CN101948771A - Bacillus amyloliquefaciens growing in disease-preventing and growth-promoting plant and application thereof - Google Patents
Bacillus amyloliquefaciens growing in disease-preventing and growth-promoting plant and application thereof Download PDFInfo
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Abstract
The invention relates to a bacillus amyloliquefaciens growing in a disease-preventing and growth-promoting plant and application thereof, relating to bacillus amyloliquefaciens and the application thereof. The bacillus amyloliquefaciens TF28 growing in the disease-preventing and growth-promoting plant belongs to bacillus, has the storage number of CGMCC No.4038 and the storage data of July 26, 2010. The application of the bacillus amyloliquefaciens TF28 comprises the following steps of: (1) preparing activated bacilli liquid; (2) inoculating the activated bacilli liquid in an NYD culture fluid to obtain a strain fermentation solution; and (3) soaking seeds in a dilution solution 50 times of the strain fermentation solution. The strain TF28 in the invention can be colonized, reproduced and transmitted in plants, can simultaneously generate two antibacterial substances, i.e. antimicrobial proteins and lipopeptide antibiotic and has the multiple efficacy of preventing diseases, stimulating growth, increasing the yield, improving the quality, widening an antibacterial spectrum, and the like. Rice seeds treated by the dilution solution 50 times of the fermentation solution of the strain TF28 reach a room-temperature bakana resistance rate over 84.6 percent.
Description
Technical field
The present invention relates to bacillus amyloliquefaciens and application thereof.
Background technology
Plant diseases is one of principal element that influences agricultural year, stable yields and high-quality production, due to illness the loss that does harm on the annual agricultural and cause accounts for the gross output value about 20%, the direct economic loss that annual due to illness evil causes is up to more than 5,000,000,000, all be to use chemical pesticide to prevent and treat for a long time, but chemical pesticide exist many drawbacks that can't overcome such as resistance, contaminate environment, pesticide residue, species diversity reduction, toxicity to man and livestock always.
Biological pesticide is because it is little to the person poultry toxicity, and environment compatibility is good, and sick worm is difficult for producing advantage such as resistance and meets the requirement of modern society to agriculture production and agricultural chemicals, has wide development space.Most bacterial strains of producing biological pesticide are that separation screening obtains from the plant rhizosphere soil microorganism, because these soil biocontrol microorganisms are subject to the influence of external environmental condition, be difficult in the competition occupy microorganism growing surely for a long time and preponderating enclosing to practise with soil, plant rhizosphere or leaf, protection effect is undesirable, thereby has greatly influenced their actual protection effect.
Endophytic bacterium is the resource microorganism that a class secondary metabolite enriches, has a extensive future, have in plant materials grow surely fast, conduction and breeding success, many endogenetic bacterias have biological functions such as fixed nitrogen, short life, drought resisting, diseases prevention, endogenetic bacteria is as potential biological and ecological methods to prevent plant disease, pests, and erosion resource, can remedy the unsettled defective of biocontrol microorganisms field efficacy in soil source, be the novel biocontrol strain of tool development potentiality.
Summary of the invention
The invention provides disease prevention growth-promoting plant endogenesis bacillus amyloliquefaciens and application thereof.
Disease prevention growth-promoting plant endogenesis bacillus amyloliquefaciens, it is disease prevention growth-promoting plant endogenesis bacillus amyloliquefaciens TF28, be bacillus amyloliquefaciens Bacillus amyloliquefaciens, belong to bacillus (Bacillus), in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, deposit number is CGMCC No.4038, and preservation date is on July 26th, 2010; Disease prevention growth-promoting plant endogenesis bacillus amyloliquefaciens TF28 is the Gram-positive aerobic bacteria, and for shaft-like, length is 1.5 μ m～2.5 μ m, and wide is 0.5 μ m～1.5 μ m, endogenous spore, atrichia; On beef-protein medium, form the oyster white bacterium colony, gauffer, the edge is irregular, and the bacterium colony umbo is carinate.
The disease prevention growth-promoting plant endogenesis bacillus amyloliquefaciens TF28 glucose fermentation sample positive, the starch hydrolysis experiment positive, the gelatin liquification test positive, V.P reacting positive, catalase reacting positive, the clark and Lubsreaction positive, the nitrate reduction test positive, the Citrate trianion utilization positive, good health length, growth pH value are 6.5～8.5 among the present invention, and growth temperature is 30～37 ℃.
Disease prevention growth-promoting plant endogenesis bacillus amyloliquefaciens among the present invention, bacterial strain number is TF28, be bacillus amyloliquefaciens Bacillus amyloliquefaciens, belong to bacillus (Bacillus), in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, deposit number is CGMCC No.4038, and preservation date is on July 26th, 2010.
The application of disease prevention growth-promoting plant endogenesis bacillus amyloliquefaciens: the bacterium liquid of one, getting the disease prevention growth-promoting plant endogenesis bacillus amyloliquefaciens TF28 of-70 ℃ of freezing preservations, streak inoculation is on the LB solid medium, cultivate 18～20h in 30 ℃ of thermostat containers, picking list colony inoculation is in the LB of 5ml liquid nutrient medium, 18～20h is cultivated in 30 ℃ of concussions, obtains activating bacterium liquid; Two: will activate bacterium liquid by 2%～5% inoculum size and be inoculated in the 500ml triangular flask that 100ml NYD nutrient solution is housed, cultivate 32～50h for 30 ℃, rotating speed is 150～250r/min, the fermentation ends bacterial concentration reaches 20～4,000,000,000/ml, is disease prevention growth-promoting plant endogenesis bacillus amyloliquefaciens TF28 bacterial strain fermentation liquor; Three, seed water in advance to be prevented and treated is soaked 5～7d, is soaked in the diseases prevention that 2d in 50 times of diluents of disease prevention growth-promoting plant endogenesis bacillus amyloliquefaciens TF28 bacterial strain fermentation liquor promptly finishes seed again.
Disease prevention growth-promoting plant endogenesis bacillus amyloliquefaciens TF28 of the present invention, be from the isolating biocontrol strain that various plants pathogenic fungies such as root rot, rice bakanae disease are had higher preventive effect of soybean root, having in various crop root colonization abilities such as soybean, paddy rice, is the biocontrol strain that a strain has development and application values.
Bacterial strain TF28 can grow, breed and conduct in that plant materials is decided at the higher level but not officially announced among the present invention, and this bacterial strain can produce antibacterial protein and two kinds of antimicrobial substances of lipopeptide antibiotic simultaneously, has multi-efficiencies such as disease prevention growth-promoting, increasing yield and improving quality, antimicrobial spectrum be wide;
In the application of disease prevention growth-promoting plant endogenesis bacillus amyloliquefaciens of the present invention, the preparation low production cost, simple to operate, easy to implement, can produce in batches and be applicable; The microbial inoculum of bacterial strain TF28 helps the ecotope that protects the fields, and reduces pesticide residue, is suitable for the requirement that China's non-polluted farm product is produced; Adopt 50 times of diluent Rice seeds treated of bacterial strain TF28 fermented liquid, rice bakanae disease greenhouse preventive effect is reached more than 84.6%, simultaneously, three kinds of pathogen of soybean root rot (sickle-like bacteria root rot, rotten mould root rot and phytophthora root rot), cucumber fusarium axysporum, muskmelon wilt, withered germ of water-melon, botrytis cinerea, capsicum early blight bacterium, soybean sclerotinia crown rot bacterium, cucumber rhizoctonia rot bacterium, fusarium moniliforme, rice blast fungus all had strong restraining effect.
Embodiment
Embodiment one: present embodiment disease prevention growth-promoting plant endogenesis bacillus amyloliquefaciens, it is disease prevention growth-promoting plant endogenesis bacillus amyloliquefaciens TF28, be bacillus amyloliquefaciens Bacillusamyloliquefaciens, belong to bacillus (Bacillus), in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, deposit number is CGMCC No.4038, and preservation date is on July 26th, 2010; Disease prevention growth-promoting plant endogenesis bacillus amyloliquefaciens TF28 is the Gram-positive aerobic bacteria, and for shaft-like, length is 1.5 μ m～2.5 μ m, and wide is 0.5 μ m～1.5 μ m, endogenous spore, atrichia; On beef-protein medium, form the oyster white bacterium colony, gauffer, the edge is irregular, and the bacterium colony umbo is carinate.
Disease prevention growth-promoting plant endogenesis bacillus amyloliquefaciens is according to " the outstanding Bacteria Identification handbook preliminary judgement of uncle is a bacillus, is accredited as bacillus (Bacillus) through the SHERLOCK microflora then in the present embodiment.
Disease prevention growth-promoting plant endogenesis bacillus amyloliquefaciens TF28 antimicrobial spectrum: adopt the face-off cultural method to determine the bacterial strain antimicrobial spectrum, there are 12 kinds for the examination pathogenic bacteria, be F.oxysporum soybean (causing the fusarium oxysporum bacterium of root rot), Rhizoctonia solani soybean (causing the dry thread Pyrenomycetes of root rot), Phythium spp. (causing the pythium spp of root rot), F.oxysporum cucumber (cucumber fusarium axysporum), F.oxysporum melonis (muskmelon wilt), Fusarium oxysporum niveum (withered germ of water-melon), Botrytis cinerea tomato (botrytis cinerea), Alternaria solani pepper (capsicum early blight bacterium), Sclerotinia sclerotiorum soybean (soybean sclerotinia crown rot bacterium), Fusariummoniliforme sheld (fusarium moniliforme), Pyricularia oryzae cay. (rice blast fungus) and R.solani cucumber (cucumber rhizoctonia rot bacterium), getting a ring activation bacterium liquid cross lines on the PDA flat board, cultivating 7d for 28 ℃ apart from inoculation line 3cm place inoculation pathogenic bacteria (bacterium piece diameter 5mm), measure antibacterial bandwidth, test-results is as shown in table 1, as seen bacterial strain TF28 all has strong restraining effect to above-mentioned pathogenic bacteria, and antibacterial bandwidth is 8.4mm～15.2mm.
Table 1
| Bacterial strain | Antibacterial bandwidth (mm) |
| F.oxysporum?soybean | 15.2±0.09 |
| Rhizoctonia?solani?soybean | 14.8±0.12 |
| Phythium?spp. | 13.2±0.04 |
| E?oxysporum?cucumber | 13.5±0.10 |
| F.oxysporum?tomato | 12.8±0.07 |
| F.oxysporum?niveum | 12.3±0.08 |
| Botrytis?cinerea?tomato | 9.8±0.07 |
| Alternaria?solani?pepper | 8.7±0.09 |
| Sclerotinia?sclerotiorum?soybean | 8.4±0.11 |
| Fusarium?moniliforme?sheld | 13.2±0.09 |
| Pyricularia?oryzae?cav. | 8.6±0.03 |
| R.solani?cucumber | 9.6±0.04 |
Determining of disease prevention growth-promoting plant endogenesis bacillus amyloliquefaciens TF28 antimicrobial substance:
1, determining of antibacterial protein: get the bacterium liquid of the bacterial strain TF28 that cultivates 48h, 4000r/mim is centrifugal, collects supernatant liquor, adds (the NH of 40% saturation ratio
4)
2SO
4, 4 ℃ are spent the night and place the precipitation antibacterial proteins, and 10000r/mim is centrifugal, collecting precipitation, with phosphoric acid buffer dissolving back dialysis desalination, after concentrating with PEG8000 ,-20 ℃ of freezing preservations in the aseptic plastic pipe of packing into; Get 10 μ l samples and adopt the inhibition situation of paper disk method mensuration to fusarium moniliforme, the result shows that bacterial strain TF28 can produce antibacterial protein, and this antibacterial protein is inhibited to rice bakanae disease;
2, determining of lipopeptide antibiotic: get the bacterium liquid of the bacterial strain TF28 that cultivates 48h, 4000r/mim is centrifugal, collects supernatant liquor, and adding concentrated hydrochloric acid to pH value is 4.0,4 ℃ of precipitations of spending the night, and 10000r/mim is centrifugal, collecting precipitation, the methyl alcohol lixiviate of dry back; Get 10 μ l samples and adopt the inhibition situation of paper disk method mensuration to fusarium moniliforme, the result shows that bacterial strain TF28 can produce lipopeptide antibiotic, and this lipopeptide antibiotic is inhibited to rice bakanae disease.
Disease prevention growth-promoting plant endogenesis bacillus amyloliquefaciens TF28 obtains from the soybean root separation in the present embodiment, and soybean is chosen from big No. 2 kinds in east, Heilongjiang Province, and its separation method is as follows:
1, soybean root being rinsed well with flowing water, got 1g as for the examination material, be cut into the 2mm segment, is that 70% alcohol and mass concentration are that 2% clorox soaks 10min and carries out surface sterilization with mass concentration successively, kills for trying the material surface microorganism;
2, in gnotobasis,, get last 1 the aseptic water washing liquid of 100 μ l and coat on the NYD solid medium and cultivate 48h with aseptic water washing 3 times for the examination material, observe and have or not bacterium colony to produce;
3, to adding the homogenate of 5ml sterilized water for the examination material, get 100 μ l and coat on the NYD solid medium, cultivate 48h for 30 ℃, the picking list bacterium colony purifying of ruling repeatedly on the NYD solid medium is till obtaining being sheerly bacterial strain;
4, will be sheerly inoculation in the NYD liquid nutrient medium, and cultivate 18h for 30 ℃, getting bacterium liquid 700 μ l, to add mass concentration be 50% glycerine 300 μ l, mixes being placed on freezing preservation in-70 ℃ of refrigerator-freezers;
Wherein step 2 purpose is whether checking can all kill for examination material surface microorganism, if there is bacterium colony to produce, then the testimonial material surface microorganism does not all kill, otherwise does not then have.
Embodiment two: what present embodiment and embodiment one were different is disease prevention growth-promoting plant endogenesis bacillus amyloliquefaciens, the glucose fermentation test positive, the starch hydrolysis experiment positive, the gelatin liquification test positive, V.P reacting positive, catalase reacting positive, the clark and Lubsreaction positive, the nitrate reduction test positive, the Citrate trianion utilization positive, good health length, growth pH value are 6.5～8.5, and growth temperature is 30～37 ℃.Other is identical with embodiment one.
Embodiment three: the application of present embodiment disease prevention growth-promoting plant endogenesis bacillus amyloliquefaciens: the bacterium liquid of one, getting the bacterial strain TF28 of-70 ℃ of freezing preservations, streak inoculation is on the LB solid medium, cultivate 18～20h in 30 ℃ of thermostat containers, picking list colony inoculation is in the LB of 5ml liquid nutrient medium, 18～20h is cultivated in 30 ℃ of concussions, must activate bacterium liquid; Two, will activate bacterium liquid by 2%～5% inoculum size and be inoculated in the 500ml triangular flask that 100ml NYD nutrient solution is housed, and cultivate 32～50h for 30 ℃, rotating speed is 150～250r/min, and the fermentation ends bacterial concentration reaches 20～4,000,000,000/ml, is bacterial strain fermentation liquor; Three, seed water in advance to be prevented and treated is soaked 5～7d, is soaked in that 2d promptly finishes in 50 times of diluents of bacterial strain fermentation liquor again.
Bacterial strain fermentation liquor is tested the prevention effect of rice bakanae disease in the present embodiment:
For the examination pathogenic bacteria: fusarium moniliforme, use the wheat culture medium culturing, treat that mycelia is covered with after, must the pathogenic bacteria powder after grinding, standby;
Reagent agent: 50 times of diluents of bacterial strain fermentation liquor, paddy rice chemical agent (25% Shi Baoke);
For studying thing: paddy rice; Kind: No. 9, loose round-grained rice;
Seed screening: screen full uniform seed;
Pathogenic bacteria inoculation: get 10g pathogenic bacteria powder, evenly be sprinkled into 50cm * 50cm seeding and growing seedling;
Experiment is handled: after earlier rice paddy seed being soaked 7d with clear water, pull out and carry out following processing again:
Handle 1: the seed that will be soaked in water soaks 2d with 50 times of diluents of bacterial strain fermentation liquor, pulls vernalization out, treats to be seeded in the pot for growing seedlings of inoculation germ 400 seeds of every alms bowl behind the just broken chest of seed;
Handle 2: blank, will directly be seeded in after the presprouting of seeds that be soaked in water in the pot for growing seedlings that does not connect germ, application rate is 400 seeds of every alms bowl;
Handle 3: the germ contrast, will directly be seeded in after the presprouting of seeds that be soaked in water in the pot for growing seedlings that connects germ, application rate is 400 seeds of every alms bowl;
Handle 4: the chemical agent contrast, the seed that was soaked in water is soaked 2d with 25% Shi Baoke, pull vernalization out, treat to be seeded in the pot for growing seedlings of inoculation germ 400 seeds of every alms bowl behind the just broken chest of seed;
Investigation: seed soaking " Invest, Then Investigate " rice seed germination rate, the " Invest, Then Investigate " seedling rate of emerging, a situation arises and growth traits in 3 leaf phases investigation bakanae disease; Experimental result is as shown in table 2, and as seen, 50 of the bacterial strain fermentation liquor of bacterial strain TF28 times of diluents are are effectively prevented and treated rice bakanae disease generation result in the present embodiment, and preventive effect is 84.6%, and is suitable with the chemical agent preventive effect;
Table 2
| Handle | Percentage of germination % | Seedling rate % | Sickness rate % | Preventive effect % |
| 1 | 90.2 | 96.5 | 6.7 | 84.6a |
| 2 | 89.8 | 94.5 | 5.8 | |
| 3 | 89.8. | 67.8 | 43.6 | |
| 4 | 89.3 | 93.4 | 5.2 | 88.1a |
Seedling rate is as shown in table 3, and from seedling rate, 50 of the bacterial strain fermentation liquor of bacterial strain TF28 times of diluent treatment group are the highest in the present embodiment, secondly is the contrast of blank and chemical agent, and the germ control group is minimum; Treatment group 1 and chemical agent treatment group 4 are wide at stem, all be better than blank 2 and germ contrast 3 on root length, radical, strain fresh weight and the wide index of leaf, and reach the significant difference level; Treatment group 1 every index is better than chemical agent treatment group 4, but both differences are not remarkable, as seen, 50 times of diluents with the bacterial strain fermentation liquor of bacterial strain TF28 in the present embodiment can be used to prevent and treat rice bakanae disease by the instead of chemical medicament as microbial inoculum, and this microbial inoculum has remarkable promoter action to paddy growth, can make cauline leaf broadening, root increases, and radical and strain fresh weight increase.
Table 3
| Handle | The long cm of strain | The wide cm of stem | The long cm of root | Radical (bar) | Strain fresh weight g | The wide cm of leaf |
| 1 | 22.3b | 0.25a | 5.3a | 18a | 0.25a | 0.42a |
| 2 | 25.8b | 0.15b | 4.8a | 14ab | 0.12b | 0.34ab |
| 3 | 32.5a | 0.09c | 3.2b | 9b | 0.08b | 0.14b |
| 4 | 20.3b | 0.21a | 4.8a | 14ab | 0.20a | 0.38a |
Embodiment four: present embodiment and embodiment three are different is that the every 1000mL of LB solid medium soaks the NaCl of the agar of powder, 20g, 10g by the yeast of the Tryptones of 10g, 5g and the water of surplus is formed in the step 1, and the pH value is 6.0～8.0.Other step and parameter are identical with embodiment three.
Embodiment five: present embodiment and embodiment four are different is that the every 1000mL of LB liquid nutrient medium soaks the NaCl of powder, 10g by the yeast of the Tryptones of 10g, 5g and the water of surplus is formed in the step 1, and the pH value is 6.0～8.0.Other step and parameter are identical with embodiment four.
Embodiment six: what present embodiment and embodiment five were different is that the every 1000mL of NYD nutrient solution is made up of the water of 8g extractum carnis, 5g yeast extract paste, 10g glucose and surplus in the step 2, and the pH value is 7.0～8.0.Other step and parameter are identical with embodiment five.
Claims (6)
1. disease prevention growth-promoting plant endogenesis bacillus amyloliquefaciens, it is characterized in that it is disease prevention growth-promoting plant endogenesis bacillus amyloliquefaciens TF28, be bacillus amyloliquefaciens Bacillus amyloliquefaciens, belong to bacillus (Bacillus), in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, deposit number is CGMCC No.4038, and preservation date is on July 26th, 2010; Disease prevention growth-promoting plant endogenesis bacillus amyloliquefaciens TF28 is the Gram-positive aerobic bacteria, and for shaft-like, length is 1.5 μ m～2.5 μ m, and wide is 0.5 μ m～1.5 μ m, endogenous spore, atrichia; On beef-protein medium, form the oyster white bacterium colony, gauffer, the edge is irregular, and the bacterium colony umbo is carinate.
2. disease prevention growth-promoting plant endogenesis bacillus amyloliquefaciens according to claim 1, it is characterized in that the disease prevention growth-promoting plant endogenesis bacillus amyloliquefaciens TF28 glucose fermentation test positive, the starch hydrolysis experiment positive, the gelatin liquification test positive, V.P reacting positive, catalase reacting positive, the clark and Lubsreaction positive, the nitrate reduction test positive, the Citrate trianion utilization positive, good health length, growth pH value are 6.5～8.5, growth temperature is 30～37 ℃.
3. the application of disease prevention growth-promoting plant endogenesis bacillus amyloliquefaciens described in claim 1, it is characterized in that the application of disease prevention growth-promoting plant endogenesis bacillus amyloliquefaciens: the bacterium liquid of one, getting the disease prevention growth-promoting plant endogenesis bacillus amyloliquefaciens TF28 of-70 ℃ of freezing preservations, streak inoculation is on the LB solid medium, cultivate 18～20h in 30 ℃ of thermostat containers, picking list colony inoculation is in the LB of 5ml liquid nutrient medium, 18～20h is cultivated in 30 ℃ of concussions, obtains activating bacterium liquid; Two: will activate bacterium liquid by 2%～5% inoculum size and be inoculated in the 500ml triangular flask that 100ml NYD nutrient solution is housed, cultivate 32～50h for 30 ℃, rotating speed is 150～250r/min, the fermentation ends bacterial concentration reaches 20～4,000,000,000/ml, is disease prevention growth-promoting plant endogenesis bacillus amyloliquefaciens TF28 bacterial strain fermentation liquor; Three, seed water in advance to be prevented and treated is soaked 5～7d, is soaked in the diseases prevention that 2d in 50 times of diluents of disease prevention growth-promoting plant endogenesis bacillus amyloliquefaciens TF28 bacterial strain fermentation liquor promptly finishes seed again.
4. the application of disease prevention growth-promoting plant endogenesis bacillus amyloliquefaciens according to claim 3, it is characterized in that the every 1000mL of LB solid medium in the step 1 soaks the NaCl of the agar of powder, 20g, 10g by the yeast of the Tryptones of 10g, 5g and the water of surplus is formed, the pH value is 7.0～8.0.
5. the application of disease prevention growth-promoting plant endogenesis bacillus amyloliquefaciens according to claim 4, it is characterized in that the every 1000mL of LB liquid nutrient medium in the step 1 soaks the NaCI of powder, 10g by the yeast of the Tryptones of 10g, 5g and the water of surplus is formed, the pH value is 7.0～8.0.
6. the application of disease prevention growth-promoting plant endogenesis bacillus amyloliquefaciens according to claim 5 is characterized in that the every 1000mL of NYD nutrient solution is made up of the water of 8g extractum carnis, 5g yeast extract paste, 10g glucose and surplus in the step 2, and the pH value is 7.0～8.0.
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