Streptomyces droomrhizosphere and application of streptomyces droomrhizosphere
By using Streptomyces rhizosphere strain 14-3 and its fermentation broth, the problem of soybean anthracnose control was solved, and effective inhibition of Anthracnose bacterium was achieved, providing a safe and environmentally friendly biological control solution.
Patent Information
- Application Number
- CN202511515634.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2025-10-11
- Filing Date
- 2025-10-22
- Publication Date
- 2026-01-20
AI Technical Summary
Current technologies lack effective control methods against Anthracnose, especially in soybean anthracnose. Chemical control leads to a high risk of drug resistance, while biological control methods are insufficient.
Streptomyces rhizosphaerihabitans strain 14-3 and its fermentation broth were used to inhibit the mycelial growth and spore germination of Anthracnose. The preparation method included fermentation on Gao's No. 1 medium, ethyl acetate extraction and DMSO dissolution, and it was applied to the prevention and control of soybean anthracnose.
It significantly inhibits the mycelial growth and spore germination of *Anthracis chinensis*, providing excellent control effects comparable to chemical control agents, with no phytotoxicity observed.
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Figure CN121362678A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of crop disease biological control, in particular to a strain of Streptomyces rhizosphaerihabitans 14-3 and its application. BACKGROUND
[0002] Colletotrichum Corda fungi are a class of economically important plant pathogenic fungi that can cause a variety of crops to have anthracnose disease, often causing serious economic losses to agricultural production. Colletotrichum truncatum is an important population in the genus Colletotrichum, which has the characteristics of fast reproduction, large spore production, latent infection and multiple re-infection, and has a high resistance risk, and is a class of difficult-to-control pathogens. Colletotrichum truncatum has more than 460 host plants, and often causes soybean, pepper and other crop anthracnose diseases.
[0003] The main measures to prevent and control anthracnose disease in production are to plant disease-resistant varieties and chemical control. However, there is currently a lack of germplasm resources with broad-spectrum resistance to anthracnose. In addition, with the increase in the frequency and length of time of fungicide use, resistant populations of anthracnose have appeared in the field.
[0004] Biological control has the advantages of safety, environmental protection and difficulty in developing resistance, and has become an important development direction for green control of plant diseases. Microorganisms with antagonistic activity and their active metabolites have great potential in inhibiting plant pathogenic fungi, and have become an important development direction for green control of plant diseases in recent years. Actinomycetes, as an important group of antagonistic microorganisms, are widely distributed and species-rich in nature, and can produce antibiotics, proteases, growth hormones and other substances, and have been widely used in agriculture, medicine and other fields. For example, guvermectin produced by Streptomyces hygroscopicus NEAU6 can effectively control rice bacterial leaf blight, tomato bacterial wilt and kiwi fruit canker disease, and has good growth-promoting effect on rice and other crops, and was registered as a new pesticide in 2021. SUMMARY
[0005] The primary purpose of the present application is to provide a strain of Streptomyces rhizosphaerihabitans 14-3, which has antagonistic effect on Colletotrichum truncatum, the main pathogen causing soybean anthracnose disease, to hinder the healthy development of the soybean industry due to the occurrence of soybean anthracnose disease.
[0006] Another object of the present application is to provide the application of the fermentation broth of Streptomyces rhizosphaerihabitans strain 14-3 in the prevention and treatment of soybean anthracnose.
[0007] The object of the present application is achieved by the following technical solutions:
[0008] The Streptomyces rhizosphaerihabitans strain 14-3 is preserved in the Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC No. 63693, and the preservation date is August 3, 2023.
[0009] The Streptomyces rhizosphaerihabitans strain 14-3 has antagonistic activity on C. truncatum which causes soybean anthracnose.
[0010] The 16s rRNA sequence of the Streptomyces rhizosphaerihabitans strain 14-3 is shown as SEQ ID NO: 1.
[0011] The Streptomyces rhizosphaerihabitans strain 14-3 has the following physiological and biochemical characteristics: the 14-3 strain can coagulate and peptonize milk, produce melanin, hydrolyze casein, esculin and cellulose, reduce nitrate, have no gelatin liquefaction ability, and cannot hydrolyze starch and urea.
[0012] The Streptomyces rhizosphaerihabitans strain 14-3 has the following physiological and biochemical characteristics: the 14-3 strain can coagulate and peptonize milk, produce melanin, hydrolyze casein, esculin and cellulose, reduce nitrate, have no gelatin liquefaction ability, and cannot hydrolyze starch and urea.
[0013] The Streptomyces rhizosphaerihabitans strain 14-3 has the following physiological and biochemical characteristics: the 14-3 strain can coagulate and peptonize milk, produce melanin, hydrolyze casein, esculin and cellulose, reduce nitrate, have no gelatin liquefaction ability, and cannot hydrolyze starch and urea.
[0014] The preparation method of the fermentation liquor of Streptomyces rhizosphaerihabitans strain 14-3, wherein the Streptomyces rhizosphaerihabitans strain 14-3 is inoculated on a Kesheng No.1 synthetic solid culture medium, the fermentation temperature is 26-28 DEG C, the fermentation culture time is 7-8 days, and then the fermentation liquor is obtained by extracting with ethyl acetate, concentrating and evaporating the extract under reduced pressure, and then dissolving the residue in DMSO.
[0015] The application of the fermentation liquor of Streptomyces rhizosphaerihabitans strain 14-3, wherein the fermentation liquor of Streptomyces rhizosphaerihabitans strain 14-3 is applied to the prevention and treatment of soybean anthracnose.
[0016] The fermentation liquor of Streptomyces rhizosphaerihabitans strain 14-3 is used for inhibiting the mycelium growth and spore germination of the soybean anthracnose pathogen.
[0017] The soybean anthracnose pathogen is C. truncatum.
[0018] Compared with the prior art, the present application has the following advantages:
[0019] Streptomyces rhizosphaerihabitans strain 14-3 has antagonistic effect on the main pathogen C. truncatum causing soybean anthracnose, the fermentation liquor can significantly inhibit the mycelium growth and spore germination of C. truncatum, and the prepared fermentation liquor has excellent prevention and treatment effect on soybean anthracnose. BRIEF DESCRIPTION OF DRAWINGS
[0020] Figure 1 It is the culture characteristics of Streptomyces rhizosphaerihabitans strain 14-3 on Kesheng No.1 medium.
[0021] Figure 2 It is the phylogenetic tree of Streptomyces rhizosphaerihabitans strain 14-3.
[0022] Figure 3Inhibition effect of Streptomyces rhizosphaerihabitans strain 14-3 on C. truncatum under face-to-face culture condition. Note: A is C. truncatum Ct9-5, B is C. truncatum Ct8-1, and CK is the control group.
[0023] Figure 4 Inhibition effect of Streptomyces rhizosphaerihabitans strain 14-3 fermentation broth on C. truncatum mycelial growth. Note: A is C. truncatum Ct9-5, B is C. truncatum Ct8-1, and CK is the control group.
[0024] Figure 5 Inhibition effect of Streptomyces rhizosphaerihabitans strain 14-3 fermentation broth on C. truncatum conidial germination. Note: A is the conidial germination of C. truncatum Ct9-5 treated with Streptomyces rhizosphaerihabitans strain 14-3 fermentation broth, B is the conidial germination of C. truncatum Ct8-1 treated with Streptomyces rhizosphaerihabitans strain 14-3 fermentation broth, and CK is the control group. DETAILED DESCRIPTION
[0025] The content of the present application will be described in detail below in combination with the drawings and examples of the specification:
[0026] Example 1: Isolation and identification of Streptomyces rhizosphaerihabitans strain 14-3.
[0027] 1. Collection of soil samples
[0028] Three soil samples were collected from Jinggang Mountain in Jiangxi Province. The surface soil was removed, and the soil samples at a depth of 5-20 cm were collected, labeled, and then brought back to the laboratory for natural air drying.
[0029] 2. Isolation of actinomycetes
[0030] Plate dilution method was used for isolation. The air-dried soil sample was ground with a mortar, and 1 g of the sample was suspended in 9 mL of sterile water. After shaking at 40°C and 180 rpm for 30 min and standing for 5 min, it was diluted by 10 times successively to prepare 10 -2 , 10 -3 , 10-4 The suspension was diluted to different concentrations, 0.1 mL of each concentration was taken and inoculated on the modified HVA medium (100-200 ppm of potassium dichromate was added) and incubated at 28°C. After 5-7 days, different single colonies were picked and purified by streaking. The purified strains were preserved in -80°C refrigerator by glycerol method.
[0031] 3. Identification of Streptomyces rhizosphaerihabitans strain 14-3
[0032] (1) Morphological characteristics observation
[0033] Streptomyces rhizosphaerihabitans strain 14-3 grew well on most of the culture media and did not produce soluble pigments, as shown in Table 1. Under optical microscope, the strain 14-3 had no septum in aerial hyphae and could produce a large amount of spores. The spore chains were straight or flexuous, and the spores were spherical or oval.
[0034] Table 1 Culture characteristics of Streptomyces rhizosphaerihabitans 14-3
[0035]
[0036] (2) Physiological and biochemical characteristics
[0037] The starch hydrolysis and nitrate reduction of Streptomyces rhizosphaerihabitans 14-3 were determined according to the methods described in the Manual of Streptomyces Identification, and the results are shown in Table 2.
[0038] Table 2 Physiological and biochemical characteristics of Streptomyces rhizosphaerihabitans 14-3
[0039]
[0040] (3) Sequence analysis
[0041] The genomic DNA of Streptomyces rhizosphaerihabitans 14-3 was extracted by using a bacterial genomic extraction kit, and 16S rRNA amplification was performed. The full-length sequence of the obtained sequence was 1354 bp. The sequence was submitted to GenBank database for BLAST comparison. According to the morphological characteristics, physiological and biochemical characteristics and 16S rRNA sequence analysis, it was concluded that the strain 14-3 was Streptomyces rhizosphaerihabitans. Figure 2 ).
[0042] Example 2: Determination of the antagonistic effect of Streptomyces rhizosphaerihabitans strain 14-3 on Colletotrichum gloeosporioides.
[0043] The antagonistic effect of Streptomyces rhizosphaerihabitans strain 14-3 on Colletotrichum gloeosporioides Ct9-5 and Ct8-1 was determined by using a plate confrontation culture method. First, strain 14-3 was inoculated on the edge of PDA medium by streaking, and 3 days later, a 5 mm test pathogenic fungus cake was inoculated in the center of the plate, and the width of the inhibition zone of strain 14-3 on the test pathogenic fungus was measured after 5 days of culture at 28°C (Table 3). The test pathogenic fungus without antagonistic bacteria was used as a control. The results showed that strain 14-3 had very strong antagonistic effect on Colletotrichum gloeosporioides Ct8-1 and Ct9-5. Figure 3 ).
[0044] Table 3 Antagonistic activity of strain 14-3 on Colletotrichum gloeosporioides
[0045]
[0046] Example 3: Inhibition of mycelial growth of Colletotrichum gloeosporioides by Streptomyces rhizosphaerihabitans strain 14-3 fermentation broth.
[0047] Strain 14-3 was activated in ISP2 medium at 28°C for 5 days. The spore solution of strain 14-3 was inoculated into Keshi synthetic No. 1 solid medium, and fermented at 26-28°C for 7-8 days. The fermentation broth was extracted with ethyl acetate for 3 times, and the extract was concentrated and evaporated under reduced pressure. Then, the extract was dissolved in DMSO to prepare a 20 mg / mL fermentation broth, which was ready for use.
[0048] The fermentation broth of strain 14-3 was taken 100 μL and added into 100 mL PDA medium cooled to about 50°C (not hot hand), mixed evenly and poured into a culture dish, a 5 mm diameter test pathogenic fungus cake was inserted into the center of the PDA medium plate, and the colony diameter was measured after 5 d culture at 28°C. The same amount of DMSO was added as a control, and the inhibition rate of the fermentation broth on the mycelial growth of Colletotrichum gloeosporioides was calculated (Table 4). The results showed that the fermentation broth of strain 14-3 had very strong inhibitory effect on the mycelial growth of Colletotrichum gloeosporioides Ct8-1 and Ct9-5 Figure 4 ).
[0049] Table 4 Inhibitory effect of fermentation broth of strain 14-3 on Colletotrichum gloeosporioides
[0050]
[0051] Example Four: Inhibitory effect of fermentation broth of Streptomyces rhizosphaerihabitans strain 14-3 on conidial germination of Colletotrichum gloeosporioides
[0052] A 5 mm diameter test pathogenic fungus cake was inserted into PDB liquid medium, and after 7 d culture at 28°C with 180 rpm shaking, the spore suspension was collected by filtration, and its concentration was adjusted to 1 × 10 5 Example Three: Inhibitory effect of fermentation broth of Streptomyces rhizosphaerihabitans strain 14-3 on mycelial growth of Colletotrichum gloeosporioides Figure 5 ).
[0053] Table 5 Inhibitory effect of fermentation broth of strain 14-3 on conidial germination of three Colletotrichum gloeosporioides
[0054]
[0055] Example Five: Application of fermentation broth of Streptomyces rhizosphaerihabitans strain 14-3 in prevention and treatment of soybean anthracnose
[0056] The test Colletotrichum gloeosporioides Ct8-1 was cultured at 28°C for 5 d, and a 1 mm diameter puncher was used to punch a cake at the edge of the colony for standby.
[0057] Soybean seeds of Mao Dou No. 3 were sown in sterile seedling substrate (the thickness of the sterile seedling substrate was about 2 cm) in a seedling tray, and the row spacing and plant spacing were both 3 cm. After sowing, about 1 cm of nutrient soil was covered, and water was sprinkled until the nutrient soil was moist. The seedlings were cultured in darkness at 23-28°C and a relative air humidity of no more than 80% for 3 days. When the soybean sprouts grew to 3-4 cm, the soybean sprouts were pulled out, and soybean sprouts with a consistent growth trend were selected, cleaned with sterile water, and used as needed.
[0058] The 14-3 KF broth in Example 3 was diluted with sterile water to 100 μg / mL and 200 μg / mL,
[0059] 95% difenoconazole technical material was diluted with sterile water to 100 μg / mL of an aqueous solution as a positive control. Then, the soybean seedlings were soaked with the above-mentioned agents for 1 h, and the treatment of soaking with sterile water was used as a negative control. After drying, the soybean seedlings were inoculated with C. gloeosporioides.
[0060] About 2 g of absorbent cotton was rolled into a cotton ball and placed in a 15 mL centrifuge tube. 2 mL of sterile water was added to each tube. After the absorbent cotton ball absorbed water, the cotton ball was placed at the bottom of the centrifuge tube and used as needed. A sterile needle was used to prick the stems of the soybean seedlings (about 1 mm from the cotyledon) to cause a small wound. The C. gloeosporioides cake was inoculated onto the wound using a sterile needle. A wet absorbent cotton strip was used to cover the cake and keep it moist. Eight soybean sprouts were inoculated in each repeat, and the treatment of inoculating a sterile agar block was used as a control. After inoculation, the soybean sprouts were placed in the centrifuge tube with the root and stem facing down, so that the base of the soybean sprout contacted the absorbent cotton ball. The centrifuge tube cap was rotated half a turn. The inoculated centrifuge tube was placed in a 25°C incubator and cultured in darkness for 3 days.
[0061] After 3 days of inoculation, the disease index was investigated according to the expansion of the lesion. The disease grading standard was as follows: 0 level, no symptoms; 1 level, the lesion area accounted for less than 10% of the stem area; 3 level, the lesion area accounted for more than or equal to 10% and less than 25% of the stem area; 5 level, the lesion area accounted for more than or equal to 25% and less than 50% of the stem area; 7 level, the lesion area accounted for more than or equal to 50% and less than 75% of the stem area; and 9 level, the lesion area accounted for more than or equal to 75% of the stem area. The disease index calculation formula was: disease index = [∑ (the number of each level of diseased plants x the relative level value) ÷ (the total number of plants surveyed x 9)] x 100. The control effect calculation formula was: control effect (%) = [(the disease index of the blank control area - the disease index of the treatment area) ÷ the disease index of the blank control area] x 100.
[0062] Table 6: The control effect of the fermentation broth of strain 14-3 on soybean anthracnose
[0063]
[0064] The results showed that the control effects of 100 μg / mL fermentation broth of strain 14-3, 200 μg / mL fermentation broth of strain 14-3 and 100 μg / mL difenoconazole on soybean anthracnose were 25.88%, 62.50% and 60.62%, respectively, and all of them were safe to soybean without phytotoxicity. The control effect of 200 μg / mL fermentation broth of strain 14-3 on soybean anthracnose was not significantly different from that of 100 μg / mL difenoconazole at 5% and 1% significant levels. Therefore, the control effect of 200 μg / mL fermentation broth of strain 14-3 on soybean anthracnose was equivalent to that of 100 μg / mL difenoconazole, which had a good application prospect.
Claims
1. A strain of Streptomyces rhizosphaerihabitans, characterized in that: The Streptomyces rhizosphaerihabitans strain 14-3 is preserved in the Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC No. 63693, and the preservation date is August 3, 2023.
2. The Streptomyces rhizosphaeraehabitans strain 14-3 according to claim 1, characterized in that: The Streptomyces rhizosphaerihabitans strain 14-3 has antagonistic effect on the main pathogen Colletotrichum truncatum causing soybean anthracnose.
3. The Streptomyces rhizosphaerihabitans strain 14-3 for use in preventing and treating soybean anthracnose according to claim 1.
4. The method of preparing a fermentation broth of Streptomyces rhizosphaerihabitans strain 14-3 according to claim 1, characterized in that: The Streptomyces rhizosphaerihabitans strain 14-3 is inoculated on the Keshi synthetic No. 1 solid culture medium, the fermentation temperature is 26-28°C, the fermentation culture time is 7-8 d, and then the fermentation liquid is obtained by using ethyl acetate extraction, vacuum concentration and evaporation, and then dissolving in DMSO.
5. The fermentation liquid of the Streptomyces rhizosphaerihabitans strain 14-3 prepared by the method of claim 4.
6. Use of the fermentation broth of Streptomyces rhizosphaerihabitans strain 14-3 according to claim 5, characterized in that: The fermentation liquid of the Streptomyces rhizosphaerihabitans strain 14-3 for use in preventing and treating soybean anthracnose.
7. Use of the fermentation broth of Streptomyces rhizosphaerihabitans strain 14-3 according to claim 6, characterized in that: The fermentation liquid of the Streptomyces rhizosphaerihabitans strain 14-3 is used for inhibiting the mycelial growth and conidial germination of the soybean anthracnose pathogen. The soybean anthracnose pathogen is Colletotrichum truncatum.