CN112920963B - Biocontrol strain and biocontrol microbial inoculum for preventing and treating cotton wilt and preparation method and application thereof - Google Patents
Biocontrol strain and biocontrol microbial inoculum for preventing and treating cotton wilt and preparation method and application thereof Download PDFInfo
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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
- A01N63/27—Pseudomonas
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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
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention relates to a biocontrol strain for preventing and treating cotton wilt, which is named as Pseudomonas alloputida Pa2, the preservation date is 2020, 11 and 9 days, and the preservation number is as follows: CGMCC NO.21149; the biocontrol strain has the advantages of simple production process, short culture time and low cost, and the biocontrol preparation for preventing and treating cotton wilt can be obtained by the strain through a simple, quick and low-cost preparation method; the biological control agent has a very obvious bacteriostatic effect, and experiments prove that the biological control agent provided by the invention has a control effect on cotton wilt of 74.48% under a greenhouse potting condition, has a control effect on cotton wilt of 70.11% under a field condition, is obviously superior to a control, and further greatly promotes the income increase of farmers.
Description
Technical Field
The invention relates to the technical field of microorganism application, in particular to a biocontrol strain and a biocontrol microbial inoculum for preventing and treating cotton wilt, and a preparation method and application thereof.
Background
The cotton wilt is one of two cotton diseases in the world, which affects cotton production in various cotton producing countries in the world to different degrees, and China is also deeply harmed by the disease. Fusarium oxysporum wilting specialized fungus (Fusarium oxysporum f.sp.vasifectum) is a pathogenic bacterium of cotton Fusarium wilt, and the fungus can easily survive under natural conditions, can produce conidia and is mainly transmitted through soil, seeds and fertilizers. Fusarium oxysporum usually only infects a few plants such as cotton, okra and kenaf, and can infect various plants such as wheat, solanaceae, leguminosae, melon and tobacco under the inoculation condition of a greenhouse or a disease nursery. The cotton wilt is a typical vascular bundle disease, and cotton can be infected during the whole growth and development period. There are many types of disease symptoms, including a withered type, a yellow type, a crinkled type, a yellow net pattern, a red leaf type and the like in a seedling stage; the bud stage has wrinkle shrinkage type, withered spot type, half-edge yellowing type, top withered type, light stalk type, etc. The disease often causes a large amount of dead seedlings in the early stage of cotton, and leaves and buds in the later stage drop off or even wither. The disease rate of the cotton wilt in the serious disaster area of China is up to 60-70%, the seedling death rate is up to 35-40%, and the yield is generally reduced by about 20-30%. The prevention and control of cotton wilt is one of the important ways to raise cotton yield.
The main methods for preventing and treating cotton wilt at present include drug prevention and treatment, scientific field management and selection of disease-resistant varieties. These methods have their own advantages and limitations. The chemical agent has a certain effect in the prevention and treatment of cotton diseases, and is widely applied to the prevention and treatment of cotton wilt. The seeds are usually soaked with diluted antiseptic 402 liquid or carbendazim suspension before sowing to kill pathogenic bacteria. Common sterilization pesticides include carbendazim, thiophanate methyl, 56% kresoxim-methyl chlorothalonil, 41% polyastatinepyrimethanil, kehuang cumic, 20% silazole prochloraz, cumin yellow factor, cotton Sanqing and the like. The chemical agent has obvious treatment effect on diseased plants and local soil in sporadic disease areas. However, chemical agents are highly toxic, most of the chemical agents are volatile, and pesticides enter human bodies for administration through mouth and respiratory tract or directly contact with the human bodies, so that acute poisoning is easily caused. Most of the chemical drugs have the properties of carcinogenesis, teratogenesis and mutagenesis, and after entering the food chain, the pesticide can be accumulated in all levels of consumers, thereby seriously harming the health of people and animals.
Scientific field management comprises crop rotation, deep ploughing in autumn, reasonable close planting, timely disease seedling removal and the like. But is influenced by planting habits and natural environment, rotation has certain difficulty in popularization and application, fusarium wilt bacteria can survive in soil for a long time, short-term rotation cannot achieve obvious control effect, and long-term rotation is not easy to implement. The field management methods such as deep ploughing in autumn and reasonable close planting cannot thoroughly kill pathogenic bacteria, and the prevention and treatment effect on cotton wilt is limited.
The application of disease-resistant varieties is an economic and effective disease prevention measure, and the application is wide at present. But the breeding period of the disease-resistant variety is longer, and the research and development cost is higher; some varieties have resistance which can not meet the production requirement; the fusarium oxysporum f.sp.cubense is easy to change and has the phenomenon of pathogenicity differentiation, and when a single disease-resistant variety is cultivated in a large area, the risk of losing disease resistance exists.
Therefore, the development cost is low, the effect is good, the environment is friendly, the method is healthy and sustainable, and the method is a difficult problem which is urgently needed to be solved in the prevention and treatment of the cotton wilt at present. Although some biocontrol bacteria for cotton wilt are researched at present, the field control effect of the biocontrol bacteria is always poor due to the special biological characteristics of the cotton wilt, and the biocontrol bacteria which are successfully popularized in a large area at the production line are not developed up to now.
Disclosure of Invention
The invention aims to solve the defects of the prior art, provides a bacterial strain capable of preventing and treating cotton wilt and a biocontrol microbial inoculum containing the bacterial strain, and can be well applied to the prevention and treatment of cotton wilt.
In order to realize the purpose of the invention, the following technical scheme is adopted:
a biocontrol strain for preventing and treating cotton wilt is named as Pseudomonas alloputida Pa2, the preservation date is 11 months and 9 days in 2020, and the preservation address is as follows: china general microbiological culture Collection center (address: west Lu No. 1 Hospital No. 3, north Chen, chaoyang, beijing) with the following preservation number: CGMCC NO.21149.
The biocontrol strain Pseudomonas alloputida Pa2 for preventing and treating cotton wilt is obtained by separating in 2019 soil around leek roots in a certain farm in Lishui area of Nanjing City.
The separation method comprises the following steps: taking the leek root soil by a 5-point sampling method, respectively filling the leek root soil into sterilized sealed plastic bags, and immediately taking the leek root soil back to a laboratory. Adding 5g of soil sample into 45mL of sterile water, carrying out shaking culture on a shaking table at 150r/min for 1h, filtering by using sterile gauze to obtain a suspension, and obtaining a 10-6-fold dilution solution by adopting a 10-fold serial dilution method. Respectively taking 100 mu L of diluent, uniformly coating the diluent on an LB flat plate, culturing the diluent in a constant temperature incubator at 28 ℃ for 48 hours, selecting single colonies with different colors, glossiness, sizes and types, streaking and purifying strains, uniformly numbering the strains, transferring the strains into an LB inclined plane culture medium, preserving the strains by using 80% sterilized glycerol, and preserving the strains at-70 ℃ for later use.
Plate confrontation method: selecting cotton wilt bacterium cake (5 mm) cultured for 2d, placing in the center of PDA plate, perforating at three symmetrical points (diameter 5 mm) at a distance of 25mm from the bacterium cake, injecting 100 μ L fermentation culture solution of test strain, and adding 100 μ L LNB as control. After culturing for 48 hours at the constant temperature of 28 ℃, observing and measuring the size of the inhibition zone, repeating the experiment three times, and selecting the strain with the largest inhibition zone, namely Pseudomonas alloputida Pa2.
Biological characteristics: gram-negative bacteria, rod-shaped or oval, the size of the bacteria is 1.5-5.0 μm long and 0.5-1 μm wide, and the bacteria have flagella, no spores and no capsules. Colony morphology: round, smooth surface, opaque, complete edge.
The biocontrol microbial inoculum containing the Pseudomonas alloputida Pa2 also belongs to the protection scope of the application, and the preparation method comprises the steps of activating the strain Pa2, culturing seeds and carrying out amplification culture.
The specific preparation method preferably comprises the following steps: carrying out shake culture on Pseudomonas alloputida Pa2 in an LB culture medium at 25-30 ℃ and 150-200 rpm for 12-24 h to obtain a seed solution; inoculating the seed liquid into a basic salt culture medium with the inoculation amount of 1%, and performing shaking culture at the temperature of 25-30 ℃ and the speed of 150-200 rpm to obtain the total viable bacteria concentration of 1 multiplied by 10 9 ～1×10 10 CFU/mL biocontrol microbial inoculum finished product.
The invention also provides application of the biocontrol strain or the biocontrol microbial inoculum in preventing and treating cotton wilt.
The specific method comprises the following steps: before use, the prepared biocontrol microbial inoculum is diluted to 1 multiplied by 10 7 CFU/mL, root irrigation treatment.
The invention has the following beneficial effects:
(1) The biocontrol microbial inoculum contains a specific strain Pseudomonas alloputida Pa2 for inhibiting the growth of pathogenic bacteria of cotton wilt, and the bacteriostatic effect is very obvious, and experiments prove that the biocontrol microbial inoculum provided by the invention has the control effect on the cotton wilt of 74.48 percent under the greenhouse potting condition and 70.11 percent under the field condition, and is obviously superior to a control.
(2) The Pseudomonas alloputida Pa2 has the advantages of simple production process, short culture time and low cost, and the biocontrol microbial inoculum for preventing and treating cotton wilt can be obtained by a simple, rapid and low-cost preparation method.
(3) The biocontrol microbial inoculum provided by the invention is a microbial control agent, is pollution-free, does not produce environmental pollution, can reduce or even not use other corresponding chemical pesticides, not only saves the expenditure of farmers, but also improves the rice quality.
(4) Experiments prove that the biocontrol microbial inoculum provided by the invention has a disease-resistant effect, and further greatly promotes the income increase of farmers.
Detailed Description
For the purpose of enhancing understanding of the present invention, the present invention will be described in further detail with reference to examples, which are provided for illustration only and are not intended to limit the scope of the present invention.
Example 1: isolation, purification and characterization of Pseudomonas alloputida Pa2
The biocontrol strain Pa2 for preventing and treating cotton wilt is obtained by separating leek root surrounding soil in a farm in Lishui area of Nanjing City in 2019.
The separation method comprises the following steps: taking the leek root soil by a 5-point sampling method, respectively filling the leek root soil into sterilized sealed plastic bags, and immediately taking the leek root soil back to a laboratory. Adding 5g of soil sample into 45mL of sterile water, carrying out shaking culture on a shaking table at 150r/min for 1h, filtering by using sterile gauze to obtain a suspension, and obtaining a 10-6-fold dilution solution by adopting a 10-fold serial dilution method. Respectively taking 100 mu L of diluent, uniformly coating the diluent on an LB flat plate, culturing the diluent in a constant temperature incubator at 28 ℃ for 48 hours, selecting single colonies with different colors, glossiness, sizes and types, streaking and purifying strains, uniformly numbering the strains, transferring the strains into an LB inclined plane culture medium, preserving the strains by using 80% sterilized glycerol, and preserving the strains at-70 ℃ for later use.
Plate confrontation method: selecting cotton wilt bacterium cake (5 mm) cultured for 2d, placing in the center of PDA plate, perforating at three symmetrical points (diameter 5 mm) at a distance of 25mm from the bacterium cake, injecting 100 μ L fermentation culture solution of test strain, and adding 100 μ L LNB as control. After culturing for 48h at the constant temperature of 28 ℃, observing and measuring the size of the antibacterial zone, repeating the experiment for three times, and selecting the strain with the largest antibacterial zone, namely Pseudomonas alloputida Pa2.
Biological characteristics: gram-negative bacteria, rod-shaped or oval, the size of the bacteria is 1.5-5.0 μm long and 0.5-1 μm wide, and the bacteria have flagella, no spores and no capsules. Colony morphology: round, smooth surface, opaque, complete edge.
Identified as Pseudomonas alloputida according to the evolutionary analysis and biological characteristics of a 16S rRNA gene system, and preserved in China general microbiological culture Collection center (address: no. 3 Xilu No. 1 Beijing of the Beijing Chaoyang district, north Chen West Lu No. 1) in 11 months and 9 days in 2020, and the preservation number of the strain is CGMCC NO.21149.
Example 2: preparation of biocontrol microbial inoculum
The preparation method of the biocontrol microbial inoculum by using the separated Pseudomonas alloputida Pa2 comprises the following steps: adding Pa2 single colony into LB liquid culture medium, shaking at 28 deg.C and 180rpm for 20 hr to obtain seed liquid with total viable bacteria concentration of about 10 6 CFU/mL; inoculating the seed solution into basal salt culture medium with 1% inoculum size, loading 50mL (250 mL triangular flask), shake culturing at 30 deg.C and 180rpm for 72h to obtain viable bacteria total concentration of 1 × 10 10 CFU/mL biocontrol microbial inoculum finished product.
LB liquid medium: 5g/L yeast extract, 10g/L tryptone, 10g/L sodium chloride, diluting to 1L with double distilled water, adjusting pH to 7.2, and adding into a container at 1 × 10 5 Pa sterilizing for 30min.
LB solid medium: 5g/L yeast extract, 10g/L tryptone, 10g/L sodium chloride, 15g/L agar, double distilled water to constant volume of 1L, adjusting pH to 7.2, and adding at 1 × 10 5 Pa sterilizing for 30min.
Basic salt culture medium: 5g of glucose, 2g of ammonium sulfate, 1g of sodium citrate, 0.2g of magnesium sulfate heptahydrate, 4g of dipotassium hydrogen phosphate and 6g of potassium dihydrogen phosphate, wherein double distilled water is added to the volume of 1L, the pH value is 5, and the pH value is 1 multiplied by 10 5 Pa sterilizing for 30min.
Example 3: plate confrontation test for detecting control effect of biocontrol microbial inoculum on cotton wilt
The biocontrol microbial inoculum prepared in the example 2 is diluted by 5 times to obtain the total viable bacteria concentration of 2 multiplied by 10 8 A biocontrol microbial inoculum of CFU/mL is prepared by transferring Fusarium oxysporum f.sp.vassinfectum (Fusarium oxysporum f.sp.vassinfectum) into PDA plate by hyphal growth rate method, activating at 25 deg.C for 96 hr, preparing bacterial cake with diameter of 5mm at the edge near to bacterial colony by using puncher, and transferring to a plate containing 0.1mL of viable bacteria at total concentration of 2 × 10 8 The PDA plate of the CFU/mL biocontrol microbial inoculum is used as a processing group, a control group is used for directly transferring cotton wilt bacteria to the PDA plate, the processing group and the control group are cultured at 25 ℃, when the colony of the control group grows to about 4/5 of the diameter of the plate, the colony diameter is measured by adopting a cross method, the average value of the colony diameters is calculated, the table 1 shows the inhibition effect of the biocontrol microbial inoculum on the growth of the tomato wilt bacteria hyphae, and the average inhibition rate of the hyphae growth is calculated according to the following formula.
The average inhibition rate of hypha growth = [ (mean diameter of control group bacterial colony-mean diameter of treatment group bacterial colony)/(mean diameter of control group bacterial colony-diameter of inoculation bacterial cake) ] × 100%.
TABLE 1 inhibitory Effect of biocontrol agents on the growth of hyphae of Fusarium oxysporum f.sp.cubense
The result shows that the biocontrol microbial inoculum prepared by Pseudomonas alloputida Pa2 has an inhibition effect of 84.57 percent on the growth of cotton fusarium wilt hyphae.
Example 4: determination of cotton fusarium wilt bacterium inhibition activity by strain Pa2
And (3) selecting a Pa2 single colony to be cultured in a liquid LB culture medium overnight under constant temperature oscillation to prepare a seed solution. Inoculating 1% of the strain in liquid LB culture medium, culturing at 28 deg.C in 180r/min constant temperature shaking incubator for 18h, and sequentially diluting the fermentation liquid concentration to 10 9 、10 8 、10 7 、10 6 、10 5 CFU/mL; cotton wilt bacterium cakes (d =5 mm) are picked up and placed in the center of a PDA flat plate,respectively sucking 1 mu L of the gradient concentration fermentation liquor by using a liquid transfer gun, placing the gradient concentration fermentation liquor at the positions of 1.5cm on two sides of a pathogenic bacteria cake, adding 1 mu L of sterile water as comparison treatment, reversely buckling and culturing the culture dish in a constant-temperature incubator at 25 ℃, and measuring the size of a bacteriostatic circle when the hyphae on the flat plate to be subjected to the comparison treatment grow to 3/4 of the diameter of the whole plate, wherein the table 2 shows the bacteriostatic activity determination value of the strain Pa2 on cotton wilt.
TABLE 2 bacteriostatic activity of strain Pa2 on cotton wilt
In the confronting culture test on the PDA plate, the width of the inhibition zone after the culture ranged from 7 to 10mm, indicating that the biocontrol strain Pa2 had a high level of antifungal activity against cotton wilt germs. The pathogenic hyphae in the healthy control plates grew vigorously and grew spread throughout the PDA plates; on the contrary, the hyphal growth of the pathogenic bacteria was inhibited by the strain Pa2 and could not break through its limitation. In the mycelium growth measurement of PDA plates of the strain Pa2 with different concentrations, the diameter of the cotton fusarium oxysporum mycelium shows a trend of decreasing firstly and then increasing along with the decrease of the concentration of the strain Pa 2; as is clear from the data in Table 2, 10 of the strain Pa2 was observed 7 The average bacteriostatic diameter of the cotton wilt pathogen is the largest under the concentration of CFU/mL.
Example 5: potted plant control effect determination of fermentation liquor of strain Pa2 on cotton wilt
Inoculating the strain Pa2 into LB culture medium, performing shaking culture at 28 deg.C and 180r/min for 24h, determining effective viable bacteria concentration by plate colony counting method, and adjusting to 10 7 cfu/mL to obtain Pa2 microbial inoculum, and storing at 4 ℃ for later use.
Inoculating the fusarium oxysporum strain in a bran culture medium, culturing at 28 ℃ for 7 days, air-drying at 30 ℃ after hyphae overgrow, and then crushing to 80 meshes to obtain the fusarium oxysporum microbial inoculum. Healthy soil is taken and potted, fusarium oxysporum inoculant and Pa2 inoculant are inoculated according to the inoculation amounts shown in the table 3, and a potting test is carried out. Fusarium oxysporum inoculant is uniformly mixed with soil before cultivation, pa2 inoculant is poured in with water after sowing, thinning is carried out when the height of cotton seedlings reaches 5cm, the seedling age is 15d, 1 strain is reserved in each pot, management is carried out according to a conventional mode, the number of diseased plants is observed and recorded at 60d after sowing, and the prevention and control effects of the Pa2 inoculant in a greenhouse on cotton blight are recorded in a table 4.
TABLE 3 design of the potting experiment
Grading standard of cotton wilt disease conditions:
grade 0, healthy cotton plants, no diseased leaves and normal growth;
1 grade, 1-2 cotton plants with leaf yellowing and wilting;
grade 2, 2 cotyledons and 1 true leaf of the cotton plant become yellow and wilted, and the leaf vein is in a yellow reticulate pattern;
grade 3, cotton plant 2 cotyledons and 2 or more true leaves become yellow and wilted, leaf vein is yellow reticulate or withered, cotton plant is dwarfed or wilted;
and 4, all leaves of the cotton plant are attacked, and the cotton plant is withered.
The disease index and the relative prevention effect are calculated according to the following formula:
disease index = ∑ (number of disease-grade plants × number of disease-grade)/(total number of plants × number of highest disease-grade)
Control effect = (disease index of control group-disease index of treatment group)/disease index of control group x 100%
TABLE 4 prevention and treatment effects of Pa2 fungicide on cotton wilt in greenhouse
As can be seen from the table 4, the Pa2 fungicide achieves 74.48 percent of greenhouse potting control effect on cotton wilt, which is obviously higher than that of the control.
Example 6: determination of field control effect of biocontrol microbial inoculum on cotton wilt
Selecting cotton seedlings with consistent growth vigor, inoculating cotton wilt germs in the two-leaf one-heart period until cotton leaves have zeroBeginning with the macula of star disease 10 7 Root irrigation treatment is carried out on cfu/mL biocontrol microbial inoculum, the biocontrol microbial inoculum is sprayed once every 15 days for 2 times, disease indexes are counted 15 days after the last spraying, the prevention and control effects are investigated, and the prevention and control effects of the biocontrol microbial inoculum in the greenhouse on cotton wilt are shown in table 5.
TABLE 5 control Effect of biocontrol microbial inoculum in greenhouse on cotton wilt
As can be seen from the data in Table 4, the field control effect of the biocontrol microbial inoculum on cotton wilt disease reaches 74.48 percent, which is obviously higher than that of a control.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (6)
1. The biocontrol strain for preventing and treating cotton wilt is named as Pseudomonas alloputida Pa2, the preservation date is 11/9/2020, and the preservation number is as follows: CGMCC NO.21149.
2. A biocontrol microbial inoculum containing the biocontrol strain of claim 1.
3. The biocontrol microbial inoculum of claim 2 wherein the total viable bacteria concentration in the biocontrol microbial inoculum is 1 x 10 9 ～1×10 10 CFU/mL。
4. A method for preparing the biocontrol microbial inoculum of claim 3, comprising the steps of: carrying out shake culture on Pseudomonas alloputida Pa2 in an LB culture medium at 25-30 ℃ and 150-200 rpm for 12-24 h to obtain a seed solution; inoculating the seed liquid into a basic salt culture medium with the inoculation amount of 1%, and performing shaking culture at the temperature of 25-30 ℃ and the speed of 150-200 rpm to obtain the total viable bacteria concentration of 1 multiplied by 10 9 ～1×10 10 CFU/mLThe finished product of the microbial inoculum.
5. The biocontrol strain as claimed in claim 1 or the biocontrol microbial inoculum as claimed in claim 2 is applied to the control of cotton wilt.
6. The use according to claim 5, wherein the prepared biocontrol microbial inoculum is diluted to 1 x 10 before use 7 CFU/mL, root irrigation treatment.
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