Burkholderia pyrrocinia L2 and application thereof
By leveraging the iron-carrier-producing ability of Burkholderia pyrogallol L2, the iron resources of pathogens are deprived, solving the control problems of anthracnose in peppers and root rot in Sichuan pepper, achieving disease suppression and plant growth promotion effects, and is applicable to a variety of crops.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-23
- Publication Date
- 2026-04-07
AI Technical Summary
Existing technologies are insufficient to effectively control anthracnose in peppers and root rot in Sichuan pepper, and chemical control leads to pesticide resistance and excessive pesticide residues, affecting crop yield and quality.
Burkholderia pyrrocinia L2 was used to deprive pathogens of iron resources through its iron-producing ability, thereby inhibiting pathogen growth. It was then applied in the rhizosphere soil of plants to promote plant growth.
It significantly reduces the incidence of diseases, improves plant germination rate and seedling vigor, and has the dual functions of disease prevention and growth promotion. It is suitable for seed treatment of high-value crops such as chili peppers and Sichuan pepper, as well as rice.
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Figure CN121801751A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial technology, and more specifically to a strain of Burkholderia pyrrole (…). Burkholderia pyrrocinia L2 and its applications. Background Technology
[0002] Anthracnose in peppers is caused by various anthrax bacteria ( Colletotrichum capsici This serious disease, caused by pesticides, is widely distributed in major chili-producing areas, often resulting in large-scale yield reductions or even crop failure, especially during the fruit enlargement and ripening stages. Current control measures rely primarily on chemical fungicides, but due to long-term, high-frequency use, the pathogens have developed significant resistance. Furthermore, excessive pesticide residues limit the sale of chilies in high-value markets.
[0003] Sichuan peppercorns ( Zanthoxylum bungeanum As an important specialty economic crop in Southwest China, Sichuan pepper has been suffering from increasingly severe root rot in recent years, manifesting as root decay, plant wilting, and even the death of the entire plant. Because this disease is closely related to the imbalance of soil microbial communities, traditional chemical control methods are difficult to achieve lasting effects, seriously impacting the stable production and quality improvement of the Sichuan pepper industry.
[0004] In recent years, biocontrol microorganisms have attracted attention due to their green, environmentally friendly, and sustainable characteristics. Therefore, screening and applying biocontrol strains with specific target inhibition capabilities to effectively control anthracnose in peppers and root rot in Sichuan pepper is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention
[0005] In view of this, the present invention provides a strain of Burkholderia pyrrole (… Burkholderia pyrrocinia L2 and its applications.
[0006] To solve the above-mentioned technical problems, this application adopts the following technical solution: A strain of Burkholderia pyrrole ( Burkholderia pyrrocinia The *Burkholderia pyrrole* L2, with accession number GDMCC NO: 66999, was deposited on September 19, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.
[0007] Another object of the present invention is to provide a microbial inoculant comprising the above-mentioned Burkholderia pyrrole L2.
[0008] Another object of the present invention is to provide the use of the above-mentioned Burkholderia pyrrole L2 or the above-mentioned microbial agents in the preparation of products for the prevention and control of anthracnose in peppers and / or SM1 strains.
[0009] Still another object of the present application is to provide the use of the above-mentioned B. pyrrocinia L2 or the above-mentioned microbial inoculant in promoting the germination and / or rooting of rice seeds.
[0010] Still another object of the present application is to provide a medicine comprising the above-mentioned B. pyrrocinia L2 and / or the above-mentioned microbial inoculant.
[0011] Still another object of the present application is to provide a method for preventing and controlling pepper anthracnose, wherein the above-mentioned B. pyrrocinia L2 or the above-mentioned microbial inoculant is sprayed on pepper plants and rhizosphere soil.
[0012] Still another object of the present application is to provide a method for preventing and controlling Zanthoxylum schinifolium root rot, wherein the above-mentioned B. pyrrocinia L2 or the above-mentioned microbial inoculant is sprayed on Zanthoxylum schinifolium plants and rhizosphere soil.
[0013] According to the above technical solution, compared with the prior art, the present application has the following beneficial effects: the present application provides a B. pyrrocinia L2 strain, which is identified by molecular identification and physiological and biochemical characteristics, has a significant siderophore production ability, can effectively deprive the essential iron resources of pathogenic bacteria in a low-iron environment, thereby inhibiting the spore germination and mycelial growth of pathogenic bacteria. The strain shows obvious antagonistic effect on pepper anthracnose pathogen and Zanthoxylum schinifolium root rot pathogen, and can significantly reduce the disease incidence.
[0014] In addition, the strain has unique advantages in plant growth promotion. Test results show that it can effectively improve the germination rate of rice seeds and the vigor of seedlings, which is speculated to be related to the secretion of siderophores, indole acetic acid (IAA) and nutrient activating substances. The strain has dual functions of disease prevention and growth promotion, and can be applied as a biocontrol agent for disease control of high economic value crops such as peppers and Zanthoxylum schinifolium, and can also be applied to seed treatment and early seedling stage of food crops such as rice, and has wide application prospects. BRIEF DESCRIPTION OF DRAWINGS
[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the following embodiment or prior art description will be briefly introduced. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the provided drawings.
[0016] Figure 1 Figure 1 is a colony morphology diagram of the strain L2 of the present application.
[0017] Figure 2 Figure 2 is a phylogenetic tree of the strain L2 of the present application.
[0018] Figure 3The strain L2 of the application is identified for the ability of producing siderophore.
[0019] Figure 4 The strain L2 of the application is identified for the ability of producing siderophore. Colletotrichum capsici The confrontation results of the strain L2 of the application on the pepper anthracnose pathogen. The confrontation results of the strain L2 of the application on the pepper anthracnose pathogen.
[0020] The confrontation results of the strain L2 of the application on the pepper anthracnose pathogen. Figure 5 The confrontation results of the strain L2 of the application on the pepper anthracnose pathogen. Fusarium solani The confrontation results of the strain L2 of the application on the pepper anthracnose pathogen. The confrontation results of the strain L2 of the application on the pepper anthracnose pathogen.
[0021] The confrontation results of the strain L2 of the application on the pepper anthracnose pathogen. Figure 6 The confrontation results of the strain L2 of the application on the pepper anthracnose pathogen. DETAILED DESCRIPTION
[0022] The technical solutions in the embodiments of the application will be clearly and completely described below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only some of the embodiments of the application, rather than all the embodiments of the application. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the application.
[0023] In the following examples, the experimental methods are all conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents and the like used in the following examples can be obtained from commercial channels, unless otherwise specified.
[0024] Example 1: Isolation and identification of strain L2 1. Isolation: 1 g of Zanthoxylum bungeanum Maxim. soil sample was mixed with 9 mL of sterile water to prepare 10 -1 to 10 -3 gradient dilutions. 100 μL of the dilutions of different gradients were uniformly coated on R2A solid medium (composition (g / L): yeast extract powder 0.5, protein peptone 0.5, casein hydrolysate 0.5, glucose 0.5, soluble starch 0.5, potassium phosphate dibasic 0.3, anhydrous magnesium sulfate 0.024, sodium pyruvate 0.3, agar 15.0, pH value 7.2 ± 0.2) to which nystatin and rifampicin antibiotics were added, and double parallel experiments were set for each group. The culture was incubated at 30°C for 48-72 h, and single colonies were picked according to the morphological characteristics such as colony size, color, transparency, etc. for three-zone streaking and purification to obtain strain L2.
[0025] 2. Identification: (1) Morphological identification: the colony morphology of strain L2 is shown in FIG. 1, and the colony is round, yellow or milk yellow, viscous, shiny and moist. Figure 1
[0026] (2) Molecular identification: 5 g of resin was added to 100 mL of sterile water and magnetically stirred until uniformly suspended. 200 μL of the resin suspension was taken to a 1.5 mL centrifuge tube, and an appropriate amount of bacteria was scraped from the third quadrant of the plate and added to the tube, vortexed for 30-60 s to mix thoroughly. Then the centrifuge tube was placed in a boiling water bath at 100 ℃ for 10 min, and after cooling to room temperature, it was centrifuged at 12850 rpm for 5 min, and the supernatant was collected as the DNA solution. The extracted DNA was used as a template for 16S rRNA gene amplification, and the PCR product containing the target band was sent to Shanghai Sangon Biological Engineering Co., Ltd. for Sanger bidirectional sequencing. ContigExpress software was used to splice the bidirectional sequencing peak map, and the abnormal bases in the low-quality segments were removed by manual correction. The corrected 16S rRNA sequence was submitted to the EzBioCloud database for similarity comparison, and the closest matching result with the known model strain was obtained. The phylogenetic tree is shown in Figure 2. Figure 2 .
[0027] 16S rRNA: , SEQ ID NO.1.
[0028] Based on the results of morphological and molecular identification, strain L2 was ultimately identified as Burkholderia pyrrole. Burkholderia pyrrocinia ).
[0029] 3. Preservation strain Burkholderia pyrrocinia L2 was deposited on September 19, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC NO: 66999 and classified as follows: Burkholderia pyrrocinia .
[0030] Example 2 Identification of iron carrier production ability of strain L2 (CAS color detection method) A single colony of L2 was picked up with a sterile inoculation loop and inoculated on the center of a CAS color detection plate (ingredients (L -1 ): chrome azurol S 60.5 mg, cetyltrimethylammonium bromide 72.9 mg, ferric chloride hexahydrate 2.645 mg, sodium dihydrogen phosphate dihydrate 295.25 mg, sodium hydrogen phosphate dodecahydrate 1213.5 mg, ammonium chloride 125 mg, potassium dihydrogen phosphate 37.5 mg, sodium chloride 62.5 mg, agar 9000 mg). The plate was placed in a 30°C constant temperature incubator and incubated for 48 h. After incubation, whether a yellow halo was formed around the colony was observed, and the diameter of the strain (d) and the halo diameter (D) were measured and recorded, and the D / d ratio was calculated.
[0031] As shown in the accompanying Figure 3 , the diameter of L2 strain was 0.6 cm (d), the halo diameter was 0.7 cm (D), and the D / d ratio was 1.2, indicating that the strain L2 could produce iron carrier.
[0032] Example 3 Identification of the bacteriostatic ability of strain L2 Pathogenic fungi: pepper anthracnose fungus Colletotrichum capsici , root rot fungus Fusarium solani .
[0033] Experimental method: Strain preparation: Before the experiment, all equipment was sterilized, and strain L2 and pathogenic fungi were activated on LB medium plates.
[0034] Fungal block preparation and inoculation: The culture medium covered with pepper anthracnose fungus (or SM1 strain) was cut into 2 mm diameter ring-shaped small blocks with sterile tools; the fungal small blocks were picked up with a sterile inoculation loop and inoculated into the center of PDA medium (ingredients (L -1 ): 300.0 g of potato infusion powder, 20.0 g of glucose, 15.0 g of agar).
[0035] L2 strain inoculation: L2 strain on LB medium was picked up with a sterile inoculation loop, and the fungal block in the center of PDA medium was used as a reference to inoculate at a distance of 2 cm.
[0036] Incubation and control setting: the inoculated medium was placed in a 30°C constant temperature incubator and incubated for 4-5 d, with 2 replicates for each group; at the same time, "only fungal inoculation" blank controls were set up for the two fungi respectively. Result observation: After the end of the culture, the bacteriostatic effect of strain L2 was observed, and the growth of the blank control group of fungi was compared.
[0037] Results are shown in the accompanying Figure 4 and accompanying Figure 5 Figures, strain L2 has a significant inhibitory effect on Colletotrichum gloeosporioides and Fusarium solani SM1, among which the d / D value of Colletotrichum gloeosporioides is 0.6 (1.2 / 2), and the d / D value of SM1 is 0.2 (0.6 / 2.6). Example 4: Promoting effect of strain L2 on rice seeds Experimental materials: strain L2, full rice seeds.
[0038] Experimental steps: (1) Preparation of bacterial solution Under sterile conditions, strain L2 was inoculated into LB medium (10.0 g of proteose peptone, 5.0 g of yeast extract powder, 5.0 g of sodium chloride, 1.0 g of glucose, and water to 1 L).
[0039] The inoculated medium was placed in a shaking incubator at 30°C and 180 rpm for 72 hours.
[0040] After the end of the culture, the OD value measuring instrument was used to adjust the concentration of the bacterial solution, so that the OD value of the bacterial solution was 0.2, ready for use. 600 =0.2, standby.
[0041] (2) Sterilization treatment of rice seeds Select full rice seeds, wash with sterile water for 3 times to complete the preliminary cleaning.
[0042] The cleaned seeds were divided into 2 groups for treatment, and 3 biological replicates were set for each group (15 seeds in one group): The first group: the seeds were soaked in the prepared L2 bacterial solution (OD 600 =0.2).
[0043] The second group: the seeds were soaked in sterile water.
[0044] Both groups of seeds were placed in a 25°C light incubator for 24 hours.
[0045] (3) Germination rate and index determination Germination rate determination: The two groups of seeds treated above were respectively laid on the culture dishes covered with wet sterile filter paper.
[0046] Put the culture dishes into a 25°C light incubator for constant temperature culture, and the culture time is 4 days.
[0047] After the end of culture, the seed germination rate was counted, and the criterion was that the radicle broke through the seed coat ≥1 mm. The calculation formula was: germination rate = number of germinated seeds / total number of seeds x 100%.
[0048] Growth-promoting index determination: The culture dish was continuously placed in a 25℃ light incubator for culture, and the cumulative culture was performed for 7 days.
[0049] After 7 days of culture, the following growth indexes were determined: Plant height: the distance from the rhizome junction to the longest leaf tip was measured.
[0050] Main root length: the longest vertical distance of the main root was measured.
[0051] Lateral root number: the number of branch roots with a length ≥1 mm on the main root was counted.
[0052] The results are shown in Table 1 and the accompanying Figure 6
[0053] Table 1: Growth-promoting effect of strain L2 on rice seeds
[0054] The various embodiments in the specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be mutually referred to.
[0055] The above description of the disclosed embodiments enables a person skilled in the art to implement or use the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. A strain of Burkholderia pyrrole ( Burkholderia pyrrocinia L2, characterized in that, The preservation number of Burkholderia pyrrole L2 is GDMCC NO: 66999.
2. A microbial inoculant, characterized in that, The microbial agent includes Burkholderia pyrrologensis L2 as described in claim 1.
3. The use of Burkholderia pyrrole-Holder L2 as described in claim 1 or the microbial agent as described in claim 2 in the preparation of products for the prevention and control of anthracnose in peppers and / or SM1 strains.
4. The use of Burkholderia pyrrole-Holder L2 as described in claim 1 or the microbial agent as described in claim 2 in promoting rice seed germination and / or rooting.
5. A drug, characterized in that, The drug comprises Burkholderia pyrogallol L2 as described in claim 1 and / or the microbial agent as described in claim 2.
6. A method for controlling anthracnose in peppers, characterized in that, The Burkholderia pyrrologenus L2 of claim 1 or the microbial agent of claim 2 is sprayed onto the chili plants and rhizosphere soil.
7. A method for controlling root rot in Sichuan pepper, characterized in that, The Burkholderia pyrrole-Holder L2 of claim 1 or the microbial agent of claim 2 is sprayed onto the Sichuan pepper plants and rhizosphere soil.