Paenibacillus terrae Pt05 and application thereof
By screening and identifying Paenibacillus subtilis Pt05, the problem of insufficient antagonistic microbial resources in the prevention and control of banana wilt disease was solved, an efficient biological control method was provided, and the control effect and environmental adaptability were enhanced.
Patent Information
- Application Number
- CN202511041387.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-28
- Publication Date
- 2025-09-16
AI Technical Summary
Existing technologies are difficult to effectively prevent and control banana wilt disease, and the breeding process of disease-resistant varieties is slow. Biological control methods have not yet been widely used, and there is a lack of stable and environmentally adaptable antagonistic microbial resources.
Paenibacillus terrae Pt05 was isolated and screened from the soil in Guangxi, and identified as Paenibacillus terrae through morphological, physiological and biochemical tests and 16S rRNA sequence analysis. Its antagonistic activity against banana Fusarium wilt and ultraviolet tolerance were verified, and it was developed as an antibacterial agent for the prevention and control of banana Fusarium wilt.
A strain Pt05 with obvious antagonistic activity against banana wilt and strong ultraviolet tolerance is provided. It significantly reduces the disease index, enhances the stability and environmental adaptability of biological control, and lays the foundation for the development of antibacterial agents.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of microbiology, and in particular relates to a Paenibacillus terrestris Pt05 and an application thereof. Background Art
[0002] Fusarium oxysporum Cuban Fusarium oxysporum f. sp. cubense Banana wilt, caused by infection with Fococcus occidentalis (Fococcus occidentalis), is currently the most devastating disease affecting the global banana industry. This disease is a typical soil-borne disease. Since its emergence, researchers and growers have tried various control methods, including soil fumigation, crop rotation, and organic soil amendments, but none have been effective. Although breeding disease-resistant varieties is the most fundamental approach to controlling soil-borne diseases, most cultivated bananas with superior agronomic traits are vegetatively propagated, and disease-resistant varieties have been developed through hybridization. Disease-resistant mutants obtained through mutation breeding often exhibit unstable agronomic traits. While transgenic breeding can ensure stable inheritance of resistance, it struggles to gain market acceptance. Consequently, the development of disease-resistant varieties has been slow, and no commercially accepted resistant varieties have yet emerged. Biological control, using living beneficial microorganisms to combat soil-borne diseases, can improve the soil microbial ecosystem, enrich the soil microbial community, prevent the development of resistance, and contribute to sustained disease suppression. Therefore, the selection of antagonistic microorganisms for banana wilt is an effective and worthy approach.
[0003] Screening and obtaining antagonistic microorganisms with biocontrol potential is an important basis for carrying out biological control work. In recent years, a variety of biocontrol bacteria with antagonistic effects on banana wilt have been reported. Sekhar et al. isolated a Pseudomonas aeruginosa GNS.13.2a from the banana stem tip that inhibited the growth of Foc; Huang Jianfeng et al. studied the control effects of a Bacillus subtilis H-2 and a Bacillus amyloliquefaciens H-7 on BFW under greenhouse conditions, which were 59.1% and 53.0%, respectively; Xu Zhizhou et al. isolated strain HQB-1 ( Burkholderia stagnalis ), after optimizing culture conditions, the strain exhibited an inhibition rate of 34.60% against Foc on petri dishes. Napitupulu et al. measured the inhibitory activity of 10 strains of Trichoderma harzianum against Foc on petri dishes, with the highest inhibition rate reaching 44%. Belgrove et al. tested 14 non-pathogenic Fusarium strains under greenhouse conditions, finding that 10 of them significantly reduced the incidence of BFW. However, due to the characteristics of the biocontrol bacteria themselves and environmental factors, relatively few biocontrol strains can consistently achieve control effects in the field.
[0004] To further explore the biocontrol bacteria resources for banana Fusarium wilt, this study isolated and screened the biocontrol bacterium Pt05 from soils of different habitats, which has obvious antagonistic activity and good control effect against banana Fusarium wilt. It was identified through morphological observation, physiological and biochemical tests and 16S rRNA sequence analysis, in order to provide new bacterial resources for the biological control of banana Fusarium wilt and lay the foundation for the subsequent development and utilization of anti-pathogen agents and fertilizers. Summary of the Invention
[0005] The present invention aims to provide a Paenibacillus terrestris Pt05 and application thereof.
[0006] The Paenibacillus sp. Pt05 provided by the present invention was isolated from soil in Guangxi and identified as Paenibacillus terrae , was deposited on July 11, 2022 in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC for short, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology, Postal Code: 510070), with the deposit number GDMCC No.62613.
[0007] The present invention also provides an antibacterial agent, which comprises the Paenibacillus terrestris Pt05.
[0008] Secondly, the present invention also provides the use of the Paenibacillus terrestris Pt05 or the antibacterial agent in preventing and treating wilt disease.
[0009] In addition, the present invention also provides the use of the Paenibacillus terrestris Pt05 or the antibacterial agent in preventing and treating banana wilt.
[0010] Compared with the prior art, the present invention has the following beneficial technical effects: The present invention isolated a strain with strong antagonistic activity against Fusarium oxysporum Cuban species 4 from soil in Guangxi. The strain was identified through morphological identification, physiological and biochemical tests, and 16S rRNA sequence analysis. The antagonistic bacteria potted control effect and ultraviolet tolerance were also measured. The obtained Pt05 was identified as Paenibacillus terrestris. Paenibacillus terrae In the antagonistic bacteria potted control effect test, strain Pt05 has obvious antagonistic activity and good control effect on banana wilt pathogen; in the test results of the antagonistic bacteria's tolerance to ultraviolet rays, strain Pt05 has relatively strong tolerance to ultraviolet rays, strong adaptability to the environment, easier survival, and exerts control effect, which can provide new bacterial resources for the biological control of banana wilt disease and lay the foundation for the subsequent development and utilization of anti-pathogenic bacteria and fertilizers.
[0011] Preservation Information Paenibacillus terraePt05, the deposit number is GDMCC No.62613, the deposit date is July 11, 2022, the deposit unit is Guangdong Microbiological Culture Collection Center (GDMCC), and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS
[0012] Figure 1 The colony and morphology of strain Pt05 under optical microscope; Figure 2 is the phylogenetic tree of strain Pt05; Figure 3 These are the results of the strain Pt05's tolerance test to ultraviolet rays. DETAILED DESCRIPTION
[0013] The present invention will be further described in detail below with reference to specific embodiments, which are intended to explain the present invention rather than to limit it.
[0014] Unless otherwise specified, the materials and reagents used in the following examples are all commercially available. The experimental methods used in the following examples are all conventional methods unless otherwise specified.
[0015] Example 1. Materials and Methods 1.1 Materials PDA medium: 200 g potatoes, 20 g glucose, 15 g agar, 1 L water, natural pH.
[0016] NA medium: 10 g peptone, 3 g beef extract, 5 g sodium chloride, 15 g agar, 1 L water, pH 7.0. The medium without agar is called NA liquid medium.
[0017] Test strain: Fusarium oxysporum Cuban specific type 4 physiological substrain Foc1402, isolated and preserved by the Institute of Plant Protection, Guangxi Academy of Agricultural Sciences.
[0018] Banana seedlings for testing: 5-leaf-old “Williams B6” banana cup seedlings with uniform growth were purchased from Nanning Xiangjie Agricultural Science and Technology Co., Ltd.
[0019] 1.2 Isolation and screening of antagonistic bacteria against banana wilt 1.2.1 Soil microbial isolation The soil was collected from the root zone of the crop at a distance of 3-10 cm from the surface using the five-point sampling method. After mixing, 2 g of the sample was weighed into a 50 mL sterile centrifuge tube, 18 mL of sterile water was added, and the soil suspension was prepared by vortexing and then diluted 10-fold.
[0020] Take 100 μL of each and dilute it to 10 -4 , 10 -5 and 10 -6 The soil suspension was spread on NA plates and incubated at 28°C. After 24 hours, single colonies with different morphologies were picked for purification and stored at 4°C for later use.
[0021] 1.2.2 Initial screening of Foc antagonistic bacteria Foc1402 was cultured on PDA medium at 28°C for 3 days. A 5 mm diameter sterile punch was used to punch a bacterial cake along the edge of the colony. This cake was transferred to the center of a PDA plate and incubated at 28°C for 24 hours. The bacteria to be tested were then streaked 2 cm from the cake. After incubation at 28°C for 4 days, strains with an inhibition zone of 3 mm or greater were selected for further screening.
[0022] 1.2.3 Rescreening of Foc antagonistic bacteria The antagonistic bacteria obtained in the initial screening were transferred to NA liquid culture medium and cultured at 28 °C with shaking at 120 r / min for 72 h. The concentration of the bacterial suspension was adjusted to 1×10 8 cfu / mL, and used as antagonistic bacteria fermentation broth.
[0023] After Foc1402 was cultured on a PDA tube slant at 28°C for 10 days, sterile water was added and gently shaken to prepare a conidia suspension of the pathogen, and the spore concentration was adjusted to 1×10 6 spores / mL, set aside.
[0024] Add a 1% by volume suspension of pathogen conidia to PDA medium cooled to 45°C, rapidly shake to mix, and pour onto a water agar plate placed in an Oxford cup. After solidification, remove the Oxford cup and incubate at 28°C for 6 hours. Add the supernatant of the fermentation of the antagonistic strain to be tested, which has been centrifuged at 5000 rpm for 3 minutes. Sterile water was used as a control. Each treatment was repeated three times. After incubation at 28°C for 3 days, the diameter of the inhibition zone was measured, and the inhibition rate was calculated. Strains with the highest inhibition rate were selected for subsequent testing.
[0025] 1.3 Identification of antagonistic strains 1.3.1 Morphological observation The selected antagonistic strains were cultured on NA medium at 28°C for 48 h. The colony characteristics such as colony shape, transparency and colony color were regularly observed and recorded, and the bacterial morphology was observed under an optical microscope.
[0026] 1.3.2 Physiological and biochemical reaction tests Physiological and biochemical reaction tests were performed according to the instructions for the Staphylococcus biochemical identification tubes and Enterobacteriaceae biochemical identification tubes provided by Hangzhou Microbiological Reagent Co., Ltd. The reaction results were determined according to the "Common Bacteria Systematic Identification Manual" and the "Berger's Bacteria Systematic Identification Manual."
[0027] 1.3.3 Molecular identification The antagonistic strain was inoculated into NA liquid medium and cultured at 28°C, shaking at 120 rpm for 16 h. 1 mL of the culture was collected by centrifugation at 12,000 rpm for 1 min. Genomic DNA was extracted using a bacterial genomic DNA extraction kit, and the 16S rRNA sequence of the test strain was amplified using universal primers. The universal primer sequences are shown in Table 1, and the amplification reaction system is shown in Table 2.
[0028] Table 1 16S rRNA primers
[0029] Table 2 PCR amplification reaction system
[0030] The PCR program was as follows: pre-denaturation at 94°C for 5 min; 30 cycles of denaturation at 94°C for 45 s, annealing at 55°C for 45 s, and extension at 72°C for 90 s; final extension at 72°C for 10 min, and storage at 4°C.
[0031] The PCR amplification products were sequenced, and the sequencing results were compared by BLAST on the NCBI database. A phylogenetic tree was constructed using the neighbor-joining method using MEGA X software.
[0032] 1.4 Determination of the control effect of antagonistic bacteria in potted plants Prepare the antagonistic bacteria fermentation liquid and pathogen conidia suspension according to the method in 1.2.3. Transplant five-leaf banana seedlings of uniform growth into plastic pots with vermiculite as the substrate. After 10 days of planting at room temperature, use a knife to wound the banana roots along the root circumference. Inoculate each banana seedling with 40 mL of spores at a concentration of 1×10 6 1×10 / mL of pathogen conidia suspension was poured into the 8 cfu / mL of an antagonistic bacterial fermentation suspension was inoculated. Seven days later, an equal volume of the antagonistic bacterial fermentation suspension at the same concentration was inoculated again. Sterile water served as a control. Each treatment was replicated three times, with six banana seedlings per replicate. Twenty-one days after inoculation, disease activity was assessed, and the disease index and control efficacy of each treatment were calculated.
[0033] The disease grade is determined based on the percentage of browning area in the longitudinal section of the banana seedling bulb. The disease grade assessment criteria are shown in Table 3.
[0034] Table 3 Disease grade assessment criteria
[0035]
[0036]
[0037] 1.5 Test of the tolerance of antagonistic strains to ultraviolet light The antagonistic strain was transferred to NA liquid medium and cultured at 28°C with shaking at 120 r / min for 72 h. The fermentation broth concentration was adjusted to 1×108 cfu / mL with sterile water and used as the antagonistic bacteria fermentation broth.
[0038] 15 mL of the biocontrol bacterial fermentation broth was placed in sterile Petri dishes equipped with a sterile magnetic stir bar and stirred using a magnetic stirrer. A 20W UV lamp, preheated for 20 minutes, was placed 50 cm above the Petri dish. The dish lid was opened and irradiated. The irradiation duration was adjusted based on the strain's tolerance to UV radiation. Unirradiated bacterial broth served as a control, and each treatment was repeated three times. The irradiated bacterial broth was diluted 10-fold in a series under red light. 100 μL of each dilution was plated onto NA plates and incubated at 28°C in the dark. After 24 hours, colonies were counted and the mortality rate was calculated.
[0039]
[0040] 2. Results and Analysis 2.1 Microbial isolation and antimicrobial screening Using the gradient dilution spread method, 345 bacterial strains were isolated from soil samples collected from different regions and habitats. Initial screening using the plate confrontation method revealed 48 strains with antagonistic activity against banana Fusarium wilt. Rescreening using the Oxford cup method yielded several strains with high inhibition rates, including strain Pt05, which exhibited an inhibition rate of 37.27%.
[0041] 2.2 Identification of antagonistic bacteria 2.2.1 Morphological identification of antagonistic bacteria Observation results are shown in Figure 1 .
[0042] Depend on Figure 1 It can be seen that strain Pt05 forms a round, off-white single colony with a low convex middle part, a rough and opaque surface, and is difficult to pick up; the bacteria are rod-shaped under a microscope.
[0043] 2.2.2 Physiological and biochemical tests of antagonistic strains Physiological and biochemical testing showed that strain Pt05 could break down glucose, sucrose, fructose, maltose, lactose, and mannose, and hydrolyze starch, but not xylose or mannitol. Gram staining, hydrogen sulfide, mannitol, nitrate reduction, ornithine, urea, and peptone water (VP) tests were negative, while methyl red (MR) was positive. The strain's main physiological and biochemical characteristics are essentially consistent with those of Paenibacillus terrestris.
[0044] 2.2.3 Molecular identification of antagonistic strains The 16S rRNA sequences of the antagonistic bacteria were compared by BLAST in GenBank. The results showed that the strain Pt05 had a similarity to that of Paenibacillus terrestris. Paenibacillus terrae The highest homology is 99.61%.
[0045] Based on the 16S rRNA of strain Pt05, the phylogenetic tree was constructed using the neighbor-joining method. Figure 2 .
[0046] Depend on Figure 2 It can be seen that the antagonistic bacteria Pt05 and two strains of Paenibacillus terrestris (MT634584, LC127093) are in the same branch, with a bootstrap support rate of 100%, which is the same as that of the other strains. Paenibacillus spp. can be clearly distinguished. Combining the morphological characteristics and physiological and biochemical characteristics of the strain, strain Pt05 was identified as Paenibacillus terrestris Paenibacillus terrae .
[0047] 2.3 Antagonistic bacteria potted control effect After 21 days of treatment, the leaves of the banana seedlings in the control group inoculated with the pathogen alone turned yellow, while those treated with the fermented suspension of the antagonistic bacteria remained green. The efficacy of the antagonistic bacteria against banana Fusarium wilt in potted plants is shown in Table 4.
[0048] Table 4 The control effect of antagonistic bacteria on banana wilt in potted plants
[0049] As shown in Table 4, the disease index of the control group was 71.43, and the disease index of strain Pt05 was 57.14, which was significantly lower than that of the control group. In terms of control effect, the control effect of the treatment with Paenibacillus terrestris Pt05 was 20.00%.
[0050] In summary, the strain Pt05 screened out in the present invention can be used to prevent and control banana wilt disease, provide a new bacterial strain resource for the biological control of banana wilt disease, and lay the foundation for the subsequent development and utilization of antibacterial agents and fertilizers.
[0051] 2.2.4 Test of the tolerance of antagonistic strains to ultraviolet light The results of the strain Pt05's tolerance to ultraviolet light are shown in Figure 3 .
[0052] from Figure 3 As can be seen, strain Pt05 is relatively tolerant to UV rays. When irradiated for 7.5 minutes, its lethality rate was 93.07%. This strong tolerance of strain Pt05 to UV rays suggests that it is more adaptable to the environment during application, making it more likely to survive and achieve effective control.
[0053] The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various exemplary embodiments and various choices and changes of the present invention. The scope of the present invention is intended to be defined by the claims and their equivalents.
Claims
1. A strain of Paenibacillus terrestris Pt05, characterized in that: The Latin name of the land bacillus is Paenibacillus terrae , deposited in Guangdong Provincial Microbiological Culture Collection Center on July 11, 2022, with the deposit number GDMCC No.62613, and its 16S rRNA is shown in SEQ ID No.
1.
2. An antibacterial agent, characterized in that: The invention comprises the Paenibacillus sp. Pt05 according to claim 1.
3. Use of the Paenibacillus sp. Pt05 according to claim 1 or the antibacterial agent according to claim 2 in preventing and treating Fusarium wilt.
4. Use of the Paenibacillus terrestris Pt05 according to claim 1 or the antibacterial agent according to claim 2 in preventing and treating banana wilt.