CN117210374B

CN117210374B - Acinetobacter petechiae T-150 for preventing and treating pine wood nematode disease and application thereof

Info

Publication number: CN117210374B
Application number: CN202311324382.9A
Authority: CN
Inventors: 牛犇; 韩洪蓥; 王浩宇; 吴迪; 张丽霞; 汪彦宏; 王传珍; 王�琦; 王思佳; 赵宇
Original assignee: Northeast Forestry University
Current assignee: Northeast Forestry University
Priority date: 2023-10-12
Filing date: 2023-10-12
Publication date: 2024-05-31
Anticipated expiration: 2043-10-12
Also published as: CN117210374A

Abstract

The invention relates to the technical field of agricultural microorganisms, in particular to a Pittrium T-150 for preventing and treating pine wood nematode diseases and application thereof. The Acinetobacter (Acinetobacter pittii) T-150 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.27755. The strain has high activity of inhibiting and killing pine wood nematodes, and when the fermentation liquor is OD ₆₀₀ =1.5 (the bacterial content is 3.1× ⁸CFU·mL^‑1), the mortality rate of the fermentation liquor to the pine wood nematodes reaches more than 90%, and the disease index of pine tree seedlings infected with the pine wood nematodes can be effectively reduced.

Description

Acinetobacter petechiae T-150 for preventing and treating pine wood nematode disease and application thereof

Technical Field

The invention relates to the technical field of agricultural microorganisms, in particular to a Pittrium T-150 for preventing and treating pine wood nematode diseases and application thereof.

Background

Pine wood nematode disease is also called pine wilt disease, and is a destructive forest disease. Since the disease is transferred into China in 1982, the disease spreads rapidly, 17 provinces (city and district) are generated in China at present, a large number of pine trees die, and serious damage is caused to pine forest resources, natural landscapes and ecological environment of China. The disease is caused by pine wood nematodes belonging to the genus Aphannaceae, the order Aphanales, the family Aphannaceae, the genus Aphanothece, is one of important worldwide plant pathogens, is also a main pathogen of pine wilt, belongs to a major foreign invasive species in China, and has been listed as an object of forest plant quarantine for the interior and exterior. In China, monochamus alternatus is a main vector insect of the pine wood nematodes.

In summer, pine wood nematodes spread throughout the tree and propagate very rapidly. When the vascular bundle cells of pine are destroyed by pine wood nematodes, the water transport is terminated, the resin flow is slowed down and finally stopped, thereby causing pine wilt and causing pine death. Pine wood nematode hosts are wide, and Chinese hosts mainly comprise Pinus plants such as Pinus koraiensis (Pinus densiflora), pinus koraiensis (Pinus thunbergii), pinus massoniana (Pinus massoniana), pinus koraiensis (Pinus ponderosa), pinus koraiensis (Pinus pinaster), pinus taeda (Pinus taeda), pinus koraiensis (Pinus elliottii), pinus koraiensis (Pinus luchuensis) and Pinus alba (Pinus bungeana).

The prevention and treatment measures of pine wilt mainly comprise physical prevention and treatment, chemical prevention and treatment and biological prevention and treatment. Phytophthora root-mean-square is a main strategy for physical control, and the strategy is simple and effective, but requires a large amount of manpower and material resources, and has high control cost. The common chemical prevention and treatment method comprises trunk injection and flying prevention, can effectively reduce the incidence rate of pine wilt, and can cause environmental pollution. Biological control has gained increasing attention as an environmentally friendly, efficient control measure. The adoption of biocontrol bacteria to block penetration of pine wood nematodes into tree bodies has proved to be an effective prevention and control means. The biocontrol bacteria with the nematicidal activity mainly originate from bacillus, streptomyces, pseudomonas and the like. Biocontrol bacteria kill nematodes by producing toxins, competing for nutrition, digesting body surface tissues of nematodes, interfering with modes of nematode behavior, mobilizing fungi to predate nematodes, and the like. Bacillus thuringiensis (Bacillus thuringiensis) (Bt) produces a variety of insecticidal chaperone crystal protein toxins, wherein the beta-and delta-endotoxins have nematode killing functions; extracellular proteases secreted by Bacillus laterosporus (Brevibacillus laterosporus) and Pseudomonas fluorescens (Pseudomonas fluorescens) and the like can degrade the body surface of nematodes and form pores, and the enzymes can continuously degrade inwards from the epidermal cells and digest internal tissues.

The biocontrol bacteria have high activity of inhibiting and killing pine wood nematodes, and the application of the biocontrol bacteria preparation for preventing and controlling pine tree wilt disease can effectively reduce the occurrence of the disease, does not pollute the environment, is not easy to produce drug-resistant pathogens, and is an important way for realizing green prevention and control of pine wood nematode diseases. The development and application of biocontrol microbial agents against pine wood nematode disease have attracted considerable attention by the excavation of biocontrol bacterial species having high-efficiency biocidal activity against pine wood nematodes.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide a Pittrium T-150 for preventing and treating pine wood nematode diseases and application thereof. The Pitts acinetobacter T-150 is separated from pine tissues, has stable and efficient activity of inhibiting and killing pine wood nematodes, has a mortality rate of more than 90 percent on the pine wood nematodes, and can effectively reduce the disease index of pine tree seedlings with pine wood nematode diseases.

In order to achieve the above purpose, the technical scheme provided by the invention is as follows:

One aspect of the invention relates to a Pitts Acinetobacter (Acinetobacter pittii) T-150 which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 27755.

The Pitts acinetobacter T-150 is separated from pine, has higher mortality rate to pine wood nematodes and can reach more than 90 percent.

The 16S rDNA sequence of the Acinetobacter petite T-150 is shown as SEQ ID No. 1.

The Acinetobacter petechiae T-150 is inoculated on an LB solid culture medium for culture, and after the culture is carried out for 24 hours at a constant temperature of 30 ℃, a bacterial colony which is nearly round, milky white and has a central microprotrusion, smooth and moist surface, luster and tidy edge is formed. As a result of observation under an optical microscope, T-150 cells were found to be short-rod-shaped and the gram stain was pink, which was a gram-negative bacterium.

The Pitts acinetobacter T-150 can generate obvious hydrolysis circles on starch and fat solid culture media, so that the strain T-150 can secrete amylase and lipase, reaction tests of hydrogen sulfide and V.P of the strain T-150 are negative, and test of methyl red and contact enzyme are positive.

Another aspect of the invention relates to a microbial formulation comprising: at least one of the Pittrium T-150, a supernatant of a culture solution of the Pittrium T-150, a fermentation product of the Pittrium T-150 or a metabolite of the Pittrium T-150.

The microbial preparation provided by the invention has the capability of killing pine wood nematodes, and can be used for preventing and treating pine wood nematode diseases.

Preferably, the microbial agent comprises a solid microbial agent or a liquid microbial agent.

Preferably, when the microbial preparation is a solid microbial preparation, the number of viable bacteria of the Pitts Acinetobacter T-150 is 1.1X10- ⁹CFU·g^-1 or more.

Preferably, when the microbial preparation is a liquid microbial preparation, the number of viable bacteria of the Pitts Acinetobacter T-150 is 1.1X10 ⁸～4.0×10⁸CFU·mL^-1.

Preferably, when the microbial agent is a solid microbial agent, the microbial agent may comprise a solid carrier including, but not limited to, one or more of talc, lignite, montmorillonite, alginate, silica, quartz powder, vermiculite, kaolin, light calcium carbonate, diatomaceous earth. Optionally, the solid microbial inoculum further comprises one or more of an antioxidant, a binder and a water-retaining agent; such antioxidants include, but are not limited to, one or more of vitamin C, vitamin E, and citric acid, for example; the binders include, but are not limited to, maltose and/or dextrin; the water-retaining agent comprises at least one of water-absorbent resin and mannitol, but is not limited to the water-absorbent resin and the mannitol.

Preferably, the microbial formulation further comprises an adjuvant.

Preferably, the auxiliary agent comprises: at least one of a dispersant, a stabilizer, a disintegrant, a solvent, or a wetting agent. In a specific embodiment, the adjuvant comprises one or more of trehalose, glycerol, buffer salts, yeast extract, white carbon black, sodium chloride, amino acids, and the like.

Preferably, the microbial formulation further comprises an agriculturally effective amount of a fertilizer and/or pesticide.

Preferably, the microbial agent is in the form of wettable powder, water dispersible granules, water suspension or dispersible oil suspension.

Another aspect of the invention relates to a method for controlling pine wood nematode disease, said Pitts Acinetobacter T-150 or said microbial preparation being administered to a subject.

The method can effectively prevent and treat pine wood nematode disease.

Another aspect of the invention relates to the use of said Pittrium T-150 or said microbial preparation for controlling plant diseases.

Preferably, the plant disease comprises pine wood nematode disease caused by pine wood nematodes.

Preferably, the pine tree comprises: one or more of black pine, pinus sylvestris, pinus koraiensis, larch, and pinus massoniana; preferably pinus sylvestris or pinus sylvestris.

Biological sample preservation information: acinetobacter petri (Acinetobacter pittii) T-150, which is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of 27755 in 2023 and 06 months and 30 days. Preservation address: the institute of microbiology, academy of sciences, china, no. 3, north Star West way 1, the Korean area of Beijing, china, postal code 100101.

Compared with the prior art, the invention has the beneficial effects that:

(1) The Pitts acinetobacter (Acinetobacter pittii) T-150 provided by the invention is separated from pine tissues, has high-efficiency activity of inhibiting and killing pine wood nematodes, has a mortality rate of more than 90% on the pine wood nematodes when the fermentation liquor is OD ₆₀₀ =1.5 (the bacterial content is 3.1×10 ⁸CFU·mL^-1), and can effectively reduce the disease index of pine wood nematode-infected pine tree seedlings.

(2) The microbial preparation provided by the invention comprises thalli of the strain T-150, culture solution supernatant thereof, fermentation products thereof or metabolites thereof, and can be compounded with common fertilizers or pesticides for preventing and treating plant diseases, in particular pine wood nematode diseases.

(3) The method for preventing and treating pine wood nematode disease provided by the invention is simple and convenient to operate, has good preventing and treating effect, and only needs to apply the Pitei Acinetobacter T-150 or the microbial preparation to a prevention object.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following description will briefly explain the drawings needed in the embodiments or the prior art description, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 shows colony morphology of Acinetobacter picoteus T-150 (scale: 2 mm);

FIG. 2 shows the result of gram staining of Acinetobacter picoteus T-150 (scale: 10 μm);

FIG. 3 shows the carbon source utilization of Acinetobacter Petertiaryanus T-150, and is prepared by: the darker the color, the stronger the strain's ability to utilize this carbon source;

FIG. 4 shows the result of staining with methylene blue solution of P.pityriasis T-150 fermentation broth, aqueous dilution of fermentation broth and supernatant of fermentation broth;

FIG. 5 is a 4,6 diamino 2 phenylindole (DAPI) staining result (scale: 200 μm) of P.pityriasis T-150 treated pine wood nematodes;

FIG. 6 shows the nematicidal activity of Acinetobacter picoteatci T-150 fermentation broth, aqueous dilutions of the fermentation broth and supernatants of the fermentation broth;

FIG. 7 is a graph showing the effect of concentration of Acinetobacter pituitosum T-150 fermentation broth and treatment time on its pine wood nematode killing activity;

FIG. 8 shows the result of a greenhouse disease prevention experiment for pine seedlings of Pinus sylvestris (a is a pine wood nematode treatment group, b is a water treatment group, c is a Pitts Acinetobacter T-150 fermentation broth and pine wood nematode treatment group, and the scale is 4 cm);

FIG. 9 shows disease index of greenhouse disease prevention experiment of pinus sylvestris seedlings;

FIG. 10 shows the disease control effect of Acinetobacter petite T-150 on pine wilt prevention in greenhouse.

Detailed Description

The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and detailed description, but it will be understood by those skilled in the art that the examples described below are some, but not all, examples of the present invention, and are intended to be illustrative of the present invention only and should not be construed as limiting the scope of the present invention. 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. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example 1 identification of Strain T-150

(1) 16S rRNA gene sequencing analysis of Strain T-150

Genomic DNA of strain T-150 was extracted and used as a template for PCR amplification of the 16S rRNA gene fragment using universal primers 27F and 1492R (27F: 5'-AGAGTTTGATCMTGGCTCAG-3'; 142R: 5 '-TACGGYTACCTTGTTACGACTT-3'). PCR reaction system: 1. Mu.L of template DNA, 1. Mu.L of primer 27F, 1. Mu.L of primer 1499R, 12.5. Mu.L of 2 XTaq Mixture, 9.5. Mu.L of ddH ₂ O, and the PCR reaction procedure is: pre-denaturation at 98℃for 5min, denaturation at 98℃for 30s, annealing at 55℃for 30s, extension at 72℃for 2min,30 cycles, 72℃for 5min. The amplified product is purified and subjected to sequencing analysis after 1% agarose gel electrophoresis detection, and the sequencing result is shown as SEQ ID No. 1.

SEQ ID No.1：

CATGCAGTCGAGCGGAGAGAGGTAGCTTGCTACTGATCTTAGCGGCGGACGGGTGAGTAATGCTTAGGAATCTGCCTATTAGTGGGGGACAACATTTCGAAAGGAATGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGATCTTCGGACCTTGCGCTAATAGATGAGCCTAAGTCGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCTGTAGCGGGTCTGAGAGGATGATCCGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGCCTTATGGTTGTAAAGCACTTTAAGCGAGGAGGAGGCTACTTTAGTTAATACCTAGAGATAGTGGACGTTACTCGCAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGATTTACTGGGCGTAAAGCGCGCGTAGGCGGCTAATTAAGTCAAATGTGAAATCCCCGAGCTTAACTTGGGAATTGCATTCGATACTGGTTAGCTAGAGTGTGGGAGAGGATGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGATGGCGAAGGCAGCCATCTGGCCTAACACTGACGCTGAGGTGCGAAAGCATGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGTCTACTAGCCGTTGGGGCCTTTGAGGCTTTAGTGGCGCAGCTAACGCGATAAGTAGACCGCCTGGGGAGTACGGTCGCAAGACTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGCCTTGACATAGTAAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTTACATACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTTTCCTTATTTGCCAGCGAGTAATGTCGGGAACTTTAAGGATACTGCCAGTGACAAACTGGAGGAAGGCGGGGACGACGTCAAGTCATCATGGCCCTTACGGCCAGGGCTACACACGTGCTACAATGGTCGGTACAAAGGGTTGCTACCTAGCGATAGGATGCTAATCTCAAAAAGCCGATCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCGGATCAGAATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTTGTGCACCAGAAGTAGCTAGCCTAACTGCAAAGAGGGCGGTACC.

The sequence obtained was subjected to BLAST search in the National Center for Biological Information (NCBI), and the sequence similarity with the 16S rRNA gene fragment of strain T-150 was Acinetobacter pittii, with homology as high as 99.93%, whereby strain T-150 could be identified as Acinetobacter pituitum, designated as Acinetobacter pituitum (Acinetobacter pittii) T-150.

(2) Morphological observations of Strain T-150

After the strain T-150 is inoculated in LB solid medium and cultured for 24 hours at a constant temperature of 30 ℃, nearly round, milky white, central microprotrusions can be observed, and colonies with smooth, moist, glossy and tidy edges can be observed (see figure 1). As a result of microscopic observation, the strain T-150 was in the form of short rods.

(3) Physiological biochemical response test of Strain T-150

The physiological and biochemical reaction test of the strain T-150 is mainly described in a common bacterial System identification handbook, wherein gram staining of the strain is pink and gram negative (see figure 2); strain T-150 is capable of producing distinct hydrolysis circles in starch and fat media, so that it is capable of secreting amylase and lipase; the hydrogen sulfide and V.P reaction test results of the strain T-150 are negative, the methyl red and contact enzyme test results are positive, and the test results are shown in table 1.

TABLE 1 results of physiological and biochemical characterization of Strain T-150

(4) Analysis of carbon Source utilization by Strain T-150

A small number of colonies of strain T-150 were picked with a gun head, placed in a sterile 1 XPBS solution and shaken uniformly, OD ₅₉₀ was adjusted to 0.1, then 150. Mu.L of the bacterial suspension was added to each well of the Biolog-ECO plate, and incubated at 30℃for 72 hours, and OD ₅₉₀ was measured every 24 hours, which revealed that strain T-150 could utilize 13 carbon sources including L-arginine, methyl pyruvate, D-xylose, L-asparagine, tween 40, L-phenylalanine, tween 80, 4-hydroxybenzoic acid, L-serine, gamma-hydroxybutyric acid, alpha-butanoic acid, D-malic acid and putrescine (see FIG. 3).

The strain T-150 can be identified as Acinetobacter petite by combining the results of 16S rRNA gene sequencing analysis, morphological observation, physiological and biochemical reaction test and carbon source utilization analysis. The strain is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No.27755.

EXAMPLE 2 determination of Acinetobacter piti T-150 pine wood nematode killing Activity

Preparation of pine wood nematode suspension:

Adding sterile water into a botrytis cinerea flat plate growing with pine wood nematodes until fungus hyphae are gone out, standing for 10min, continuously sucking and spitting suspension liquid by using a 5mL pipetting gun, suspending the pine wood nematodes in the sterile water, transferring the pine wood nematodes suspension liquid to a 15mL centrifuge tube, standing for 15-20 min, removing the supernatant, retaining about 1.5mL of pine wood nematodes suspension liquid, adding 400 mu L of streptomycin sulfate (5 g/L), fixing the volume to 10mL by using the sterile water, standing for 15-20 min after gently shaking, removing the supernatant, retaining about 1.5mL of nematodes suspension liquid, adding sterile water to wash streptomycin sulfate on the surface of the pine wood nematodes for many times, retaining about 1.5mL of pine wood nematodes suspension liquid, sucking 20 mu L of the pine wood nematodes suspension liquid after shaking and mixing, investigating the quantity of pine wood nematodes under a body microscope, and adjusting the concentration to 4-6 heads/mu L.

Preparation of Strain T-150:

a. Preparation of Strain T-150 fermentation broth: activating the Pitts Acinetobacter strain T-150 stored in a-80 ℃ ultralow temperature refrigerator on a Lysozyme Broth (LB) solid culture medium by a three-zone streaking method, culturing for 24 hours in an inverted mode in a 30 ℃ constant temperature incubator, picking single colonies growing on an LB plate, inoculating the single colonies into a Brain Heart Infusion (BHI) liquid culture medium, placing the single colonies in a shaking table at 30 ℃ and 200rpm, and culturing for 24 hours in a shaking table. The bacterial suspension concentration was adjusted to OD ₆₀₀ = 1.5 with BHI broth.

B. preparation of strain T-150 fermentation broth supernatant: centrifuging the strain T-150 fermentation liquor at 13000rpm for 3min to obtain supernatant which is the strain T-150 fermentation liquor supernatant.

C. Preparation of an aqueous dilution of the fermentation broth of strain T-150: the Pitts Acinetobacter strain T-150 stored in an ultralow temperature refrigerator at the temperature of minus 80 ℃ is activated on an LB solid culture medium by a three-zone streaking method, the incubator at the constant temperature of 30 ℃ is used for inverted culture for 24 hours, single bacterial colonies growing on an LB flat plate are picked up and inoculated into a BHI liquid culture medium, and the BHI liquid culture medium is placed in a shaking table at the temperature of 30 ℃ and at the speed of 200rpm and shake-cultured for 24 hours. The concentration of the bacterial suspension was adjusted to OD ₆₀₀ = 1.5 with sterile water.

And (3) detecting the activity of killing pine wood nematodes:

taking 96-well plates, adding 40 mu L of a sample to be tested of the strain T-150 (comprising fermentation liquor, fermentation liquor supernatant or fermentation liquor water diluent) and 20 mu L of pine wood nematode suspension into each well, setting four repetitions of each sample, culturing for 24 hours in a constant temperature incubator at 25 ℃, adding 200 mu L of sterile water into each well to wash the pine wood nematodes, repeating the steps for three times, adding 4 mu L of methylene blue solution or 4, 6-diamino-2-phenylindole (DAPI) dye, dyeing for 4 hours, washing the dye with 200 mu L of sterile water, observing the nematode morphology under a bulk microscope or a laser confocal microscope, dying the nematode bodies to be stiff and blue (see fig. 4 and 5), curling the bodies of the living nematodes without dyeing, and calculating the corrected mortality of the pine wood nematodes according to the following formula, wherein the result is shown in fig. 6.

The result shows that the fermentation liquor of the Pitezobacter T-150 has stable high nematicidal activity, and the average corrected mortality rate of the pine wood nematodes is 88% after 24 hours of co-culture; the supernatant and the water dilution of the fermentation broth of the strain T-150 also show a certain activity of killing pine wood nematodes, but the activity of killing lines is obviously weaker than that of the fermentation broth, and after 24 hours of co-culture, the average corrected mortality rate of the pine wood nematodes is 75.8% and 61.2%, respectively (see figure 6).

Different broth concentrations (OD ₆₀₀, 1.0, 1.5 and 2.0 respectively) and times of co-cultivation with pine wood nematodes (12 h, 24h, 36h and 48 h) were set to determine optimal conditions for P.pituitari T-150 to kill pine wood nematodes. The results show that the strain T-150 fermentation broth with OD ₆₀₀ of 1.5 (the bacterial content of 3.1X10- ⁸CFU·mL^-1) is co-cultured with the pine wood nematodes for 24 hours, and the corrected death rate of the pine wood nematodes reaches 92.8% (see FIG. 7), which can be determined as the optimal condition for killing the pine wood nematodes by the strain T-150.

Example 3 greenhouse disease prevention experiment of Pinus sylvestris seedlings

Soaking the pinus sylvestris seeds in 5% potassium permanganate solution for 1h, and sterilizing the surfaces of the seeds. The potassium permanganate on the seed surface was washed with sterile water until the eluent was colorless. The treated seeds were wrapped with moist sterile gauze, placed in petri dishes and germinated in a 30 ℃ incubator. Black soil and vermiculite were mixed at 3:1, and sterilizing for standby. Sowing the germinated seeds in sterilized soil, performing dark circulation at 23+/-2 ℃ for 16h and light/8 h, and culturing for 30d at 60% -80% humidity. After emergence, watering is carried out once every 2 d. When the pinus sylvestris seedling is 4-6cm high and has 30-50 needle leaves, the pinus sylvestris seedling is used for evaluating the disease prevention effect. Cutting a wound on phloem of a trunk (2-3 cm away from the root) of a seedling by using a syringe needle, installing 1mL of the syringe needle at the wound, fixing the needle by using a small wood stick with the length of about 10cm and a sealing film, wrapping the wound, adding 100 mu L (30 heads/mu L) of pine wood nematode suspension into the syringe needle, and spraying 5mL of to-be-detected bacterial liquid with OD ₆₀₀ = 1.5 to the pinus sylvestris seedling. After the nematodes are inoculated for 14d, the disease index of each treatment is investigated and counted with reference to published disease grades (ganA functional analysis in the pine wood nematode killing by enterobacter ludwigii, 20230103, chinese biological control theory, see page 7, line 5 to line 11 in particular), and the disease preventing effect is calculated. The calculation formula of the disease index and the disease prevention effect is as follows:

The pine needle seedlings inoculated with the pine wood nematode suspension are used as positive control, and the pine needle seedlings inoculated with the pine wood nematode suspension and the strain T-150 fermentation broth are used as treatment groups, and the blank control is the pine needle seedlings treated by sterile water. The results of the greenhouse disease prevention experiments show that the disease index of the positive control group is 74.8, the disease symptoms of the treatment group are obviously reduced, the disease index is 34, the fermentation liquor of the strain T-150 can obviously reduce the disease index of pine wood nematode disease of the pinus sylvestris seedlings under the greenhouse condition, the occurrence of the pine wood nematode disease is effectively reduced, and the disease prevention effect is 54.5% (see figure 10). In conclusion, under the greenhouse condition, the acinetobacter pituitarioides T-150 disclosed by the invention can effectively prevent and treat pine wood nematode diseases.

While the invention has been illustrated and described with reference to specific embodiments, it is to be understood that the above embodiments are merely illustrative of the technical aspects of the invention and not restrictive thereof; those of ordinary skill in the art will appreciate that: modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some or all of the technical features thereof, without departing from the spirit and scope of the present invention; such modifications and substitutions do not depart from the spirit of the corresponding technical solutions; it is therefore intended to cover in the appended claims all such alternatives and modifications as fall within the scope of the invention.

Claims

1. Acinetobacter petri (Acinetobacter pittii) T-150 for preventing and treating pine wood nematode diseases is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of 27755.

2. A microbial preparation comprising the Acinetobacter petite T-150 and/or a bacterial fermentation broth thereof according to claim 1.

3. The microbial preparation of claim 2, wherein the microbial preparation comprises a solid microbial agent or a liquid microbial agent.

4. The microbial preparation according to claim 2, wherein when the microbial preparation is a solid microbial preparation, the number of viable bacteria of the Acinetobacter petri T-150 is 1.1X10- ⁹CFU·g^-1 or more.

5. The microbial preparation according to claim 2, wherein when the microbial preparation is a liquid microbial preparation, the microbial preparation contains 1.1x10 ⁸~4.0×10⁸CFU·mL^-1 viable bacteria of the acinetobacter pituitarius T-150.

6. The microbial formulation of claim 2, wherein the microbial formulation further comprises an adjuvant.

7. The microbial formulation of claim 6, wherein the adjuvant comprises: at least one of a dispersant, a stabilizer, a disintegrant, a solvent, or a wetting agent.

8. The microbial formulation of claim 2, further comprising an agriculturally effective amount of a fertilizer and/or a pesticide.

9. A method for controlling pine wood nematode disease, characterized in that the microbial preparation of the acinetobacter pituitarioides T-150 according to claim 1 or any one of claims 2 to 8 is applied to a control subject.

10. Use of the acinetobacter pituitus T-150 of claim 1 or the microbial preparation of any of claims 2-8 for controlling pine wood nematode disease caused by pine wood nematodes.

11. The use according to claim 10, wherein the pine wood comprises: one or more of black pine, pinus sylvestris, pinus koraiensis, larch, and pinus massoniana.

12. The use according to claim 10, wherein the pine material is pinus sylvestris or pinus sylvestris.