CN114705662B - Method for high-throughput screening of biocontrol bacteria of pine wood nematode disease - Google Patents
Method for high-throughput screening of biocontrol bacteria of pine wood nematode disease Download PDFInfo
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Abstract
The invention provides a method for screening biocontrol bacteria of pine wood nematode disease in high flux, and relates to the technical field of biology. The method comprises the steps of mixing and culturing the nematode suspension of the pine wood nematodes with the bacterial suspension of a strain to be detected, washing the mixture with sterile water to remove bacterial liquid, and performing biological control bacteria primary screening on the nematode body staining of the pine wood nematodes; then the pinus sylvestris seedlings are utilized to carry out secondary screening on the biocontrol bacterial disease prevention effect; wherein the dyeing solution used for the primary screening is a methylene blue solution and/or a 4,6-diamino-2-phenylindole solution. The invention provides a method for screening pine wood nematode disease biocontrol bacteria with high throughput, low cost and high accuracy, and provides technical support for screening pine wood nematode disease biocontrol bacteria and biological control.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for screening biocontrol bacteria of pine wood nematode disease in a high-throughput manner.
Background
Pine nematode disease caused by pine nematodes is a global forest disease that can harm 70 conifers, of which 57 pine species and 13 non-pine species. The large-area occurrence of the pine wood nematode disease causes serious damage to pine forest resources, natural landscapes and ecological environment.
For the prevention and treatment of the pine wood nematode disease, the traditional method adopts smoking, burning, felling, pesticide spraying, chemical pesticide injection on the tree trunk and the like. These methods would entail huge economic losses and environmental burdens, as well as increased tolerance to bursaphelenchus xylophilus. A novel biological control method, namely screening suitable biocontrol bacteria to produce an environment-friendly microbial preparation, is one of the best methods for controlling pine wilt.
In the prior art, the reported screening method of pine wood nematode disease biocontrol bacteria is mainly to co-culture bacteria and pine wood nematodes and judge the death and the vitality of the pine wood nematodes according to the form and the behavior of the pine wood nematodes, thereby determining whether the bacteria have the ability of killing the pine wood nematodes. However, this method has the following drawbacks: firstly, the judgment of the death condition of the pine wood nematodes is not accurate enough, and some nematodes which are stiff and do not move actually do not die, so that false positive bacteria are often screened out; secondly, the method has low flux, each sample needs to be observed under a body type microscope by human eyes, and the energy is wasted; thirdly, the bacteria screened by the method often cannot achieve good practical application effect, because the prevention and the control of the pine wood nematode disease need to be started from the combined action of three aspects of pine, nematode and bacteria, and only the capability of killing the pine wood nematode by the bacteria is considered to be too high. In addition, the reported greenhouse experiments for preventing and controlling the pine wood nematode disease mostly adopt pine seedlings growing for 3 years or 10 years, the biocontrol effect can be displayed only after 1 to 3 months, the experiment period is extremely long, a large amount of manpower and material resources are consumed, the greenhouse space and the environment are also higher in requirement, and the biocontrol bacterium screening efficiency of the pine wood nematode is extremely low.
Therefore, there is a need to provide a new method for screening bacteria for preventing and controlling bursaphelenchus xylophilus disease, especially a high-efficiency, accurate and high-throughput screening method.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a method for screening the pine wood nematode disease biocontrol bacteria, which has high flux, low cost and high accuracy and provides technical support for screening and biological control of the pine wood nematode disease biocontrol bacteria.
The technical scheme provided by the invention is as follows:
the invention provides a method for screening pine wood nematode disease biocontrol bacteria at high flux, which comprises the steps of carrying out mixed culture on a pine wood nematode worm suspension and a bacterial suspension of a strain to be tested, washing with sterile water to remove bacterial liquid, carrying out primary screening on the pine wood nematode worm body for biocontrol bacteria, and then carrying out secondary screening on biocontrol bacteria disease prevention effects by utilizing pinus sylvestris seedlings; wherein the staining solution used for prescreening is methylene blue solution and/or 4,6-diamino-2-phenylindole (DAPI) solution.
In the screening process of the pine wood nematode disease biocontrol bacteria, after the insect suspension of the pine wood nematodes and the bacterial suspension of the strain to be tested are mixed and cultured for a period of time, the bacteria with the pine wood nematode disease biocontrol function can show the effect of killing the pine wood nematodes to cause death of the pine wood nematodes. According to the invention, the condition of nematode death is judged in a clearer and more accurate manner by dyeing with methylene blue solution and/or 4,6-diamino-2-phenylindole solution, so that the statistical error generated in the prior art method when the result is observed by naked eyes under a body type mirror can be remarkably reduced (for example, the nematode which does not move straightly or is just paralyzed is counted as death).
In addition, the invention also combines the disease prevention effect of the biocontrol bacteria to re-screen, screens and verifies the application effect of the screened bacteria, namely, the biocontrol bacteria of the pine wood nematode disease are screened by evaluating the interaction effect of the pine wood, the nematode and the bacteria under the actual condition, the screening efficiency is higher, and the screening result can more accurately evaluate the biocontrol effect of the pine wood nematode disease of the tested bacteria.
In one embodiment, the prescreening comprises co-culturing the selected strain with the pine wood nematode after a first prescreening experiment using a methylene blue solution, followed by a second prescreening experiment using a 4,6-diamino-2-phenylindole solution.
In one embodiment, the final concentration of the methylene blue solution is 0.15 to 0.25mg/mL, preferably 0.2mg/mL; the 4,6-diamino-2-phenylindole solution has a final concentration of 4-6 μ g/mL, preferably 5 μ g/mL.
In one embodiment, the condition for mixed culture of the insect suspension of the pine wood nematode and the bacterial suspension of the strain to be tested is as follows: culturing at 25 deg.c for 18-24 hr; preferably 24 hours.
In one embodiment, the concentration of the insect suspension of Bursaphelenchus xylophilus is 40-50 head Bursaphelenchus xylophilus/20 μ L; the viable count of the bacterial suspension of the bacterial strain to be detected is 10 12 CFU/mL。
In one embodiment, the adding volume ratio of the insect suspension of the bursaphelenchus xylophilus to the bacterial suspension of the strain to be detected is 1.8-2.5; preferably 1:2.
In one embodiment, the pine wood nematode suspension is a sterile pine wood nematode suspension; the bacterial suspension is fermentation liquor obtained after the monoclonal strain is cultured in a liquid LB culture medium.
In a specific embodiment, the nematodes are washed with sterile water containing 2% streptomycin sulfate, and the suspension of the nematodes is adjusted with sterile water to 40-50 head nematodes per 20 μ L for use.
In a specific embodiment, the test strain is activated by three-line plate-drawing method, cultured at 30 ℃ for 16 hours, the isolated monoclonal strain is inoculated in 5mL liquid LB medium, shake-cultured at 200rpm and 30 ℃ for 24 hours, the fermentation broth is collected, and the concentration is adjusted to OD 600 The value was 1.0 (bacteria content 10) 12 CFU/mL) for use.
In one embodiment, after staining with the methylene blue solution, using a scanner to image, and after graying the image, mortality and corrected mortality are calculated; after the 4,6-diamino-2-phenylindole solution is used for dyeing, a fluorescence microscope is used for imaging, and the mortality and the corrected mortality are calculated according to the fluorescence intensity;
preferably, when the methylene blue solution is used for dyeing, the dyeing condition is dark condition at 25 ℃, and the dyeing time is 3.5-4.5 hours; preferably 4 hours;
preferably, when the 4,6-diamino-2-phenylindole solution is used for dyeing, the dyeing condition is dark condition at 25 ℃, and the dyeing time is 0.5-2 hours; preferably 1 hour;
more preferably, after dyeing, washing is performed with sterile water to remove bacteria liquid and dye.
According to the invention, the methylene blue stained nematodes are matched with the scanner, so that on one hand, the accuracy of visual observation can be improved, and on the other hand, the mortality can be calculated and corrected by processing the scanning image of the scanner through image parameters (such as gray values). In addition, the method can also select DAPI to dye the nematodes, so that the death condition of the nematodes can be judged more clearly and accurately, the accuracy of the original method is greatly improved, and the difference of fluorescence intensity of DAPI dyeing results can be quantified, so that the mortality can be calculated more efficiently and corrected. The invention can also combine the 2 modes to carry out primary screening and secondary primary screening so as to further increase the screening accuracy.
In one embodiment, the mortality and corrected mortality are calculated according to the following formulas:
in one specific embodiment, in performing the primary screening experiment, a positive control zone, a target test zone, a negative control zone, and a calibration control zone are provided on a 96-well plate, with five well replicates per treatment.
For example, 40. Mu.L of bacterial suspension and 20. Mu.L of insect suspension are added to each well. The bacterial suspension added into the positive control group is Bacillus pumilus (Bacillus pumilus) YLT40 with a known good pine wood nematode killing effect, which is provided by the institute of genetic and physiological research of the academy of agriculture and forestry, hebei province, the bacterial suspension added into the negative control group is Bacillus polymyxa (Paenibacillus polymyxa) M-1 with a known low pine wood nematode killing activity, which is provided by the plant pathology department of Chinese agriculture university, and the correction control area is 40 mu L of sterile water and 20 mu L of worm suspension for calculating and correcting the mortality rate.
In one embodiment, after incubation in 96-well plates for 24 hours at 25 ℃, the nematicidal reaction is terminated and then staining is performed; before dyeing, sterile water can be used for cleaning to remove bacteria liquid; after dyeing, the dye can be removed by washing with sterile water again.
When the methylene blue solution is used for dyeing, the final concentration of the methylene blue solution is 0.2mg/mL, and the dyeing is carried out for 4 hours under the dark condition at the temperature of 25 ℃; scanning and imaging a 96-well plate by using a scanner, wherein dead nematodes in a stiff state are stained blue, and live nematodes which are not bent are not stained as can be seen from the scanned images; graying the image, using the gray values, calculating mortality and correcting mortality.
When stained with 4'6-diamino-2-phenylindole (DAPI), the final concentration of the DAPI solution was 5. Mu.g/mL, and staining was performed for 1 hour at 25 ℃ in the dark; the dead nematodes can be observed to generate fluorescence due to staining at the 488nm excitation light wavelength by using a fluorescence micro-mirror, and the live nematodes do not stain so as not to generate fluorescence; mortality and corrected mortality were calculated from the quantified fluorescence intensity.
In one embodiment, the biological control bacteria disease prevention effect rescreening comprises the steps of adopting 1-month-old pine seedlings, spraying a bacterial liquid to be tested and injecting a pine wood nematode suspension, and determining the pine wood nematode prevention effect of the biological control bacteria according to the seedling disease index. The method adopts pinus sylvestris seedlings of 1 month old, and the disease prevention effect of the biocontrol bacteria can be observed within 9 days. The screening experiment of the disease prevention effect of the biological control bacteria is carried out, and the prevention and control effect of the pine wood nematode disease of the screened biological control bacteria can be further determined and evaluated.
In one embodiment, a positive control group, a negative control group and a calibration control group are also set in the primary screen; wherein the bacterial suspension added into the positive control group is Bacillus pumilus YLT40, the bacterial suspension added into the negative control group is Paenibacillus polymyxa M-1, and the bacterial suspension is replaced by sterile water for the correction control group.
In one embodiment, the biocontrol bacteria disease control effect rescreen further comprises injecting sterile water at the wound as a blank control and injecting a pine wood nematode suspension at the wound as a negative control.
In a specific embodiment, 5mL of OD is sprayed on 1 month old Pinus sylvestris seedlings 600 And =1.0, cutting a wound on the bast of the seedling of the pine tree, and injecting 100 μ L of 30 head/μ L of the pine wood nematode suspension.
In a specific embodiment, 100 μ L of sterile water is injected at the wound as a blank; 100 μ L of bursaphelenchus xylophilus suspension was injected at the wound as a negative control. After 9 days of inoculation, corresponding disease indexes are obtained according to disease grade classification.
In a specific embodiment, the disease index is calculated according to the following formula:
in addition, the invention also provides a method for preventing and controlling the pine wilt disease, and the biocontrol bacteria screened by the method are used for preventing and controlling. In some cases, the bursaphelenchus xylophilus-killing strain screened under laboratory conditions cannot necessarily prevent the bursaphelenchus xylophilus disease in the practical application process, which may be caused by the interaction of pine, nematode and bacteria, or the influence of the living environment of nematode and bacteria. The invention can further screen the strains which really have the function of preventing and controlling the pine wood nematode disease by utilizing the bio-control bacteria disease prevention effect of the pinus sylvestris seedlings.
Has the advantages that:
according to the technical scheme, the observation and calculation method of the death condition of the nematodes is highly simplified by combining the innovation of the dyeing and the statistical method (by matching the methylene blue dyed nematodes with a scanner and/or by combining DAPI dyeing with fluorescence intensity analysis), the accuracy is improved, and the efficiency of screening the biocontrol bacteria of the bursaphelenchus xylophilus disease is increased;
according to the invention, 1-month-old pine seedlings are adopted, and the disease prevention effect of the pine wilt disease of the biocontrol bacteria can be observed in 9 days, so that the greenhouse experiment period is greatly shortened, the greenhouse experiment cost is reduced, and the greenhouse experiment efficiency is improved;
the method starts from the biocontrol bacteria, the pine wood nematodes and the pine trees together, realizes high-flux screening of the bacteria for preventing and treating the pine wood nematode disease, and can efficiently screen and obtain the strains with the real biocontrol effect on the pine wood nematode disease.
The method disclosed by the invention is subjected to repeated experiments and actual screening practices of a plurality of different strains, and the result shows that the method has high accuracy and good repeatability for screening the biocontrol bacteria of the bursaphelenchus xylophilus disease; and the screening time is greatly saved, and the labor cost is reduced.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a process for primary screening of biocontrol bacteria against Bursaphelenchus xylophilus disease according to an embodiment of the present invention;
FIG. 2 is an evaluation of a screening model for biocontrol bacteria against bursaphelenchus xylophilus disease (nematode corrected mortality results for positive and negative control groups);
FIG. 3 is a graph of the staining of bacterially treated Bursaphelenchus xyfolus with methylene blue according to an embodiment of the invention (wherein prior to image processing, the result is not shown by the scanner, the dead nematodes are blue, and after gray scale processing of the image, only the dead nematodes are shown), scale bar, 2mm;
FIG. 4 is the result of staining of bacterially treated Bursaphelenchus xyfolus with DAPI (dead nematodes fluoresced and surviving nematodes did not fluoresce), scale bar, 2mm;
FIG. 5 is the result of the experiment of the disease prevention effect of pine wilt disease of green-house seedling for biological prevention of bacteria, with a scale of 5cm;
FIG. 6 is a disease classification of pine wilt disease of greenhouse seedlings, scale bar, 5cm;
FIG. 7 is a primary screening result of pine wood nematode killing activity test on 308 strains of bacteria using the established pine wood nematode disease biocontrol bacteria screening model;
FIG. 8 is a secondary primary screening result of 30 high-efficiency pine wilt disease biocontrol strains obtained by primary screening;
FIG. 9 shows the corrected mortality of 30 high-efficiency pine wood nematode disease biocontrol strains for killing pine wood nematodes;
FIG. 10 shows the results of rescreening the disease prevention effect of the pine wilt disease of 30 high-efficiency pine wilt disease biocontrol strains of greenhouse seedlings;
FIG. 11 shows disease indexes of pine wilt disease of 30 high-efficiency pine wilt disease biocontrol strains of greenhouse seedlings;
FIG. 12 shows the species ratio of 30 high-efficiency pine wood nematode biocontrol strains.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 screening model establishment of biocontrol bacteria against pine wilt disease
1. Culture of Bursaphelenchus xylophilus
The pine wood nematode is from Zhejiang agriculture and forestry university forestry and biotechnology institute Guo Kai auxiliary professor's gift.
(1) Mixing 10. Mu.L of 10 8 The CFU/mL Botrytis cinerea spore suspension (purchased from Shanghai bacteria center, AS 3.3789) is inoculated to the center of a PDA plate, and cultured in a constant temperature incubator at 25 ℃ for 4 days until hyphae grow over the whole PDA plate for later use.
(2) The pine wood nematode suspension with the concentration of 2500 heads/mL is connected onto a well grown fungus plate and grows at 25 ℃ until the area of the nematode reaches 2/3 of a culture dish (about 5 days of culture), so that the fungus hyphae are obviously reduced, the surface of a fungus colony is covered by oily substances, and a large amount of nematodes can be observed under a 4-fold microscope of a body type microscope. After the culture is completed, the fungus plate with the pine wood nematodes cannot be placed for too long, otherwise the activity of the nematodes is reduced and even the fungi die in a large amount.
(3) 10mL of sterile water was added to the nematode-bearing fungal plates and soaked for 1h. Transferring the nematode suspension into a 15mL centrifuge tube, standing for precipitation, and discarding the supernatant. Adding 5mL of sterile water containing 2% streptomycin sulfate, mixing, sterilizing nematode, treating for 5min, standing, removing supernatant, and washing with sterile water for 3 times.
(4) Taking 1 50mL acid burette, vertically fixing on an iron support, filling 49mL0.3% sodium carboxymethyl cellulose (CMC) solution, slowly adding 1mL pine wood nematode water solution (about 7000 nematodes) from the orifice, and standing for 12h. Taking 25mL of the upper end of the burette, centrifuging for 5min (2000 r/min), and removing supernatant to obtain second-instar pine wood nematode larvae.
(5) And (3) dropwise adding the second-instar pine wood nematode larvae onto a fresh fungus flat plate, and obtaining fourth-instar pine wood nematodes after 48 hours. Add 10mL of sterile water to the plate and soak for 1h. Transferring the four-year-old pine wood nematode suspension into a 15mL centrifuge tube, standing for precipitation, and discarding the supernatant. Adding 5mL sterile water containing 2% streptomycin sulfate, mixing, sterilizing the pine wood of four ages, treating for 5min, standing, discarding supernatant, and washing with sterile water for 3 times. Sterile water is used for adjusting the insect suspension of the four-year-old pine wood nematodes to 40-50 heads per 20 mu L for later use.
2. Preparation of the bacterial suspension
Activating the strain to be tested stored at-80 deg.C on LB solid plate by three-line plate-drawing method, culturing at 30 deg.C for 16 hr, inoculating the separated monoclonal strain to 5mL liquid LB culture medium, shake culturing at 200rpm and 30 deg.C for 24 hr, collecting the fermentation liquid, adjusting the concentration to OD 600 The value was 1.0 (bacteria content 10) 12 CFU/mL) for use.
3. Screening and evaluation of biocontrol bacteria of pine wood nematode disease
Setting a positive control area, a target test area, a negative control area and a correction control area on a 96-well plate, wherein five Kong Chongfu are processed in each treatment; adding 40 mu L of bacterial suspension and 20 mu L of insect suspension into each hole;
adding a bacterial suspension into the positive control group to obtain the bacillus pumilus YLT40 with a known good effect of killing the pine wood nematodes, adding a bacterial suspension into the negative control group to obtain the paenibacillus polymyxa M-1 with known no pine wood nematode killing activity, and calculating a correction control area by using 40 mu L of sterile water and 20 mu L of insect suspension for calculating the correction mortality;
the prepared test plate was incubated at 25 ℃ for 20 hours.
After 20 hours, the well plate was placed in a plate washer, washed 3 times with sterile water, the bacterial solution was removed, and the nematicidal reaction was terminated.
And (3) counting the nematode-corrected mortality of the positive control group and the negative control group by using a formula:
σ p, σ n are positive and negative control-corrected mortality variance, respectively; μ p, μ n, positive and negative control corrected mortality means, respectively, and the Z-factor (Z-factor) of the model was calculated. The theoretical value of the Z-factor is 1, which is the main parameter for evaluating the quality of the test method. If the Z-factor is between 0.5 and 1, the experiment has high reliability; if the Z-factor is less than 0.5, the test is a critical test and the reliability is low. The Z-factor of the screening model is 0.653, which shows that the screening condition and the measuring method of the experiment have high reliability and can be used for large-scale experiments. FIG. 2 is a graph of nematode corrected mortality for positive and negative controls.
4. Accurate visualization of pine wood nematode disease biocontrol bacterium thread killing activity result
4.1. Methylene blue dyeing method
And in the primary screening stage, after the thread killing reaction is stopped, adding methylene blue solution with the final concentration of 0.2mg/mL, dyeing for 4 hours in dark at 25 ℃, and dyeing the pine wood nematodes. If the nematode dies, the permeability of the nematode cells is increased, and methylene blue solution can enter the nematode cells to dye the nematode cells into blue.
Using a plate washer, the plate was washed three times with sterile water to remove excess dye from the well plate. Using a scanner, 96-well plate scans were imaged and from the scan it can be seen that dead nematodes in a stiff state were stained blue, while live nematodes without bending were not stained (fig. 3, left panel).
The scan was opened with Photoshop software, adjusting the threshold color level to between 180-190, which only shows dead nematodes stained blue. And opening the processed picture by using image J software, selecting a region needing to be analyzed in the picture by using a gray scale measurement function, obtaining a gray scale value, and calculating the mortality and correcting the mortality by using the gray scale value (figure 3, right picture). The ratio of the total gray value to the gray value of a single nematode is the number of dead nematodes, and the number of dead nematodes plus the number of surviving nematodes is the total number of the nematodes to be tested. The ratio of the number of dead nematodes to the number of test nematodes is the mortality of the nematodes.
The method is convenient and fast, the experimental results can be processed in batches through a computer, and after dyeing is finished, all the experimental results of the 96-well plate can be obtained within half an hour. In the prior method, manual operation is carried out under a body type microscope, 2-3 hours are needed for obtaining all experimental results of a 96-well plate, and meanwhile, the error of the results is reduced due to the difference of manual visual counting. And judging the death and survival condition of the nematodes and the inaccuracy thereof through the stiffness (death) and bending (survival) of the nematodes.
Fig. 1 shows a primary screening process of biocontrol bacteria of pine wood nematode disease, which mainly comprises cultivation of pine wood nematodes, preparation of bacterial suspension, mixed cultivation of bacterial suspension and nematode suspension in a 96-well plate, methylene blue staining, nematode cleaning, nematode scanning and observation and statistics.
4.2.DAPI staining method
In order to more accurately detect the pine wood nematode killing activity of the pine wood nematode disease biocontrol bacteria, the strains with good primary screening effect are subjected to secondary primary screening. Culturing with nematode at 25 deg.C for 24 hr in 96-well plate, stopping killing reaction, and staining Bursaphelenchus xyfolus co-cultured with bacteria with 4'6-diamino-2-phenylindole (DAPI) to obtain DAPI solution with final concentration of 5.0 μ g/mL.
After 1 hour of dyeing, the cells were washed 3 times with sterile water to remove the dye. Using a confocal laser microscope (Zeiss, LSM 800), fluorescence of dead nematodes due to staining was observed at 353nm of excitation light and 465nm of absorption light, while fluorescence of live nematodes was not observed (FIG. 4). Using confocal laser microscopy picture analysis software (ZEN _ lite), selecting the region to be analyzed in the map, obtaining the fluorescence intensity value of the region, calculating the mortality rate and correcting the mortality rate according to the quantified fluorescence intensity. Only displaying dead nematodes in the fluorescence field, wherein the ratio of the total signal intensity value of the fluorescence field to the signal intensity value of the fluorescence field of the single nematode is the number of the dead nematodes; and the bright field displays all the nematodes to be tested, and the ratio of the total intensity value of the bright field signals to the intensity value of the single nematode bright field signals is the number of the nematodes to be tested. The ratio of the number of dead nematodes to the number of all nematodes is the mortality of the nematodes. By using the method, the death condition of the nematodes can be judged more accurately and clearly, and the accurate visualization of the screening result is realized.
Example 2 experiment for testing disease prevention effect of biocontrol bacteria for pine wood nematode disease in greenhouse seedlings
1. Culture of pinus sylvestris seedlings
The pinus sylvestris seeds are soaked in 5 percent potassium permanganate solution for 1 hour, and the seeds are subjected to surface sterilization. Washing with sterile water to colorless, and removing the potassium permanganate solution on the surface of the seeds. Wrapping the sterilized seeds with sterile gauze, wetting the gauze with sterile water, and standing at 30 deg.C for germination. After 48 hours, the germinated seeds were picked, loaded in sterile soil, cultured in a greenhouse at 25 ℃ for 30 days, and the seedlings were watered every 3 days.
2. Experiment of biological control bacteria disease prevention effect of pine wood nematode disease
Spraying 5mL of OD to Pinus sylvestris seedlings 600 Bacterial suspension to be tested of = 1.0. Strain YLT40 was used as a positive control, strain M-1 was used as a negative control, and sterile water was used as a blank control (FIG. 5). Cutting a wound on the phloem of the trunk part of the pine seedling at a distance of 2-3 cm from the root by using an injection needle, installing a 1mL syringe needle at the wound, fixing the needle and wrapping the wound by using a small wooden stick of about 10cm and a sealing film, and injecting 100 muL of 30 head/muL pine wood nematode suspension into the syringe needle.
Each group treated 10 pinus sylvestris seedlings. After 9 weeks of inoculation, corresponding disease indices were obtained according to disease classification (fig. 6).
3. Grading severity of pine wood nematode disease of pinus sylvestris seedlings
Grade 1, the seedlings are slightly deformed, the stems are still plump but begin to yellow, the branches are dehydrated and bent, the tips of the branches turn yellow, and the whole seedlings are dehydrated and present the phenomenon of branch curling.
Grade 2, the seedlings are deformed, the stems are dehydrated, slightly shriveled and yellowed, branches are shrunk and shrunk to have the phenomena of shriveling and shrinking, the tips are withered and yellow, the whole seedlings are obviously dehydrated and slightly withered.
Grade 3, withered and yellow stem, obvious branch bending, low verticality, severe shrinkage, severe water loss of the whole tree and impending blight.
Grade 4, withered and yellow stem, shriveled and withered and yellow branch bending, complete water loss of the whole tree seedling, withered and dead tree seedling.
EXAMPLE 3.308 Primary screening of strains of bacteria
And (3) carrying out pine wood nematode killing activity test on 308 strains of bacteria collected and separated in the laboratory by utilizing the established high-throughput screening pine wood nematode disease biocontrol bacteria model. 30 high-efficiency bursaphelenchus xylophilus biocontrol strains are obtained by primary screening through methylene blue staining, the corrected mortality rate of the strains exceeds 85% (figure 7), and the strains still have high-efficiency nematicidal activity by secondary primary screening through a DAPI staining method (figure 8) (figure 9).
In order to further verify the disease prevention effect of the pine wilt disease, the 30 strains and the pine nematode are inoculated into pinus sylvestris seedlings together to carry out rescreening on the disease prevention effect of the pine wilt disease. According to the disease prevention effect (figure 10), the disease index of the pinus sylvestris seedlings (figure 11) is calculated, which shows that the pinus sylvestris seedlings have strong effect of killing pine wood nematodes and show excellent biological prevention effect.
We further classified these strains and found that they belong to 21 bacteria of 7 genera including bacillus, paenibacillus, pseudomonas, enterobacter, chryseobacterium, stenotrophomonas, pantoea (fig. 12), and in the published studies, these bacteria were all prokaryotes with potential wood nematode prevention, and this screening result provided stock material for biological control of wood nematode disease, and the adopted method for high throughput screening of wood nematode disease biocontrol bacteria provided technical support for screening and biological control of wood nematode disease biocontrol bacteria.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (12)
1. A method for screening pine wood nematode disease biocontrol bacteria at high flux is characterized by comprising the steps of carrying out mixed culture on a pine wood nematode worm suspension and a bacterial suspension of a strain to be tested in a 96-well plate for 24 hours, washing with sterile water to remove bacterial liquid, carrying out primary screening on the pine wood nematode worm body for biocontrol bacteria, and completing secondary screening of biocontrol bacteria disease prevention effect within 9 days by utilizing pinus sylvestris seedlings; wherein the primary screening comprises the steps of performing a primary screening experiment by using a methylene blue solution, then performing co-culture on the screened strains and the pine wood nematodes again, and performing a secondary screening experiment by using a 4,6-diamino-2-phenylindole solution;
after the methylene blue solution is used for dyeing, imaging by using a scanner, and calculating the mortality and correcting the mortality after graying the image; after the 4,6-diamino-2-phenylindole solution is used for dyeing, a fluorescence microscope is used for imaging, and the mortality and the corrected mortality are calculated according to the fluorescence intensity;
a positive control group, a negative control group and a correction control group are arranged in the preliminary screening; wherein the bacterial suspension added to the positive control group is Bacillus pumilus (YLT) YLT40, the bacterial suspension added to the negative control group is Bacillus polymyxa (Paenibacillus polymyxa) M-1, and the correction control group replaces the bacterial suspension with sterile water;
the biological control bacteria disease prevention effect re-screening comprises the steps of adopting pinus sylvestris seedlings with the age of 1 month, spraying a bacterial liquid to be detected and injecting a pine wood nematode suspension, and determining the pine wood nematode prevention effect of the biological control bacteria according to the disease indexes of the seedlings.
2. The method of claim 1, wherein the final concentration of the methylene blue solution is 0.15 to 0.25mg/mL; the final concentration of the 4,6-diamino-2-phenylindole solution is 4-6 mug/mL.
3. The method of claim 1, wherein the final concentration of the methylene blue solution is 0.2mg/mL; the final concentration of the 4,6-diamino-2-phenylindole solution is 5 μ g/mL.
4. The method according to claim 1, wherein the mixed culture conditions of the insect suspension of the Bursaphelenchus xylophilus and the bacterial suspension of the strain to be tested are as follows: culturing at 25 deg.c for 18-24 hr.
5. The method of claim 4, wherein the concentration of the insect suspension of Bursaphelenchus xylophilus is 40-50 head Bursaphelenchus xylophilus/20 μ L; the viable count of the bacterial suspension of the bacterial strain to be detected is 10 12 CFU/mL。
6. The method according to claim 1, wherein the addition volume ratio of the bursaphelenchus xylophilus suspension to the bacterial suspension of the strain to be tested is 1.8-2.5.
7. The method of claim 1, wherein the ratio of the volume of the insect suspension of Bursaphelenchus xylophilus to the bacterial suspension of the test strain is 1:2.
8. The method according to claim 1, wherein when the methylene blue solution is used for dyeing, the dyeing is performed under a dark condition at 25 ℃ for 3.5 to 4.5 hours; when the solution of 4,6-diamino-2-phenylindole is used for dyeing, the dyeing condition is that the dyeing is carried out for 0.5 to 2 hours at 25 ℃ in the dark.
9. The method according to claim 1, wherein when the staining is performed using the methylene blue solution, the staining conditions are 25 ℃ for 4 hours in the dark;
when staining was performed using 4,6-diamino-2-phenylindole solution, the staining conditions were 25 ℃ in the dark for 1 hour.
10. The method of claim 1, wherein after the staining, washing with sterile water is performed to remove bacteria and dyes.
11. The method of claim 1 or 2, wherein said bio-control bacterial disease prevention effect rescreening further comprises injecting sterile water at the wound as a blank control and injecting a bursaphelenchus xylophilus suspension at the wound as a negative control.
12. A method for controlling pine wilt disease, characterized in that said biocontrol bacteria screened by the method of any one of claims 1-11 are used for control.
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