CN116286365A - Screening method of bacillus for preventing and treating crop fusarium wilt - Google Patents
Screening method of bacillus for preventing and treating crop fusarium wilt Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P3/00—Fungicides
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
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Abstract
The invention discloses a screening method of bacillus for preventing and treating crop fusarium wilt, which mainly comprises the steps of firstly treating a soil sample at 80 ℃, eliminating other bacteria, only retaining bacillus, and then selecting a colony with vigorous growth on an LB culture medium containing fusaric acid, namely, the bacillus with fusaric acid resistance; and then the control effect on crop fusarium wilt is identified through bacteriostasis test and field test. The screening method has strong purposefulness, high screening efficiency and low cost; secondly, the strain obtained by the screening method is resistant to fusaric acid, so that the inhibition effect of the fusaric acid on biocontrol bacteria is avoided; in addition, the strain screened by the invention has good control effect on crop fusarium wilt, and has good control effect on different specialization types of fusarium oxysporum, and the control spectrum is wide; in addition, the fermentation broth using the strain of the invention also significantly promotes the improvement of yield and quality.
Description
Technical Field
The invention relates to the field of screening methods of biocontrol microorganisms, in particular to a screening method of bacillus for preventing and treating crop fusarium wilt.
The background technology is as follows:
fusarium oxysporum (Fusarium oxysporum) is a soil-borne pathogenic fungus which is distributed worldwide, has a wide host range, can cause the occurrence of over 100 plant blight diseases of melons, solanaceae, bananas, cottons, leguminosae, flowers and the like, and causes serious loss to agricultural production.
At present, the method for preventing and treating fusarium oxysporum-induced fusarium wilt mainly comprises the following steps: (1) rotation and continuous cropping. Because chlamydospores formed by pathogenic bacteria can survive in soil for a long time, the chlamydospores are difficult to effectively prevent and treat the fusarium wilt through agricultural measures such as crop rotation and continuous cropping. (2) chemical control. The chemical agent can not directly act on pathogenic bacteria, so that the control effect is poor, and the chemical control also has the problems of environmental pollution and the like. (3) grafting. Grafting is a relatively effective measure for preventing and treating fusarium wilt, but planting grafted seedlings not only increases the agricultural cost, but also reduces the quality and taste of fruits. (4) biological control. Biological control has the advantages of strong specialization, long lasting drug effect, difficult generation of drug resistance, environmental friendliness and the like, so that the biological control is one of the most economical and effective measures for controlling crop wilt and is more and more paid attention to people.
The Bacillus is a genus of bacteria, can generate various antibacterial active substances, has the characteristics of stress resistance, high temperature resistance, quick reviving, stronger secretase and the like, and can survive under the aerobic and anaerobic conditions; under the conditions of nutrition deficiency, drought and the like, spores are formed, and the spores can be germinated again into nutrients under proper conditions, so that bacillus is an important resource for developing microbial bactericides. The current method for screening bacillus mainly comprises the steps of firstly adopting antibacterial activity measurement to obtain antagonistic bacteria, then obtaining effective biocontrol bacteria (Zhang Han and the like, chinese biological control journal of China 2021,37 (05): 1058-1065, sun Zhengxiang and the like, university of Yangtze river (natural science edition) 2021,18 (04): 114-120) through greenhouse potting test and field plot test, wherein the method is time-consuming and labor-consuming, has low probability of obtaining effective biocontrol bacteria, and cannot meet the requirement of high flux.
Fusaric acid (Fusaric acid) is the main toxin produced by fusarium oxysporum, can cause wilting and death of crops, has strong antagonistic activity on bacillus, and can inhibit growth and colonization of bacillus rhizosphere by fusarium oxysporum existing in a large amount in plant rhizosphere by producing fusarium oxysporum, thereby inhibiting the control effect of biocontrol bacteria (Bacon CW et al J Appl Microbiol 2006;100 (1): 185-94). Therefore, bacillus which can not only resist fusaric acid but also have stronger antagonistic activity to fusarium oxysporum is screened, and the bacillus is expected to be a biocontrol bacterium for effectively preventing and controlling crop fusarium wilt. The studies by Hideoshi Toyoda et al (Toyoda, phytophology HJ.1988) have found that Fusarium acid inhibits the growth of Pseudomonas solanacearum, and that a mutant of Pseudomonas solanacearum can decompose Fusarium acid and thus develop resistance to Fusarium acid, which is effective in controlling tomato blight caused by fusarium.
At present, reports of rapid screening of biocontrol bacillus by taking tolerant fusaric acid as an index are not found.
Disclosure of Invention
The invention aims to solve the problems of low screening efficiency, poor effect and the like in the existing bacillus screening technology, and provides a bacillus screening method for preventing and treating crop fusarium wilt.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention discloses a screening method of bacillus for preventing and treating crop fusarium wilt, which comprises the following steps:
(1) Acquisition of bacillus: at the locus of crop blight, soil samples of healthy plant rhizosphere were collected, and then, according to 1: adding the soil sample into distilled water according to the weight ratio of 10, oscillating for 30min at 30 ℃ and 180rpm, taking out, and standing for 30min at room temperature; then taking 1mL of supernatant, placing the supernatant at 80 ℃ for 15min, and cooling the supernatant at room temperature to obtain a bacillus soil suspension in a spore state;
(2) Isolation of fusarium acid tolerant bacillus: diluting the soil suspension obtained in the step (1) by 100 times with distilled water; taking 100 mu L of diluted soil suspension, uniformly coating the diluted soil suspension on LB culture medium containing 20 mu g/mL of fusaric acid, culturing for 12 hours at 37 ℃, and selecting a colony with vigorous growth, namely bacillus capable of tolerating the fusaric acid;
(3) Antibacterial activity assay: testing the antibacterial activity of the fusarium acid-tolerant bacillus obtained in the step (2) on target bacteria by using crop fusarium wilt bacteria as the target bacteria through a flat plate counter culture method, and selecting antagonistic bacteria with strong antibacterial activity (antibacterial zone is more than 0.5 cm);
(4) And (3) carrying out greenhouse evaluation and field evaluation on the antagonistic bacteria obtained in the step (3) according to a conventional method, and selecting a strain with the control effect of more than 60%, namely the bacillus which is resistant to fusaric acid and has the control effect on crop fusarium wilt.
According to the screening method, the LB medium containing the fusarium acid in the step (2) comprises the following components in percentage by weight: 1% of tryptone, 1% of yeast extract, 0.5% of sodium chloride, 1% of agar powder, 20 mug/mL of fusaric acid as a final concentration and the balance of distilled water.
The preparation method of the LB culture medium containing the fusaric acid comprises the following steps: comprising the following steps: preparing LB culture medium according to conventional method, adding Fusarium acid solution at 50deg.C before solidification to make the final concentration of Fusarium acid 20 μg/mL, and shaking thoroughly to prepare flat plate.
Fusarium acid can be purchased in the market (such as Sigma company), dissolved in ethanol to prepare 20mg/mL Fusarium acid solution, and stored at-20deg.C.
According to the above screening method, the plate-opposing culture method in the step (3) is as follows:
(a) Inoculating target bacteria on a PDA plate by taking crop fusarium wilt bacteria as the target bacteria, and culturing for 7 days at a constant temperature of 25 ℃ until pathogenic bacteria grow on the whole culture dish;
(b) Taking the fungus blocks with the diameter of 6mm in the step (a) by using a puncher, and inoculating the fungus blocks in the center of another PDA culture medium; and (3) inoculating the fusarium acid-tolerant bacillus obtained in the step (2) of the screening method at a position 2.5cm away from the bacterial mass by using a sterilized toothpick, standing and culturing for 5-7d at 25 ℃, and selecting antagonistic bacteria with a bacteriostasis zone of more than 0.5cm, namely bacillus which has bacteriostasis activity on crop fusarium wilt and is tolerant to the fusarium acid.
The crop fusarium wilt is pathogenic bacteria fusarium oxysporum of crop fusarium wilt, and the pathogenic bacteria of different crop fusarium wilt are different fusarium oxysporum specialization; such as tomato specialization, cucumber specialization, watermelon specialization, etc.
According to the screening method, the crop fusarium wilt in the step (3) refers to tomato fusarium wilt, watermelon fusarium wilt, cucumber fusarium wilt or the like.
The pathogenic bacteria of the tomato blight is Fusarium oxysporum tomato specialization (Fusarium oxysporum f.sp. lycopersici Snyder et Hansen).
The pathogenic bacteria of watermelon Fusarium wilt are Fusarium oxysporum watermelon specialization (Fusarium oxysporum. Sp. Hiveum (e.f. smith) Wollen).
The pathogenic bacteria of cucumber fusarium wilt are fusarium oxysporum cucumber specialization (Fusarium oxysporum (schl.) f.sp.
According to the above screening method, the method for evaluating a greenhouse in the step (4) is as follows:
(a) Preparing biocontrol bacterium fermentation liquid: inoculating bacillus obtained in the screening method into 5mL of liquid LB culture medium, carrying out shaking culture for 12h at 180rpm and 37 ℃, transferring the bacillus into 200mL of LB liquid culture medium according to the weight percentage ratio of 1%, carrying out shaking culture for 48h at 180rpm and 37 ℃ to obtain biocontrol bacteria fermentation broth, and calculating the concentration of thallus (CFU/mL) in the fermentation broth by using a plate counting method;
(b) Accelerating germination and seedling raising of crop seeds, and applying 3mL of biocontrol bacteria fermentation liquor to the root of each seedling when two leaves are in one heart;
(c) Collecting pathogenic bacteria Fusarium oxysporum pieces with diameter of 6mm with a puncher, inoculating into a liquid culture medium of PDB, culturing at 25deg.C and 180rpm for 5 days, filtering with sterilized gauze, counting spores, mixing 1L pathogenic bacteria liquid with 20kg sterilized soil in a soil mixer, and final concentration of 5.0X10 5 Spores/gram soil; filling sterilized soil containing pathogenic bacteria into a small flowerpot;
(d) Transplanting seedlings: transplanting seedlings after applying biocontrol bacteria fermentation liquor for 3 days, aligning roots in the seedling transplanting process, cutting fibrous roots (two centimeters are left) by scissors, performing root injury treatment, transplanting the seedlings into a small flowerpot, and setting three repetitions of each treatment; applying a second biocontrol bacterium fermentation liquor three days after seedling transplanting, using the same dosage as the first biocontrol bacterium fermentation liquor, watering and observing on time, surveying the illness state after 10 days, and calculating the illness state index and the prevention and treatment effect; selecting a strain with a control effect of 60% -100%; wherein the calculation formulas of the disease index and the prevention and treatment effect are as follows:
disease index = Σ (number of disease plants at each stage×representative value at each stage)/(total number of investigation×representative value at highest stage) ×100;
control effect (%) = [ (control disease index-treatment disease index)/control disease index ] ×100.
Compared with the prior art, the invention has the advantages and beneficial effects that: (1) The method has strong screening purpose and high screening efficiency. The antagonistic bacteria obtained by the conventional antibacterial activity measurement not only comprise bacillus, but also comprise other bacteria, and the quantity of the antagonistic bacteria is large, so that the workload of a later greenhouse potting test is large; the method of the invention firstly processes the collected sample at 80 ℃ to kill negative bacteria, only retains bacillus, and greatly reduces the quantity of antagonistic bacteria screened by the conventional method, thereby greatly reducing the scale of greenhouse potting experiments and greatly reducing the workload, so the method of the invention has high screening efficiency and low cost. (2) The method of the invention takes the fusaric acid as the selective pressure to screen and obtain the fusaric acid-resistant biocontrol bacteria, thereby avoiding the inhibition effect of the fusaric acid secreted by rhizosphere fusarium on the biocontrol bacteria and ensuring that the biocontrol bacteria obtained by screening have high and stable control effect. (3) The strain screened by the method has good prevention effect on different specialization types of fusarium oxysporum, and has wide prevention and control spectrum. (4) The bacillus screened by the method has good effect of preventing and treating crop fusarium wilt, and remarkably improves the yield and the quality.
Drawings
FIG. 1 is a bar graph of tomato yield treated with strain B31, 30833, F68.
Detailed Description
The invention is further illustrated by the following specific examples, which do not limit the scope of the invention in any way. Unless otherwise indicated, the methods described in the examples below are all conventional methods and the reagents are all conventional biochemical reagents.
EXAMPLE 1 screening test of Fusarium acid-resistant Bacillus of the invention
The method is carried out according to the following steps:
(1) 48 parts of soil samples are collected in the rhizosphere of healthy plants at the place where cucumber, tomato and watermelon fusarium wilt occur, 1g of each soil sample is collected, the soil samples are respectively added into a triangular flask filled with 10mL of distilled water, shake is carried out for 30min at 30 ℃ and 180rpm, and the soil samples are taken out and then are stood for 30min at room temperature; 1mL of the supernatant was further taken and subjected to treatment at 80℃for 15 minutes, and cooled at room temperature, to obtain soil suspensions of Bacillus in the form of spores, respectively.
(2) Diluting the soil suspension obtained in the step (1) by 100 times with distilled water; 100 mu L of diluted soil suspension is taken, uniformly coated on LB culture medium (LB culture medium is prepared according to a conventional method) containing 20 mu g/mL of fusaric acid, cultured for 12 hours at 37 ℃, and the colony with vigorous growth is selected, namely bacillus capable of tolerating the fusaric acid.
(3) Determination of antibacterial activity:
(a) Cucumber fusarium wilt bacteria (fusarium oxysporum cucumber specialization (schl.) f.sp.cucuminum owen), watermelon fusarium wilt bacteria (fusarium oxysporum f.sp.hiveum (e.f.smith) Wollen)) and tomato fusarium wilt bacteria (fusarium oxysporum tomato specialization (fusarium oxysporum f.sp.lycopersicum Hansen)) saved by plant protection research institute of the university of river north are used as target bacteria, and the target bacteria are inoculated on PDA plates respectively and cultivated at constant temperature of 25 ℃ for 7d until the pathogenic bacteria grow in the whole culture dish;
(b) Taking the fungus blocks with the diameter of 6mm in the step (a) by using a puncher, and inoculating the fungus blocks in the center of another PDA culture medium; inoculating the fusarium acid-tolerant bacillus obtained in the step (2) of the screening method with a sterilized toothpick at a position 2.5cm away from the fungus block, standing and culturing for 5-7d at 25 ℃, and selecting antagonistic bacteria with a bacteriostatic zone of more than 0.5 cm; as a result, 68 strains of bacillus which have antibacterial activity on three fusarium oxysporum specialization types and are tolerant to fusaric acid were obtained; the bacterial strains named 30833, B31 and F68 (the bacteriostasis of which is shown in table 1) are included, and the 3 bacterial strains have good bacteriostasis effect as can be seen from table 1.
TABLE 1 test results (cm) of part of the Bacillus of the invention which was resistant to Fusarium acid and antagonistic to Fusarium oxysporum
EXAMPLE 2 preparation of Fusarium acid-resistant Bacillus fermentation broth obtained by the present invention
The method is carried out according to the following steps:
inoculating the fusarium acid-resistant bacillus obtained in the step 1 in 5mL of liquid LB culture medium respectively, and carrying out shaking culture at 180rpm and 37 ℃ for 12 hours; transferring the strain into 200mL of liquid LB culture medium according to the proportion of 1% by weight, culturing for 48h at 180rpm and 37 ℃ in an oscillating way to obtain biocontrol bacteria fermentation liquid, and calculating the concentration of bacteria (CFU/mL) in the fermentation liquid by using a plate counting method.
Example 3 greenhouse control test of Fusarium acid resistant Strain of the invention on cucumber fusarium wilt
The process is carried out as follows
(1) Sprouting and seedling raising of cucumber (Zhongnong No. 6, high-sensitivity cucumber fusarium wilt) seeds: soaking cucumber seeds in warm water at 55deg.C for 30min, washing with clear water, soaking in clear water for 8 hr, wrapping with wet gauze, germinating in a 25 deg.C incubator overnight, and culturing seedlings in vermiculite.
(2) When two leaves and one heart of cucumber grow out, the first biocontrol bacterium is applied, and 3mL of biocontrol bacterium fermentation broth prepared in the example 2 is applied to the root of each seedling.
(3) Preparation of pathogenic bacteria FOC 1-2-11 (cucumber specialization) bacterial liquid and soil mixing: taking Fusarium oxysporum pieces with diameter of 6mm with a puncher, inoculating into a PDB liquid culture medium, shaking and culturing at 25deg.C and 180rpm for 5 days, filtering with sterilized gauze, counting spores, mixing 1L pathogenic bacteria liquid with 20kg sterilized soil in a soil mixer, and final concentration of 5.0X10 5 Spores/gram soil, placed in a small flowerpot.
(4) Transplanting seedlings: transplanting seedlings after the biocontrol bacteria are grafted for 3 days, aligning roots of cucumber seedlings in the seedling transplanting process, cutting fibrous roots (two centimeters are left) by scissors, performing root injury treatment, transplanting the seedlings into a small flowerpot, and setting three repetitions of each treatment.
(5) Applying secondary biocontrol bacteria: the second biocontrol bacteria fermentation liquor is applied three days after seedling transplanting, the usage amount is the same as that of the first biocontrol bacteria fermentation liquor, watering and observing are carried out on time, and the disease condition is investigated after 10 days.
Grading standard of cucumber fusarium wilt in seedling stage (reference to standard reported by Zhou Gongmei, mao Aijun, etc.): level 0: no symptoms; stage 1: yellowing of cotyledons, no wilting; 2 stages: wilting cotyledons or slightly wilting plants; 3 stages: obvious wilting or dwarfing of plants; 4 stages: plants die.
Disease index = Σ (number of disease plants at each stage×representative value at each stage)/(total number of investigation×representative value at highest stage) ×100.
Control effect (%) = [ (control disease index-treatment disease index)/control disease index ] ×100.
As a result, 5 strains of the strain have high control effect on cucumber fusarium wilt, wherein the control effects of 30833, B31 and F68 on cucumber fusarium wilt are 85.72%, 87.53% and 86.19% respectively. The 3 strains are proved to have high control effect on cucumber fusarium wilt.
Example 4 greenhouse efficacy test of Fusarium acid resistant Strain of the invention against tomato blight
The method is carried out according to the following steps:
(1) Accelerating germination and seedling raising of tomato seeds (Namei, high-sensitivity tomato fusarium wilt): the tomato seeds are washed by clear water, wrapped by wet gauze after washing, sprouted overnight in a temperature box at 25 ℃, and then grown in vermiculite.
(2) When tomatoes grow into two leaves and one heart, applying first biocontrol bacteria, diluting 150mL of biocontrol bacteria fermentation liquid prepared in the example 2 by one time with distilled water, and filling the biocontrol bacteria fermentation liquid into a seedling tray.
(3) Preparation of pathogenic bacterium FQ 143 (tomato specialization) bacterial liquid: punching Fusarium oxysporum pieces with diameter of 6mm with a puncher, inoculating into PDB culture medium, shaking culture at 25deg.C and 180rpm for 5d, filtering with sterilized gauze, and counting spores with concentration of 4.×10 7 Spores/ml were placed in a flowerpot.
(4) Transplanting seedlings: transplanting seedlings after the biocontrol bacteria are grafted for 3 days, aligning roots of tomato seedlings in the seedling transplanting process, cutting fibrous roots (two centimeters are left) by scissors, performing root injury treatment, soaking the roots below pathogenic bacteria liquid for 60 minutes, and transplanting the seedlings into a flowerpot, wherein three repetitions of treatment are arranged.
(5) The disease was investigated for the first time after about 7 days and for the second time after two weeks by on-time watering observation.
Grading standard of tomato wilt in seedling stage (disease investigation is carried out with reference to the standard reported by Bai Minzhan, zheng Guibin, etc.): level 0: no symptoms; stage 1:1 or 2 cotyledons are obviously yellowing to fall off; 2 stages: 3 or 4 leaves of Zhen She Bianhuang, leaf wilting and sagging; 3 stages: 5 or 6 true She Bianhuang, or true leaf wilting and sagging; 4 stages: the whole plant seriously wilts to die.
Disease index = Σ (number of disease plants at each stage×representative value at each stage)/(total number of investigation×representative value at highest stage) ×100.
Control effect (%) = [ (control disease index-treatment disease index)/control disease index ] ×100.
As a result, the prevention and treatment effects of the fusarium acid-resistant strains 30833, B31 and F68 of the invention on tomato fusarium wilt are 91.95%, 97.12% and 90.69% respectively; the three strains are proved to have good control effect on tomato wilt.
Example 5 greenhouse control test of Fusarium acid resistant Strain of the present invention on watermelon fusarium wilt
The method is carried out according to the following steps:
(1) Accelerating germination and seedling raising of watermelon seeds (early-season disease 84-24, high-sensitivity watermelon fusarium wilt): soaking watermelon seeds in 55 deg.C warm water for 30min, stirring, washing with clear water, soaking in clear water for 8 hr, wrapping with wet gauze, germinating in 25 deg.C incubator overnight, and culturing in vermiculite.
(2) When the watermelon grows out with two leaves and one heart, the first biocontrol bacterium is applied, and 3mL of biocontrol bacterium fermentation liquid prepared in the example 2 is applied to the root of each seedling.
(3) Preparation of pathogenic bacterium XG0010 (watermelon specialization) bacterial liquid and soil mixing: punching Fusarium oxysporum with diameter of 6mm with a puncher, inoculating into PDB culture medium, shaking culture at 25deg.C and 180rpm for 5d, filtering with sterilized gauze, counting spores, mixing 1L bacterial liquid with 20kg sterilized soil in a soil mixer, and final concentration of 5.0X10 5 Spore/gram of soil is placed in a flowerpot.
(4) Transplanting seedlings: transplanting seedlings after the biocontrol bacteria are grafted for 3 days, aligning the roots of the watermelon seedlings in the seedling transplanting process, cutting fibrous roots (two centimeters are left) by scissors, performing root injury treatment, transplanting the watermelon seedlings into a flowerpot, and setting three repetitions of each treatment.
(5) Applying secondary biocontrol bacteria: the second biocontrol bacteria fermentation liquor is applied three days after seedling transplanting, the usage amount is the same as that of the first biocontrol bacteria fermentation liquor, watering and observing are carried out on time, and the disease condition is investigated after 10 days.
Grading standard of seedling stage watermelon fusarium wilt (reference to standard reported by Wen, wang Xiqing and the like for disease investigation): level 0: the plants grow normally; stage 1: leaf or stem wilting area accounts for 1/4 or less than 1/4 of the whole plant; 2 stages: leaf or stem wilting area accounts for 1/2 of the whole plant; 3 stages: leaf or stem wilting area accounts for 3/4 of the whole plant; 4 stages: the whole plant wilts and dies.
Disease index = Σ (number of disease plants at each stage×representative value at each stage)/(total number of investigation×representative value at highest stage) ×100.
Control effect (%) = [ (control disease index-treatment disease index)/control disease index ] ×100.
As a result, the prevention and treatment effects of the strains 30833, B31 and F68 of the invention on the watermelon fusarium wilt are 88.92%, 92.09% and 89.61%, respectively, and the results show that the prevention and treatment effects of the three strains of the invention on the watermelon fusarium wilt are good.
Example 6 field control and yield test of the inventive strains B31, 30833 and F68 against tomato blight
1. Test materials
1. The tomato variety is Pu Lin Wangsi.
2. The test biocontrol strains were 30833, B31 and F68.
2. Test method
The test was carried out seriously in the greenhouse tomato blight of the Qingding city, xingxing county, hebei province. Before tomato transplanting, 20mL of biocontrol bacteria fermentation liquid of strains B31, 30833 and F68 prepared in example 2 is added into each tomato seedling, and transplanting is carried out after 12h of treatment. Respectively applying B31, 30833 and F68 biocontrol bacteria fermentation liquid (with the concentration of 1 hundred million CFU/mL) prepared according to the example 2 in a drip irrigation mode after transplanting, wherein the application amount is 500L/mu; each treatment was performed in 5 rows and 4 replicates. The water treatment was set as a control. And (3) investigating the incidence rate of fusarium wilt in the flowering and fruiting period of tomatoes, and measuring and comparing the tomato yield and the red fruit rate in the biocontrol bacteria treatment area and the non-treatment area after the growing period is finished.
The classification standard, disease index and prevention and treatment effect calculation formula of tomato wilt are the same as that of example 4.
As a result (see Table 2 and FIG. 1), the field control effects of strains B31, 30833 and F68 on tomato blight were 75.53%, 74.73% and 77.37%, respectively. Tomato yields increased by 17.23%, 23.87%, 25.28% after B31, 30833 and F68 treatments, respectively, and the three strains also promoted fruit redness, with increased redness rates of 35.60%, 38.02%, 42.24%, respectively. The results show that the strains 30833, B31 and F68 have good field control effect on tomato wilt, obviously increase tomato yield and improve tomato fruit quality.
TABLE 2 control effect of B31, 30833 and F68 on tomato blight in greenhouse
| Strain name | Index of disease condition | Preventing effect (%) |
| F68 | 12.28 | 77.37 |
| 30833 | 13.28 | 74.73 |
| B31 | 13.28 | 75.53 |
| CK | 54.27 | - |
Claims (8)
1. A screening method of bacillus for controlling crop wilt, comprising the steps of:
(1) Acquisition of bacillus: at the locus of crop blight, soil samples of healthy plant rhizosphere were collected, and then, according to 1: adding the soil sample into distilled water according to the weight ratio of 10, oscillating for 30min at 30 ℃ and 180rpm, taking out, and standing for 30min at room temperature; then taking 1mL of supernatant, placing the supernatant at 80 ℃ for 15min, and cooling the supernatant at room temperature to obtain a bacillus soil suspension in a spore state;
(2) Isolation of fusarium acid tolerant bacillus: diluting the soil suspension obtained in the step (1) by 100 times with distilled water; taking 100 mu L of diluted soil suspension, uniformly coating the diluted soil suspension on LB culture medium containing 20 mu g/mL of fusaric acid, culturing for 12 hours at 37 ℃, and selecting a colony with vigorous growth, namely bacillus capable of tolerating the fusaric acid;
(3) Antibacterial activity assay: testing the antibacterial activity of the fusarium acid-tolerant bacillus obtained in the step (2) on target bacteria by using crop fusarium wilt bacteria as the target bacteria through a flat plate counter culture method, and selecting antagonistic bacteria with strong antibacterial activity (antibacterial zone is more than 0.5 cm);
(4) And (3) carrying out greenhouse evaluation and field evaluation on the antagonistic bacteria obtained in the step (3) according to a conventional method, and selecting a strain with the control effect of more than 60%, namely the bacillus which is resistant to fusaric acid and has the control effect on crop fusarium wilt.
2. The screening method according to claim 1, wherein the composition of the LB medium containing Fusarium acid in the step (2) and the weight percentage thereof are: 1% of tryptone, 1% of yeast extract, 0.5% of sodium chloride, 1% of agar powder, 20 mug/mL of fusaric acid as a final concentration and the balance of distilled water.
3. The screening method according to claim 1, wherein the plate-opposing culture method in the step (3) is as follows:
(a) Inoculating target bacteria on a PDA plate by taking crop fusarium wilt bacteria as the target bacteria, and culturing for 7 days at a constant temperature of 25 ℃ until pathogenic bacteria grow on the whole culture dish;
(b) Taking the fungus blocks with the diameter of 6mm in the step (a) by using a puncher, and inoculating the fungus blocks in the center of another PDA culture medium; and (3) inoculating the fusarium acid-tolerant bacillus obtained in the step (2) of the screening method at a position 2.5cm away from the bacterial mass by using a sterilized toothpick, standing and culturing for 5-7d at 25 ℃, and selecting antagonistic bacteria with a bacteriostasis zone of more than 0.5cm, namely bacillus which has bacteriostasis activity on crop fusarium wilt and is tolerant to the fusarium acid.
4. The screening method according to claim 1, wherein the crop wilt in the step (3) is tomato wilt, watermelon wilt or cucumber wilt.
5. The screening method according to claim 4, wherein the pathogenic bacteria of tomato blight is Fusarium oxysporum tomato specialization (Fusarium oxysporum f.sp.lycopersici Snyder et Hansen).
6. The screening method according to claim 4, wherein the pathogenic bacteria of watermelon fusarium wilt is fusarium oxysporum watermelon specialization (Fusarium oxysporum f.sp.hiveum (e.f.smith) Wollen).
7. The screening method according to claim 4, wherein the pathogenic bacteria of cucumber fusarium wilt is fusarium oxysporum cucumber specialization (Fusarium oxysporum (schl.) f.sp.
8. The method according to claim 1, wherein the method for evaluating a greenhouse in the step (4) is as follows:
(a) Preparing biocontrol bacterium fermentation liquid: inoculating bacillus obtained in the screening method of claim 1 into 5mL of liquid LB culture medium, culturing for 12 hours at 180rpm and 37 ℃ in an oscillating way, transferring into 200mL of LB liquid culture medium according to a weight percentage ratio of 1%, culturing for 48 hours at 180rpm and 37 ℃ in an oscillating way, obtaining biocontrol bacteria fermentation broth, and calculating the concentration of bacteria (CFU/mL) in the fermentation broth by using a plate counting method;
(b) Accelerating germination and seedling raising of crop seeds, and applying 3mL of biocontrol bacteria fermentation liquor to the root of each seedling when two leaves are in one heart;
(c) Collecting pathogenic bacteria Fusarium oxysporum pieces with diameter of 6mm with a puncher, inoculating into a liquid culture medium of PDB, culturing at 25deg.C and 180rpm for 5 days, filtering with sterilized gauze, counting spores, mixing 1L pathogenic bacteria liquid with 20kg sterilized soil in a soil mixer, and final concentration of 5.0X10 5 Spores/gram soil; filling sterilized soil containing pathogenic bacteria into a small flowerpot;
(d) Transplanting seedlings: transplanting seedlings after applying biocontrol bacteria fermentation liquor for 3 days, aligning roots in the seedling transplanting process, cutting fibrous roots (two centimeters are left) by scissors, performing root injury treatment, transplanting the seedlings into a small flowerpot, and setting three repetitions of each treatment; applying a second biocontrol bacterium fermentation liquor three days after seedling transplanting, using the same dosage as the first biocontrol bacterium fermentation liquor, watering and observing on time, surveying the illness state after 10 days, and calculating the illness state index and the prevention and treatment effect; selecting a strain with a control effect of 60% -100%; wherein the calculation formulas of the disease index and the prevention and treatment effect are as follows:
disease index = Σ (number of disease plants at each stage×representative value at each stage)/(total number of investigation×representative value at highest stage) ×100;
control effect (%) = [ (control disease index-treatment disease index)/control disease index ] ×100.
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