CN111647540A - Method for obtaining biocontrol bacteria for preventing and treating continuous cropping plant root diseases - Google Patents
Method for obtaining biocontrol bacteria for preventing and treating continuous cropping plant root diseases Download PDFInfo
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Abstract
The invention provides an acquisition method of biocontrol bacteria for preventing and controlling continuous cropping plant root diseases, belonging to the technical field of soil-borne disease prevention and control, and the acquisition method comprises the following steps: obtaining rhizosphere soil of a continuous cropping plot and rhizosphere soil of a non-continuous cropping plot; separating and identifying the flora types of the continuous cropping soil and the non-continuous cropping soil; comparing the continuous cropping flora with the non-continuous cropping flora, and screening out lost flora in the continuous cropping flora, namely biocontrol bacteria for preventing and treating the continuous cropping plant root diseases; and verifying the bacteriostasis of the biocontrol bacteria. The method can effectively obtain the biocontrol bacteria for preventing and treating the root diseases of the continuous cropping plants. The biocontrol bacteria obtained by the method can improve the rhizosphere colonization ability of microorganisms, enhance the antibacterial spectrum, have good disease control effect, and provide support for effectively researching and developing effective disease prevention microbial inoculum, thereby fundamentally breaking through the bottleneck that the biocontrol bacteria is difficult to popularize commercially.
Description
Technical Field
The invention relates to a method for obtaining biocontrol bacteria for preventing and controlling continuous cropping plant root diseases, and belongs to the technical field of soil-borne disease prevention and control.
Background
Continuous cropping obstacles are a phenomenon commonly occurring in many plants, and refer to the phenomena of poor crop growth, pest and disease damage and yield reduction even if normal cultivation management measures are adopted after the same plant or closely related plants are continuously cropped. The method is limited to the farmland area and the agricultural industrialized development, and the intensive production phenomenon of a plurality of crops is common, so that the problem of continuous cropping obstacle is prominent. Continuous cropping often causes the aggravation of soil-borne fungal diseases, wherein root diseases are the most serious diseases of continuous cropping and are important factors for restricting local crop production.
For soil-borne diseases, soil pathogens must first break through the rhizosphere area and then infect the roots to cause the diseases, so the rhizosphere area is a 'protective wall' for controlling the occurrence of the soil-borne diseases of crops. Wherein, the rhizosphere microorganism is superior to the antagonism function of the specific microorganism population in controlling the occurrence of the soil-borne diseases through the population or diversity, and is a general mechanism for maintaining the root health of the crops. At present, a method for scientifically judging the rhizosphere biocontrol bacteria is lacked.
Disclosure of Invention
The invention aims to provide a method for acquiring biocontrol bacteria for preventing and treating continuous cropping plant root diseases.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for acquiring biocontrol bacteria for preventing and treating continuous cropping plant root diseases, which comprises the following steps:
1) obtaining rhizosphere soil of a plot with continuous cropping time more than or equal to 5 years as continuous cropping soil, and obtaining rhizosphere soil of a non-continuous cropping plot of the same type as the continuous cropping soil as the non-continuous cropping soil;
2) separating and identifying the flora types of the continuous cropping soil and the non-continuous cropping soil to respectively obtain a continuous cropping flora and a non-continuous cropping flora;
3) comparing the continuous cropping flora with the non-continuous cropping flora, and screening out the flora existing in the non-continuous cropping flora and lost in the continuous cropping flora to obtain biocontrol bacteria for preventing and treating continuous cropping plant root diseases;
4) and verifying the bacteriostasis of the biocontrol bacteria.
Preferably, the non-continuous cropping plots in step 1) include plots for which different crops are planted in approximately 5-year rotation.
Preferably, the method for separating and identifying said flora type in step 2) comprises a microbial culture method and a molecular biological sequencing method.
Preferably, step 4) further comprises, before verifying the bacteriostatic activity of the biocontrol bacteria: recombining the biocontrol bacteria, wherein the number of the strains in each combination is more than or equal to 2 and less than or equal to the number of the strains of all the biocontrol bacteria; the concentration of each bacterium was the same in each combination.
Preferably, the method for verifying the bacteriostasis of the biocontrol bacteria in the step 4) comprises a laboratory bacteriostasis test and/or a field test.
Preferably, the laboratory bacteriostatic test comprises detecting the inhibition effect of the biocontrol bacteria on the hyphae of the pathogenic bacteria and/or detecting the influence of the biocontrol bacteria on the spore germination of the pathogenic bacteria.
Preferably, the field test comprises the steps of supplementing biocontrol bacteria to rhizosphere of continuous cropping plants in a plot with continuous cropping time of more than or equal to 5 years, and checking bacteriostasis.
Preferably, the method for replenishing biocontrol bacteria preferably comprises the steps of soaking plant seeds with the biocontrol bacteria and checking the bacteriostasis; and/or, the biocontrol bacteria and the decomposed organic fertilizer are mixed and applied to crops to check the antibacterial activity.
The invention has the beneficial effects that: the invention provides a method for acquiring biocontrol bacteria for preventing and treating continuous cropping plant root diseases, which comprises the following steps: obtaining rhizosphere soil of a continuous cropping plot and rhizosphere soil of a non-continuous cropping plot; separating and identifying the flora types of the continuous cropping soil and the non-continuous cropping soil; comparing the continuous cropping flora with the non-continuous cropping flora, and screening out lost flora in the continuous cropping flora, namely biocontrol bacteria for preventing and treating the continuous cropping plant root diseases; and verifying the bacteriostasis of the biocontrol bacteria. The method can effectively obtain the biocontrol bacteria for preventing and treating the root diseases of the continuous cropping plants. The biocontrol bacteria obtained by the method can improve the rhizosphere colonization ability of microorganisms, enhance the antibacterial spectrum, have good disease control effect, and provide support for effectively researching and developing effective disease prevention microbial inoculum, thereby fundamentally breaking through the bottleneck that the biocontrol bacteria is difficult to popularize commercially.
Detailed Description
The invention provides a method for acquiring biocontrol bacteria for preventing and treating continuous cropping plant root diseases, which comprises the following steps:
1) obtaining rhizosphere soil of a plot with continuous cropping time more than or equal to 5 years as continuous cropping soil, and obtaining rhizosphere soil of a non-continuous cropping plot of the same type as the continuous cropping soil as the non-continuous cropping soil;
2) separating and identifying the flora types of the continuous cropping soil and the non-continuous cropping soil to respectively obtain a continuous cropping flora and a non-continuous cropping flora;
3) comparing the continuous cropping flora with the non-continuous cropping flora, and screening out the flora existing in the non-continuous cropping flora and lost in the continuous cropping flora to obtain biocontrol bacteria for preventing and treating continuous cropping plant root diseases;
4) and verifying the bacteriostasis of the biocontrol bacteria.
Firstly, obtaining rhizosphere soil of a plot with continuous cropping time more than or equal to 5 years as continuous cropping soil, and obtaining rhizosphere soil of a non-continuous cropping plot of the same type as the continuous cropping soil as the non-continuous cropping soil; the plots with continuous cropping time more than or equal to 5 years and the non-continuous cropping plots are preferably adjacent plots; the non-continuous cropping plots preferably comprise plots for which nearly 5 years of rotation crop different crops; the method for obtaining the continuous cropping soil and/or the non-continuous cropping soil preferably comprises a root shaking method; the plants planted in the plots with continuous cropping time of more than or equal to 5 years are preferably peanuts.
After continuous cropping soil and non-continuous cropping soil are obtained, the invention separates and identifies the flora types of the continuous cropping soil and the non-continuous cropping soil, and respectively obtains continuous cropping flora and non-continuous cropping flora; the method for separating and identifying the flora type preferably comprises a microorganism culture method and a molecular biology sequencing method; the microorganism culture method preferably includes: determining flora capable of being cultured (beef extract peptone culture medium) in continuous cropping soil and non-continuous cropping soil by adopting a dilution coating and plate streaking method; the molecular biological sequencing method preferably comprises determining the flora in continuous cropping soil and non-continuous cropping soil using high throughput sequencing.
After continuous cropping flora and non-continuous cropping flora are obtained, the invention compares the continuous cropping flora with the non-continuous cropping flora, screens out the flora existing in the non-continuous cropping flora and lost in the continuous cropping flora, and obtains the biocontrol strain for preventing and treating the continuous cropping plant root disease. In the specific implementation process of the invention, flora existing in the non-continuous cropping flora and lost in the continuous cropping flora is obtained according to the abundance change of the OTU, and the flora which can be obtained by culturing in the continuous cropping soil and the non-continuous cropping soil is determined by combining the dilution coating and plate streaking methods, so that the biocontrol strain for preventing and treating the continuous cropping plant root disease is obtained.
After the biocontrol strain for preventing and treating the continuous cropping plant root disease is obtained, the invention verifies the bacteriostasis of the biocontrol strain.
Before verifying the bacteriostasis of the biocontrol bacteria, the biocontrol bacteria are preferably recombined, the number of the strains in each combination is more than or equal to 2 and less than or equal to the number of the strains of all the biocontrol bacteria, the concentration of each strain in each combination is the same, and the total effective viable count in each combination is preferably 1 × 1010～9×1010cfu/g, preferably each bacterium in each combination is separately activated to OD600nmObtaining a bacterial solution, centrifuging, collecting a precipitate, and resuspending the precipitate by using a sterile technique; the culture medium used for the activation culture preferably includes NB medium.
In the present invention, the method for verifying the bacteriostasis of the biocontrol bacteria preferably comprises a laboratory bacteriostasis test and/or a field test; the laboratory bacteriostatic test preferably comprises the steps of detecting the inhibition effect of the biocontrol bacteria on the hyphae of the pathogenic bacteria and/or detecting the influence of the biocontrol bacteria on the spore germination of the pathogenic bacteria; the field test preferably comprises the steps of supplementing biocontrol bacteria to rhizosphere of continuous cropping plants in a plot with continuous cropping time of more than or equal to 5 years, and checking bacteriostasis.
In the invention, the method for replenishing biocontrol bacteria preferably comprises the steps of soaking plant seeds with the biocontrol bacteria, and checking the bacteriostasis; and/or, the biocontrol bacteria and the decomposed organic fertilizer are mixed and applied to crops to check the antibacterial activity.
In the invention, the mass ratio of the biocontrol bacteria to the plant seed soaking is preferably (1: 2) - (1: 5); the mass ratio of the biocontrol bacteria to the decomposed organic fertilizer is preferably (1: 10) - (1: 15); the application mode of the compound material of the biocontrol bacteria and the decomposed organic fertilizer preferably comprises root area fertilization and basal application, and the application amount is preferably 50 kg/mu-100 kg/mu.
In the present invention, the method for detecting the inhibitory effect of biocontrol bacteria on the hyphae of pathogenic bacteria preferably comprises a plate opposing method; the method for detecting the influence of the biocontrol bacteria on the germination of the pathogenic bacteria spores preferably comprises a 96-well plate culture method.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the 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
1. Method of producing a composite material
Selecting continuous cropping soil and non-continuous cropping soil of the tested peanuts;
performing pot culture experiments, and performing separation and identification on rhizosphere bacteria of peanuts;
determining 7 strains of bacteria with rhizosphere deletion of continuous cropping peanuts;
preparing 7, 4 and 2 bacterial combinations of peanut rhizosphere;
determining the disease inhibiting performance of the combination of 7 strains, 4 strains and 2 strains of bacteria in the peanut rhizosphere;
determining the disease inhibiting performance of 7 strains of bacteria combination and continuous cropping peanut bacteria in the peanut rhizosphere;
performing root irrigation application on 7 strains of the peanut rhizosphere bacteria combination;
the seed soaking application is carried out on the combination of 7 strains, 4 strains and 2 strains of bacteria in the peanut rhizosphere.
2. Test soil
The experimental field of peanuts with continuous cropping time of more than 5 years is selected as test soil, the experimental field of corn, soybean and peanut rotation in nearly five years is selected as control experimental soil, and the pathogenic bacteria of the test is peanut root rot Fusarium (Fusarium oxysporum) ACCC 36194.
3. Obtaining peanut rhizosphere bacteria
Firstly, the soil around the potted peanut plants is removed by a small steel shovel and carefully pulled outGently shaking the root system of the peanut to remove large soil blocks, and then collecting the soil on the surface of the peanut rhizosphere by using a sterile brush; taking 1g of peanut rhizosphere soil, putting the peanut rhizosphere soil into a 50mL sterile conical flask, adding 9mL sterile water, shaking at 25 ℃ for 30min at 180r/min, and horizontally placing a table top for 10 min. 1mL of the supernatant was aspirated and the fungus was removed with a filter of 5 μm pore size to prepare 10-1And (5) suspending the soil bacteria for later use. Subsequent dilution 10-2、10-3、10-4、10-5And 10-6Serial gradient bacterial suspensions, 200. mu.L of 10 each-4、10-5、10-6The soil suspension of concentration was evenly spread on NA medium, each concentration gradient was repeated 3 times, and incubated in the dark at 25 ℃ for 2 d. Selecting single colonies from a plate with a proper concentration gradient according to the shape, color and size of the bacteria, respectively selecting 80 strains of bacteria from a peanut rhizosphere bacterial plate in continuous cropping, and carrying out plate streaking and purification on each single colony in an NA culture medium by using an inoculating needle for 3 times to obtain pure colonies, numbering and storing. Pure fine colonies were picked up by an inoculating needle in NB medium and cultured at 25 ℃ at 180r/min for 24 h. And extracting the DNA of the bacteria by adopting a DNA extraction kit. The extracted DNA was subjected to PCR amplification of 16S rDNA. And detecting the fragment length of the PCR amplification product by using 1% agarose gel electrophoresis, then sending the PCR amplification product to Shanghai biological engineering technology service company Limited for sequencing, comparing the obtained bacterial sequences through an NCBI website database, and selecting a 16S rDNA sequence with the highest similarity. And performing phylogenetic analysis by using MEGA software to determine the taxonomic position of the strain to be identified.
4. Obtaining key bacteria of continuous cropping peanut rhizosphere deletion
The components of partial peanut rhizosphere bacteria are analyzed by adopting a flat plate culturable method, and 80 strains of bacteria are respectively separated from continuous cropping peanuts and crop rotation peanuts. 51 strains of bacteria were identified in the continuous cropping peanut rhizosphere, 69 strains of bacteria were identified in the alternate cropping peanut rhizosphere, and the compositions of culturable bacteria in the continuous cropping and alternate cropping peanut rhizosphere were obtained (table 1).
TABLE 1 composition of bacteria isolated from peanut rhizosphere by plating and streaking
In addition, sterile blades are used from 10-1Surface bacteria were scraped from the treated bacterial plates in 1.5mL centrifuge tubes, plate bacterial DNA was extracted using Fast DNA SPIN Kit for soil Kit, quality checked and then sent to the Bio Inc for high throughput sequencing. The composition of all culturable bacteria in the rhizosphere of peanuts was analyzed (Table 2).
TABLE 2 high throughput sequencing identified peanut rhizosphere culturable bacterial composition
Note that R: rotating the peanut rhizosphere to culture a bacterial plate; c: the continuous cropping peanut rhizosphere can be used for culturing bacterial plates.
The strains lost in the continuous cropping peanut rhizosphere are Pantoea (Pantoea sp.), Pseudobacillus (Fictibacillus sp.), Enterobacter (Enterobacter sp.), Paenibacillus (Paenibacillus sp.), Sarcina gemmifera (Sporosarcina sp.), Bacillus (Lysinibacillus sp.) and Pseudomonas aeruginosa (Pseudomonas sp.) according to the tables 1 and 2.
5. Key bacterium combination for obtaining continuous cropping peanut rhizosphere deletion
Respectively activating 7 continuous cropping peanut deletion strains by NB culture medium, centrifuging for 5min at 3000g, removing bacterial fermentation liquid), adding sterile water to adjust bacterial concentration to OD600nm1, spare. The strains are randomly combined and mixed according to 7 strains, 4 strains and 2 strains, and the 7 strains are combined and treated by 1; the 4 strains are combined for 8 treatments; there were 10 treatments with 2 combinations of bacteria. The same concentration of each strain was ensured in each treatment. Sucking 1mL of each strain into a 10mL sterile centrifuge tube, adding sterile water to ensure that the concentration of each strain in each treatment is the same, and reversing the strains for several times to mix the bacterial suspension for later use.
6. Method for determining inhibition ability of key bacterial composition on pathogenic bacteria
The direct inhibition effect of the bacterial suspensions of the 7-bacterium combination, the 4-bacterium combination and the 2-bacterium combination on fusarium hyphae is detected by adopting a plate direct confrontation method, and the influence of the bacterial suspensions of the 7-bacterium combination, the 4-bacterium combination and the 2-bacterium combination on fusarium spore germination is detected by adopting a 96-well plate culture method (Table 3).
Table 3 a combination of 7, 4 and 2 peanut rhizosphere bacteria was prepared as follows:
note: the effective viable count of each bacterium in each bacterium combination is the same. A: pantoea (Pantoea sp.), B (Pseudobacillus sp.), C (Enterobacter sp.), D (Paenibacillus sp.), E (Sporosarcina sp.), F (Lysinibacillus sp.), G (Pseudomonas aeruginosa sp.), etc.).
Influence of strain combination on fusarium hypha growth: 10 mu L of bacterial liquid to be detected is inoculated on the NA culture medium, and each plate is connected with 3 parts. Co-culturing at 25 deg.C in dark for 48h, and adding sterile water to control group. After pre-culturing for 24h, adding a fusarium cake with the diameter of 5mm in the middle of the culture medium. And co-culturing at 25 ℃ in the dark for 3-7 days, and measuring the growth diameter of the fungi.
Secondly, the influence of different strain combinations on fusarium spore germination: and (3) taking 100 mu L of the diluted mixed bacterial suspension with different combinations into a sterile 96-hole, additionally adding 100 mu L of fusarium spore suspension, adding sterile water into a control group, co-culturing for 24h, observing the spore germination condition under a microscope, and calculating the bacteriostasis rate and the spore germination rate (table 4). As shown in Table 4, the inhibition ability against pathogenic bacteria was gradually enhanced with the increase of the bacterial population combinations, and among them, 7 mixed bacterial populations showed the best inhibition effect against pathogenic bacteria.
TABLE 4 peanut rhizosphere 7, 4 and 2 bacterial combinations for disease control
Note: the bacteriostasis rate is (treatment-blank control)/blank control multiplied by 100%; the spore germination rate is the number of germinated spores/total spores multiplied by 100%
7. Determination of pathogenicity of peanut rhizosphere 7-strain bacterial combination and continuous cropping peanut bacteria
Respectively activating 7 strains of peanut rhizosphere by NB culture medium, culturing at 25 deg.C in dark for 24 hr, centrifuging at 3000g for 5min, discarding bacteria fermentation liquid, adding sterile water to adjust different bacteria concentrations to OD600nm1. 200. mu.L of each strain was put in a 2mL sterile tube and turned upside down several times to mix them thoroughly for use. Weighing 5g of continuous cropping peanut rhizosphere soil sample, putting the continuous cropping peanut rhizosphere soil sample into a triangular flask containing 45mL of sterile water, and oscillating for 30min at 25 ℃. Standing on a table for 10min, sucking supernatant of 5mL soil suspension, removing fungi with filter membrane with pore diameter of 5 μm, and preparing into 10-concentration solution-1And (5) suspending the soil bacteria for later use.
Preparing NA culture medium, placing sterilized filter paper of 2cm × 4cm on one side, flattening, inoculating 200 μ L10-1The soil bacterium suspension is cultured for 48 hours at 25 ℃ in the dark. 100 mu L of peanut rhizosphere 7 strain mixed solution is added on the surface of the filter paper, and a fusarium cake with the thickness of 5mm is added on the other side. Sterile water was added to the control group. And (3) co-culturing at 25 ℃ in the dark for 3-7 days, and observing the growth diameter of the fungi (table 5). It can be seen from table 5 that the bacterial colony combination of the invention can significantly improve the inhibition ability to pathogenic bacteria by supplementing the bacterial colony combination in the continuous cropping peanut rhizosphere, and the inhibition rate is improved by 82.4%.
TABLE 5 peanut rhizosphere 7 strain combinations and continuous cropping peanut rhizosphere bacteria inhibition rates
Note: b: continuous cropping of peanut rhizobacteria; b + F: the continuous cropping peanut rhizosphere bacteria +7 strains of peanut rhizosphere bacteria. The bacteriostasis rate is (processing-blank control)/blank control multiplied by 100%
8. Method for determining root irrigation application of key bacterium combination
Firstly, disinfecting peanut seeds by using 0.1 percent (mass percentage) of mercuric chloride solution, sowing the aseptic peanut seeds in aseptic vermiculite for 3d, then pulling out the peanut seedlings from the aseptic vermiculite, washing the peanut seedlings clean by aseptic water, and selecting peanuts with consistent growth vigor in a flowerpot filled with the aseptic vermiculite. Placing in an artificial illumination incubator for 10 days, and supplementing equivalent sterile water according to the surface humidity of vermiculite every day.
Inoculating fusarium hypha in PDB culture medium at 180rmin-1Culturing at 25 deg.C in dark for 72h, filtering cultured Fusarium suspension with 4 layers of sterile lens-wiping paper to obtain spore suspension, and diluting with sterile water to concentration of 1 × 108Each mL-1And then standby. Simultaneously, 7 peanut rhizosphere strains, 180rmin, are respectively cultured by utilizing NB culture medium-1Culturing at 25 deg.C in dark for 48h, 3000g, centrifuging for 5min, and diluting with sterile water to obtain bacterial liquid with OD concentration600nmEach strain was pipetted 20mL into a 500mL sterile beaker for a total of 140mL and mixed until needed.
The experiment was set to 3 treatments: b + F treatment; f, processing; and (5) CK processing. After the peanuts are transplanted and cultured for 10 days, 10mL of 7 continuous cropping lost strain mixed liquor and 10mL of fusarium spore suspension are added to each peanut in the B + F treatment, 10mL of sterile water and 10mL of fusarium spore suspension are added to each peanut in the F treatment, and 20mL of sterile water is added to each peanut in the CK treatment. After further culturing in the artificial incubator for 10 days, plant samples were collected and the onset of root rot was determined (Table 6). As can be seen from Table 6, when the bacterial population composition (B + F) of the present invention was supplemented with the inoculated pathogenic bacteria treatment, the occurrence of diseases was significantly suppressed, and the incidence of diseases was reduced by 87.5% as compared with the treatment (F) with the inoculated pathogenic bacteria alone.
TABLE 6 peanut rhizosphere 7-strain bacterium combination for root irrigation application indoor peanut morbidity
Note: CK: sterile water treatment; b + F: 7 peanut rhizosphere bacteria combination and fusarium spore suspension; f: fusarium spore suspension. The incidence rate is (number of infected plants/total number of investigated plants) × 100%.
9. Method for determining seed soaking application of key bacterial composition
The field test selects the peanut soil for continuous cropping for more than 5 years. The experiment was started after peanut harvest with 14 treatments, 8 replicates for CK, 8 replicates for 7 bacteria combinations, 4 treatments for 4 bacteria combinations, 4 replicates for each treatment, 8 treatments for 2 bacteria combinations, 2 replicates for each treatment, and 6 wells of peanuts for one (table 7). Each repeat was ridged and spaced, with 15cm spacing between the wells.
TABLE 7 preparation of combinations of 7, 4 and 2 strains of bacteria in the peanut rhizosphere
Note: the effective viable count of each bacterium in each bacterium combination is the same. A: pantoea (Pantoea sp.), B (Pseudobacillus sp.), C (Enterobacter sp.), D (Paenibacillus sp.), E (Sporosarcina sp.), F (Lysinibacillus sp.), G (Pseudomonas aeruginosa sp.), etc.).
Firstly, 7 strains of bacteria are respectively cultured in NB culture medium, OD600nm of each strain is adjusted to be 1 by sterile water, each strain is respectively absorbed into a 100mL sterile centrifuge tube, and the strains are mixed in equal proportion concentration in different treatments, and the final concentration of the bacteria-conserving liquid is the same. And (3) selecting peanut seeds with consistent growth, soaking the peanut seeds in each treatment, sowing the seeds in a field plot after 12 hours, collecting plant samples after the peanuts grow for 30 days, and determining the disease condition of the root rot (table 8). It can be seen from table 8 that the control effect on plant root diseases is gradually enhanced with the increase of the flora combinations, wherein the control effect on 7 mixed flora is the best.
TABLE 8 determination of the control of floral root rot by combinations of 7, 4 and 2 strains of bacteria in the peanut rhizosphere
Note: the incidence rate is (number of infected plants/total number of investigated plants) × 100%; disease index ∑ (number of diseased plants at each stage × disease-grade value)/(total number of investigated plants × highest-grade value) × 100
Example 2
1. Method of producing a composite material
Selecting continuous cropping soil and non-continuous cropping soil of cotton to be tested;
performing pot experiment, and performing separation and identification on rhizosphere bacteria of cotton;
determining 7 strains of bacteria deleted in the rhizosphere of the continuous cropping cotton;
preparing a combination of 7 strains, 4 strains and 2 strains of bacteria at the cotton rhizosphere;
determining the disease inhibition of the combination of 7 strains, 4 strains and 2 strains of bacteria in the cotton rhizosphere;
determining the disease inhibiting performance of 7 strains of bacteria combination and continuous cropping cotton bacteria at the rhizosphere of cotton;
the root irrigation application is carried out on the combination of 7 strains of bacteria at the rhizosphere of cotton.
2. Test soil
The experimental field of cotton for 15 years of continuous cropping is selected as test soil, the experimental field of corn, soybean and cotton which are crop rotation in nearly five years is selected as control experimental soil, and the pathogenic bacteria of the test is cotton fusarium wilt (F.oxysporum.sp.vassinfectum (Atk.)).
3. Obtaining cotton rhizosphere bacteria
Firstly, removing soil around potted cotton plants by using a small steel shovel, carefully pulling out the cotton, slightly shaking the root system to remove large soil blocks, and then collecting the soil on the surface of the peanut rhizosphere by using a sterile brush; 1g of cotton rhizosphere soil is put into a 50mL sterile conical flask, 9mL sterile water is added, the temperature is 25 ℃, 180r/min, the shaking is carried out for 30min, and the table top is horizontally placed for 10 min. 1mL of the supernatant was aspirated and the fungus was removed with a filter of 5 μm pore size to prepare 10-1And (5) suspending the soil bacteria for later use. Subsequent dilution 10-2、10-3、10-4、10-5And 10-6Serial gradient bacterial suspensions, 200. mu.L of 10 each-4、10-5、10-6The soil suspension of concentration was evenly spread on NA medium, each concentration gradient was repeated 3 times, and incubated in the dark at 25 ℃ for 2 d. Selecting single bacterial colony from the plate with proper concentration gradient according to the shape, color and size of the bacteria, respectively selecting 50 strains of bacteria from the rhizosphere bacteria plate of the continuous cropping cotton, and using an inoculating needle to obtain each single bacterial colony in an NA culture mediumPlates were streaked 3 times to obtain pure colonies, numbered and stored. Pure fine colonies were picked up by an inoculating needle in NB medium and cultured at 25 ℃ at 180r/min for 24 h. And extracting the DNA of the bacteria by adopting a DNA extraction kit. The extracted DNA was subjected to PCR amplification of 16S rDNA. And detecting the fragment length of the PCR amplification product by using 1% agarose gel electrophoresis, then sending the PCR amplification product to Shanghai biological engineering technology service company Limited for sequencing, comparing the obtained bacterial sequences through an NCBI website database, and selecting a 16S rDNA sequence with the highest similarity.
4. Obtaining key bacteria of continuous cropping cotton rhizosphere deletion
With sterile blades from 10-1Surface bacteria were scraped from the treated bacterial plates in 1.5mL centrifuge tubes, and the plate bacterial DNA was extracted using the FastDNA SPIN Kit for soil Kit, quality checked, and then sent to the Bio-Corp for high throughput sequencing. The composition of all culturable bacteria in the cotton rhizosphere was analyzed (Table 9).
TABLE 9 high throughput sequencing identified Cotton rhizosphere culturable bacterial compositions
Note that R: rotating to culture bacteria plate in cotton rhizosphere; c: the continuous cropping cotton rhizosphere can be used for culturing bacterial plates.
Further adopting a plate separation culture method to identify related bacterial strains from the rhizosphere of the crop rotation cotton. In combination with the sequencing results of table 1, the continuous cropping cotton rhizosphere loss strains were bacillus (bacillaceae sp.), brevundimonas (caulobactraceae sp.), xanthomonas (xanthmonas sp.), acinetobacter (acinetobacter sp.), chitin phage (chitinophaceae sp.), you (optiututaceae sp.) and acidmicrobe (acidimiacross sp.).
5. Key bacterium combination for obtaining rhizosphere deletion of continuous cropping cotton
Respectively activating 7 continuous cropping cotton deletion strains by NB culture medium, centrifuging for 5min at 3000g, removing bacterial fermentation liquid), adding sterile water to adjust bacterial concentration to OD600nm1, stand by. The strains are randomly combined and mixed according to 7 strains, 4 strains and 2 strains, and the 7 strains are combined and treated by 1; the 4 strains are combined for 8 treatments; there were 10 treatments with 2 combinations of bacteria. The same concentration of each strain was ensured in each treatment. Sucking 1mL of each strain into a 10mL sterile centrifuge tube, adding sterile water to ensure that the concentration of each strain in each treatment is the same, and reversing the strains for several times to mix the bacterial suspension for later use.
6. Method for determining inhibition ability of key bacterial composition on pathogenic bacteria
The direct inhibition effect of the bacterial suspensions of the 7-bacterium combination, the 4-bacterium combination and the 2-bacterium combination on the cotton wilt hyphae is detected by adopting a flat plate direct confrontation method, and the influence of the bacterial suspensions of the 7-bacterium combination, the 4-bacterium combination and the 2-bacterium combination on the cotton wilt spore germination is detected by adopting a 96-well plate culture method (Table 10).
Table 10 cotton rhizosphere 7, 4 and 2 bacterial combinations were prepared as follows:
note: the effective viable count of each bacterium in each bacterium combination is the same. Bacillus (Bacillaceae sp.), B Brevundimonas (Caulobacter sp.), C Xanthomonas (Xanthomonas sp.), D Acinetobacter (Acinetobacter sp.), E Chitinophaga (Chitinophagaceae sp.), F Haemophilus (Opitutaceae sp.) and G acidibacterium (Acidiflores sp.).
It can be seen from table 11 that the inhibitory activity against pathogenic bacteria is gradually enhanced with the increase of the combinations of the bacterial colonies, and among them, 7 mixed bacterial colonies showed the best inhibitory effect against pathogenic bacteria.
TABLE 11 disease inhibition by combination of 7, 4 and 2 strains of cotton rhizosphere bacteria
Note: the bacteriostasis rate is (treatment-blank control)/blank control multiplied by 100%; the spore germination rate is the number of germinated spores/total spores multiplied by 100%
7. Determination of pathogenicity of 7 strains of bacteria combination and continuous cropping cotton bacteria in cotton rhizosphere
Respectively activating 7 strains of cotton rhizosphere with NB culture medium, culturing at 25 deg.C in dark for 24 hr, centrifuging at 3000g for 5min, discarding bacteria fermentation liquid, adding sterile water to adjust different bacteria concentrations to OD600nm1. 200. mu.L of each strain was put in a 2mL sterile tube and turned upside down several times to mix them thoroughly for use. Weighing 5g of continuous cropping cotton rhizosphere soil sample, placing the sample into a triangular flask containing 45mL of sterile water, and oscillating for 30min at 25 ℃. Standing on a table for 10min, sucking supernatant of 5mL soil suspension, removing fungi with filter membrane with pore diameter of 5 μm, and preparing into 10-concentration solution-1And (5) suspending the soil bacteria for later use.
Preparing NA culture medium, placing sterilized filter paper of 2cm × 4cm on one side, flattening, inoculating 200 μ L10-1The soil bacterium suspension is cultured for 48 hours at 25 ℃ in the dark. 100 mu L of peanut rhizosphere 7 strain mixed solution is added on the surface of the filter paper, and a fusarium cake with the thickness of 5mm is added on the other side. Sterile water was added to the control group. And (4) co-culturing at 25 ℃ in the dark for 3-7 days, and observing the growth diameter of the fungi (Table 12). From table 12, it can be seen that the bacterial flora combination of the present invention can significantly improve the inhibition ability against pathogenic bacteria in the rhizosphere of continuous cropping cotton, and the inhibition rate is improved by 72%.
TABLE 12 bacteriostatic ratio of cotton rhizosphere 7 strain combinations and continuous cropping peanut rhizosphere bacteria
Note: b: continuous cropping of cotton rhizosphere bacteria; b + F: combination of continuous cropping cotton rhizosphere bacteria +7 strains of cotton rhizosphere bacteria. The bacteriostasis rate is (processing-blank control)/blank control multiplied by 100%
8. Method for determining root irrigation application of key bacterium combination
Firstly, disinfecting cotton seeds by using 0.1 percent (mass percentage) of mercuric chloride solution, sowing the sterile cotton seeds in sterile vermiculite for 3d, then pulling out the cotton seedlings from the sterile vermiculite, washing the cotton seedlings clean by using sterile water, and selecting cotton with consistent growth in a flowerpot filled with the sterile vermiculite. Placing in an artificial illumination incubator for 10 days, and supplementing equivalent sterile water according to the surface humidity of vermiculite every day.
Picking cotton wilt hypha and inoculating in PDB culture medium for 180r min-1Culturing at 25 deg.C in dark for 72h, filtering cultured Fusarium suspension with 4 layers of sterile lens-wiping paper to obtain spore suspension, and diluting with sterile water to concentration of 1 × 108Each mL-1And then standby. Simultaneously, 7 cotton rhizosphere strains, 180rmin, are respectively cultured by utilizing NB culture medium-1Culturing at 25 deg.C in dark for 48h, 3000g, centrifuging for 5min, and diluting with sterile water to obtain bacterial liquid with OD concentration600nmEach strain was pipetted 20mL into a 500mL sterile beaker for a total of 140mL and mixed until needed.
The experiment was set to 3 treatments: b + F treatment; f, processing; and (5) CK processing. After the cotton is transplanted and cultured for 10 days, 10mL of 7 continuous cropping lost strain mixed liquor and 10mL of fusarium spore suspension are added to each cotton in the B + F treatment, 10mL of sterile water and 10mL of fusarium spore suspension are added to each cotton in the F treatment, and 20mL of sterile water is added to each cotton in the CK treatment. After further culturing in the artificial incubator for 20 days, plant samples were collected and the onset of blight was determined (Table 13). As can be seen from Table 4, the bacterial population composition (B + F) of the present invention significantly suppressed the occurrence of diseases when supplemented with the bacterial pathogen inoculation treatment, and the incidence of diseases was reduced by 87.5% as compared with the bacterial pathogen inoculation treatment (F) alone.
TABLE 13 cotton rhizosphere 7-strain bacterium combination for root irrigation application indoor cotton wilt disease incidence
Note: CK: sterile water treatment; b + F: combining 7 cotton rhizosphere bacteria and fusarium spore suspension; f: fusarium spore suspension. The incidence rate is (number of infected plants/total number of investigated plants) × 100%.
The embodiments show that the rhizosphere disease inhibiting capability is enhanced by using the key bacterium combination deleted from the rhizosphere of the continuous cropping plant, the rhizosphere protective wall is effectively constructed to inhibit the infection of pathogenic bacteria to the root of the plant, and the bacterial strain separated and screened from the rhizosphere of the crop plant is used for biological control. Firstly, indoor screening of rhizosphere lost strains of continuous cropping plants, and verification of bacteriostatic activity of different strain combinations; and then, the inoculation application is carried out indoors and in the field, so that the incidence rate of the plant soil-borne diseases can be effectively reduced. The method has the advantages of simplicity, easy implementation, easy popularization and application, low cost and remarkable bacteriostatic effect.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (8)
1. A method for obtaining biocontrol bacteria for preventing and treating continuous cropping plant root diseases comprises the following steps:
1) obtaining rhizosphere soil of a plot with continuous cropping time more than or equal to 5 years as continuous cropping soil, and obtaining rhizosphere soil of a non-continuous cropping plot of the same type as the continuous cropping soil as the non-continuous cropping soil;
2) separating and identifying the flora types of the continuous cropping soil and the non-continuous cropping soil to respectively obtain a continuous cropping flora and a non-continuous cropping flora;
3) comparing the continuous cropping flora with the non-continuous cropping flora, and screening out the flora existing in the non-continuous cropping flora and lost in the continuous cropping flora to obtain biocontrol bacteria for preventing and treating continuous cropping plant root diseases;
4) and verifying the bacteriostasis of the biocontrol bacteria.
2. The method for obtaining as claimed in claim 1, wherein the non-continuous cropping plots in step 1) include plots of approximately 5-year rotation of different plants.
3. The method for obtaining according to claim 1, wherein the method for separating and identifying the flora type in step 2) comprises a microbial culture method and a molecular biological sequencing method.
4. The acquisition method according to claim 1, further comprising in step 4) before verifying the bacteriostatic activity of the biocontrol bacteria: recombining the biocontrol bacteria, wherein the number of the strains in each combination is more than or equal to 2 and less than or equal to the number of the strains of all the biocontrol bacteria; the concentration of each bacterium was the same in each combination.
5. The obtaining method according to claim 1, wherein the method for verifying the bacteriostasis of the biocontrol bacteria in step 4) comprises a laboratory bacteriostasis test and/or a field test.
6. The method of claim 4, wherein the laboratory tests for bacteriostatic activity comprise testing the inhibitory effect of biocontrol bacteria on hyphae of pathogenic bacteria and/or testing the effect of biocontrol bacteria on spore germination of pathogenic bacteria.
7. The method for obtaining the plant fungi of claim 4, wherein the field test comprises the step of checking the bacterial inhibition of the rhizosphere anaplerosis biocontrol bacteria of the continuous cropping plants in the plots with the continuous cropping time being more than or equal to 5 years.
8. The method for obtaining the biocontrol bacteria of claim 7, wherein the method for restoring the biocontrol bacteria preferably comprises the steps of soaking plant seeds with the biocontrol bacteria, and checking the biocontrol bacteria for bacteriostasis; and/or, the biocontrol bacteria and the decomposed organic fertilizer are mixed and applied to crops to check the antibacterial activity.
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| CN115872812A (en) * | 2022-12-01 | 2023-03-31 | 贵州省植物保护研究所 | Combination of compound microbial fertilizer |
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| CN112314626B (en) * | 2020-11-09 | 2022-04-01 | 青岛农业大学 | Application of biological stimulin for improving activity of continuous cropping salvia miltiorrhiza seedlings |
| CN115872812A (en) * | 2022-12-01 | 2023-03-31 | 贵州省植物保护研究所 | Combination of compound microbial fertilizer |
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