CN115725423A

CN115725423A - Phytophthora and bacterium symbiotic system and analysis method and application thereof

Info

Publication number: CN115725423A
Application number: CN202211292468.3A
Authority: CN
Inventors: 高瑞芳; 卢小雨; 汪莹; 张伟锋; 章桂明; 王颖
Original assignee: Shenzhen Customs Animal and Plant Inspection and Quarantine Technology Center
Current assignee: Shenzhen Customs Animal and Plant Inspection and Quarantine Technology Center
Priority date: 2022-10-21
Filing date: 2022-10-21
Publication date: 2023-03-03

Abstract

The application discloses a symbiotic system of phytophthora and bacteria, and an analysis method and application thereof. In the symbiotic system of Phytophthora and bacteria, the Phytophthora is Phytophthora syringae, phytophthora cactorum or Phytophthora fragaria var. Rubi, and the bacteria are Pandoraea sputterum or Achromobacter insoletus. The application researches and discovers a symbiotic system of phytophthora and bacteria at first, and provides a new scheme and way for preventing and treating plant diseases caused by phytophthora. Phytophthora belongs to the class of Oomycetes, and the application rate firstly discovers the phenomenon of symbiotic bacteria in fungi of the class of Oomycetes and has important significance for the research of the symbiotic system of the fungi and the bacteria.

Description

Phytophthora and bacterium symbiotic system and analysis method and application thereof

Technical Field

The application relates to the technical field of fungus and bacterium symbiosis, in particular to a phytophthora and bacterium symbiosis system and an analysis method and application thereof.

Background

Within the plant kingdom, the machinery of plant intracellular symbiotic bacteria and eukaryotes is widely known, and the most studied is the mycorrhiza, a reciprocal symbiont of fungi and plant roots. In addition to symbiotic systems of plant roots and fungi, it has been shown that some fungi and bacteria can also constitute symbiotic systems, for example, some fungi of the phylum coccidioidomycota (Glomeromycota), the subdivision mucormycotina (Mucoromycotina), the phylum Ascomycota (Ascomycota) and the phylum Basidiomycota (Basidiomycota) are symbiotic.

Research shows that in a symbiotic system of fungi and bacteria, the bacteria can promote nutrient absorption of the symbiotic system and influence the growth of fungal hyphae, cytoplasm formation, vacuole morphology, cell wall formation, liquid matrix and pigment granulation. Moreover, bacteria in symbiotic systems are associated with vegetative propagation, production of toxins to cause disease, improved adaptation, increased viability and disease resistance.

At present, except for symbiosis of part of the fungi in Glomeromycota, mucor cortina, ascomycota and Basidiomycota, no symbiotic system of the fungi in other phyla or classifications is found. However, the symbiotic system is a ubiquitous natural phenomenon, and the development of more symbiotic systems, especially the symbiotic systems of fungi or bacteria related to plants, has important significance for promoting plant growth, preventing and treating diseases and the like.

Disclosure of Invention

The application aims to provide a novel phytophthora and bacteria symbiosis system and an analysis method and application thereof.

The following technical scheme is adopted in the application:

one aspect of the present application discloses a symbiotic system of Phytophthora and bacteria, wherein the Phytophthora in the symbiotic system is Phytophthora fragaraense var. Rubi, phytophthora syringae or Phytophthora cactorum, and the bacteria in the symbiotic system is Pandoraea sputrum or Achromobacter insoletus.

It should be noted that, the application researches and discovers the symbiotic system of Phytophthora and bacteria at first, and proves the symbiotic system of Phytophthora and bacteria through a large number of verification and exclusion experiments, and finally confirms that three species of Phytophthora variegata var. Rubi, phytophthora cactorum or Phytophthora sorasyrinae have symbiosis with two kinds of bacteria of Pandoraea sputrum or Achromobacter insidolitus. The symbiotic system of the phytophthora and the bacteria provides a new scheme and a new way for preventing and treating plant diseases caused by the phytophthora.

In one implementation of the present application, the symbiont system is a symbiont system of Phytophthora fragrou var. Rubi or Phytophthora cactorum, and Pandoraea sputorum; alternatively, a symbiotic system of Phytophthora syringae and Achromobacter insoleus.

It should be noted that, phytophthora fragraniae var. Rubi or Phytophthora cactorum and Pandoraea sputrum symbiotic system; a symbiotic system of Phytophthora syringae and Achromobacter insidatus; this is only a specific symbiotic system case found in one implementation of the present application, and does not exclude other combinations between these three phytophthora species and two bacteria.

In one implementation of the present application, pandoraea sputorem is Pandoraea sputorem strain CUB2S; achromobacter insidious is Achromobacter inside strain Sn1.

It should be noted that Pandoraea sputoreum strain CUB2S and Achromobacter insolens strain Sn1 are only specific strains of Pandoraea sputoreum and Achromobacter insolens determined by sequence alignment in an implementation manner of the present application.

The application also discloses an analysis method of a phytophthora and bacteria symbiotic system, which comprises a separation culture step, a fluorescence microscope and/or transmission electron microscope observation step and a nucleic acid sequencing verification step; separating and culturing, namely taking hyphae of a phytophthora sample to be analyzed, dispersing the hyphae in water to break the hyphae, then centrifuging, taking supernatant, coating the supernatant on a nutrient agar plate, culturing for at least 10 hours at constant temperature, and observing whether a bacterial sample organic matter exists on the nutrient agar plate; a step of observing by a fluorescence microscope and/or a transmission electron microscope, which comprises the step of observing hyphae with bacterial organic matters by the fluorescence microscope or the step of observing by the transmission electron microscope, and further confirming whether symbiotic bacteria exist in a phytophthora sample to be analyzed; and (3) nucleic acid sequencing verification, which comprises the steps of respectively extracting DNA of hyphae of a phytophthora sample to be analyzed and DNA of the bacterial organic matter, carrying out ITS and 16S rRNA sequencing on the DNA of the hyphae, carrying out 16S rRNA sequencing on the DNA of the bacterial organic matter, further verifying the existence of a phytophthora and a bacterial symbiotic system through the ITS and 16S rRNA sequencing of the DNA of the hyphae, comparing sequencing results, and determining a specific species or strain of phytophthora in the phytophthora sample to be analyzed and a specific species or strain of bacteria in the symbiotic system.

It should be noted that, after finding that the Phytophthora and the bacteria have symbiosis, the research of the application creatively develops a method capable of accurately and effectively analyzing the symbiotic system of the Phytophthora and the bacteria, and finally confirms that the Phytophthora is Phytophthora fragaragiae var. Rubi, phytophthora cactorum or Phytophthora raraysoragene and the bacteria is Pandoraea sputrum or Achromobacter insidolitus through the method of the application. It is understood that the use of the present analytical method does not exclude the possibility to develop further phytophthora and bacteria symbiotic systems.

In one implementation of the present application, the step of separating and culturing further comprises repeatedly blowing hyphae to break the hyphae with a pipette after the hyphae are dispersed in water.

In an implementation manner of the application, the method further comprises the step of centrifuging in two rounds when supernatant is obtained through centrifugation, centrifuging in two rounds for no more than 5min at a speed of no more than 500rpm in a first round, obtaining the supernatant after centrifugation in the first round, then centrifuging the supernatant in the first round in a second round, wherein the speed of centrifugation in the second round is at least 4000rpm, the centrifugation time is no less than 20min, obtaining the supernatant after centrifugation in the second round, and coating the supernatant after centrifugation in the first round and the supernatant after centrifugation in the second round on a nutrient agar plate for constant-temperature culture.

In one implementation of the present application, the temperature of the isothermal culture is 30 ℃.

In one implementation of the present application, the fluorescence microscope observation includes transferring the hyphae to 0.85% NaCl solution, after dispersing uniformly, taking the mixed solution on the slide, adding nucleic acid dye for dyeing, and then observing with the fluorescence microscope.

In an implementation manner of the application, add nucleic acid dye to carry out dyeing, specifically include, add SYTO9 green fluorescence nucleic acid dye to add the same amount of propidium iodide dye, incubate under the dark condition for at least 15min, add ProLong Gold anti reagent, incubate for at least 20min, then carry out fluorescence microscope observation under 480/500nm wavelength condition.

In one implementation manner of the application, the observation by a transmission electron microscope comprises the steps of transferring a phytophthora sample to be analyzed cultured by a solid culture medium or hyphae thereof to a centrifuge tube of 2.5-4% glutaraldehyde solution, fixing the sample at 0-4 ℃ for more than 4 hours, pouring off the glutaraldehyde solution, and washing the sample for at least 3 times with PBS Buffer with 0.1M and pH 7.2, wherein the PBS Buffer is used for washing for at least 10min each time; adding 1% osmic acid to treat the sample to become black, and fixing the sample for 1-2h; pouring off osmic acid, washing with 0.1M PBS Buffer at pH 7.2 for at least 3 times, each time for at least 10min; dehydrating the sample with ethanol solution of gradient concentration for at least 10min, and treating with 100% ethanol for at least 15min for at least 2 times; then, the process is shifted to acetone treatment for at least 2 times, and each time lasts for at least 15min; treating the sample for 2-4h by using a mixed solution of an embedding medium and acetone in a volume ratio of 1; embedding the processed sample by using a mould, polymerizing for 24 hours at 45 ℃, and then polymerizing for 24 hours at 60 ℃ to obtain an embedded sample; and (3) trimming the embedded sample into a required size and a required section by using a trimmer, cutting a section with the thickness of 70-100nm by using a slicer, carrying out double dyeing on the section by using a uranyl acetate solution and a lead citrate solution, airing, and carrying out transmission electron microscope observation.

In one implementation manner of the application, before transferring the phytophthora to be analyzed sample cultured in the solid culture medium or the mycelium thereof to a centrifugal tube of 2.5-4% glutaraldehyde solution, the method further comprises cutting 0.5-1.0 mm ³ The sample block is cultured by a solid culture medium of a phytophthora sample to be analyzed or hypha thereof, and is centrifuged into a mass with the size of 1-2 mm on the premise of not damaging the appearance of the sample ³ 。

In one implementation of the present application, the number of sample pieces cultured in a solid medium of a phytophthora sample to be analyzed or the mycelium thereof is small or is difficult to form by centrifugation to 1 to 2mm ³ In the case of the pellet of (4), pre-embedding is carried out using an agar block.

In one implementation of the present application, the sample is dehydrated by an ethanol solution with a gradient concentration, specifically including sequentially dehydrating the sample by 30%, 50%, 70%, 80%, 90% and 95% ethanol, each concentration is treated for at least 10min, centrifuging after the treatment is completed, removing the supernatant, and then adding ethanol with the next concentration to perform dehydration.

In one implementation manner of the present application, the analysis method of the present application further comprises identifying the bacteria-like organic substance by using a bacteria identification system or a bacteria identification kit, and determining a specific species or strain of bacteria in the symbiotic system.

The application also discloses application of the symbiotic system of the phytophthora and the bacteria in prevention and treatment of plant diseases caused by the phytophthora.

The beneficial effect of this application lies in:

the application researches and discovers a symbiotic system of phytophthora and bacteria at first, and the symbiotic system provides a new scheme and way for preventing and treating plant diseases caused by phytophthora. Phytophthora belongs to the class of Oomycetes, and the application rate firstly discovers the phenomenon of symbiotic bacteria in fungi of the class of Oomycetes and has important significance for the research of the symbiotic system of the fungi and the bacteria.

Drawings

FIG. 1 is a graph showing the results of isolated culture of bacteria in the examples of the present application;

FIG. 2 is a fluorescent photomicrograph of hyphae in the examples of the present application;

FIG. 3 is a transmission electron microscope image of hyphae in the examples of the present application;

FIG. 4 is a transmission electron microscope image of the isolated bacteria in the examples of the present application;

FIG. 5 is a graph of the results of 10 days of inoculation on seedlings of Dendrobium officinale in the examples of the present application;

FIG. 6 is a graph of the observation results of 20 days of seeding for Dendrobium officinale seedlings in the example of the present application;

FIG. 7 is a graph of the observation results of 30 days of seeding for Dendrobium officinale seedlings in the examples of the present application.

Detailed Description

Historically, many scientific experiments have been found by chance. The application finds the situation of the phytophthora symbiotic bacteria unexpectedly when carrying out the phytophthora genome sequencing analysis. The phenomenon is proved through a large number of verification and elimination experiments, and a symbiotic system of three epidemic mold species and two bacteria is discovered through research. This is the phenomenon of first finding symbiotic bacteria in oomycetes, and oomycetes are the lowest bacteria found in symbiotic systems.

The present application will be described in further detail with reference to specific examples. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application.

Example one

1. Materials and methods

1. Bacterial isolation

In this example, 84 strains of Phytophthora were subjected to an endophytic bacteria-searching formula, and the strain information was as shown in Table 1, inoculated and cultured on a V8 juice plateThe above. These species are all from the netherlands fungal biodiversity research center CBS, american model culture collection bank ATCC, and british IMI species resource bank. After hypha formation, 100. Mu.L of ddH was taken out ₂ In the O centrifuge tube, hyphae were repeatedly blown out using a 1mL pipette tip to rupture and release bacteria. After two rounds of centrifugation, the first round of centrifugation is performed at a low speed of 500rpm/32g for 5min, the second round of centrifugation is performed at a low speed of 4000rpm/2060g for 20min, and after each round of centrifugation, the supernatant is coated on a nutrient agar plate and cultured overnight at 30 ℃. The nutrient agar plate formulation used in this example was: 5.0g of peptone, 3.0g of meat extract, 15.0g of agar and 1000mL of water.

TABLE 1 Phytophthora test information

In table 1 "No." indicates the number of the species, for example "4" indicates that the laboratory has 4 strains of the species, each strain corresponding to a different number, host and source.

2. Fluorescence microscopic photograph

Transfer 0.5mL of hyphae to an EP tube containing 0.5mL of 0.85% NaCl, spread 10. Mu.L of the mixture on a glass slide, add 0.5. Mu.L of SYTO9 green fluorescent nucleic acid dye for staining, add equal amount of propidium iodide dye [ Live/Dead Back light background visualization Kit (Molecular Probes-L7007) ], incubate for 15min in the dark, add ProLong Gold antigen reagent (Molecular Probes-P36935) for fluorescence enhancement, incubate for 20min,480/500nm wavelength, and observe under a Zeiss LSM 510Meta Lascope Microscope.

3. Hypha and bacterial DNA extraction

The hypha DNA extraction method uses a plant fungus DNA extraction kit produced by QINGEN company, and extracts according to the method provided by the kit, and the operation method is as follows:

(1) Scraping hyphae from a plate culture medium, avoiding scraping the hyphae to the culture medium as much as possible, and grinding the hyphae by liquid nitrogen;

(2) Adding 400 mu LBuffer AP1 and 4 mu L RNase A, whirling, mixing uniformly, and carrying out water bath at 65 ℃ for 10min, wherein the process is reversed for 2-3 times;

(3) Taking out the centrifuge tube, directly adding 130 μ LBuffer AP2, shaking and mixing uniformly, and culturing on ice for 5min;

(4) Transferring the supernatant into QIA shredder mini spin colomn, recording the volume number, purple tube, 20000g (14000 rpm), and centrifuging for 2min;

(5) Discarding the column part, adding 1.5 times volume of Buffer AP3/E into the filtered liquid, and mixing (filtrate);

(6) Placing 650 μ L of the mixed solution into a DNeasy mini spin column (white tube), centrifuging for 1min, wherein the volume is not less than 6000g (not less than 8000 rpm), removing the filtrate, and repeating the steps on the rest sample (mixed solution);

(7) Putting spin column into a new collection tube of 2mL, adding 500 μ L Buffer AW at 6000g (8000 rpm) or more, centrifuging for 1min, and discarding the filtrate (lower part);

(8) Then 500. Mu.L Buffer AW, 20000g (14000 rpm) are added, and the mixture is centrifuged for 2min; note that: when spin column is taken, the spin column cannot contact the filtrate in the next step; transferring the spin column into a new 1.5mL or 2mL centrifuge tube, adding 100 muL/50 muL Buffer AE for elution, standing at room temperature for 5min, and centrifuging at a temperature of more than or equal to 6000g for 1min; this operation is repeated.

The preparation of bacterial DNA adopts Axygen bacterial DNA preparation kit, and the operation method is as follows:

(1) Collecting the cell suspension by using a 1.5mL centrifuge tube, centrifuging at 14000rpm for 5min, and then discarding the supernatant;

(2) Adding 350 mu L of deionized water or PBS suspension cells, and uniformly mixing by oscillation;

(3) Adding 0.8 mu of LRNaseA, vortexing and oscillating for 15 seconds, and standing for 1 minute at room temperature;

(4) Adding 150 μ L Buffer C-L and 8 μ L proteinase K, immediately vortex and shake for 1min, centrifuging for a short time, and placing in 56 deg.C water bath for 10min;

(5) Adding 350 mu L of Buffer P-D, and uniformly mixing by vortex oscillation for 30 s; centrifuging at 120000rpm for 10min;

(6) Placing the DNA preparation tube in a 2mL centrifuge tube, and centrifuging at 12000rpm for 1min;

(7) Discarding the filtrate, adding 500 μ L Buffer W1, and centrifuging at 12000rpm for 1min;

(8) Discarding the filtrate, adding 700 μ L Buffer W2, centrifuging at 12000rpm for 1min, and repeating for 1 time;

(9) Discarding the filtrate and centrifuging for 1min;

(10) Put into a clean 1.5mL centrifuge tube, 120. Mu.L of H is added ₂ And O, standing for 1min, and centrifuging for 1min.

4.PCR

ITS and 16S rRNA gene amplification is carried out on the hypha DNA of the fungus, the accuracy of the fungus and the existence of symbiotic bacteria are verified, and the 16S rRNA gene is amplified by the DNA of the bacteria. Fungal ITS fragments were amplified using the universal primers ITS4 and ITS 5. The reaction conditions are 95 ℃/10min,95 ℃/1min,58 ℃/1min,72 ℃/90s,35cycles,72 ℃/5min. The PCR reaction system used was a 25. Mu.L reaction system comprising 2.5. Mu.L of 10 XBuffer (containing Mg) ²⁺ ) 2.5. Mu.L of dNTPs (2.5 mM), 0.5. Mu.L of each of the upstream and downstream primers, 0.15. Mu.L of ExTaq enzyme (5U/. Mu.L), 0.75. Mu.L of DNA template, ddH ₂ O make up to 25. Mu.L.

ITS4, seq ID No.1:5' TCCTCCGCTTATTGATATGC-3

ITS5, seq ID No.2:5 'GGAAGTAAAAGTCGTAACAAGG-3'.

The 16S rRNA gene was amplified using universal primers B27f and U1492 r. The reaction conditions are 95 ℃/10min,93 ℃/1min,50 ℃/1min,72 ℃/70s,30cycles,72 ℃/5min. The PCR reaction system used was a 25. Mu.L reaction system comprising 2.5. Mu.L of 10 XBuffer (containing Mg) ²⁺ ) 2.5. Mu.L of dNTPs (2.5 mM), 0.5. Mu.L of each of the upstream and downstream primers, 0.15. Mu.L of ExTaq enzyme (5U/. Mu.L), 0.75. Mu.L of DNA template, ddH ₂ O make up 25. Mu.L.

B27f, seq ID No.3:5 '-AGAGAGTTTGATCTMTGGCTCAG-3'

U1492r is Seq ID No.4:5 'ACGGCTACCTTGTTTACGACT-3'.

5. Sequencing and alignment

The PCR products were sent to the Kyoto Liuhua Dagen Co., ltd, beijing to perform sequencing on an ABI (Applied Biosystems, foster City, CA) sequencer. In order to ensure the accuracy of sequencing, forward and reverse bidirectional sequencing is adopted. Sequences were spliced using BioEdit and the collated sequences were submitted to BLAST for homology alignment. Phylogenetic trees were constructed using Mega5.0 software.

6. Transmission electron microscope imaging

Section preparation and staining:

(1) Material taking: wiping grease with alcohol cotton by a sharp blade, and quickly cutting to obtain the grease with the size of 0.5-1.0 mm ³ The sample block of (1); bacteria and fungi cultured by the solid culture medium are centrifuged into a mass with the size of 1-2 mm on the premise of not damaging the appearance of a sample ³ (ii) a A small number of samples, particularly small in number or difficult to agglomerate, can be pre-embedded with agar blocks;

(2) Front fixed block: rapidly transferring the sample into centrifuge tubes added with 4% glutaraldehyde solution, filling more than 20 sample blocks in each centrifuge tube, and fixing for more than 4h at 4 ℃;

(3) Rinsing: the fixative was decanted off and washed 3 times with 0.1M PBS Buffer pH 7.2, 10mins each time;

(4) Post-fixing: add 1% osmic acid until the sample turns black, fix sample for 2h;

(5) Rinsing: the fixative was decanted off and washed 3 times with 0.1M PBS Buffer pH 7.2, 10mins each time;

(6) And (3) dehydrating: dehydrating the sample with ethanol solutions of gradient concentrations, including six concentrations of 30%, 50%, 70%, 80%, 90% and 95%, each concentration for 10min, and then treating with 100% ethanol for 2 times, 15min each time, wherein the process cannot suck the liquid too dry and slightly leave the liquid;

(7) And (3) transition: finally, the process is shifted to acetone treatment for 2 times, each time for 15min;

(8) And (3) infiltration: treating the sample with a mixed solution of an embedding machine and acetone (V/V = 1/3) for 3h, treating the sample with a mixed solution of an embedding agent and acetone (V/V = 1/1) for 3h, treating the sample with a mixed solution of an embedding agent and acetone (V/V = 3/1) for 12h, treating the sample with a pure embedding agent for 24h, replacing the pure resin once, and replacing the tube;

(9) Embedding the sample subjected to the permeation treatment by using a mold, polymerizing for 24 hours at 45 ℃, and then polymerizing for 24 hours at 60 ℃ to obtain an embedded sample; taking out and placing in a dryer for storage for later use, and slicing;

(10) Ultrathin section and staining: the embedding block is trimmed into a proper size and a proper section by a trimmer, a slice with the thickness of 70nm is obtained in an ultrathin slicer, double staining is carried out by a uranyl acetate solution and a lead citrate solution, and observation can be carried out in a transmission electron microscope after airing.

Taking a picture by a transmission electron microscope:

the transmission electron microscope used is a Tecnai 12 analytical transmission electron microscope produced by FEI company in the Netherlands, and the debugging steps of the instrument and equipment are as follows:

(1) Opening a TecniUser Interface main program of the software, and opening GatanDigitalMicrograph shooting software;

(2) Inspecting the state of an electron microscope: in TecnaiUser Interface software and a Setup-Vacuum control panel, the pressure indication conditions of Gun, column and Camera are all green until normal; in TecniUser Interface software and a Setup-High Tension control panel, under a normal condition, a High Tension indication value is yellow, and a High pressure indication value is 200Kv;

(3) Filling liquid nitrogen: blocking a window of the projection chamber by using a baffle, wearing gloves, carefully pouring liquid nitrogen into the Dewar flask, slowly extending the copper braid into the Dewar flask, and arranging the Dewar flask on a bracket; filling the liquid nitrogen in the bottle and covering the bottle with a cover;

(4) Loading a sample: selecting a sample rod, taking down the front sleeve, checking whether the tip of the sample rod and the clamp are clean and dry, and keeping one hand against the tail end of the sample rod to ensure that the sample rod cannot move out of the sleeve; inserting a tool into the hole in front of the clamp, lifting the clamp to the maximum possible angle, and loading a sample to be tested into the sample rod with the front side of the sample facing downwards; carefully lower the clamp onto the sample with a tool and ensure that the sample remains in the correct position; the sample safety clamps must be carefully lowered, otherwise, the sample and clamps can be damaged; the sample rod is rotated by 180 degrees, and the sleeve is tapped to ensure that the sample cannot fall off;

(5) Inserting a sample rod: horizontally holding the tail end of the sample rod, aligning a positioning pin on the sample rod with a slit on the sample table, and slowly inserting the sample rod until the sample rod cannot be inserted continuously; at the moment, a red light on the sample table is on, a mechanical pump starts to pre-pump vacuum at the air lock of the sample, and the sample rod slowly slides into the electron microscope under the action of vacuum suction;

(6) Adding a filament current: before the operation is started, in the TecnaiUser Interface software Setup, a Filament indication bar should be gray; when the partial vacuum value of the lens barrel is reduced to be below 20, the Filament is automatically added to a preset state by clicking the Filament indicator light, and the Filament indicator bar is yellow;

the photographing step is as follows:

(1) Opening Col.valves, confirming that the vacuum value of the lens barrel part is reduced to be below 20, clicking a Col.valves indication bar, and changing yellow to gray; yellow for off, grey for on; valves stands for V7 and V4 vacuum valves, which isolate the column from the electron gun and the column from the camera chamber, respectively.

(2) Finding a sample and focusing; after the Col.Valves is opened, the electron beam and the sample image can be seen, at this time, an interested area is usually found at a lower multiple, and then the image is focused clearly by turning to a higher multiple; the method for adjusting the optimal height of the sample comprises the following steps: the Focus button is used for fine tuning by first pressing the EucentricFocus button on the right operating panel of the desktop and then trying to Focus by adjusting the height Z. Before photographing, the image is adjusted to be in an under-focus state (the image edge is bright).

(3) Paraxial directions; before taking a picture, the electronic optical path system needs to be adjusted, which is called as a closed axis; in TecniUser Interface → Tune → Direct Alignments, the following alignment operation (alignment at SA magnification) can be done:

gun Tilt: find a position without sample, adjust mul.x and Y (multi-function knob) respectively to minimize exposure time (electron beam brightest).

Gun Shift: in the position without sample, first point Beam Shift, spot size is adjusted to9, and Mul.X and Y are adjusted (multifunctional buttons), and the electron Beam is adjusted to the center of the fluorescent screen; then, the point Gun Shift, spot size is adjusted to 3, and Mul.X and Y (multi-function buttons) are adjusted to adjust the electron beam to the center of the screen. The adjustment is repeated for several times. Finally, the spot size is adjusted back to 1.

Beam Tilt PPX and Beam Tilt PPY: in the position without sample, mul.x and Y (multi-function knob) were adjusted to bring the two electron beam spots that were rocked together. PPX and PPY are adjusted in two directions respectively.

Rotation Center: the magnification is increased to hundreds of thousands of times, a characteristic position on the sample is found, the sample is placed in the center of the fluorescent screen, the small screen is lifted, the selected characteristic image is observed by means of the ocular lens, and the shaking of the characteristic image is minimized by adjusting mul.x and Y (multifunctional buttons).

(4) Astigmatism elimination

In the TenaiUser Interface → Tune → Stigmator control panel, three kinds of lens astigmatism can be eliminated, which are:

(1) objective-astigmatism (most important) of the Objective lens. Finding an amorphous region on the sample, increasing the magnification to several hundred thousand times, collecting the dynamic image (Search mode) with the aid of a CCD camera, adjusting the Focus button to Focus the image clearly (under Focus), and then performing dynamic FFT (digital micrograph → Process → Live → FFT). Adjust mul.x and Y (multi-function knob) to make the amorphous ring in FFT as round as possible. This is a shortcut for eliminating astigmatism of the objective lens, but the precision is not very high.

(2) Condensor is Condenser astigmatism. In image mode, mul.x and Y (multi-function buttons) are adjusted to make the electron beam spot circular.

(3) Diffraction: diffraction astigmatism. In the diffraction mode, mul.x and Y (multi-function buttons) are adjusted to round the transmission spot.

(5) And recording and storing the image. The film is used for photographing, after Exposure conditions are set, the Exposure button on the left operation panel is pressed, the fluorescent screen is automatically lifted, the film is automatically guided into the light path by the film feeding box, and Exposure is started. After exposure, the negative film is automatically transmitted back to the film receiving box, and the fluorescent screen is put down. At this time, the related information such as the film number is recorded in time.

2. Results and discussion

1. Symbiotic bacteria isolation

The endophyte status was screened from 84 strains of 60 species of phytophthora. The results showed that bacterial cultures were isolated from the hyphae of three strains of phytophthora p.rubi (No. cbs 109892), p.cacorum (No. acccc 36421) and p.syringae (No. cbs 132.23), and the results are shown in fig. 1. In fig. 1, a is a p.rubi culture picture, B is a p.cactorum culture picture, and C is a p.syringae culture picture. The white globular colonies in the image were tentatively designated as bacterial-like organisms (BLOS).

2. Fluorescence microscopy of fungal-bacterial symbiont

The observation of the hyphae of 84 strains revealed that bacterial symbiosis was also found in P.rubi (No. CBS 109892), P.cacorum (No. ACCCC36421) and P.syringae (No. CBS 132.23). The results of fluorescence microscopic observation of three strains of phytophthora are shown in fig. 2. In fig. 2, a is an observation view of p.rubi, B is an observation view of p.cactorum, and C is an observation view of p.syringae. The results in FIG. 2 show that the linear fluorescent spots in the hyphae were clearly seen in P.rubi (No. CBS 109892), and the released fluorescent spots were clearly seen along the hyphae and neatly along the hyphae at the hypha breaks. After hypha rupture is seen in P.syringae (No. CBS 132.23), a large amount of floccules appear in the environmental system, releasing contents for the hypha. Tiny individuals of the dense hemp appear in the environment, fluoresce and swim. Slightly large spherical substances appear in the hyphae, and the floccules form a conglobation structure. Minute individuals are also found within the hyphae and move about. Similar to the phenomenon of P.syringum (No. ACCCC36421), the tip of the hypha can be seen to emit fluorescent conglobation structure, and the environment has a few large spherical substances which are similar to the release of large flocculent conglobation substances in the hypha.

3. Transmission electron microscope observation of fungus-bacterium symbiotic system

The presence of fungal hyphae and the morphology of the isolated bacteria of endosymbiotic bacteria were observed using transmission electron microscopy. The results of observing three strains of phytophthora, p.rubi (No. cbs 109892), p.cacorum (No. acccc36421) and p.syringae (No. cbs 132.23) are shown in fig. 3. Fig. 3 shows an observation view of p.rubi, an observation view of p.sectorum, and an observation view of p.syringae. The results in fig. 3 show the p. Rubi endophytic bacteria, vacuoles, cytoplasm, hyphal flagella, hyphal wall, etc. microstructures. Endogenous bacteria are of different sizes and shapes. There are irregular ellipsoids, ovals, spindles, and also some irregular shapes. The size range is 0.3-1 μm, and the particles are scattered in cytoplasm. In the case of endophytic bacteria in p.cactorum, the volume of the endophytic bacteria is slightly larger, the diameter is slightly larger, about 1 μm, and more pictures show that the quantity of the endophytic bacteria is less, and generally only 1-2 bacteria exist in one hypha. The endophytic bacteria in P.syringae have various shapes, the shapes and the sizes are similar to those of the bacteria in P.rubi, but 1-2 endophytic bacteria exist in each hypha.

And (4) taking a picture of the separated bacteria-like organic matters through a transmission electron microscope, wherein the picture is used for determining bacteria in a fungus endosymbiotic system by reference on one hand and mastering morphological characteristics of the bacteria more deeply on the other hand. The results are shown in FIG. 4. In fig. 4, panel a is an observation view of a bacterial-like organic matter isolated from p.rubi, panel B is an observation view of a bacterial-like organic matter isolated from p.cactorum, and panel C is an observation view of a bacterial-like organic matter isolated from p.syringae. It can be seen that the three bacteria have similar shapes and sizes, the diameter of 200nm-1 μm is spherical, ellipsoidal or spindle-shaped, and there are also a few indefinite forms.

4. Comparison of symbiotic bacteria

The 16S rRNA genes of the hyphal symbiota and the separated endosymbiont bacteria are amplified respectively, and the result shows that the 16S rRNA gene sequence of each symbiota is 100 percent similar to the 16S rRNA gene sequence of the separated symbiont bacteria. That is, the 16S rRNA gene sequencing results of nucleic acids extracted from p.rubi hyphae were 100% identical to the 16S rRNA gene sequencing results of nucleic acids extracted from bacteria-like organisms isolated from p.rubi hyphae; the 16S rRNA gene sequencing results of nucleic acids extracted from p.cactorum hyphae were 100% identical to the 16S rRNA gene sequencing results of nucleic acids extracted from bacterial-like organic matter isolated from p.cactorum hyphae; the 16S rRNA gene sequencing results of nucleic acids extracted from p.syringae hyphae were 100% identical to those of nucleic acids extracted from bacterial-like organisms isolated from p.syringae hyphae.

The result of the 16S rRNA gene sequence was compared with the data in the NCBI database, and the result showed that the symbiotic bacteria of p.rubi and p.cactorum had very high similarity to Pandoraea particulate strain CUB2S, 99% and 100%, respectively. The sympathogenic P.syringae has the highest similarity to Achromobacter insolens strain Sn1, which is 98%. The statistical results are shown in table 2. Neither of these two bacteria has been reported in other fungus-bacteria symbiota, that is, they were first discovered as fungus symbiotic bacteria.

TABLE 2 comparison of symbiotic bacteria

Fungal symbiota	Highest similarity strain	Login number	Similarity of the two
				Phytophthora rubi	Pandoraea sputorum strain CUB2S	JQ 839144	99
P.cactorum	Pandoraea sputorum strain CUB2S	JQ 839144	100
				P.syringae	Achromobacter insolitus strain Sn1	KC 683714.1	98

5. Phylogenetic analysis of symbiotic systems

The family, which is also the group of commensal bacteria which have been reported to find the most frequent occurrence of the commensal bacterial group, and which are both pathogenic bacteria, is probably not accidental, is also the most common group of commensal bacteria, according to the alignment of the 16S rRNA genes of the bacteria and phylogenetic analysis, and the bacteria isolated from P.rubi and P.cacorum belong to the genus Pandoraea, the kindrea of which are the sibles of Burkholderia and Candidatus glomeribacteria, which belong to Burkholderia ceae. Meanwhile, the fungal group with symbiotic bacteria is also subjected to phylogenetic analysis based on the ITS fragment to reveal the diversity and evolutionary relationship of the symbiotic fungi, and the result shows that the fungal species with symbiotic relationship, different from bacteria, are various and widely exist in Basidiomycota, ascomycota, glomeromycota and Zygomycota, while the Oomycota fungal phytophthora discovered in the research of the embodiment is discovered for the first time. Oomycota is the least evolutionary, belonging to the kingdom of bacteria.

6. Discussion of the related Art

Although fungal and bacterial symbiosis systems are found in other fungi, bacterial symbiosis of three strains of Phytophthora in this example was first found, and actually, bacterial symbiosis was first found in the class of Oomycetes to which Phytophthora belongs.

In addition, the bacteria Pandoraeasputorum and Achromobacter insoletus which are found to be symbiotic with three strains of phytophthora in the embodiment are also found to be symbiotic with fungi for the first time; the symbiosis of the two bacteria with other species, such as plants, insects and the like is not reported, and the symbiosis of the two bacteria is firstly found in the test.

The class oomycetes differ greatly from other fungi and it is believed that oomycetes are a group of organisms that are morphologically and functionally similar to fungi and are virtually unrelated to fungi. The main differences are: (1) the nutriment of the oomycete is diploid, and other fungi are haploid; (2) the main component of the cell wall of the oomycete is cellulose and lacks obvious layers, while the other components of the cell wall of the fungus are chitin; (3) sexual reproduction of oomycetes is oogametophy, producing oospores, which are rare among other fungi, and so on. In the eighth edition of the fungal dictionary, oomycetes are classified as Trichophyton, which is an intermediate between prokaryotes and fungi. The classification status of oomycetes is controversial. With respect to symbiotic mechanisms, scientists have proposed hypotheses and hypotheses that endogenic bacteria play a role in the evolution of their hosts. Organelles such as mitochondria, chloroplasts, plastids and the like can be traced to bacterial precursors thereof, and genetic analysis of the precursor symbiotic genome and the eukaryotic genome reveals that the two exist in a homologous relationship. Oomycetes are evolved intermediate species, all of which have led to the thinking that bacteria play a role in the process of oomycete evolution. This hypothesis has also been reported in other bacteria-fungi symbiota. The origin of critical intercellular organelles such as mitochondria, chloroplasts and plastids can be traced back to bacterial progenitors, the genomes of the progenitors endosymbionts have been stripped to the core, and many of the basic functions are now encoded by the host eukaryotic cells. Therefore, the experiment takes the lead to research and discover the symbiotic system of phytophthora and bacteria, and has important significance on the research of oomycete evolution.

Example two

Virulence validation was performed against phytophthorafragiae var. Rubi and its isolated bacterium Pandoraea sputrum. The following pathogenicity figures are negative controls, the bacterium Pandoraea sputeroum and the symbiotic system in that order.

1. Pathogenic force to plant

Non-toxic dendrobium officinale seedlings are used as inoculation materials and are respectively inoculated into a tissue culture medium in a culture medium inoculation mode.

Inoculating an oomycete bacterial symbiotic system by using an oomycete hypha suspension, culturing in a V8 liquid culture medium, shaking at 25 ℃ and 130rpm for 5d, taking out hyphae, rinsing with sterile water for 1-2 times, blowing and crushing the mycelium into hypha sections with the average length of 0.2-0.5cm, adding sterile water to prepare the hypha suspension, and mixing uniformly for later use.

The bacteria were inoculated with the cell suspension, and after 12 hours of plating, the concentration of the bacterial liquid was observed under a microscope and prepared to be 1X 10 with sterile water ⁵ And (5) mixing the CFU/mL bacterial solution uniformly for later use.

In this example, a negative control group, a bacterial group and a symbiotic system group were provided. The negative control group adopts sterile water, the bacteria group adopts the prepared bacterial suspension, and the symbiotic system group adopts the prepared hypha suspension, namely the symbiotic system.

Observations and photographs were made on 10 days, 20 days and 30 days of culture, respectively, and the results are shown in fig. 5 to 7. Fig. 5 is a photograph taken for 10 days of inoculation, fig. 6 is a photograph taken for 20 days of inoculation, and fig. 7 is a photograph taken for 30 days of inoculation. In fig. 5 to 7, from left to right are a negative control, the bacterium Pandoraea sputoum and a symbiotic system, respectively.

As seen in the inoculation process, at the 10 th day, the isolated bacterium Pandoraea sputrum and the symbiotic system inoculated seedlings all show symptoms, and have the characters of leaf edge yellowing, leaf tip wilting, slight blackening and the like. At 20d, the inoculation symptoms worsened, the leaf margins and leaf tips became yellow, and the leaves developed rotting symptoms. At 30d, the inoculation symptoms are severe, and the leaves are almost completely yellow, dry and wilted to death.

2. Infectivity to animals

SPF-grade BALB/c mice, 6-8 weeks old, males were used. Animals were acclimated for 3 days, on a free water diet, at room temperature (23 +/-1) ° c, humidity (60 +/-10)%. After the Balb/c mice are treated by nasal drip with the strain culture solution, the lung tissues of the mice are observed 1 day, 3 days, 5 days, 10 days, 15 days, 20 days and 30 days after the treatment respectively, and a camera shoots the pictures of the lung tissues. The control group was treated with sterile water in the same manner.

The infection process can be seen, lesion symptoms appear in the lung tissues of the 5d mice, the blood forming symptoms appear in the lungs of the mice infected by the isolated bacteria Pandoraea sputocrum and symbiotic system, and grayish white lesions can be seen after the bacteria infection. At 20d, the lesion color deepens and becomes more intense.

The results show that the pathogenicity of plant pathogenic bacteria, i.e. oomycetes, is related to symbiotic bacteria, and the discovery provides a new thought and direction for preventing and treating related diseases.

The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. It will be apparent to those skilled in the art from this disclosure that many more simple deductions or substitutions can be made without departing from the spirit of the disclosure.

Claims

1. A symbiotic system of phytophthora and bacteria, which is characterized in that: the Phytophthora in the symbiotic system is Phytophthora fragaraca var. Rubi, phytophthora cactorum or Phytophthora syringae, and the bacteria in the symbiotic system are Pandoraea sputrum or Achromobacter insolens.

2. The symbiotic system according to claim 1, characterized in that: the symbiont system is a symbiont system of Phytophthora fragaria var. Rubi or Phytophthora cactorum and Pandoraea sputoreum;

or the symbiont is a symbiont system of Phytophthora syringae and Achromobacter insoleus.

3. The symbiotic system according to claim 1 or 2, characterized in that: the Pandoraea sputoreum is Pandoraea sputoreum strain CUB2S;

the Achromobacter insidious is Achromobacter inside strain Sn1.

4. An analysis method of a phytophthora and bacteria symbiotic system is characterized in that: comprises a separation culture step, a fluorescence microscope and/or transmission electron microscope observation step and a nucleic acid sequencing verification step;

the separation culture step comprises the steps of taking hyphae of a phytophthora sample to be analyzed, dispersing the hyphae into water to break the hyphae, then centrifuging, taking supernatant, coating the supernatant on a nutrient agar plate, culturing for at least 10 hours at constant temperature, and observing whether bacterial-like organic matters exist on the nutrient agar plate;

the step of observing by using a fluorescence microscope and/or a transmission electron microscope comprises the steps of observing hyphae with bacterial organic matters by using the fluorescence microscope or observing by using the transmission electron microscope, and further confirming whether symbiotic bacteria exist in a phytophthora sample to be analyzed;

the nucleic acid sequencing verification step comprises the steps of respectively extracting DNA of hyphae of a phytophthora sample to be analyzed and DNA of the bacterial organic matter, carrying out ITS and 16S rRNA sequencing on the DNA of the hyphae, carrying out 16S rRNA sequencing on the DNA of the bacterial organic matter, further verifying the existence of a phytophthora and a bacterial symbiotic system through the ITS and 16S rRNA sequencing of the DNA of the hyphae, comparing sequencing results, and determining specific species or strains of phytophthora in the phytophthora sample to be analyzed and specific species or strains of bacteria in the symbiotic system.

5. The analytical method of claim 4, wherein: the step of separating and culturing also comprises repeatedly blowing hyphae with a liquid-transfering gun to break the hyphae after the hyphae are dispersed in water;

preferably, the method further comprises the steps of centrifuging in two rounds when supernatant is obtained through centrifugation, centrifuging for no more than 5min in the first round at the speed of no more than 500rpm to obtain supernatant of the first round, then centrifuging the supernatant of the first round at the speed of at least 4000rpm for no less than 20min in the second round to obtain supernatant of the second round, and coating the supernatant of the first round and the supernatant of the second round on a nutrient agar plate for constant-temperature culture;

preferably, the temperature of the isothermal culture is 30 ℃.

6. The analytical method of claim 4, wherein: the fluorescence microscope observation comprises transferring the hyphae into 0.85% NaCl solution, dispersing uniformly, taking the mixed solution on a slide, adding nucleic acid dye for dyeing, and observing by adopting a fluorescence microscope;

preferably, the nucleic acid dye is added for dyeing, and specifically, the method comprises adding SYTO9 green fluorescent nucleic acid dye and equal amount of propidium iodide dye, incubating for at least 15min under dark conditions, adding ProLong Gold antigen reagent, incubating for at least 20min, and observing under a fluorescence microscope under 480/500nm wavelength condition.

7. The analytical method of claim 4, wherein: the transmission electron microscope observation comprises the steps of transferring a phytophthora sample to be analyzed cultured by a solid culture medium or hyphae thereof into a centrifugal tube of 2.5-4% glutaraldehyde solution, fixing the sample at 0-4 ℃ for more than 4h, pouring off the glutaraldehyde solution, and washing the sample for at least 3 times by using PBS Buffer with 0.1M and pH 7.2, wherein each time lasts for at least 10min; adding 1% osmic acid until the sample turns black, fixing the sample for 1-2h; pouring off osmic acid, washing with 0.1M PBS Buffer at pH 7.2 for at least 3 times, each time for at least 10min; dehydrating the sample with ethanol solution with gradient concentration for at least 10min, and treating with 100% ethanol for at least 2 times for at least 15min; then, the process is shifted to acetone treatment for at least 2 times, and each time lasts for at least 15min; treating the sample for 2-4h by using a mixed solution of an embedding agent and acetone in a volume ratio of 1; embedding the processed sample by using a mould, polymerizing for 24 hours at 45 ℃, and then polymerizing for 24 hours at 60 ℃ to obtain an embedded sample; and (3) trimming the embedded sample into a required size and a required section by using a trimmer, cutting a section with the thickness of 70-100nm by using a slicer, carrying out double dyeing on the section by using a uranyl acetate solution and a lead citrate solution, airing, and carrying out transmission electron microscope observation.

8. The analytical method of claim 7, wherein: before transferring the phytophthora sample to be analyzed or the mycelium thereof cultured in the solid culture medium to a centrifuge tube of 2.5-4% glutaraldehyde solution, cutting 0.5-1.0 mm ³ The solid culture medium of the phytophthora sample to be analyzed or the hypha thereof cultures the sample block, and the sample block is centrifuged into a mass with the size of 1-2 mm on the premise of not damaging the appearance of the sample ³ ；

Preferably, the amount of the sample pieces cultured in the solid medium of the Phytophthora specimen or its hyphae to be analyzed is small or difficult to form 1 to 2mm by centrifugation ³ Under the condition of the clusters, the agar blocks are adopted for pre-embedding;

preferably, the sample is dehydrated by ethanol solution with gradient concentration, specifically comprising the steps of dehydrating the sample by ethanol with concentration of 30%, 50%, 70%, 80%, 90% and 95% in sequence, wherein each concentration is treated for at least 10min, centrifuging after the treatment is finished, removing supernatant, and adding ethanol with the next concentration for dehydration.

9. The assay method according to any one of claims 4 to 8, wherein: and identifying the bacteria-like organic matter by using a bacteria identification system or a bacteria identification kit to determine the specific species or strain of the bacteria in the symbiotic system.

10. Use of the symbiota according to any one of claims 1 to 3 for the control of plant diseases caused by phytophthora.