Biocontrol bacterium for corm rot of saffron and application of biocontrol bacterium

Abstract

The invention discloses a stigma croci corm rot biocontrol bacterium and application thereof, and belongs to the field of biological control of plant diseases. The Xiahong peanut antibacterial agent is obtained by being separated from an orris plant saffron healthy corm, and is preserved in China General Microbiological Culture Collection Center (CGMCC) on June 30, 2025, the address of the China General Microbiological Culture Collection Center is No.1 Yard 3, Beichen West Road, Chaoyang District, Beijing, and the preservation number of the Xiahong peanut antibacterial agent is CGMCC No.35040. The biocontrol bacterium and the metabolite of the biocontrol bacterium have an obvious prevention and treatment effect on the stigma croci corm rot disease, and meanwhile, the biocontrol bacterium and the metabolite of the biocontrol bacterium have wide antibacterial activity and have wide application prospects in the field of biological prevention and treatment of stigma croci and other plant fungus diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological control of plant diseases, and specifically relates to a strain of Birkholderia biformis for controlling saffron corm rot Burkholderia ambifaria ZCLAB-HRB88 and its application in controlling saffron corm rot. BACKGROUND

[0002] Saffron (Crocus sativus L.) belongs to the medicinal plant of Iridaceae, is a triploid, mainly relies on corm for asexual reproduction, is also named as safflower and saffron, and the dried stigma is used as a medicine and has the effects of activating blood circulation to remove blood stasis, cooling blood to detoxify and relieving depression and tranquilizing. Long-term adherence to taking saffron can improve human immunity and has high medicinal value. Saffron corm rot is a common fungal disease during saffron planting, and it is currently generally believed that the disease is a soil-borne disease caused by Fusarium oxysporum f. sp. crotalariae (Foc) and mainly harms saffron corms. Crocus sativus ‌Fusarium oxysporum After the pathogenic bacteria invade the plant host, the plant cell wall can be degraded, the cell membrane permeability can be changed, the growth of the plant corm and root can be inhibited, and the water absorption of the host plant is hindered, so that the corms are rotted and the plants are wilted. The pathogenic bacteria mainly overwinter in the form of mycelium and chlamydospores with the host disease residues in the soil and can survive in the soil for more than 6-8 years, so that it is very difficult to control saffron corm rot.

[0003] At present, the control of saffron corm rot mainly relies on field crop rotation and chemical control. Although the use of chemical agents has a quick effect, it threatens the health of humans and animals and the ecological environment, and long-term and large-scale use of chemical agents can also lead to the emergence of drug-resistant strains, further increasing the difficulty of disease control. Biological control, on the one hand, has gradually attracted widespread attention in the control of plant diseases due to its environmental friendliness, difficulty in pathogenic bacteria to develop drug resistance and sustainable development; on the other hand, due to the fact that the biological control strains are generally isolated from plants or soil, can form a good symbiotic relationship with the host and co-evolve with the host and pathogenic bacteria, and have a long control effect and broad application prospect. However, the domestic reported strains for controlling saffron corm rot are few, mainly concentrated in Trichoderma (Hypocrea) sp. and Bacillus sp., which seriously limits the control of saffron corm rot and the development of biocontrol agents in saffron production. Trichoderma Bacillus

[0004] Therefore, it is of great significance to mine high-efficiency biocontrol microbial resources for the control of diseases and the development of biocontrol agents. SUMMARY

[0005] In order to solve the above problems, the present application provides a biocontrol bacterial Birkholderia biformis ZCLAB-HRB88 Burkholderia ambifaria ​​​ZCLAB-HRB88 and application thereof in preventing and treating saffron corm rot disease.

[0006] In a first aspect, the present application provides a biocontrol bacterium, which is isolated from healthy saffron corm tissue and preserved in China General Microbiological Culture Collection Center on June 30, 2025, with a preservation number of CGMCC No.35040.

[0007] In a second aspect, the present application provides an application of the biocontrol bacterium in inhibiting growth of pathogenic bacteria.

[0008] Further, the pathogenic bacteria are one or more of Fusarium oxysporum F. oxysporum ), Aspergillus niger A. tamarii ), Aspergillus niger A. niger ), Fusarium graminearum F. commune ), Fusarium solani F. solani ).

[0009] In a third aspect, the present application provides a metabolite of the biocontrol bacterium, which is produced in a metabolic process of the biocontrol bacterium.

[0010] In a fourth aspect, the present application provides a preparation method of the metabolite of the biocontrol bacterium, which comprises the following steps: fermenting and culturing the biocontrol bacterium. Preferably, the method further comprises the following step: removing the bacterial cells in the fermentation broth obtained through the fermentation and culturing. Preferably, the bacterial cells are removed by centrifugation.

[0011] In a fifth aspect, the present application provides a biocontrol preparation, which comprises the biocontrol bacterium, the metabolite of the biocontrol bacterium or the metabolite prepared by the preparation method.

[0012] In a sixth aspect, the present application provides an application of the biocontrol bacterium, the metabolite or the biocontrol preparation in preventing and treating saffron corm rot disease.

[0013] Preferably, the saffron corm rot disease is caused by infection of Fusarium oxysporum.

[0014] Preferably, the biocontrol preparation is applied to the saffron plant by a root irrigation method.

[0015] Compared with the prior art, the present application has the following beneficial effects:

[0016] (1) The present application provides a new biocontrol bacterium B. zclab-hrb88 Burkholderia ambifariaZCLAB-HRB88, the strain can have significant inhibitory effect on multiple pathogenic bacteria, including Fusarium oxysporum, Aspergillus niger, Aspergillus niger, Fusarium graminearum, Fusarium solani; (2) Compared with other biocontrol bacteria, the biocontrol bacteria B. biformis in the application has the advantages of Burkholderia ambifaria ZCLAB-HRB88 has strong inhibitory effect on Fusarium oxysporum and Aspergillus niger, and produces a wider inhibition zone in the plate confrontation experiment, so that the biocontrol bacteria in the application have more excellent inhibitory and control effects; (3) The biocontrol bacteria provided in the application have inhibitory ability; (4) The biological preparation in the application can be a fermentation broth supernatant of the biocontrol bacteria, so that the complex steps of maintaining the number and activity of live bacteria and recovering the strain can be omitted, and the biological preparation has higher chemical stability, application convenience and effect consistency; in addition, the fermentation supernatant does not contain live bacteria for safety consideration, and potential ecological risks caused by introducing exogenous live bacteria into the environment are avoided. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The B. biformis in the application Burkholderia ambifaria ZCLAB-HRB88 and the pathogenic bacterium Fusarium oxysporum of saffron corm rot F. oxysporum ) confrontation culture picture; Figure 2 The B. biformis in the application Burkholderia ambifaria ZCLAB-HRB88 morphological identification; Figure 3 The B. biformis in the application Burkholderia ambifaria ZCLAB-HRB88 16S rRNA gene sequence PCR amplification agarose gel electrophoresis result picture; Figure 4 The B. biformis in the application Burkholderia ambifaria ZCLAB-HRB88 phylogenetic tree (NJ method) constructed based on 16S rRNA gene sequence; Figure 5 The B. biformis in the application Burkholderia ambifaria ZCLAB-HRB88 confrontation culture picture of saffron pathogenic bacterium Aspergillus niger A. tamarii ); Figure 6 The B. biformis in the application Burkholderia ambifaria ZCLAB-HRB88 confrontation culture picture of saffron pathogenic bacterium Aspergillus niger A. niger ); Figure 7B. parafarraria in the present application Burkholderia ambifaria ZCLAB-HRB88 against the pathogen of Atractylodes macrocephala Koidz K. Sphaeria atra (Pers.) Fr. F. commune Picture of confrontation culture; Figure 8 B. parafarraria in the present application Burkholderia ambifaria ZCLAB-HRB88 against the pathogen of Atractylodes macrocephala Koidz K. Sphaeria atra (Pers.) Fr. F. solani Picture of confrontation culture; Figure 9 B. parafarraria in the present application under scanning electron microscope Burkholderia ambifaria ZCLAB-HRB88 against the pathogen of Atractylodes macrocephala Koidz K. Sphaeria atra (Pers.) Fr. F. oxysporum Picture of confrontation culture; Figure 10 B. parafarraria in the present application Burkholderia ambifaria Picture of potting prevention and treatment effect of ZCLAB-HRB88 against the pathogen of Atractylodes macrocephala Koidz K. Sphaeria atra (Pers.) Fr. DETAILED DESCRIPTION

[0018] The present application will be further described in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and devices used in the present application are conventional reagents, methods and devices in the technical field.

[0019] The biocontrol bacteria described in the present application are isolated from healthy "No. 1 Crocus sativus L." corm tissues, and the corm samples are collected in Jiande City, Zhejiang Province (29°32'18'' N, 119°36'11'' E, 190 feet above sea level).

[0020] Example 1: Isolation and screening of strains After the membrane coating of the corm of Crocus sativus L. was removed, it was washed under running water for 30 min to remove the surface sand; under sterile conditions, the corm was soaked in 75 vol% ethanol for 30 s, washed with sterile water for 3 times, 1 min each time to clean, and then soaked in 15 wt% sodium hypochlorite solution for 20 min (during which the corm was shaken to ensure full contact with the solution and ensure sufficient disinfection), washed with sterile water for 5 times, 5 min each time to clean; after disinfection, the corm was dried with sterile absorbent paper, cut into 1-2 cm 2, the thickness is 1-2 mm, inoculated in Luria-Bertani (LB) solid medium (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar 15-20 g / L, natural pH, 121℃ sterilization for 20 min), 30℃ constant temperature incubator for 1-2 days, after the bulb slice around the bacteria grow, using inoculation ring dipped in the new LB solid plate to make streak dilution purification, until the streak growth of single colony morphology is consistent, clear boundary, can be preliminary determined that the bacteria has been completely purified. The purified strain uses liquid LB medium (LB solid medium without agar) to expand culture and then stored in a glycerol tube, which is stored at -80℃ for long-term preservation.

[0021] Strain identification of example 2 1. Morphology and physiological and biochemical identification As shown in Figure 2 , the left graph in Figure 2 is the colony morphology of the strain obtained in example 1 of the application on LB solid medium; Figure 2 the middle graph is the gram staining graph of the strain obtained in example 1 of the application, Figure 2 and the right graph is the morphology of the strain obtained in example 1 under scanning electron microscope. After the strain obtained in example 1 is cultured on LB solid medium at 30℃ for 24h, the colony surface is wet and smooth, and is yellow to bright yellow; under optical microscope observation, gram staining is red, which belongs to gram-negative bacteria without spores; under scanning electron microscope observation, the bacteria are short rod-shaped. Physiological and biochemical identification is carried out according to the “Berger Bacteria Identification Manual”, and the results are shown in Table 1.

[0022] Table 1: Physiological and biochemical characteristics of the strain

[0023] 2. Molecular biology identification The 16S rRNA gene sequence of the strain obtained in example 1 is amplified using bacterial identification universal primers 27F / 1492R, and a 928bp-long gene sequence (as shown in SEQ ID NO. 1) is obtained. Blast homologous comparison is carried out, and a phylogenetic tree (NJ method) is constructed using MEGA 7, which finds that it has high sequence similarity with B. bilipha IHBB 1073 (sequence ID: KF475799.1) Figure 3 and Figure 4 . Combined with morphological identification and physiological and biochemical characteristics, the strain is finally identified as B. bilipha Burkholderia ambifaria , which is named as B. bilipha Burkholderia ambifaria ZCLAB-HRB88.

[0024] Example 3 Burkholderia biformis Burkholderia ambifaria Inhibition of different pathogenic fungi by ZCLAB-HRB88 1. The antagonistic activity of bacterial strains was screened by using the flat-plate confrontation method. The purified bacteria were inoculated in 2 ml liquid LB medium, and the bacterial suspension (OD600=0.1, 5x10 5 cfu / mL) was obtained after 12-18 hours of expansion culture at 30°C and 200 rpm. 5 μl of the bacterial suspension was added to one side of the PDA plate, and a pathogenic fungus cake was inoculated on the other side. The plate was incubated in a constant temperature incubator at 25°C for 3-5 days. The results are shown in Figure 1 . Burkholderia ambifaria The Burkholderia biformis ZCLAB-HRB88 strain can significantly inhibit the growth of Fusarium oxysporum F. oxysporum . Figure 1 The left side is a blank control (CK), and the right side is the confrontation culture of the pathogenic fungus and the biocontrol fungus.

[0025] Further observation by scanning electron microscopy found that, compared with the control, the Burkholderia biformis ZCLAB-HRB88 strain can cause the Fusarium oxysporum Burkholderia ambifaria mycelium to shrink and wrinkle, resulting in inhibition of its growth (see F. oxysporum ). Figure 9 .

[0026] 2. The flat-plate confrontation method is the same as described above. In addition to Fusarium oxysporum F. oxysporum , the Burkholderia biformis ZCLAB-HRB88 strain can also significantly inhibit the growth of Aspergillus alliaceus Burkholderia ambifaria and Aspergillus niger A. tamarii . A. niger Figure 5 Figure 6 , and can also significantly inhibit the growth of the common pathogenic fungus Fusarium solani and the common pathogenic fungus Fusarium solani f. sp. arthracti F. commune . F. solani Figure 7 Figure 8 .

[0027] Example 4 Burkholderia biformis Burkholderia ambifaria Determination of the control effect of ZCLAB-HRB88 on crocus corm rot Burkholderia biformis Burkholderia ambifaria Verification of the potting control effect of ZCLAB-HRB88 1. Burkholderia biformis Burkholderia ambifaria ​​​​Preparation of ZCLAB-HRB88 fermentation broth: The strain was inoculated into 2 ml of liquid LB medium and cultured at 30℃ with shaking at 200 rpm for 12-16 h to obtain seed culture. The seed culture was then inoculated into 100 ml of liquid LB medium at a ratio of 1% for expansion culture for 12 h (fermentation broth cell concentration 1×10⁻⁶). 9 (cfu / ml), centrifuged at 12000 rpm for 15 min to remove bacterial cells and collect the supernatant for later use.

[0028] 2. Fusarium oxysporum ( F. oxysporum Preparation of spore suspension: Fusarium oxysporum ( F. oxysporum Inoculated onto PDA plates and pre-cultured at 25°C for 5 days. Spores were washed away with sterile water containing 0.05% Tween 80 (v / v) to adjust the spore concentration to 10. 5 per ml.

[0029] 3. The surface disinfection treatment of the bulbs was the same as in Example 1. The planting substrate (nutrient soil: perlite: vermiculite = 3:1:1) was sterilized at 121℃ three times. The disinfected bulbs were planted in the sterile substrate. A total of 4 treatments were set up: (1) inoculation with sterile water as a blank control (CK); (2) inoculation with Burkholderia bifidum. Burkholderia ambifaria ZCLAB-HRB88 fermentation broth (T1); (3) Burkholderia bifidum Burkholderia ambifaria ZCLAB-HRB88 fermentation broth and Fusarium oxysporum ( F. oxysporum (4) Inoculate with Fusarium oxysporum (Spore suspension 1:1) (T2); F. oxysporum Spore suspension. After inoculation, the spores were cultured in a greenhouse and treated every 2 days with 5 ml each time. The disease index was statistically analyzed after 7 days.

[0030] The disease index is calculated using the formula: Disease Index = [Σ(Number of diseased plants × Representative value) / Total number of plants × Representative value of the highest disease level] × 100%.

[0031] Table 2 Burkholderia bifidum Burkholderia ambifaria The effect of ZCLAB-HRB88 on the prevention and control of saffron bulb rot

[0032] Based on the above results, it can be seen that *Burkholderia bifidum* Burkholderia ambifaria The supernatant of the fermentation broth of ZCLAB-HRB88 also has a high-efficiency control effect on saffron rot, which also indicates that the metabolites in the supernatant of the fermentation broth are also effective active ingredients for the prevention and control of saffron rot.

Claims

1. A biocontrol bacterium, characterized in that, Isolated from the healthy corm tissue of Crocus sativus L., preserved in China General Microbiological Culture Collection Center on June 30, 2025, and the preservation number is CGMCC No. 35040.

2. Use of the biocontrol bacteria of claim 1 for inhibiting pathogenic bacteria, characterized in that, The pathogenic fungi are one or more of Fusarium oxysporum, Aspergillus niger, Aspergillus niger, Fusarium graminearum and Fusarium solani.

3. Metabolites of the biocontrol bacteria of claim 1, characterized in that, Metabolites of the biocontrol bacteria of claim 1.

4. The method of preparing a metabolite according to claim 3, characterized in that, The biocontrol bacteria are fermented.

5. The production method according to claim 4, characterized by, The bacteria in the fermentation broth obtained by the fermentation are removed.

6. The production method according to claim 5, wherein The bacteria are removed by centrifugation.

7. A biocontrol formulation, characterized in that, The biocontrol bacteria of claim 1, the metabolites of the biocontrol bacteria of claim 3, or the metabolites of the biocontrol bacteria prepared by the method of any one of claims 4-6.

8. The biocontrol bacteria of claim 1, the metabolites of the biocontrol bacteria of claim 3, or the biocontrol preparation of claim 7 for use in preventing and treating the corm rot disease of Crocus sativus L.

9. Use according to claim 8, characterized in that, The corm rot disease of Crocus sativus L. is caused by the infection of Fusarium oxysporum.

10. Use according to claim 8, characterized in that, The biocontrol preparation is applied to the plant of Crocus sativus L. by root irrigation.

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