CN112195120A - Biocontrol strain G2C3 for preventing and treating powdery mildew of strawberries and application thereof - Google Patents
Biocontrol strain G2C3 for preventing and treating powdery mildew of strawberries and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of agricultural microorganisms, and relates to a biocontrol strain G2C3 for preventing and treating strawberry powdery mildew and application thereof. The biocontrol strain G2C3 is identified as Bacillus subtilis and is preserved in China general microbiological culture Collection center (CGMCC) in 26 days 7 and 7 months 2018, and the preservation number of the strain is CGMCC NO. 16164. The bacterial strain has obvious effect of preventing and treating powdery mildew of strawberry, and can reach 60.0%. Meanwhile, field test results show that the biocontrol microbial inoculum G2C3 can obviously prevent and treat powdery mildew of strawberry, the prevention and treatment effect is 55.60%, and the yield increase effect is 38.46%. Therefore, the strain and the microbial inoculum prepared by the strain can be applied to the aspects of preventing and treating powdery mildew of strawberries and increasing yield.
Description
Technical Field
The invention belongs to the field of agricultural microorganisms, and relates to a biocontrol strain G2C3 for preventing and treating powdery mildew of strawberries and application thereof.
Background
Powdery mildew is an important disease caused by powdery mildew and frequently occurs on field crops of fruits and vegetables such as strawberries, cucumbers, wheat and the like. Has the characteristics of short incubation period, frequent infection and strong infectivity. Powdery mildew pathogenic bacteria can be infected in the whole growth period of crops. With the development of facility agriculture, powdery mildew diseases become serious year by year.
The current main prevention and treatment method is chemical pesticide prevention and treatment, but the long-term frequent use of a large amount of chemical pesticides causes soil pollution and causes serious environmental pollution; the resistance of powdery mildew is enhanced, and pesticide residue causes food safety problem. Although the cultivation of antiviral seedlings is an ideal way for preventing and treating powdery mildew, the cultivation period is long and the powdery mildew resistance inheritance is complex, so that a breeding line with stable resistance is difficult to breed. Therefore, research and development of a new safe, environment-friendly and more effective microbial agent for controlling powdery mildew of various crops such as strawberries and the like is a need for improving quality and increasing yield in agriculture, and is a need for safe production and sustainable development in agriculture.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a biocontrol strain G2C3 for effectively preventing powdery mildew.
The invention also aims to provide a microbial inoculum prepared by the strain.
Another object of the present invention is to provide the use of the strain.
The purpose of the invention is realized by the following technical scheme:
a biological control strain G2C3 for preventing and treating strawberry powdery mildew is identified to belong to Bacillus subtilis and is preserved in China general microbiological culture Collection center (CGMCC) in 26 months and 7 in 2018, and the strain preservation number is CGMCC NO. 16164.
The biocontrol microbial inoculum prepared by the strain G2C3 for preventing and controlling strawberry powdery mildew.
The preparation method of the biocontrol microbial inoculum comprises the following steps: the biocontrol strain G2C3 for preventing and controlling powdery mildew disease is subjected to shake cultivation for 28h at 32 ℃ and 180r/min in an LB liquid culture medium, and then is centrifuged for 15min at 4000 r/min. Diluting with sterile water to obtain bacterial liquid with effective viable count of 1 × 109-1010CFU/mL。
Of course, the preparation of the biocontrol microbial inoculum by using the biocontrol strain G2C3 for preventing and treating powdery mildew of strawberries is not limited to the above method. All methods which can culture G2C3 in large quantity and can keep the strawberry powdery mildew resistance activity can be used for preparing the biocontrol preparation claimed by the invention.
The biocontrol strain G2C3 is applied to preventing and treating powdery mildew of strawberries and increasing yield.
The biocontrol microbial inoculum is applied to preventing and treating powdery mildew of various crops such as strawberries and the like and increasing yield.
Has the advantages that:
the invention relates to a biocontrol microbial inoculum specially developed for strawberry powdery mildew. Because the microbial preparation completely avoids a series of problems caused by applying chemical pesticides, the microbial preparation is beneficial to green and pollution-free production of planting crops such as strawberries, greatly reduces the consumption of the chemical pesticides, can save expenses, reduces the residue of the chemical pesticides, protects the safety of agricultural products, and really realizes the production of organic products.
Greenhouse experiments show that: the biocontrol strain G2C3 can effectively prevent and treat powdery mildew of strawberries, and the prevention and treatment effect reaches 69.69%. The field experiment result shows that the biocontrol strain G2C3 has remarkable effect of preventing and treating strawberry powdery mildew, and the prevention and treatment effect is 51.27-54.56%; the yield increasing effect of the microbial inoculum on strawberries is more than 30%.
Biological sample preservation information
The strain G2C3, Bacillus subtilis, was deposited in the China general microbiological culture Collection center in 7 months and 26 months in 2018 at the deposition address: no. 3 of Xilu No.1 of Beijing, Chaoyang, and the preservation number of the strain is CGMCC NO. 16164.
Drawings
FIG. 1 is a plate culture of Bacillus subtilis G2C3 according to the present invention.
FIG. 2 is a cell morphology of Bacillus subtilis G2C3 in the present invention.
FIG. 3 is a phylogenetic tree of Bacillus subtilis G2C3 according to the present invention.
Detailed Description
The invention is further illustrated by the following figures and examples.
Example 1 screening and identification of Strain G2C3
The screening method comprises the following steps: the bacillus subtilis G2C3 is obtained by separating rhizosphere soil of strawberries in a strawberry planting greenhouse in the Zhu river city. Adding 5g of strawberry rhizosphere soil into 45g of sterile water, shaking in a shaking table at 180r/min for 30min, and standing for 10min to obtain 10-1A soil dilution; will 10-1The soil diluent is diluted in a gradient way, 10 are respectively taken-3，10-5，10-7And (3) plating 100uL of the dilution solution with gradient on an LB plate, culturing for 48h at 37 ℃, selecting a single colony, inoculating the single colony on a new LB plate, and repeating the steps for multiple times to obtain the purified strain. The purified strain was stored in a-80 ℃ refrigerator with 40% glycerol.
The identification method comprises the following steps: the strain is identified through morphological characteristics, physiological and biochemical experiments and 16S rDNA sequence analysis.
Morphological characteristics: gram-positive bacteria, rod-like, often present singly or in pairs, are aerobic. The colony is opaque, regular round, with spread surface and irregular edge, and is generally 0.5-1.2mm in size.
The main physiological and biochemical characteristics are as follows:
TABLE 1 major physiological and biochemical characteristics of Strain G2C3
16S rRNA gene amplification and sequence analysis:
culturing bacillus subtilis G2C3 in LB culture medium to logarithmic phase, centrifuging at 37 ℃ and 12000r/min for 5min to collect thalli, using genome DNA rapid extraction kit of Botryobo bioengineering (Dalian) Limited company to extract genome DNA, then using the extracted DNA as a template, using bacterial 16S rRNA to amplify a universal primer 27F: 5 '-AGAGTTTGATCMTGGCTCAG-3'; 1492R: 5'-GGTTACCTTGTTACGACTT-3', respectively; A16S rDNA gene fragment was amplified from the genomic DNA. The PCR product is sent to Shanghai biological engineering Co., Ltd for sequencing. Homology comparison of the determined 16S rRNA gene sequences was performed by BLAST software, and the strain was identified as Bacillus subtilis as shown in Table 2.
Table 2G2C3 sequencing alignment results
Example 2G2C3 preparation of an anti-microbial agent
Inoculating Bacillus subtilis G2C3(CGMCC NO.16164) into LB liquid culture medium, shake culturing at 37 deg.C for 18 hr, sampling every 2 hr, measuring OD at 600nm, and culturing when OD is 0.5-0.8 to obtain the bacterial liquid as seed liquid. Inoculating the seed solution into LB fermentation culture solution with an inoculum size of 0.5%, fermenting at 30 deg.C and 180rpm for 24 hr, with dissolved oxygen content of 20% and ventilation volume of 5-8m3The tank pressure is 0.05-0.1KPa, and the pH is maintained at 7.0. Centrifuging the bacterial liquid at 4000r/min for 15min after fermentation is finished, diluting the obtained precipitate into a microbial inoculum by using sterile water, wherein the effective viable count in the finished microbial inoculum is 1 multiplied by 109-1010CFU/mL。
Example 3 Effect control test of G2C3 on strawberry powdery mildew disease under greenhouse conditions
The strawberry variety is "Happy incense", the bacterial agent prepared in example 2 is taken and diluted with water until the final concentration of the bacterial liquid is 5X 107CFU/mL, the root dipping and transplanting are carried out at the 4-leaf stage, the leaf surface spraying is carried out, and the spraying standard is that each leaf is distributed by mist-shaped liquid drops so as not to fall. Inoculating powdery mildew of strawberry on the 7 th day after the treatment of the biocontrol microbial inoculum. The same treatment was performed 2 nd day 15 after the 1 st treatment. The control group was prepared by inoculating only powdery mildew of strawberry and spraying no biocontrol agent. Each treatment had 24 strawberries, 3 in parallel. Placing strawberry at 28 deg.C, and irradiating with light 1Culturing under greenhouse condition of 2h/2 h. Investigating disease grade, calculating disease severity and preventing and treating effect.
The disease grade standard of powdery mildew is as follows:
level 0: no disease spots;
level 1: the lesion area accounts for less than 5% of the whole leaf area;
and 3, level: the lesion area accounts for 6 to 15 percent of the whole leaf area;
and 5, stage: the lesion area accounts for 16 to 25 percent of the whole leaf area;
and 7, stage: the lesion area accounts for 26-50% of the whole leaf area;
and 9, stage: the lesion area accounts for more than 50% of the whole leaf area;
disease index of 100X Sigma (number of disease leaves in each stage x corresponding stage value)/(total number of survey leaves x highest disease stage number)
The control effect (%) is 100% × (control disease index-treatment disease index)/control disease index
The investigation result of the 1 st treatment of the biocontrol microbial inoculum G2C3 for 30 days shows that the control effect of the biocontrol strain G2C3 on the powdery mildew disease of strawberry reaches 60.0 percent, and the result is shown in Table 3.
TABLE 3 biocontrol microbial inoculum G2C3 biocontrol effect on strawberry powdery mildew disease (30d)
Note: the result is the statistical effect of the control group after the treatment of the anti-microbial agent G2C3 for 30 days and the attack of the control group for 15 days.
Example 4 sunlight greenhouse field test verifies the control effect and yield increase effect of the biocontrol microbial inoculum G2C3 on strawberry powdery mildew
The experimental site: sunlight greenhouse for strawberries in Taiping ditch of Taiping town in Zhuang river
The strawberry variety is "happy fragrant". The field test was divided into 2 treatment groups in total. Treatment group 1 was the G2C3 biocontrol agent prepared in example 2, and treatment group 2 was a control group administered with clear water. Each treated 3 cells, 60 seedlings per cell. The pesticide application method is page spraying, and the spraying standard is as follows: each leaf is divided by mist-like dropletsCloth, so as not to fall. The spray concentration is 5 × 107CFU/mL. After the 1d treatment, the same treatment was performed every 15 d. The growth conditions of the seedlings are observed every day, and the disease index and the prevention and treatment effect are calculated and shown in Table 4. The control effect of the biological control bactericide G2C3 on the powdery mildew disease of the strawberries after the strawberries are transplanted for 45 days reaches 55.60%.
TABLE 4 biocontrol microbial inoculum G2C3 biocontrol effect on strawberry powdery mildew disease (45d)
Note: the result is the statistical effect of the control group after the treatment of the anti-microbial agent G2C3 for 30 days and the attack of the control group for 15 days.
At the same time, we performed statistics of yield. The result shows that compared with the control group, the yield increasing effect of the biocontrol microbial inoculum G2C3 on strawberries is 38.46%, and the weight of a single fruit is obviously higher than that of the control group. The results are shown in Table 5.
TABLE 5 Effect of biocontrol agent G2C3 on strawberry yield
Claims (4)
1. A biocontrol strain G2C3 for preventing and treating powdery mildew of strawberry is identified to belong to Bacillus subtilis, and is preserved in China general microbiological culture Collection center in 7-month and 26-month days in 2018, wherein the strain preservation number is CGMCC NO. 16164.
2. A method for preparing a biocontrol microbial inoculum is characterized by comprising the following steps: the biocontrol strain G2C3 for preventing and controlling powdery mildew disease is cultured in LB liquid culture medium at 32 ℃ for 28h at 180r/min in a shaking table, and then centrifuged for 15min at 4000 r/min. Diluting with sterile water to obtain bacterial liquid with effective viable count of 1 × 109-1010CFU/mL。
3. The biocontrol strain G2C3 for preventing and treating strawberry powdery mildew disease according to claim 1, wherein the strain is applied to preventing and treating strawberry powdery mildew disease and increasing yield.
4. The method for preparing the biocontrol microbial inoculum according to claim 2, wherein the biocontrol microbial inoculum prepared by the method is applied to preventing and treating powdery mildew of strawberry and increasing yield.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116179367A (en) * | 2023-01-30 | 2023-05-30 | 江苏农林职业技术学院 | Strawberry disease biocontrol bacterium JSNL-B118 and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102250814A (en) * | 2011-07-20 | 2011-11-23 | 范成明 | Bacillus subtilis and use thereof in bio-control preparation for preventing and controlling powdery mildew |
| CN104403970A (en) * | 2014-11-27 | 2015-03-11 | 青岛蔚蓝生物集团有限公司 | Bacillus subtilis and application thereof in plant disease control |
| CN104872200A (en) * | 2015-04-21 | 2015-09-02 | 苏州市新泾村农业基地专业合作社 | Biological pesticide suitable for strawberry powdery mildew and preparation method of biological pesticide |
| CN105145309A (en) * | 2015-09-23 | 2015-12-16 | 山东农业大学 | Integrated ecological strawberry cultivation method for reducing pest and disease damage and improving fruit quality |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102250814A (en) * | 2011-07-20 | 2011-11-23 | 范成明 | Bacillus subtilis and use thereof in bio-control preparation for preventing and controlling powdery mildew |
| CN104403970A (en) * | 2014-11-27 | 2015-03-11 | 青岛蔚蓝生物集团有限公司 | Bacillus subtilis and application thereof in plant disease control |
| CN104872200A (en) * | 2015-04-21 | 2015-09-02 | 苏州市新泾村农业基地专业合作社 | Biological pesticide suitable for strawberry powdery mildew and preparation method of biological pesticide |
| CN105145309A (en) * | 2015-09-23 | 2015-12-16 | 山东农业大学 | Integrated ecological strawberry cultivation method for reducing pest and disease damage and improving fruit quality |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116179367A (en) * | 2023-01-30 | 2023-05-30 | 江苏农林职业技术学院 | Strawberry disease biocontrol bacterium JSNL-B118 and application thereof |
| CN116179367B (en) * | 2023-01-30 | 2023-08-25 | 江苏农林职业技术学院 | Strawberry disease biocontrol bacterium JSNL-B118 and application thereof |
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Application publication date: 20210108 |