, Beichenyuan, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.
[0032] Table 1: Alignment results of SEQ ID NO. 1 sequence
[0033]
[0034] Example 2: Inhibition effect of Cunninghamella echinulata JSAFC 2240 on Colletotrichum gloeosporioides
[0035] (1) On the super-clean bench, a 4mm-diameter mycelium cake of G. cingulata JSAFC 1340 was inoculated on one side of a PDA culture medium plate, and on the other side, no C. echinulata JSAFC 2240 was inoculated as a control group, and on the other side, a 4mm-diameter mycelium cake of C. echinulata JSAFC 2240 was inoculated as an experimental group.
[0036] (2) The experimental group and the control group of step (1) were cultured in an incubator at 25°C under inverted culture, and the culture conditions were 8h of light followed by 16h of darkness, for a total of 7 days of culture.
[0037] (3) The colony radius of G. cingulata was measured by the plate confrontation method, and the inhibitory effect of C. echinulata JSAFC 2240 on G. cingulata was observed.
[0038] The results are shown in Table 1. Figure 2 As shown in Table 1, the colony radius of G. cingulata in the blank control group was 3.91cm; in the experimental group, the average radius of the G. cingulata pathogen was only 1.02cm as determined by the plate confrontation method, and the inhibition rate was 73.91%, indicating that C. echinulata JSAFC 2240 had a significant inhibitory effect on G. cingulata.
[0039] Example 3: Effect of C. echinulata JSAFC 2240 on the growth of rosemary
[0040] (1) A puncher was used to take a 5mm-diameter mycelium cake of strain C. echinulata JSAFC 2240 and inoculate it in a 9cm-diameter culture dish containing 15ml of PDA culture medium, and the culture dish was cultured at 25°C and 65% humidity in the dark for 7 days.
[0041] (2) Under sterile conditions, the mycelium was scraped off, washed with sterile water, and filtered with gauze, and then the spore concentration was adjusted to 10 5 / ml.
[0042] (3) A 20cm-diameter pot was selected, and 1 healthy rosemary seedling with uniform growth was transplanted into each pot, and the experiment was set up in 2 groups (a control group and a treatment group), with 10 replicates for each treatment. The experimental group was: each pot was irrigated with 5mL of C. echinulata JSAFC 2240 spore suspension; the control group was: each pot was irrigated with 5mL of sterile water.
[0043] (4) Water each group of rosemary seedlings every 2 days. After 30 days, count the plant heights of the rosemary seedlings and observe the growth and development of the roots of the rosemary seedlings.
[0044] Plant height results as Figure 3 and shown in Table 2.
[0045] Table 2 Statistical results of rosemary plant height
[0046]
[0047] The numerical expression of plant height in the table is mean ± standard error.
[0048] from Figure 3 As shown in Table 2, the plant height and rosemary leaf growth of the experimental group were significantly better than those of the control group, indicating that Cunninghamella echinulata JSAFC 2240 promoted the growth of rosemary. Figure 4 This is a comparison of the roots of the experimental group and the control group, indicating that Cunninghamella echinulata JSAFC 2240 also has a growth-promoting effect on the rosemary root system.
Claims
1. A Cunninghamella echinulata, characterized in that The strain number of the Cunninghamella echinulata is JSAFC 2240, which was deposited in the General Microbiology Center of the China Culture Collection Administration on May 6, 2025, with the deposit number CGMCC No.41984.
2. The Cunninghamella echinulata according to claim 1, wherein The nucleotide sequence of the ITS gene of Cunninghamella echinops is shown in SEQ ID NO.
1.
3. A biocontrol agent, characterized in that: The active ingredient of the biocontrol fungus agent is the Cunninghamella echinulata according to any one of claims 1 to 2.
4. Use of the Cunninghamella echinulata according to any one of claims 1 to 2 and the biocontrol agent according to claim 3 in inhibiting the growth of Colletotrichum gloeosporioides.
5. Use of the Cunninghamella echinulata according to any one of claims 1 to 2 and the biocontrol fungus agent according to claim 3 in promoting the growth of rosemary.
6. The use according to claim 5, characterized in that The application is specifically to promote the plant height growth, leaf increase and root growth of rosemary by irrigating the rosemary roots with a spore suspension of Cunninghamella echinulata JSAFC 2240.
7. The use according to claim 6, characterized in that The preparation method of the spore suspension is as follows: (1) taking the biocontrol fungus Cunninghamella spp. JSAFC 2240, picking up hyphae with an inoculating needle under sterile conditions, inoculating the hyphae with a sterilized PDA medium, and culturing for 5-7 days; (2) scraping the hyphae under sterile conditions, washing the hyphae with sterile water and filtering with gauze; (3) adjusting the spore concentration with sterile water under sterile conditions.
8. The use according to claim 7, characterized in that The culture conditions in step (1) are 25° C., 65% humidity, and dark culture.
9. The use according to claim 6, characterized in that The concentration of the spore suspension is 10 5 / ml.
10. The use according to claim 6, characterized in that The spore suspension was applied at a rate of 5 mL / time / 2 days for 30 days.