CN118638698A - Pseudomonas radicaldarius and application thereof in preventing and treating kiwi fruit bacterial canker - Google Patents
Pseudomonas radicaldarius and application thereof in preventing and treating kiwi fruit bacterial canker Download PDFInfo
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Abstract
The invention belongs to the technical field of microorganisms, and discloses pseudomonas rhizophila and application thereof in preventing and treating bacterial canker of kiwi fruits. The invention provides a Pseudomonas radicals, named as Pseudomonas radicals Pseudomonas rhizophila Z, which is preserved in China general microbiological culture Collection center (CGMCC) No.31088. The supernatant, fermentation product, filtrate or extract of the pseudomonas radicalyx Pseudomonas rhizophila Z98 and/or the culture thereof provided by the invention has an inhibiting effect on a plurality of plant pathogenic bacteria such as pseudomonas syringae kiwi pathogenic varieties, pseudomonas syringae bean pathogenic varieties, pseudomonas syringae clove pathogenic varieties, pseudomonas syringae tomato pathogenic varieties, citrus canker, apple tree canker, tobacco red star germ, phytophthora capsici and the like.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and discloses pseudomonas rhizophila and application thereof in preventing and treating bacterial canker of kiwi fruits.
Background
Among plant diseases, fungi cause the greatest effect in crop yield reduction, followed by phytopathogenic bacteria and viruses. At present, the main control means for plant fungal diseases or plant bacterial diseases are still chemical control, but the chemical control has the defects of pesticide residues, environmental pollution, pathogen resistance and the like, and the application of the chemical control is gradually limited. In contrast to chemical control, the use of benign microorganisms as "biocontrol agents" to combat plant diseases caused by pathogens has been widely studied. Biological control is safe to the environment and can protect plants from being damaged. Biocontrol is therefore considered the most sustainable and viable pesticide alternative as part of the integrated management of pathogens. Biological control is paid more and more attention at present based on the biological control, and the bacterial strain with biological control potential is excavated and screened, so that the biological control has important research significance in preventing and controlling diseases.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention provides a pseudomonas rhizophila strain and application thereof in preventing and treating bacterial canker of kiwi fruits. Compared with the prior art, the invention has the advantages of providing the pseudomonas rhizophila which has broad-spectrum inhibition effect on plant fungal diseases or pathogenic bacteria of plant bacterial diseases, and is particularly suitable for preventing and treating bacterial canker of kiwi fruits.
In one aspect, the invention relates to a Pseudomonas radicaldarius strain with the following preservation information:
strain name: z98;
Classification naming: pseudomonas radicaldarius Pseudomonas rhizophila;
the time of collection of the preservation center: 2024, 06, 26;
preservation certificate issuance time: 2024, 07, 05;
preservation unit: china general microbiological culture Collection center (CGMCC);
Preservation number: CGMCC No.31088.
Address: the institute of microbiology, national academy of sciences, china, the area North Star, west way 1, 3, beijing, chaoyang.
Further, in the Pseudomonas radiata provided by the invention, the nucleotide sequence of the 16S rDNA of the Pseudomonas radiata is shown as SEQ ID NO:1, the gyrB nucleotide sequence of which is shown as SEQ ID NO: 2.
In another aspect, the invention relates to a biocontrol agent comprising a supernatant, fermentation product, filtrate or extract of said pseudomonas radicaldarius and/or a culture thereof.
The spores or pre-spores of pseudomonas radicalii Pseudomonas rhizophilaZ98, or any of its active variants provided herein, or combinations of hyphae, pre-spores, and/or spores, can be formulated into hyphae pastes, wettable powders, hyphae agglomerates, dust, granules, slurries, dry powders, aqueous or oily-based liquid products, and the like. Such formulations will comprise the pseudomonas radicaldarius Pseudomonas rhizophilaZ98 provided herein, or active variants thereof, and/or compositions derived therefrom, as well as carriers and other agents. The formulations may be used in a variety of methods as disclosed elsewhere herein.
The various compositions and formulations disclosed herein may comprise an amount of hyphae of pseudomonas radicalyx Pseudomonas rhizophilaZ98, or an active variant thereof, or spores or pre-spores, or a combination of hyphae, pre-spores, and/or spores; and/or may comprise an amount of a composition derived from pseudomonas radicalyx Pseudomonas rhizophilaZ98 or any of its active variants. Such amounts may include at least about 10 4 CFU/mL to about 10 11 CFU/mL, at least about 10 5 CFU/mL to about 10 11 CFU/mL, about 10 5 CFU/mL to about 10 10 CFU/mL, about 10 5 CFU/mL to about 10 12 CFU/mL, About 10 5 CFU/mL to about 10 6 CFU/mL, about 10 6 CFU/mL to about 10 7 CFU/mL, About 10 7 CFU/mL to about 10 8 CFU/mL, about 10 8 CFU/mL to about 10 9 CFU/mL, About 10 9 CFU/mL to about 10 10 CFU/mL, A strain concentration of about 10 10 CFU/mL to about 10 11 CFU/mL or about 10 11 CFU/mL to about 10 12 CFU/mL. In other embodiments, the strain concentration comprises at least about 10 4 CFU/mL, at least about 10 5 CFU/mL, at least about 10 6 CFU/mL, at least about 10 7 CFU/mL, At least about 10 8 CFU/mL, at least about 10 9 CFU/mL, at least about 10 10 CFU/mL, at least about 10 11 CFU/mL, At least about 10 12 CFU/mL. The strain may be formed in any desired formulation type at the concentrations described above, including, for example, in a liquid formulation, a wettable powder, a spray-dried formulation, a hyphal paste, wettable granules or a freeze-dried formulation.
As used herein, "supernatant" refers to the liquid that remains when pseudomonas radicalyx Pseudomonas rhizophilaZ98 is grown in liquid medium or harvested from solid medium into another liquid and removed by centrifugation, filtration, sedimentation, or other means known in the art. In some embodiments, the supernatant may be diluted with another substance, such as water, buffer, fresh medium, and/or formulation. The diluted supernatant is still considered to be the supernatant of the present invention.
As used herein, "filtrate" refers to the liquid from the fermentation culture of pseudomonas stutzeri Pseudomonas rhizophilaZ98 passing through the membrane via the liquid medium. The filtrate may comprise a concentrated amount of the active compound or metabolite compared to the concentration of the active compound or metabolite in the fermentation culture or supernatant.
As used herein, "extract" refers to a liquid material that is removed from the pseudomonas stutzeri Pseudomonas rhizophilaZ98,98 fermentation broth by a solvent (e.g., water, detergent, buffer, and/or organic solvent) and separated from pseudomonas stutzeri Pseudomonas rhizophilaZ98 by centrifugation, filtration, or other methods known in the art. The extract may comprise a concentrated amount of the active compound or metabolite compared to the concentration of the active compound or metabolite in the fermentation culture of pseudomonas rhizophila Pseudomonas rhizophilaZ prior to extraction. Alternatively, the filtrate or extract may then be diluted with another composition, such as water, buffer, fresh medium, and/or formulation. Such diluted filtrates or extracts are still considered to be the filtrates and extracts of the invention.
As used herein, "metabolite" or "metabolite" refers to a compound, substance, or by-product produced by fermentation of pseudomonas stutzeri Pseudomonas rhizophila Z98. An effective compound or metabolite is a compound that is present in the supernatant, a fermentation culture comprising pseudomonas stuffinis Pseudomonas rhizophila Z98, or pseudomonas stuffinis Pseudomonas rhizophila Z98, which, when applied in an effective amount to a target plant or the space in which the target plant is located, can improve any target agronomic trait of the plant, or control pathogenic bacteria causing fungal or bacterial plant disease.
In another aspect, the invention relates to the use of the Pseudomonas radicaldarius or the biocontrol agent in the control of plant fungal diseases or plant bacterial diseases.
Further, in the application provided by the invention, the host plant of the plant fungal disease or plant bacterial disease is one of kiwi, kidney bean, clove, tomato, citrus, apple, tobacco and capsicum.
Further, in the application provided by the present invention, the pathogenic bacteria of the plant fungal disease or plant bacterial disease is at least one of Pseudomonas syringae pathogenic variety Pseudomonas syringae actinidiae, pseudomonas syringae pathogenic variety Pseudomonas syringae Keisslar, pseudomonas syringae pathogenic variety Pseudomonas syringae, pseudomonas syringae pathogenic variety Pseudomonas syringae, pseudomonas canker, xanthomonas citri sp.
Further, in the application provided by the invention, the pseudomonas radicaldarius or the biocontrol agent is used for preventing and treating bacterial canker of kiwi fruits.
In another aspect, the present invention relates to a method for controlling a fungal disease or a bacterial disease of a plant, comprising: such that the Pseudomonas radiata or the biocontrol agent acts on the pathogenic bacteria of the plant fungal disease or plant bacterial disease and/or their habitat.
Further, in the method provided by the present invention, the Pseudomonas radiata or the biocontrol agent is applied to pathogenic bacteria having or at risk of developing a plant fungal disease or a plant bacterial disease.
Further, in the method provided by the invention, the pathogenic bacteria of the plant fungal disease or plant bacterial disease is at least one of Pseudomonas syringae pathogenic variety Pseudomonas syringae, actinidiae, pseudomonas syringae pathogenic variety Pseudomonas syringae, pseudomonas canker, xanthomonas citri, pseudomonas citri, apple tree rot pathogen VALSA MALI, alternaria tabacum ALTERNARIA ALTERNATE (Fries) Keisslar and Phytophthora capsici Phytophthora capsici.
Further, in the method provided by the invention, the pseudomonas radicaldarius or the biocontrol agent is used for preventing and treating bacterial canker of kiwi fruits.
Compared with the prior art, the technical scheme provided by the invention has at least the following beneficial effects or advantages.
The present invention provides a strain of Pseudomonas radiata Pseudomonas rhizophilaZ98, a strain or modified strain thereof, an active variant thereof and/or a composition derived therefrom, which can be used with any plant species or habitat thereof for the purpose of controlling plant fungal diseases or plant bacterial diseases. The above control of plant fungal diseases or plant bacterial diseases may be, but is not limited to: preventing infection of a plant by a pathogenic bacterium of a plant fungal disease or a plant bacterial disease, treating a plant that has been infected by a pathogenic bacterium of a plant fungal disease or a plant bacterial disease, controlling bacterial source formation in a plant, or pathogenic habitat of a plant fungal disease or a plant bacterial disease, controlling bacterial source formation in a risk area of a pathogenic bacterium that may produce a plant fungal disease or a plant bacterial disease.
The Pseudomonas radicals Pseudomonas rhizophilaZ, biocontrol agents or methods disclosed herein can be used to control one or more pathogenic bacteria of a plant fungal disease or plant bacterial disease, which may be, but are not limited to, a species selected from the group consisting of: the pathogenic bacteria are Pseudomonas syringae, actinidia chinensis pathogenic variety Pseudomonas syringae actinidiae Pseudomonas syringae pathogenic variety Pseudomonas pseudolaricina, pseudomonas syringae pathogenic variety Pseudomonas syringae, syringae Pseudomonas syringae tomato pathogenic variety pseudopseudomonadsyringaepv. Mat, citrus canker Xanthoscintitosubsp. Citri, apple tree rot pathogen VALSA MALI, alternaria tabacum ALTERNARIA ALTERNATE (Fries) Keisslar, phytophthora capsici Phytophthora capsici.
The invention discloses that the pseudomonas radicalyx Pseudomonas rhizophilaZ98,98 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
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a colony morphology of Pseudomonas radiata Pseudomonas rhizophilaZ.rhizogenes 98 on LB solid medium.
FIG. 2 is a diagram showing the morphology of Pseudomonas radiata Pseudomonas rhizophilaZ.radiata cells 98 under a scanning electron microscope and a transmission electron microscope (negative staining).
FIG. 3 is a phylogenetic tree of Pseudomonas radicals Pseudomonas rhizophilaZ98 constructed based on the Maximum likehood method.
FIG. 4 is a graph showing the results of the culture of Pseudomonas radiata Pseudomonas rhizophilaZ98 in a medium containing PsaM and other Psa strains, pseudomonas pathogenic varieties, leptosphaeria citri, leptosphaeria pomace, alternaria tabaci, phytophthora capsici.
FIG. 5 is a test of the inhibitory effect of Pseudomonas radiata Pseudomonas rhizophilaZ98 on Psa strain in vitro. In FIG. 5, M228-GFP represents that only M228 bacterial liquid was permeated; Z98+M228 represents that the Z98 bacterial liquid is permeated firstly, and the M228 bacterial liquid is permeated after 12 hours; M228+Z98 represents that the M228 bacterial liquid is permeated first, and the Z98 bacterial liquid is permeated after 12 hours; h 2 O represents treating the leaf with sterile water only.
FIG. 6 is a test of the inhibitory effect of Pseudomonas radicalophila Pseudomonas rhizophilaZ98 on Psa strain in vitro shoots. In FIG. 6, a represents inoculating only M228 bacteria solution; b represents that the Z98 bacterial liquid is inoculated firstly, and the M228 bacterial liquid is inoculated after 1 d; c represents that the M228 bacterial liquid is inoculated firstly, and the Z98 bacterial liquid is inoculated after 1 d.
FIG. 7 shows leaf changes of Pseudomonas radicalyx Pseudomonas rhizophilaZ.radicals at 0-25 days after inoculation of the kiwi leaves with P.radicals Pseudomonas rhizophilaZ.radicals.
Detailed Description
The following describes the technical aspects of the present invention with reference to examples, but the present invention is not limited to the following examples. The experimental methods and the detection methods in each embodiment are conventional methods unless otherwise specified; the reagents and materials can be purchased in the market unless specified otherwise, and the percentages and thousandths in the following examples are mass percentages and mass thousandths unless specified otherwise.
Example 1
This example provides the acquisition of Pseudomonas radicalophila Pseudomonas rhizophilaZ98 and its physiological properties.
Isolation of strains: pseudomonas radicalyx Pseudomonas rhizophilaZ98 is collected from the rhizosphere of Yang Lingou Liu Huangbao village kiwi fruits in Yangyang City of Shaanxi, and is stored in a plastic self-sealing bag after soil collection. 1 g rhizosphere soil samples are taken and placed into a sterilized 250mL triangular flask, and 99mL sterile water is added. 10min of the suspension A was obtained by shaking on a shaker at a rotation speed of 200 rpm/min and standing. Under aseptic conditions, 1mL of suspension A was mixed with 99mL of sterile water to obtain suspension B. mu.L of suspension B was aspirated and coated on LB plates. 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 a strain with obvious inhibition effect on kiwi fruit bacterial canker pathogenic bacteria, wherein the number of the strain is Z98, which is hereinafter referred to as Z98 strain. The Z98 strain shows remarkable inhibition effect on a wild type M228 strain of pseudomonas syringae actinidia pathogenic variety (Pseudomonas syringae, psa). The Z98 strain was placed in a freezer containing 50% glycerol (1:1) and stored at 80 ℃.
Colony morphology: the colony morphology of the Z98 strain on LB solid medium is shown in FIG. 1. As shown in FIG. 1, the colony of the Z98 strain is shown on an LB solid medium plate as a colony with a diameter of 1.5-2.2 mm, and is round, convex, and glossy in surface, and is semitransparent, irregular in edge, dark yellow and casein. As shown in FIG. 2, the results of the scanning electron microscope and the transmission electron microscope show that the strain Z98 has a short rod shape and has flagella at both ends.
Example 2
This example provides a taxonomic identification of Pseudomonas radicalyx Pseudomonas rhizophilaZ.radicals 98.
The Z98 strain DNA selected in example 1 was extracted according to the procedure of the bacterial genome total DNA extraction kit (Omega Bio-Tek D3350), the 16S rDNA and gyrB genes were PCR amplified, and the amplified products were 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 the Z98 strain is shown as SEQ ID NO:1, the gyrB nucleotide sequence of which is shown as SEQ ID NO: 2.
BLAST comparison is carried out on the sequencing result in NCBI database to obtain a 16S rDNA sequence and a gyrB sequence of a plurality of strains with similar genetic relationship, genes are combined through PhyloSuite software, then MEGA X software is used for carrying out multi-sequence comparison and trimming alignment, and a phylogenetic tree is constructed based on Maximum likehood method (figure 3), so that the classification status of the Z98 strain is determined. As can be seen from FIG. 3, the Z98 strain was classified as Pseudomonas radicals (Pseudomonas rhizophila) and was designated as Pseudomonas radicals Pseudomonas rhizophilaZ98.
Example 3
This example provides an inhibition test of Pseudomonas radicalyx Pseudomonas rhizophilaZ.radicals 3838 against a variety of plant pathogenic bacteria.
Taking in-dish inhibition effect test of Z98 strain on kiwi fruit bacterial canker pathogen M228 (Pseudomonas syringae mutant Pseudomonas syringae actinidiae) as an example. Preparation of LB liquid Medium: mixing tryptone 10g, yeast powder 5g, naCl 10g, and distilled water 1L, sterilizing in high pressure steam sterilizing pot (121deg.C, 20 min), and sterilizing to obtain LB liquid culture medium. Preparing M228 bacterial liquid: and (3) streaking and activating the M228 on the LB plate, picking an M228 single colony, inoculating the M228 single colony to a sterilized LB liquid culture medium, and shaking and culturing at 28 ℃ until OD 600 = 0.1 to obtain an M228 bacterial liquid. Preparing a bacteria-containing plate: after 50-fold dilution of M228 with OD600 ≡ 0.1, the plates were plated with 5mL M228, spread and left to stand for 3 minutes. Preparing pseudomonas radicalyx Pseudomonas rhizophilaZ and 98 bacterial liquid: and streaking and activating Pseudomonas radiata Pseudomonas rhizophilaZ to 98 on an LB plate, and shaking and culturing at 28 ℃ until OD 600 =0.1 to obtain a Z98 bacterial liquid. And 5 mu L of the bacterial liquid is sucked and dripped into the center of the prepared bacteria-containing flat plate, the flat plate which is only inoculated with pathogenic bacteria M228 is used as a control, the culture is repeated for 3 times at 28 ℃ for 2d, and the diameter of a bacteriostasis ring is measured by a crisscross method.
The experimental results are shown in fig. 4, and the clear transparent inhibition zone appears in the center of the flat plate in fig. 4. The diameter of the inhibition zone is 19 mm, which shows that the pseudomonas radicalyx Pseudomonas rhizophilaZ has remarkable antagonism on kiwi fruit bacterial canker pathogenic bacteria. Meanwhile, the Pseudomonas radiata Pseudomonas rhizophilaZ98 has obvious inhibition effect on Pseudomonas radiata bean pathogenic variety Pseudomonas pseudolaricola 1448A (Paph 1448A), pseudomonas syringae clove pathogenic variety Pseudomonas syringae B728A (Pseudomonas B728A), pseudomonas syringae tomato pathogenic variety Pseudomonas pseudolaris D.matoDC3000 (Pst DC 3000), pseudomonas citri Xanthomonas sp (Xcc), apple tree rot germ VALSA MALI-8, alternaria tabacum ALTERNARIA ALTERNATE (Fries) Keisslar, phytophthora capsici Phytophthora capsici and the like.
The supernatant, fermentation product, filtrate or extract of the pseudomonas radicalyx Pseudomonas rhizophilaZ98 and/or the culture thereof has broad-spectrum inhibition effect on various plant pathogenic bacteria (bacteria or fungi) and can be used for preventing and controlling plant fungal diseases or bacterial diseases of various host plants such as kiwi fruits, beans, clove, tomatoes, oranges, apples, tobaccos, peppers and the like. The following examples are further presented with kiwi fruit, kiwi fruit bacterial canker pathogen M228 (Pseudomonas syringae kiwi fruit pathogenic variety Pseudomonas syringae. Actinidiae) and kiwi fruit bacterial canker.
Example 4
The present example provides the inhibition effect of the pseudomonas radicalyx Pseudomonas rhizophilaZ98 on M228 on the in vitro kiwi fruit leaf.
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;
M228-GFP: only M228 bacterial liquid is permeated;
z98+ M228: the Z98 bacterial liquid is permeated first, and the M228 bacterial liquid is permeated after 12 hours.
M228+z98: firstly, the M228 bacterial liquid is permeated, and then the bacterial liquid is permeated after 12 hours.
The preparation method of the Z98 bacterial liquid comprises the following steps: and (3) carrying out activation culture on the pseudomonas radicalyx Pseudomonas rhizophilaZ98 on an LB culture medium flat plate for 24 hours, picking a single colony, placing 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 Z98 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 the test results in FIG. 6, under the in vitro condition, the occurrence of the kiwi fruit leaves treated with M228-GFP is heavier, the disease spot area treated with Z98+M228 is obviously smaller than that treated with M228-GFP, and the disease spot area treated with M228+Z98 is smaller than that treated with M228-GFP but larger than that treated with Z98+M228, which indicates that the Z98 strain has remarkable prevention effect on kiwi fruit canker and is better than treatment effect. The embodiment shows that the pseudomonas radicalyx Pseudomonas rhizophilaZ98 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 5
The present example provides the inhibition effect of the pseudomonas radicalyx Pseudomonas rhizophilaZ.radicalyx 98 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: only M228 bacterial liquid is permeated;
treatment b: the Z98 bacterial liquid is permeated first, and the M228 bacterial liquid is permeated after 12 hours.
Treatment c: firstly, the M228 bacterial liquid is permeated, and after 12 hours, the Z98 bacterial liquid is permeated.
The preparation method of the Z98 bacterial liquid comprises the following steps: and (3) streaking and activating the pseudomonas radicalyx Pseudomonas rhizophilaZ98 on an LB culture medium plate for 24 hours, picking a single colony, placing the single colony into a test tube filled with 5mL of LB liquid culture medium, shaking and culturing the single colony in a constant-temperature shaking incubator at 28 ℃ for 24 hours, and adjusting the OD 600 value to be 0.3 to obtain the Z98 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 6. As can be seen from fig. 6, under the condition of ex vivo, the disease of the kiwi fruit branches treated with a is heavier, 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, and the disease spot length of the kiwi fruit branches treated with c is obviously smaller than that of the kiwi fruit branches treated with a and slightly larger than that of the kiwi fruit branches treated with b. The embodiment shows that the pseudomonas radicalyx Pseudomonas rhizophilaZ98 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.
Example 6
The embodiment provides a leaf variation condition of the pseudomonas radicalyx Pseudomonas rhizophilaZ.radicalyx 98 after inoculating on kiwi fruit leaves for 0-25 days.
The Z98 bacterial liquid (the concentration is 1X 10 5 CFU/mL) is quantitatively sprayed to the sterilized 3 month tissue culture Miao Shemian (10 mL standard watering cans on the front and back sides of the leaves respectively with 5 pumps), and the plants are cultivated in a 25 ℃ incubator by taking water treatment as a control. Health status recordings were made on discs with diameters of 1 cm at random, respectively, 3d, 5 d, 11 d, 15 d, 17 d, 21 d, 25 d. This example shows that Pseudomonas radicalophila Pseudomonas rhizophilaZ.radicals 98 did not affect healthy growth of plants within 25 days after inoculation.
As described above, the basic principles, main features and advantages of the present invention are better described. The above examples and descriptions are merely illustrative of preferred embodiments of the present invention, and the present invention is not limited to the above examples, and various changes and modifications to the technical solution of the present invention by those skilled in the art should fall within the scope of protection defined by the present invention without departing from the spirit and scope of the present invention.
Claims (8)
1. The Pseudomonas radicals is named as Pseudomonas radicals Pseudomonas rhizophila Z to 98 and is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.31088.
2. A biocontrol agent comprising the supernatant, fermentation product, filtrate or extract of pseudomonas radicaltrop and/or a culture thereof of claim 1.
3. The use of the biocontrol agent of pseudomonas stutzeri according to claim 1 or claim 2 for controlling plant fungal diseases or plant bacterial diseases, wherein the pathogenic bacteria of plant fungal diseases or plant bacterial diseases are at least one of pseudomonas syringae kiwi pathogenic variety Pseudomonas syringae pv. actinidiae, pseudomonas syringae bean pathogenic variety Pseudomonassyringae pv. phaseolicola, pseudomonas syringae clove pathogenic variety Pseudomonassyringae pv. syringae, pseudomonas syringae tomato pathogenic variety Pseudomonassyringae pv. totaro, citrus canker Xanthomonascitri subsp.
4. The use according to claim 3, wherein the host plant of the plant fungal disease or plant bacterial disease is one of kiwi, kidney bean, clove, tomato, citrus, apple, tobacco, capsicum.
5. The use according to claim 3, characterized in that the pseudomonas radicalis or the biocontrol agent is used for the control of bacterial canker in kiwi fruits.
6. A method for controlling a fungal or bacterial plant disease, comprising: allowing the pseudomonas radicaldarius of claim 1 or the biocontrol agent of claim 2 to act on pathogenic bacteria of said plant fungal disease or plant bacterial disease and/or their habitat;
The pathogenic bacteria of the plant fungal disease or plant bacterial disease are at least one of pseudomonas syringae kiwi pathogenic variety Pseudomonas syringae pv. actinidiae, pseudomonas syringae bean pathogenic variety Pseudomonassyringae pv. phaseolicola, pseudomonas syringae clove pathogenic variety Pseudomonassyringae pv. syringae, pseudomonas syringae tomato pathogenic variety pseudomonadsyringaepv, kmato, citrus canker Xanthomonascitri subsp.
7. The method of claim 6, wherein the pseudomonas rhizophila or the biocontrol agent is applied to a pathogen having or at risk of developing a plant fungal disease or a plant bacterial disease.
8. The method according to claim 6, wherein the pseudomonas radicaldarius or the biocontrol agent is used for the control of bacterial canker in kiwi fruits.
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