Kabatiella bupleuri strain for producing anthraquinone compound and application thereof

Abstract

The application discloses a Kabatiella bupleuri strain for producing anthraquinone compounds and application, and a Kabatiella bupleuri strain TSYW-49 CGMCC NO.25421 for producing anthraquinone compounds. Experiments prove that the Kabatiella bupleuri strain for producing anthraquinone compounds can produce anthraquinone compounds, extracellular fermentation extracts of the strain have significant bacteriostatic activity on various plant pathogenic bacteria, the strain can be used for producing biological pesticides, and has important application value.

Description

Technical Field

[0001] This invention belongs to the field of plant pathogen control technology, and more specifically relates to a Kabatiella bupleuri strain that produces anthraquinone compounds and its applications. Background Technology

[0002] Anthraquinones refer to those formed at C9, C6, and C7. 10 Anthraquinone compounds, containing a carbonyl group, belong to the fused-ring organic compound family and have wide applications in the textile, dyeing, pharmaceutical, and pesticide industries. Industrially, anthraquinone dyes hold an important position in the synthetic dye field, and they are also used as papermaking cooking agents and raw materials for hydrogen peroxide production. In the pharmaceutical field, anthraquinone compounds generally possess various pharmacological effects such as antitumor, antibacterial, antioxidant, diuretic, and hemostatic properties. For example, sennoside has a laxative effect, and mitoxantrone can be used to treat acute leukemia and malignant lymphoma. In agriculture, dicyanantrone is used for the control of fruit tree diseases and pests.

[0003] Anthraquinones are commonly found in plants of the Polygonaceae, Fabaceae, Rubiaceae, and Rhamnaceae families, and are also metabolic products of lichens and fungi. Studies have shown that some microorganisms can produce anthraquinones. For example, emodin, besides being extracted from medicinal plants like rhubarb and Polygonum cuspidatum, can also be produced using Aspergillus ochraceus or Fusarium solani, although the yield is low. In addition, the herbicidal herbicidal longinosporin is synthesized by Curvularia glabra, and aspergillin and emodin methyl ether found in Aspergillus glaucus are both anthraquinones. Fusarium solani can produce erythromycin, Fusarium oxysporum can produce aflatoxin, and Fusarium oxysporum can produce bicardiin, which inhibits the growth of other fungi. *Plasmodium styracifolium* can infect dead wood, causing it to turn blue-green; the pigment is pyromycin. However, the yields of anthraquinones produced by these strains are low, directly limiting their production and application. Because anthraquinones generally have antibacterial properties, they have potential value as biopesticides, and screening high-yielding strains of anthraquinones from the environment has become a current research hotspot. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a Kabatiella bupleuri strain that produces anthraquinone compounds.

[0005] A second objective of this invention is to provide an extracellular fermentation extract of the Kabatiella bupleuri strain TSYW-49CGMCC NO.25421, which produces anthraquinone compounds.

[0006] A third objective of this invention is to provide the application of the above-mentioned extracellular fermentation extract in the preparation of plant pathogen inhibitors.

[0007] The technical solution of this invention is summarized as follows:

[0008] A Kabatiella bupleuri strain TSYW-49CGMCC NO.25421 that produces anthraquinone compounds.

[0009] The extracellular fermentation extract of the Kabatiella bupleuri strain TSYW-49CGMCC NO.25421, which produces anthraquinone compounds.

[0010] The application of the above-mentioned extracellular fermentation extract in the preparation of plant pathogen inhibitors.

[0011] Plant pathogens are preferably *Anthracis acuminata*, *Anthracis niger*, *Smuts*, or *Red yeast*.

[0012] Advantages of this invention:

[0013] Experiments have shown that the Kabatiella bupleuri strain of the present invention, which produces anthraquinone compounds, can produce anthraquinone compounds. Its extracellular fermentation extract has significant antibacterial activity against a variety of plant pathogens and can be used to produce biopesticides, thus having important application value. Attached Figure Description

[0014] Figure 1 This is a colony morphology diagram of strain TSYW-49;

[0015] Figure 2 Microscopic morphological image of strain TSYW-49;

[0016] Figure 3 The graph shows the color change of the extracellular extract of strain TSYW-49 in the Borntrager reaction.

[0017] Figure 4 The antibacterial effect of the extracellular fermentation products of strain TSYW-49 was evaluated; among them, 1-8: 0.151 g / L, 0.188 g / L, 0.378 g / L, 0.755 g / L, 1.51 g / L, 3.02 g / L, 6.04 g / L, 12.08 g / L; A: negative control;

[0018] Figure 5 This is a microscopic photograph of the crystals formed by the extracellular secretion of products from strain TSYW-49 that are insoluble in water. Detailed Implementation

[0019] This invention discloses a Kabatiella bupleuri strain producing anthraquinone compounds, isolated and screened from nature. Morphological observation revealed this strain to be yeast-like, and ITS sequence analysis showed its closest phylogenetic relationship to Kabatiella bupleuri, confirming its identity as Kabatiella bupleuri. The screened strain was designated TSYW-49. It was deposited on July 27, 2022, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO.25421. The address of the depository is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0020] Example 1: Isolation of bacterial strains

[0021] Plant leaves were collected from the environment (Shaoxing City, Zhejiang Province, China) as samples. These samples were immersed in PBS buffer and vortexed to wash away surface microorganisms, obtaining a microbial enrichment solution. The microbial enrichment solution was subsequently diluted 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, and 1000-fold with PBS buffer. 100 μL of each dilution was spread onto YPD solid medium and incubated at 30°C for 120 h.

[0022] Select single colonies from the above plates that are red in color and have a significant inhibitory effect on surrounding strains, and isolate and purify them by streaking them on the above YPD solid medium. Repeat this step 2 to 3 times until pure cultured microorganisms are obtained.

[0023] PBS buffer: Weigh 8g NaCl, 0.2g KCl, 1.44g Na₂HPO₄, and 0.24g KH₂PO₄, dissolve them in 800ml distilled water, adjust the pH of the solution to 7.4 with HCl, and finally add distilled water to a final volume of 1L. Autoclave (121℃, 20min), cool to room temperature, add penicillin and streptomycin to a final concentration of 100ug / ml, and store at 4℃.

[0024] YPD solid medium: 1% yeast extract, 2% peptone, 2% glucose, 1.5% agar powder.

[0025] A strain producing extracellular red pigment was screened from the vegetation of Fuzhishan Scenic Area, Lingnan Township, Shangyu District, Shaoxing City, Zhejiang Province (latitude: 29°44'58.2"N, longitude: 120°58'24.48"E), and named TSYW-49. The extracellular active compound produced by this strain has a significant inhibitory effect on the surrounding fungi.

[0026] Example 2: Colony Morphology Observation

[0027] The strain TSYW-49 obtained in Example 1 was streaked onto YPD solid medium plates and incubated at 30°C for 4 days. The colony diameter was 20–30 mm. Initially, the colony surface was smooth, moist, and viscous, but later it appeared wrinkled and loose in texture. With increasing incubation time, the colony color gradually changed from milky white to dark red, and in the later stages, the reverse side of the plate showed a white-red or dark red color. Figure 1 Microscopic morphological observation revealed that the single-celled bacterial cells resembled yeast cells, while the multicellular cells exhibited septate, branched hyphae. Figure 2 ).

[0028] Example 3: Molecular biological identification of the strain

[0029] 1) Inoculate TSYW-49 strain into YPD liquid medium, culture at 30℃ and 220 rpm for 48 h with shaking, and collect the bacterial cells by centrifugation;

[0030] 2) Genomic DNA was extracted using the TIANGEN yeast genome extraction kit (DP307);

[0031] 3) The ribosomal ITS rDNA sequence of strain TSYW-49 was amplified using universal primers for the ITS sequence (ITS1: 5'-TCCGTAGGTGAACCTGCGG-3' SEQ ID NO.2, ITS4: 5'-TCCTCCGCTTATTGATATGC-3' SEQ ID NO.3). The PCR reaction system consisted of 25 μL: 12.5 μL of 2×Taq Mix, 1 μL each of ITS1 and ITS4 primers, 1 μL of genomic DNA, and 9.5 μL of deionized water. PCR conditions were: 95℃ for 3 min pre-denaturation; 95℃ for 15 s denaturation, 55℃ for 15 s annealing, 72℃ for 1 min extension, 35 cycles; and a final extension at 72℃ for 5 min. After the reaction, the PCR product was recovered from an agarose gel and sequenced. Sequencing was performed by Qingke Biotechnology Co., Ltd., using ITS1 and ITS4 primers. After rigorous verification, the DNA fragment sequence shown in SEQ ID NO.1 was obtained.

[0032] Sequence alignment of the above sequencing results with the GenBank database revealed that the ITS sequence of strain TSYW-49 showed the highest homology with the ITS sequence of strain Kabatiella bupleuri CBS 131304, indicating that this strain is Kabatiella bupleuri.

[0033] DNA sequence analysis showed that TSYW-49 belongs to Kabatiella bupleuri. The selected strain was named Kabatiella bupleuri, strain number TSYW-49, and was deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 27, 2022.

[0034] Example 4

[0035] YPD solid medium 1L: Dissolve 10g yeast powder, 20g peptone, and 20g glucose in an appropriate amount of deionized water, add 15g agar powder, stir, and add deionized water to 1L.

[0036] Seed culture medium 1L: 10g yeast powder, 20g peptone, 20g glucose, dissolved in an appropriate amount of deionized water, then add deionized water to 1L.

[0037] Seed activation and culture:

[0038] First, sterilize the culture medium and pour the YPD solid culture medium into a disposable sterile agar plate, and wait for it to solidify into a solid agar plate culture medium;

[0039] After thawing the TSYW-49 strain frozen at -80℃, it was streaked onto a solid plate medium and then incubated at 30℃ for 2 days to activate it. Uniformly sized and normally colored single clones were picked from the solid plate medium and inoculated into seed culture medium. The culture was then carried out at 30℃ and 220 rpm for 3 days to obtain fresh seed culture.

[0040] Example 5

[0041] Preparation of fermentation medium (3L): 312.78g sucrose, 25.47g peanut meal powder, 1.02g ammonium chloride, 4.5g disodium hydrogen phosphate, 1.5g magnesium sulfate heptahydrate, 6mg manganese sulfate monohydrate, 15mg calcium chloride, add deionized water to 3L.

[0042] The cultured Kabatiella bupleuri seed culture (prepared in Example 4) was inoculated into a fermentation medium for fermentation. During the logarithmic phase, the tank pressure was 0.05 MPa, the tank temperature was 29°C, the air flow rate was 8 L / min, and the stirring speed was 500 r / min. The pH was adjusted to 5 with 5% ammonia solution. From the time the fermentation entered the stationary phase until the end of fermentation, the tank pressure was 0.02 MPa, the tank temperature was 29°C, the air flow rate was 2 L / min, and the stirring speed was 150 r / min. The pH was adjusted to 3.5 with 5% potassium acetate solution, and sucrose solution was added to control the sucrose content in the fermentation medium at 20 g / L. After 9 days of fermentation, Kabatiella bupleuri fermentation broth was obtained.

[0043] Example 6

[0044] The method for extracting anthraquinone compounds from Kabatella bupleuri fermentation broth (prepared in Example 5) includes the following steps:

[0045] 1) Centrifuge the Kabatiella bupleuri fermentation broth at a centrifugal force of 6000×g and collect the insoluble matter in the fermentation broth;

[0046] 2) Extract the insoluble matter with ethyl acetate (ratio 4 L: 1 kg) by centrifugation at 6000 × g to obtain the extract; adjust the pH to 10, a precipitate will form, centrifuge at 6000 × g, and collect the precipitate; redissolve the precipitate with a 1.2% sodium hydroxide aqueous solution, centrifuge at 6000 × g, and collect the liquid; adjust the pH to 5.5, crystals will precipitate, centrifuge at 6000 × g, collect the crystals, dry them, and obtain the crystals. See Figure 5 .

[0047] Example 7: Identification of TSYW-49 extracellular fermentation extract

[0048] The extracellular fermentation extract of strain TSYW-49 was prepared into solutions with pH values ​​ranging from 1 to 14 using ethyl acetate. Borntrager colorimetric reaction revealed that the solutions were yellow when pH < 4, changed from red to purple at pH 4–8, and formed a gray flocculent precipitate at pH > 8. Figure 3 Based on the above reaction, the compound was initially determined to be an anthraquinone compound.

[0049] Example 8: Determination of the antibacterial effect of the extracellular fermentation extract of strain TSYW-49

[0050] 1) Activation culture of indicator bacteria

[0051] Anthrax fussiforme, Anthrax niger, Ustilago maydis, or Rhodotorula rubrum were selected as indicator bacteria and inoculated into YPD liquid medium. The medium was incubated at 30°C with shaking at 220 rpm for 72 hours until the bacterial culture became turbid (OD). 600 =4.0).

[0052] 3) Determination of antibacterial effect

[0053] The inhibition zone method was used for determination: the activated culture broths of *Anthracis niger*, *Anthracis aureus*, *Ustilago maydis*, and *Rhodotorula rubrum* were diluted with fresh YPD liquid medium to an OD value of [missing value]. 600The culture was brought to approximately 1.0, and then 100 μL of bacterial suspension was added dropwise to 20 mL of melted (approximately 40°C) YPD solid medium. After mixing thoroughly, the mixture was poured into a petri dish. Once solidified, different concentrations of the extracellular fermentation extract solution of TSYW-49 strain were added to the surface of the medium, with sterile water used as a negative control. After the addition was complete, the liquid on the surface of the medium was dried in a clean bench, and the plates were then incubated upright at 30°C for 72 h to observe the antibacterial activity. The experimental results showed that the extracellular fermentation extract of TSYW-49 strain had significant inhibitory effects on *Anthracis niger*, *Anthracis aureus*, *Ustilago maydis*, and *Rhodotorula rubrum*. Figure 4 ).

[0054] (The plant pathogen was purchased in March 2022 from Ningbo Mingzhou Biotechnology Co., Ltd., Zhejiang, China, https: / / www.mingzhoubio.com / )

[0055] Experiments have shown that the extracellular fermentation extract of the TSYW-49 strain has significant antibacterial activity against a variety of plant pathogens and can be used to produce biopesticides, thus having important application value.

Claims

1. A strain TSYW-49 that produces anthraquinone compounds, characterized in that... The strain is Kabatiella bupleuri The strain has the preservation number CGMCC NO.25421.

2. An extracellular fermentation extract, characterized in that: The extracellular fermentation extract is the extracellular fermentation extract of the strain described in claim 1.

3. The application of the extracellular fermentation extract as described in claim 2 in the preparation of plant pathogen inhibitors, wherein the plant pathogen is *Anthracis acuminata* or *Anthracis niger*.

Images