CN104531559B - Bacillus amyloliquefaciens Lh 1 and its application - Google Patents
Bacillus amyloliquefaciens Lh 1 and its application Download PDFInfo
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Abstract
The present invention relates to microbial control field, specially a kind of bacillus amyloliquefaciens Lh 1 and its application, bacillus amyloliquefaciens（bacillusamyloliquefaciens subsp）Lh 1, deposit number is CGMCC No.8548, in preventing and treating cotton verticillium wilt, soft rot of Chinese cabbage, bacterial wilt, peach rot, canker of apple fruit, Phytophthora capsici disease, application in terms of graw mold of tomato, the present invention is combined with the chitosan oligosaccharide of the fertile economic benefits and social benefits of medicine using bacillus amyloliquefaciens Lh 1 and the microecological microbial agents of Lh 1 is made, viable count is high, consumption is small, simple production process, cost is low, double action with disease prevention growth-promoting, by nursery verification experimental verification, reduce the incidence of disease of crops, especially for the preventing and treating of cotton verticillium wilt, the incidence of disease is down to 6%, prevention effect is notable；Further, since be biological bacterial strain preparation, completely not because of the brought a series of problems that uses of chemical pesticide, thus be conducive to the No-harmful apple orchard of crops, peasant can without or reduce the consumption of chemical pesticide, greatly save spending.
Description
Technical field
The present invention relates to microbial control field, specially a kind of bacillus amyloliquefaciens Lh-1 and its application.
Background technology
Known, bacillus amyloliquefaciens can produce a series of metabolite in the growth course of its own, these
Metabolite can widely suppress the activity of crop pathogenic fungi and bacterium, be carried out using bacillus amyloliquefaciens effective
Biological control, safe and efficient pollution-free, potentiality to be exploited is huge.The bacillus amyloliquefaciens synthesis microbial inoculum viable bacteria reported at present
Number is low, and antibacterial material extracts inconvenience, such as：CN100999667 discloses a kind of vegetable field soil improving agent and preparation method thereof
With application, emphasize as soil conditioner, the proportioning and the proportioning of solid fungicide carrier of strain in microbial inoculum, but microbial inoculum individually ferments
Viable count is only 1,000,000,000/ml.Its zymotic fluid bacterium number is substantially relatively low, and consumption is too big in actual applications, and integrated cost is higher.CN
Antibacterial material produced by 102485880A discloses a bacillus amyloliquefaciens and its application, but bacterial strain exists only in cell
Interior, extraction process is cumbersome, production cost increase, and not in the application of field actual production.
In addition, the low molecular weight product that chitosan oligosaccharide is good water solubility, function is big, bioactivity is high, can improve plant
The immunocompetence of thing in itself, effectively improves crop yield, prevents and treats the beneficial bacterium in pest and disease damage, propagation soil and bio-fertilizer, is praised
Not to be the agricultural chemicals of agricultural chemicals, not being the chemical fertilizer of chemical fertilizer, the special role of the fertile economic benefits and social benefits of this medicine of chitosan oligosaccharide determines it in agricultural
The extensive use in field.
Cotton verticillium wilt is to endanger one of Major Diseases of Cotton Production, is referred to as " cotton cancer ", is cotton growth hair
Most universal, the important disease that loss is serious occurs during educating.In modern agricultural production, because the excess of chemical pesticide makes
With causing pathogenic bacteria resistance to drugs to strengthen, topsoil unbalance of structure land pollution, the problems such as cotton quality yield declines is increasingly serious.
With the enhancing of people's environmental consciousness, microorganism formulation is because of its highly effective and safe, the advantage such as environmental protection, using more and more wider.But
It is that up to the present, there are no bacillus amyloliquefaciens and chitosan oligosaccharide combination application in cotton verticillium wilt and other Chinese cabbages
The preventing and treating of Bacteria erwinia, peach rotten pathogenic bacteria, Valsa mali etc..
The content of the invention
It is an object of the invention to provide a kind of bacillus amyloliquefaciens Lh-1 and its application.
The present invention adopts the following technical scheme that realization：A kind of bacillus amyloliquefaciens
（bacillusamyloliquefaciens subsp）Lh-1, deposit number is CGMCC No.8548, Main Biological
For Gram-positive, thalline is in shaft-like, amphitrichous, and with motility, aerobic growth can form endogenous spore, in PDA
It is in orange red opaque colony on culture medium, surface folding, edge is irregular.Depositary institution is Chinese microorganism strain preservation pipe
Reason committee common micro-organisms center, address is BeiChen West Road, Chaoyang District, BeiJing City I institutes 3, and preservation date is 2013 12
The moon 6.
A kind of bacillus amyloliquefaciens Lh-1 rots in preventing and treating cotton verticillium wilt, soft rot of Chinese cabbage, bacterial wilt, peach
Application in terms of disease, canker of apple fruit, Phytophthora capsici disease, graw mold of tomato.
The microecological microbial agent being made using described bacillus amyloliquefaciens Lh-1.
The microecological microbial agent being made using bacillus amyloliquefaciens Lh-1, the viable bacteria content of the bacillus amyloliquefaciens is
8×109 cfu/g。
Using the preparation method of the bacillus amyloliquefaciens Lh-1 microecological microbial agents being made, comprise the following steps：（1）By ice
The bacillus amyloliquefaciens Lh-1 streak inoculations of case preservation are in PDA solid slope culture mediums, and 31 DEG C of cultures are activated for 48 hours；Will
Strain after activation is inoculated in PDA liquid medium, 31 DEG C of concussion and cultivates 24 hours, and bacterium solution is standby as seed liquor；（2）Expand
The raw material and percentage by weight of big fermenting and producing are as follows：Corn flour 2%, soya-bean cake 0.5%, fish meal 0.5%, brown sugar 1%, sodium chloride
0.1%, remaining is water, and raw material is dissolved in into water by proportioning adds in fermentation tank, high pressure steam sterilization, by step（1）Obtained seed
Liquid is inoculated into fermentation tank, and inoculum concentration accounts for the 2-5% of raw material cumulative volume, mixing speed 180r/min, 31 DEG C, pH6.8, dissolved oxygen phase
To value 20%-40%, ferment 36-48 hours；（3）When viable count reaches 8 × 10 in fermentation tank9During cfu/g, chitosan oligosaccharide powder is added
Enter step（2）It is well mixed in obtained zymotic fluid, the addition of chitosan oligosaccharide powder accounts for step（2）Gained zymotic fluid gross weight
Bacillus amyloliquefaciens Lh-1 microecological microbial agents are made in 1%-2%, bottling.
In above-mentioned microecological microbial agent, bacillus amyloliquefaciens (Bacillusamyloliquefaciens subsp) Lh-1
With broad-spectrum antibacterial activity, to verticillium dahliae（Verticillium dahliae）, Chinese cabbage soft rot bacteria（Erwinia carotovora）, ralstonia solanacearum（Ralstonia solanacearum）, peach rotten pathogenic bacteria（Valsa leucostoma）、
Valsa mali（Valsa mali Miyabe et Yamada）, phytophthora blight of pepper（Phytophthora capsici）, kind
Eggplant botrytis cinerea（Phytophthora infestans）Stronger inhibitory action can be produced, is especially had to verticillium dahliae
Inhibitory action strongly, thus show this Antagonistic Fungi bioactivity there is wide spectrum, it is efficient the characteristics of；Chitosan oligosaccharide is existing
Known products, can change soil flora, promote beneficial microorganism growth, chitosan oligosaccharide also can induce the disease resistance of plant, right
A variety of fungies, bacterium and virus produce immune and killing action, and the present invention has effect of disease prevention growth-promoting concurrently using chitosan oligosaccharide.
The present invention is combined with the chitosan oligosaccharide of the fertile economic benefits and social benefits of medicine using bacillus amyloliquefaciens Lh-1 and Lh-1 microecological microbial agents is made,
Viable count is high, and consumption is small, and simple production process, cost is low, the double action with disease prevention growth-promoting, by nursery verification experimental verification,
The incidence of disease of crops is greatly reduced, especially for the preventing and treating of cotton verticillium wilt, the incidence of disease is down to 6%, and prevention effect shows
Write；Further, since being biological bacterial strain preparation, brought a series of problems is not used because of chemical pesticide completely, thus is had
Beneficial to the No-harmful apple orchard of crops, peasant can without or reduce the consumption of chemical pesticide, greatly save spending, Er Qieyou
Beneficial to the outlet of agricultural product.
Brief description of the drawings
Fig. 1 is the phylogenetic tree collection of illustrative plates built based on 16S rDNA partial sequences；
Fig. 2 is Lh-1 growth curve chart.
Embodiment
The present invention is further elaborated with reference to embodiments, but is not used to limit the present invention.
Embodiment 1：
Bacillus amyloliquefaciens of the present invention (Bacillusamyloliquefaciens subsp) LH-1 separation and mirror
It is fixed
1st, sample collection
Drying 24 is small under the conditions of 100g, 60 DEG C are sampled at from Shanxi Shanxi Academy of Agricultural Sciences experimental plot upper soll layer to 15cm
When, take 1g to do separation and use.
2nd, the separation of bacterial strain and resistance screening
(1) strain dilutes：Weigh 1g to be dissolved into the triangular flask equipped with 100ml sterile purified waters, soil suspension is made
Liquid, then gradient dilution is to 10-9Soil Slurry.
(2) strain separating and screening:Soil Slurry 50ul and Liquid Culture the verticillium dahliae 50ul of 3 days is drawn to add
Enter into the PDA culture medium cooled down, even spread, place and culture 24h is inverted in 30 DEG C of incubators, observe and select Antagonistic Fungi
Fall and (suppress the big bacterium colony of circle)；
(3) antagonistic strain is purified:Institute's choosing colony is subjected to line purifying on PAD culture mediums, then by obtained pure bacterium
Strain carries out antagonism checking with verticillium dahliae, obtains one plant of stronger bacterium bacterial strain to verticillium dahliae antagonistic ability,
It is named as Lh-1.
3rd, the identification of bacterial strain
Morphology, Physiology and biochemistry identification and Phylogenetic Analysis are carried out to bacterial strain LH-1.
Bacterial strain LH-1 Gram-positives, nitrate reduction test is positive, catalase reacting positive, Starch Hydrolysis reaction
The positive, glucose fermentation experiment is positive, and Starch Hydrolysis experiment is positive.Microscope hypothallus is in shaft-like, amphitrichous, with motion
Property, aerobic growth can form endogenous spore, be in orange-yellow opaque colony in PDA culture medium, and surface folding, edge is not advised
Then.The bacterial strain 5-45 DEG C can on the culture mediums such as LB culture mediums, PDA culture medium flourish.The bacterium laboratory is most suitable
Culture medium is PDA culture medium, 31 DEG C of optimum growth temperature, most suitable Ph values 6.8.
Exponential phase of growth thalline is collected, genomic DNA is extracted with genome DNA extracting reagent kit, is used using it as template
16S rDNA universal primers expand 16S rDNA genetic fragments.Forward primer：5’-AGTTTGATCCTGGCTCA-3’；Reversely draw
Thing：5’-TACCTTGTTACGACTTCA-3’.PCR reactions use 25uL systems：10 × buffer solution 2.5uL；2 uL （25mmol/
L）MgCl2；4 uL（1.25 mmol/L）dNTP；2 uL（100 umol/L）Primer；0.4 uL（5U/uL）Taq enzyme；DNA profiling
1μL(0.05-1μg/μL)；The uL of deionized water 13.1.PCR amplification conditions：94 DEG C of min of pre-degeneration 3;94 DEG C of denaturation 1
min;50 DEG C of 1 min of annealing；72 DEG C of extension 3min, totally 30 circulations, 72 DEG C of 5 min of extension.The PCR primer of amplification is pure
It is connected and Transformed E .coli DH5 α with pMD18-T carriers after change, extracting has the plasmid of 16S rDNA insertions and transfers to Shanghai to give birth to work
Bioisystech Co., Ltd is sequenced, and will determine the 16SrDNA of obtained 16SrDNA sequence bacterium related in ncbi database
It is compared with Clustal X1.8, and chadogram (Fig. 1) is built using Mega4.0.
According to the result of study to the morphology of bacterial strain, cultural characteristic, Physiology and biochemistry and 16S rDNA, bacterial strain LH-1 identifications
For bacillus amyloliquefaciens (Bacillusamyloliquefaciens subsp).Bacillus amyloliquefaciens Lh-1 is micro- in China
Biological inoculum preservation administration committee common micro-organisms center (BeiChen West Road, Chaoyang District, BeiJing City I institutes 3, the Chinese Academy of Sciences
Institute of microbiology) preservation registration number be CGMCC No.8548.
4th, bacillus amyloliquefaciens Lh-1 growth kinetics research
Growth kinetics research to bacillus amyloliquefaciens Lh-1 will give birth to for the scale fermentation of Lh-1 microecological microbial agents
Production provides basic data.Strain after being activated through PDA solid slope culture mediums is inoculated in PDA liquid medium, 31 DEG C, shake
Swing culture.0, sampling in 4,8,12,16,20,24,28,32 hours, with 752 ultraviolet specrophotometer (λ=6
10 nm) OD values are determined, make Lh-1 growth curve chart（Fig. 2）
The bacillus amyloliquefaciens Lh-1 antimicrobial spectrums of embodiment 2 are determined
By verticillium dahliae（Verticillium dahliae）, Chinese cabbage soft rot bacteria（Erwinia carotovora）, ralstonia solanacearum（Ralstonia solanacearum）, peach rotten pathogenic bacteria（Valsa leucostoma）、
Valsa mali（Valsa mali Miyabe et Yamada）, phytophthora blight of pepper（Phytophthora capsici）, kind
Eggplant botrytis cinerea（Phytophthora infestans）As indicator bacteria, bacillus amyloliquefaciens Lh-1 fungistatic effects are determined.Tool
Body method is as follows:Aseptically, 48h, dilution 10 will be cultivated5Times 50ul bacillus amyloliquefaciens Lh-1 bacterium solution even spreads
Culture 48h is inverted onto PDA plate culture medium, in 31 DEG C of incubators.Chloroform is fumigated after bacterium colony 20min inactivations, with containing indicator bacteria
Soft agar by thin one layer of the paving in bacterium colony surface, continue to cultivate 24h, often processing is repeated 3 times, and determines antibacterial index.Antibacterial index=suppression
Bacterium loop diameter/Lh-1 colony diameters.
The antibacterial assessment of indices of the Lh-1 of table 1
As a result show that bacillus amyloliquefaciens Lh-1 produces stronger inhibitory action to above-mentioned indicator bacteria, especially to cotton yellow
The germ that withers has an inhibitory action strongly, thus show this Antagonistic Fungi bioactivity there is wide spectrum, it is efficient the characteristics of.
Embodiment 3
Bacillus amyloliquefaciens (Bacillusamyloliquefaciens subsp) Lh-1 microecological microbial agents preparation
（1）By in the Lh-1 strains streak inoculation of -80 DEG C of Storage in refrigerator to PDA solid slope culture mediums, 31 DEG C are cultivated 48
Hour activation.Strain after activation is inoculated in PDA liquid medium, 31 DEG C of concussion and cultivates 24 hours, bacterium solution is used as seed
Liquid is standby；（2）The raw material and percentage by weight for expanding fermenting and producing are as follows：Corn flour 2%, soya-bean cake 0.5%, fish meal 0.5% is red
Sugar 1%, sodium chloride 0.1%, remaining is water, and raw material is dissolved in into water by proportioning adds in fermentation tank, high pressure steam sterilization, by step
（1）Obtained seed liquor is inoculated into fermentation tank, and inoculum concentration accounts for the 2-5% of raw material cumulative volume, preferably 3%, mixing speed 180r/
Min, 31 DEG C, pH6.8, dissolved oxygen relative value 20%-40% ferment 36-48 hours；（3）When viable count reaches 8 × 10 in fermentation tank9
During cfu/g, chitosan oligosaccharide powder is added into step（2）It is well mixed in obtained zymotic fluid, the addition of chitosan oligosaccharide powder accounts for step（2）
The 1%-2% of gained zymotic fluid gross weight, preferably 1.5%, bacillus amyloliquefaciens Lh-1 microecological microbial agents are made in bottling.
Embodiment 4
Bacillus amyloliquefaciens (Bacillusamyloliquefaciens subsp) Lh-1 microecological microbial agents nursery
Experiment
Experiment sets 5 processing（Table 2）, using the method for feeding block seedlings raising, base fertilizer applicating ratio is 1%, nutrition in nutritive cube
It is transplanted to during alms bowl nursery to 2-3 piece true leaves in the basin equipped with 5kg soil, every group of basin of processing 50.Base fertilizer applicating ratio is 1% in basin, place
Nutrient difference urea, calcium superphosphate and potassium chloride polishing between reason.Pathogen inoculation method in basin alms bowl：By cotton verticillium wilt cause of disease
An armful sub- suspension for bacterium is well mixed dress basin with potting soil, and (ultimate density is 104Cfu/g soil)；The pure bacterium solutions of Lh-1（The pure bacterium solution is
The pure bacterium solution of chitosan oligosaccharide is not added with embodiment 3 after lower tank）Inoculation method：Bacterium solution is well mixed with the soil in basin to reach
107Cfu/g soil；Lh-1 microecological microbial agent inoculation methods：Microbial inoculum is well mixed with the soil in basin to reach 107Cfu/g soil
（Microorganism in microbial inoculum has 8 × 109Cfu/g, is added in basin soil in proportion, viable bacteria content in microbial inoculum is met requirement）；Basin
Alms bowl experiment harvested after 50d.
The nursery test process table of table 2.
Prevention effect：
After the cotton plant harvest of all processing, with strain number of falling ill, the incidence of disease and every group of total fresh weight of processing are index, weigh anti-
Control effect（Table 3）,
The incidence of disease=（Morbidity strain number/every group of total strain number）x100%.
The prevention effect table of table 3
As seen from the above table, in the blank control of experimental group 1, because seed may carry pathogenic bacteria, there is 1 plant of morbidity.Experimental group 2
98% is up to due to adding the verticillium wilt pathogen incidence of disease, and the total fresh weight of contrast experiment's group 1 declines substantially.Verticillium wilt is added in experimental group 3
Bacterium obtains good prevention effect with Lh-1, and the incidence of disease drops to 22% by 98%.Verticillium wilt pathogen and chitosan oligosaccharide are added in experimental group 4
There is certain prevention effect, although the incidence of disease is higher than experimental group 3, but non-disease plant growing way is very well in experimental group 4, total fresh weight
With group 3 quite, illustrate that chitosan oligosaccharide has effect of disease prevention growth-promoting concurrently.We use the Lh-1 that Lh-1 is combined with chitosan oligosaccharide in experimental group 5
Microecological microbial agent is prevented and treated verticillium wilt, and the incidence of disease is down to 6%, and total fresh weight is higher than the blank control 3360g of experimental group 1, prevents
Sick growth-promoting effect is notable.
Claims (3)
1. a kind of bacillus amyloliquefaciens（bacillus amyloliquefaciens subsp）Lh-1, deposit number is
CGMCC No.8548, Main Biological is that Gram-positive, thalline is in shaft-like, amphitrichous, good with motility
Oxide growth, can form endogenous spore, be in orange red opaque colony in PDA culture medium, and surface folding, edge is irregular.
2. a kind of bacillus amyloliquefaciens Lh-1 according to claim 1 preventing and treating cotton verticillium wilt, soft rot of Chinese cabbage,
Application in terms of bacterial wilt, peach rot, canker of apple fruit, Phytophthora capsici disease or graw mold of tomato.
3. the microecological microbial agent that bacillus amyloliquefaciens Lh-1 according to claim 1 is made, it is characterized in that micro- life
The preparation method of state microbial inoculum, comprises the following steps：
（1）By in the bacillus amyloliquefaciens Lh-1 streak inoculations of Storage in refrigerator to PDA solid slope culture mediums, 31 DEG C are cultivated 48
Hour activation；Strain after activation is inoculated in PDA liquid medium, 31 DEG C of concussion and cultivates 24 hours, bacterium solution is used as seed
Liquid is standby；
（2）The raw material and percentage by weight for expanding fermenting and producing are as follows：Corn flour 2%, soya-bean cake 0.5%, fish meal 0.5%, brown sugar
1%, sodium chloride 0.1%, remaining is water, and raw material is dissolved in into water by proportioning adds in fermentation tank, high pressure steam sterilization, by step（1）
Obtained seed liquor is inoculated into fermentation tank, and inoculum concentration accounts for the 2-5% of raw material cumulative volume, mixing speed 180r/min, 31 DEG C,
PH6.8, dissolved oxygen relative value 20%-40%, ferment 36-48 hours；
（3）When viable count reaches 8 × 10 in fermentation tank9During cfu/g, chitosan oligosaccharide powder is added into step（2）In obtained zymotic fluid
Well mixed, the addition of chitosan oligosaccharide powder accounts for step（2）Solution starch gemma is made in the 1%-2% of gained zymotic fluid gross weight, bottling
Bacillus Lh-1 microecological microbial agents.
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| CN102604864A (en) * | 2012-03-09 | 2012-07-25 | 陕西加伦多作物科学有限公司 | Microbial bactericide and application thereof |
| CN103131655A (en) * | 2013-03-06 | 2013-06-05 | 江苏苏滨生物农化有限公司 | Bacillus amyloliquefaciens K-8 and bactericide thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102604864A (en) * | 2012-03-09 | 2012-07-25 | 陕西加伦多作物科学有限公司 | Microbial bactericide and application thereof |
| CN103131655A (en) * | 2013-03-06 | 2013-06-05 | 江苏苏滨生物农化有限公司 | Bacillus amyloliquefaciens K-8 and bactericide thereof |
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| 内生解淀粉芽孢杆菌Ｘ－２７８发酵条件的优化;魏娇洋等;《北方园艺》;20140331(第5期);第106-110页 * |
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| 高岭土、壳聚糖和几丁质作为Fo47 菌剂填料的研究;田晓丽等;《中国生物防治》;20101130;第26卷;第73-79页 * |
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