CN114703094A - Bacillus pseudomycoides and application thereof in preparation of plant pathogenic bacteria bacteriostat - Google Patents
Bacillus pseudomycoides and application thereof in preparation of plant pathogenic bacteria bacteriostat Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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Abstract
The invention belongs to the technical field of microorganisms, and particularly relates to pseudomonas pseudofungoides FJH2 and application thereof in preparation of a plant pathogenic bacteria bacteriostatic agent. The strain has the functions of resisting plant pathogenic bacteria and the like; can grow in the pH of 6.0-8.0, the salt concentration of 1-6% and the temperature of 20-40 ℃, has good inhibition effect on several plant pathogenic bacteria, wherein the inhibition rate on fusarium oxysporum is as high as 91.47%, and has potential application value in preparation of biocontrol bacteria.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to pseudomonas pseudofungoides and application thereof in preparation of a plant pathogenic bacterium bacteriostatic agent.
Background
Fusarium is widely present in soil and plants, mainly acts on plant roots and root-tuber junctions to destroy vascular bundle tissues, and generates metabolites such as enzymes and phenols in the metabolic process to influence the growth and development of host plants. The plant blight occurs most seriously in summer, and infected plants are mainly manifested by growth retardation, yellow leaves, withered and fallen leaves death, rhizome decay and the like. At present, no chemical pesticide which is very effective for treating the blight exists in agriculture, so that the wilt treatment mainly depends on a biological prevention and treatment method. Research shows that the pseudomonas stutzeri can produce plasmin and increase potassium absorption of crops, but reports on the bacterium in bacteriostasis are few.
The selected pseudomonas stutzeri has a good inhibition effect on fusarium oxysporum and fusarium solani, and the inhibition rate is very high and reaches 91.47%; can play a certain role in preventing and treating plant diseases and has great potential for developing biocontrol bacteria.
Disclosure of Invention
The invention aims to provide a bacillus pseudomycoides (Bacillus pseudomycoides) FJH2, wherein the preservation number of the bacteria is as follows: CCTCC NO: M2022049.
The invention also aims to provide application of the pseudomonas mycoides in preparation of the plant pathogenic bacteria bacteriostatic agent.
In order to achieve the purpose, the invention adopts the following technical measures:
the applicant separates a strain of pseudomonas stutzeri from soil samples in the city of great wall of Shanxi Weinan city, has an antibacterial effect on pathogenic bacteria such as fusarium oxysporum and fusarium solani, has a high antibacterial rate, is identified as the pseudomonas stutzeri, and is delivered to the China center for type culture collection at 1 month and 10 days in 2022 for storage, and the address is Wuhan university in Wuhan, China, and is named after classification: bacillus pseudomycoides FJH2 with the preservation number: CCTCC NO: M2022049.
The colony morphology of the isolated Bacillus pseudomycoides (Bacillus pseudomycoides) on LB solid medium is shown in FIG. 1, and the colony morphology is off-white and relatively flat root. The physiological and biochemical manifestations are gram-positive staining, non-hydrolysable starch, non-hydrolysable cellulose, negative in methyl red test, negative in acetyl methyl alcohol test, and positive in catalase.
The application of the pseudomonas pseudomycoides FJH2 in preparing the plant pathogenic bacteria bacteriostatic agent is provided, wherein the plant pathogenic bacteria are banana Fusarium oxysporum F.sp.Cubense, Fusarium oxysporum, Penicillium brasiliensis, Fusarium solani, Fusarium chlamydosporium, Penicillium gluerucum and trichoderma.
Compared with the prior art, the invention has the following characteristics:
the invention discloses a pseudomycosis bacillus for the first time, which has good bacteriostatic effect on several pathogenic bacteria causing plant blight.
Drawings
FIG. 1 is a diagram showing the culture characteristics of Bacillus pseudomycoides FJH 2.
FIG. 2 is a schematic diagram showing the inhibitory effect of Bacillus pseudomycoides FJH2 on Fusarium oxysporum.
FIG. 3 is a schematic diagram showing the inhibitory effect of Bacillus pseudomycoides FJH2 on Fusarium solani.
FIG. 4 is a schematic diagram showing the inhibitory effect of Bacillus pseudomycoides FJH2 on Mucor.
Detailed Description
The technical scheme of the invention is a conventional technology if not particularly specified; the reagents or materials, if not specifically mentioned, are commercially available.
Example 1:
isolation and identification of Bacillus pseudomycoides:
2.5g of soil sample from Typha county, Weinan, Shanxi was placed in 50ml of sterile water and shaken at 30 ℃ and 250rpm for 1 hour, after which 500. mu.l of the soil suspension was subjected to gradient dilution in a PA flask containing 4.5ml of sterile water. Get 10-1、10-2、10-3、10-4、10-5The diluted soil suspensions are respectively added into a culture medium for coating, the growth condition of the plate bacterial colony is observed after 24 hours, 48 hours, 72 hours and 96 hours of culture, all the grown bacterial colonies are respectively picked out for carrying out an antibacterial activity experiment; selecting bacteria with high bacteriostasis rate and wide bacteriostasis spectrum; the Bacillus pseudomycoides FJH2 was finally selected.
(1) Morphological characteristics
The colony morphology of the isolated Bacillus pseudomycoides on LB solid medium is shown in FIG. 1, and the colony is in the form of off-white relatively flat root.
(2) Physiological and biochemical experiment
The physiological and biochemical manifestations are gram staining positive, non-hydrolysable starch, non-hydrolysable cellulose, negative in methyl red test, negative in acetyl methyl alcohol test, and positive in catalase.
(3) Gene identification of Bacillus pseudomycoides
Extracting the genome DNA of the strain to be detected by using a bacterial DNA extraction kit, and referring to the instruction for the specific operation steps. And (3) amplifying the bacteria by using the extracted bacteria genome DNA as a template, wherein a primer for amplifying 16S rRNA:
27F:5＇-gtttgatcctggctcag-3＇
1492R:5＇-tacggctaccttgttacgactt-3＇；
the sequence is subjected to homology comparison analysis through a Blast16SrDNA sequence, and the result analysis shows that the sequence has the highest similarity with the 16SrDNA gene sequence of the strain Bacillus pseudomycin found by the strain, belongs to a strain of Bacillus pseudomycoides, and the strain is delivered to a China center for type culture collection at 1 month and 10 days 2022, and the address is university of Wuhan, China, and is classified and named as follows: bacillus pseudomycoides FJH2 with the preservation number as follows: CCTCC NO: M2022049.
Example 2:
tolerance testing of pseudomonas:
(1) experiment of pH resistant range:
inoculating 2% Bacillus pseudofungoides seed solution into LB culture medium with different initial pH respectively, culturing at 30 deg.C and 200r/min, culturing for 16 hr, sampling, and measuring OD600. The initial pH gradient of the medium was set to 3, 4, 5, 6, 7, 8, 9, 10 (initial 0D is 0).
LB culture medium: 10g of peptone, 5g of yeast extract powder, 10g of NaCl, 1000mL of distilled water, pH7.0, and high-pressure steam sterilization at 121 ℃ for 20 min.
| PH | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| OD600 | 0 | 0 | 0 | 4.712 | 5.356 | 4.504 | 0 | 0 |
(2) Salt tolerance test:
inoculating 2% of Bacillus pseudomycoides seed solution into LB culture medium with different salt concentration, culturing at 30 deg.C and 200r/min for 16 hr, sampling, and measuring OD600. The salt concentration gradient of the medium was set at 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% (0 for initial 0D).
LB culture medium: 10g of peptone, 5g of yeast extract powder, 10g of NaCl, 1000mL of distilled water, pH7.0 and high-pressure steam sterilization at 121 ℃ for 20 min.
| Salt concentration | 1％ | 2％ | 3％ | 4％ | 5％ | 6％ | 7％ | 8％ |
| OD600 | 3.298 | 3.51 | 4.5 | 3.122 | 2.248 | 1.516 | 0 | 0 |
(3) Growth temperature experiment:
inoculating 2% Bacillus pseudomycoides seed solution into LB culture medium, shake culturing at different temperatures for 200r/min, culturing for 16 hr, sampling, and measuring OD600. The temperature gradient of the medium was set at 20 deg.C, 28 deg.C, 37 deg.C, 40 deg.C, 45 deg.C (0 as initial 0D).
| Temperature (. degree.C.) | 20℃ | 25℃ | 30℃ | 35℃ | 40℃ | 45℃ |
| OD600 | 0.3 | 2.728 | 6.28 | 5.176 | 4.892 | 0 |
Example 3:
inhibition of phytopathogens by pseudomonas mycoides:
the inhibition ability of the pseudomonas mycoides on pathogenic bacteria is measured by a bacteriostasis loop method. Pseudomycosis fungoides was activated with LB, and the seed solution was diluted appropriately and applied to PDA plates, and sterile water was applied as a control. Perforating a PDA plate full of pathogenic fungi by using a gun head, selecting fungi blocks, inversely inoculating the fungi blocks on the PDA plate coated with the pseudomycosis fungoides, culturing the PDA plate at 37 ℃ for 4 days, measuring the diameter of a fungus colony, and calculating the bacteriostasis rate, thereby analyzing the bacteriostasis effect.
The plant pathogenic bacteria selected in the experiment include banana Fusarium oxysporum f.sp.cubense, Fusarium oxysporum, Penicillium brasiliensis, Fusarium solani, Fusarium chlamydosporium, Penicillium gluerucum and Mucor mucora.
PDA culture medium: 200g of potato, 20g of glucose and 1000mL of distilled water, wherein the pH is natural pH, and the potato is sterilized by high-pressure steam at 115 ℃ for 30 min.
Note: d, control plate colony diameter (mm); d, experimental plate colony diameter (mm).
The pseudomonas has good control effect on fusarium oxysporum, and the inhibition rate of pathogenic bacteria causing the plant diseases reaches 91.47%; the compound preparation has certain inhibition effect on fusarium solani and mucor, and the inhibition rate reaches 69.48% and 59.76%. Has poor inhibition effect on penicillium, fusarium chlamydosporia, fusarium oxysporum and fusarium solani. The bacteriostatic ratio for each bacterium is shown in the following table.
The embodiment shows that the invention provides the bacillus pseudomycoides FJH2, which has good control effect on the plant blight caused by fusarium oxysporum and fusarium solani.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be construed as the protection scope of the present invention.
Sequence listing
<110> Shandong Yetian Biotechnology Ltd
<120> a strain of Bacillus pseudomycoides and application thereof in preparation of plant pathogenic bacteria bacteriostat
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gtttgatcct ggctcag 17
<210> 2
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tacggctacc ttgttacgac tt 22
Claims (2)
1. A separated pseudomycosis bacillus (B.pseudomycoides)Bacilluspseudomycoides) The preservation number of the pseudomonas is as follows: CCTCC NO: M2022049.
2. A use of Bacillus pseudomycoides according to claim 1 in the preparation of a bacteriostatic agent for a phytopathogen selected from the group consisting of banana vascular wilt (Byssochlamus paradisi)Fusarium oxysporum F.sp. Cubense) Fusarium oxysporum (F.), (Fusarium oxysporum) Penicillium brasiliensis (II)Penicillium brasilianum) Fusarium solani (F.), (Fusarium solani) Fusarium chlamydosporia (C.), (B.), (C.), (C.), (C.) (C.) (C)Fusarium chlamydosporum) Penicillium (A) and (B)Penicillium glaucum) And Mucor: (Mucor）。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117431191A (en) * | 2023-12-19 | 2024-01-23 | 中国农业大学 | Bacillus pseudomycoides HDCP1 and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117431191A (en) * | 2023-12-19 | 2024-01-23 | 中国农业大学 | Bacillus pseudomycoides HDCP1 and application thereof |
| CN117431191B (en) * | 2023-12-19 | 2024-03-12 | 中国农业大学 | Bacillus pseudomycoides HDCP1 and application thereof |
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Inventor after: Huang Li Inventor after: Su Shanzhu Inventor after: Yang Shaozhou Inventor after: Liu Yonggao Inventor after: Ding Xiangqi Inventor after: Chen Zhenmin Inventor after: Yan Xunbo Inventor after: Zhu Qiuyan Inventor after: Qi Hongyue Inventor after: Feng Ke Inventor after: Geng Yuefeng Inventor after: Zhang Xinlong Inventor after: Jia Zhongqing Inventor before: Huang Li Inventor before: Su Shanzhu Inventor before: Yang Shaozhou Inventor before: Liu Yonggao Inventor before: Ding Xiangqi Inventor before: Chen Zhenmin Inventor before: Yan Xunbo Inventor before: Zhu Qiuyan Inventor before: Qi Hongyue Inventor before: Feng Ke Inventor before: Geng Yuefeng Inventor before: Zhang Xinlong Inventor before: Jia Zhongqing |
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