CN117165492B - Bacillus bailii and application thereof - Google Patents
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- CN117165492B CN117165492B CN202311297782.5A CN202311297782A CN117165492B CN 117165492 B CN117165492 B CN 117165492B CN 202311297782 A CN202311297782 A CN 202311297782A CN 117165492 B CN117165492 B CN 117165492B
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Abstract
The invention belongs to the technical field of agricultural microorganisms, and provides bacillus bailii (Bacillus velezensis) JNS-1 with a preservation number of CGMCC No.22531. The bacillus belay JNS-1 and the fermentation liquor thereof can be used for preparing medicaments or fertilizers for preventing and treating strawberry diseases. The strain is applied to strawberry production, can solve the problems of serious root disease prevention and control, easy pollution of underground water due to chemical prevention and control and unsatisfactory prevention and control effect.
Description
Technical Field
The invention belongs to the technical field of agricultural microorganisms, and particularly relates to bacillus beijerinckii and application thereof in preventing and controlling strawberry diseases.
Background
The strawberry is mainly cultivated in facilities, the environment is relatively shielded, root diseases are serious due to successive year planting, and death is caused. For root diseases, the prevention and control effect of spraying medicines on the overground part is limited, and groundwater is easy to be polluted if soil treatment operation is improper. Meanwhile, the chemical bactericide is frequently and unreasonably used, and the drug resistance of target pathogenic bacteria is easy to generate.
Biological control has become a research hot spot in recent years due to the advantages of no toxicity, environmental friendliness and the like. However, the alternative biocontrol microbial agents in the market at present have poor colonization capability in the environment, which often results in poor biocontrol effect. Moreover, the pathogenic bacteria of strawberry anthracnose and root rot are complex, and antagonistic strains aiming at novel root rot pathogenic bacteria such as trichoderma pseudodisc and red-shell bacteria are not reported.
Disclosure of Invention
Aiming at the problem that the novel strawberry root rot pathogen antagonistic bacteria are lack at the present stage, the invention provides bacillus beijerinus which can antagonize colletotrichum Colletotrichum gloeosporioides, fusarium oxysporum Fusarium oxysporium, trichosanilis-like Neopestalotipsis and red-shell bacteria Calonectria and has excellent control effect on strawberry colletotrichum, fusarium wilt and root rot caused by the pathogenic bacteria.
The invention further aims to provide a preparation containing the biocontrol bacteria, which is used for preventing and controlling root diseases in strawberry production and can solve the problems of serious death of plants, easy pollution of underground water due to chemical prevention and control and unsatisfactory prevention and control effect.
In order to achieve the above purpose, the present invention adopts the following technical scheme.
Bacillus bailii (Bacillus velezensis) JNS-1 with the preservation number of CGMCC No.22531.
The bacillus beleiensis JNS-1 can be used for preparing medicaments or fertilizers for preventing and treating strawberry diseases.
Preferably, the strawberry diseases are strawberry anthracnose, strawberry blight and strawberry root rot.
Preferably, the strawberry disease is caused by fusarium oxysporum Fusarium oxysporum of strawberry fusarium wilt, colletotrichum gloeosporioides Colletotrichum gloeosporioides of strawberry anthracnose pathogen and polyrhiza fungi Neopestalotipsis of root rot pathogen or radicicol Calonectria.
A pesticide or fertilizer comprising the bacillus belicus JNS-1.
In the pesticide or fertilizer, the concentration of the bacillus beijerinus JNS-1 is (5-10) multiplied by 10 8 cfu/g or (5-10) multiplied by 10 8 cfu/mL.
In order to expand the spectrum of control or to increase the efficacy, the pesticide may also contain other active ingredients; the active ingredient may be a natural extract or a living microorganism which may be a chemically synthesized pesticide, an animal or plant or a microorganism.
The pesticide may also contain inert fillers such as emulsifiers, dispersants, wetting agents, disintegrants, uv absorbers, fillers, etc. for ease of application or to extend shelf life.
The invention has the following advantages:
The bacillus belay (Bacillus velezensis) JNS-1 provided by the invention can effectively antagonize novel strawberry soil-borne diseases, and is found by indoor potting experiments and field experiments to have good control effect, simple use method and convenient popularization. The preparation containing the JNS-1 can solve the problems of serious death, easy pollution of underground water due to chemical prevention and control and unsatisfactory prevention effect caused by novel root diseases when being applied to strawberry production.
Biological preservation information
Bacillus belicus (Bacillus velezensis) JNS-1 was deposited at China general microbiological culture Collection center (CGMCC) of China Committee for culture Collection of microorganisms at about 14 months of 2021, with a deposit address of CGMCC No.22531, a microbiological institute of China, national institute of sciences, no. 3, xiyang area North Star, beijing, city.
Drawings
FIG. 1 is a scanning electron microscope picture of JNS-1 strain;
FIG. 2 shows the inhibitory effect of JNS-1 strain on different strawberry diseases; wherein a is colletotrichum gloeosporioides, b is fusarium oxysporum, c is trichoderma pseudodisc, and d is erythromyces.
Detailed Description
The present invention will be further described with reference to examples and drawings, but the present invention is not limited to the examples.
EXAMPLE 1 isolation and characterization of Bacillus bailii Bacillus velezensisJNS-1
The strain is obtained by separating soil within 5 cm around the rhizosphere of a robust plant in a Jin Niushan base strawberry greenhouse of a fruit tree institute of Shandong province in Taian City of Shandong province Mount Tai. Collecting the soil sample, air-drying and sieving with a2 mm sieve. 5.0 g air-dried soil sample is weighed into a 100mL triangular flask, 45 mL sterile water is added, 10min is oscillated by vortex, and the sample suspension with the mass concentration of 10 -1 is prepared. The sample suspension was serially diluted to obtain a concentration of 10 -2、10-3、10-4、10-5、10-6. 100 mu L of sample suspension with the concentration of 10 -4-10-6 is respectively taken on a flat plate of LB culture medium (NaCl 10.0 g/L, tryptone 10.0 g/L, yeast extract powder 5.0 g/L, pH value is regulated to 7.0 by NaOH solution, agar is 20 g/L), the mixture is uniformly coated, and the mixture is subjected to dark culture at 33 ℃ for 1-2 d. Single colony is selected, streaked on a new LB culture medium plate for culture, separation and purification.
The strain JNS-1 with the largest inhibition zone is obtained by screening the strains by adopting the strawberry anthracnose fungus colletotrichum gloeosporioides Colletotrichum gloeosporioides as indicator bacteria through a counter culture method, and is taken as a target strain to be identified:
After activation of the strain, genomic DNA was extracted and amplified with 27F and 1492R primers to give a 16S gene fragment (SEQ ID NO: 1) of 1433bp in length. The 16SrDNA sequences were aligned by BLAST and the Bacillus strain most similar to the above strains. By adopting MEGA5.10 software and adopting UPGMA algorithm to construct phylogenetic tree, the strain and Bacillusvelezensis belong to the same genetic branch, the relatedness reaches 99.93%, the screened strain can be determined to be bacillus bailii Bacillus velezensis, and a scanning electron microscope picture of the strain is shown in figure 1.
The JNS-1 strain is preserved in the China general microbiological culture Collection center (CGMCC) for 5 months and 14 days in 2021, and has the address of North Star Xway No. 1, no. 3 in the Korean region of Beijing, the preservation name of Bacillus belicus JNS-1, the classification name of Bacillus velezensis and the preservation number of CGMCC No.22531.
Example 2 JNS-1 inhibition of growth of pathogenic bacteria of strawberry root disease
According to the conventional operation in the field, fusarium oxysporum Fusarium oxysporum of strawberry fusarium wilt, colletotrichum Colletotrichum gloeosporioides of strawberry anthracnose pathogenic bacteria, hirsutella pseudolarix Neopestalotipsis of root rot pathogenic bacteria and red-shell bacteria Calonectria are separated and verified by adopting a Koch rule.
On an ultra-clean workbench, inoculating the pathogenic bacteria cake with the diameter of 7 mm on the center of a PDA culture medium plate, culturing in darkness at 25 ℃ for 24: 24h, picking single bacterial colony of bacillus beleiensis JNS-1 described in example 1 by using a sterile toothpick, inoculating 1 point on the upper, lower, left and right 4 directions at the position 2cm away from the center of the bacterial cake, sealing by using a preservative film, and culturing in darkness at 25 ℃ for 3-4 d. Sterile water was inoculated as a control. When the colony of the control group grows to 1/2 to 2/3 of the culture dish, the diameter of each treated colony is measured by adopting a crisscross method, and the antagonism effect is observed.
TABLE 1 inhibition ratio of Bacillus bailii JNS-1 to strawberry diseases
The bacteriostasis circles of the bacillus belicus JNS-1, which are opposite to the plates of the strawberry gum spore anthrax Colletotrichum gloeosporioides, the fusarium oxysporum Fusarium oxysporum, the like-disc hirsutella sinensis Neopestalotipsis and the like-red-shell fungus Calonectria, are respectively shown in the figure 2, and the bacteriostasis circles are clearly visible; as can be seen from the data in Table 1, the inhibition rates of JNS-1 against each pathogenic bacterium were 55.1%, 59.2%, 53.7% and 52.9%, respectively, and the inhibition effects were very remarkable.
Example 3 JNS-1 preparation of liquid and solid formulations
Bacillus belicus JNS-1 obtained in example 1 was inoculated into a 250 mL triangular flask containing 150 mL LB liquid medium (NaCl 10.0 g/L, tryptone 10.0 g/L, yeast extract 5.0 g/L, pH value was adjusted to 7.0 with NaOH solution), and cultured in a dark shaker at 30℃and 150 r/min for 36 h to obtain a primary fermentation broth with a cell concentration of 20X 10 8 cfu/mL.
Inoculating the primary fermentation liquor into a 100L fermentation tank filled with 50L LB liquid culture medium according to an inoculation proportion of 10%, stirring at a speed of 300r/min, fermenting at a temperature of 33 ℃, and culturing at a dissolved oxygen content of 50% -60% for 24 h to obtain a secondary fermentation liquor, wherein the concentration of thalli is 10 multiplied by 10 8 cfu/mL.
Inoculating the secondary fermentation liquor into a large-scale fermentation tank with 1000L LB liquid culture medium and 2000L volume according to an inoculation proportion of 2%, wherein the stirring speed is 300 r/min, the fermentation temperature is 33 ℃, the dissolved oxygen amount is 50% -60%, and the culture is 24h, so that the tertiary fermentation liquor is obtained, and the thallus concentration is 2 multiplied by 10 8 cfu/mL.
Standing the fermentation liquor for 12 h, centrifuging the fermentation liquor at the upper 50% at 2000rpm for 20 min, taking precipitate, mixing with the fermentation liquor at the lower 50%, adding 0.1% xanthan gum and 0.1% sodium benzoate, and stirring thoroughly to obtain liquid microbial inoculum with thallus concentration of 5× 8 cfu/mL.
Standing the fermentation broth for 24 h, and collecting precipitate. The supernatant was centrifuged at 2000rpm for 15 min to obtain a precipitate. And combining the precipitate thalli. 0.5 Adding 0.25 kg potassium fulvate, 0.25 kg diatomite and 0.5 kg humic acid into the kg precipitate, fully stirring, uniformly mixing, drying at 55-60 ℃ until the water content is less than or equal to 15%, and sieving with a 1mm sieve to obtain the solid microbial inoculum, wherein the concentration of the microbial inoculum is 5 multiplied by 10 8 cfu/g.
EXAMPLE 4 JNS-1 potted plant efficacy evaluation of liquid and solid formulations against strawberry root disease
Filling a special substrate for strawberries into a plastic flowerpot with the diameter of 15 cm, and transplanting 3-leaf 1-heart-period 'Chapter-Ji' variety strawberry seedlings. Each pot was inoculated with 1 dish of each pathogen described in example 2, cultured for 7d hours, inoculated with hyphae, spores, medium and mixed well. Liquid microbial inoculum treatment groups 10 mL and 20 mL of the liquid microbial inoculum described in example 3 were irrigated at the rhizosphere of each strawberry plant, respectively. Solid microbial inoculant treatment groups, 10g and 20 g of the solid microbial inoculant described in example 3 were broadcast on the rhizosphere of each strawberry strain. The control group was untreated. Each treatment was repeated 3 times, 10 pots each. The incidence of each strawberry root disease was investigated and the relative control was calculated using conventional cultivation management at 15 d and 30 d post-treatment.
The potting control effect of the JNS-1 strain liquid and solid preparation on strawberry root diseases is shown in tables 2-5:
TABLE 2 JNS-1 potted plant control Effect of liquid and solid formulations on anthracnose caused by Alternaria strawberry
The JNS-1 liquid preparation has 83.33 percent of control effect on anthracnose 15 d caused by colletotrichum gloeosporioides infection according to the dosage treatment of 10 mL per strain and 20 mL per strain, and 68.75 percent and 75.00 percent of control effect on 30 d respectively. The JNS-1 solid preparation has 75.00 percent of control effect and 83.33 percent of control effect on anthracnose 15 d caused by colletotrichum gloeosporioides infection according to the dosage treatment of 10 g per plant and 20 g per plant, and has 81.25 percent of control effect and 87.50 percent of control effect on 30 d.
TABLE 3 JNS-1 potted plant control Effect of liquid and solid formulations on Fusarium oxysporum Fragaricum caused fusarium wilt
The JNS-1 liquid preparation has 80.00% and 100.00% of control effect on fusarium oxysporum of 15 d and 75.00% of control effect on fusarium oxysporum of 30d according to the dosage of 10 mL/plant and 20 mL/plant. The JNS-1 solid preparation has 80.00 percent and 90.00 percent of control effect on fusarium oxysporum of 15 d and 87.50 percent of control effect on 30d of control effect on fusarium oxysporum of strawberry according to the dosage of 10 g per plant and 20g per plant.
TABLE 4 JNS-1 potted plant control Effect of liquid and solid preparations on Fusarium oxysporum of strawberry species
The JNS-1 liquid preparation has the control effects of 66.67 percent and 77.78 percent respectively on root rot caused by the Mucor pulmonale of strawberries according to the dosage of 10 mL per strain and 20mL per strain, and the control effects of 34 percent and 82.35 percent respectively on 30 d. The JNS-1 solid preparation has the control effects of 66.67 percent and 77.78 percent respectively on root rot caused by the Mucor pulmonale of strawberries according to the dosage of 10 g per strain and 20g per strain, and the control effects of 30 d percent and 70.59 percent and 82.35 percent respectively.
TABLE 5 JNS-1 potted plant control Effect of liquid and solid preparations on fusarium wilt caused by Chilli strawberry
The JNS-1 liquid preparation has the control effects of 15 d and 30 d of respectively 60.00% and 90.00% on root rot caused by red-shell fungus of strawberry according to the dosage of 10 mL/strain and 20 mL/strain, and 66.67% and 83.33% respectively. The JNS-1 solid preparation has the control effects of 15 d and 30 d of 70.00 percent and 80.00 percent respectively and 75.00 percent and 83.33 percent respectively on root rot caused by red-shell fungus of strawberry according to the dosage of 10g per plant and 20g per plant.
Example 5 JNS-1 evaluation of field control efficacy of liquid and solid formulations against strawberry root disease
And selecting a greenhouse with serious root diseases such as strawberry, fusarium wilt, anthracnose, root rot and the like which are planted continuously for many years, and transplanting 3-leaf 1-heart-period 'Chapter-Ji' variety strawberry seedlings. Liquid microbial inoculum treatment groups 10 mL and 20 mL of the liquid microbial inoculum prepared in example 3 were irrigated on the rhizosphere of each strawberry. Solid microbial inoculum treatment group, the solid microbial inoculum prepared in example 3 was broadcast 10 g and 20 g respectively on the rhizosphere of each strawberry. The control group was untreated. Each treatment was repeated 3 times, 1 repeat per cell, area 10m 2, and about 120 strawberry seedlings were planted. The mixed incidence of strawberry root disease was investigated at 15 d and 30 d post-treatment using conventional cultivation management, and the relative control was calculated.
Table 6 JNS-1 field control Effect of liquid and solid formulations on strawberry root disease
The JNS-1 liquid preparation has the control effect of 75.00 percent and 87.96 percent respectively on 15 d mixed diseases of strawberry roots according to the dosage of 10 mL per plant and 20 mL per plant, and the control effect of 78.17 percent and 85.91 percent respectively on 30 d. The JNS-1 solid preparation has the control effects of 76.85 percent and 87.04 percent on root diseases according to the dosage of 10 g per plant and 20 g per plant, and the control effects of 78.87 percent and 85.21 percent on root diseases according to the dosage of 30 d.
Claims (5)
1. Bacillus bailii (Bacillus velezensis) JNS-1 with the preservation number of CGMCC No.22531.
2. Use of bacillus beijerinus JNS-1 according to claim 1 for preparing a medicament or fertilizer for controlling strawberry diseases;
the strawberry diseases are strawberry anthracnose, strawberry fusarium wilt and strawberry root rot;
The strawberry diseases are caused by fusarium oxysporum Fusarium oxysporum of strawberry fusarium wilt, colletotrichum gloeosporioides Colletotrichum gloeosporioides of strawberry anthracnose pathogen and polyrhiza fungi Neopestalotipsis of root rot pathogen or red shell fungus Calonectria.
3. A pesticide or fertilizer comprising bacillus belgium JNS-1 of claim 1.
4. A pesticide or fertiliser according to claim 3, characterised in that the concentration of bacillus bailii JNS-1 is (5-10) x 10 8 cfu/g or (5-10) x 10 8 cfu/mL.
5. A pesticide or fertiliser according to claim 3 characterised in that the pesticide further comprises an inert filler.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100976760B1 (en) * | 2009-11-18 | 2010-08-18 | 충청남도 | Bacillus velezensis nsb-1 strain, composition for control anthracnose and control method of anthracnose with same |
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| CN108587938A (en) * | 2018-03-29 | 2018-09-28 | 山东省果树研究所 | It is a kind of prevention strawberry droop bacillus amyloliquefaciens and its application |
| CN111172080A (en) * | 2020-02-24 | 2020-05-19 | 山东农业大学 | Bacillus belgii and application thereof |
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