CN103805538B - A kind of complex micro organism fungicide and its application in terms of leaf tobacco production - Google Patents
A kind of complex micro organism fungicide and its application in terms of leaf tobacco production Download PDFInfo
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- CN103805538B CN103805538B CN201310747674.3A CN201310747674A CN103805538B CN 103805538 B CN103805538 B CN 103805538B CN 201310747674 A CN201310747674 A CN 201310747674A CN 103805538 B CN103805538 B CN 103805538B
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Abstract
The invention discloses a kind of complex micro organism fungicide, it is to be mixed to get using the nutrient solution and yeast bacteria culture fluid of bacterial strain P22 in proportion, in the nutrient solution and yeast bacteria culture fluid of the bacterial strain P22, bacterial strain concentration is all higher than 200,000,000/mL, in the nutrient solution and yeast bacteria culture fluid of bacterial strain P22 after mixture mix bacterium agent concentration >=200,000,000/mL.Complex micro organism fungicide of the present invention is applied to leaf tobacco production, can reduce tobacco leaf nicotine content, improves tobacco leaf overall quality, improves the ratio of first-class tobacco leaf, coordinates nutrition, improves yield of tobacco；Cigarette strain disease-resistant resistance is improved, the purpose of biological control is reached, with important application value.
Description
Technical field
The present invention relates to complex micro organism fungicide and tobacco leaf production technical field, more particularly, to a kind of composite microbial
Thing microbial inoculum and its application in terms of leaf tobacco production.
Background technology
With the intensification carried out in a deep going way with cigarette consumption person to Smoking is harmful to your health awareness of scientific and technological emerging cigarette theory, society
Substantial change can be there has also been to the quality requirement of tobacco leaf, people must constantly seek raising tobacco leaf overall quality, coordinate nutrition,
The ratio of first-class tobacco leaf is improved, yield of tobacco is improved.Leaf tobacco production is the strong production procedure of a technical comparison, on the whole
It is that three points of seven points of kinds are baked, good tobacco leaf degree quality is constructed on the sound tobacco basis provided in leaf tobacco production.
Nicotine is the important component of tobacco.Nicotine can be addicting or produce dependence（Be most difficult to give up drug addiction it
One）, people are generally difficult to contain oneself, and reusing nicotine also increases heart speed and raising blood pressure and reduce appetite.Greatly
The nicotine of dosage can cause vomiting and nausea, and when serious, people can be dead.Nicotine would generally be contained in tobacco, this makes many
Smoker cannot give up the major reason of craving for tobacco.So tobacco leaf overall quality is improved during leaf tobacco production, first-class cigarette is improved
The ratio of leaf, the nicotine content coordinated nutrition, improve yield of tobacco, effectively reduce tobacco leaf become the emphasis of research.
The content of the invention
The technical problem to be solved in the present invention is to overcome the shortcomings of microbial bacterial agent application technology in existing tobacco leaf planting, is carried
For a kind of complex micro organism fungicide.
The invention solves the problems that another technical problem be to provide the preparation method of the complex micro organism fungicide.
A present invention also technical problem to be solved is to provide the application of the complex micro organism fungicide.
The purpose of the present invention is achieved by the following technical programs：
A kind of complex micro organism fungicide is provided, is mixed with using the nutrient solution and yeast bacteria culture fluid of bacterial strain P22
Arrive, in the nutrient solution and yeast bacteria culture fluid of the bacterial strain P22, bacterial strain concentration is all higher than 200,000,000/mL, the nutrient solution of bacterial strain P22 and
In yeast bacteria culture fluid after mixture mix bacterium agent concentration >=200,000,000/mL；The nutrient solution and yeast bacteria culture fluid of the bacterial strain P22
It is 1 according to volume ratio:1～3:1；The bacterial strain P22 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms
Center, preservation date are August in 2010 23, plant name：Defect shortwave monad, Latin literary fame are referred to asBrevundimonas diminuta, bacterial strain name P22, preserving number are CGMCC NO.4122.
Preferably, the nutrient solution and yeast bacteria culture fluid of the bacterial strain P22 is 1 according to volume ratio:1、2:1 or 3:1 mixing.
It is 2 that most preferred mixed proportion is the volume ratio of the nutrient solution and yeast bacteria culture fluid of bacterial strain P22:1.
The saccharomycete preferably saccharomyces cerevisiae.
Invention also provides the preparation method of the complex micro organism fungicide, comprises the following steps：
S1. ferment Amplification Culture bacterial strain P22 and saccharomycete respectively, makes final bacteria concentration >=2 of nutrient solution of each bacterial classification
Hundred million/mL, respectively obtains bacterial strain P22 nutrient solutions and yeast bacteria culture fluid；
S2. by bacterial strain P22 nutrient solutions and yeast bacteria culture fluid, aseptic mixture obtains complex micro organism fungicide in proportion.
Wherein, the fermentation Amplification Culture described in S1 is comprised the following steps：
S11. actication of culture：1 ring bacterial strain P22, saccharomycetic lawn are taken to fluid nutrient medium from slant preservation pipe respectively
Middle culture.Specifically take in 1 ring bacterial strain P22, saccharomycetic lawn to 30mL fluid nutrient mediums from slant preservation pipe respectively, 37
DEG C, 200rpm cultivate 24 hours；
S12. Shaking culture：Draw during S1 culture gained bacterium solutions are inoculated in shaking flask and cultivate.Specifically drawn with sterile pipette
The bacterium solution for taking S1 activation is inoculated in shaking flask, and inoculum concentration is 1%；Cultivation temperature is 37 DEG C, and rotating speed is 200rpm, and incubation time is
24h；
S13. fermentation tank culture：The cultured shaking flasks of S2 are inoculated into fermentation tank Amplification Culture, make final cell concentration >=2
Hundred million/mL.The cultured shaking flasks of specifically S2 are inoculated into fermentation tank Amplification Culture, and vaccination ways are flame inoculation, and inoculum concentration is 1%；
Cultivation temperature is 37 DEG C, and rotating speed is 180rpm, and throughput is 1:1, tank pressure is 0.02MPa, and incubation time is 24～36h, is made most
Whole cell concentration >=200,000,000/mL；
Also include step S14：S14. viable count is determined：The solid medium of sterilizing is poured in aseptic culture dish while hot
（Each culture dish fills about 20mL）Flat board is made in cooling, and bacterium solution is according to gradient dilution to 10-8, 10 are taken successively-8、10-7、10-6、10-5
The each 0.1mL of bacterium solution is repeated 3 times in flat board central authorities, each concentration, and aseptic coating rod coating is uniform, and 37 DEG C of culture 24h take clump count
In 30～300 flat board as flat board is counted, bacterium solution viable count is calculated；
Wherein, the training of fermentation tank culture described in the culture medium of Shaking culture, S13 described in fluid nutrient medium, S12 described in S11
Foster base preparation method is：25 weight portion bean sprouts add 75 parts by weight of deionized water to boil 20 minutes, and filter cleaner supplies volume, plus
Enter the dissolving of 5 weight portion glucose to mix, to 7.0,121 DEG C sterilize 30 minutes adjustment pH value, obtain final product.
The preparation method of the slant medium described in S11 is：25 weight portion bean sprouts add 75 parts by weight of deionized water to boil 20
Minute, filter cleaner supplies volume, adds 5 weight portion glucose, the dissolving of 2 weight portion agar powders to mix, adjusts pH value to 7.0,
121 DEG C sterilize 30 minutes, obtain final product.
S14 prepares bacterial strain flat board counting culture medium（Solid medium）Method be：25 weight portion bean sprouts add 75 weight
Part deionized water is boiled 20 minutes, and filter cleaner supplies volume, adds 5 weight portion glucose, the dissolving of 2 weight portion agar powders mixed
Even, to 7.0,121 DEG C sterilize 30 minutes adjustment pH value, obtain final product.
Present invention simultaneously provides application of the composite bacteria agent in terms of leaf tobacco production is promoted.
The application includes being applied to reduce tobacco leaf nicotine content, increasing blade area, suppress the composite bacteria agent
In terms of nicotine formation, coordination cured tobacco leaf chemical composition, the presentation quality for improving tobacco leaf and/or the upper medium tobacco leaf ratio of raising
Using.
The coordination cured tobacco leaf particularly preferably coordinates tobacco leaf top chemical composition.
The application improves the biological control of cigarette strain disease-resistant resistance in also including leaf tobacco production in terms of.
The application process is：Spray by the complex micro organism fungicide or containing the liquid for meeting microbial bacterial agent
In the tobacco leaf surface of cigarette strain.
Preferably, it is according to microbial inoculum by the complex micro organism fungicide water：Water is 1:100～1:300 volume ratio is dilute
Spray in tobacco leaf surface after releasing mixing.Preferably mixed volume ratio is complex micro organism fungicide：Water=1:200.
Preferably, the liquid containing the complex micro organism fungicide was sprayed in tobacco leaf surface in the 5th day after tobacco leaf is pinched.
Preferably, it is described to spray process carries out dripping to blade face.
Preferably, the process that sprays is carried out in the afternoon in fine day.Rainy day and fine day high noon is avoided to spray.
Beneficial effect of the present invention：
The invention provides a kind of composite bacteria agent, simply, preparation cost is relatively low for preparation process, but lives with good application
Property, during being applied to tobacco leaf planting, tobacco leaf nicotine content can be reduced, promote tobacco leaf cells growth and divide
Split, increase blade area, suppress nicotine formation, coordination cured tobacco leaf to be particularly upper tobacco leaf chemical composition, improve the outer of tobacco leaf
Appearance quality improves the significantly technique effect such as upper medium tobacco leaf ratio.
The composite bacteria agent is sprayed application to and colonize after tobacco leaf surface in tobacco leaf by the bacterium source that composite bacteria agent of the present invention is adopted
Easily, survival rate is high；Viable bacteria well affects tobacco leaf by the enzyme of own growth metabolic activity and secretion after tobacco leaf surface is colonized
Cell growth metabolic condition.
The present invention through long-felt, using bacterial strain P22 and saccharomycete scientific compatibility, will not be mutual between the bacterial strain of selection
Suppress, colonize growth, can also be complementary on functional effect, overcome various bacterium in prior art and be combined and easily occur between bacterial strain
Mutually suppress, affect the defect of growth.
Microbial inoculum of the present invention is while leaf tobacco production is promoted, moreover it is possible to effectively play the work for improving cigarette strain disease-resistant resistance
With reaching the purpose of biological control.
Specific embodiment
The present invention is further illustrated with reference to specific embodiment.Unless stated otherwise, adopt in the embodiment of the present invention
Raw material and method are the routinely commercial raw material in this area and conventional use of method.
Embodiment 1
The present invention is promoted tobacco leaf cells division from fresh and alive tobacco leaf surface separation screening viable bacteria, expands tobacco leaf area, suppression
The bacterial strain P22 that tobacco curing alkali is formed, the bacterial strain P22 are preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms
The heart, preservation date are August in 2010 23, plant name：Defect shortwave monad, Latin literary fame are referred to asBrevundimonas diminuta, bacterial strain name P22, preserving number are CGMCC NO.4122.
Physical resource referring also to 201110024393.6 patent application document of Application No. in regard to the bacterial strain P22
Description.The saccharomycete that the present embodiment is adopted is for saccharomyces cerevisiae（Buy Shandong Kang Yuan bio tech ltd）, but and therefore
The scope of the invention is limited, the conventional yeasts bacterium in other sources can be adopted.
The composite bacteria agent is prepared according to following steps method：
S1. ferment Amplification Culture bacterial strain P22 and saccharomycete respectively, makes final bacteria concentration >=2 of nutrient solution of each bacterial classification
Hundred million/mL.Respectively obtain bacterial strain P22 nutrient solutions and yeast bacteria culture fluid；
S11. actication of culture：1 ring bacterial strain P22, saccharomycetic lawn are taken from slant preservation pipe respectively to train to 30mL liquid
In foster base, 37 DEG C, 200rpm cultivate 24 hours；
S12. Shaking culture：The bacterium solution that S1 activation is drawn with sterile pipette is inoculated in shaking flask, and inoculum concentration is 1%；Culture
Temperature is 37 DEG C, and rotating speed is 200rpm, and incubation time is 24h；
S13. fermentation tank culture：The cultured shaking flasks of S2 are inoculated into fermentation tank Amplification Culture, and vaccination ways are flame inoculation,
Inoculum concentration is 1%；Cultivation temperature is 37 DEG C, and rotating speed is 180rpm, and throughput is 1:1, tank pressure is 0.02MPa, and incubation time is 24
～36h, makes final cell concentration >=200,000,000/mL；The nutrient solution and saccharomycetic nutrient solution of P22 are respectively obtained.
To ensure quality, also including viable count determination step：
S14. viable count is determined：The solid medium of sterilizing is poured in aseptic culture dish while hot（Each culture dish is filled about
20mL）Flat board is made in cooling, and bacterium solution is according to gradient dilution to 10-8, 10 are taken successively-8、10-7、10-6、10-5The each 0.1mL of bacterium solution is in flat
Plate central authorities, each concentration are repeated 3 times, and aseptic coating rod coating is uniform, and 37 DEG C of culture 24h take the flat board that clump count is 30～300
As flat board is counted, bacterium solution viable count is calculated.
Wherein, the training of fermentation tank culture described in the culture medium of Shaking culture, S13 described in fluid nutrient medium, S12 described in S11
Foster base preparation method is：25 weight portion bean sprouts add 75 parts by weight of deionized water to boil 20 minutes, and filter cleaner supplies volume, plus
Enter the dissolving of 5 weight portion glucose to mix, to 7.0,121 DEG C sterilize 30 minutes adjustment pH value, obtain final product.
S14 prepares bacterial strain inclined-plane and plate count culture medium（Solid medium）Method be：25 weight portion bean sprouts add
75 parts by weight of deionized water are boiled 20 minutes, filter cleaner, supply volume, add 5 weight portion glucose, 2 weight portion agar powders
Dissolving is mixed, and to 7.0,121 DEG C sterilize 30 minutes adjustment pH value, obtain final product.
S2. P22 nutrient solutions are pressed:Yeast bacteria culture fluid is 2:The aseptic mixture of volume ratio of 1 mixing obtains complex microorganism
Microbial inoculum.Mixture can be carried out in aseptic stainless steel mixture tank.
After testing, each strain bacteria concentration >=200,000,000/mL in the complex micro organism fungicide that the present embodiment is prepared, nutrient solution are mixed
Concentration with rear mix bacterium agent >=200,000,000/mL.
Embodiment 2 other with embodiment 1, wherein S2 is by P22 nutrient solutions:Yeast bacteria culture fluid is 1:The volume of 1 mixing
The aseptic mixture of ratio obtains complex micro organism fungicide.
Embodiment 3 other with embodiment 1, wherein S2 is by P22 nutrient solutions:Yeast bacteria culture fluid is 3:The volume of 1 mixing
The aseptic mixture of ratio obtains complex micro organism fungicide.
Embodiment 4 other with embodiment 1, wherein S2 is by P22 nutrient solutions:Yeast bacteria culture fluid is 1.2:The body of 1 mixing
The aseptic mixture of product ratio obtains complex micro organism fungicide.
The application of 5 composite bacteria agent of embodiment
The complex micro organism fungicide that mixture is obtained is with water respectively with 1:200 volume ratio dilution is sprayed in tobacco leaf after mixing
Surface.Spray the time pinch for tobacco leaf after 5 days spraying effects it is best.Spraying method：Bacterium solution after dilution is mixed is sprayed in cigarette
Leaf surface, the amount of spraying are dripped with blade face and are advisable；Spraying to be preferably selected is carried out in the afternoon in fine day, it is to avoid rainy day and the spray of fine day high noon
Apply.
Test effect：Test site：Wuchuan Guizhou tobacco science and technology demonstration；For trying flue-cured tobacco cultivars：K326
This test sets six process：（1）1 liquid bacterial agent of test group（Composite microbial bacteria obtained in 1 methods described of embodiment
Agent and water are according to 1:Liquid bacterial agent obtained by 200 dilution proportions）；（2）2 liquid bacterial agent of test group（2 methods described of embodiment is obtained
Complex micro organism fungicide and water according to 1:Liquid bacterial agent obtained by 200 dilution proportions）；（3）3 liquid bacterial agent of test group（Implement
Complex micro organism fungicide obtained in 3 methods described of example and water are according to 1:Liquid bacterial agent obtained by 200 dilution proportions）；（4）Test group
4 liquid bacterial agents（Complex micro organism fungicide obtained in 4 methods described of embodiment and water are according to 1:Liquid obtained by 200 dilution proportions
Microbial inoculum）；（5）Clear water is compareed；（6）Bacterial strain P22 nutrient solution control groups.Experimental design adopts randomized complete-block design, totally 3 areas
Group.Planting density is 1100 plants/acre, spacing in the rows 0.5m, line-spacing 1.2m, sets 1 protection row between each process.Various cultivation management measures are pressed
Manage with delicacy according to local high yield and high quality flue-cured tobacco cultivation technology.Each process when tobacco leaf reaches technical maturity is harvested, using three sections
Formula baking process is toasted.Test effect is as shown in table 1.
Table 1
As seen from the experiment, spray complex micro organism fungicide of the present invention may advantageously facilitate tobacco leaf be particularly upper tobacco leaf bake
Cigarette tobacco leaf cells grow and divide and blade area is increased, is dramatically increased yield and economic benefit；Effectively improve tobacco leaf into
Ripe degree and oil, its aesthetic quality are significantly improved, and the sucking quality of tobacco leaf is preferably improved, and tobacco leaf utilizability is further carried
Rise, improve aroma quality and perfume quantity, keep appropriate concentration and strength, reduce miscellaneous gas, improve pleasant impression；Spray upper back tobacco leaf cigarette
Alkali, total nitrogen and protein content have declined, and potassium chlorine coordinates tobacco leaf internal chemical composition than rising, and improve tobacco leaf and are particularly
Upper tobacco leaf quality.Spray nicotine content in tobacco leaf is can obviously reduce using complex micro organism fungicide of the present invention.
6 Comparison study of embodiment is tested
Complex micro organism fungicide that 1 mixture of embodiment is obtained and water are with 1:50、1:100、1:200、1:300、1:400
Volume ratio is sprayed in tobacco leaf surface after diluting mixing respectively.Spray the time pinch for tobacco leaf after 5 days spraying effects it is best.The side of spraying
Method：Bacterium solution after dilution is mixed is sprayed in tobacco leaf surface, and the amount of spraying is dripped with blade face and is advisable；Spray and be preferably selected at the fine day noon
After carry out, it is to avoid rainy day and fine day high noon spray.
Test effect：Test site：Wuchuan Guizhou tobacco science and technology demonstration；For trying flue-cured tobacco cultivars：K326
Experimental design adopts randomized complete-block design, totally 3 district's groups.Planting density is 1100 plants/acre, spacing in the rows 0.5m,
Line-spacing 1.2m, sets 1 protection row between each process.Various cultivation management measures are managed meticulously according to local high yield and high quality flue-cured tobacco cultivation technology
Reason.Each process when tobacco leaf reaches technical maturity is harvested, and is toasted using three-phase-drying for tobacco technique.Test effect is as shown in table 2.
Table 2
The present invention through long-felt, using bacterial strain P22 and saccharomycete scientific compatibility, will not be mutual between the bacterial strain of selection
Suppress, colonize growth, on functional effect can also complementation, played significant synergistic effect, overcome various in prior art
The compound defect for easily occurring mutually suppressing, affecting growth between bacterial strain of bacterium.The complex micro organism fungicide is applied to into tobacco leaf
Production, can reduce tobacco leaf nicotine content, improve tobacco leaf overall quality, improve the ratio of first-class tobacco leaf, coordinate nutrition, improve cigarette
Leaf-making quantity；Cigarette strain disease-resistant resistance is improved, the purpose of biological control is reached, with important application value.
Claims (9)
1. a kind of complex micro organism fungicide for being applied to promote leaf tobacco production and/or improve cigarette strain disease-resistant resistance, its feature exists
In, it is to be mixed with and obtain using the nutrient solution and yeast bacteria culture fluid of bacterial strain P22, the nutrient solution and yeast of the bacterial strain P22
In bacteria culture fluid, bacterial strain concentration is all higher than 200,000,000/mL, mix bacterium agent after the nutrient solution and yeast bacteria culture fluid mixture of bacterial strain P22
Concentration >=200,000,000/mL；
The nutrient solution and yeast bacteria culture fluid of the bacterial strain P22 is 1 according to volume ratio:1～3:1 mixing；
The bacterial strain P22 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preservation date is
On August 23rd, 2010, plants name：Defect shortwave monad (Brevundimonas diminuta), preserving number is CGMCC
NO.4122。
2. complex micro organism fungicide according to claim 1, it is characterised in that the nutrient solution and yeast of the bacterial strain P22
Bacteria culture fluid is 1 according to volume ratio:1、2:1 or 3:1 mixing.
3. complex micro organism fungicide according to claim 2, it is characterised in that the nutrient solution and yeast of the bacterial strain P22
Bacteria culture fluid is 2 according to volume ratio:1 mixing.
4. complex micro organism fungicide according to claim 1, it is characterised in that the saccharomycete is saccharomyces cerevisiae.
5. complex micro organism fungicide described in any one of Claims 1 to 4 is promoting leaf tobacco production and/or raising cigarette strain disease-resistant degeneration-resistant
Property biological control in terms of application, it is characterised in that be according to 1 by the microbial inoculum and water:Spray after 200 volume ratio mixing
In tobacco surface.
6. the preparation method of complex micro organism fungicide described in any one of Claims 1 to 4, it is characterised in that comprise the following steps：
S1. ferment Amplification Culture bacterial strain P22 and saccharomycete respectively, make the final bacteria concentration of nutrient solution of each bacterial classification more than 200,000,000/
ML, obtains bacterial strain P22 nutrient solutions and yeast bacteria culture fluid；
S2. by bacterial strain P22 nutrient solutions and yeast bacteria culture fluid, mixture obtains complex micro organism fungicide in proportion.
7. the preparation method of complex micro organism fungicide according to claim 6, it is characterised in that the fermentation described in S1 expands training
Support and comprise the following steps：
S11. actication of culture：Train in taking 1 ring bacterial strain P22, saccharomycetic lawn to fluid nutrient medium respectively from slant preservation pipe
Support；
S12. Shaking culture：Draw during S11 culture gained bacterium solutions are inoculated in shaking flask and cultivate；
S13. fermentation tank culture：The cultured shaking flasks of S12 are inoculated into fermentation tank Amplification Culture, make final cell concentration more than 200,000,000/
mL。
8. the preparation method of complex micro organism fungicide according to claim 7, it is characterised in that fluid nutrient medium described in S11,
The culture medium of Shaking culture described in S12, the preparation method of the culture medium of fermentation tank culture described in S13 are：25 weight portion bean sprouts add
75 parts by weight of deionized water are boiled 20 minutes, filter cleaner, supply volume, add the dissolving of 5 weight portion glucose to mix, adjust pH
It is worth to 7.0,121 DEG C sterilize 30 minutes, obtain final product；
The preparation method of the culture medium on inclined-plane described in S11 is：25 weight portion bean sprouts add 75 parts by weight of deionized water to boil 20 minutes,
Filter cleaner, supplies volume, adds 5 weight portion glucose, the dissolving of 2 weight portion agar powders to mix, adjusts pH value to 7.0,121 DEG C
Sterilizing 30 minutes, obtains final product.
9. the preparation method of complex micro organism fungicide according to claim 7, it is characterised in that further comprising the steps of：
S14. viable count is determined：The solid medium of sterilizing pours into cooling in aseptic culture dish while hot and makes flat board, and coating is equal
Even, culture, the flat board for taking clump count 30～300 calculate bacterium solution viable count as flat board is counted；
The preparation method of the solid medium is：25 weight portion bean sprouts add 75 parts by weight of deionized water to boil 20 minutes, filter
Slagging-off, supplies volume, adds 5 weight portion glucose, the dissolving of 2 weight portion agar powders to mix, adjusts pH value to 7.0,121 DEG C of sterilizings
30 minutes, obtain final product.
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| CN102191193B (en) * | 2011-01-21 | 2012-10-10 | 福州农博士生物技术有限公司 | Brevundimonas diminuta as well as fermentation method, preparation and application thereof |
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