CN116162577A - Bacillus mucilaginosus and application thereof - Google Patents
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Abstract
The invention belongs to the technical field of crop control, and particularly relates to bacillus mucilaginosus and application thereof. The bacillus mucilaginosus is bacillus mucilaginosus @Bacillus mucilaginosus) HBTG859, accession no: the CCTCC NO is M20221745, and the strain can be used for preventing and controlling diseases such as citrus blue mold, citrus canker, citrus yellow dragon disease, citrus root-knot nematode and the like. Has no cross resistance with the existing bactericides and pesticides. 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 due to natural environment, and has wide applicationAnd (3) prospect.
Description
Background
Citrus (Citrus) belongs to Rutaceae, and is a generic name of mandarin orange, fructus Citri Junoris, fructus Citri Tangerinae, fructus Aurantii Immaturus, etc. The main cultivation area variety, climate conditions, cultivation management habit, and different production levels cause serious penicilliosis, canker, nematodiasis and the like. The prevention and control of citrus diseases mainly takes chemical bactericides as main materials, however, the frequent use of chemical pesticides causes the drug resistance of diseases, environmental pollution and pesticide residues, and seriously damages food and environmental safety. The microbial agent is not easy to generate drug resistance, has no pesticide residue and environmental pollution, has a regulation and control effect on the soil microbial community structure, and inhibits the occurrence of diseases. Based on the characteristics, the microbial agent is widely used for preventing and controlling crop diseases.
At present, the application of bacillus mucilaginosus in agriculture is reported, and the growth promotion and yield increase are mainly achieved. Few reports on bacteriostasis and disease prevention and control are available. Wang Bacillus mucilaginosus CX-7 has been reported to have excellent antibacterial activity against verticillium cotton, fusarium wilt cotton, rhizoctonia solani, rhizoctonia cerealis, gibberella wheat and trichoderma viride. Hu Liangliang et al report that Bacillus mucilaginosus PS04 has good antibacterial activity against Rhizoctonia solani. Li Wenpeng et al report that Bacillus mucilaginosus D4B1 and XDB1 have good antibacterial activity against tobacco gray mold.
The citrus planting is mainly carried out on acid soil in the south, and bacillus is used as a microorganism, and the acid soil is unfavorable for survival and propagation, so that the bacillus for citrus disease prevention and control also needs to have a certain tolerance to acid. Therefore, the screening of bacillus with good prevention and control effects on citrus diseases and certain tolerance to acidity is of great significance. Zhang Xiaobo et al report that Bacillus mucilaginosus hn1, hn2, hn3, hn4 and hn5 are incapable of growing under pH5 conditions, but have a certain salt tolerance, and are capable of growing under 2% NaCl conditions, but are incapable of growing at a low temperature of 10 ℃.
The bacillus mucilaginosus HBTG859 provided by the invention is a bacillus which is separated from citrus rhizosphere soil and has good control effect on citrus diseases, has good control effect on citrus blue mold, canker and root knot nematode, has good colonization capability on citrus roots, has certain acid resistance, and has wide application prospect.
Disclosure of Invention
The invention aims to provide an isolated bacillus which is bacillus mucilaginosus HBTG859 and has a preservation number of CCTCC NO: M20221745.
Another object of the present invention is to provide a method for preparing a liquid fermentation broth of Bacillus mucilaginosus HBTG859.
The final object of the invention is to provide the application of bacillus mucilaginosus HBTG859 in the preparation of citrus blue mold, canker, yellow dragon disease and root knot nematode disease.
In order to achieve the above object, the present invention adopts the following technical measures:
the applicant isolated a bacillus strain from healthy citrus plant rhizosphere soil of citrus nematode severe disease field, identified as bacillus mucilaginosus (Bacillus mucilaginosus), and sent to the chinese collection of typical cultures for preservation at 11/09 of 2022, classically named: bacillus mucilaginosus HBTG859 and 859; the preservation number is CCTCC NO: M20221745; location: chinese, university of martial arts, martial arts.
Bacillus mucilaginosus HBTG859 grew well on the LA culture medium colonies, which were transparent jelly-like (FIG. 1). The protection scope of the invention comprises:
use of bacillus mucilaginosus (Bacillus mucilaginosus) HBTG859 for controlling citrus diseases, such as citrus blue mold, citrus canker and/or citrus yellow dragon.
Use of bacillus mucilaginosus (Bacillus mucilaginosus) HBTG859 for the control of citrus nematodiasis.
Application of bacillus mucilaginosus (Bacillus mucilaginosus) HBTG859 in preparation of citrus disease biocontrol agents;
application of bacillus mucilaginosus (Bacillus mucilaginosus) HBTG859 in preparation of preparation for preventing and treating citrus nematodes.
In the application, the HBTG859 is used for preventing and treating the citrus nematode disease by root irrigation of citrus with a preparation containing HBTG859;
in the above application, HBTG859 is used in citrus diseases by spraying the formulation containing HBTG859 on the part that is subject to attack by pathogens.
Use of bacillus mucilaginosus (Bacillus mucilaginosus) for inhibiting plant pathogenic bacteria selected from the group consisting of verticillium dahliae (Verticillium dahliae), fusarium oxysporum (Fusarium oxysporum), botrytis cinerea (Botrytis cinerea), and/or rhizoctonia solani (Rhizoctonia solani).
Compared with the prior art, the invention has the following advantages:
(1) Compared with the prior art, the bacillus mucilaginosus provided by the invention has the biological control functions of resisting bacteria and nematodes, and has no cross 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.
(3) Bacillus mucilaginosus screened by the invention also has the characteristics of acid resistance, low temperature resistance and colonization in citrus root systems.
Drawings
FIG. 1 colony morphology of HBTG859 strain.
FIG. 2 shows the schematic of the antibacterial activity of HBTG859.
FIG. 3HBTG859 schematic representation of the control of citrus green mold.
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 of bacillus mucilaginosus HBTG 859:
the applicant isolated a bacillus strain from the rhizosphere soil of healthy citrus plants in citrus nematode severe disease fields, which was identified by whole genome sequencing in combination with physiological and biochemical as belonging to bacillus mucilaginosus (Bacillus mucilaginosus) (fig. 1, table 1), and sent to the chinese collection of typical cultures for preservation at 11-09 of 2022, and named after classification: bacillus mucilaginosus (Bacillus mucilaginosus) HBTG859; the preservation number is CCTCC NO: M20221745; address: chinese, university of martial arts, martial arts.
In the present invention, bacillus mucilaginosus is referred to as HBTG859.
Morphological characteristics: milky white, purulent, viscous, and round colonies.
Physiological and biochemical characteristics of the strain: as in Table 1
Table 1: physiological and biochemical characteristics of strain HBTG859
Example 2:
fermentation of bacillus mucilaginosus HBTG859 (the percentages in the following culture medium formulas are mass percentages):
seed liquid preparation: activating HBTG859 strain from the inclined plane, inoculating single colony into NB liquid culture medium, shake culturing at 30deg.C with liquid loading amount of 1L/3L and 200r/min for 18 hr.
20000L tank fermentation: the culture medium is mannose 2%, corn steep liquor 1%, mgSO 4 ·7H 2 O，0.15％,CaCO 3 0.1%, sample loading 10000L, initial pH 7.5, culture temperature 36 ℃, 5L seed solution, ventilation 200m 3 After fermentation time of 36h, the number of spores in the fermentation broth was measured to be 5.0X10 8 cfu/ml。
Example 3:
antibacterial activity of bacillus mucilaginosus HBTG859 on pathogenic bacteria of penicillium citrinum and prevention and control effect on penicillium citrinum:
strawberry gray mold (Botrytis cinerea), watermelon fusarium wilt (Fusarium oxysporum), pepper epidemic disease (Phytophthora capici), rice sheath blight (Rhizoctonia solani) and eggplant verticillium (Verticillium dahliae) including penicillium citrinum pathogen (Penicillium italicum) are activated from the inclined plane to the PDA plate for standby. In vitro bacteriostasis test was performed by the counter culture method, sterilized filter paper sheets were placed on both ends of the PDA plate, and 10. Mu.L of HBTG859 fermentation broth prepared in example 2 was then dropped. And (3) after 24 hours, inoculating the pathogenic bacteria dish by using a 4mm puncher, and taking the inoculated sterile water as a blank control, wherein the distance between the pathogenic bacteria and the filter paper sheet is 25mm. And after 5d, observing a bacteriostasis zone between biocontrol bacteria and penicillium citrinum pathogenic bacteria, and determining the bacteriostasis activity. And counting the diameters of all treated and control colonies after other pathogenic bacteria are compared and grow on the flat plate, and calculating the bacteriostasis rate.
Antibacterial ratio = [ (control colony diameter-treated colony diameter)/(control colony diameter-bacterial cake diameter) ] ×100%
Culturing Penicillium citrinum pathogenic bacteria (Penicillium italicum) at 28deg.C for 10d to produce spore, and regulating with sterile water to 1×10 6 The spores/ml are reserved, fresh citrus is taken, a 3mm multiplied by 3mm wound is pricked at the waist of the fruit by a scalpel, 20 mu L of HBTG859 fermentation liquor diluted 20 times is dripped at the wound, the control is clear water treatment, and 20 mu L of spore suspension is dripped after natural air drying. Each treatment was repeated 3 times, 15 fruits each. And (3) carrying out moisture preservation culture at 25 ℃, counting control and treatment lesion diameters after inoculation for 7d, and calculating the control effect.
Control = (control plaque diameter-treatment plaque diameter/control plaque diameter) ×100;
in vitro antibacterial tests show that the HBTG859 strain has good antibacterial activity on penicillium citrinum and the like (figure 2), and obvious antibacterial zones exist between biocontrol bacteria and pathogenic bacteria.
In addition, HBTG859 strain has good antibacterial activity against strawberry gray mold (Botrytis cinerea), watermelon fusarium wilt (Fusarium oxysporum) and eggplant verticillium wilt (Verticillium dahliae), and poor antibacterial activity against pepper epidemic disease (Phytophthora capici) and rice sheath blight (Rhizoctonia solani) (table 3). HBTG859 fermentation broth has good control effect on citrus blue mould, and control effect 80.21% (figure 3).
TABLE 3 in vitro antibacterial Activity of HBTG859 Strain
Example 4:
HBTG859 strain has in-vitro activity on citrus nematodes and effect of preventing and controlling citrus root-knot nematodes
Ex vivo test: taking the root of southSterilizing ovum of the meloidogyne incognita with 2% sodium hypochlorite, and incubating with sterile water to obtain second-instar meloidogyne for use; HBTG859 fermentation broth prepared in example 2 was added to adjust the spore count to 1X 10 8 cfu/mL, with clear water as a blank, 50 hundred million cfu/g Bacillus mucilaginosus (North sea Qiang Biotechnology Co., ltd.) product strain as a strain control, were subjected to single-strain separation, and fermentation according to example 2, the spore count was adjusted to 1X 10 8 cfu/mL; incubation at 20℃and observation of nematode survival under an inverted microscope at 24h, 48h and 72h, respectively; the nematode death rate is counted according to the following formula:
nematode mortality = (number of nematode deaths/number of nematodes tested) ×100%
Nematode corrected mortality = [ (treated nematode mortality-control mortality)/1-control mortality ] ×100%
The in vitro test result shows that after 72 hours, HBTG859 has obvious insecticidal effect on the in vitro correction mortality rate of root-knot nematodes being 84.33%, and the mortality rate of nematodes after treatment of Bacillus mucilaginosus of North sea Qiang Xinxing biotechnology Co is 0.
The field test was carried out in Xinfeng county, ganzha, jiangxi province, the citrus variety to be tested was sugar orange, 5 years old, the HBTG859 fermentation broth prepared according to example 2 was root-irrigated 200 times, 15L each plant was root-irrigated, root-irrigated again at 15d intervals, water was irrigated in contrast, each treatment was repeated 3 times, and each treatment was repeated 5 trees. And counting the root knot indexes of each treatment for 3 months and calculating the control effect.
Counting the occurrence of diseases according to the disease classification standard: level 0, no root knot; 1 grade, root knot accounts for 1% -10% of the total root system; 2-stage, wherein root knots account for 11% -25% of the total root system; 3, the root knot accounts for 26 to 50 percent of the total root system; 4, the root knot accounts for 51% -75% of the total root system; grade 5, root knot accounts for 69% -90% of the total root system, grade 6: root knot accounts for 91-100% of the total root system.
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 HBTG859 has a certain prevention and control effect on citrus root-knot nematodes, and the prevention effect reaches 57.98% (Table 3).
Table 3HBTG859 microbial inoculum has effect on citrus root knot nematode field control
| Treatment of | Root knot index | Preventing effect (%) |
| HBTG859 | 21.94 | 57.98 |
| Control | 52.21 | - |
Example 5:
HBTG859 strain for preventing and controlling citrus canker
The field test was carried out in Xinfeng county, ganzhou, jiangxi province, the test citrus variety was sugar orange, 5 years old, and the spore count was adjusted to 1×10 according to the HBTG859 fermentation broth prepared in example 2 8 cfu/mL, 50 hundred million cfu/g Bacillus mucilaginosus (North sea Qiang Xin Biotechnology Co., ltd.) product strain was used as strain control, single strain isolation was followed by fermentation as in example 2, and the number of spores was adjusted to 1X 10 8 cfu/mL. The fermentation broth was diluted 100-fold, sprayed (sprayed to the page) on foliage and fruit, sprayed 2 nd times at 7d intervals, applied 3 times together, and sprayed with water as a control. The field cells are arranged according to random groups, the area of each treatment cell is 2 strains, and the treatment cells are repeated for 3 times. Each of whichThe district surveys 2 trees, each tree samples according to five points of east, west, south, north and middle, each point surveys 2 summer tips randomly, and records the leaf numbers of each stage on the summer tips.
The leaf (fruit) classification method is as follows: level 0: no disease; stage 1: 1-5 lesions per leaf (fruit); 3 stages: 6-10 lesions are formed on each leaf (fruit); 5 stages: each leaf (fruit) has 11-15 lesions; 7 stages: 16-20 lesions are formed on each leaf (fruit); stage 9: there are 21 lesions and more per leaf (fruit).
Disease index (%) = Σ (number of diseased leaves (fruits) at each stage×grade)/(number of total leaves (fruits) under investigation×highest representative grade) ×100.
Control effect = (control disease index-treatment disease index)/control root knot index x 100%
The test result shows that the disease index of the citrus ulcer is obviously reduced by spraying the HBTG859 microbial inoculum, the control effect reaches 65.56%, and the control effect of the control strain is only 10.20%.
TABLE 3HBTG859 microbial inoculum for controlling citrus canker
| Treatment of | Index of disease condition | Preventing effect (%) |
| HBTG859 | 14.59 | 65.56 |
| Control strain | 38.04 | 10.20 |
| Clear water control | 42.36 | - |
Example 6: colonisation ability of HBTG859 strain at citrus root system:
HBTG859 was subjected to resistance mutant screening on plates containing different gradient concentrations of rifampicin LA to obtain resistance mutant strains, using the bacillus mucilaginosus (north sea qiang biotechnology limited) product strain of example 4 as a control strain, and resistance mutants were prepared and fermented as in example 2. 2 bacillus mucilaginosus strains are regulated to 1 hundred million cfu/mL in fermentation liquor concentration, citrus seedling roots are irrigated with root, 10L/plant is irrigated with citrus roots after 14d, the root is repeatedly washed with sterile water for 3 times, 1g of the root is sheared and weighed, and the root is coated with the mixture containing 300 mu g.mL after grinding and dilution -1 After 24h incubation at 37 ℃, the ability of HBTG859 and control bacillus mucilaginosus to colonize citrus root systems was determined.
The test result shows that the colonization amount of HBTG859 in citrus root system is 9.8X10 6 cfu/g, while the control bacillus mucilaginosus citrus root system colonization amount is 0, HBTG859 contributes to the prevention and control of citrus nematode disease in citrus such colonization capacity.
Example 7:
acid resistance, salt and alkali resistance and low temperature resistance determination of HBTG859 strain
HBTG859 strain was used as in example 2, and the fermentation broth concentration was adjusted to 1 hundred million cfu/mL, and Bacillus mucilaginosus (North Hai Qiang Biotech Co., ltd.) product strain was used as strain control, and fermentation was performed in the same fermentation method and the fermentation broth concentration was adjusted to 1 hundred million cfu/mL for use.
The 2 kinds of fermentation liquids are used as seed liquid and are inoculated into a triangular flask of LB culture medium with the PH of 4, the volume of the culture medium is 100mL, the inoculation amount is 100 mu L, and the test is repeated for 3 times. The colony count was determined after shaking culture at 30℃and 200r/min for 18 hours.
The 2 fermentation broths were used as seed liquid and inoculated into triangular flasks containing LB medium with NaCl 0.1%, 0.25%, 0.5% and 1%, the medium volume being 100mL, the inoculum size being 100. Mu.L, and the test set being repeated 3 times. The colony count was determined after shaking culture at 30℃and 200r/min for 18 hours.
The fermentation broth of example 2 was streaked on LB plates, and then, after using Bacillus mucilaginosus (North sea Qiang Biotechnology Co., ltd.) product strains as strain controls, they were placed at 0℃and 2℃and 4℃and 6℃and 8℃and 10℃respectively, and 30℃were used as controls, and after 48 hours, whether or not growth was observed.
The test result shows that under the condition of an acid culture medium, the spore number of HBTG859 after culture is 3.4X10 7 cfu/mL, and the number of spores of control Bacillus mucilaginosus is 7.7X10 5 cfu/mL, HBTG859 is more beneficial to the colonization and propagation of the acid-resistant acid in the acid soil of citrus planting. Growth of 2 strains was inhibited in the presence of 0.5% NaCl (Table 4), growth stopped and salt was more sensitive.
HBTG has good low temperature resistance, grows at a low temperature of 4 ℃ and the control strain cannot grow (Table 5), and the low temperature resistance is beneficial to propagation in soil in a low-temperature environment and better exerts the performance of the HBTG.
TABLE 4 salt tolerance of HBTG859 strains
TABLE 5 Low temperature resistance of HBTG859 Strain
Claims (9)
1. Isolated bacillus mucilaginosusBacillus mucilaginosus) HBTG859, and the preservation number of bacillus mucilaginosus is CCTCC NO: M20221745.
2. Use of bacillus mucilaginosus according to claim 1 for controlling citrus diseases.
3. Use according to claim 2, said citrus diseaseHarmful citrus green moldPenicillium italicum) Citrus canker and/or citrus yellow dragon.
4. Use of bacillus mucilaginosus according to claim 1 for controlling citrus nematode disease.
5. The use of bacillus mucilaginosus according to claim 1 for the preparation of a biocontrol agent against citrus diseases.
6. Use of bacillus mucilaginosus according to claim 1 for the preparation of a formulation for controlling citrus nematodes.
7. The use according to claim 2, wherein said bacillus mucilaginosus is used for controlling citrus diseases by spraying a part which is eroded by pathogens and contains bacillus mucilaginosus according to claim 1.
8. The use according to claim 4, wherein the bacillus mucilaginosus is used for controlling citrus nematodiasis by root irrigation of citrus with a preparation containing the bacillus mucilaginosus according to claim 1.
9. Use of bacillus mucilaginosus according to claim 1 for inhibiting plant pathogenic bacteria, said plant pathogenic bacteria being verticillium dahliae (Verticillium dahliae), fusarium oxysporum (Fusarium oxysporum), botrytis cinerea (Botrytis cinerea), and/or rhizoctonia solani (Rhizoctonia solani).
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| CN117004508B (en) * | 2023-05-22 | 2024-02-09 | 湖北同光生物科技有限公司 | Bacillus composite microbial inoculum and application thereof in citrus disease prevention and control |
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