Streptomyces griseoaurantii capable of preventing and treating konjak southern blight and application of streptomyces griseoaurantii

Abstract

The invention relates to streptomyces griseoaurantii ZDY1-19 capable of preventing and treating konjak southern blight, which is preserved in China General Microbiological Culture Collection Center (CGMCC) on September 27, 2024, and has a preservation number of CGMCC No.32086. The invention also relates to application of the streptomyces griseoaurantii ZDY1-19 in preventing and treating the konjak southern blight. The streptomyces griseoaurantiacus ZDY1-19 strain disclosed by the invention has a very strong inhibition effect on pathogenic bacteria of konjak southern blight, and fermentation liquor of the streptomyces griseoaurantiacus ZDY1-19 strain contains various metabolites with antibacterial effects. The strain has a relatively strong inhibition effect on sclerotium production and sclerotium germination of sclerotium rolfsii, and the inhibition rates can respectively reach 85.89% and 81.67%. The prevention and treatment effect of the konjak potted plant treated by the ZDY1-19 fermentation liquor is 64.36%. The bacterial strain is separated from konjak soil, has natural harmonious compatibility with a konjak planting environment, and has no toxic or side effect and no residue on soil ecology compared with chemical pesticides, so that the bacterial strain has potential commercial development and application values in biological prevention and control practice of diseases.

Description

Technical Field

[0001] The invention relates to the field of crop disease prevention and control, and more particularly to a streptomyces glauca capable of preventing and controlling konjac white rot and application thereof. Background Art

[0002] Konjac white rot is an important soil-borne disease that has occurred in konjac planting areas in recent years. It is also the second largest killer after soft rot. In areas where konjac is continuously planted, the incidence of the disease in general fields is 0%-14%, and the number of fields with serious disease is more than 42%, which seriously affects the yield and quality of konjac and causes great losses to farmers.

[0003] In the early stage of white rot, small irregular dark brown spots like water stains first appear at the base of the konjac stems, then gradually turn brown and rot, showing a wet rot state. The above-ground leaves wilt, and then the plant falls over. A large number of white silky hyphae are produced in the diseased part, which in the later stage are formed into rapeseed-like sclerotia that are initially milky white, gradually turn into light yellow, and finally turn into tea brown to dark brown.

[0004] At present, in actual production, the prevention and control of konjac diseases mainly relies on chemical control, which is relatively simple and quick to operate, with low cost and quick results. However, excessive use of chemical agents can easily lead to consequences such as drug resistance and pesticide residues in the soil, ultimately resulting in poor prevention and control effects, harm to ecological security and human health, and other problems.

[0005] With the further development of scientific research, it is found that biocontrol microorganisms have unique advantages in overcoming the resistance of plant diseases, reducing the pollution of chemical fungicides to the environment, ecological damage and the residues of chemical pesticides in agricultural and sideline products. The biological pesticides prepared by the secondary metabolites of biocontrol bacteria have the characteristics of no pollution, no residue, not easy to make pests develop resistance, high compatibility with the environment, and safety for humans and animals. And the research on using the metabolites produced by microorganisms to treat plant pathogens and achieve biological control of diseases is currently a hot topic. The biocontrol potential of secondary metabolites produced by actinomycetes is mainly reflected in the inhibitory effect on the growth of pathogenic mycelium and infection. More and more actinomycetes derived from various plant endogenous or different extreme environments have been discovered, and the types of metabolites produced by actinomycetes will become more and more abundant. Therefore, vigorously carrying out research on biological control is an important way to achieve sustainable agricultural development and has important practical significance for production. Summary of the invention

[0006] To solve the above problems, the present invention provides a gray-orange Streptomyces that can prevent and treat konjac white rot, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on September 27, 2024, with the deposit number CGMCC No.32086.

[0007] The present invention also provides the use of the streptomyces griseo-orange in preventing and treating konjac southern blight.

[0008] The present invention also provides a method for preventing and controlling konjac white rot, comprising the step of applying the fermentation liquid of the streptomyces griseo-orange to the soil where crops are planted.

[0009] In a specific embodiment, the method comprises the following steps:

[0010] S1: culturing the Streptomyces griseo-orange to prepare a fermentation liquid or a sterile fermentation liquid of the Streptomyces griseo-orange;

[0011] S2: applying the fermentation liquid of Streptomyces griseoaurantifolia or the sterile fermentation liquid to the soil where the crop is planted.

[0012] In a specific embodiment, in S1, the Streptomyces griseoaurantifolia is cultured in Gao's medium No. 1.

[0013] In a specific embodiment, the fermentation broth of Streptomyces griseoaurantifolia in S1 is prepared by the following steps:

[0014] S11: inoculating the Streptomyces griseo-orange into Gao's liquid culture medium No. 1 for cultivation;

[0015] S12: Cultivate at 28° C. and 180 rpm for 5-7 days to obtain the fermentation liquid of Streptomyces griseoaurantifolia.

[0016] In a specific embodiment, the sterile fermentation broth of Streptomyces griseoaurantifolia described in S1 is obtained by removing cells and filtering the fermentation broth of Streptomyces griseoaurantifolia.

[0017] In a specific embodiment, in S2, the crop is irrigated with the fermentation liquid of Streptomyces griseoaurantifolia or the sterile fermentation liquid.

[0018] The gray orange Streptomyces ZDY1-19 strain of the present invention has a strong inhibitory effect on the pathogenic bacteria of konjac white rot, and its fermentation liquid contains a variety of metabolites with antibacterial effects. The strain has a strong inhibitory effect on the generation of sclerotia of white rot and the germination of sclerotia, and the inhibition rates can reach 85.89% and 81.67% respectively. The konjac potted plant control effect treated with the ZDY1-19 fermentation liquid is 64.36%. Because the strain is separated from konjac soil, it has natural harmony and compatibility with the konjac planting environment, and compared with chemical pesticides, it has no toxic side effects on soil ecology and no residue, therefore, it has potential commercial development and application value in the biological control practice of diseases.

[0019] Biomaterial Deposit

[0020] The purified Streptomyces griseo-orange ZDY1-19 of the present invention was deposited on September 27, 2024 at the China General Microbiology Center (CGMCC) of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 32086. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the colony morphology of Streptomyces griseo-orange ZDY1-19 after 7 days of culture.

[0022] Figure 2 This is the colony morphology of Streptomyces griseo-orange ZDY1-19 after 14 days of culture.

[0023] Figure 3 This is the aerial hyphae morphology of Streptomyces griseo-orange ZDY1-19.

[0024] Figure 4 This is the spore chain morphology of Streptomyces griseo-orange ZDY1-19.

[0025] Figure 5 The results show that Streptomyces griseo-orange ZDY1-19 has an inhibitory effect on Saccharomyces rolfsii.

[0026] Figure 6 This is the effect of the fermentation filtrate of Streptomyces griseoaurantifolia ZDY1-19 on the mycelial morphology of Sclerotium rolfsii.

[0027] Figure 7 The results show that Streptomyces griseo-orange ZDY1-19 has an inhibitory effect on the sclerotia production of Sclerotium rolfsii.

[0028] Figure 8 The inhibitory effect of Streptomyces griseo-orange ZDY1-19 on the germination of sclerotia of Sclerotium rolfsii.

[0029] Fig. 9 The results show that Streptomyces glauca ZDY1-19 has a preventive and therapeutic effect on konjac white rot. DETAILED DESCRIPTION

[0030] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0031] 1. Identification and preservation of actinomycete ZDY1-19

[0032] Our team collected field soil from cultivated konjac in Zhongdu Town, Pingshan County, Yibin City, and isolated multiple strains. After initial and rescreening, they finally identified the actinomycete ZDY1-19, which has a good preventive and therapeutic effect on konjac white rot, through an indoor potted inoculation test of living plants.

[0033] The strain ZDY1-19 was cultured on Gao's No. 1 culture plate at 28°C for 7 days, and a single colony was obtained as shown in the following figure. Figure 1 As shown; after 14 days of culture, the single colony photo is as shown Figure 2 shown.

[0034] Insert the sterilized slide obliquely into the plate at 30℃, incubate it upside down in a 28℃ incubator for 7 days, remove the cover glass, drop a little distilled water on the slide, and observe the strain under an optical microscope. The hyphae morphology is as follows: Figure 3 As shown, the spore chain morphology is as follows Figure 4 shown.

[0035] The genomic DNA of the strain was extracted, and the bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO: 1,) and 1492R (5'-TACGGCTACCTTGACGAC TT-3', SEQ ID NO: 2). The genomic DNA of strain ZDY1-19 was used as a template, and PCR amplification was performed after electrophoresis detection and sequencing to obtain the 16S rDNA sequence as shown in SEQ ID NO: 3. The obtained sequence results were compared with Blast in NCBI, and the 16S rDNA sequences of related strains were selected. The phylogenetic tree was constructed using MEGA11 software, and the strain was identified as Streptomyces griseaurantiacus in combination with morphological characteristics.

[0036] The above-mentioned strain was deposited on September 27, 2024 at the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number: CGMCC No.32086.

[0037] 2. Preparation of fermentation broth of Streptomyces griseo-orange ZDY1-19 and analysis of metabolites

[0038] Two ZDY1-19 bacterial cakes with a diameter of 5 mm were inoculated into a 300 mL Erlenmeyer flask containing 100 mL of liquid Gao's No. 1 medium, and placed in a constant temperature shaker at 28°C and 180 r / min for 5 days to obtain the fermentation broth. The fermentation broth was poured into a sterile 50 mL centrifuge tube, centrifuged at 8000 rpm for 15 min, and the supernatant was filtered with a 0.22 μm filter to obtain a sterile fermentation filtrate. The sterile fermentation broth was subjected to product analysis, and the results are shown in Table 1, which showed that it contained a variety of antibacterial metabolites, including cycloheximide, campesterol, menthol, carvone, hypoxanthine, propionic acid, pantothenic acid, etc.

[0039] Table 1 Main antibacterial active substances produced by strain ZDY1-19

[0040]

[0041] 3. Streptomyces griseo-orange ZDY1-19 for the prevention and control of pathogens

[0042] 3.1 Antibacterial activity of Streptomyces griseo-orange ZDY1-19 against pathogens

[0043] The plate confrontation method was used to determine the effect of antagonistic bacteria on the mycelial growth of pathogenic fungi. The ZDY1-19 strain was streaked and inoculated on Gao's No. 1 medium. After inverted culture at 28℃ for 5 days, a 5mm bacterial cake was taken for use. A 5mm bacterial cake of the ZDY1-19 strain was placed on the PDA plate at 25mm on both sides of the center and inverted culture at 28℃ for 2 days. A 5mm bacterial cake of white rot was taken and placed in the center of the plate inoculated with ZDY1-19 bacterial cake. The control group was inoculated with white rot cake in the center of the PDA plate without ZDY1-19 bacterial cake. Each treatment was repeated 3 times. After 3 days of culture in a constant temperature incubator at 28℃, the colony diameter of white rot was measured and the inhibition rate was calculated.

[0044] The results are as follows Figure 5 As shown, ZDY1-19 has a significant antibacterial effect.

[0045] 3.2 Inhibitory effect of sterile fermentation broth of Streptomyces glauca ZDY1-19 on the nuclei of Sclerotium rolfsii

[0046] Pour 32mL of PDA medium melted and cooled to 50℃ into a 50mL centrifuge tube, add 8mL of fermentation filtrate, mix well and pour evenly into 3 90mm sterile culture dishes to prepare a drug-containing plate containing 20% ​​fermentation filtrate; the control is 32mL PDA medium inoculated with 8mL of Gao's No. 1 liquid medium. Wait for the plate to be completely cooled and solidified before use. Inoculate a 5mm konjac white rot fungus cake into the center of the plate containing 20% ​​ZDY1-19 strain fermentation filtrate, place it in a 28℃ constant temperature incubator, and culture in the dark for 30 days. Take out the sclerotia from the culture dishes respectively, dry them until the weight no longer changes, and weigh them, record the dry weight of sclerotia of each dish, and calculate the inhibition rate.

[0047] The results of scanning electron microscopy observations are as follows Figure 6 As shown in the figure, the sterile fermentation liquid of Streptomyces griseo-orange ZDY1-19 can cause the mycelium of S. rolfsii to shrink and become deformed, and the sterile fermentation liquid of Streptomyces griseo-orange ZDY1-19 also showed a significant inhibition on the production of sclerotia ( Figure 7 ), the inhibition rate can reach 85.89%.

[0048] 3.3 Inhibitory effect of sterile fermentation broth of Streptomyces glauca ZDY1-19 on germination of Sclerotium rolfsii

[0049] In a plate containing 20% ​​of the fermentation filtrate of the ZDY1-19 strain, 20 sclerotia of S. rolfsii of equal size were placed, and the sclerotia were evenly distributed in 4 rows and 5 columns in the dish. The plate was placed in a constant temperature incubator at 28°C and cultured in the dark. The germination of sclerotia was recorded every 24 hours and the inhibition rate was calculated.

[0050] The results are as follows Figure 8 As shown, the sterile fermentation broth of Streptomyces griseoaurantifolia ZDY1-19 exerted a strong inhibitory effect on sclerotial germination, with an inhibition rate of up to 81.67%.

[0051] 3.4 Control effect of Streptomyces grisea ZDY1-19 fermentation liquid on konjac white rot

[0052] The potted experiment was carried out by inoculating konjac with needle inoculation. ZDY1-19 bacterial cake was inoculated into Gao's No. 1 liquid culture medium and cultured in a shaker at 28°C and 180 rpm for 7 days to obtain a concentration of 10 8 CFU / mL of ZDY1-19 strain fermentation liquid. Select seed taro of the same size and culture at 25℃. Wait for the plants to fully expand their leaves for 10 days before use. Treatment group: irrigate the roots with ZDY1-19 strain fermentation liquid, 30mL per plant, and apply the same amount of water to the control group, 12 plants per group, and repeat the experiment 3 times. After 2 days of root irrigation treatment, use an inoculation needle to puncture the base of each konjac stem and inoculate the bacterial cake of white rot fungus. After 7 days, investigate the disease situation and calculate the control effect.

[0053] The results are as follows Fig. 9 As shown, the fermentation liquid of Streptomyces griseoaurantifolia ZDY1-19 has a good control effect on white rot, and the control effect of konjac potted plants treated with ZDY1-19 fermentation liquid is 64.36%.

[0054] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A Streptomyces glaucae for preventing and treating konjac white rot, characterized in that: It was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 27, 2024, with the deposit number CGMCC No.32086.

2. Use of the Streptomyces glauca described in claim 1 in preventing and treating Sclerotium rot of konjac.

3. A method for preventing and treating konjac white rot, characterized in that, The method comprises the step of applying the fermentation liquid of Streptomyces griseoaurantifolia according to claim 1 to soil for planting crops.

4. The method according to claim 3, characterized in that The following steps are involved: S1: culturing the Streptomyces griseo-orange to prepare a fermentation liquid or a sterile fermentation liquid of the Streptomyces griseo-orange; S2: applying the fermentation liquid of Streptomyces griseoaurantifolia or the sterile fermentation liquid to the soil where the crop is planted.

5. The method according to claim 4, characterized in that In S1, the Streptomyces griseo-orange is cultured in Gao's medium No.

1.

6. The method according to claim 5, characterized in that The Streptomyces griseo-orange fermentation broth described in S1 is prepared by the following steps: S11: inoculating the Streptomyces griseo-orange into Gao's liquid culture medium No. 1 for cultivation; S12: Cultivate at 28° C. and 180 rpm for 5-7 days to obtain the fermentation liquid of Streptomyces griseoaurantifolia.

7. The method according to claim 6, characterized in that S1 The sterile fermentation broth of Streptomyces griseo-orange is obtained by removing cells and filtering the fermentation broth of Streptomyces griseo-orange.

8. The method according to claim 4, characterized in that In S2, the crops are irrigated with the fermentation liquid of Streptomyces griseo-orange or the sterile fermentation liquid.

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