Chinese yam endophytic bacterium and application thereof

Abstract

The invention discloses a Chinese yam endophytic bacterium and application thereof, and belongs to the technical field of biological control of plant diseases. The Chinese yam endophytic bacterium is Burkholderia cenocepacia, the strain name is SMDR8, the Chinese yam endophytic bacterium is preserved in the China Center for Type Culture Collection (CCTCC) on August 31, 2023, the preservation number is CCTCC M 20231555, the 16S rDNA sequence fragment of the SMDR8 is 1448 bp, and the nucleotide sequence of the SMDR8 is shown as SEQ ID No.1. The Chinese yam endophytic bacterium can be used for preparing the Chinese yam endophytic bacterium. The SMDR8 disclosed by the invention has a certain antagonistic effect on colletotrichum gloeosporioides of Chinese yam in a plate confrontation experiment; the SMDR8 biocontrol microbial inoculum is diluted and then sprayed on Chinese yam leaves of a planting base, and after 45 days, the disease index of an experimental group is obviously lower than that of a control group. Compared with chemical pesticides, the pesticide composition has the advantages of no pollution, no residue, no toxicity and higher safety to the environment, and provides a new strategy for preventing and treating the Chinese yam anthracnose.

Description

Technical Field

[0001] The present invention relates to the technical field of biological control of plant diseases, and more specifically to an endophytic bacterium of Chinese yam and its application. Background Art

[0002] Chinese yam ( Dioscorea alata ), also known as Huai yam, is an annual or perennial herbaceous vine of the genus Dioscorea in the family Dioscoreaceae, and is widely cultivated in China. As one of the functional foods with both medicinal and edible properties, Chinese yam has a soft and glutinous taste and is rich in nutrients. It contains various functional active ingredients such as polysaccharides, polyphenols, and amino acids, and has functions such as antioxidant, anti-tumor, hypoglycemic, hypolipidemic, and immune function regulation.

[0003] Colletotrichum is a fungal disease with a wide distribution range, many host plants, and strong infectivity. It harms various crops such as citrus, strawberries, sorghum, and corn, thus causing anthracnose. With the continuous expansion of the planting scale, the increase in the planting years, and the continuous accumulation of pathogenic bacteria in the Chinese yam planting area, the occurrence of Chinese yam anthracnose is becoming more and more serious. Due to the occurrence of anthracnose, there are frequent cases of significant reduction or even complete loss of Chinese yam production. At first, Chinese yam anthracnose mainly infects the leaves and vines of Chinese yam. After the disease occurs, the lesions continue to expand until the whole plant is affected, and finally the vines wither and the whole plant dies.

[0004] Currently, in production, 77% copper hydroxide wettable powder, 50% thiram wettable powder, 70% mancozeb wettable powder and other agents are often used to control Chinese yam anthracnose, and at the same time, measures such as field cultivation management (crop rotation, removal of diseased residues in the field, reasonable close planting, etc.) are combined. However, the long-term use of chemical pesticides will cause pathogenic bacteria to develop drug resistance, which in turn leads to an increase in the amount of pesticides used by growers, and further enhances the drug resistance of pathogenic bacteria, forming a vicious cycle. The control efficiency of Chinese yam anthracnose by single chemical control is low, and it poses a great threat to the environment and food safety. Summary of the Invention

[0005] The present invention provides an endophytic bacterium of Chinese yam and its application, aiming to solve the problems that the existing single chemical agent has low control efficiency for Chinese yam anthracnose and poses a great threat to the environment and food safety.

[0006] The present invention adopts the following technical scheme: An endophytic bacterium of Chinese yam, which is Burkholderia cepacia Burkholderia cenocepacia, the strain name is SMDR8, which was deposited at the China Center for Type Culture Collection (CCTCC) on August 31, 2023, with the deposit number CCTCC M20231555. The 16S rDNA sequence fragment of SMDR8 is 1448 bp, and the nucleotide sequence is shown in SEQ ID No.1.

[0007] Application of the activated culture strain of the above SMDR8 in antagonizing Colletotrichum gloeosporioides of Dioscorea opposita.

[0008] Application of the above SMDR8 in preparing a biocontrol agent.

[0009] Application of the biocontrol agent of the above SMDR8 in controlling Colletotrichum gloeosporioides of Dioscorea opposita.

[0010] The concentration of the above biocontrol agent is OD 600 = 0.2.

[0011] Preparation method of the above biocontrol agent, comprising the following steps: (1) Activate the SMDR8 strain on an LB plate medium; (2) After the strain activation in step (1), pick a single colony and inoculate it into an LB liquid medium for culture to obtain a seed solution of SMDR8; (3) Expand the culture of the SMDR8 seed solution obtained in step (2) in an LB liquid medium to prepare a biocontrol agent.

[0012] From the above description of the present invention, compared with the prior art, the present invention has the following advantages: SMDR8 of the present invention has a certain antagonistic effect on Colletotrichum gloeosporioides in the plate confrontation experiment; and after diluting the SMDR8 biocontrol agent and spraying it on the leaves of Dioscorea opposita in the planting base, the disease index of the experimental group is significantly lower than that of the control group after 45 d. Compared with chemical pesticides, it is pollution-free, residue-free, and non-toxic, and has high safety for the environment. It is a green prevention and control measure, providing a new strategy for controlling Colletotrichum gloeosporioides of Dioscorea opposita. Description of the Drawings

[0013] Figure 1 Schematic diagram of the situation of SMDR8 of the present invention antagonizing Colletotrichum gloeosporioides of Dioscorea opposita on a plate.

[0014] Figure 2 Schematic diagram of the comparison of the situation of the biocontrol agent of SMDR8 of the present invention in controlling Colletotrichum gloeosporioides of Dioscorea opposita with a water experiment.

[0015] Figure 3 Schematic diagram of the growth morphology of SMDR8 of the present invention on an LB plate medium.

[0016] Figure 4This is the 16S rDNA phylogenetic tree of SMDR8 of the present invention. Detailed implementation manners

[0017] The following describes the detailed implementation manners of the present invention with reference to examples. To fully understand the present invention, many details are described below. However, for those skilled in the art, the present invention can be implemented without these details. For well-known components, methods, and processes, no further detailed description will be given below.

[0018] An endophytic bacterium of Chinese yam of the present invention is Burkholderia cepacia Burkholderia cenocepacia , with the strain name of SMDR8, which was deposited at the China Center for Type Culture Collection (CCTCC) on August 31, 2023, with the deposit number of CCTCC M 20231555, and the deposit address is Wuhan University.

[0019] The application of the above SMDR8 in the preparation of a biocontrol agent, and the dosage form is a liquid preparation. The preparation method of the biocontrol agent for preventing and treating anthracnose of Chinese yam of the present invention includes the following steps: (1) Activate SMDR8 on an LB plate medium. The LB medium has a pH value of 7.0. The components of the LB plate medium (1 L) include: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and 18 g of agar powder.

[0020] (2) After activating the strain in step (1), pick a single colony and inoculate it into an LB liquid medium for cultivation. The LB liquid medium has a pH value of 7.0. The components of the LB liquid medium (1 L) include: 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride. The cultivation method is shaking cultivation on a shaker to obtain a seed liquid of Burkholderia cepacia SMDR8. The shaking rate of the shaker is 180 rpm, the cultivation temperature is 30°C, and the cultivation time is 16 h.

[0021] (3) Expand and cultivate the SMDR8 seed liquid obtained in step (2) in an LB liquid medium to prepare a biocontrol agent. The volume ratio of the SMDR8 seed liquid to the LB liquid medium is 1:100. The cultivation method is shaking cultivation on a shaker. The shaking rate of the shaker is 180 rpm, the cultivation temperature is 30°C, and the cultivation time is 24 h.

[0022] Cultivation method of Colletotrichum gloeosporioides f. sp. dioscoreae: Activate Colletotrichum gloeosporioides f. sp. dioscoreae on a PDA plate medium. The pH of the PDA plate medium is the natural value. The components of the PDA plate medium (1 L) include: 200 g of peeled potato, 20 g of glucose, and 20 g of agar.

[0023] Determination of the control effect of SMDR8 strain on Colletotrichum gloeosporioides f. sp. dioscoreae: (1) Plate confrontation experiment: Inoculate a 2-day-old Colletotrichum gloeosporioides (diameter 5 mm) cake in the center of a PDA plate medium. Symmetrically spot-inoculate the activated antagonistic strain around the cake in a cross shape, with each point 4 cm away from the Colletotrichum gloeosporioides. Repeat 3 times. Use a plate inoculated only with Colletotrichum gloeosporioides as a control. Incubate in the dark at 28 °C in an incubator. After 14 days, measure the width of the inhibition zone, which is 1.4 cm. See Figure 1 .

[0024] (2) Determination of field control effect: The spray method was used to conduct the field control effect determination experiment. The concentrations of the above biocontrol agents were OD 600 = 0.2, OD 600 = 0.4, and OD 600 = 0.6. Dilute 100 times and spray an equal amount of water as a control treatment (CK). Spray the next time after an interval of 14 days. After 30 days of 3 treatments, count the disease index and control effect. Investigate 1 m 2 of yam leaves to determine its disease index. Disease index = ∑(diseased leaves * relative grade) / (total number of investigated diseased leaves * highest disease grade) * 100; Control effect (%) = (control group disease index - treatment group disease index) / control group disease index * 100.

[0025] Dilute the culture solutions of the biocontrol agents with OD 600 of 0.2, 0.4, and 0.6 respectively by 100 times and spray them on the yam leaves. The results show that the disease index of the yam leaves sprayed with the biocontrol agent is significantly lower than that of the water control treatment. See Figure 2 (In the figure: the left side is the control effect diagram of SMDR8, and the right side is the water control). Among them, the disease index of the treatment with OD 600 = 0.2 is 14.90, and the control effect is 59.92%. Therefore, the most suitable application condition is OD 600 = 0.2 diluted 100 times. The disease index and control effect results are shown in Table 1.

[0026] Table 1 Disease index and control effect of yam after spraying SMDR8 for 45 days treatment disease index control effect (%) CK (spraying water) 37.18±2.08 a —— <![CDATA[OD 600 =0.2]]> 14.90±1.10 c 59.92 <![CDATA[OD 600 =0.4]]> 17.91±2.35 bc 51.83 <![CDATA[OD 600 =0.6]]> 21.65±1.40 b 41.77 Identification of the strain: The identification method of the endophytic Burkholderia cepacia in yam with antagonistic effect against Colletotrichum gloeosporioides in yam includes the following steps: (1) Morphological observation of the SMDR8 strain: Culture the SMDR8 strain on an LB solid medium for 2 days and observe its colony morphology. The colony is white, round, with regular edges, smooth surface, opaque, and moist and viscous (see Figure 3 ); Gram staining is negative, and the bacteria are rod-shaped.

[0027] (2) Determination of 16S rDNA sequence of SMDR8 strain: A single SMDR8 colony growing on LB plate was selected and cultured on a shaker at a shaking rate of 180 rpm, a culture temperature of 30°C, and a culture time of 16 h to obtain a bacterial suspension. Centrifuge at 1000 rpm for 10 min, take the supernatant, and extract DNA. PCR amplification of the strain was performed using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3').

[0028] The reagents added to the amplification reaction system (20 μL) were as follows: the template was the genomic DNA of strain SMDR8, 2×Rapid MasterMix 10 μL, Primer 27F 1 μL, Primer 1492R 1 μL, dd H 2 O 7 μL, DNA template 1 μL.

[0029] PCR program: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 1 min, 30 cycles, and extension at 72°C for 10 min.

[0030] (3) 6 μL of the amplified product was taken for 1.5% (m / V) agarose gel electrophoresis. The gel was placed in 1×TAE buffer and electrophoresed at 120 V for 30 min. The PCR product of the target band of about 1500 bp was sent to Shanghai Biotechnology Co., Ltd. for sequencing. The results were sequenced by DNAMAN and compared with the known sequences in the NCBI database by BLAST. It was found that the SMDR8 strain was similar to Burkholderia cepacia Burkholderia cenocepacia The homology of the two genes is 99.86%. The phylogenetic tree was constructed using MEGA 7.0 software and the Neighbor-Joining method (see Figure 4 ).

[0031] The SMDR8 of the present invention has a 16S rDNA sequence fragment of 1448 bp, and the nucleotide sequence is shown in SEQ ID No.1.

[0032] The above is only a specific implementation of the present invention, but the design concept of the present invention is not limited to this. Any non-substantial changes to the present invention using this concept shall be deemed as an infringement of the protection scope of the present invention.

Claims

1. An endophytic bacterium of Chinese yam, characterized by: The endophytic bacteria of Chinese yam are Burkholderia cepacia Burkholderia cenocepacia The strain name is SMDR8, which was deposited in the China Center for Type Culture Collection (CCTCC) on August 31, 2023, with the deposit number CCTCC M 20231555. The 16S rDNA sequence fragment of SMDR8 is 1448 bp, and the nucleotide sequence is shown in SEQ ID No.

1.

2. The endophytic bacteria of yam according to claim 1, characterized in that: The activated culture strain of SMDR8 is used to antagonize yam anthracnose pathogens.

3. The endophytic bacteria of Chinese yam according to claim 1, characterized in that: The application of SMDR8 in the preparation of biocontrol agents.

4. The endophytic bacteria of Chinese yam according to claim 3, characterized in that: The application of the SMDR8 biocontrol agent in preventing and controlling yam anthracnose.

5. The endophytic bacteria of Chinese yam according to claim 1, characterized in that: The concentration of the biocontrol agent is OD 600 =0.

2.

6. The endophytic bacteria of yam according to claim 1, characterized in that: The preparation method of the biocontrol agent comprises the following steps: (1) SMDR8 strain was activated on LB plate medium; (2) After the strain in step (1) is activated, a single colony is picked and inoculated into LB liquid culture medium for cultivation to obtain a seed solution of Burkholderia cepacia SMDR8; (3) The SMDR8 seed solution obtained in step (2) is cultured in LB liquid culture medium to obtain a biocontrol agent.

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