Burkholderia sp. DCF01 and application thereof

Abstract

The invention discloses a Burkholderia sp. DCF01 and application of the Burkholderia sp. DCF01, the disclosed strain is named as Burkholderia sp., and the preservation number of the strain is CGMCC (China General Microbiological Culture Collection Center) No.31529. The bacterial strain has obvious bacteriostatic activity on important crop pathogenic bacteria such as colletotrichum spinosus, alternaria alternata, botrytis cinerea, apple cyst, low-temperature-suitable fruit rot fungi and the like, and fermentation liquor of the bacterial strain has a good prevention and control effect on apple tree canker and apple anthracnose bitter rot.

Description

Technical Field

[0001] The present invention belongs to the technical field of agricultural microorganisms, and particularly relates to a Burkholderia bacterium and its application in the field of biological control microorganisms for plant diseases. Background Art

[0002] Burkholderia is a genus of Gram-negative bacteria widely present in water, soil, plants and the human body. The genus currently contains more than 100 species, and some species have good biocontrol application value. In recent years, research has shown that Burkholderia has obvious application effects in aspects such as phosphorus solubilization, growth promotion, and disease prevention.

[0003] CN110669686B discloses a Burkholderia strain (CGMCC No. 16428), which has obvious antibacterial activity against plant pathogenic bacteria such as Fusarium graminearum, Botrytis cinerea, and Rhizoctonia solani. Summary of the Invention

[0004] The object of the present invention is to provide a biocontrol bacterial strain that has excellent broad-spectrum antagonistic effects against plant pathogenic bacteria and can effectively control apple anthracnose bitter rot and apple tree canker.

[0005] The Burkholderia strain DCF01 of the present invention is taxonomically named Burkholderia contaminans; the strain is deposited in the General Microbiology Center of the China Center for Type Culture Collection (address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, postcode: 100101), the deposit date is August 2, 2024, and the deposit number is CGMCC No. 31529.

[0006] The present invention also provides the fermentation product of the above strain, which is a fermentation broth containing the Burkholderia DCF01 strain. The fermentation broth has obvious antibacterial effects on a variety of plant pathogenic fungi. Optionally, the fermentation product is prepared by fermenting the Burkholderia DCF01 with an LB liquid medium.

[0007] The present invention also provides the uses of the Burkholderia DCF01 strain and its fermentation product: inhibiting the growth of plant pathogenic bacteria. The plant pathogenic bacteria are Colletotrichum fructicola, Alternaria alternata, Botrytis cinerea, Cytospora mali, and Languo psychrtropha.

[0008] Meanwhile, the present invention also provides the application of Burkholderia sp. DCF01 and its fermentation products in preventing and treating apple anthracnose bitter rot or / and apple tree canker.

[0009] The Burkholderia sp. DCF01 strain of the present invention can inhibit the growth of various plant pathogens such as Colletotrichum fructicola, Alternaria alternata, Botrytis cinerea, Cytospora mandshurica, and low-temperature fruit rot fungi, and has good biocontrol effects on apple anthracnose and apple tree canker. This strain has good application prospects in the development of microbial pesticides. Description of the Drawings

[0010] Figure 1 Colony morphology of Burkholderia sp. DCF01 strain on PDA and LB media; A is PDA medium, and B is LB medium.

[0011] Figure 2 In-dish antibacterial effect of Burkholderia sp. DCF01 strain on Colletotrichum fructicola on PDA medium.

[0012] Figure 3 Antagonistic effect diagram of Burkholderia sp. DCF01 strain on five plant pathogens on PDA medium.

[0013] Figure 4 Biocontrol effect diagram of Burkholderia sp. DCF01 strain on anthracnose bitter rot of Gala apple fruits under non-wounded inoculation conditions (10 days after inoculation).

[0014] Figure 5 Biocontrol effect diagram of Burkholderia sp. DCF01 strain on anthracnose bitter rot of Gala apple fruits under wounded inoculation conditions; A is the fruit symptom 6 days after inoculation, and B is the statistical chart of lesion diameter.

[0015] Figure 6 Biocontrol effect diagram of Burkholderia sp. DCF01 strain on canker of Gala apple branches; A is the disease symptom of branches 10 days after inoculation, and B is the statistical chart of branch lesion diameter. Detailed Embodiments

[0016] Unless otherwise specified, scientific and technical terms in this article are understood according to the understanding of those of ordinary skill in the relevant fields. Unless otherwise stated, reagents, materials, etc. used in the following examples are all commercially available products.

[0017] Example 1: Isolation and Identification of Burkholderia sp. DCF01 Strain of the Present Invention

[0018] (1) Source of Soil Samples

[0019] Select the rhizosphere soil of Salvia miltiorrhiza from Shipulipu Village, Miganqiao Town, Fengxiang District, Baoji City, Shaanxi Province (107°27'31.731"E, 34°31'6.242"N) collected in 2022 for the isolation of rhizosphere bacteria and the screening of antagonistic bacteria.

[0020] (2) Isolation of rhizosphere bacteria of Salvia miltiorrhiza

[0021] The dilution isolation method was used to isolate the rhizosphere bacteria of Salvia miltiorrhiza; the dilution isolation medium was beef extract peptone solid medium (3 g of beef extract, 10 g of peptone, 10 g of sodium chloride, 20 g of agar, 1 L of distilled water, pH 7.0 - 7.2); during isolation, healthy root tissues of Salvia miltiorrhiza were selected, and the attached soil was removed by rinsing with tap water. 100 mg of root tissues were suspended in 1 mL of sterile water and vortexed. The rinsing solution was aspirated and diluted to 10 -3 、10 -4 、10 -5 concentrations, spread on the beef extract peptone solid medium, and the plate was incubated in a constant temperature incubator at 37°C for 36 h. The colonies were picked and streaked and purified on the beef extract peptone solid medium. The purified strains were stored in a -80°C refrigerator.

[0022] (3) Screening of antagonistic bacteria

[0023] Using the pathogen Colletotrichum fructicola causing apple anthracnose bitter rot (provided by the Fungal Research Laboratory of Northwest A&F University) as the indicator target strain, antagonistic bacteria were screened from the bacterial strains isolated and purified in the previous step (2); the screening of antagonistic activity was carried out on a potato dextrose agar medium plate (200 g of potato, 20 g of dextrose, 20 g of agar, 1 L of distilled water, pH about 5.6); through steps such as primary screening and re-screening, 1 bacterial strain with better antibacterial effect was obtained, numbered DCF01; among them, both the primary screening and re-screening adopted the confrontation culture method in the dish. A mycelial disc of Colletotrichum fructicola strain was punched from the edge of the PDA colony and inoculated in the center of the PDA medium. The bacteria to be screened were streaked about 2 cm on both sides of the inoculation point; the plate was placed at 25°C for incubation. After 5 days, a ruler was used to measure the vertical and horizontal diameters of the Colletotrichum fructicola colony to judge the antibacterial rate of the bacteria to be screened. The re-screening adopted the same experimental steps as the primary screening.

[0024] (4) Identification of strain DCF01

[0025] Based on the colony morphological characteristics of the strain and 16S rRNA gene sequence analysis, species identification of strain DCF01 was carried out.

[0026] After the strain grew on the beef extract peptone solid medium for 24 h, the colonies were round, milky white, with a flat and moist surface. Using the primers 27F (5’-AGAGTTTGATCMTGGCTCAG-3’) and 1492R (5’-TACGGYTACCTTGTTACGACTT-3’), the strain DCF01 was amplified and sequenced. The obtained sequence length was 1304 bp. The specific sequence is shown in SIQ ID NO.1. BLAST alignment with the GenBank database found that the similarity with Burkholderia sp. was 100%. It was identified as Burkholderia sp.

[0027] Figure 1 Figure 4 shows the colony morphology of Burkholderia sp. strain DCF01 of the present invention after streaking on PDA and LB media for 2 days. A is the PDA medium, and B is the LB medium.

[0028] Example 2: Antagonistic effect of Burkholderia sp. strain DCF01 of the present invention

[0029] Colletotrichum fructicola, Alternaria alternata, Botrytis cinerea, Cytospora mali, and Languo psychrtropha (all strains used in this study were provided by the Fungal Research Laboratory of Northwest A&F University) were respectively inoculated on PDA medium and cultured at room temperature. Mycelial blocks with a diameter of 5 mm were punched out and inoculated on PDA medium. Strain DCF01 was also inoculated on the PDA medium at the same time. According to the amplification rate of the pathogen colonies, the inhibition rate was calculated to judge whether Burkholderia sp. strain DCF01 had an inhibitory effect.

[0030] Figure 2 、 3 The results shown in Figure 5 indicate that Burkholderia sp. strain DCF01 has an obvious inhibitory effect on the tested strains, indicating that Burkholderia sp. strain DCF01 of the present invention has broad-spectrum antibacterial activity.

[0031] Example 3: Prevention and control effect of Burkholderia sp. strain DCF01 of the present invention on apple anthracnose bitter rot

[0032] Burkholderia sp. strain DCF01 was inoculated in beef extract peptone liquid medium and cultured at 37 °C and 160 rpm for 1 day. The fermentation broth was collected and reserved. Healthy apple fruits were selected and treated with immersion in water and immersion in the fermentation broth respectively for 10 minutes. After 24 h of fruit immersion, they were respectively inoculated with a conidial suspension of Colletotrichum fructicola, the pathogen of apple bitter rot (1x 106 (number / mL), inoculate apples with moisturizing treatment for 10 days, observe and record the disease occurrence results.

[0033] Figure 4 The results shown indicate that the pretreatment with the fermentation broth of Burkholderia sp. DCF01 strain significantly reduced the disease severity of apple bitter rot, suggesting that the strain has good control effects.

[0034] Further select healthy apple fruits, prick the fruit surface with inoculation needles, drop 10 μL of clear water and the fermentation broth of DCF01 strain respectively, air dry and then inoculate with the cake of Colletotrichum fructicola; inoculate apples with moisturizing treatment for 6 days, observe and record the disease occurrence results.

[0035] Figure 5 The results show that after inoculation in the control treatment, obvious expanding rotten tissues were produced around the inoculation points on the fruits (average diameter 2.61 cm, n = 10), while after treatment with the fermentation broth, the expansion of rotten tissues was not obvious (average diameter 0.49 cm, n = 10), and the inhibition rate of lesion expansion was 81.3%. This further supports that the fermentation broth of DCF01 strain can effectively control apple bitter rot.

[0036] Example 4: Control effect of Burkholderia sp. DCF01 strain on apple canker

[0037] Inoculate Burkholderia sp. DCF01 strain in beef extract peptone liquid medium, shake culture at 37 °C and 160 rpm for 1 day, collect the fermentation broth for standby. Select healthy one-year-old apple branches, spray clear water and the fermentation broth respectively, air dry and then prick and inoculate with the cake of apple canker pathogen (the relevant pathogen is provided by the Fungal Research Laboratory of Northwest A&F University). Inoculate apple branches with moisturizing treatment for 10 days, observe and record the disease occurrence results.

[0038] Figure 6 The results show that after inoculation in the control treatment, the canker lesions expanded significantly from the inoculation points (average diameter 3.80 cm, n = 7), while after treatment with the fermentation broth, the expansion of rotten tissues was not obvious (average diameter 0.82 cm, n = 7), and the inhibition rate of lesion expansion was 79.4%. This indicates that the fermentation broth of DCF01 strain can effectively control apple canker.

[0039]

[0040]

Claims

1. A Burkholderia strain DCF01, named Burkholderia contaminans, with a deposit number of CGMCC No.31529.

2. A fermentation product, characterized in that The fermentation product is produced by fermentation of the Burkholderia DCF01 according to claim 1.

3. The fermentation product according to claim 2, characterized in that The fermentation medium is selected from PDA medium, LB medium or beef extract peptone liquid medium.

4. Use of the Burkholderia DCF01 or its fermentation product according to claim 1 for preparing an antibacterial agent for plant pathogens.

5. The use according to claim 3, characterized in that: The plant pathogenic bacteria are Colletotrichum fructicola, Alternaria alternata, Botrytis cinerea, Cytospora mali and / or Languo psychrtropha.

6. The Burkholderia DCF01 or its fermentation product according to claim 1 is used for preparing a method for preventing and treating apple tree rot and / or apple anthracnose bitter rot.

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