CN112760230B - Gliocladium roseum, gliocladium roseum bacterial liquid and application thereof in preventing and treating sunflower sclerotium disease - Google Patents
Gliocladium roseum, gliocladium roseum bacterial liquid and application thereof in preventing and treating sunflower sclerotium disease Download PDFInfo
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- CN112760230B CN112760230B CN202011621356.9A CN202011621356A CN112760230B CN 112760230 B CN112760230 B CN 112760230B CN 202011621356 A CN202011621356 A CN 202011621356A CN 112760230 B CN112760230 B CN 112760230B
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- 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
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- 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
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
Abstract
The invention relates to gliocladium roseum, gliocladium roseum bacterial liquid and application of the gliocladium roseum bacterial liquid in preventing and treating sunflower sclerotinia rot. The gliocladium roseum MES72701 adopted by the invention has the following preservation unit name: china general microbiological preservation management center, preservation number: CGMCC 3.11519; the invention provides gliocladium roseum bacterial liquid, wherein the content of gliocladium roseum spores is 1.8 multiplied by 10 7 2.3X 10 units/mL 7 The preparation method comprises the following steps: selecting fresh mycelial of gliocladium roseum after activated culture, inoculating the mycelial to a plate containing a solid PDA culture medium, and then placing the mycelial in a constant temperature incubator at 25-30 ℃ for culturing for 12-16 days; then 20-30mL of sterile water containing tween 80 was added to the petri dish, the spores were washed off with an applicator, filtered, and diluted with an 8-12 fold gradient. The gliocladium roseum bacterial liquid provided by the invention has an obvious antagonistic effect on sclerotinia causing sunflower sclerotiorum, can prevent or reduce the occurrence of sunflower sclerotinia, promotes the stable yield and the yield increase of sunflower, and improves and enhances the yield and the quality of sunflower.
Description
Technical Field
The invention belongs to the technical field of biological control of plant diseases, and particularly relates to gliocladium roseum, gliocladium roseum bacterial liquid and application of the gliocladium roseum bacterial liquid in control of sunflower sclerotium diseases.
Background
Sunflower is an important oil crop in China, and in recent years, sunflower producing areas in China are greatly damaged by sunflower sclerotiniose, and even sunflower failure in production and harvest is caused seriously. Affecting the yield and quality improvement of the sunflower and hindering the development of the sunflower industry.
Helianthus annuus sclerotiniose (sclerotitinoia sclerotiorum (lib.) debry) is called Sclerotinia sclerotiorum, and belongs to the Ascomycotina fungus. The germs live through the winter in soil, in disease residues and between seeds by using sclerotium. Pathogenic bacteria can grow at 0-37 deg.C, and the optimal growth temperature is 25 deg.C. Sunflower sclerotinia can occur throughout the growth period, causing stem, stem base, flower disc and kernel rot. There are 4 symptoms of root rot type, stem rot type, leaf rot type and flower rot type, wherein the damage of root rot type and flower rot type is serious. In spring, the stems are rotten and heavy in low temperature and rain, and the flower disc is rotten and heavy in flowering phase. The continuous cropping field soil has large sclerotium amount and heavy diseases.
The conventional control method adopts crop rotation, planting of disease-resistant varieties and removal of burnt field disease residues for prevention and germ diffusion, and adopts chemical pesticide such as procymidone, dimethachlon for seed dressing, and spraying of sulpiride or benralin during flowering phase to control diseases. Usually, the operation is more tedious and chemical pesticide can cause problems such as pesticide residue, influences the promotion of sunflower output and quality, and chemical prevention and control can cause environmental pollution, is not conform to the road and the direction of green sustainable development.
The microorganisms can effectively prevent and control the occurrence of plant diseases, and the prevention and control of the plant diseases by utilizing antagonistic microorganisms accords with the development direction of green prevention and control. Therefore, what kind of microorganism is adopted to effectively control sunflower sclerotinia sclerotiorum is a problem to be solved.
Disclosure of Invention
The invention aims to provide gliocladium roseum and gliocladium roseum bacterial liquid and application thereof in preventing and treating sunflower sclerotium disease. The gliocladium roseum bacterial liquid provided by the invention has obvious antagonistic effect on Sclerotinia sclerotiorum (lib.) debry causing sunflower Sclerotinia sclerotiorum, can prevent or reduce the occurrence of sunflower Sclerotinia sclerotiorum, promotes the stable production and the yield increase of sunflower, and improves and enhances the yield and the quality of sunflower.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides application of gliocladium roseum in preventing and treating sunflower sclerotium disease. Through retrieval, the applicant does not find a related way of preventing and treating sunflower diseases by adopting gliocladium roseum, the gliocladium roseum is creatively used for preventing and treating sunflower sclerotinia, the prevention effect is obvious, the gliocladium roseum is green and environment-friendly, the use amount of pesticides can be reduced, the yield and the quality of agricultural products can be improved, and the prospect of large-area popularization and use is very wide.
As a further improvement of the above scheme: preferably, the Gliocladium roseum is Gliocladium roseum (Clinostacchys rosea) MES72701, depository name: china general microbiological preservation management center, preservation number: CGMCC 3.11519, storage address: xilu No. 1 Hospital No. 3, beijing, chaoyang, north. The gliocladium roseum MES72701 strain is purchased from China General Microbiological Collection Center (CGMCC) in 12 months in 2019, and is preserved in a refrigerator at 4 ℃ and minus 80 ℃ after activated culture. Gliocladium roseum is Gliocladium roseum of Gliocladiales of Aphyllophorales of Deuteromycota. On the PDA plate, the hyphae are originally white loose creeping hyphae, the aerial hyphae are undeveloped, the hyphae turn pink, and orange-red pigment is generated at the bottom of the colony.
The invention provides a gliocladium roseum bacterial liquid, wherein the active ingredient comprises the gliocladium roseum.
Preferably, the content of gliocladium roseum spores in gliocladium roseum bacterial liquid is 1.8 multiplied by 10 7 2.3X 10 units/mL 7 One per mL.
The invention provides application of the gliocladium roseum bacterial liquid in preventing and treating sunflower sclerotium diseases.
The invention provides a preparation method of gliocladium roseum bacterial liquid, which comprises the following steps: selecting fresh mycelial of gliocladium roseum after activated culture, inoculating the mycelial to a plate containing a solid PDA culture medium, and then placing the mycelial in a constant temperature incubator at 25-30 ℃ for culturing for 12-16 days; then 20-30mL of sterile water containing tween 80 was added to the petri dish, the spores were washed down with an applicator, and filtered, and 8-12 fold gradient dilution was performed. In the invention, at least 3 plates are inoculated.
Preferably, the solid PDA culture medium formula comprises: 20g/L of glucose, 200g/L of potato, 19g/L of agar and 1000mL of water; in sterile water containing Tween 80, the mass concentration of the Tween 80 is 0.02 to 0.08 percent; the filtration is carried out by adopting gauze, and the number of the layers of the gauze is four. The pH value of the solid PDA culture medium adopted by the invention does not need to be adjusted, and the preparation is completed according to the proportion; the preparation method of the PDA culture medium refers to the preparation of a conventional potato glucose culture medium.
The invention provides gliocladium roseum bacterial liquid prepared by the method.
The invention provides application of the gliocladium roseum bacterial liquid in preventing and treating sunflower sclerotium diseases.
The application method of the gliocladium roseum bacterial liquid in preventing and treating sunflower sclerotium disease provided by the invention comprises the following steps: the gliocladium roseum bacterial liquid is prepared according to the following steps of 1: (1-5) diluting with water, and uniformly watering the plant roots.
The technical scheme provided by the invention has the following beneficial effects: (1) The gliocladium roseum and gliocladium roseum bacterial liquid provided by the invention has obvious antagonistic effect on Sclerotinia sclerotiorum (lib.) debry causing sunflower Sclerotinia sclerotiorum, can prevent or reduce the occurrence of sunflower Sclerotinia sclerotiorum, promotes the stable yield and the income of sunflower, and improves and increases the yield and the quality of the sunflower; (2) The gliocladium roseum bacterial liquid is potted in seedling stage to prevent and treat sunflower sclerotinia rot, the prevention and treatment effect reaches more than 80%, the prevention and treatment effect is obvious, the gliocladium roseum bacterial liquid belongs to a green and environment-friendly preparation, the usage amount of pesticides can be reduced, the yield and the quality of agricultural products can be improved, and the gliocladium roseum bacterial liquid has a very wide prospect of large-area popularization and application.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
FIG. 1 is a graph showing the antagonistic effect of Gliocladium virens on Sclerotinia solanacearum in example 2 of the present invention;
FIG. 2 is a graph showing the effect of a control inoculated with only Helianthus annuus sclerotiniose in example 2 of the present invention.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby.
The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
The sources of the strains related to the invention are as follows:
(1) The sunflower Sclerotinia sclerotiorum used by the invention is obtained by separating and purifying the sclerotium in the field with the incidence of Nemontage erythropolis by the applicant, and is identified as the sunflower sclerotiniose (lib.) debry) through 16S rDNA;
(2) The gliocladium roseum (Clonostachys rosea) MES72701 used in the invention is purchased from China general microbiological culture Collection center (CGMCC) in 2019 and 12 months, and the name of the preservation unit is as follows: china general microbiological preservation management center, preservation number: CGMCC 3.11519, storage address: xilu No. 1 Hospital No. 3, beijing, chaoyang, north. After activation culture, the cells were stored in a refrigerator at 4 ℃ and-80 ℃.
Example 1: preparation of Gliocladium roseum MES72701 bacterial liquid
The embodiment provides a preparation method of gliocladium roseum MES72701 bacterial liquid, which comprises the following steps.
1. Inoculation and culture
Selecting fresh MES72701 mycelium subjected to activation culture, inoculating onto solid PDA culture medium, inoculating into 3 plates, and culturing in a constant temperature incubator at 28 deg.C for 15 days. The PDA culture medium formula is as follows: 20g/L glucose, 200g/L potato, 19g/L agar, 1000mL water and natural pH; the preparation method of the PDA culture medium refers to the preparation of a conventional potato glucose culture medium.
2. Preparation of spore liquid
After 15 days, 25mL of sterile water (containing 0.05% tween 80) was added to each dish, the spores were washed off with an applicator and filtered through four layers of gauze for 10-fold gradient dilution.
3. Spore content detection
The spore concentration in the spore suspension was counted using a hemocytometer. The final spore suspension was measured at a concentration of 2X 10 7 One per mL.
Example 2: bacteriostatic test of gliocladium roseum MES72701 bacterial liquid on sunflower sclerotinia rot
1. Time and place of experiment: 12 months in 2019 in a laboratory of a company research and development center.
2. The test method comprises the following steps:
(1) Source of test strain
The sunflower Sclerotinia sclerotiorum is separated from a sunflower Sclerotinia sclerotiorum disease onset field in Nemadura akaara, is cultured and purified by company developers, is finally identified as the sunflower Sclerotinia sclerotiorum (lib.) debry through 16S rDNA, and is identified as strong pathogenicity through pathogenicity determination.
Gliocladium roseum bacterial liquid is obtained by inoculating and culturing the strain described in example 1 and preparing spore liquid.
(2) Plate antagonism experiment
PDA solid plates were prepared, and 200. Mu.L of the spore suspension prepared in example 1 was pipetted and spread evenly on PDA plates in triplicate with sterile water as a control. And culturing the culture dish in a constant-temperature incubator at 28 ℃, beating a fresh sclerotinia sclerotiorum hypha block by using a puncher after 2 days, placing the sclerotinia sclerotiorum hypha block on a culture medium coated with gliocladium roseum spore solution and a control culture medium, measuring the growth radius of the sclerotinia sclerotiorum hypha of the treated and controlled strains of the sunflower in the incubator at 25 ℃ after the sclerotinia sclerotiorum hypha on the control plate grows over the whole plate, and calculating the bacteriostasis rate.
Bacteriostatic rate = (control colony growth radius-treated colony growth radius) × 100/control colony growth radius.
(3) Antagonistic Effect
The experiment shows that the antagonism effect of gliocladium roseum MES72701 on sunflower sclerotinia sclerotiorum reaches 80.79%; the average colony radius for the control was 4.5cm and the average colony radius for the inoculated Gliocladium roseum was 2.8cm (as shown in FIGS. 1 and 2). The gliocladium roseum MES72701 has obvious inhibition effect on sunflower sclerotinia sclerotiorum and has biocontrol potential for preventing and treating sunflower sclerotinia sclerotiorum.
Example 3: potted plant control effect of gliocladium roseum MES72701 bacterial liquid on sunflower sclerotinia sclerotiorum
1. Test agent
(1) Microbial agent
Gliocladium roseum MES72701 bacterial solution (MES 72701 spore suspension) prepared in example 1, having a spore content of 2X 10 7 One per mL.
(2) Chemical agent
A50% wettable powder of procymidone (Shandong Haichi Biochemical Co., ltd.) in 2000 times volume.
(3) Blank control
Clear water treatment was set as a blank control.
2. Time and place of experiment
The test was carried out in the nursery room of the company 12 months in 2019.
3. Test method
Processing and setting: the sunflower variety is Longshigai No. 2, 5 treatments are set in the test, and the treatments are respectively the original concentration, 1-time dilution, 3-time dilution and 5-time dilution of the spore suspension, no bacteria control addition of clear water and 2000-time treatment of 50% procymidone wettable powder in example 1.
The experimental method comprises the following steps: taking 4 sunflower seeds for each treatment, putting the seeds into a culture dish, adding 10mL of pure water to immerse the seeds, immersing the seeds in a 30 ℃ culture box, planting the seeds in a seedling pot to grow after the sunflower seeds are exposed, and thinning and reserving one seedling after seedling emergence. The change was observed. Pouring 40mL of tap water or 40mL of spore suspension with each concentration every 5 days, and uniformly pouring the spore suspension on the roots of the plants. The effect was observed after 30 days.
4. Test results
The test results are shown in table 1, and the results show that the disease index (8.16-8.35) of the treatment by adding gliocladium roseum MES72701 fungal powder is remarkably lower than the disease index (87.29) of a blank control treatment, the disease index (7.25) of the treatment by adding gliocladium roseum MES72701 fungal powder is not remarkably different from the disease index (7.25) of the treatment by adding a chemical agent, and the control effect of MES72701 on sunflower sclerotinia sclerotiorum reaches 90.20% -91.49%. The gliocladium roseum MES72701 can be used as a biocontrol strain for preventing and treating sunflower sclerotinia sclerotiorum and has wide development and utilization prospects.
TABLE 1 control of Helianthus annuus sclerotinia by different treatments
| Treatment of | Index of disease condition | Control effect (%) |
| MES72701 spore suspension: the water ratio is 1:1 | 8.16bc | 91.49 |
| MES72701 spore suspension: the water ratio is 1:3 | 8.25bc | 90.65 |
| MES72701 spore suspension: the water ratio is 1:5 | 8.35bc | 90.20 |
| Pythium ultimum | 7.25b | 92.69 |
| Blank control | 87.29a | 0.00 |
The above is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, it can be made several improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be regarded as the inventive content of the present invention.
Claims (2)
1. The application of gliocladium roseum in preventing and treating sunflower sclerotium disease is gliocladium roseum MES72701, and the preservation unit name is as follows: china general microbiological preservation management center, preservation number: CGMCC 3.11519.
2. An application of Gliocladium roseum bacterial liquid in preventing and treating sunflower sclerotium disease, wherein the bacterial liquid comprises the Gliocladium roseum MES72701 spores in claim 1, and the content of the Gliocladium roseum spores MES72701 in the bacterial liquid is 1.8 x 10 7 2.3X 10 units/mL 7 one/mL.
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