Streptomyces Venezuralensis and application thereof

Abstract

The invention discloses a streptomyces Venezuelae strain cao-82, which is streptomyces Venezuelae, is preserved in the Guangdong Microbial Culture Collection Center, and has a preservation number of GDMCC No: 64953. The streptomyces Venezuelae strain cao-82 is a streptomyces Venezuelae strain cao-82 and is preserved in the Guangdong Microbial Culture Collection Center. Studies show that the streptomyces spinosus cao-82 has bacteriostatic activity on fungal plant diseases such as fusarium tsaoko, and a plate buckling test shows that the volatile component of the streptomyces can effectively inhibit the growth of the fusarium tsaoko, and the highest inhibition rate reaches 88.3%.

Description

Technical Field

[0001] The invention belongs to the technical field of biological pesticides and plant disease prevention and control, and particularly relates to a Venezuelan Streptomyces strain and application thereof in inhibiting plant pathogens. Background Art

[0002] Amomum villosum is one of the major varieties of Chinese medicinal materials for both medicinal and edible use. It is a perennial evergreen clump of herbaceous plants belonging to the subgenus Amomum villosum of the genus Amomum of the family Zingiberaceae. It is mainly distributed in some counties and cities in Nujiang Prefecture, Wenshan Prefecture, and Honghe Prefecture in Yunnan Province, my country. In recent years, many places in Yunnan Province have made efforts to make Amomum villosum into an agricultural product with local characteristics to increase the source of income for growers. With the expansion of the planting area and the imperfect supporting management, its diseases show a trend of local spread, with wilt disease as the main disease, which is a type of soil-borne disease caused by complex fungi mainly composed of Fusarium oxysporum. The current means of preventing and controlling Amomum villosum wilt disease are mainly chemical control, which will cause certain drug damage to the plants, kill beneficial microorganisms, cause pathogens to develop drug resistance, and the product safety is not guaranteed; while microbial control has the advantages of being environmentally friendly, safe, and quickly inhibiting the growth and reproduction of pathogenic microorganisms.

[0003] Microbial control, as a means of disease control, is to use the interaction between biological species to inhibit another or another type of organism with one or a type of organism, thereby preventing and controlling diseases. Streptomyces is an important soil microorganism that plays an important role in agricultural production. As a bacterium that produces soil bactericidal substances, it can play an important ecological role in agricultural production. The production of Streptomyces and its application in agriculture is an important direction for agricultural development, and it is of great significance to promote the transformation of agricultural production methods and improve agricultural production efficiency. Isolating and screening Streptomyces with biocontrol functions from the rhizosphere soil of Amomum villosum, and evaluating their biocontrol functions and biological characteristics are important for the next step of developing special biocontrol agents and fertilizers for Amomum villosum and reducing the application of chemical pesticides.

[0004] At present, there are few patents on the research of special microbial agents for amomum villosum diseases. There is only patent number CN 115044481 B. Yang Peiwen et al. reported that they randomly found a strain of Trichoderma and compounded it with organic carbon fertilizer, oil cake, bagasse, tea residue and nitrogen, phosphorus and potassium compound fertilizer, which had a good control effect on amomum villosum. However, there is a problem that the Trichoderma used was not isolated from the amomum villosum field, and it is uncertain whether it can effectively colonize after being applied to the amomum villosum field. In addition, the patent did not provide pictures of the biocontrol effect of Trichoderma, making it difficult to objectively evaluate the biocontrol effect of Trichoderma. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention proposes a Venezuelan Streptomyces and its application in inhibiting plant pathogens. The Streptomyces provided by the present invention is isolated from the healthy rhizosphere soil next to the grass fruit root rot disease plants in Maji Township, Fugong County, Nujiang Prefecture, and has good field colonization. The obtained Streptomyces showed good biocontrol function in confrontation culture, screening of fermentation liquid, and plate punching method activity test on the distribution of the three main pathogens of grass fruit wilt, providing an important basis for the application of the biocontrol agent in production.

[0006] The technical solution of the present invention is as follows:

[0007] The present invention first provides a Venezuelan Streptomyces strain, which is classified and named as Streptomyces venezuelae, and is preserved in Guangdong Microbiological Culture Collection Center with a preservation number of GDMCC No: 64953, a preservation date of February 5, 2024, and a preservation address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0008] The present invention also provides a fermentation broth of the Venezuelan Streptomyces, and the preparation method of the fermentation broth comprises the following steps: inoculating the Venezuelan Streptomyces into an ISP2 culture medium for fermentation culture; the ISP2 culture medium is composed of the following components: 10.0 g / L malt extract, 4.0 g / L glucose, 4.0 g / L yeast powder, and water.

[0009] Preferably, the fermentation culture conditions are 28°C to 30°C, shaking constant temperature culture for 5 to 7 days.

[0010] On the other hand, the present invention also provides the use of the Venezuelan Streptomyces and / or its fermentation products in inhibiting the growth of tsaoko pathogens.

[0011] Preferably, the pathogenic fungi include at least one of Fusarium graminearum, Fusarium mangiferae and Fusarium proliferatum.

[0012] Preferably, the fermentation product comprises volatile and / or non-volatile components produced by fermentation.

[0013] Preferably, the fermentation product comprises the fermentation broth.

[0014] Beneficial effects of the present invention:

[0015] The Venezuelan Streptomyces cao-82 provided by the present invention is derived from the rhizosphere soil of the tsaoko, which ensures that the strain can be effectively colonized and survive after being returned to the tsaoko planting site, laying a foundation for the practicality of developing microbial agents in the future.

[0016] The present invention is the first to use Venezuelan Streptomyces to carry out biological control of the tsaoko pathogen, and explores a fermentation process suitable for the Streptomyces cao-82, laying a foundation for its later production of bacterial agents.

[0017] The biocontrol bacterium cao-82 has the function of antagonizing the pathogenic fungi of Amomum villosum by releasing volatile and non-volatile components.

[0018] The plate confrontation experiment showed that the strain had a good inhibitory effect on the grass fruit pathogen Fusarium graminearum, Fusarium mangiferae and Fusarium proliferatum, with inhibition rates of 49.1%, 56.7% and 53.3% respectively. The plate-matching experiment showed that the inhibition rates of the three pathogens were 52.8%, 88.3% and 66.7% respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : Morphology of Streptomyces cao-82 strain.

[0020] Figure 2 : Growth morphology of three grass fruit pathogens on PDA medium.

[0021] Figure 3 :The morphology of Streptomyces cao-82 grown on different culture media.

[0022] Figure 4 : The results of confrontation between Streptomyces cao-82 and the pathogenic fungi of Amomum villosum.

[0023] Figure 5 : Scanning electron micrograph of mango malformation disease Fusarium solani.

[0024] Figure 6 : Experimental results of the double plate method. The left side is the experimental group (upper: F. mangiferae, lower: cao-82), and the right side is the blank group (upper: F. mangiferae, lower: ISP4).

[0025] Figure 7 :The antibacterial effect of Streptomyces cao-82 fermentation broth on different culture media. DETAILED DESCRIPTION

[0026] In order to better understand the technical content of the present invention, the present invention is further described below through specific embodiments.

[0027] Example 1 Isolation, screening and identification of Streptomyces venezuelae cao-82 strain

[0028] (1) Preparation of culture medium

[0029] Gao's medium No. 1: prepared in the following mass volume ratio: soluble starch 20.0 g / L, sodium chloride 0.5 g / L, ferrous sulfate 0.01 g / L, potassium sulfate 1.0 g / L, potassium nitrate 1.0 g / L, magnesium sulfate 0.5 g / L, potassium dichromate 50 μg / mL, agar 15 g / L, pH 7.2-7.4.

[0030] PDA medium was prepared in the following mass volume ratio: potato 200.0 g / L, glucose 20.0 g / L, agar 18.0 g / L, pH 5.6±0.2.

[0031] Use ready-made ISP4 medium.

[0032] (2) Sample collection and processing

[0033] The rhizosphere soil 8-10 cm below the ground surface was collected from the Tsaoko planting field in Zuokanzhi Village, Shiyueliang Township, Fugong County, Nujiang Prefecture, Yunnan Province, China.

[0034] (3) Strain isolation and purification

[0035] The modified Gao's medium No. 1 was selected as the plate for isolating actinomycetes. The isolation process was as follows: 1 g of soil sample was weighed into a sterile test tube, 10 ml of sterile water was added, and the mixture was shaken at 28°C and 200 rpm for 30 min, which was recorded as 10. The multiple dilutions were used to obtain 10 -1 , 10 -2 , 10 -3 Dilute the soil sample solution at different concentrations. Take 100ul of each concentration and spread it on the plate, repeat three times. Culture at 28℃ for 14 days, pick representative single colonies with different morphological characteristics, separate and purify them on ISP4 medium, and obtain an antagonistic Streptomyces strain that can effectively inhibit multiple pathogens, named cao-82. After purification, freeze in 25% glycerol tubes at -80℃.

[0036] (4) Identification results of strain cao-82

[0037] ① The morphological characteristics of the strains are shown in Table 1:

[0038] Table 1 Morphological characteristics of strains

[0039]

[0040] ②Inoculate the purified cao-82 on ISP1, ISP2, ISP3, ISP4, ISP5, and ISP7 culture media to observe its growth morphology. Figure 3 .

[0041] ③ The purified cao-82 was inoculated on ISP4 plates and cultured at 28°C for 5 days. The plates with bacteria were sent to the Wuhan Branch of Beijing Liuhe BGI Gene Technology Co., Ltd. for sequencing and strain identification. The results showed that the strain was Streptomyces venezuelae.

[0042] The strain cao-82 of the present invention is named as Streptomyces venezuelae under the preservation classification, and is preserved in Guangdong Microbiological Culture Collection Center, with the preservation number as GDMCC No: 64953 and the preservation date as February 5, 2024.

[0043] Nucleotide sequence of strain cao-82 (SEQ ID NO.1)

[0044]

[0045] Example 2 Antibacterial test of Streptomyces cao-82 strain against tsaoko pathogenic fungi

[0046] (1) Plant pathogenic fungi tested

[0047] Fusarium graminearum, Fusarium mangiferae and Fusarium proliferatum all originate from the pathogenic tissue of Amomum villosum. Their pathogenicity to Amomum villosum was verified through multiple inoculation experiments.

[0048] (2) Culture medium

[0049] ISP4 medium

[0050] (3) Test method

[0051] Plate confrontation method: activate the three test grass fruit pathogenic fungi plates, pick up hyphae with a sterile inoculation loop, and spot-inoculate on the center point of the ISP4 medium plate. Use a sterile inoculation loop to evenly spot four points of Cao-82 Streptomyces at a distance of 2 cm from the pathogens. Select Streptomyces-fungus co-culture, and use the ISP4 plate with only the pathogens in the center point as the control group. Keep it at a constant temperature of 28°C and invert it for culture. Record the culture results. Calculate the hyphae growth inhibition rate after 7 days of confrontation culture. Mycelium growth inhibition rate = (control colony growth diameter - treatment colony growth diameter) / control colony growth diameter × 100%.

[0052] After 15 days of co-cultivation, the hyphae of Streptomyces mango deformity were collected from the normal growing and confrontation treated strains for scanning electron microscopy to observe the hyphae morphology.

[0053] Double plate method: The upper culture dish is PDA medium, inoculated with the grass fruit pathogen, and the lower ISP4 medium is inoculated with Streptomyces cao82. After 2 days of pre-culture of Streptomyces, the grass fruit pathogen is inoculated in PDA medium and placed on the ISP4 culture dish coated with Streptomyces cao82, which is sealed with sealing film. After 5 days of static culture at 28℃ in the dark, the mycelium diameter is measured and the inhibition rate is calculated using the same calculation formula as above.

[0054] (4) Test results

[0055] The inhibition rate of the strain against Fusarium graminearum was 49.1%, against Fusarium graminearum causing mango malformation was 56.7%, and against Fusarium solani was 53.3%. The growth of the pathogenic fungi of the tsaoko in the co-culture group was significantly inhibited compared with that in the control group during the same period. Streptomyces cao-82 showed a broad spectrum of antibacterial activity against the tested pathogenic fungi of the tsaoko. For details, see Figure 4 .

[0056] Scanning electron microscope was used to observe the hyphae of mango deformity disease Fusarium. It can be seen that the hyphae of the control group were smooth and uniform; the hyphae of the plate confrontation treatment were seriously deformed and broken, and the hyphae appeared sunken and flat, which led to the growth of hyphae being hindered, and even stopped growing. For details, see Figure 5 .

[0057] The plate test showed that Streptomyces cao-82 exhibited different degrees of gas inhibition activity against the three pathogens. The inhibition rate against Fusarium graminearum was 52.8%, against Fusarium solani causing mango malformation was 88.3%, and against Fusarium solani was 66.7%.

[0058] Example 3 Screening of Streptomyces cao-82 fermentation broth (antipathogenic fungi test)

[0059] (1) Preparation of each liquid culture medium

[0060] Improved SGCY medium: sucrose 20.0 g / L, glucose 10.0 g / L, chelating amino acids 0.1 g / L, yeast powder 5.0 g / L, MOPS propanesulfonic acid 5.0 g / L, K SO 0.25 g / L, MgCl·6H O 1.0 g / L, ultrapure water 1.0 L.

[0061] Improved SCAS medium: soluble starch 40.0 g / L, complexed amino acids 5.0 g / L, KH PO 0.5 g / L, MgSO · 7HO 0.5 g / L, FeSO · 7HO 0.5 g / L, ultrapure water 1.0 L.

[0062] ISP2: malt extract 10.0 g / L, glucose 4.0 g / L, yeast powder 4.0 g / L, ultrapure water 1.0 L.

[0063] ISP4 medium without agar powder.

[0064] (2) Screening test of Streptomyces cao-82 fermentation broth

[0065] The strain cao-82 was inoculated on an ISP4 plate and cultured at 28°C for 5 to 7 days. A single bacterial block was picked and inoculated into a 500-mL shake flask containing 200 mL of each liquid culture medium. The culture was shaken at 28°C and 200 rpm for 5 to 7 days, and each fermentation broth was filtered with a sterile filter.

[0066] Take the activated mango deformity disease Fusarium plate, use a sterile puncher (Φ5mm) to make a bacterial cake for later use, inoculate it on the center of the PDA medium plate, use a sterile puncher (Φ5mm) to punch four holes at a spacing of 2.0cm, take the same filtered fermentation liquid from each plate, take 110ul from each hole, and use sterile water as a control group. Culture at a constant temperature of 28℃ for 6 days.

[0067] (3) Test results

[0068] The most suitable formula for the fermentation broth of Streptomyces cao-82 is ISP2, which has a more obvious effect of inhibiting the growth of pathogenic fungi, with an inhibition rate of 32.5%.

[0069] In summary, the Streptomyces cao-82 strain provided by the present invention has a significant antibacterial effect on the main pathogens of tsaoko, Fusarium graminearum, Fusarium mangiferae, and Fusarium proliferatum, providing a new strain resource for the biological control of tsaoko diseases.

[0070] The above descriptions are only some embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall fall within the protection scope of the present invention.

Claims

1. A strain of Streptomyces venezuelae, characterized in that The Venezuelan Streptomyces is deposited in Guangdong Microbiological Culture Collection Center with the deposit number of GDMCC No: 64953.

2. The fermentation broth of Streptomyces venezuelae according to claim 1, characterized in that The method for preparing the fermentation liquid comprises the following steps: inoculating Streptomyces venezuelae into ISP2 medium for fermentation culture; the ISP2 medium is composed of the following components: 10.0 g / L malt extract, 4.0 g / L glucose, 4.0 g / L yeast powder, and water.

3. The fermentation broth according to claim 2, characterized in that The fermentation culture conditions are 28°C to 30°C and shaking constant temperature culture for 5 to 7 days.

4. Use of the Streptomyces venezuelae and / or its fermentation products according to claim 1 in inhibiting the growth of pathogenic bacteria of Amomum villosum.

5. The use according to claim 4, characterized in that: The pathogenic fungi include at least one of Fusarium graminearum, Fusarium mangiferae and Fusarium proliferatum.

6. The use according to claim 4, characterized in that: The fermentation product includes volatile and / or non-volatile components produced by fermentation.

7. The use according to claim 4, characterized in that: The fermentation product comprises the fermentation broth according to any one of claims 2 to 3.

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