CN102433273A - Bacillus subtilis HBY-11 and application method thereof - Google Patents
Bacillus subtilis HBY-11 and application method thereof Download PDFInfo
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- CN102433273A CN102433273A CN2011103623782A CN201110362378A CN102433273A CN 102433273 A CN102433273 A CN 102433273A CN 2011103623782 A CN2011103623782 A CN 2011103623782A CN 201110362378 A CN201110362378 A CN 201110362378A CN 102433273 A CN102433273 A CN 102433273A
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Abstract
The invention provides bacillus subtilis HBY-11 and an application method thereof in decomposition of nutrient, improvement of biological characters of crops and improvement of disease resistance of soil so as to improve the yield and the quality of the crops.
Description
Technical field:
The invention belongs to the agricultural microorganism formulation art, be specifically related to a bacillus subtilis HBY-11 and an application method thereof.
Background technology:
Mikrobe has effect on a plurality of nutrient functions such as the decomposition soil organism, fixed nitrogen, phosphorus decomposing, molten potassium; Genus bacillus also has good little ecological regulatory function simultaneously; Be applied to agricultural soil, can reduce the generation of plant pest, enhancing crop resistance etc.At present; The production of multifunctional agricultural microbiobacterial agent is used for achieving the goal through using multiple mikrobe to mix to make usually; But there are a lot of shortcomings such as cost is high, technical difficulty is big in the production of mixing microorganisms microbial inoculum and use; The seed selection of multi-functional single bacterial classification and use, significant for the Industry Promotion of agricultural microorganism microbial inoculum.
Summary of the invention:
The purpose of this invention is to provide a bacillus subtilis HBY-11 and decomposing nutrient, the biological character of improvement crop, improve the soil, disease-resistant, thereby improve the application method on the yield and quality of crop.
One bacillus subtilis HBY-11 deposit number is CGMCC No.5184.
Described subtilis HBY-11 is applied to decompose nutrient; Especially for a minute phosphorus decomposing.
Described subtilis HBY-11 be applied to improve crop biological character, improve the soil, disease-resistant, improve the yield and quality of crop; Described crop comprises Chinese cabbage or capsicum.
Subtilis HBY-11 of the present invention checks it to separate the nutrient effect through liquid phosphorus decomposing, soil phosphorus decomposing, dull and stereotyped phosphorus decomposing and pot experiment.The result shows: liquid phosphorus decomposing, soil phosphorus decomposing and pot experiment data all reach the significance statistical significance through statistical study.The dull and stereotyped phosphorus decomposing test that point connects this bacterium forms the obvious transparent circle, and the D/d peak is 3.0, and minimum value also reaches 2.53, more than 4 types of tests show all that this genus bacillus has and significantly separate the nutrient effect to have very big research and development and be worth.
Studied the influence of inoculation HBY-11 through pot experiment to Chinese cabbage growth yield and quality.The result shows: compare with contrast, plant height, range of extension, fresh weight increase by on a year-on-year basis and are respectively 16.4%, 17.3%, 50.8%; Nitrate salt, reducing sugar, Vc and 3-protein d PS variance analysis: P＜0.01 has significant difference; The content of total potassium has increased by 1/3 times than CK, and total nutrient has increased by 6.3% on year-on-year basis; The available phosphorus of potted plant soil and available potassium increase 11.0mg/kg and 42.1mg/kg respectively than soil before potted plant.Inoculation HBY-11 bacterium fluid power enough improve Plantula Brassicae chinensis biological character, improve the soil, disease-resistant, can also improve the yield and quality of Chinese cabbage during results.Plot experiment shows: HBY-11 uses with fertilizer has significant difference to the Chinese cabbage raising the output.Simultaneously, plot experiment shows that the nutrient that HBY-11 uses yield of hot pepper, biological shape, quality and plant and fruit with fertilizer also has significant difference.
Subtilis HBY-11 of the present invention is in the institute of microbiology of Chinese state AS of on the August 30th, 2011 of No. 3, No. 1 institute at North Star West Road, Chaoyang District, BeiJing, China city; China Committee for Culture Collection of Microorganisms common micro-organisms center; Carried out preservation; Classification called after subtilis (Bacillus subtilis) HBY-11, preserving number is: CGMCC No.5184.
Description of drawings:
Fig. 1 is the aspect graph of bacterial strain of the present invention;
A is strain morphology figure among the figure, for shaft-like;
B gives birth to figure in the gemma;
C is the electromicroscopic photograph of bacterial strain;
D is the flagella staining figure of bacterial strain;
Fig. 2 decomposes the nutrient picture for bacterial strain of the present invention;
A is the dissolving of inorganic phosphorus among the figure;
B is the dissolving of organophosphorus;
The antibiotic picture of Fig. 3 bacterial strain of the present invention;
A is to the restraining effect of ralstonia solanacearum;
B is to the restraining effect of flavus;
Fig. 4 is the Chinese cabbage growthhabit comparison diagram of inoculation bacterium liquid of the present invention and inactivated bacterial liquid;
Fig. 5 is the growth situation map of the Chinese cabbage of inoculation bacterium liquid of the present invention in plot experiment;
Fig. 6 is 4 kinds of situation pepper plant growth conditions figure.
Embodiment:
Be intended to further specify the present invention below in conjunction with embodiment, and unrestricted the present invention.
Embodiment 1
The screening of bacterial strain
The screening of bacterial strain of the present invention: the obtaining of pedotheque → selective medium just screens → rule partition method to carry out purifying → bacterial strain to bacterial strain and screens again → functional study → identification of strains → bacterial strain applied research → bacterial strain preservation.
Bacterial strain of the present invention is preserved substratum: glucose 10.0g Carnis Bovis seu Bubali cream 5.0g peptone 10.0g sodium-chlor 5.0g water 1000ml pH 7.0～7.2.The bacterium liquid that the present invention uses also be with this substratum at 28-30 ℃, 200 rev/mins, need not illumination, cultivate and prepared in 36 hours.
Several kinds of culture medium culturing below bacterial strain of the present invention can also be used:
Inorganic phosphorus liquid nutrient medium: ammonium sulfate 0.5g, Repone K 0.3g, Presfersul 0.03g, tricalcium phosphate 5g, glucose 10g, sodium-chlor 0.3g, sal epsom 0.3g, manganous sulfate 0.03g, water 1000ml PH:7.0～7.5.
Organophosphorus bacterial liquid substratum: glucose 10g, sodium-chlor 0.3g, sal epsom 0.3g, manganous sulfate 0.03g, Yelkin TTS 0.2g, ammonium sulfate 0.5g, Repone K 0.3g, ferrous sulfate 0.03g, lime carbonate 5g, water 1000ml PH:7.0～7.5
Silicate liquid substratum: sucrose 5g Sodium phosphate, dibasic 2g sal epsom 0.5g iron(ic)chloride 0.05g lime carbonate 0.1g feldspar in powder 0.5g water 1000ml PH:7.2～7.4.
The agar of adding 2% is corresponding solid medium in the aforesaid liquid substratum, can supply inclined-plane and slab to use.
Strain culturing condition of the present invention: temperature 28-30 ℃, 200 rev/mins, need not illumination, cultivate and just can be applied to Chinese cabbage or capsicum in 36 hours.
Bacterial strain Physiology and biochemistry experimental result of the present invention is following:
This genus bacillus (little Huang) bacterium colony that on beef-protein medium, is white in color, cell dia is 0.622 μ m, can utilize D-glucose, D-wood sugar, D-N.F,USP MANNITOL to produce acid; Can hydrolyzed starch; V-P reaction is positive, can not hydrolysis tyrosine, can not form indoles; Can not utilize propionic salt, meet the essential characteristic of subtilis, bacillus amyloliquefaciens.And the bacillus cereus cell dia can not utilize D-wood sugar, D-N.F,USP MANNITOL to produce acid greater than 1.0 μ m, can hydrolysis utilize tyrosine; Bacillus licheniformis can utilize propionic salt; Bacillus pumilus can not hydrolyzed starch; Bacillus firmus can not utilize Citrate trianion; The bacillus thuringiensis cell dia all is not inconsistent with experimental result greater than 1.0um etc.
The 16Sr DNA analysis of bacterial strain of the present invention
Sequencing results: (1411? Letters)
Accession number is JN609382 in GenBank, analyzes the similarity of this bacterial strain and subtilis the highest (99%) through BLAT.
The result shows that this bacterium is subtilis (Bacillus subtilis), called after subtilis HBY-11 bacterial strain.
Embodiment 2
The function of subtilis HBY-11 bacterial strain
Through liquid phosphorus decomposing, soil phosphorus decomposing, dull and stereotyped phosphorus decomposing and pot experiment, the check genus bacillus separate the nutrient effect.The result shows: liquid phosphorus decomposing, soil phosphorus decomposing and pot experiment data all reach the significance statistical significance through statistical study.The budder dull and stereotyped phosphorus decomposing test of spore bacillus of point forms the obvious transparent circle, and the D/d peak is 3.0, and minimum value also reaches 2.53, more than 4 types of tests show all that this genus bacillus has and significantly separate the nutrient effect to have very big research and development and be worth.
Bacterial strain of the present invention is also apparent in view in the effect of antibiosis, sees the restraining effect of Fig. 3 a to ralstonia solanacearum; 3b is to the restraining effect of flavus.
Embodiment 3
The Chinese cabbage test
The Chinese cabbage pot experiment is transplanted on December 27th, 2010 and is gathered on March 8th, 2011, and vegetative period is 40d altogether.2 processing, blank soil+genus bacillus inactivated bacterial liquid of the present invention are established in test; Blank soil+bacillus living liquid of the present invention; Repeat for 5 times., Chinese cabbage seed transplants after sending seedling; The Chinese cabbage seedling (the Chinese cabbage seedling is two leaves wholeheartedly) of high size such as transplanting respectively; Transplant the back every strain Plantula Brassicae chinensis pouring 50ml zero(ppm) water is made normal root water, transplant after seven days every strain Plantula Brassicae chinensis is inoculated 5ml living bacterial liquid or inactivated bacterial liquid, flow in the soil along root.The Chinese cabbage growing period is not executed any fertilizer, but the work of watering zero(ppm) water and removing worm's ovum, the biological character of 40d post analysis Chinese cabbage acts on the influence of soil to the Plantula Brassicae chinensis growth thereby analyze genus bacillus.Studied the influence of inoculation HBY-11 through pot experiment to Chinese cabbage growth yield and quality.The result shows: the inoculation HBY-11 can improve Plantula Brassicae chinensis biological character, improve the soil, disease-resistant, can also improve the yield and quality of Chinese cabbage during results.
By shown in the table 1, draw the pot experiment biological character data that Plantula Brassicae chinensis carries out behind 40d through pot experiment.Plant height, range of extension, fresh weight all have remarkable effect, compare with contrast, increase by on a year-on-year basis to be respectively 16.4%, 17.3%, 50.8% [year-on-year growth rate is: (test group-control group)/control group], and leaf is wide slightly to be increased, and the number of blade is very nearly the same.This shows that HBY-11 can promote plant growth, improve crop yield, the biological character of improvement crop.
Table 1HBY-11 is to the influence of Chinese cabbage biological character
By shown in the table 2, to the variance analysis of the Plantula Brassicae chinensis value of averaging, plant height is compared p＜0.01 with CK, has extremely significantly statistical significance.The wide p of leaf＞0.05 does not have statistical significance.Range of extension p＜0.01.Has extremely significantly statistical significance.Number of blade p＞0.05 does not have statistical significance.Fresh weight p＜0.05 has statistical significance.This shows that HBY-11 acts on soil has remarkable effect to the growth of Plantula Brassicae chinensis, significantly improve plant height, leaf is wide and the fresh weight of plant.
Table 2HBY-11 is to the MV variance table (
n=5) of Chinese cabbage biological character influence
Annotate: with connect inactivated bacterial liquid and compare:
△P＜0.05,
▲P＜0.01
Can know that by table 3 potted plant Chinese cabbage over-ground part plant has been carried out the mensuration of each item index, and the content of organic matter, pH, total nitrogen, total phosphorus has basically no variation.The content of total potassium has increased by 1/3 times than CK, and total nutrient has increased by 6.3% on year-on-year basis, explains that HBY-11 can improve total potassium and the total nutrient amount in the Chinese cabbage plant, improves the quality of crop.
Table 3HBY-11 is to the influence of Chinese cabbage plant nutrition
Nitrate salt, reducing sugar, Vc and protein are the indexs of estimating Chinese cabbage nutritional quality and health.The nitrate salt Excessive Intake can influence people's self health, and the nitrate salt major part of needed by human body all comes from vegetables, and the content that China's vegetable nitrate allows is 432mg kg
-1(bright appearance) could guarantee the healthy and safe of human body.Vc helps the enhancing body immunological competence, and carbohydrate and protein also are the necessary nutritive substances of human growth.Therefore the influence of studying HBY-11 dialogue vegetable matter aspect gears to actual circumstances.By shown in the table 4, the content of the nitrate content of Chinese cabbage of inoculation HBY-11 has lowered 37.7%, explains that HBY-11 can effectively reduce the absorption accumulation of nitrate salt, helps absorption and trans-utilization to nitric nitrogen.Reducing sugar, Vc content and Protein content are increased by 33.3%, 10.1% and 20.5% respectively.Explain that HBY-11 helps reducing sugar, Vc and the proteinic increase in the Chinese cabbage.Adopt the DPS variance analysis, compare with contrast, nitrate salt, reducing sugar, Vc and protein: P＜0.01 has extremely significant difference.
The influence (
n=5) of table 4HBY-11 dialogue vegetable matter
Annotate: compare with the CK group:
△P＜0.05,
▲P＜0.01
Find out from the result's (table 5) who handles soil; Under the prerequisite of not applying fertilizer contrast potted plant before soil and potted plant back soil, the value of organic matter, alkali-hydrolyzable nitrogen, living bacteria count descends, alkali-hydrolyzable nitrogen is reduced to 126.6mg/kg and 85.3mg/kg by 223.5mg/kg before; The Soil Nitrogen nutrient that wherein connects the HBY-11 living bacterial liquid descends little; The decline that connects inactivated bacteria is big, and the absolute magnitude of the nitrogen that the Plantula Brassicae chinensis plant the absorbs bacterium of taking over a job is many, connects lacking of inactivated bacteria; Soil is described by the activation of the function yeast of inoculation institute, can more quick-acting nitrogen nutrient be provided than control group.
Microbe number in the soil and organic content also demonstrate different features, and organic matter drops to 18.5g/kg and 15.2g/kg by 22.7g/kg, connects the living bacterial liquid group than the high 3.3g/kg that connects inactivated bacterial liquid, and potted plant preceding living bacteria count is 1.12 * 10
8Individual/g, be respectively 3.91 * 10 in Chinese cabbage harvesting back contrast and test
7Individual/g and 3.67 * 10
6Individual/g, effective viable bacteria ratio of inoculation function yeast connects little 1 one magnitude of inactivated bacterial liquid group, and than little 2 one magnitude of test initial stage soil, its reason possibly be: 1. environment change and inoculation beneficial functions bacterium can suppress the growth of other flora, impel bacterium quantity to descend; 2. the consumption positive correlation of the quantity of function yeast and the soil organism; 3. the ability of the indigenous microorganism in the ability force rate soil of the function yeast activation soil nutrient of test group interpolation is strong, can activate soil than control group and offer the more nitrogen of plant, phosphorus, potassium nutrition.
The content of available phosphorus and available potassium increases 11.0mg/kg and 42.1mg/kg on year-on-year basis than soil before potted plant behind the inoculation living bacterial liquid, explains that HBY-11 has the effect of significantly separating mineral phosphorus, potassium; Discharging more titanium pigment potassium when can ensure the plant healthy growth again goes in the middle of the soil; The quality of improving the soil promotes the trophic structure of crop simultaneously.Soil can also increase 11.6mg/kg before available potassium was tested after the inoculation inactivated bacterial liquid, and its reason possibly be because the effect of plant root can discharge soluble potassium in soil.In sum, HBY-11 has tangible improving effect to soil.
Table 5HBY-11 dialogue nappy is planted the influence of soil physico-chemical property
When Chinese cabbage is transplanted and to grow to 18d, the cabbage leaf back side appearance in various degree of control group the acarid worm's ovum, and the worm's ovum phenomenon is not found at the cabbage leaf table back side of test group.After one week, control group can occur by the mashed blade of insect bite gradually, and blade also begins to change into deep green, and is attended by the appearance of yellow blade.And the roomy bright green that is of the blade that connects living bacterial liquid does not have mashed leaf, no worm's ovum.
Plot experiment was sowed on May 10th, 2011, and on June 20th, 2011 gathered, and front and back are 40d altogether, handled 1: blank, execute 5% inactivated bacterial liquid of the present invention; Handle 2: execute 5% living bacterial liquid of the present invention; Handle 3: apply biological organic fertilizer 200kg/667m
2+ 5% inactivated bacterial liquid of the present invention; Handle 4, apply biological organic fertilizer 200kg/667m
2+ 5% living bacterial liquid of the present invention.Meter produces after 40 days.Plot experiment shows that HBY-11 uses with fertilizer Chinese cabbage raising the output is had significant difference.
Shown in following table 6; Handling 2 (connecing living bacterial liquid) compares with processing 1 (connecing inactivated bacterial liquid): p＞0.05, and not statistically significant, reason possibly be because preceding crop also is without fertilizer treatment; The rapid available phosphorus element is absorbed by the preceding crop utilization basically in the soil; Though and HBY-11 can decompose inorganic phosphorus, it is required that its decomposition gained nutrient does not catch up with the Chinese cabbage growth far away, so effect of increasing production is not obvious.Handling 3 (inactivated bacterial liquid+biological organic fertilizers) compares with processing 1: p＞0.05, not statistically significant.Handling 4 (living bacterial liquid+biological organic fertilizers) compares with processing 2: p＜0.05, fertilizer treatment has significant difference, can be effectively after HBY-11 being described and fertilizer combining even bring into play its phosphorus decomposing ability efficiently.Handle 4 with handle 1 relatively: p＜0.01, have utmost point significant difference, can increase production Chinese cabbage after drawing fertilizer and living bacterial liquid and being used and have the effect of utmost point significance.
Table 6HBY-11 is to the influence (
n=5) of Chinese cabbage plot experiment output
Embodiment 4
The capsicum test
The capsicum plot experiment was sowed on July 23rd, 2011, and on August 18th, 2011 transplanted capsicum, treated to apply microbial inoculum of the present invention again after the capsicum seedling is survived fully, and on October 10th, 2011 gathered, and front and back are 78d altogether.4 processing are established in test altogether, 3 repetitions.Handle 1: blank, execute 5% inactivated bacterial liquid of the present invention; Handle 2: execute 5% living bacterial liquid of the present invention; Handle 3: apply refined organic fertilizer 200kg/667m
2+ 5% inactivated bacterial liquid of the present invention; Handle 4, apply refined organic fertilizer 200kg/667m
2+ 5% living bacterial liquid of the present invention, meter produces after 78 days.Plot experiment shows that the nutrient that HBY-11 uses yield of hot pepper, biological shape, quality and plant and fruit with fertilizer has significant difference.
To the influence of pepper plant The data DPS software analysis HBY-11 to the capsicum biological character, by following table 7 explanations, HBY-11 uses with fertilizer has effect extremely significantly to output and biological shape.
Table 7HBY-11 is to the influence of capsicum biological character
Compare with processing 1: zero: p＜0.05, ●: p＜0.01
Compare with processing 2: △: p＜0.05, ▲: p＜0.01
Compare with processing 3: ☆: p＜0.05, ★: p＜0.01
After 4 kinds of processing, pepper plant growth conditions figure sees Fig. 6.
HBY-11 effect soil is to the influence of capsicum quality, and like following table 8 explanations, HBY-11 uses separately and uses with refined organic fertilizer has the significance effect to the capsicum quality-improving.
Table 8HBY-11 is to the influence of fruit quality
| Chlorophyll (mg/g) | Vc content (mg/100g) | Reducing sugar amount (g/100g) | |
| Handle 1 | 119.1±8.6 | 86.5±3.4 | 2.32±0.01 |
| Handle 2 | 160.2±4.9● | 98.7±4.8● | 2.53±0.01● |
| Handle 3 | 176.3±0.8●▲ | 105.3±6.1● | 2.67±0.03●▲ |
| Handle 4 | 189.4±5.3●▲☆ | 116.7±4.2●▲☆ | 3.00±0.08●▲★ |
Compare with processing 1: zero: p＜0.05, ●: p＜0.01
Compare with processing 2: △: p＜0.05, ▲: p＜0.01
Compare with processing 3: ☆: p＜0.05, ★: p＜0.01
HBY-11 effect soil is applied to soil like following table 9HBY-11 and executes with refined organic fertilizer the influence of capsicum nutrient
Use plant and fruit nutrient are had significant difference.
Table 9HBY-11 is to the influence of nutrient
Compare with processing 1: zero: p＜0.05, ●: p＜0.01
Compare with processing 2: △: p＜0.05, ▲: p＜0.01
Compare with processing 3: ☆: p＜0.05, ★: p＜0.01.
Claims (5)
1. a bacillus subtilis HBY-11 is characterized in that, deposit number is CGMCC No.5184.
2. the application method of the described subtilis HBY-11 of claim 1 is characterized in that, is used to decompose nutrient.
3. the application method of subtilis HBY-11 according to claim 2 is characterized in that, is used for the branch phosphorus decomposing.
4. the application method of the described subtilis HBY-11 of claim 1 is characterized in that, be used to improve crop biological character, improve the soil, disease-resistant, improve the yield and quality of crop.
5. the application method of subtilis HBY-11 according to claim 4 is characterized in that, described crop comprises Chinese cabbage or capsicum.
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| CN102876603A (en) * | 2012-09-23 | 2013-01-16 | 江苏丘陵地区镇江农业科学研究所 | Bacillus amyloliquefaciens Mg116 and application thereof |
| CN112625947A (en) * | 2020-12-16 | 2021-04-09 | 江苏省中国科学院植物研究所 | Bacillus subtilis capable of dissolving phosphorus strongly, carbon-based microbial compound fertilizer thereof and application of bacillus subtilis |
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| CN101899405A (en) * | 2010-01-20 | 2010-12-01 | 辛伟成 | Bacillus subtilis GU and application in preparing efficient biological compound phosphate fertilizer thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102876603A (en) * | 2012-09-23 | 2013-01-16 | 江苏丘陵地区镇江农业科学研究所 | Bacillus amyloliquefaciens Mg116 and application thereof |
| CN112625947A (en) * | 2020-12-16 | 2021-04-09 | 江苏省中国科学院植物研究所 | Bacillus subtilis capable of dissolving phosphorus strongly, carbon-based microbial compound fertilizer thereof and application of bacillus subtilis |
| CN112625947B (en) * | 2020-12-16 | 2021-08-03 | 江苏省中国科学院植物研究所 | Bacillus subtilis capable of dissolving phosphorus strongly, carbon-based microbial compound fertilizer thereof and application of bacillus subtilis |
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Application publication date: 20120502 |