CN105238723A - Bacillus amyloliquefaciens strain for control of crop greensickness and microbial agent thereof - Google Patents
Bacillus amyloliquefaciens strain for control of crop greensickness and microbial agent thereof Download PDFInfo
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Abstract
The invention belongs to the field of agricultural biological control, and particularly discloses a Bacillus amyloliquefaciens strain HMB27684 for control of crop greensickness, and the strain has a preservation number of CGMCC No.11456. The present invention also discloses a microbial agent prepared from the strain, and application thereof to control of crop greensickness. The invention provides an efficient microbe for the control of greensickness of cotton, eggplant and other crops, has average control effect of more than 70% on cotton and eggplant, and has strong pertinency; the microbial agent of the invention is safe to human and livestock, and has little environmental pollution; in addition, the strain has long effective period on the control of greensickness on cotton and eggplant, and does not produce drug resistance. The invention has the advantages of simple preparation method, low cost and easy usage.
Description
Technical field
The invention belongs to Strategies of Agricultural Bio-control field, be specifically related to a kind of bacillus amyloliquefaciens preventing and treating crop verticillium, and the microbiobacterial agent containing this bacillus amyloliquefaciens, also relate to them and prevent and treat the application in crop verticillium.
Background technology
The crop verticillium such as cotton, eggplant caused by Garden Dahlia Verticillium (Verticilliumdahaliae) is a kind of soil-borne disease endangering seriously and be difficult to control.This disease is on the rise in Cotton in China, the harm of eggplant main producing region at present.Cotton verticillium wilt at China's population outbreak regularly, annual by its sown areas of cotton affected more than 4,000 ten thousand mu, cause the cotton underproduction about 10% ~ 20%, there is serious plot and cause the underproduction up to 60% ~ 70% in disease throughout the year.Verticillium often causes the whole strain withered death of eggplant, is close to total crop failure total crop failure.
About the prevention and controls of the crop such as cotton, eggplant verticillium, owing to lacking stable effective resisting verticillium gene, the poor effect of breeding for disease resistance; Then there is the shortcomings such as preventive effect is not good enough, contaminate environment, destruction agricultural ecological balance in chemical prevention; Comparatively effectively crop rotation for many years in cultural control measure, but under specific agricultural environment condition in the past, have a large population and a few land and substitute cotton, eggplant high price crop species deficient, be difficult to apply.Biological control due to have environmental pollution little, person poultry safety, specialization are developed immunity to drugs by force, not easily, act on the lasting period long, the advantages such as available protecting natural enemy, production cost be low can day by day be subject to the favor of people.
Genus bacillus (Bacilliussp) is a kind of biocontrol bacteria being subject to extensive concern.With wide, the easily separated cultivation of its distribution, the features such as the stronger brood cell of resistance, storage period be long and easy to use can be produced, become a kind of desirable Biocontrol microorganism.Because of genus bacillus can produce in sprout born of the same parents, have extremely strong anti-adversity ability, compare the bio-control factors of other types, be more conducive to the production of microbial inoculum, survive in formulation environment, surely grow and breeding.Therefore, screening the genus bacillus that pathogenic bacteria is inhibited is one of most effectual way of preventing and treating concerned plant disease.
At present, the genus bacillus for preventing and treating crop verticillium mainly contains subtilis, Bacillus licheniformis, Amado Azar base sub-genus bacillus, bacillus pumilus, bacillus amyloliquefaciens, bacillus polymyxa etc., achieves significant prevention effect.But due to diversity and the synchronous evolution of pathogenic bacteria, still need to find new bacterial strain and be applied to control crop verticillium.
Through retrieval, do not find the report about Bacillus amyloliquefaciens strain HMB27684 of the present invention.
Summary of the invention
The object of the invention is to provide a kind of Bacillus amyloliquefaciens strain for preventing and treating crop verticillium, the advantages such as this bacterial strain has efficiently, fungicidal spectrum is wide.
Another object of the present invention is to provide the microbiobacterial agent utilizing above-mentioned bacillus amyloliquefaciens to produce.
The present invention the 3rd object is the preparation method providing mentioned microorganism microbial inoculum.
The present invention the 4th object is to provide the purposes of above-mentioned Bacillus amyloliquefaciens strain in control crop verticillium.
The present invention the 5th object is to provide the purposes of mentioned microorganism microbial inoculum in control crop verticillium.
The present invention the 6th object is the authentication method providing above-mentioned Bacillus amyloliquefaciens strain.
The present invention the 7th object is the authentication method providing mentioned microorganism microbial inoculum.
The present invention is achieved through the following technical solutions:
A kind of bacillus amyloliquefaciens (Bacillusamyloliquefaciens) bacterial strain HMB27684, oneself is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica) on September 29th, 2015, and deposit number is CGMCCNo.11456.
Utilize the microbiobacterial agent that above-mentioned bacillus amyloliquefaciens HMB27684 produces, its activeconstituents is bacillus amyloliquefaciens HMB27684 thalline.
Mentioned microorganism microbial inoculum, the viable count of its HMB27684 is greater than 17.0 × 10
8cfu/mL.
Mentioned microorganism microbial inoculum can be liquid preparation or pulvis.
The preparation method of mentioned microorganism microbial inoculum, comprises the steps:
(1) actication of culture: the HMB27684 bacterial strain of cryopreservation is activated on LB plate culture medium, picking list bacterium colony, on LB slant medium, is cultivated 10 ~ 16 hours, is obtained the bacterial strain of activation at 25 ~ 35 DEG C;
(2) seed liquor preparation: the inoculation activated with aseptic transfering loop scraping one ring step (1) is in 100mLLB liquid nutrient medium, 25 ~ 35 DEG C, shaking speed be the condition of 150 ~ 220rpm under cultivate 10 ~ 16 hours, obtain seed liquor;
(3) fermentation culture: according to volume ratio be 1 ~ 3% ratio the seed liquor of step (2) is linked in Semen Maydis powder soybean powder medium (pH value is 7.0), temperature be 25 ~ 35 DEG C, rotating speed be 150 ~ 220rpm condition bottom fermentation cultivate 40 ~ 50h, obtain fermented liquid;
(4) detect thalline and brood cell's quantity in fermented liquid, when ripe brood cell accounts for 90% of brood cell and thalline sum in fermented liquid, stop fermentation culture; Gained is the liquid preparation of HMB27684 bacterial strain.
Described LB plate culture medium, LB slant medium and LB liquid nutrient medium are all conventionally prepared.
LB plate culture medium described in above-mentioned preparation method's step (1) or LB slant medium, its moiety and weight ratio thereof are: Tryptones 8 ~ 12g, yeast extract 4 ~ 6g, sodium-chlor 4 ~ 6g, agar powder 12 ~ 18g, water 1000mL.
LB liquid nutrient medium described in above-mentioned preparation method's step (2), its moiety and weight ratio thereof are: Tryptones 8 ~ 12g, yeast extract 4 ~ 6g, sodium-chlor 4 ~ 6g, water 1000mL.
Described Semen Maydis powder soybean powder medium described in above-mentioned preparation method's step (3), its moiety and weight percent thereof are: Semen Maydis powder 1.0 ~ 3.0%, analysis for soybean powder 1.0 ~ 3.0%, NaCl0.1 ~ 1.0%, MnSO
4h
2o0.5 ~ 1.0%, all the other are water.
The preparation method of described Semen Maydis powder soybean powder medium, according to weight percent by Semen Maydis powder, analysis for soybean powder, NaCl and MnSO
4h
2o mixes, then adds water, and regulates pH to stir.
The application of described bacillus amyloliquefaciens HMB27684 in control crop verticillium.
The application of mentioned microorganism microbial inoculum in control crop verticillium.
Crop described in above-mentioned application refers to cotton or eggplant.
The using method of mentioned microorganism microbial inoculum: it is 10 that above-mentioned gained microbiobacterial agent is diluted with water to viable bacteria body number
8cfu/mL, by seed-soaking half an hour before cotton, eggplant sowing; Or by above-mentioned gained microbiobacterial agent titanium pillaring solution, be made into bacillus amyloliquefaciens HMB27684 pulvis, dress seed according to pesticide-seeds ratio 1:10 before cotton, eggplant sowing, all can reach the object of prevention cotton, eggplant verticillium wilt generation.
The screening and separating process of HMB27684 bacterial strain
HMB27684 bacterial strain is separated to obtain from the Cotton Soil of Jingzhou City of Hubei Province.In April, 2013 is 5 collection soil samples from cotton field, Jingzhou City of Hubei Province, taking 1g after mixing is put in the sterilizing triangular flask of 250mL, add 100mL sterilized water, be put on shaking table, 170r/min vibrates 30min, leaves standstill 2h, getting supernatant liquor 10mL adds in 50mL sterile centrifugation tube, 80 DEG C of waters bath with thermostatic control 30 minutes, then get 1mL and add sterilized water 9mL, 10mL10
-3times soil microorganisms suspension, is diluted to 10 by soil supension then
-4, 10
-5, 10
-6times diluent, get each concentration microorganism suspension 200 μ L and be applied on LB culture medium flat plate, each concentration repeats 3 times, at 30 DEG C of constant temperature culture 1d-3d, carries out the abstraction and purification of bacterium.And with cotton, eggplant verticillium wilt for target, carried out the screening of biocontrol microorganisms by double-layer agar technique, pot experiment method.Result therefrom filters out one has obvious prevention effect bacterial strain to cotton, eggplant verticillium wilt, names as HMB27684.
The taxonomic identification of HMB27684 bacterial strain:
(1) identification by morphological characters
It is shaft-like that LB substratum is cultivated thalline, produces brood cell after cultivating 10h, and raw in brood cell, oval, cyst does not expand, and acid-fast stain is negative, without parasporal crystal, can move, flagellum Zhousheng.On nutrient agar plate, Initial stage of culture bacterium colony light oyster white, purulence shape, circular, neat in edge, bacterium colony protuberance is steamed bun shape, surface wettability; Late stage of culture bacterium colony is faint yellow, and edge is irregular, and surface drying has fold; Streak culture on nutrient agar slopes, linearly shape; Static gas wave refrigerator in liquid medium within, surface forms white mycoderm.These morphological specificitys and " common bacteria system identification handbook " (eastern elegant pearls etc. are write. Science Press .2001) in the bacillus morphological specificity that describes basically identical, tentatively judge that bacterial strain HMB27684 belongs to bacillus (Bacillus).
(2) 16SrDNA Sequence Identification is utilized to classify
With the genomic dna of HMB27684 for template, with F27 and R1492 for primer pair 16SrDNA carries out pcr amplification, described primer sequence is:
F27：5’AGAGTTTGATCATGGCTCAG3’；(SEQIDNo:1)
R1492：5’GGCTACCTTGTTACGACTT3’；(SEQIDNo:2)
The amplification reaction system of 16SrDNA is 50 μ L:10 × PCRBuffer (Mg
2+) 5 μ L; DNTPMixture (2.5mM) 5 μ L; Taq (5U/ μ L) 1 μ L, F27 (10 μm of ol/L) 1 μ L, R1492 (10 μm of ol/L) 1 μ L; The genomic dna 50ng of HMB27684; ddH
2o complements to 50 μ L.The reaction conditions of PCR is 95 DEG C of 5min; 95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1.5min, 30 circulations; 72 DEG C of 10min.Gained pcr amplification product is carried out gel electrophoresis, delivers the order-checking of Shanghai Sheng Gong biotechnology company limited, obtain the 16SrDNA sequence (see SEQIDNo:3) of HMB27684.The 16SrDNA sequence of gained HMB27684 is carried out tetraploid rice in Genbank, and the 16SrDNA homology of results strain HMB27684 and bacillus reaches 99%; Phylogenetic tree construction (see Fig. 1), together with HMB27684 is aggregated to bacillus, illustrates that HMB27684 belongs to bacillus (Bacillus).
(3) gyrB gene order is utilized to identify classification
With HMB27684 genomic dna for template, utilize genus bacillus gyrB gene degenerated primer gyrB-F and gyrB-R to carry out pcr amplification for primer, obtain pcr amplification product; The sequence of wherein said gyrB-F and gyrB-R primer is:
gyrB-F：5’TTGRCGGHRGYGGHTATAAAGT3’；(SEQIDNo:4)
gyrB-R：5’TCCDCCSTCAGARTCWCCCTC3’；(SEQIDNo:5)
The pcr amplification reaction system of gyrB is 50 μ L:10 × PCRBuffer (Mg
2+) 5 μ L; DNTPMixture (2.5mM) 5 μ L; Taq (5U/ μ L) 1 μ L; GyrB-F (10 μm of ol/L) 1 μ L, gyrB-R (10 μm of ol/L) 1 μ L; HMB27684 genomic dna 50ng; ddH
2o complements to 50 μ L.The reaction conditions of PCR is 95 DEG C of 5min; 95 DEG C of 30s, 55 DEG C of 45s, 72 DEG C of 1min, 30 circulations; 72 DEG C of 10min.Amplified production is delivered the order-checking of Shanghai Sheng Gong biotechnology company limited, obtain the gyrB gene order (see SEQIDNo:6) of HMB27684 bacterial strain.The gyrB gene order of the HMB27684 bacterial strain of acquisition is carried out tetraploid rice in Genbank, utilizes MEGA software (MolecularEvolutionaryGeneticsAnalysis, Molecular Evolutionary Genetics analysis) phylogenetic tree construction simultaneously.Found that the gyrB gene homology of HMB27684 and bacillus amyloliquefaciens is the highest; Phylogenetic tree construction (see Fig. 2), together with HMB27684 bacterial strain is aggregated to bacillus amyloliquefaciens, illustrates that HMB27684 is bacillus amyloliquefaciens (Bacillusamyloliquefaciens), and is a new strains.
The result of comprehensive above morphological specificity, the comparative analysis of 16SrDNA and gyrB gene homology, known HMB27684 belongs to bacillus amyloliquefaciens (Bacillusamyloliquefaciens), and different with existing Bacillus amyloliquefaciens strain, be a new strains.
The authentication method of above-mentioned Bacillus amyloliquefaciens strain HMB27684, comprise with the genomic dna of test strains for template, with F27 and R1492 for primer pair carries out pcr amplification, if gained pcr amplification product is the nucleotide sequence shown in SEQIDNo:3, be HMB27684 bacterial strain.
The authentication method of above-mentioned Bacillus amyloliquefaciens strain HMB27684, comprise with the genomic dna of test strains for template, with gyrB-F and gyrB-R for primer carries out pcr amplification, if gained pcr amplification product is the nucleotide sequence shown in SEQIDNo:6, be HMB27684 bacterial strain
The authentication method of mentioned microorganism microbial inoculum, comprise with the genomic dna extracted in microbial inoculum to be measured for template, with F27 and R1492 for primer pair carries out pcr amplification, if gained pcr amplification product is the nucleotide sequence shown in SEQIDNo:3, be HMB27684 microbiobacterial agent.Or with the genomic dna extracted in microbial inoculum to be measured for template, with gyrB-F and gyrB-R for primer carries out pcr amplification, if gained pcr amplification product is the nucleotide sequence shown in SEQIDNo:6, be HMB27684 microbiobacterial agent.
The advantage that the present invention has and beneficial effect: (1) the present invention provides an efficient microorganism for verticillium such as crop such as control cotton, eggplant etc., opens an effective controlling way; (2) drug effect of bacillus amyloliquefaciens HMB27684 of the present invention to cotton, eggplant verticillium wilt is high, and average preventive effect is more than 70.0%, and with strong points; (3) microbiobacterial agent of the present invention is to people, animal safety, does not have environmental pollution; (4) utilize the inventive method to prevent and treat cotton, eggplant verticillium wilt not easily to develop immunity to drugs; (5) preparation method of the present invention is simple, cost is low, use is simple.
Accompanying drawing explanation
Fig. 1 is the phylogeny tree graph of the HMB27684 bacterial strain according to the acquisition of 16SrDNA sequence.
Fig. 2 is the phylogeny tree graph of the HMB27684 bacterial strain according to the acquisition of gyrB gene order.
Embodiment
Come clearly to explain the present invention further with specific embodiment below, but be construed as limiting the invention never in any form.Experimental technique in following embodiment, if no special instructions, is ordinary method; Percentage composition in following embodiment, if no special instructions, is weight percentage.
The preparation of embodiment 1HMB27684 microbiobacterial agent
Carry out in accordance with the following steps:
(1) actication of culture: by be stored in-80 DEG C Bacillus amyloliquefaciens strain HMB27684 (this bacterial strain oneself is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on September 29th, 2015, deposit number is CGMCCNo.11456) at LB plate culture medium, at LB plate culture medium, (its moiety and weight ratio thereof are: Tryptones 10g, yeast extract 5g, sodium-chlor 5g, agar powder 15g, water 1000mL) on activate at 30 DEG C, at LB slant medium, (its moiety and weight ratio thereof are picking list bacterium colony: Tryptones 10g, yeast extract 5g, sodium-chlor 5g, agar powder 15g, water 1000mL) at 30 DEG C cultivate 12 hours, the bacterial strain that must activate,
(2) preparation of seed liquor: (its moiety and weight ratio thereof are: Tryptones 10g to make LB liquid nutrient medium according to a conventional method, yeast extract 5g, sodium-chlor 5g, water 1000mL), in 250mL triangular flask, load LB nutrient solution 100mL, high pressure moist heat sterilization, drop to after room temperature until temperature, access the bacterial strain of activation in a transfering loop step (1) in every bottle, 30 DEG C, carry out shaking culture 12 hours under the condition of shaking speed 190rpm, obtain seed liquor;
(3) preparation of Semen Maydis powder soybean powder medium: according to weight percent by Semen Maydis powder 1.5%, analysis for soybean powder 2.0%, NaCl0.5%, MnSO
4h
2o0.6% is added to the water, and is uniformly mixed, and obtains Semen Maydis powder soybean powder medium; Be sub-packed in 500mL triangular flask, every bottle of 200mL; At 121 DEG C, sterilizing is carried out 30 minutes to Semen Maydis powder soybean powder medium, then cool to 30 DEG C for subsequent use;
(4) fermentation culture: inoculation step (2) gained seed liquor 2mL in step (3) gained every bottle Semen Maydis powder soybean powder medium 200mL; 30 DEG C, carry out fermentation culture 36h under shaking speed 180rpm condition, sampled from triangular flask every 30 minutes later and carry out microscopy, brood cell in the visual field and total thalline number are counted, and calculates brood cell and lead (brood cell leads (%)=ripe brood cell's number/(ripe brood cell's number+thalline number) × 100); Brood cell leads when reaching 90% and stops fermentation culture; Fermentation culture 48h, obtains the liquid preparation of bacillus amyloliquefaciens HMB27684 altogether.
Embodiment 2 bacillus amyloliquefaciens HMB27684 is to the restraining effect of Verticillium Dahliae spore germination
(1) test period and place: in June, 2013 carries out in biological and ecological methods to prevent plant disease, pests, and erosion laboratory.
(2) test method:
(1) for examination Verticillium Dahliae source: Verticillium Dahliae WX-1 bacterial strain picks up from Xingtai City Wei County, Hebei province cotton verticillium wilt diseased plant; through Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie's separation and purification; department of plant pathology of Plant Protection College, Hebei Agricultural Univ. is accredited as Garden Dahlia Verticillium (Verticilliumdahaliae), and Pathogenic Tests shows as High pathogenicity.
(2) dull and stereotyped determination test:
First Verticillium Dahliae WX-1 is activated on PDA flat board, cultivate after 7 days at colony edge region picking 4-5 ferfas silk block (irregular shape, about 15mm
2), in access PDB nutrient solution, cultivate 4 days (25 DEG C, 170 revs/min) at constant-temperature table.Liquid culture is used double-layer sterile filtered through gauze in aseptic operating platform, obtains Verticillium Dahliae spore suspension (10
8individual spore/milliliter).2mL Verticillium Dahliae spore suspension is added in 100mLPDA substratum (45-50 DEG C), make PDA after mixing dull and stereotyped.On PDA flat board, genus bacillus HMB27684 is surveyed in some reception, cultivate 3 days afterwards observation inhibition zone transparency (+++ represent completely transparent; ++ represent translucent; + represent opaque) and measure inhibition zone size.
Result (see table 1) bacillus amyloliquefaciens HMB276384 of the present invention has strongly inhibited effect to Verticillium Dahliae spore germination, produces bright inhibition zone, antibacterial circle diameter 1.8mm.Illustrate that bacillus amyloliquefaciens HMB27684 has significant restraining effect to Verticillium Dahliae, there is the Biocontrol Potential of control cotton verticillium wilt.
Table 1HMB27684 bacterial strain is to the restraining effect test-results of Verticillium Dahliae spore germination
| Strain name | Transparent circle brightness | Transparent circle diameter (mm) |
| HMB27684 | +++ | 1.8 |
Embodiment 3 bacillus amyloliquefaciens HMB27684 tests the restraining effect of Verticillium Dahliae mycelial growth
(1) test period and place: in July, 2013 carries out in biological control laboratory.
(2) test method:
(1) for examination Verticillium Dahliae source: Verticillium Dahliae WX-1 bacterial strain picks up from Xingtai City Wei County, Hebei province cotton verticillium wilt diseased plant; through Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie's separation and purification; department of plant pathology of Plant Protection College, Hebei Agricultural Univ. is accredited as Garden Dahlia Verticillium (Verticilliumdahaliae), and Pathogenic Tests shows as High pathogenicity.
(2) dull and stereotyped determination test:
First Verticillium Dahliae WX-1 is activated on PDA flat board, cultivates in colony edge region after 7 days, with punch tool (
) punch and make bacterium sheet, by the switching of Verticillium Dahliae bacterium sheet in the dull and stereotyped central authorities of another PDA, again the bacillus amyloliquefaciens HMB27684 point that embodiment 1 step (1) activates is connected on apart from indicator bacterium sheet 2.0 centimeters, if blank (not putting the Verticillium Dahliae growing state connecing HMB27684 bacterial strain).25 DEG C of constant temperature culture, when blank is about to cover with whole culture dish, measure contrast increment (colony radius) and the process increment (the Developing restraint radius after inoculation HMB27684) of Verticillium Dahliae, antagonistic action bacteriostasis rate represents.Calculation formula:
Bacteriostasis rate (%)=(contrast increment-process increment)/contrast increment × 100.
The inhibiting rate of result (see table 2) bacillus amyloliquefaciens HMB27684 of the present invention to Verticillium Dahliae reaches 85.00%; Transparent antibacterial bandwidth 11.0 millimeters; Illustrate that bacillus amyloliquefaciens HMB27684 has obvious restraining effect to Verticillium Dahliae, there is the Biocontrol Potential of control cotton verticillium wilt.
Table 2HMB27684 bacterial strain is to the antagonism test result of Verticillium Dahliae
| Strain name | Contrast increment (mm) | Process increment (mm) | Bacteriostasis rate (%) |
| HMB27684 | 30.0 | 4.5 | 85.00 |
Embodiment 4 bacillus amyloliquefaciens HMB27684 tests the prevention effect of cotton verticillium wilt
(1) test process:
(1) microbiobacterial agent: HMB27684 liquid preparation prepared by embodiment 1; Dilute with water 100 times.
(2) medicament contrast: 1,000,000,000 brood cell/gram subtilis wettable powders (Baoding Ke Lvfeng biochemical technology company limited) alive; Dilute with water 100 times.
(3) blank: clear water
(2) test method: this test is carried out in Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie's disease flocking biocontrol laboratory.Cotton variety selects Jifeng 106, and HMB27684 liquid preparation 100 times of water diluents that before sowing prepared by Application Example 1 are soaked seed half an hour; Dilute 100 times of seed soaking using " 1,000,000,000 brood cell/gram subtilis wettable powder alive " to contrast as medicament half an hour; Using Seed soaking half an hour as blank.After planting normally cultivate.When cotton seedling grows to two panels true leaf, cut the spore suspension (107 spore/milliliters) of root inoculation Verticillium Dahliae WX-1 bacterial strain.Continue to be cultured to when blank is fully fallen ill and investigate disease index, calculate prevention effect.
Table 3 bacillus amyloliquefaciens HMB27684 is to cotton verticillium wilt efficiency test result
| Process | Disease index | Preventive effect (%) |
| HMB27684 | 21.39b | 74.30 |
| 1000000000 brood cell/gram subtilis wettable powders alive | 23.69b | 71.54 |
| Blank | 83.23a | -- |
Result (see table 3) bacillus amyloliquefaciens of the present invention HMB27684 has good prevention effect to cotton verticillium wilt, preventive effect can reach 74.30%, and the effect (71.54%) of preventing and treating cotton verticillium wilt with the microbial bactericide of formally registering is suitable.Illustrate that bacillus amyloliquefaciens HMB27684 of the present invention and microbiobacterial agent thereof have good prevention effect to cotton verticillium wilt.
Embodiment 5 bacillus amyloliquefaciens HMB27684 tests the prevention effect of cotton verticillium wilt
(1) test process:
(1) microbiobacterial agent: HMB27684 liquid preparation prepared by embodiment 1; Calcium carbonate is added, preparation HMB27684 pulvis according to 1:1 Billy.
(2) medicament contrast: 1,000,000,000 brood cell/gram subtilis wettable powders (Baoding Ke Lvfeng biochemical technology company limited) alive.
(3) blank: clear water
(2) test method: this test is carried out in biological and ecological methods to prevent plant disease, pests, and erosion laboratory.HMB27634 liquid preparation embodiment 1 prepared adds calcium carbonate according to 1:1 Billy, preparation HMB27634 pulvis.Cotton variety selects Jifeng 106, and before sowing, the HMB27634 pulvis of application preparation is dressed seed according to pesticide-seeds ratio 1:10; Contrast as medicament using " 1,000,000,000 brood cell/gram subtilis wettable powder alive " according to pesticide-seeds ratio 1:10 seed dressing; Not process seed as blank.After planting normally cultivate.When cotton seedling grows to two leaf one new film true leaves, cut the spore suspension (10 of root inoculation Verticillium Dahliae WX-1 bacterial strain
7individual spore/milliliter).Continue to be cultured to when blank is fully fallen ill and investigate disease index, calculate prevention effect.
Result (see table 4) bacillus amyloliquefaciens of the present invention HMB27684 has good prevention effect to cotton verticillium wilt, preventive effect can reach 68.30%, and the effect (69.67%) of preventing and treating cotton verticillium wilt with the microbial bactericide of formally registering is suitable.Illustrate that bacillus amyloliquefaciens HMB27684 of the present invention and microbiobacterial agent thereof have good prevention effect to cotton verticillium wilt.
Table 4 bacillus amyloliquefaciens HMB27684 is to cotton verticillium wilt efficiency test result
| Process | Disease index | Preventive effect (%) |
| HMB27684 | 20.13b | 68.30 |
| 1000000000 brood cell/gram subtilis wettable powders alive | 19.26b | 69.67 |
| Blank | 63.51a | -- |
Embodiment 6 bacillus amyloliquefaciens HMB27684 tests the prevention effect of eggplant verticillium wilt
(1) test process:
(1) microbiobacterial agent: HMB27684 liquid preparation prepared by embodiment 1; Dilute with water 100 times.
(2) medicament contrast: 1,000,000,000 brood cell/gram subtilis wettable powders (Baoding Ke Lvfeng biochemical technology company limited) alive; Dilute with water 100 times.
(3) blank: clear water
(2) test method:
(1) to wither bacterium source for examination eggplant Huang: the eggplant Huang bacterium RY-1 bacterial strain that withers picks up from Hengshui City Raoyang County, Hebei province eggplant verticillium wilt diseased plant; through Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie's separation and purification; department of plant pathology of Plant Protection College, Hebei Agricultural Univ. is accredited as Garden Dahlia Verticillium (Verticilliumdahaliae), and Pathogenic Tests shows as High pathogenicity.
(2) pot experiment: this test is carried out in biological and ecological methods to prevent plant disease, pests, and erosion laboratory.Eggplant Varieties selects the black long eggplant of Han Yu, and HMB27684 liquid preparation 100 times of water diluents that before sowing prepared by Application Example 1 are soaked seed half an hour; Dilute 100 times of seed soaking using " 1,000,000,000 brood cell/gram subtilis wettable powder alive " to contrast as medicament half an hour; Using Seed soaking half an hour as blank.After planting normally cultivate.When cotton seedling grows the 5th true leaf, cut the spore suspension (10 of the yellow bacterium RY-1 bacterial strain that withers of root inoculation eggplant
7individual spore/milliliter).Continue to be cultured to when blank is fully fallen ill and investigate disease index, calculate prevention effect.
Result (see table 5) bacillus amyloliquefaciens HMB27684 has good prevention effect to eggplant verticillium wilt, and preventive effect can reach 72.23%, is better than the effect (68.08%) of the microbial bactericide control eggplant verticillium wilt of formal registration.Illustrate that bacillus amyloliquefaciens HMB27684 of the present invention and microbiobacterial agent thereof have good prevention effect to eggplant verticillium wilt.
Table 5 bacillus amyloliquefaciens HMB27684 is to eggplant verticillium wilt efficiency test result
| Process | Disease index | Preventive effect (%) |
| HMB27684 | 16.88b | 72.23 |
| 1000000000 brood cell/gram subtilis wettable powders alive | 19.38b | 68.08 |
| Blank | 60.83a | -- |
Embodiment 7 bacillus amyloliquefaciens HMB27684 tests the prevention effect of eggplant verticillium wilt
(1) test process:
(1) microbiobacterial agent: HMB27684 liquid preparation prepared by embodiment 1; Calcium carbonate is added, preparation HMB27684 pulvis according to 1:1 Billy.
(2) medicament contrast: 1,000,000,000 brood cell/gram subtilis wettable powders (Baoding Ke Lvfeng biochemical technology company limited) alive.
(3) blank: untreated
(2) test method:
(1) to wither bacterium source for examination eggplant Huang: the eggplant Huang bacterium RY-1 bacterial strain that withers picks up from Hengshui City Raoyang County, Hebei province eggplant verticillium wilt diseased plant; through Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie's separation and purification; department of plant pathology of Plant Protection College, Hebei Agricultural Univ. is accredited as Garden Dahlia Verticillium (Verticilliumdahaliae), and Pathogenic Tests shows as High pathogenicity.
(2) pot experiment: this test is carried out in biological and ecological methods to prevent plant disease, pests, and erosion laboratory.HMB27634 liquid preparation embodiment 1 prepared adds calcium carbonate according to 1:1 Billy, preparation HMB27634 pulvis.Eggplant Varieties selects the black long eggplant of Han Yu.Select and rich support substrate seedling soil, high pressure steam sterilization 2 hours, to be paved with the seedling pan of nursery soil as chassis, every cave field planting 1 strain eggplant seedling in seedling pan, for subsequent use when being cultured to 3-4 sheet true leaf.Bacterium inoculated by hypha block of being withered by eggplant Huang, in PDB nutrient solution, at 28 DEG C, is cultivated 4 days for 170 revs/min, is filtered mycelia and obtain conidium, access in aseptic seedling medium, mixing, make the bacterium conidium content that withers of Huang in soil reach 10
6individual/gram soil.Band soil bacteria is loaded the flowerpot of diameter 15 centimetres, in flowerpot, the position of field planting eggplant seedling applies 1.5 grams, the HMB27634 pulvis of preparation, field planting one strain eggplant seedling in every basin.During transplanting, picking-up taper seedling pan makes the eggplant shoot root tip break and is beneficial to pathogen infection.Then cover tape soil bacteria.With 1,000,000,000 brood cells that spread manuer in holes/gram subtilis wettable powder 1.5 grams for medicament contrast, directly transplant eggplant seedling as blank using medicament of not spreading manuer in holes.After transplanting, normal cultivation, investigates disease index when fully falling ill to blank, calculates prevention effect.
Result (see table 6) bacillus amyloliquefaciens HMB27684 has good prevention effect to eggplant verticillium wilt, and preventive effect can reach 75.02%, and the effect (78.33%) of preventing and treating eggplant verticillium wilt with the microbial bactericide of formally registering is suitable.Illustrate that bacillus amyloliquefaciens HMB27684 of the present invention and microbiobacterial agent thereof have good prevention effect to eggplant verticillium wilt.
Table 6 bacillus amyloliquefaciens HMB27684 is to eggplant verticillium wilt efficiency test result
| Process | Disease index | Preventive effect (%) |
| HMB27684 | 18.3b | 75.02 |
| 1000000000 brood cell/gram subtilis wettable powders alive | 15.8b | 78.33 |
| Blank | 73.3a | --- |
Claims (10)
1. bacillus amyloliquefaciens (Bacillusamyloliquefaciens) bacterial strain HMB27684, oneself is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on September 29th, 2015, and deposit number is CGMCCNo.11456.
2. the microbiobacterial agent utilizing the bacillus amyloliquefaciens HMB27684 described in claim 1 to produce, is characterized in that its activeconstituents is bacillus amyloliquefaciens HMB27684 thalline.
3., according to microbiobacterial agent according to claim 2, it is characterized in that for liquid preparation or pulvis.
4. the preparation method of microbiobacterial agent according to claim 2, is characterized in that comprising the steps:
(1) activated on LB plate culture medium by the HMB27684 bacterial strain of cryopreservation, picking list bacterium colony, on LB slant medium, is cultivated 10 ~ 16 hours, is obtained the bacterial strain of activation at 25 ~ 35 DEG C;
(2) inoculation activated with aseptic transfering loop scraping one ring step (1) in 100mLLB liquid nutrient medium, 25 ~ 35 DEG C, shaking speed cultivates 10 ~ 16 hours under being the condition of 150 ~ 220rpm, obtains seed liquor;
(3) according to volume ratio be 1 ~ 3% ratio the seed liquor of step (2) is linked in Semen Maydis powder soybean powder medium, temperature be 25 ~ 35 DEG C, rotating speed be 150 ~ 220rpm condition bottom fermentation cultivate 40 ~ 50h, obtain fermented liquid;
(4) detect thalline and brood cell's quantity in fermented liquid, when ripe brood cell accounts for 90% of brood cell and thalline sum in fermented liquid, stop fermentation culture; Gained is the liquid preparation of HMB27684 bacterial strain.
5. according to preparation method according to claim 4, it is characterized in that moiety and the weight ratio thereof of the LB plate culture medium described in its step (1) or LB slant medium are: Tryptones 8 ~ 12g, yeast extract 4 ~ 6g, sodium-chlor 4 ~ 6g, agar powder 12 ~ 18g, water 1000mL; Moiety and the weight ratio thereof of the LB liquid nutrient medium described in its step (2) are: Tryptones 8 ~ 12g, yeast extract 4 ~ 6g, sodium-chlor 4 ~ 6g, water 1000mL.
6. according to preparation method according to claim 4, it is characterized in that moiety and the weight percent thereof of the described Semen Maydis powder soybean powder medium described in its step (3) are: Semen Maydis powder 1.0 ~ 3.0%, analysis for soybean powder 1.0 ~ 3.0%, NaCl0.1 ~ 1.0%, MnSO
4h
2o0.5 ~ 1.0%, all the other are water; PH value is 7.0.
7. the application of bacillus amyloliquefaciens HMB27684 according to claim 1 in control crop verticillium; Described crop refers to cotton or eggplant.
8. the application of microbiobacterial agent according to claim 2 in control crop verticillium; Described crop refers to cotton or eggplant.
9. the authentication method of Bacillus amyloliquefaciens strain HMB27684 according to claim 1, it is characterized in that with the genomic dna of test strains for template, with F27 and R1492 for primer pair carries out pcr amplification, if gained pcr amplification product is the nucleotide sequence shown in SEQIDNo:3, be HMB27684 bacterial strain.
10. the authentication method of Bacillus amyloliquefaciens strain HMB27684 according to claim 1, it is characterized in that with the genomic dna of test strains for template, with gyrB-F and gyrB-R for primer carries out pcr amplification, if gained pcr amplification product is the nucleotide sequence shown in SEQIDNo:6, be HMB27684 bacterial strain.
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| CN115960788A (en) * | 2022-12-31 | 2023-04-14 | 石河子大学 | Special microbial inoculum for preventing and treating cotton verticillium wilt |
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Effective date of registration: 20200416 Address after: No. 302, unit 2, building 2, No. 40, Xianfeng street, Xinshi District, Baoding City, Hebei Province 071000 Patentee after: Qi Yongxin Address before: 071000 No. 118 Fuxing West Road, Hebei, Baoding Patentee before: BAODING WEIKONG BIOTECHNOLOGY Co.,Ltd. |