Streptomyces 70-1 for resisting cotton verticillium wilt and application thereof

Abstract

The invention discloses a strain of streptomyces syringae 70-1 and application of the strain of streptomyces syringae 70-1, and belongs to the technical field of biocontrol of plant diseases. The streptomyces sp. 70-1 has been preserved in Guangdong Microbial Culture Collection Center (GDMCC) on November 6, 2025, and the preservation number is GDMCC NO.67234. The streptomyces sp. 70-1 has been preserved in Guangdong Microbial Culture Collection Center (GDMCC). The streptomyces sp. 70-1 has a remarkable inhibition effect on the cotton verticillium wilt caused by verticillium dahliae, can effectively relieve the occurrence of the cotton verticillium wilt, and has a very good biological control effect. The compound can be used for developing and preparing biopesticide aiming at the verticillium wilt disease of crops, and a new thought and a new method are provided for biologically preventing and treating the verticillium wilt of the crops.

Description

Technical Field

[0001] This invention belongs to the field of plant disease biocontrol technology, specifically relating to a Streptomyces strain 70-1 resistant to cotton Verticillium wilt. Streptomyces syringium ) and its applications. Background Technology

[0002] Cotton is an important economic crop, and cotton fiber is a crucial raw material for the textile industry. In my country, cotton growth is threatened by more than 40 diseases, among which Verticillium wilt is one of the most serious, severely impacting cotton yield. Verticillium wilt was first discovered and reported in 1914 on upland cotton in Virginia, USA. It was introduced to my country in 1935 when Swarovski cotton was introduced from the United States, and gradually spread. In the 1960s, Verticillium wilt appeared in major cotton-growing areas of my country. After the 1990s, the disease caused a major outbreak nationwide, resulting in the loss of 100 million kg of lint in 1993 alone. It subsequently caused several more major outbreaks in my country's main cotton-producing areas, affecting an annual area of ​​3 million hectares. 2 The annual economic loss reached as high as 1.2 billion yuan.

[0003] Verticillium dahliae is the main pathogen causing cotton wilt. Verticillium dahliae ) and Verticillium spp. ( Verticillium abboatrum Verticillium dahliae is a soil-borne fungal vascular disease of cotton. In my country, the pathogen of cotton Verticillium dahliae is transmitted through the soil, invading the vascular tissue of plants and causing the disease. It is characterized by a wide host range, rapid spread, large area of ​​damage, and strong environmental adaptability. The main pathogenic mechanism of Verticillium dahliae is the blockage of xylem vessels and toxin production. When the fungus invades the plant, the hyphae block the xylem vessels, affecting the transport of water and nutrients within the plant. However, the above-ground parts have strong transpiration and respiration, causing water imbalance within the plant, resulting in symptoms such as leaf wilting and yellowing, leading to poor plant development, and in severe cases, the death of the entire cotton plant. The average yield loss caused by cotton Verticillium dahliae is approximately 10-35%, and it can also reduce fiber quality.

[0004] Currently, the control of cotton Verticillium wilt mainly involves four aspects: first, the breeding and planting of Verticillium wilt-resistant varieties; second, agricultural control techniques such as rational crop rotation and deep plowing; third, chemical pesticide control; and fourth, biological control. Research on disease-resistant breeding lacks breakthrough highly resistant varieties due to the lack of natural Verticillium wilt-resistant germplasm and the tendency for transgenic resistant varieties to degenerate. The use of chemical pesticides, while inhibiting pathogenic microorganisms, also suppresses the growth of beneficial microorganisms and has adverse environmental impacts. Therefore, biological control technologies, with their environmentally friendly and green advantages, are receiving increasing attention, with biocontrol bacteria being an important part of biological control. The earliest biocontrol bacteria studied for Verticillium wilt were actinomycetes, mainly focusing on Streptomyces (…). StreptomycesThe experiment is carried out on Streptomyces sp. Streptomyces is a gram-positive filamentous actinomycete and widely active in soil. According to statistics, 70% of antibiotics produced by microorganisms are produced by actinomycetes, and more than 70% of the antibiotics are produced by Streptomyces. As an efficient antibiotic, the antibiotic produced by actinomycetes has a wide application in the field of agriculture, such as Jinggangmycin for preventing and treating wheat, rice, corn, and vegetable diseases caused by Rhizoctonia solani; Zhongshengmycin for preventing and treating cabbage soft rot, rice white leaf blight, and cucumber angular spot.

[0005] Therefore, searching for effective biocontrol strains and control methods is the development direction and effective way for preventing and treating crop Verticillium wilt. SUMMARY

[0006] In order to solve the above problems, the application discloses a kind of Streptomyces sp. 70-1 for resisting cotton Verticillium wilt and application thereof, which has a good inhibitory effect on crop Verticillium wilt disease and can be developed and utilized as a biological pesticide.

[0007] The application provides a kind of Streptomyces sp. 70-1 for resisting cotton Verticillium wilt ( Streptomyces syringium ), which is classified and named as Streptomyces syringium , and is preserved in Guangdong Microbial Culture Collection Center on November 6, 2025, with the preservation number GDMCC NO. 67234. The 16S rDNA sequence of the Streptomyces sp. 70-1 is shown as SEQ ID NO. 1. The Streptomyces sp. can effectively prevent and treat crop Verticillium wilt disease.

[0008] The application further provides a kind of biocontrol agent containing the Streptomyces sp. 70-1. The preparation method of the biocontrol agent is specifically as follows: the Streptomyces sp. 70-1 is inoculated into 50 mL of ISP-2 culture solution, cultured at 30 DEG C and 180 rpm / min for 3 days, then transferred into 1 L of ISP-2 fermentation medium at a volume ratio of 3:100 under the same conditions for expansion culture for 7 days, and the obtained fermentation liquor is the biocontrol agent of the Streptomyces sp. 70-1.

[0009] Preferably, the concentration of the Streptomyces sp. Streptomyces syringium in the biocontrol agent is preferably 1 x 10 8 cfu / mL.

[0010] The application further provides the application of the biocontrol agent of the Streptomyces sp. 70-1 in preparing a preparation for preventing and treating cotton Verticillium wilt caused by Verticillium dahliae. In the plate antagonistic test, the inhibition rate of the Streptomyces sp. 70-1 on Verticillium dahliae is 69.63%. The fermentation liquor supernatant of the Streptomyces sp. 70-1 can effectively inhibit the growth of Verticillium dahliae.

[0011] The experimental results of the embodiment show that the streptomycete 70-1 strain can effectively inhibit Verticillium dahliae Vd991 to cause Verticillium wilt in the greenhouse experiment. The disease index of the cotton in the experimental group is 75.83, and the disease index of the cotton in the control group is 89.16, which is obviously reduced, and the growth of the cotton in the experimental group is obviously better than that in the control group.

[0012] The application further provides application of the biocontrol agent of the streptomycete 70-1 in preparation of a preparation for plant diseases caused by Fusarium graminearum and / or Rhizoctonia solani.

[0013] Compared with the prior art, the application has the beneficial effects that: The streptomycete 70-1 strain has high efficient antagonistic effect on Verticillium dahliae, can be used for preventing and treating cotton Verticillium wilt caused by Verticillium dahliae infection, and has good antagonistic effect on Fusarium graminearum and Rhizoctonia solani, and can be used for preventing and treating plant diseases caused by infection of the pathogens. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 It is a plate antagonistic effect diagram of the streptomycete 70-1 on Verticillium dahliae Vd991; Figure 2 It is a plate antagonistic effect diagram of the streptomycete 70-1 on Fusarium graminearum; Figure 3 It is a plate antagonistic effect diagram of the streptomycete 70-1 on Rhizoctonia solani; Figure 4 It is a colony morphology diagram of the streptomycete 70-1 on the ISP-2 medium; Figure 5 It is a phylogenetic tree of the streptomycete 70-1 and adjacent strains according to 16S rDNA sequences; Figure 6 It is an activity test diagram of the fermentation broth supernatant of the streptomycete 70-1 for inhibiting the growth of Verticillium dahliae; Figure 7 It is a prevention and treatment effect diagram of the fermentation supernatant of the streptomycete 70-1 on cotton TM-1 Verticillium wilt. DETAILED DESCRIPTION

[0015] Embodiment 1: Isolation and screening of the streptomycete 70-1 strain Sample collection: In the cotton production area of Xinjiang, soil samples of cotton roots are collected, bagged and stored, and taken back to the laboratory for strain isolation. The soil samples are isolated by the dilution coating plate method. 10 g of soil is put into a conical flask, 150 mL of physiological saline is added, and the sample is shaken at 28 DEG C and 180 rpm for 20 min, and then the supernatant is gradient diluted to 10 -2 , 10 -3The plate was coated, and the plate was cultured at 28°C in the dark for 5 days. Single colonies with different morphologies, sizes, and colors were picked and purified. After 3-5 days of culture, the single colonies were picked with a toothpick and placed in 900 μL of ISP-2 liquid medium (the formula of ISP-2 liquid medium / L is: malt extract 10 g, anhydrous glucose 4 g, yeast extract 4 g, PH=7.2). The mixture was cultured at 28°C with 180 rpm shaking for 3-5 days, then 900 μL of 50% glycerol was added, mixed well, and stored in a -80°C freezer.

[0016] Antagonistic strain screening: plate inhibition zone test was used. PDA medium (the formula of PDA medium / L is: potato 200 g, glucose 20 g, agar 15 g) was used to activate the pathogenic fungus L. digitata Vd991, and the culture was incubated at 28°C for 7 days. The sample was collected and stored in ISP-2 solid medium.

[0017] A punch was used to punch a 6 mm diameter circular block of well-grown pathogenic fungus from the outer edge of the colony, with the mycelium facing down. The activated strain was inoculated on one side of the PDA plate, 4 cm from the center of the block, with three replicates in each group. The same size of pathogenic fungus was taken and inoculated in the middle of the PDA plate with the mycelium facing down as the control group. The activity of the isolated strains against L. digitata was evaluated according to the size of the inhibition zone. The calculation process of the inhibition rate is: Inhibition rate = [(R1-R2) / R1] x 100%; Where R1 is the colony radius of the control group of pathogenic fungus; R2 is the colony radius of the pathogenic fungus in the direction of inoculation of the antagonistic strain.

[0018] The results of the screening of antagonistic strains are shown in Table 1. As shown in Table 1, Streptomyces 70-1 has a strong inhibitory effect on L. digitata, with an inhibition rate of 69.63%. The plate antagonism of Streptomyces 70-1 is shown in Figure 1 .

[0019] Table 1 Inhibition rate of some antagonistic strains Example 2 Plate antagonistic activity of Streptomyces 70-1 strain against other plant pathogenic fungi After 3 days of streak culture of Streptomyces 70-1 strain at a distance of 2.5 cm from the center of the plate, a punch was used to punch a 6 mm diameter circular block of well-grown pathogenic fungus from the outer edge of the colony, with the mycelium facing down. The plate was inoculated in the center of the PDA plate, with three replicates. The control group was a plate inoculated with only pathogenic fungus.

[0020] 28℃ culture, after the control group of strains grow full plate to detect 70-1 strain on fusarium graminearum, rhizoctonia solani antagonistic activity. By Figure 2 , Figure 3 It can be seen that compared with the control group, after inoculation of 70-1 strain, the growth of two kinds of pathogens is obviously inhibited, and the colony morphology is obviously different from the normal strain. 70-1 strain has good antagonistic effect on the two types of pathogens.

[0021] Example 3 Identification of Streptomyces 70-1 strain Streptomyces 70-1 gram staining positive, aerial hyphae light yellow, basal filament yellow brown. After 7 days of culture in ISP2 medium, 7.0 mm colonies were observed on the culture, the colonies were light yellow, pigment producing, approximately circular, wrinkled surface, not easy to pick up, the morphological structure was shown in Figure 4 The 16S rDNA sequence of 70-1 strain is shown in SEQ ID NO. 1, and it is identified as Streptomyces by phylogenetic tree comparison analysis. Streptomyces syringium The phylogenetic tree constructed according to the 16S rDNA sequence is shown in Figure 5 .

[0022] Example 4 Preparation of biocontrol agent containing Streptomyces 70-1 strain Inoculate 70-1 strain single colony into 50 mL of ISP-2 culture solution, and cultivate at 30℃, 180 rpm for 3d to obtain seed liquid. The seed liquid is transferred into a 1 L conical flask containing ISP-2 culture solution at a volume ratio of 3:100, and cultivated at 30℃, 180 rpm for 7d to obtain 70-1 fermentation liquid. After fermentation, the fermentation liquid is gradient diluted, plated and detected for the number of colonies, and the concentration of the bacterial liquid is adjusted to 1×10 8 cfu / mL to obtain the biocontrol agent of Streptomyces 70-1.

[0023] The formula of ISP-2 medium per liter is: malt extract 10g, glucose 4g, yeast extract 4g, PH adjusted to 7.2, 1000mL water.

[0024] Example 5 Determination of antibacterial activity of Streptomyces 70-1 strain fermentation liquid Preparation of verticillium dahliae v991 spore solution: take fresh activated verticillium dahliae blocks and place them in liquid sabouraud medium, cultivate at 25℃, 220 rpm for 3d, filter with sterile gauze, obtain verticillium dahliae spore solution, dilute the spore concentration to 1×10 7 / mL with sterile water, and store in 4℃ refrigerator for standby.

[0025] The fermentation broth of biocontrol bacteria strain 70-1 obtained in Example 4 was centrifuged at 12,000 rpm for 5 min, and the supernatant was collected. The supernatant was filtered through a 0.22 μm microporous membrane to obtain a sterile supernatant for later use. Dahlia verticillata spore solution was added to PDA medium at 40°C until the final spore concentration reached 10. 5 Species / mL. Pour PDA medium containing Verticillium dahliae spores into 6 cm diameter plates. After cooling and solidification, punch a hole in the center of the plate using an 8 mm diameter punch. Add 200 μL of the prepared sterile supernatant to each well, using the culture medium as a control group. Observe the inhibition zone after static incubation at 25℃ for 3 days. The test results are as follows. Figure 6 As shown, a clear inhibition zone was observed in the experimental group.

[0026] Example 6: Biocontrol effect of Streptomyces strain 70-1 against cotton Verticillium wilt Preparation of Verticillium dahliae Vd991 spore suspension: Verticillium dahliae mycelial blocks were placed in liquid Czapek's medium and cultured at 25℃ and 220 rpm for 3 days with shaking. The mixture was then filtered through four layers of gauze to obtain Verticillium dahliae spore suspension. The spore suspension concentration was diluted to 1×10⁻⁶ with sterile water. 6 Quantity / mL, for later use.

[0027] Mix the potting soil and vermiculite in a 3:1 ratio, water thoroughly, and sow the cotton TM-1 seeds. Cultivate the seedlings until they have two true leaves. Then, use a mixed inoculation method to inoculate the cotton seedlings by watering the roots after root damage: 20 ml of the solution at a concentration of 1×10⁻⁶. 8 The experimental group consisted of a mixture of Streptomyces 70-1 biocontrol agent at CFU / mL and 20 ml of Verticillium dahliae spore solution. The concentration in 20 ml was 1×10⁻⁶. 8 A control group was prepared by mixing 20 ml of ISP-2 culture medium with *Streptomyces 70-1* at CFU / mL. The plants were cultured in a 28℃ greenhouse for 16 hours of light followed by 8 hours of darkness. The growth and disease incidence of cotton seedlings were observed daily. After 30 days of treatment, the disease index and control efficacy were calculated. The calculation process for the disease index is as follows: The cotton plants are classified into five levels based on the number of diseased leaves on each plant: Level 0: No diseased leaves on the plant; Level 1: 25% or less of the leaves on the plant are diseased; Level 2: 25% or more but less than or equal to 50% of the leaves on the plant are diseased; Level 3: 50% or more but less than or equal to 75% of the leaves on the plant are diseased; Level 4: 75% or more of the leaves on the plant are diseased.

[0028] Disease index = ×100; dc represents the disease severity level of the plant; nc represents the number of diseased plants at each severity level; nt represents the total number of plants.

[0029] Experimental results showed that in a replicate experiment with a sample size of 30, after 30 days of treatment, the disease index of cotton in the experimental group was 75.83, while that in the control group was 89.16, significantly reducing the disease index. Furthermore, compared to the control group, the disease index of cotton in the experimental group was significantly lower. The growth was significantly better than that of the control group. Figure 7 This result indicates that strain 70-1 can effectively control cotton Verticillium wilt.

[0030] The above embodiments are only used to illustrate the design concept and features of the present invention, and their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. The protection scope of the present invention is not limited to the above embodiments. Therefore, all equivalent changes or modifications made based on the principles and design ideas disclosed in the present invention are within the protection scope of the present invention.

Claims

1. A strain of Streptomyces sp. 70-1, which is resistant to cotton Verticillium wilt, characterized in that, The taxonomic name of the said Streptomyces 70-1 is Streptomyces syringium , which was deposited with the Guangdong Microbial Culture Collection Center on November 6, 2025, and has the accession number GDMCC NO. 67234.

2. The Streptomyces sp. 70-1 of claim 1, wherein, The 16S rDNA sequence of the Streptomyces syringium 70-1 is shown as SEQ ID NO.

1.

3. A biocontrol agent prepared from the Streptomyces syringium 70-1 according to any one of claims 1-2.

4. The preparation method of the biocontrol agent according to claim 3, characterized in that, Specifically, the following steps are used: the Streptomyces syringium single colony 70-1 is inoculated into 50 mL of ISP-2 culture solution, and cultured at 30°C and 180 rpm / min for 3 days, then transferred into 1 L of ISP-2 fermentation culture solution at a volume ratio of 3:100, and expanded cultured under the same conditions for 7 days, and the obtained fermentation solution is the biocontrol agent of Streptomyces syringium 70-1.

5. The preparation method of the biocontrol agent according to claim 4, characterized in that, The concentration of Streptomyces 70-1 in the biocontrol agent is 1 x 10 8 cfu / mL.

6. Use of the biocontrol agent of Streptomyces syringium 70-1 according to claim 3 in the preparation of a preparation for preventing and treating cotton Verticillium wilt caused by Verticillium dahliae.

7. Use of the biocontrol agent of Streptomyces syringium 70-1 according to claim 3 in the preparation of a preparation for plant diseases caused by Fusarium graminearum and / or Rhizoctonia solani.

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