CN117965405A - Pseudomonas eastern lake and application thereof in plant disease control - Google Patents

Pseudomonas eastern lake and application thereof in plant disease control Download PDF

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CN117965405A
CN117965405A CN202410390792.1A CN202410390792A CN117965405A CN 117965405 A CN117965405 A CN 117965405A CN 202410390792 A CN202410390792 A CN 202410390792A CN 117965405 A CN117965405 A CN 117965405A
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pseudomonas
eastern
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donghuensis
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CN117965405B (en
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黄丽丽
王娜娜
梁怡非
陈思圯
何维鹏
刘巍
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Shenzhen Research Institute Of Northwest University Of Agriculture And Forestry Science And Technology
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • 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
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/27—Pseudomonas
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • C12R2001/38—Pseudomonas

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Abstract

The invention belongs to the technical field of microorganisms, and discloses pseudomonas eastern lake and application thereof in plant disease control. The invention separates and screens a strain of eastern lake pseudomonas Pseudomonas donghuensis HYBS-267 from healthy branches of kiwi fruits, the preservation number is CGMCC NO.29933, and the nucleotide sequence of 16S rDNA is shown as SEQ ID NO. 1. The pseudomonas Donghu Pseudomonas donghuensis HYBS-267 has remarkable inhibition effect on pathogenic varieties of pseudomonas syringae and kiwi fruits, and is expected to be developed into a safe and efficient biocontrol microbial inoculum for preventing and controlling bacterial canker of kiwi fruits.

Description

Pseudomonas eastern lake and application thereof in plant disease control
Technical Field
The invention belongs to the technical field of microorganisms, relates to separation, identification and application of functional bacteria, and in particular relates to pseudomonas eastern lake and application of pseudomonas eastern lake in plant disease control.
Background
Along with the gradual increase of the planting area of the kiwi fruits, the disease problem is increasingly developed. Bacterial canker of kiwifruits is one of the most extensive diseases of kiwifruits, and is caused by Pseudomonas syringae pathogenicity (Psa), and has the characteristics of high transmission speed, strong pathogenicity, high control difficulty and the like. At present, the disease is prevented and treated by using preparations such as kasugamycin, zhongshengmycin and other antibiotics or copper King, copper hydroxide and the like, but the chemical prevention and treatment are extremely easy to cause the enhancement of Psa resistance, and residues of the preparations have negative effects on human bodies and the environment, so that the food safety requirements of consumers are not met.
With the rapid progress of modern biotechnology, biological control is coming as a development opportunity as an environment-friendly plant disease management mode. The biological control has the advantages of safety, environmental protection, high efficiency, difficult generation of drug resistance and the like, ensures the safety of food and maintains ecological health. Compared with the traditional chemical pesticides, the biological pesticides have generally lower residue or even no residue, and the biological pesticides are used for reducing or replacing the use of the chemical pesticides, so that the influence of the chemical pesticides on the ecological environment and human health can be effectively solved from the source, and the sustainable development of society and economy is facilitated.
The research shows that bacteria or fungi inhibiting pathogenic microorganisms can be separated from the inside or rhizosphere of plants and can be prepared into microbial agents, so that biological control effects are achieved. Therefore, the bacterial strain with biocontrol potential is separated and screened, the bacterial canker of the kiwi fruits is controlled by utilizing a biocontrol mode, the method has important significance for the healthy development of the kiwi fruit industry, and the method has wide application prospect.
Disclosure of Invention
The application aims to provide more alternative solutions for preventing and treating bacterial canker of kiwi fruits. To achieve the technical purpose, the inventor screens out a Pseudomonas eastern lake Pseudomonas donghuensisHYBS-267 strain, and the preservation information is as follows:
Strain name: pseudomonas eastern lake
Latin name: pseudomonas donghuensis A
Strain number: HYBS-267
Preservation mechanism: china general microbiological culture Collection center (China Committee for culture Collection of microorganisms)
The preservation organization is abbreviated as: CGMCC
Preservation agency address: north Star West Lu No.1, 3, china academy of sciences of microorganisms in the Korean region North Star of Beijing
Preservation date: 2024, 03, 04
Preservation number: CGMCC No.29933
Specifically, the Pseudomonas eastern lake Pseudomonas donghuensisHYBS-267 is isolated from healthy kiwi fruit branches of Mei County Chen Jiaogong kiwi fruit plantation in Baoko, shaanxi, and the isolation and purification of the strain are described in the specific embodiment of the invention. The morphology of the cultured bacterial colony of the Pseudomonas tobermori is known by observing, and the bacterial colony of the Pseudomonas tobermori Pseudomonas donghuensisHYBS-267 is convex on the surface, irregular on the edge, light yellow and special odor.
Further, the nucleotide sequence of the 16S rDNA of the Pseudomonas Donghuensis Pseudomonas donghuensisHYBS-267 is shown as SEQ ID NO. 1.
Further, it is known from plate-stand tests, in-vitro leaf discs and in-vitro shoot tests that the Pseudomonas tobermori Pseudomonas donghuensisHYBS-267 show good inhibitory activity against Pseudomonas syringae kiwi disease-causing varieties (Psa).
Based on the above, the invention further provides application of the Pseudomonas eastern lake Pseudomonas donghuensisHYBS-267 in kiwi fruit disease control. In particular, the Pseudomonas putida Pseudomonas donghuensisHYBS-267 is used for preventing and treating bacterial canker of kiwi fruits caused by pathogenic varieties of the kiwi fruits of Pseudomonas syringae.
In addition, the invention also provides a microbial inoculum for preventing and treating bacterial canker of kiwi fruits, and the active ingredients of the microbial inoculum comprise thalli and/or fermentation liquor of the pseudomonas eastern lake Pseudomonas donghuensisHYBS-267.
The invention also claims the application of the pseudomonas eastern lake Pseudomonas donghuensisHYBS-267 in preparing the bacterial canker prevention and treatment microbial inoculum for kiwi fruits.
Compared with the prior art, the invention has the following beneficial effects:
The invention separates and screens a strain of Pseudomonas eastern lake Pseudomonas donghuensisHYBS-267 from kiwi fruit branches, and the nucleotide sequence of 16S rDNA is shown as SEQ ID NO. 1. The pseudomonas Donghthened Pseudomonas donghuensisHYBS-267 is environment-friendly, has no hidden danger of food safety, and does not produce harmful residues. The invention provides a strain with biocontrol function for the development of novel biological bactericides, and has important potential application value in the field of plant disease control, especially in the control of kiwi fruit bacterial canker.
The Pseudomonas eastern lake Pseudomonas donghuensisHYBS-267 has remarkable effect of inhibiting Psa, can be used for preventing and treating bacterial canker of kiwi fruits caused by Psa, and is an excellent biocontrol microorganism.
The invention discloses that the pseudomonas donghuensis Pseudomonas donghuensisHYBS-267 has remarkable inhibition effect on kiwi fruit bacterial canker for the first time, and provides important material accumulation and theoretical basis for biological control of kiwi fruit bacterial canker.
Drawings
FIG. 1 is a colony morphology of Pseudomonas eastern lake Pseudomonas donghuensisHYBS-267 on LB solid medium.
FIG. 2 is a diagram showing the morphology of the cells of Pseudomonas Donghuensis Pseudomonas donghuensisHYBS-267 under an optical microscope.
FIG. 3 is a phylogenetic tree of Pseudomonas putida Pseudomonas donghuensisHYBS-267 constructed based on the NJ method.
FIG. 4 is a graph showing the results of culturing the cakes of Pseudomonas Doteichthys Pseudomonas donghuensisHYBS-267 in a medium containing PsaM.
FIG. 5 is a graph showing the inhibitory effect of Psa strains of different origins after co-cultivation with Pseudomonas Donghuensis Pseudomonas donghuensisHYBS-267. In FIG. 5, M228, G65, G23, ML2, G25, H214, H79, H117 represent strains of Psa of different origin.
FIG. 6 is a test of the inhibitory effect of P.eastern lake Pseudomonas donghuensisHYBS-267 on Psa strain in vitro. In FIG. 6, H 2 O represents treating the leaf with only sterile water; treatment a represents permeation of only M228 bacterial liquid; treatment b shows that HYBS-267 bacteria liquid is permeated first, and after 12 hours, M228 bacteria liquid is permeated.
FIG. 7 is a test of the inhibitory effect of P.eastern lake Pseudomonas donghuensisHYBS-267 on Psa strain in vitro shoots. Treatment a represents inoculating only M228 bacterial liquid; treatment b represents inoculating HYBS-267 bacteria solution first, and inoculating M228 bacteria solution after 1 d.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
This example provides the isolation, purification and colony growth morphology of Pseudomonas eastern lake Pseudomonas donghuensisHYBS-267.
The Pseudomonas Donghu Pseudomonas donghuensisHYBS-267 is collected from healthy kiwi fruit branches of Mei County Chen Jiaoguang kiwi fruit plantation in Baoko city of Shaanxi at 2022, 11 months and 24 days, and the branches are collected and then stored in a plastic self-sealing bag.
Taking kiwi fruit branches, grinding, re-suspending with sterile water, taking suspension, diluting to different concentrations, uniformly coating on an LB solid culture medium flat plate, and setting 3 repeats for each concentration. The plates were placed in a constant temperature incubator at 28℃for 48 hours.
When single colony grows on the flat plate, single colony with different forms is picked up, inoculated to new LB solid culture medium and then cultured, and the process is repeated until a plurality of culture media with single colony grow are obtained.
Selecting thalli growing on a single colony culture medium, carrying out a plate counter experiment, repeating each single colony for 3 times, and screening out strains with obvious inhibition effect on kiwi fruit bacterial canker pathogenic bacteria, wherein the number of the strains is HYBS-267.HYBS-267 strain showed remarkable inhibitory effect on wild type M228 strain of Pseudomonas syringae pathogenic strain of Kiwi berry (Pseudomonas syringae, psa). The HYBS-267 cells were placed in a freezing tube (1:1) containing 50% glycerol and stored at-80 ℃.
The colony morphology of HYBS-267 strain on LB solid medium is shown in figure 1. As shown in FIG. 1, the colony of HYBS-267 strain is characterized by raised surface, irregular edge, pale yellow color and special smell on LB solid medium plate. The HYBS-267 strain is known to be gram negative bacteria by a gram staining method, and the observation result of an optical microscope is shown in figure 2.
Example 2
This example provides a taxonomic identification of Pseudomonas eastern lake Pseudomonas donghuensisHYBS-267.
The HYBS-267 strain DNA selected in example 1 was extracted according to the procedure of the bacterial genome total DNA extraction kit (Omega Bio-Tek D3350), PCR amplification was performed on 16S rDNA, and the amplified product was detected by 1.2% agarose gel electrophoresis to determine the target band. The gel was recovered according to the gel recovery kit (Omega Bio-Tek D2500) protocol and the gel recovery product was sequenced (Optimum Sonchi Co., ltd.). The nucleotide sequence of the 16S rDNA of HYBS-267 strain is shown as SEQ ID NO. 1.
And (3) BLAST comparison is carried out on the sequencing result in an NCBI database to obtain a 16S rDNA sequence of a plurality of strains with similar genetic relationship, the MEGA 11 software is used for carrying out multi-sequence comparison and trimming alignment, and then a phylogenetic tree (figure 3) is constructed based on an NJ method, so that the classification status of HYBS-267 strains is determined. As can be seen from FIG. 3, the HYBS-267 strain is classified as one with Pseudomonas eastern lake (Pseudomonas donghuensis) HYS, and thus is named as Pseudomonas eastern lake Pseudomonas donghuensisHYBS-267.
Example 3
The embodiment provides an in-dish inhibition effect test of the pseudomonas eastern lake Pseudomonas donghuensisHYBS-267 on kiwi fruit bacterial canker pathogenic bacteria M228.
In the embodiment, a plate counter method is adopted to test the inhibition effect of the eastern lake pseudomonas Pseudomonas donghuensisHYBS-267 on the kiwi fruit bacterial canker pathogenic bacteria M228.
Inoculating bacterial canker pathogen M228 of kiwi fruit with LB liquid culture medium, shaking culture at 28deg.C at 220rpm/min for 24 hr until OD 600 is about 1.0. Preparing LB culture medium containing 1% agar, cooling to about 45deg.C, adding the shake-cultured M228 strain solution at a volume ratio of 5% into the culture medium, mixing, and pouring into a bacteria-containing plate for use.
Streaking and activating Pseudomonas Donghai Pseudomonas donghuensisHYBS-267 on LB solid culture medium plate, taking bacterial cake with puncher after the activation culture, placing it on the above prepared bacteria-containing plate, placing 3 bacterial cakes on each plate, and culturing at 28deg.C for 48 hr. The culture results are shown in FIG. 4, and as can be seen in FIG. 4, a distinct zone of inhibition is produced around each cake. The diameter of each inhibition zone is measured by a crisscross method, and the average value of the measurement results is taken. The diameter of the inhibition zone is 22mm. From this, it is clear that Pseudomonas Donghuensis Pseudomonas donghuensisHYBS-267 has a significant inhibitory effect on the growth of M228.
Example 4
This example provides the inhibitory effect of Pseudomonas eastern lake Pseudomonas donghuensisHYBS-267 on Psa of different origin.
Number of test Psa in this example: m228, G65, G23, ML2, G25, H214, H79, H117 strains.
The test method was the same as in example 3. The experimental results are shown in FIG. 5. As can be seen from FIG. 5, the Psa strains from different sources show remarkable Psa inhibition effect after co-culturing with the Pseudomonas Donghuensis Pseudomonas donghuensisHYBS-267, which indicates that the Pseudomonas Donghuensis Pseudomonas donghuensisHYBS-267 has broad-spectrum anti-Psa activity and good biocontrol potential.
Example 5
The embodiment provides the inhibition effect of the Pseudomonas eastern lake Pseudomonas donghuensisHYBS-267 on M228 on the isolated kiwi fruit leaves.
The kiwi fruit leaves used in the embodiment are healthy leaves of kiwi fruits of the red sun variety. Fresh kiwi fruit leaves are washed with tap water to remove surface dust, soaked in sodium hypochlorite solution with the concentration of 6 per mill for 5min, and washed with sterile water for 3 times. Placing the washed kiwi fruit leaves on filter paper for airing; a leaf disk with the diameter of 16mm is prepared by a sterilized puncher, and the main vein is avoided during punching.
The test was performed using a vacuum infiltration atraumatic inoculation method. Vacuum infiltration conditions were 0.1MPa, 15s. After infiltration, the culture is washed for 3 times by using sterile water, and then is placed on a water agar culture medium plate prepared in advance, and is cultured for 3d at the temperature of 16 ℃ in a climatic incubator.
Test group and treatment mode of each group:
Blank group: infiltrating the leaf disc with sterile water;
Treatment a: only M228 bacterial liquid is permeated;
Treatment b: the HYBS-267 bacterial liquid is permeated first, and the M228 bacterial liquid is permeated after 12 hours.
HYBS-267 bacterial liquid preparation method: and (3) carrying out activation culture on the pseudomonas eastern lake Pseudomonas donghuensisHYBS-267 on an LB culture medium flat plate, picking a single colony after 24 hours, putting the single colony into a test tube filled with 5mL of LB liquid culture medium, shaking culture for 24 hours at 28 ℃ in a constant-temperature shaking incubator, and adjusting the OD 600 value to be=0.1 to obtain HYBS-267 bacterial liquid.
The preparation method of the M228 bacterial liquid comprises the following steps: and (3) performing activation culture on the M228 thalli on an LB culture medium flat plate, picking a single colony after 48 hours, putting the single colony into a test tube filled with 5mL of LB liquid culture medium, shaking culture for 24 hours at 28 ℃ in a constant-temperature shaking culture box, and adjusting the OD 600 value to be=0.1 to obtain the M228 bacterial liquid.
Photographing and recording the disease conditions of each group of leaf discs, measuring and calculating the disease spot area by using imageJ software, and recording the result. As shown in fig. 6, in vitro condition, the disease of the kiwi fruit leaf of the treatment a is heavier, the disease spot area of the treatment b is obviously smaller than that of the treatment a, and the inhibition effect is obvious. The embodiment shows that the Pseudomonas Donghuensis Pseudomonas donghuensisHYBS-267 has remarkable inhibition effect on the further expansion of Psa on leaves, and can be used for preventing and treating bacterial canker of kiwi fruits.
Example 6
The example provides the inhibitory effect of P.eastern lake Pseudomonas donghuensisHYBS-267 on M228 on isolated kiwi fruit branches.
The kiwi fruit branches used in the embodiment are healthy kiwi fruit branches of red sun variety. Collecting healthy branches of current-year-old kiwi fruits, shearing the healthy branches into proper lengths, cleaning the healthy branches with ddH 2 O for three times, sterilizing the healthy branches for 20 minutes in a dark place by using a 6 per mill sodium hypochlorite solution, flushing the healthy branches with sterile water for 3 times, airing the healthy branches, and sealing two ends of the branches with paraffin. A wound of about 2mm by 1mm was cut on the shoots with a sterile scalpel, and bacterial liquid was added dropwise to the wound, 5 shoot replicates were set, and the whole test was replicated 3 times. The inoculated branches are placed in a climatic incubator for culturing 40d at 16 ℃.
Test group and treatment mode of each group:
Treatment a: inoculating PsaM228,228 bacterial liquid only;
Treatment b: inoculating HYBS-267 bacteria liquid, and inoculating M228 bacteria liquid after 1 d.
HYBS-267 bacterial liquid preparation method: and (3) streaking and activating the pseudomonas eastern lake Pseudomonas donghuensisHYBS-267 on an LB culture medium plate for culturing, picking a single colony after 24 hours, putting the single colony into a test tube filled with 5mL of LB liquid culture medium, shaking and culturing for 24 hours at 28 ℃ in a constant-temperature shaking incubator, and adjusting the OD 600 value to be 0.3, thus obtaining HYBS-267 bacterial liquid.
The preparation method of the M228 bacterial liquid comprises the following steps: and (3) performing activation culture on the M228 thalli on an LB culture medium flat plate, picking a single colony after 48 hours, putting the single colony into a test tube filled with 5mL of LB liquid culture medium, shaking and culturing for 24 hours at 28 ℃ in a constant-temperature shaking incubator, and adjusting the OD 600 value to be=0.3 to obtain the M228 bacterial liquid.
Photographs were taken to record the onset of shoots from each test group, as shown in figure 7. As can be seen from fig. 7, the disease of the kiwi fruit branches treated with a is heavier under the condition of ex vivo, and the disease spot length of the kiwi fruit branches treated with b is obviously smaller than that of the kiwi fruit branches treated with a. The embodiment shows that the Pseudomonas Donghuensis Pseudomonas donghuensisHYBS-267 has remarkable inhibition effect on the further expansion of Psa on branches, and can be used for preventing and treating bacterial canker of kiwi fruits.
The embodiments described above are only some, but not all, embodiments of the invention. The detailed description of the embodiments of the invention is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments obtained without inventive effort by a person skilled in the art, which are related deductions and substitutions made by the person skilled in the art under the condition of the inventive concept, are within the scope of protection of the present invention.

Claims (7)

1. The Pseudomonas Donghu Pseudomonas donghuensis HYBS-267 is characterized in that the Pseudomonas Donghu Pseudomonas donghuensis HYBS-267 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC NO.29933.
2. The pseudomonas eastern lake Pseudomonas donghuensis HYBS-267 of claim 1 wherein the 16S rDNA nucleotide sequence of pseudomonas eastern lake Pseudomonas donghuensis HYBS-267 is shown in SEQ ID No. 1.
3. The pseudomonas eastern lake Pseudomonas donghuensis HYBS-267 of claim 1 or 2, wherein the pseudomonas eastern lake Pseudomonas donghuensis HYBS-267 has an inhibitory effect on pseudomonas syringae kiwi pathogenic varieties.
4. The use of pseudomonas eastern lake Pseudomonas donghuensis HYBS-267 in the control of kiwi fruit diseases as claimed in claim 1.
5. The use according to claim 4, wherein said pseudomonas eastern lake Pseudomonas donghuensis HYBS-267 is used for controlling bacterial canker in kiwi caused by a pseudomonas syringae kiwi pathogen.
6. A biocontrol microbial agent, wherein the active ingredient of the microbial agent comprises the bacterial cells of Pseudomonas eastern lake Pseudomonas donghuensis HYBS-267 and/or the fermentation broth thereof according to claim 1.
7. The biocontrol microbial agent of claim 6, wherein said biocontrol microbial agent is used for controlling bacterial canker of kiwi fruits.
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