CN105838649A - Bacillus amyloliquefaciens HCS and application thereof to sugarcane production - Google Patents
Bacillus amyloliquefaciens HCS and application thereof to sugarcane production Download PDFInfo
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- CN105838649A CN105838649A CN201610328203.2A CN201610328203A CN105838649A CN 105838649 A CN105838649 A CN 105838649A CN 201610328203 A CN201610328203 A CN 201610328203A CN 105838649 A CN105838649 A CN 105838649A
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Abstract
The invention relates to bacillus amyloliquefaciens HCS (Human Chorionic Somatomammotrophin) which is preserved in the China General Microbiological Culture Collection Center (CGMCC) on September 19th, 2012, wherein the preservation number is CGMCC No. 6591. The bacillus amyloliquefaciens HCS provided by the invention is applied to the sugarcane production. Tissue culture seedling heel-in, field planting and seed-stem soaking tests indicate that the bacillus amyloliquefaciens HCS plays a good role in inhibiting sugarcane root diseases and a better role in promoting the rooting and the growth of sugarcanes; a bacterial strain is indicated to be a biocontrol strain having a potential significance in preventing and treating the production of the sugarcanes.
Description
Technical field
The invention belongs to microbial technology field, be specifically related to a kind of bacillus amyloliquefaciens HCS and in sugarcane production
On application, this bacterial strain can wide spectrum preventing and treating the dissimilar disease of plant and during sugarcane tissue culture seedling provisonal planting, field planting promote
Caulis Sacchari sinensis takes root, grow and hestening rooting during the plantation of Caulis Sacchari sinensis kind stem, increase tiller and improve yield.
Background technology
Chemical pesticide is the valuable source of controlling plant diseases, but owing to too much, mistake uses, have cause residual toxicity,
Environmental pollution and the fraud of ecological disruption.It is badly in need of finding a kind of that can substitute, safer and more effective prevention and controls.Although a lot of biological and ecological methods to prevent plant disease, pests, and erosions
Preparation has been shown to have preventive and therapeutic effect, but only has a few and be applied to produce, and major part is still in research and exploratory stage.
Being applied to the biological prevention and control agent produced, every kind of biological prevention and control agent typically can only prevent and treat the disease of a few type, does not have wide
Spectrum prevention and control capability, promotes the DeGrain of plant growing.
Caulis Sacchari sinensis (Saccharum officinarum L.) is the topmost sugar crop of China, Ye Shi world main energy sources
Crop, cane suger yield accounts for more than the 90% of China's sugar total output.Caulis Sacchari sinensis is asexually propagated crop, is also that perennial root is planted for many years
Training crop, plants especially perennial root plantation for many years and subjects to repeatedly infecting and endangering of some pathogens.As far back as 20 century 70s
End, Brazil etc. state begin to sugarcane health seedling is studied, the mid-80, health seedling produce succeeded, and
Promote the use of in sugarcane production.Domestic jasmine duckweeds perhaps etc. begin through the method training of tissue culture and Bud culture in the 90's
Educate sugarcane health seedling, to remove mosaic of sugarcane and ratoon stunting disease, it is possible to increase sugarcane yield more than 15%, Sucrose Content contains
Amount improves more than 0.5%.At present, sugar-cane tissue culture seedlings produces is to utilize suitable synthetic medium and the artificial condition such as illumination, temperature
Under, by inducing the incubation steps such as callus, adventitious bud, adventitious root, eventually forming complete plant, its seedlings root is inhaled
Receiving indifferent, resistance is poor, heels in survival rate and only has about 70%, and sometimes even less than 50%, the most all it is because
The bottle single plant of Seedling take root number less, two grades of root systems are undeveloped, heel in after due to root disease and do not adapt to external environment and dead
Die.Although it is affected by various factors to heel in shoot survival percent, but ensure that the health of root of Seedling and development degree are of paramount importance.
Summary of the invention
Present invention aim to overcome that prior art defect, it is provided that a kind of bacillus amyloliquefaciens HCS and raw Caulis Sacchari sinensis
Application on product, this bacterial strain can be used for preventing and treating Caulis Sacchari sinensis root disease, and can be effectively facilitated sugarcane production, take root, it is ensured that root system
Healthy and flourishing.
For achieving the above object, the present invention adopts the following technical scheme that
One bacillus amyloliquefaciens bacterial strain, named bacillus amyloliquefaciens (Bacillus amyloliquefaciens)
HCS, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on the 19th in JIUYUE in 2012, address: north
North Star West Road, Jing Shi Chaoyang District 1 institute 3；Deposit number: CGMCC NO.6591.
Bacillus amyloliquefaciens provided by the present invention (Bacillus amyloliquefaciens) HCS bacterial strain, separates
From the Among the Sugarcane root soil in sugarcane district of Hainan Province of China, through morphology, cultural colony, customary physiological is biochemical and 16S rDNA sequence is reflected
Fixed, this bacterial strain is a new strains of bacillus amyloliquefaciens (Bacillus amyloliquefaciens), named Xie Dian
Afnyloliquefaciens (Bacillus amyloliquefaciens) HCS.
Bacillus amyloliquefaciens provided by the present invention (Bacillus amyloliquefaciens) HCS's is substantially biological
Characteristic is: bacterium colony is circular, filbert, bacterium colony central color is slightly deep, protuberance, smooth surface；Thalli morphology is shaft-like, end is round；Leather
Lan Shi is positive, produces spore, and peritrichous, tool mobility, Sporangium does not expands, spore oval.
The training method of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) the HCS bacterial strain of the present invention
For:
1) solid culture: inoculation on LB solid medium, cultivates 24 hours in 37 DEG C of constant incubators；
2) liquid culture: inoculation in LB fluid medium, at a temperature of 37 DEG C, the shaking table of rotating speed 170rpm is cultivated
36 hours.
The invention provides bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HCS in sugarcane production
In application.
Concrete, refer to that bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HCS is in Caulis Sacchari sinensis diseases prevention, rush
Take root, application in growth promotion, such as: it promotes that during sugarcane tissue culture seedling provisonal planting, field planting Caulis Sacchari sinensis takes root, grows and Caulis Sacchari sinensis kind
Hestening rooting during stem plantation, increase tiller and improve the aspect such as yield.
A kind of diseases prevention in sugarcane production, growth-promoting root, somatotrophic biological preparation, its active component is that aforesaid preservation is compiled
Number it is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HCS of CGMCC NO.6591.With institute of the present invention
The biological preparation that bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HCS is active component provided also belongs to
Protection scope of the present invention.The when of needs, said preparation can comprise common carrier and adjuvant prepared by biological preparation.
The present invention heels in test by tissue cultured seedling and finds: bacillus amyloliquefaciens (Bacillus
Amyloliquefaciens) root disease when suppression is heeled in by HCS, promotes to heel in seedling rooting and growth all has well effect
Really, up to the 100% and processed root hair heeling in Seedling of preventive effect of preventing and treating root disease is flourishing, healthy and strong and average height comparison
According to high about 3cm.
The present invention is found by the test of tissue cultured seedling survival after transplant: the tissue cultured seedling well developed root system processed, the mean tillering number ratio
Comparison improves about 25%.
The present invention plants after soaking Caulis Sacchari sinensis kind stem with the HCS bacteria suspension of different extension rates, and the survey result after 40 days is sent out
Existing: emergence rate exceeds 30% than comparison, and root fibrous root is normal, does not send out referring now to the downright bad speckle on root fibrous root, averagely
Aspect ratio comparison exceeds about 10cm, shows that it has preventing and treating root disease and growth-promoting root, somatotrophic effect.
To sum up, the present invention is by the screening and separating to sugarcane district Among the Sugarcane root Soil Microorganism, it is thus achieved that solve starch spore bar
Bacterium (Bacillus amyloliquefaciens) HCS, is heeled in by tissue cultured seedling, field planting and plant the different tests such as stem immersion
Research, result shows: bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HCS can effectively prevent and treat Caulis Sacchari sinensis
Root disease during production, and Caulis Sacchari sinensis can be effectively facilitated take root and promote sugarcane production.Show bacillus amyloliquefaciens
(Bacillus amyloliquefaciens) HCS is the biocontrol bacterial strain that a strain has potential using value in sugarcane production.
The present invention is existing by state natural sciences fund (31471555), Hainan Province's Natural Science Fund In The Light (314120) and country
Generation agricultural Sugarcane Industry technical system is provided with funds support.
Detailed description of the invention
Below in conjunction with embodiment, the detailed description of the invention of the present invention is described in further detail.Following example are used for
The present invention is described, but is not limited to the scope of the present invention.The test method of unreceipted actual conditions in the following example, generally
According to normal condition, or according to the condition proposed by manufacturer.
The screening of embodiment 1 plant pathogenic fungi antagonistic strain and qualification thereof
Bacterial strain screening: weigh 5 grams of rhizosphere soils (Rhizosphere Soil of Caulis Sacchari sinensis base, Huang Tong town, China Lingao County, Hainan Province Caulis Sacchari sinensis plant
Earth), it is placed with in the triangular flask of 100ml sterilized water, after the 30min that acutely vibrates on shaking table, stands 15min.Take 1ml to be diluted to
10-4、10-5、10-6Concentraton gradient, takes 0.1ml from each Concentraton gradient suspension, is applied to LB culture medium (formula with aseptic painting rod
For: every liter contains peptone 10g, yeast powder 5g, NaCl 10g, agar powder 12g, pH7.0～7.2) on flat board, 37 DEG C of constant temperature trainings
Supporting carton upside down to cultivate, Continuous Observation 3 days, the bacterial strain that picking colony form is different purification storage of again ruling is standby.Good
PDA plate surface coating Caulis Sacchari sinensis pine root fungus spore, after drying, connects screening after purification with sterile toothpick point in superclean bench
Bacterial strain carry out opposite culture, the bacterial strain of screening bacteriostasis the strongest (bacteriostatic activity is the strongest), result obtains a strain to Caulis Sacchari sinensis root
Maize ear rot bacterium has the bacterial strain of the strongest antagonism, is bacillus amyloliquefaciens (Bacillus by this Strain Designation
Amyloliquefaciens) HCS.Within 19th, it is preserved in China Committee for Culture Collection of Microorganisms in JIUYUE in 2012
Common micro-organisms center, address: North Star West Road 1, Chaoyang District, city of BeiJing, China institute 3, deposit number: CGMCC No.6591.
The Basic Biological Character of bacterial strain: bacterium colony is circular, filbert, bacterium colony central color is slightly deep, protuberance, smooth surface；
Thalli morphology is shaft-like, end is round；Gram-positive, produces spore, and peritrichous, tool mobility, Sporangium does not expands, spore oval
Shape.
The mensuration of bacterial strain 16S rDNA conserved sequence: the bacillus amyloliquefaciens (Bacillus that screening is obtained
Amyloliquefaciens) genomic DNA of HCS bacterial strain is template, with bacterial 16 S rDNA sequence universal primer P0、P6Make
For primer, PCR amplifies the product fragment of about about 1.5kb, and sequencing result shows that the sequence length amplified is 1516 alkali
Base (see sequence table)；By this sequence by the Internet comparison (http://www. ncbi.nlm. nih. gov/blast/B
Last. cgi), the homology of the 16S rDNA partial sequence of the fragment sequence of amplification and bacillus amyloliquefaciens reach 99% with
On, determine that this bacterial strain is attributed to bacillus amyloliquefaciens (Bacillus amyloliquefaciens).
Embodiment 2 bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HCS prevents heeling in tissue cultured seedling
Disease, growth-promoting root and the research of growth
With bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HCS be spawn culture obtain biological preparation (with
Under be weight percentage):
1. solid culture: the bacillus amyloliquefaciens (Bacillus amyloliquefaciens) of preservation state will be in
HCS is inoculated in solid medium (1% tryptone, 0.5% yeast extract, 1%NaCl, 1.2% agar powder, pH7.0.), in
37 DEG C of incubators are cultivated 1 day, makes bacterial strain activate.
2, liquid culture: by bacillus amyloliquefaciens (Bacillus amyloliquefaciens) the HCS inoculation of activation
In the 500ml triangular flask equipped with fluid medium (1% tryptone, 0.5% yeast extract, 1%NaCl, pH7.0.), every bottle
Dress 200ml, at 37 DEG C, the shaking table of rotating speed 170rpm is cultivated 36h, obtains liquid biological preparation, and be diluted to 10-1、
10-2、10-3、10-4、10-5、10-6、10-7Seven Concentraton gradient, it is thus achieved that corresponding biological preparation bacteria suspension.
Hole tray heels in tissue cultured seedling: by relatively uniform tissue cultured seedling field planting hole tray, by the biological system of above-mentioned variable concentrations gradient
Agent bacteria suspension pours into as water at all, pours into as comparison using clear water.Test sets 8 process altogether, and each process is repeated 4 times, often
Individual it is repeated as 32 caves ().Process 1 to processing 7 respectively with 10-1、10-2、10-3、10-4、10-5、10-6、10-7The life of Concentraton gradient
Thing preparation bacteria suspension pours into as water at all, processes 8 and compares for clear water.Above-mentioned variable concentrations gradient is again poured into after 7 days
Biological preparation bacteria suspension and clear water, remaining management is identical.
Investigate after 30 days, observe root incidence, count root of hair quantity, measure plant height.Result shows (to be shown in Table
1), having a little black speck with the tissue cultured seedling root of the process 7 of biological preparation bacteria suspension process, the root of the process 8 of clear water comparison is black
Speckle number has exceeded 50%, and it is pure white, without black speck that other process all shows as root fibrous root.The root of hair quantity simultaneously counted shows: process
The root of hair number of 3 process 8 compareed to process 7 and clear water is all more than processing 1 and processing 2, and bacteria suspension concentration that may be higher presses down
Make the growth of root.The measurement result of plant height shows: process 5 the highest, secondly for process 4, process 6 and process 7, clear water comparison with
Process 3 quite, process 1, to process 2 minimum.By analyzing: when hole tray heels in tissue cultured seedling, expenditure is 10-5Biological preparation
Bacteria suspension can reach diseases prevention, growth-promoting root and the effect of growth when processing simultaneously.
Table 1 hole tray heels in Seedling survey result after 30 days
。
Field planting tissue cultured seedling is prevented by embodiment 3 bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HCS
Disease, growth-promoting root and the research of growth
This test utilizes laboratory to carry out during field-transplanting after the routine that base produces heels in Seedling 30 days, and test sets 4 process,
Processing 1 to processing 4 respectively concentration is 10-3、10-4、10-5Biological preparation bacteria suspension and clear water comparison, each process repeat
4 times, each repetition 1000 strain.Detailed description of the invention is, pours into the biological preparation bacteria suspension of variable concentrations gradient after field planting immediately
With clear water as determining root water, remaining all management is identical.
Investigate after 50 days.Result shows (see Table 2), by the planting stock of variable concentrations biological preparation bacteria suspension process
Fibrous root number is significantly more than clear water comparison, by concentration 10-3Biological preparation bacteria suspension process plant height apparently higher than 10-4、10-5Place
Reason and clear water control treatment.And by often repeating to dig out 10 (clumps), to root disease, a situation arises that the survey showed that:
Only by concentration 10-3The root that processes of biological preparation bacteria suspension without obvious black scab, the root black scab that clear water processes
Reach more than 30%.Investigate all tiller situations to show, by concentration 10-3The tiller number that processes of biological preparation bacteria suspension most, ratio is clearly
Water comparison is high by 26.39%.
Table 2 hole tray heels in the Seedling field planting land for growing field crops survey result of 50 days
。
Kind of a stem plantation is prevented by embodiment 4 bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HCS
Disease, growth-promoting root and the research of growth
With the biological preparation bacteria suspension of clear water and variable concentrations gradient by planting after soaking Caulis Sacchari sinensis kind stem, it is correlated with after 40 days
Investigation.Test sets 4 process, and processing 1 to process 4 being respectively concentration is 10-2、10-3、10-4Biological preparation bacteria suspension
And clear water comparison, each process is repeated 4 times, the double bud kind stem of each repetition 1000 sections.
Result shows (see Table 3): by concentration 10-2Biological preparation bacteria suspension process emergence rate than clear water control treatment
Emergence rate exceeds 30%, and root fibrous root is normal, does not find the downright bad speckle occurred on clear water comparison root fibrous root, mean height
Spend and exceed about 10cm than clear water comparison, show that it has preventing and treating root disease and growth-promoting root, effect of growth.
3 kinds of stems of table soak the survey result after planting 40 days
。
In sum: bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HCS is that a strain is from Caulis Sacchari sinensis
The bacterial strain of rhizosphere, it is in close relations with plant root, and many soil biography phytopathy originals are had good inhibiting effect, taken into account
Promote plant establishment and plant growing, again apply and environment is also not result in environmental pollution, there is good DEVELOPMENT PROSPECT.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For Yuan, on the premise of without departing from the technology of the present invention principle, it is also possible to make some improvements and modifications, these improvements and modifications
Also should be regarded as protection scope of the present invention.
SEQUENCE LISTING
<110>China tropic Agriculture Academy Sciences tropic Biotechnology Research Institute
Hainan Medical College
<120>bacillus amyloliquefaciens HCS and the application in sugarcane production thereof
<130>
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1516
<212> DNA
<213> Bacillus amyloliquefaciens
<400> 1
gagagtttga tcctggctca ggacgaacgc tggcggcgtg cctaatacat gcaagtcgag 60
cggacagatg ggagcttgct ccctgatgtt agcggcggac gggtgagtaa cacgtgggta 120
acctgcctgt aagactggga taactccggg aaaccggggc taataccgga tggttgtttg 180
aaccgcatgg ttcagacata aaaggtggct tcggctacca cttacagatg gacccgcggc 240
gcattagcta gttggtgagg taacggctca ccaaggcaac gatgcgtagc cgacctgaga 300
gggtgatcgg ccacactggg actgagacac ggcccagact cctacgggag gcagcagtag 360
ggaatcttcc gcaatggacg aaagtctgac ggagcaacgc cgcgtgagtg atgaaggttt 420
tcggatcgta aagctctgtt gttagggaag aacaagtgcc gttcaaatag ggcggcacct 480
tgacggtacc taaccagaaa gccacggcta actacgtgcc agcagccgcg gtaatacgta 540
ggtggcaagc gttgtccgga attattgggc gtaaagggct cgcgggcggt ttcttaagtc 600
tgatgtgaaa gcccccggct caaccgggga gggtcattgg aaactgggga acttgagtgc 660
agaagaggag agtggaattc cacgtgtagc ggtgaaatgc gtagagatgt ggaggaacac 720
cagtggcgaa ggcgactctc tggtctgtaa ctgacgctga ggagcgaaag cgtggggagc 780
gaacaggatt agataccctg gtagtccacg ccgtaaacga tgagtgctaa gtgttagggg 840
gtttccgccc cttagtgctg cagctaacgc attaagcact ccgcctgggg agtacggtcg 900
caagactgaa actcaaagga attgacgggg gcccgcacaa gcggtggagc atgtggttta 960
attcgaagca acgcgaagaa ccttaccagg tcttgacatc ctctgacaat cctagagata 1020
ggacgtcccc ttcgggggca gagtgacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt 1080
gagatgttgg gttaagtccc gcaacgagcg caacccttga tcttagttgc cagcattcag 1140
ttgggcactc taaggtgact gtcggtgaca aaccggagga aggtggggat gacgtcaaat 1200
catcatgccc cttatgacct gggctacaca cgtgctacaa tggacagaac aaagggcagc 1260
gaaaccgcga ggttaagcca atcccacaaa tctgttctca gttcggatcg cagtctgcaa 1320
ctcgactgcg tgaagctgga atcgctagta atcgcggatc agcatgccgc ggtgaatacg 1380
ttcccgggcc ttgtacacac cgcccgtcac accacgagag tttgtaacat ccgaagtcgg 1440
tgaggtaacc tttatggagc cagccgccga aggtgggaca gatgattggg gtgaagtcgt 1500
aacaaggtag ccgtag 1516
Claims (4)
1. a bacillus amyloliquefaciens bacterial strain, named bacillus amyloliquefaciens (Bacillus amyloliquefaciens)
HCS, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on the 19th in JIUYUE in 2012, and preservation is compiled
Number: CGMCC NO.6591.
2. bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HCS described in claim 1 is in sugarcane production
In application.
3. bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HCS described in claim 1 prevents Caulis Sacchari sinensis
Application in disease, growth-promoting root, growth promotion.
4. diseases prevention in sugarcane production, growth-promoting root, a somatotrophic biological preparation, its active component is described in claim 1
The bacillus amyloliquefaciens that deposit number is CGMCC NO.6591 (Bacillus amyloliquefaciens) HCS.
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| MAGDALENA KRÖBER等: "Effect of the strain Bacillus amyloliquefaciens FZB42 on the microbial community in the rhizosphere of lettuce under field conditions analyzed by whole metagenome sequencing", 《FRONTIERS IN MICROBIOLOGY》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110692480A (en) * | 2019-11-11 | 2020-01-17 | 广西壮族自治区农业科学院 | Method for increasing sugarcane yield and prolonging perennial root life by obliquely cutting and reseeding sugarcane |
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