CN116496924B - Bacillus rugosus YC25 and application thereof in prevention and control of tobacco diseases - Google Patents
Bacillus rugosus YC25 and application thereof in prevention and control of tobacco diseases Download PDFInfo
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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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G13/00—Protecting plants
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/45—Tobacco
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
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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
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- A01N63/22—Bacillus
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Abstract
The invention belongs to the field of agricultural microorganisms and disclosesBacillus rugosus YC25 and application thereof in prevention and control of tobacco diseases, wherein the preservation number of the strain is CCTCC NO: M20221894. The strain is reported for the first time and can be used for preventing and controlling tobacco black shank, root rot, damping off, brown spot, anthracnose, gray mold and nematodes at the same time, and has no cross resistance with the existing bactericides and pesticides. Compared with the existing chemical synthesis pesticide control, the pesticide has the advantages of high efficiency, low toxicity, no environmental pollution, difficult generation of drug resistance and the like because the pesticide is derived from natural environment, and has wide application prospect.
Description
Technical Field
The invention belongs to the field of agricultural microorganisms, and particularly relates to Bacillus rugosus YC and application thereof in prevention and control of tobacco diseases.
Background
Due to the limited cultivated land and the driving of economic benefits, continuous cropping is a main planting mode in tobacco planting areas because of continuous cropping planting habit in most tobacco areas. The tobacco planting soil is gradually degraded due to long-term continuous cropping and large-scale use of chemical fertilizers, and the like, and the problems of reduced productivity, aggravated diseases, reduced tobacco quality and the like are solved. A large number of researches show that the soil nutrient and the soil microorganism zone system of the tobacco field are unevenly changed due to long-term continuous cropping, the diversity and uniformity of soil bacteria are reduced, mass propagation of soil-borne pathogenic bacteria and the activity of soil enzymes are reduced, and the method is extremely unfavorable for the growth and high-quality production of crops. Tobacco black shank, bacterial wilt and root-knot nematodes are the main soil-borne diseases of tobacco, commonly occurring in tobacco areas, and resulting in substantial yield loss. The disease scope and the disease degree of fusarium root rot are in an ascending trend, and serious plots cause the extinction. In addition, the tobacco soil-borne diseases are mixed, so that the difficulty in control is increased, and the damage of the soil-borne diseases is further aggravated. The prevention and control of tobacco soil-borne diseases mainly depend on chemical bactericides, which are easy to cause environmental pollution and pathogen resistance, and destroy tobacco rhizosphere microbial communities, and researches show that the occurrence of the soil-borne diseases is directly related to the diversity of soil bacteria and the change of population structures. Biological control is taken as a novel disease control measure, not only has the effects of preventing diseases and promoting growth of plants, but also can effectively overcome the problems of pesticide residues and pathogen resistance in production. In addition, the microbial agent has the function of regulating and controlling plant rhizosphere microbial community structures, and forms a soil microbial community structure which is unfavorable for disease occurrence, namely 'bacteriostatic soil'.
According to the invention, a new bacillus Bacillus rugosus YC strain is separated out to solve the problems, and has good control effects on tobacco black shank, root rot, damping-off and root-knot nematodes, and good colonization capability on tobacco root systems. And has the effects of preventing and controlling diseases such as brown spot, anthracnose, gray mold and the like of the overground part diseases of the tobacco.
Disclosure of Invention
The invention aims to provide an isolated bacillus which is Bacillus rugosus YC and has a preservation number of CCTCC NO: M20221894. The Bacillus rugosus YC can be used for preventing and treating tobacco diseases such as tobacco black shank, root rot, damping off, brown spot, anthracnose, gray mold and the like.
It is another object of the present invention to provide the use of Bacillus rugosus YC25 in the manufacture of plant pathogen inhibitors or nematicides, in particular for the manufacture of medicaments for the control of tobacco diseases.
In order to achieve the above purpose, the invention adopts the following technical scheme:
an isolated Bacillus isolated from tobacco rhizosphere identified as Bacillus rugosus, which was sent to the chinese collection for preservation at month 07 of 2022, classed designation: bacillus rugosus YC25; the preservation number is CCTCC NO: M20221894; location: university of martial arts in chinese.
The strain grew well on NA medium, pale yellow single colony (fig. 1).
The scope of the invention also includes a broth of Bacillus rugosus YC25, and the media used in obtaining the broth includes, but is not limited to, LB media, or media: 16-19g/L of sweet potato powder, 16-19g/L of yeast powder, 4-6g/L of corn steep liquor, 0.6-1.0g/L of magnesium sulfate, 0.1-0.3g/L of monopotassium phosphate and pH value of 6.5-7.
The protection content of the invention comprises:
bacillus rugosus YC25 or its fermentation broth or its fermentation supernatant for preventing and treating tobacco diseases;
in the above application, the preparation containing Bacillus rugosus YC or its fermentation liquid or its fermentation supernatant is used for root irrigation treatment or direct spraying.
Bacillus rugosus YC25 or its fermentation liquid or its fermentation supernatant in the preparation of biocontrol agent for tobacco diseases;
in the above applications, the tobacco diseases include, but are not limited to, diseases caused by phytophthora nicotianae (Phytophthora nicotianae), fusarium sp., rhizoctonia solani (Rhizoctonia solani), alternaria sp., tobacco anthrax (Colletotrichum nicotianae) and Botrytis cinerea and/or tobacco nematodes.
Compared with the prior art, the invention has the following advantages:
(1) The invention provides a Bacillus rugosus, which is reported for the first time to be used for preventing and treating tobacco black shank, root rot, damping off, brown spot, anthracnose, gray mold and nematode at the same time, has certain acid resistance, root system and smoke Tian Dingshi capability, and has no interactive drug resistance with the existing bactericides and pesticides.
(2) Compared with the existing chemical synthesis pesticide, the pesticide has the advantages of high efficiency, low toxicity, no environmental pollution, difficult generation of drug resistance and the like because the pesticide is derived from natural environment.
Drawings
FIG. 1Bacillus rugosus YC25 bacterial colony morphology schematic.
Figure 2Bacillus rugosus YC25 whole genome alignment.
Figure 3Bacillus rugosus YC25 in vitro bacteriostatic effect.
Fig. 4Bacillus rugosus YC25 shows the effect of the microbial inoculum for preventing and treating tobacco black shank.
Detailed Description
For a better explanation of the present invention, the main content of the present invention is further elucidated below in conjunction with the specific examples, but the content of the present invention is not limited to the following examples only. The technical scheme of the invention is conventional technology in the field unless specifically stated, and the reagents or materials are commercially available unless specifically stated.
Example 1:
acquisition and identification of Bacillus rugosus YC25
An isolated Bacillus strain, isolated from the tobacco rhizosphere soil of the fei county, which is characterized by full genome sequencing in NCBI comparison and biochemical identification as belonging to Bacillus rugosus (fig. 1, fig. 2, table 1, table 2), was sent to the chinese collection for preservation at 12 months 07 of 2022, and named after classification: bacillus rugosus YC25; the preservation number is CCTCC NO: M20221894; location: university of Chinese Wuhan
In the present invention, bacillus rugosus YC25 is simply referred to as YC25.
Table 1: physiological and biochemical characteristics of strain YC 25-enzyme activity and oxidation of carbon source
+: a positive reaction; -: a negative reaction;
table 2: physiological and biochemical characteristics of strain YC 25-acid production by using carbon source
+: a positive reaction; -: a negative reaction; w: weak positive reaction example 2:
bacillus rugosus YC25 in vitro bacteriostatic activity of tobacco disease pathogenic bacteria
The pathogenic bacteria of tobacco fungus diseases are activated from the inclined plane to the PDA plate, and are cultivated for 6 days at 30 ℃ for standby. YC25 single colonies were transferred to NB medium and shake cultured at 35℃and 180rpm for 12 hours. And 10 mu L of fermentation liquor is dripped at two ends of a PDA flat plate, a 4mm puncher is used for inoculating the pathogenic bacteria dish after 24 hours, the culture medium dish is used as a blank control, and the distance between the pathogenic bacteria and the YC25 bacteria dish is 25mm. After the control was grown on the plate, the diameters of the treated and control colonies were counted, and the antibacterial rate was calculated, and the pathogenic bacteria were as shown in Table 3.
Antibacterial ratio = [ (control colony diameter-treated colony diameter)/(control colony diameter-bacterial cake diameter) ] ×100%
The in vitro antibacterial test shows that YC25 has good antibacterial activity on pathogenic bacteria such as phytophthora nicotianae, fusarium oxysporum, rhizoctonia solani, botrytis cinerea, tobacco anthracis, alternaria alternata and the like, which are pathogenic bacteria of tobacco diseases (table 3, figure 3).
Table 3: YC25 in vitro antibacterial Activity
| Pathogenic bacteria | Bacteriostasis rate (%) |
| Phytophthora nicotianae (Phytophthora nicotianae) | 73.33 |
| Fusarium oxysporum (Fusarium oxysporum) | 70.41 |
| Rhizoctonia solani (Rhizoctonia solani) | 78.33 |
| Botrytis cinerea | 87.91 |
| Alternaria alternata (Alternaria alternata) | 80.34 |
| Tobacco anthracis (Colletotrichum nicotianae) | 72.09 |
Example 3:
bacillus rugosus YC25 for preventing and controlling tobacco black shank
The YC25 slant strain is inoculated into an LB liquid culture medium after being activated at 30 ℃, and is cultured for 16 hours at the temperature of 30 ℃ and the rotation speed of a shaking table of 180r/min, and is used as seed for shake flask fermentation. Seeds were inoculated into shake flask fermentation medium at 1% (v/v) and incubated at 30℃and 180r/min until spores were shed 20% at the end of fermentation, the spores being 140 hundred million cfu/mL, for the following examples.
The fermentation medium consisted of: 18g/L of sweet potato powder, 18g/L of yeast powder, 5.00g/L of corn steep liquor, 0.8g/L of magnesium sulfate, 0.20g/L of monopotassium phosphate and pH7.
Tobacco seeds are directly sown in a 32-hole tray, conventionally cultured in a greenhouse, transplanted in a seedling pot after 15d, and used for potting experiments after 30 d. Activating Phytophthora nicotianae from inclined plane to PDA plate, propagating on V8 plate, culturing at 28deg.C for 12d, brushing spores, taking out in refrigerator at 4deg.C for half an hour, and adjusting spores to 10 with blood cell counting plate 5 Spores/ml were kept ready for use.
And (3) root-filling the YC25 fermentation liquor, wherein the root-filling amount is 20 ml/plant, and inoculating phytophthora nicotianae spores after 48 hours, and 3 ml/plant. Treatment was repeated 3 times, 15 seedlings each, fresh water as a control, and after 15 days, the control and treatment indices were counted and the incidence was calculated.
Tobacco black shank disease index grading standard refers to the national tobacco industry standard of the people's republic of China (YC/T39-1996)
Disease index = Σ (number of plants at each stage×grade)/(total number of plants investigated×highest representative grade) ×100.
Control effect = (control disease index-treatment disease index)/control disease index x 100%
Potted plant biological test results show that the YC25 microbial inoculum has 100 percent of prevention effect on tobacco black shank (figure 4).
Example 4:
bacillus rugosus YC25 for preventing and controlling tobacco root rot
The test site is located in the Xianyang jujube yang of Hubei, the soil ph is 4.5, the variety CX14, the YC25 microbial inoculum prepared in the example 3 is diluted by 200 times, root irrigation treatment is carried out after tobacco transplanting and seedling reviving, the 2 nd root irrigation is carried out at intervals of 20d, the root irrigation amount is 200 ml/plant, the 3 rd root irrigation is carried out at intervals of 20d, and the root irrigation amount is 200 ml/plant. The blank cell treatments were randomly arranged, each treatment was repeated 4 times, and the cell area of each repetition was 50m 2 。
Counting after the contrast is fully developed, investigating each cell by adopting a five-point sampling method, checking 5-10 plants each, and recording and investigating total plant number and plant number of each stage of diseases.
The grading method comprises the following steps: level 0: the whole plant has no pathology; stage 1: mild vascular discoloration with no external symptoms; 2 stages: moderate vascular discoloration, no external symptoms; 3 stages: the lower leaves turn yellow and gradually wilt; 4 stages: most wilting; 5 stages: plants die.
Disease index = Σ (number of plants at each stage×grade)/(total number of plants investigated×highest representative grade) ×100.
Control effect = (control disease index-treatment disease index)/control disease index x 100%
The test result shows that the YC25 microbial inoculum has good prevention and control effect on tobacco root rot, and the field prevention effect reaches 66.38% (Table 4).
TABLE 4YC25 microbial inoculum field effect for preventing and controlling tobacco root rot
| Treatment of | Index of disease condition | Preventing effect (%) |
| YC25 | 13.04 | 66.38 |
| Control | 38.79 | - |
Example 5:
bacillus velezensis YC25 control of tobacco nematodes
The test is in the date yang city of Hubei province, soil ph is 4.5, root YC25 microbial inoculum is irrigated 200 times after 10d of tobacco transplanting and field planting, root irrigation is carried out once every 10d, 200 mL/plant is irrigated, water is irrigated in a comparison mode, each treatment is repeated for 3 times, and each repetition is 40m 2 Root irrigation is carried out for 3 times, root knot indexes of all treatments are counted before harvesting, and control effect is calculated.
The occurrence of tobacco diseases is investigated according to the classification and investigation methods of tobacco diseases and insect pests of GB/23222-2008.
Root knot index = Σ (number of plants at each stage×grade)/(total number of plants investigated×highest representative grade) ×100.
Control effect = (control root knot index-treated root knot index)/control root knot index x 100%
The test result shows that the YC25 microbial inoculum has good prevention and control effect on tobacco root-knot nematodes, and the prevention and control effect reaches 63.14 (table 5) table 5: YC25 microbial inoculum has field control effect on tobacco root knot nematode
| Treatment of | Root knot index | Preventing effect (%) |
| YC25 | 11.67 | 63.14 |
| Control | 31.66 | - |
Example 6:
bacillus velezensis YC25 ability of tobacco root to colonise
Resistant mutant screening was performed on YC25 and Bacillus belicus CY30 (CN 110564646A) on LB plates containing different gradient concentrations of rifampicin to obtain resistant mutant strains, and the obtained resistant strains were identical to the original strains in terms of the remaining properties, except that the resistant strains were made to have rifampicin resistance, and fermentation was performed on the resistant mutant strains as in example 3.
Culturing tobacco in seedling pot to 6 leaf stageDiluting YC25 and CY30 fermentation liquor (140 hundred million cfu/mL) 5 times, irrigating roots, 10 mL/plant, taking out plants after 14d, repeatedly flushing roots with sterile water for 3 times, shearing roots and weighing 1g, grinding and diluting, and coating the roots on a medium containing 300 mu g.mL -1 After 24h incubation at 37 ℃, the ability of YC25 and CY30 to colonize tobacco roots was determined.
The result shows that the colonization amount of YC25 at the root of tobacco is 8.74 multiplied by 10 6 cfu/g, whereas CY30 is only 3.2X10 2 cfu/g. This ability of YC25 to colonize helps control tobacco soil-borne diseases.
Claims (6)
1. Isolated strainBacillus rugosusYC25 strain with a preservation number of CCTCC NO: M20221894.
2. A fermentation broth prepared from the strain of claim 1, said broth comprising a viable bacteria of the strain of claim 1.
3. The broth of claim 2, wherein the medium used in obtaining the broth is LB medium, or medium: 16-19g/L of sweet potato powder, 16-19g/L of yeast powder, 4-6g/L of corn steep liquor, 0.6-1.0g/L of magnesium sulfate, 0.1-0.3g/L of monopotassium phosphate and pH value of 6.5-7.
4. The method of claim 1Bacillus rugosusUse of YC25 or the fermentation liquor of claim 2 for preventing and treating tobacco diseases, wherein the tobacco diseases are prepared from phytophthora nicotianaePhytophthora nicotianae) Fusarium (Fusarium)Fusariumsp.. And Rhizoctonia solaniRhizoctonia solani) Alternaria alternata (L.) A.Alternaria sp.. And tobacco anthracnoseColletotrichum nicotianae) Botrytis cinereaBotrytis cinerea) And/or diseases caused by tobacco nematodes.
5. The method according to claim 4, wherein the composition comprisesBacillus rugosusYC25 or a formulation of the fermentation broth of claim 2, for root irrigation of tobaccoAnd (3) either direct spraying.
6. The method of claim 1Bacillus rugosusApplication of YC25 or fermentation liquor of claim 2 in preparation of biocontrol agent for tobacco diseases, wherein the tobacco diseases are prepared from phytophthora nicotianaePhytophthora nicotianae) Fusarium (Fusarium)Fusariumsp.. And Rhizoctonia solaniRhizoctonia solani) Alternaria alternata (L.) A.Alternaria sp.. And tobacco anthracnoseColletotrichum nicotianae) Botrytis cinereaBotrytis cinerea) And/or diseases caused by tobacco nematodes.
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| CN103224897A (en) * | 2013-04-11 | 2013-07-31 | 四川省烟草公司凉山州公司 | Bacillussubtilis for tobacco black shank prevention and control |
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| CN103224897A (en) * | 2013-04-11 | 2013-07-31 | 四川省烟草公司凉山州公司 | Bacillussubtilis for tobacco black shank prevention and control |
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