CN111418610A - Application of bacillus laterosporus in killing fly maggot and its culture method - Google Patents
Application of bacillus laterosporus in killing fly maggot and its culture method Download PDFInfo
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- CN111418610A CN111418610A CN202010386193.4A CN202010386193A CN111418610A CN 111418610 A CN111418610 A CN 111418610A CN 202010386193 A CN202010386193 A CN 202010386193A CN 111418610 A CN111418610 A CN 111418610A
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- bacillus laterosporus
- killing
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
Abstract
The invention belongs to the technical field of microorganism mosquito and fly killing, and particularly relates to application of bacillus laterosporus in fly and fly killing, and a culture method of the bacillus laterosporus. The application of the bacillus laterosporus in killing fly maggots, the application in mosquitocides, mosquitocides and mosquitocide and the application in preparing feed with the function of killing fly maggots are all the protection scope of the invention. The bacillus laterosporus fungicide has the beneficial effects that (1) the bacillus laterosporus fungicide is used in livestock breeding or livestock breeding, and has high safety and strong practicability; (2) the bacillus laterosporus fungicide provided by the invention can be directly applied to killing of fly maggots in livestock and poultry breeding manure, so that the death rate of the fly maggot adults reaches 77.2 percent, and the death rate of larvae reaches 87.9 percent; (3) the bacillus laterosporus fungicide can also be applied to feeds, so that the death rates of adult mosquitoes and flies and larvae are respectively 95.8 percent and 100 percent.
Description
Technical Field
The invention belongs to the technical field of microorganism mosquito and fly killing, and particularly relates to application of bacillus laterosporus in fly and fly killing, and a culture method of the bacillus laterosporus.
Background
In the process of livestock and poultry breeding or livestock breeding, mosquitoes and flies are easy to breed, and more than 300 common mosquitoes and flies exist at present; common flies include housefly, green fly, chrysomyia megacephala and blowfly. In summer with high temperature and high humidity, because flies and mosquitoes breed in large quantity, the spread of animal epidemic diseases is accelerated, the safety hazard of the current epidemic disease outbreak is formed, a plurality of adverse effects are brought to the breeding industry, meanwhile, the flies and the mosquitoes spread various diseases of human beings, the mosquito-fly-: 1. preventing the livestock from resting and reducing the yield of the livestock; 2. carrying germs and spreading diseases; 3. threatens the health of personnel and reduces the working efficiency.
With regard to livestock breeding or livestock breeding, currently, mainly adopted measures are environmental control, feed proportioning, chemical methods, such as combining limewater and pesticide cleaning, such as killing with chemical agents; the wide application of chemical pesticides to kill mosquitoes causes serious environmental pollution problems, and the use of chemical pesticides in animal husbandry also affects the safety of the farming. In addition, biological methods such as growing flowers and plants, and culturing natural enemies such as spiders, geckoes, frogs, and the like are also used; the biological methods described above, however, have limited effectiveness. Regarding the biological mosquito killing agent, Suxiaoqing published article "a novel biological mosquito killing agent Pythium biocinum preparation", which discloses: the existing mainly-combed microbial mosquito killing agent is Bacillus thuringiensis Israel variant and Bacillus sphaericus, and the two kinds of mosquito killing bacteria have many advantages, such as good mosquito killing effect, easy production, storage and application, but also have defects, such as short duration of Bti, easy induction of mosquito resistance by Bs and the like.
The prior invention mainly has the following problems and disadvantages:
1. the prior invention does not aim at fly maggots generated by excrement of the breeding industry;
2. most of the drugs are used in a spray form, and only can expel the mosquito and fly on the air and contact surface, but cannot solve the fly larvae in the excrement;
therefore, in order to overcome the defects of the above schemes, a safe and effective biological mosquito-killing agent, especially a microbial mosquito-killing agent, which can be applied to livestock and poultry breeding or livestock breeding needs to be invented.
Disclosure of Invention
In order to solve the technical problems, the invention provides a microbial mosquito killing agent, namely bacillus laterosporus, which can be directly used as a fly killing agent to solve the problem of fly maggots in livestock and poultry breeding plants; and also provides a culture method of the bacillus laterosporus.
The application of the bacillus laterosporus fungicide in killing fly maggots; and the application of the bacillus laterosporus fungicide in killing fly maggots in an animal farm or a livestock and poultry breeding plant; the application of the bacillus laterosporus fungicide in preparing a mosquito killing agent, a mosquito killing medicine and mosquito killing water for killing mosquitoes and flies in an animal farm or a livestock and poultry breeding plant is within the protection range of the invention.
The application can add the bacillus laterosporus into livestock and poultry manure to ensure that the spore concentration is 6 × 108cfu/g feces.
The manure is any one of chicken manure, pig manure, cow manure, and sheep manure, but is not limited to the above types.
The application of the bacillus laterosporus microbial inoculum in preparing the feed with the function of killing fly maggots is also within the protection scope of the invention.
The microbial inoculum of the invention is applied to the preparation of the feed, and the bacillus laterosporus is added into the feed to ensure that the concentration of spores is1～9×1010cfu/g feed.
The 16sRNA sequence of Bacillus laterosporus is shown in SEQ ID No. 1.
The culture method of the bacillus laterosporus microbial inoculum comprises the following steps:
(1) selecting a Brevibacillus laterosporus inclined plane mother strain to carry out shake flask seed culture, wherein 8 1000m L shake flasks with the liquid loading amount of 400m L are inoculated on one inclined plane, the culture temperature is 32-39 ℃, the rotating speed is 180-220 r/min, the culture time is 18-24 h, and the formula of the used culture medium comprises 1% of sodium chloride, 1% of peptone, 0.5% of yeast extract and the pH value is 7.0;
(2) seed tank culture: the inoculation amount is 5-10%; the temperature is 35-39 ℃; stirring at a speed of 150-400 r/min; the ventilation volume is 3-4 m3The culture medium is prepared from corn flour 60-70 g/L, fish peptone 10-15 g/L, sodium chloride 4-6 g/L, magnesium sulfate 0.1-0.25 g/L2PO42～4g/L；
(3) Fermentation culture: the inoculation amount is 5-10%; the temperature is 35-39 ℃; stirring at a speed of 150-400 r/min; the ventilation volume is 3-4 m3The culture medium is prepared from corn flour 60-70 g/L, fish peptone 10-15 g/L, sodium chloride 4-6 g/L, magnesium sulfate 0.1-0.25 g/L2PO42～4g/L；
(4) And (4) carrying out spray drying on the fermentation liquor obtained in the step (3) by using a spray tower according to a conventional method to obtain a powdery bacillus laterosporus microbial inoculum.
The sequence of the 16sRNA of the bacillus laterosporus provided by the invention is shown as a sequence 1.
The invention has the beneficial effects that:
(1) the bacillus laterosporus fungicide provided by the invention is used in livestock breeding or livestock breeding, has high safety and strong practicability, and provides a novel method and thought for applying the microbial fungicide to killing mosquitoes and flies;
(2) the bacillus laterosporus fungicide provided by the invention can be directly applied to killing of fly maggots in livestock and poultry breeding manure, so that the death rate of the fly maggot adults reaches 77.2 percent, and the death rate of larvae reaches 87.9 percent;
(3) the bacillus laterosporus fungicide can also be applied to feeds, so that the death rates of adult mosquitoes and flies and larvae are respectively 95.8 percent and 100 percent.
Detailed Description
The present invention will now be further described with reference to specific embodiments in order to enable those skilled in the art to better understand the present invention.
Example 1
The method for culturing the bacillus laterosporus microbial inoculum comprises the following steps:
(1) selecting a Brevibacillus laterosporus inclined plane mother strain to carry out shake flask seed culture, wherein 8 shake flasks with the volume of 1000m L and the liquid loading of 400m L are inoculated on one inclined plane, the culture temperature is 35 ℃, the rotating speed is 200r/min, the culture time is 20 hours, and the formula of the used culture medium comprises 1 percent of sodium chloride, 1 percent of peptone, 0.5 percent of yeast extract and the pH value is 7.0;
(2) seed tank culture: the inoculation amount is 8 percent; the temperature is 37 ℃; the stirring speed is 240 r/min; the ventilation volume is 3.5m3The culture medium is prepared from corn flour 60 g/L, fish peptone 10 g/L, sodium chloride 4 g/L, and magnesium sulfate 0.1 g/L2PO42g/L；
(3) Fermentation culture: the inoculation amount is 8 percent; the temperature is 37 ℃; stirring at 300 r/min; ventilation volume 4m3The culture medium is prepared from corn flour 60 g/L, fish peptone 10 g/L, sodium chloride 4 g/L, and magnesium sulfate 0.1 g/L2PO42g/L；
(4) And (4) carrying out spray drying on the fermentation liquor obtained in the step (3) by using a spray tower according to a conventional method to obtain a powdery bacillus laterosporus microbial inoculum.
Example 2
The method for culturing the bacillus laterosporus microbial inoculum comprises the following steps:
(1) selecting a Brevibacillus laterosporus inclined plane mother strain to carry out shake flask seed culture, wherein 8 shake flasks with the volume of 1000m L and the liquid loading of 400m L are inoculated on one inclined plane, the culture temperature is 38 ℃, the rotating speed is 210r/min, the culture time is 24h, and the formula of the used culture medium comprises 1 percent of sodium chloride, 1 percent of peptone, 0.5 percent of yeast extract and the pH value is 7.0;
(2) seed tank culture: amount of inoculation8 percent; the temperature is 38 ℃; stirring at a speed of 200 r/min; ventilation volume 4m3The culture medium is prepared from corn flour 65 g/L, fish peptone 13 g/L, sodium chloride 5 g/L, and magnesium sulfate 0.15 g/L2PO43g/L；
(3) Fermentation culture: the inoculation amount is 8 percent; the temperature is 38 ℃; stirring at a speed of 180 r/min; ventilation volume 4m3The culture medium is prepared from corn flour 65 g/L, fish peptone 13 g/L, sodium chloride 5 g/L, and magnesium sulfate 0.15 g/L2PO43g/L；
(4) And (4) carrying out spray drying on the fermentation liquor obtained in the step (3) by using a spray tower according to a conventional method to obtain a powdery bacillus laterosporus microbial inoculum.
Example 3
The method for culturing the bacillus laterosporus microbial inoculum comprises the following steps:
(1) selecting a Brevibacillus laterosporus inclined plane mother strain to carry out shake flask seed culture, wherein 8 shake flasks with the volume of 1000m L and the liquid loading of 400m L are inoculated on one inclined plane, the culture temperature is 36 ℃, the rotating speed is 200r/min, the culture time is 22h, and the formula of the used culture medium comprises 1 percent of sodium chloride, 1 percent of peptone, 0.5 percent of yeast extract and the pH value is 7.0;
(2) seed tank culture: inoculating 9 percent; the temperature is 35 ℃; stirring at a speed of 400 r/min; ventilation volume 3m3The culture medium is prepared from corn flour 70 g/L, fish peptone 15 g/L, sodium chloride 6 g/L, and magnesium sulfate 0.25 g/L2PO44g/L；
(3) Fermentation culture: the inoculation amount is 5 percent; the temperature is 35 ℃; the stirring speed is 150 r/min; ventilation volume 3m3The culture medium is prepared from corn flour 70 g/L, fish peptone 15 g/L, sodium chloride 6 g/L, and magnesium sulfate 0.25 g/L2PO44g/L；
(4) And (4) carrying out spray drying on the fermentation liquor obtained in the step (3) by using a spray tower according to a conventional method to obtain a powdery bacillus laterosporus microbial inoculum.
Example 4
4.1 the application of the Brevibacillus laterosporus microbial inoculum in killing mosquitoes and flies in livestock and poultry excrement, which comprises the following specific steps: the brevibacillus laterosporus agent in the embodiment 1-3 is directly mixed with fresh excrement, the number of fly maggots in the excrement is analyzed and detected after 1 day, and the contrast example is that the excrement without the brevibacillus laterosporus agent is directly detected.
TABLE 1 mortality of adults after fly maggot extermination by Brevibacillus laterosporus agents of different concentrations (%)
| Additive concentration (hundred million cfu/g feces) | 0 | 1×108 | 2×108 | 3×108 | 4×108 | 5×108 | 6×108 |
| Example 1 | 0.64 | 45.3 | 54.1 | 63.5 | 74.3 | 88.5 | 98.7 |
| Example 2 | 0.72 | 44.8 | 54.4 | 63.1 | 74.2 | 87.9 | 98.4 |
| Example 3 | 0.69 | 45.6 | 53.9 | 63.8 | 73.8 | 88.8 | 98.9 |
TABLE 2 Larvae mortality after fly maggot extermination (%)
As can be seen from tables 1 and 2 above, when the concentration of the Brevibacillus laterosporus agent reaches 6 × 108When the concentration reaches 4 × 10, the killing effect on fly maggot imagoes is good and reaches more than 98.4 percent8In time, the effect of killing the larvae is better, and the killing effect of the three embodiments reaches 100 percent.
4.2 the application of the Brevibacillus laterosporus microbial inoculum in the feed has the following mortality rate for killing fly maggots:
mixing different amounts of solid product Brevibacillus laterosporus microbial inoculum with feed, feeding 817 broiler chickens, detecting fly maggot quantity in feces every other day, wherein the control example is an application example of a feed group without adding Brevibacillus laterosporus microbial inoculum, and the spore concentration in each gram of feed is respectively 0, 1 × 107，1×108，1×109，1×1010cfu/g；
TABLE 3 mortality of adults after fly maggot extermination (%)
TABLE 4 Lawsonia laterosporus bacteria agent with different concentrations applied to feed for killing fly maggot and then larval mortality (%)
As can be seen from tables 3 and 4 above, the concentration of the Brevibacillus laterosporus agent reaches 1 × 1010When the pesticide is used, the killing rate of the pesticide on adults can reach about 77 percent; the killing rate of the larva can reach about 87.3 percent; the application effect is better on the whole, but the killing effect on mosquitoes and flies is slightly worse than that of 4.1 which directly puts the organic fertilizer into excrement.
4.3 about the application of commercial fly products and the Brevibacillus laterosporus of the invention in excrement, after 1 day, the killing rate of adult and larva of fly is detected, and the results are as follows:
TABLE 5 direct application of Brevibacillus laterosporus agents to the mortality of adult fecal insects (%)
As can be seen from the table 5, the mosquito and fly killing effect of the commercial products is obviously lower than that of the products of the invention, and the result of the test group 1 shows that the death rate of the imagoes or the larvae after the brevibacillus laterosporus fungicide is added reaches 95 percent and 100 percent; after the commercial product is added, the death rate of adults and larvae is only 60.3 percent and 12.8 percent.
Example 5
The application of the brevibacillus laterosporus microbial inoculum in preparing the mosquito killing agent comprises the following steps of dissolving a proper amount of the brevibacillus laterosporus microbial inoculum in 5L water, and uniformly stirring at the rotating speed of 25r/min to obtain the mosquito killing agent.
The mosquito killing agent is added into excrement according to the dosage of 4.1 in example 4, and more than 90% of fly maggot larvae in the livestock excrement are killed through detection; more than 85% of adults.
Example 6
The application of the brevibacillus laterosporus microbial inoculum in preparing the feed comprises the following specific steps:
taking a proper amount of the brevibacillus laterosporus microbial inoculum of the invention, and uniformly mixing the brevibacillus laterosporus microbial inoculum with feed to ensure that the concentration of spores in each gram of feed is 1 × 1010cfu/g, and then feeding livestock and poultry, compared with the control example, detecting fly maggots in pig manure, and finding that the death rate of adults reaches about 77.1 percent and the death rate of larvae reaches about 82.4 percent.
Sequence listing
<110> Shandongtianrun and bioengineering Co Ltd
Application of bacillus laterosporus in killing fly maggot and culture method thereof
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>1151
<212>RNA
<213> Bacillus laterosporus (L atin Name Bacillus laterosporus L aubach)
<400>1
cagaggcggc acagcaacga gcggaccgag ggagcgcccc aagagcggcg gacggggaga 60
acacggagaa ccgccgaaga cgggaaacac gggaacacgg gcaaaccaga ggggaaccgc 120
aggcagacaa aaaggggccg gcaccacaca gaggacccgc ggcgcaagca ggggaggaac 180
ggccaccaag gcaacgagcg aagccgaccc agaggggaac ggccacacgg gacgacacac 240
ggcacagacc cacgggaggc agcagaggga acccgcaagg acgaaagcga cggagcaacg 300
ccgcggagga gaaggacgaa cgaaagccgg agggaagaac aaggccgcaa aagggcggca 360
ccgacggacc aaccagaaag ccacggcaac acgaccagca gccgcagaaa cgaggggcaa 420
gcggccggaa agggcgaaag gccgcaggcg gcaagcgagg aaagccaccg gccaaccggg 480
gagggcagga aacggggaac gaggaagaag aggagaggga accacggagc gggaaagcga 540
gagagggaga acaccagggc gaaggcgacc cggcgaacga cgcgaggagc gaaagcggga 600
ggcgaacagg aagaacccgg agccacgccg aaacgagagg caaggagggg gccgcaccag 660
gcgcagcaac gcaaagcacc cgccggggag acgcgcaaga cgaaaccaaa ggaagacgga 720
gcacgccaag cggggagcag ggaacgaagc aacgcgaaga accaccaggc gacacccgac 780
accagagaag gacgcccccg gcggcagagg acagggggca gggcgcagcc ggcggagagg 840
ggaagcccgc aacgagcgca acccgacagg ccagcacagg ggcaccaagg gacgccggga 900
caaaccggag gaagggggga gacgcaaaca cagccccaga ccgggcacac acggcacaag 960
gacagaacaa agagcagcga accgcgagga agccaaccca caaacgccag cggacgcagc 1020
gcaaccgacg cggaagcgga acgcagaacg cggacagcag ccgcgggaaa cgcccgggcc 1080
gacacaccgc cgcacaccac gagaggacac ccgaagcgag aggaaccagg agccagccgc 1140
agaagacgcc a 1151
Claims (9)
1. The application of the bacillus laterosporus microbial inoculum in killing fly maggots.
2. The application of the bacillus laterosporus fungicide in killing fly maggots in an animal farm or a livestock and poultry breeding plant.
3. The application of the bacillus laterosporus fungicide in preparing a mosquito killing agent, a mosquito killing medicine and mosquito killing water for killing mosquitoes and flies in an animal farm or a livestock and poultry breeding plant.
4. The use as claimed in any one of claims 1 to 3, wherein the Bacillus laterosporus preparation is added to livestock and poultry manure to make the spore concentration greater than or equal to 6 × 108cfu/g feces.
5. The use according to claim 4, wherein the manure is any one of chicken manure, pig manure, cow manure, sheep manure.
6. The application of the bacillus laterosporus microbial inoculum in preparing the feed with the function of killing fly maggots.
7. The use of claim 6, wherein the Bacillus laterosporus is added to the feed to a spore concentration of 1 to 9 × 1010cfu/g feed.
8. The bacillus laterosporus of claim 1, wherein the bacillus laterosporus 16sRNA sequence is as shown in seq id No. 1.
9. The Bacillus laterosporus of claim 1, wherein the Bacillus laterosporus bacterial agent is cultured by the following method:
(1) selecting a Brevibacillus laterosporus inclined plane mother strain to carry out shake flask seed culture, wherein 8 1000m L shake flasks with the liquid loading amount of 400m L are inoculated on one inclined plane, the culture temperature is 32-39 ℃, the rotating speed is 180-220 r/min, the culture time is 18-24 h, and the formula of the used culture medium comprises 1% of sodium chloride, 1% of peptone, 0.5% of yeast extract and the pH value is 7.0;
(2) seed tank culture: the inoculation amount is 5-10%; the temperature is 35-39 ℃; stirring at a speed of 150-400 r/min; the ventilation volume is 3-4 m3The culture medium is prepared from corn flour 60-70 g/L, fish peptone 10-15 g/L, sodium chloride 4-6 g/L,magnesium sulfate 0.1-0.25 g/L2PO42～4 g/L；
(3) Fermentation culture: the inoculation amount is 5-10%; the temperature is 35-39 ℃; stirring at a speed of 150-400 r/min; the ventilation volume is 3-4 m3The culture medium is prepared from corn flour 60-70 g/L, fish peptone 10-15 g/L, sodium chloride 4-6 g/L, magnesium sulfate 0.1-0.25 g/L2PO42-4 g/L；
(4) And (4) carrying out spray drying on the fermentation liquor obtained in the step (3) by using a spray tower according to a conventional method to obtain a powdery bacillus laterosporus microbial inoculum.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100003227A1 (en) * | 2006-09-15 | 2010-01-07 | Universita' Degli Studi Di Sassari | Brevibacillus Laterosporus Strain Compositions Containing the Same and Method for the Biological Control of Dipters |
| CN104994739A (en) * | 2012-09-24 | 2015-10-21 | 林肯大学 | New strains of side brevibacillus laterosporus of biocontrol agent as plant injurious insect, specially Lepidoptera and diptera |
| CN107669616A (en) * | 2017-11-02 | 2018-02-09 | 吕丽娅 | A kind of preparation method of long-acting mosquito-driving floral water |
| CN109007273A (en) * | 2018-06-06 | 2018-12-18 | 广东谷盈生物科技产业研究院有限公司 | A kind of multi-cultur es composite microbial feed additive and its application |
-
2020
- 2020-05-09 CN CN202010386193.4A patent/CN111418610A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100003227A1 (en) * | 2006-09-15 | 2010-01-07 | Universita' Degli Studi Di Sassari | Brevibacillus Laterosporus Strain Compositions Containing the Same and Method for the Biological Control of Dipters |
| CN104994739A (en) * | 2012-09-24 | 2015-10-21 | 林肯大学 | New strains of side brevibacillus laterosporus of biocontrol agent as plant injurious insect, specially Lepidoptera and diptera |
| CN107669616A (en) * | 2017-11-02 | 2018-02-09 | 吕丽娅 | A kind of preparation method of long-acting mosquito-driving floral water |
| CN109007273A (en) * | 2018-06-06 | 2018-12-18 | 广东谷盈生物科技产业研究院有限公司 | A kind of multi-cultur es composite microbial feed additive and its application |
Non-Patent Citations (2)
| Title |
|---|
| L. RUIU ET AL: "Administration of Brevibacillus laterosporus spores as a poultry feed additive to inhibit house fly development in feces: A new eco-sustainable concept", 《POULTRY SCIENCE》 * |
| 蒲蛰龙: "《昆虫病理学》", 31 August 1994, 广东科技出版社 * |
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Application publication date: 20200717 |