CN102965299A - Fermentation process of Bacillus pumilus LD-b1 and its application in control of plant diseases - Google Patents
Fermentation process of Bacillus pumilus LD-b1 and its application in control of plant diseases Download PDFInfo
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Abstract
本发明提供一种可用于植物病害生物防治的云雾龙胆内生菌及其发酵工艺，属于生物农药技术领域。本发明所述菌株为短小芽孢杆菌(Bacillus pumilus)LD-b1，已于2012年5月14日保藏在中国微生物菌种保藏管理委员会普通微生物中心，保藏号为CGMCC No.6112。混菌琼脂平板打孔法试验表明，菌株LD-b1可抑制多种植物病害真菌的生长。LD-b1能够显著抑制苹果腐烂病菌对苹果果实的侵蚀；温室盆栽试验中，LD-b1对黄瓜褐斑病菌的防治效果达到81.2％。将LD-b1在种子罐中培养24小时至对数生长期，按5％接种量接入生产罐，控制初始pH 7.5，发酵温度30℃，溶氧5％以上，补料分批发酵68小时，菌量可达1010cfu/mL以上。菌株LD-b1生长快，抗菌谱广，在植物病害生物防治方面具有很好的应用前景。
The invention provides an endophytic gentiana gentiana which can be used for biological control of plant diseases and a fermentation process thereof, belonging to the technical field of biological pesticides. The bacterial strain described in the present invention is Bacillus pumilus LD-b1, which has been preserved in the General Microorganism Center of China Committee for the Collection of Microbial Cultures on May 14, 2012, and the preservation number is CGMCC No.6112. The hole-punching test on the mixed bacteria agar plate showed that the strain LD-b1 could inhibit the growth of various plant disease fungi. LD-b1 can significantly inhibit the erosion of apple fruit by apple rot fungus; in the greenhouse pot experiment, the control effect of LD-b1 on cucumber brown spot fungus reached 81.2%. Cultivate LD-b1 in the seed tank for 24 hours to the logarithmic growth phase, connect it to the production tank according to the inoculation amount of 5%, control the initial pH 7.5, ferment the temperature at 30°C, dissolve oxygen above 5%, and ferment in batches for 68 hours , the amount of bacteria can reach more than 10 10 cfu/mL. Strain LD-b1 grows fast and has a broad antibacterial spectrum, which has a good application prospect in the biological control of plant diseases.
Description
Technical field
The present invention relates to the biocontrol strain of a strain controlling plant diseases, comprise its fermentation manufacturing technique and the application in biocontrol of plant disease thereof, belong to biological pesticide technical field.
Background technology
Isolate the first strain endophyte so far from ryegrass seed from Vogl in 1898, the research of endophyte has had the history in more than 100 year.Studies show that in recent years, all there is endophyte in nearly all plant, and they are grown in the tissue under the plant epidermis, mainly comprise bacterium, fungi and actinomycetes etc.Estimate to have at least hundreds thousand of kinds of endophytes to survive in the vascular plant cell or in the particular surroundings in intercellular substance according to Dreyfuss and Chapela.The plant resources of China is extremely abundant, but owing to many reasons such as opening up wasteland in recent years, herd, afforest, excavate, a lot of wild plant resource subjects to severe risks of damage.The cloud and mist rough gentian is Gentianaceae Gentiana perennial root woody plant, cures mainly gastritis, trachitis, urethritis, smallpox and acne rash, is that minority is classified as the focused protection kind not by the valuable ingredient of traditional Chinese medicine of systematic study by country in the agueweed.Separation and research and development to cloud and mist rough gentian endophyte are conducive to protect the threatened plant endophyte, and the new microorganism resource of exploitation biocontrol of plant disease.
Plant diseases is one of principal element that affects agriculture production, and present control induces germ to develop immunity to drugs to use chemical pesticide as main easily; And the chemical bactericide contaminate environment, the eubiosis of microorganism species in the destruction farmland, residual agricultural chemicals not only affects agricultural product quality, and harm humans is healthy.The biological control method of Plant diseases is safe and effective, more and more causes people's concern.But from soil and plant rhizosphere separation screening to Plant diseases biocontrol microorganisms field planting difficulty in plant materials, with the competition of disease bacterium in be difficult to occupy advantage.Endophytic bacterium occupies favourable ecological niche, is not subject to environmental influence, is colonizated in the plant materials easily, can invade by anti-microbial pathogen, has significant advantage in biocontrol of plant disease.Genus bacillus is common endogenetic bacteria kind, can produce the multiple antimicrobial substances such as bacitracin, ring grease, amino acids, nucleic acid, bacteriophage-like particle, can suppress the various plants pathogenic bacteria; And genus bacillus has advantages such as growth is fast, nutrition simple, strong stress resistance, be fit to very much be developed to biocontrol fungicide.
Summary of the invention
The purpose of this invention is to provide the bacillus pumilus bacterial strain with controlling plant diseases effect, efficient biocontrol fungicide is prepared in fermentation, can prevent and treat better the farmland plant disease, reduces chemical pesticide to the pollution of environment.
The present invention is that separation screening has the bacterium of the anti-Plant diseases fungi activity of wide spectrum to a strain from the stem of cloud and mist rough gentian, and through being accredited as bacillus pumilus (Bacillus pumilus), the bacterial strain code name is LD-b1.Its biological property is: Gram-positive, somatic cells size 0.5 μ m～0.6 μ m * 1.5 μ m～2.1 μ m; Catalase, Citrate trianion utilization, casein hydrolysis, Vitamin C2 are hydrolyzed, V.P is positive; Oxydase, urase, H2S generation, indoles generation, nitrate reduction, gelatine liquefication, arginine dihydrolase, Starch Hydrolysis, M.R. are negative; The carbon source that can utilize has: glucose, sucrose, lactose, pectinose, wood sugar, semi-lactosi, N.F,USP MANNITOL, N-acetyl-glycosamine, gluconate; Can not utilize starch, dextrin, melibiose, rhamnosyl and sorbyl alcohol.Bacterial strain LD-b1 now is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and preservation date is on May 14th, 2012, and preserving number is CGMCC No.6112.
The fermentation manufacturing technique flow process of biocontrol strain LD-b1 of the present invention is: slant strains → shaking flask bacterial classification → seeding tank → production tank → product.
The detailed step of biocontrol strain LD-b1 fermentative production is among the present invention:
On the solid slant culture base, culture medium prescription (g/L) is with the preservation of bacteria strain streak inoculation: sucrose 10, peptone 2, extractum carnis 3, yeast extract paste 0.5, agar 15, pH7.5; Cultivate 36～48h for 30 ℃, form lawn.
With the 5mL sterilized water activated spawn is washed from the test tube slant, in the access seed culture medium, culture medium prescription (g/L) is: sucrose 15, peptone 5, yeast powder 0.2, dipotassium hydrogen phosphate 0.5, sal epsom 0.05, sodium-chlor 0.1, pH7.5; The liquid seed culture medium of the bottled 100mL of 250mL triangle, postvaccinal triangular flask place on the rotary shaking table, and 150r/min, 30 ℃ of cultivation 24～30h make shake-flask seed liquid.
The substratum of seeding tank is identical with the substratum of shake-flask seed, and fermentation condition is: liquid amount 70%, inoculum size 5%, 30 ℃ of leavening temperatures, air flow 1: 1, tank pressure 0.2kg/cm
2, mixing speed 200～600r/min, dissolved oxygen be more than 10%, fermentation time 24 hours, and the bacterium amount can reach 10
8Cfu/mL.
The culture medium prescription (g/L) of producing tank is: sucrose 25, soybean cake powder 3, ammonium sulfate 2.5, yeast powder 0.2, Secondary ammonium phosphate 0.8, sal epsom 0.05, ferrous sulfate 0.02, sodium-chlor 0.1, pH7.5; Fermentation condition is: liquid amount 70%, inoculum size 5%, 30 ℃ of leavening temperatures, air flow 1: 1, tank pressure 0.2kg/cm
2, mixing speed 100～300r/min, dissolved oxygen ferment and added respectively 10g/L sucrose twice in 30 hours and 45 hours more than 5%, fermentation time 68 hours, and the bacterium amount can reach 10
10Cfu/mL.
Bacterial strain LD-b1 is to the biological control effect of Plant diseases fungi among the present invention
The test of the dull and stereotyped punch method of mixed bacterio-agar shows, bacterial strain LD-b1 all has significant inhibition to the growth of the Plant diseases fungies such as Cucumber Target Leaf Spot bacterium, botrytis cinerea pers, cucumber sclerotiorum, Valsa mali, wilt of eggplant bacterium, fusarium graminearum, Alternaria brassicae and botrytis cinerea, and wherein the antagonistic action to Cucumber Target Leaf Spot bacterium and Valsa mali is the strongest.Bacterial strain LD-b1 can suppress Valsa mali significantly to the erosion of fruit on Apple; In the greenhouse pot culture test, LD-b1 reaches 81.2% to the prevention effect of Cucumber Target Leaf Spot bacterium.
Description of drawings
Fig. 1 bacterial strain LD-b1 is to the antagonistic action of canker of apple fruit bacteria growing.
Fig. 2 bacterial strain LD-b1 is to the antagonistic action of Cucumber Target Leaf Spot bacteria growing.
Fig. 3 bacterial strain LD-b1 on Apple to the restraining effect of Valsa mali.
The pot experiment of Fig. 4 bacterial strain LD-b1 control Cucumber Target Leaf Spot evil.
Embodiment
The invention will be further described below in conjunction with specific embodiment, and following examples are intended to illustrate the present invention rather than limitation of the invention further.
Embodiment 1
The separation of bacillus pumilus LD-b1
Get one section stem of fresh cloud and mist rough gentian, aseptic water washing 2 times is used the aseptic filter paper suck dry moisture; In 70% ethanolic soln, soaked 2 minutes, soaked 30 seconds again aseptic water washing 3 times with 0.1% mercuric chloride.Under aseptic condition, with blade crust is pruned, and be cut into segment about 1 centimetre, place the screening and culturing primary surface, cultivated 4～8 days for 30 ℃.After flat board had bacterium to grow, the plate streaking purifying obtained the pure growth of separation of bacterial bacterial strain.
Embodiment 2
The morphological feature of bacterial strain LD-b1
LD-b1 on the solid medium flat board 30 ℃ cultivated 1 day, form the oyster white opaque colony, the bacterium colony sub-circular, the edge is irregular, there is pimple the centre, diameter 0.6～1.1cm does not secrete pigment; The somatic cells of bacterial strain LD-b1 is rod-short, big or small 0.5 μ .m～0.6 μ m * 1.5 μ m～2.1 μ m, and Gram-positive has gemma.
The physiological and biochemical property of bacterial strain LD-b1
The growth temperature range of bacterial strain LD-b1 is 8～50 ℃, pH scope 5.5～8.5, NaCl concentration range 0～7%; The carbon source that can utilize has: glucose, sucrose, lactose, pectinose, wood sugar, semi-lactosi, N.F,USP MANNITOL, N-acetyl-glycosamine, gluconate; Can not utilize starch, dextrin, melibiose, rhamnosyl and sorbyl alcohol; The catalase of bacterial strain LD-b1, Citrate trianion utilization, casein hydrolysis, Vitamin C2 are hydrolyzed, V.P is positive, oxydase, urase, H
2S generation, indoles generation, nitrate reduction, gelatine liquefication, arginine dihydrolase, Starch Hydrolysis, M.R. are negative.
Pcr amplification and the sequencing of the 16S rDNA gene of bacterial strain LD-b1
Bacterial strain LD-b1 is inoculated in seed culture medium, and 150r/min, 30 ℃ of shaking tables were cultivated centrifugal collection thalline 24 hours, wash 1 time with 0.5mol/LNaCl, Eddy diffusion adds N,O-Diacetylmuramidase and SDS broken wall, extract genomic dna with phenol-chloroform method, extract bacterial genomes DNA with the UNIQ-10 test kit.Obtain the goal gene fragment with bacterial 16 S rDNA universal primer 27F (5 '-GAGAGT TTGATCCTGGCTCAG-3 ') and 1541R (5 '-AAGGAGGTGATCCAGCCGCA-3 ') amplification.To the 16S rDNA sequence amplification of bacterial strain LD-b1, obtain the sequence of 1444bp after the order-checking, specifically shown in SEQ ID No:1.The sequence registration number of this sequence in GenBank/EMBL/DDBJ is JF932295, sequential analysis shows that the homology of bacterial strain LD-b1 and Bacillus pumilus KNUC389 and Bacillus pumilusKNUC235 is the highest, be respectively and reach 99.8% and 99.7%, combining form is learned and the physiological and biochemical property index, determines that bacterial strain LD-b1 is bacillus pumilus (Bacillus pumilus).Bacterial strain LD-b1 now is stored in China Committee for Culture Collection of Microorganisms common micro-organisms center (Beijing), and preservation date is on May 14th, 2012, and preservation registration number is CGMCC No.6112.
Embodiment 3
The fermentation manufacturing technique of bacterial strain LD-b1
Slant medium (g/L): sucrose 10, peptone 2, extractum carnis 3, yeast extract paste 0.5, agar 15, pH7.5,121 ℃ of sterilization 30min.
Seed culture medium (g/L): sucrose 15, peptone 5, yeast powder 0.2, dipotassium hydrogen phosphate 0.5, sal epsom 0.05, sodium-chlor 0.1, pH7.5,121 ℃ of sterilization 30min.
Fermention medium (g/L): sucrose 25, soybean cake powder 3, ammonium sulfate 2.5, yeast powder 0.2, Secondary ammonium phosphate 0.8, sal epsom 0.05, ferrous sulfate 0.02, sodium-chlor 0.1, pH7.5,121 ℃ of sterilization 30min.
To protect good bacterial classification streak inoculation on the solid slant culture base, cultivate 36～48h for 30 ℃, with the 5mL sterilized water activated spawn is washed from the test tube slant, in the access seed culture medium, the liquid seed culture medium of the bottled 100mL of 250mL triangle, postvaccinal triangular flask places on the rotary shaking table, and 150r/min, 30 ℃ of cultivation 24～30h make shake-flask seed liquid.
Seed tank culture condition: liquid amount 70%, inoculum size 5%, 30 ℃ of leavening temperatures, air flow 1: 1, tank pressure 0.2kg/cm
2, mixing speed 200～600r/min, dissolved oxygen be more than 10%, fermentation time 24 hours.
Fermentor cultivation condition: liquid amount 70%, inoculum size 5%, 30 ℃ of leavening temperatures, air flow 1: 1, tank pressure 0.2kg/cm
2, mixing speed 100～300r/min, dissolved oxygen ferment and added respectively 10g/L sucrose twice, fermentation time 68 hours in 30 hours and 45 hours more than 5%.
Embodiment 4
Bacillus pumilus LD-b1 is to the antagonistic effect of pathogenic fungi growth
With Cucumber Target Leaf Spot bacterium, botrytis cinerea pers, cucumber sclerotiorum, Valsa mali, wilt of eggplant bacterium, fusarium graminearum, Alternaria brassicae, botrytis cinerea activation and enlarged culturing, dilution makes 10
6The suspension of spore/mL, get the 0.2mL spore suspension and be mixed and made into flat board with sterilization improvement Martin substratum 15mL, after solidifying, 6mm diameter punch tool with the bacterium of going out punches at each flat board, remove agar and Xiang Kongzhong adding 0.1mL bacillus pumilus LD-b1 fermented supernatant fluid in the hole, behind 28 ℃ of cultivation 4d, measure antibacterial circle diameter.The result shows that bacterial strain LD-b1 all has significant inhibition to the growth of the Plant diseases fungies such as Cucumber Target Leaf Spot bacterium, botrytis cinerea pers, cucumber sclerotiorum, Valsa mali, wilt of eggplant bacterium, fusarium graminearum, Alternaria brassicae and botrytis cinerea, and wherein the antagonistic action to Cucumber Target Leaf Spot bacterium and Valsa mali is the strongest.
Embodiment 5
Bacillus pumilus LD-b1 on Apple to the antagonistic effect of Valsa mali
With red fuji apple 70% alcohol disinfecting, after drying, with the wound of inoculating needle at an apple thorn 6mm (deeply) * 4mm (diameter), add respectively LD-b1 fermented supernatant fluid and the sterilized water of 200 μ L, inoculate respectively 10 after 2 hours
5The Valsa mali 200 μ L of spore/mL, fruit is put in the fruit tray, fruit tray wraps up with preservative film, placed 6 days for 25 ℃, the scab diameter of contrast fruit is 17.5mm, the scab diameter of fruit is 2.4mm after the LD-b1 sample preparation, only is 13.7% of contrast, and the result shows that the antibacterial substance that endophyte LD-b1 produces can suppress Valsa mali significantly to the erosion of fruit.
Embodiment 6
Bacillus pumilus LD-b1 is to the potted plant prevention effect of Cucumber Target Leaf Spot bacterium
Choose the consistent cucumber seedling of growth, first LD-b1 bacterium liquid 2mL is evenly sprayed on cucumber leaves, inoculate 10 behind the 8h
6The Cucumber Target Leaf Spot bacterium spore suspension 2mL of spore/mL, do not spray the negative control that is treated to of germ only to spray water, only to spray the positive control that is treated to of Cucumber Target Leaf Spot bacterium spore liquid, room temperature keeps 25～28 ℃, humidity keeps 80%～90%, 3 repetitions of every processing, each repeats 5 strain cucumber seedlings, carries out the investigation of disease level.
The Cucumber Target Leaf Spot Seriousness gradation standard is as follows:
0 grade: without scab; 1 grade: lesion area accounts for below 5% of whole leaf area; 3 grades: lesion area accounts for 5%～25% of whole leaf area; 5 grades: lesion area accounts for 26%～50% of whole leaf area; 7 grades: lesion area accounts for 51%～75% of whole leaf area; 9 grades: lesion area accounts for more than 75% of whole leaf area.
The method of calculation of disease index and preventive effect are as follows:
Spraying the observations of biocontrol microorganisms LD-b1 fermented liquid after 3 days shows, the disease index of positive control is 58.92, the disease index that sprays the biological and ecological methods to prevent plant disease, pests, and erosion agent is 11.08, LD-b1 is 81.2% to the preventive effect of Cucumber Target Leaf Spot, illustrates that the antibacterial substance of biocontrol microorganisms LD-b1 generation can significantly suppress the generation of Cucumber Target Leaf Spot.
Claims (6)
1. the cloud and mist rough gentian endophyte of a strain controlling plant diseases, be gram-positive bacillus pumilus (Bacillus pumilus), the bacterial strain code name is LD-b1, this bacterial strain is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on May 14th, 2012, and preserving number is CGMCC No.6112.
2. the biological and ecological methods to prevent plant disease, pests, and erosion agent of controlling plant diseases, comprise bacillus pumilus LD-b1 or from the antagonistic activity material in its source as effective constituent.
3. biological and ecological methods to prevent plant disease, pests, and erosion agent claimed in claim 2, wherein Plant diseases comprises Cucumber Target Leaf Spot, gray mold of cucumber, cucumber timberrot, canker of apple fruit, wilt of eggplant, wheat scab, Chinese cabbage black spot and graw mold of tomato.
4. the culture medium prescription of bacillus pumilus LD-b1:
Seed culture medium (g/L): sucrose 15, peptone 5, yeast powder 0.2, dipotassium hydrogen phosphate 0.5, sal epsom 0.05, sodium-chlor 0.1, pH7.5,121 ℃ of sterilization 30min.
Fermention medium (g/L): sucrose 25, soybean cake powder 3, ammonium sulfate 2.5, yeast powder 0.2, Secondary ammonium phosphate 0.8, sal epsom 0.05, ferrous sulfate 0.02, sodium-chlor 0.1, pH7.5,121 ℃ of sterilization 30min.
5. the seed tank culture condition of bacillus pumilus LD-b1:
Liquid amount 70%, inoculum size 5%, 30 ℃ of leavening temperatures, air flow 1: 1, tank pressure 0.2kg/cm
2, mixing speed 200～600r/min, dissolved oxygen be more than 10%, fermentation time 24 hours.
6. the fermentor cultivation condition of bacillus pumilus LD-b1:
Liquid amount 70%, inoculum size 5%, 30 ℃ of leavening temperatures, air flow 1: 1, tank pressure 0.2kg/cm
2, mixing speed 100～300r/min, dissolved oxygen ferment and added respectively 10g/L sucrose twice, fermentation time 68 hours in 30 hours and 45 hours more than 5%.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103289906A (en) * | 2013-05-23 | 2013-09-11 | 都晓伟 | Gentiana manshurica kitag endophytic fungi and application thereof |
| CN104140938A (en) * | 2014-07-16 | 2014-11-12 | 新疆农业大学 | Antagonism bacterial strain preventing and treating fruit tree rot and application of antagonism bacterial strain |
| CN104232529A (en) * | 2014-08-29 | 2014-12-24 | 湖北省生物农药工程研究中心 | Process for preparing viable bacillus pumilus preparation |
| CN107629983A (en) * | 2015-04-08 | 2018-01-26 | 河北农业大学 | Soybean endophyte and application thereof |
| CN109161479A (en) * | 2018-08-15 | 2019-01-08 | 延安大学 | A kind of drug and preparation method thereof for preventing and treating apple tree canker |
| CN115287234A (en) * | 2022-08-11 | 2022-11-04 | 马鞍山盘石生物技术有限公司 | Novel application of bacillus pumilus LYMC-3 and fermentation liquor thereof and preparation method of fermentation liquor |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009022399A1 (en) * | 2007-08-10 | 2009-02-19 | Yoshichika Kameyama | Novel microorganism having gastric juice promoting action and composition secreted by the same |
-
2012
- 2012-08-16 CN CN2012102903742A patent/CN102965299A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009022399A1 (en) * | 2007-08-10 | 2009-02-19 | Yoshichika Kameyama | Novel microorganism having gastric juice promoting action and composition secreted by the same |
Non-Patent Citations (2)
| Title |
|---|
| 黄海东等: "云雾龙胆内生菌的分离鉴定及抗菌活性分析", 《微生物学通报》, vol. 37, no. 7, 20 July 2010 (2010-07-20), pages 1017 - 1021 * |
| 黄海东等: "龙胆内生菌的分离鉴定及其对苹果腐烂病菌的拮抗作用", 《食品工业科技》, vol. 33, no. 6, 31 March 2012 (2012-03-31), pages 215 - 218 * |
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| CN103289906A (en) * | 2013-05-23 | 2013-09-11 | 都晓伟 | Gentiana manshurica kitag endophytic fungi and application thereof |
| CN103289906B (en) * | 2013-05-23 | 2014-12-10 | 都晓伟 | Gentiana manshurica kitag endophytic fungi and application thereof |
| CN104140938A (en) * | 2014-07-16 | 2014-11-12 | 新疆农业大学 | Antagonism bacterial strain preventing and treating fruit tree rot and application of antagonism bacterial strain |
| CN104232529A (en) * | 2014-08-29 | 2014-12-24 | 湖北省生物农药工程研究中心 | Process for preparing viable bacillus pumilus preparation |
| CN107629983A (en) * | 2015-04-08 | 2018-01-26 | 河北农业大学 | Soybean endophyte and application thereof |
| CN107629983B (en) * | 2015-04-08 | 2020-02-14 | 河北农业大学 | Soybean endophyte and application thereof |
| CN109161479A (en) * | 2018-08-15 | 2019-01-08 | 延安大学 | A kind of drug and preparation method thereof for preventing and treating apple tree canker |
| CN115287234A (en) * | 2022-08-11 | 2022-11-04 | 马鞍山盘石生物技术有限公司 | Novel application of bacillus pumilus LYMC-3 and fermentation liquor thereof and preparation method of fermentation liquor |
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