CN106834195A - A kind of screening of anti-booth vegetable grey mold bacillus subtilis and bacterial preparation process - Google Patents
A kind of screening of anti-booth vegetable grey mold bacillus subtilis and bacterial preparation process Download PDFInfo
- Publication number
- CN106834195A CN106834195A CN201710220444.XA CN201710220444A CN106834195A CN 106834195 A CN106834195 A CN 106834195A CN 201710220444 A CN201710220444 A CN 201710220444A CN 106834195 A CN106834195 A CN 106834195A
- Authority
- CN
- China
- Prior art keywords
- grey mold
- booth vegetable
- bacillus subtilis
- vegetable
- screening
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
-
- 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
- C12N1/02—Separating microorganisms from their culture media
-
- 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
- C12N3/00—Spore forming or isolating processes
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Ash arrhizus bacteria host range is wide, is one of crops Major Diseases source, is wasted time and energy using agricultural measures preventing and treating at present and effect is undesirable, and grey mold easily develops immunity to drugs to chemical agent, and chemopreventive effects decline even to be lost.Biological control have the advantages that it is safe and environment-friendly, be grey mold preventing and treating important development direction, have huge application prospect.The present invention provides screening and the bacterial preparation process of a kind of anti-booth vegetable grey mold bacillus subtilis, a kind of anti-booth vegetable grey mold bacillus subtilis is filtered out from green house of vegetables soil, growth of the bacterial strain to grey mold has strong inhibitory action, can be used for the biological control of grey mold disease.And the preparation of microbial inoculum is realized by scale fermentation processes, it is applied to the preventing and treating of booth vegetable grey mold.
Description
Technical field
The invention belongs to biotechnology and field of biological control, more particularly to a kind of anti-booth vegetable grey mold bacillus subtilis
The screening of bacterium and bacterial preparation process.
Background technology：
Gray mold is a kind of global Major Diseases in vegetables production, and cause of disease is mainly the pathogen of Botrytis cinerea
(Boytrytiscinerea) Deuteromycotina fungi, is belonged to, its sexual generation belongs to Ascomycotina (Ascomycotina) disk
Grape in Gammaproteobacteria (Discomycetes) Helotiales (Helotiales) Sclerotiniaceae (Sclerotiniaceae) embraces cup fungi
Category (Botrytinia).It can endanger the various vegetables such as tomato, strawberry, pears, onion, grape and melon and fruit.Pathogen is more to be lost from opening
Female flower leaching people, cause petal to rot, and grow glaucous mould layer, young fruit is softened rapidly, part of damage stops life
It is long, rotten or come off, etching or decayed fruit be attached on stem cause stem's disease, it is serious when bottom stem rot rotten make top withered
Extremely.Botrytis cinerea not only influences the plant in growth period, and it does not show symptom on the plant of growth sometimes, but in latent
State after harvesting fruit storage and transportation in, due to the high humidity in hutch, temperature is low and fall ill on a large scale,
So that agricultural economy is subject to massive losses.
Preventing and treating at present to ash arrhizus bacteria is main based on chemical prevention, is aided with biological control and agricultural measures preventing and treating.By
It is easy to develop immunity to drugs in botrytis cinerea, and it is main with fruit as target during dispenser, directly consumer is constituted a threat to so that change
The method for learning preventing and treating increasingly can not be big well-established.Moreover substantial amounts of applying pesticides are also caused to agroecological environment
Very big negative effect.And agricultural measures preventing and treating input is big time-consuming takes a lot of work and effect is undesirable.Therefore in the urgent need to a kind of high
Effect, the prophylactico-therapeutic measures of low toxicity, by the disease damage control below economic threshold values.Biological control is due to its low stain, low-residual
Characteristic and highlight advantage, the emphasis as current research screens the beneficial bacterial strain and its metabolite of anti-grey mold to prevent and treat ash
Mould is the direction of agricultural sustainable development, is had broad application prospects.
The content of the invention
Goal of the invention：
The present invention for botrytis cinerea to tomato etc. murrain it is huge, industrialized agriculture and chemical prevention and control method effect have
The present situation of limit, screening efficient grey mold antagonistic strain is used for the biological control of grey mold disease.
Technical scheme：
The technical solution adopted by the present invention is as follows：
Soil, vegetable root, leaf, flower, fruit sample are gathered from vegetable cultivations such as Shandong, Hebei, Jiangxi, Beijing.Using flat
Plate culture and the method for picking single bacterium colony, separate grey mold and bacterium from these fresh samples.By will be isolated thin
Bacterium and botrytis cinerea antagonistic effect, screening anti-grey mold microorganism is used for grey mold biological control.Then ash can be suppressed to what is be separated to
Mould microorganism, and identified, and then microbial inoculum is prepared by scale fermentation, it is applied to the preventing and treating of booth vegetable grey mold.
Specific embodiment
Embodiment 1：It is prepared by the screening of booth vegetable grey mold and spore
Soil, vegetable root, leaf, flower, 84 parts of fruit sample are gathered from vegetable cultivations such as Shandong, Hebei, Jiangxi, Beijing.Adopt
It is placed in after collection fresh sample in 4 DEG C of Refrigerator stores, one week and completes microorganism separation.Soil and vegetables root grey mold separation method
For：Take 3-5g soil or be placed in 100ml sterilized waters with the vegetable root that the scissors sterilized is shredded, at room temperature 150rpm concussions 1h
Afterwards, 10 are diluted to sterilized water-1、10-2、10-3、10-4、10-5、10-6.、10-7、10-8Eight gradients, it is 10 to take extension rate-4、
10-5、10-6、10-7、10-8Sample 0.1ml coat PDA solid mediums ((potato 200g, glucose 20g, agar powder 10
~15g, distilled water 1000mL)) on, 27 DEG C are cultivated 5 days, are then peeled off identification.The preparation of grey mold spore suspension：Reflected separating
Grey mold after fixed is seeded on PDA inclined-planes, and 27 DEG C are cultivated 6 days, are rinsed spore with sterilized water, with 4 layers of sterile gauze mistake
Filter, obtains spore suspension, and it is 100 or so every milliliter to adjust spore concentration with sterilized water.
Embodiment 2：The primary dcreening operation of anti-botrytis cinerea bacillus
Soil, vegetable root, leaf, flower, 84 parts of fruit sample are gathered from vegetable cultivations such as Shandong, Hebei, Jiangxi, Beijing.Adopt
It is placed in after collection fresh sample in 4 DEG C of Refrigerator stores, one week and completes microorganism separation.Soil and vegetables root grey mold separation method
For：Take 3-5g soil or be placed in 100ml sterilized waters with the vegetable root that the scissors sterilized is shredded, at room temperature 150rpm concussions 1h
Afterwards, 10 are diluted to sterilized water-1、10-2、10-3、10-4、10-5、10-6.、10-7、10-8Eight gradients, it is 10 to take extension rate-4、
10-5、10-6、10-7、10-8Sample 0.1ml coat LB culture mediums (dusty yeast 5g, peptone 10g, sodium chloride 10g, distilled water
1 000mL；Solid adds 10~15g agar powders) on flat board, carton upside down culture, routine observation colony growth are cultivated at 25-30 DEG C
Situation, 24-48h bacterium colonies grow, the different bacterium colony of picking colony form on LB test tube slants, in 25-30 DEG C of incubator culture
24-48h, obtains 466 plants of bacteriums, is placed in 4 DEG C of Refrigerator stores standby.
Embodiment 3：The secondary screening of anti-botrytis cinerea bacillus
The preparation of bacterial suspension：Isolated bacterium is inoculated into LB fluid nutrient mediums respectively, 200rmp, 30 DEG C
Shaking table culture 48h.The culture of grey mold spore：0.1ml grey mold spore suspensions are coated on PDA plate, after culture 12h, flat
Plate center is put into paper dish, and 25 μ l bacterial suspensions are added on paper dish, and the size of inhibition zone is observed and determined in 30 DEG C of cultures.By
Screening obtains bacterial strain ZZ-302 strong inhibitory action to grey mold, and inhibition zone reaches 32mm.Identified by Physiology and biochemistry, the bacterium
Strain is bacillus subtilis (Bacillus subtilis).
It is prepared by the anti-booth vegetable grey mold bacillus subtilis microbial agent of embodiment 4
Bacterial strain ZZ-302 is inoculated in LB fluid nutrient mediums, 200rmp, 30 DEG C of shaking table culture 48h first.Then it is inoculated in
10L fermentation tanks, rotating speed 200-300rpm, 30 DEG C of temperature cultivates 12-24h, collects microbial inoculum, by method applications such as irrigation, sprayings
In green house of vegetables.
<110>Chinese Academy Of Sciences Process Engineering Research Institute
<120>A kind of screening of anti-booth vegetable grey mold bacillus subtilis and bacterial preparation process
<130> 2017
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1446
<212> DNA
<213> Bacillus subtilis zz-302
<400> 1
cagagggtgc tatacatgca agtcgagccc acagatggga gcttgctccc tgatgttagc 60
ggcggacggg tgagtaacac gtgggtaacc tgcctgtaag actgggataa ctccgggaaa 120
ccggggctaa taccggatgg ttgtctgaac cgcatggttc agacataaaa ggtggcttcg 180
gctaccactt acagatggac ccgcggcgca ttagctagtt ggtgaggtaa cggctcacca 240
aggcgacgat gcgtagccga cctgagaggg tgatcggcca cactgggact gagacacggc 300
ccagactcct acgggaggca gcagtaggga atcttccgca atggacgaaa gtctgacgga 360
gcaacgccgc gtgagtgatg aaggttttcg gatcgtaaag ctctgttgtt agggaagaac 420
aagtgccgtt caaatagggc cccaccttga cggtacctat ccagaaagcc acggctttct 480
acgtgccagc agccgcggta atacgtaggt ggcaagcgtt gtccggaaaa attgggcgta 540
aagggctcgc aggcggtttc ttaagtctga tgtgattgcc cccggctcaa ccggggaggg 600
tcattggaaa ctggggaact tgagtgcaga agaggagagt ggaattccac gtgtagcggt 660
gaaatgcgta gagatgtgga ggaacaggag tggcgaaggc gactctctgg tctgtaactg 720
acgctgagga gcgaaagcgt ggggagcgaa caggattaga taccctggta gtccacgccg 780
taaacgatga gtgctaagtg ttagccggtt tccgcccctt agtgctgcag ctaacgcatt 840
aagcactccg cctggggagt acggtcccaa gactgaaact caaaggaatt gacgggggcc 900
cgcacaagcg gtggagcatg tggtttaatt cgaagcaacg cgaagttcct taccaggtct 960
tgacatcctc tgacaatcct agagatagga cgtggccttc gggggcagag tgacaggtgg 1020
tgcatggttg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa 1080
cccttgatct tagttgccag cattcagttg ggcactctaa ggtgactgcc ggtgacaaac 1140
cggaggaacc tggggatgac gtcaaatcat catgccccaa atgacctggg ctacacacgt 1200
gctacaatgg acagaacaaa gggcagcgaa accgcgaggt taagccaatc ccacaaatct 1260
gttctcagaa cggatcgcag tctgcaactc gactgcgtga agctggaatc gctagtaatc 1320
gcggatcagc atgccgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc 1380
acgagagttt gtaacacccg aagtcggtga ggtttccttt atggagccag ccgccgaagg 1440
ggacag 1446
Claims (6)
1. a kind of screening of anti-booth vegetable grey mold bacillus subtilis and bacterial preparation process.
2. according to claim 1 prepared by the screening of booth vegetable grey mold and spore：Booth vegetable soil or vegetable root are put
In 100-500ml sterilized waters, 150-250rpm concussions 1-3h, 10 are diluted to sterilized water at room temperature-1-10-8Eight gradients,
Then PDA solid mediums are inoculated in, and 27 DEG C are cultivated 5-8 days, spore are rinsed with sterilized water, are filtered with sterile gauze,
Obtain spore suspension.
3. anti-booth vegetable grey mold bacillus subtilis screening according to claim 1：By booth vegetable soil or vegetable root
It is placed in 100-500ml sterilized waters, 150-250rpm concussions 1-3h, 10 are diluted to sterilized water at room temperature-1-10-8Eight ladders
Degree, coats beef extract-peptone solid medium, and carton upside down culture 24-48h is cultivated at 25-35 DEG C, and bacterium is placed in into 4 DEG C of ice
Case is saved backup.
4. the 16SRNA gene orders of anti-booth vegetable grey mold bacillus subtilis such as SEQ ID NO according to claim 1:
Shown in 1.
5. anti-booth vegetable grey mold bacillus subtilis screening according to claim 1：The ash that will be prepared according to claim 2
Mould spore suspension is coated on PDA solid mediums, after culture 12-36h, paper dish is put into flat board center, and 25 μ are added on paper dish
The bacterial suspension that l is prepared according to claim 3,30 DEG C of cultures, observes and determines the size of inhibition zone.
6. anti-booth vegetable grey mold bacillus subtilis microbial agent preparation method according to claim 1：The fermented and cultured of bacterial strain
Base：Tryptone:10-20g/L, yeast extract:10-20g/L, sodium chloride:5-10g/L, rotating speed 200-300rpm, temperature
25-35 DEG C, cultivate 12-24h.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710220444.XA CN106834195A (en) | 2017-04-06 | 2017-04-06 | A kind of screening of anti-booth vegetable grey mold bacillus subtilis and bacterial preparation process |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710220444.XA CN106834195A (en) | 2017-04-06 | 2017-04-06 | A kind of screening of anti-booth vegetable grey mold bacillus subtilis and bacterial preparation process |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106834195A true CN106834195A (en) | 2017-06-13 |
Family
ID=59146510
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710220444.XA Pending CN106834195A (en) | 2017-04-06 | 2017-04-06 | A kind of screening of anti-booth vegetable grey mold bacillus subtilis and bacterial preparation process |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106834195A (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104195071A (en) * | 2014-08-06 | 2014-12-10 | 江苏农林职业技术学院 | Bacillus subtilis B33 and application thereof |
| CN104232514A (en) * | 2014-08-13 | 2014-12-24 | 江苏农林职业技术学院 | Bacillus subtilis and application thereof |
| CN105132312A (en) * | 2015-08-17 | 2015-12-09 | 河南科技学院 | Bacillus subtilis and application thereof as well as microbial fungicide containing bacillus subtilis and preparation method of microbial fungicide |
| CN105907680A (en) * | 2016-05-27 | 2016-08-31 | 河北省科学院生物研究所 | Bacillus subtilis J-5 as well as biological agent and application thereof |
| CN105907662A (en) * | 2016-04-08 | 2016-08-31 | 中国农业大学 | Bacillus subtillis and application thereof |
| CN106190920A (en) * | 2016-08-08 | 2016-12-07 | 湖南农业大学 | Bacillus subtilis YN145 and application thereof |
-
2017
- 2017-04-06 CN CN201710220444.XA patent/CN106834195A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104195071A (en) * | 2014-08-06 | 2014-12-10 | 江苏农林职业技术学院 | Bacillus subtilis B33 and application thereof |
| CN104232514A (en) * | 2014-08-13 | 2014-12-24 | 江苏农林职业技术学院 | Bacillus subtilis and application thereof |
| CN105132312A (en) * | 2015-08-17 | 2015-12-09 | 河南科技学院 | Bacillus subtilis and application thereof as well as microbial fungicide containing bacillus subtilis and preparation method of microbial fungicide |
| CN105907662A (en) * | 2016-04-08 | 2016-08-31 | 中国农业大学 | Bacillus subtillis and application thereof |
| CN105907680A (en) * | 2016-05-27 | 2016-08-31 | 河北省科学院生物研究所 | Bacillus subtilis J-5 as well as biological agent and application thereof |
| CN106190920A (en) * | 2016-08-08 | 2016-12-07 | 湖南农业大学 | Bacillus subtilis YN145 and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| 吴永汉等: "浙南地区蔬菜灰霉病菌抗药性研究及田间药效试验", 《浙江农业科学》 * |
| 师宝忠等: "黄瓜灰霉病拮抗菌的筛选鉴定及其抑菌特性研究", 《河北省科学院学报》 * |
| 李丽梅等: "抗黄瓜灰霉病的枯草芽孢杆菌筛选及其抑茵活性的初步研究", 《天津科技大学学报》 * |
| 杜立新等: "枯草芽孢杆菌BS-208和BS-209菌株防治番茄灰霉病研究", 《农药学学报》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105820981B (en) | The preparation and application of one plant height ground bacillus microbial inoculum | |
| CN104630121B (en) | Bacillus amyloliquefaciens plant subspecies ZFH-3 microbial bacterial agents and its application | |
| CN101525587B (en) | Streptomyces strain and application thereof | |
| CN104745483B (en) | A kind of Paecilonyces variotii strain SJ1 and its application | |
| CN105734000A (en) | Paenibacillus polymyxa NSY50 with capabilities of promoting growth and preventing diseases | |
| NL2029438B1 (en) | Strain of camellia sinensis endophyte penicillium ehrlichii and its application | |
| CN104560827B (en) | A kind of biocontrol actinomycetes bacterial strain for preventing and treating tobacco bacterial wilt and its application | |
| CN106172507B (en) | Applications of the bacillus subtilis HMB19198 in prevention powdery mildew of cucumber | |
| CN103224904B (en) | Rhodopseudomonas strain, biocontrol microbial inoculum and biocontrol fermentation liquid, and corresponding preparation methods and application thereof in controlling phytophthora blight of pepper | |
| CN101503708B (en) | Cultivation fermentation method of Bacillus coagulans antimycotics active substance | |
| CN105462881B (en) | A kind of Paenibacillus polymyxa and its application for being used to prevent crop verticillium wilt | |
| CN104593283A (en) | Bacillus amyloliquefaciens DPPG-74 and its use in disease prevention and growth promotion | |
| CN108148794A (en) | A kind of the bacillus subtilis DYr3.3 and preparation method and application of broad-spectrum antibacterial activity | |
| CN104651260A (en) | Brevibacillus brevis BBC-3 and application thereof as well as preparation method of microbial inoculum of brevibacillus brevis | |
| CN104894018A (en) | Luna Lin Haresii streptomycete and application thereof | |
| CN111172060A (en) | Bacillus with banana vascular wilt prevention and treatment function and preparation method and application thereof | |
| CN105176831A (en) | Penicillum oxalicum, and applications thereof | |
| CN107267412A (en) | Methylotrophic bacillus and its application | |
| CN104195071A (en) | Bacillus subtilis B33 and application thereof | |
| CN105316266A (en) | Bacillus amyloliquefaciens LX-J1 and application thereof | |
| CN101928673A (en) | Trichoderma harzianum | |
| CN107142229B (en) | It is a kind of prevent and treat capsicum epidemic disease biocontrol actinomycetes bacterial strain and its application | |
| CN103525725B (en) | Pseudomonas syringae and application thereof in prevention of tomato gray mold | |
| CN104726384A (en) | Bacillus subtilis for prohibiting rhizoctonia solani of potatoes | |
| CN100539854C (en) | The fruit and vegetable preserving antistaling agent that contains recombinant chitinase |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20170613 |
|
| WD01 | Invention patent application deemed withdrawn after publication |