CN102787084A - Bacillus methylotrophicus 4-L-16 for prevention and control of banana vascular wilt and its application - Google Patents
Bacillus methylotrophicus 4-L-16 for prevention and control of banana vascular wilt and its application Download PDFInfo
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Abstract
The invention discloses a strain bacillus methylotrophicus 4-L-16 for prevention and control of banana vascular wilt. The strain has a preservation name of bacillus methylotrophicus 4-L-16, and is classified and named as Bacillus methylotrophicus 4-L-16. The preservation organization is China Center for Type Culture Collection, the preservation date is June 26, 2012, and the preservation number is CCTCC NO:M2012248. The strain has a strong antagonistic effect on No.1 and No.4 physiological races of banana vascular wilt, and other banana common disease categories. The invention also discloses application of the strain in preparation of drugs with a banana vascular wilt prevention and control function, as well as a microbiological preparation containing the strain. The microbiological preparation not only has a better antagonistic effect on a variety of banana pathogenic bacteria, but also has a facilitating effect on plant growth.
Description
Technical field
The present invention relates to a kind of bacterial strain and application thereof that prevents and treats banana blight, be specifically related to a kind of methylotrophy type Bacillus strain 4-L-16 and application thereof of preventing and controlling banana fusarium wilt.
Background technology
Banana blight is by sharp born of the same parents sickle-like bacteria Cuba specialized form (Fusarium oxysporium f.sp.cubense; FOC) the crushing soil-borne disease that causes of pathogenic fungi; From 1874 first since Australia finds; Once causing big area harm in 1896 in Panama, and reached gradually all over the world afterwards, is an insurmountable so far world technology difficult problem.At present; In banana main producing regions such as China Guangdong, Hainan, Guangxi, Yunnan and Fujian, banana blight is that No. 4 physiological strains by FOC cause, the dwarf banana blight is that No. 1 physiological strain by FOC causes; Morbidity any of several broadleaf plants garden sickness rate is generally at 30-50%; The grave illness district can reach more than 90%, causes the banana underproduction even total crop failure, is a main limiting factor on the banana production.
So far this banana blight still there are not ideal disease-resistant variety and chemical agent, therefore, in conjunction with cultivation step; Use the microorganism strains that this disease is had antagonistic effect; Thereby suppress soil banana blight quantity, change the microbial ecological structure of soil, promote the banana growth; Improve the banana broad spectrum resistance, become a kind of ideal bio-control method.
At present antagonism bacterium source is that separation and screening through soil, the mikrobe of plant own obtains, a little less than this method obtains antagonistic microbe and has the field planting ability usually, shortcoming such as breeding coefficient is low, antagonism bacterium antimicrobial spectrum is narrower.Particularly separating the antagonism bacterium obtain in the soil often can not a large amount of field planting in the plant body, and the prevention and control effect is not remarkable.Therefore adopt other approach to obtain to be easy to breeding, the wide antagonism bacterium of antibacterial range is particularly important.
Summary of the invention
First technical problem to be solved by this invention is to provide a kind of methylotrophy type Bacillus strain 4-L-16 of preventing and controlling banana fusarium wilt; This strains separation is in the fermented liquid of organic and brown sugar water; Be easy to the field planting breeding; Antibacterial range is extensive, and multiple pathogenic bacterias such as No. 1, No. 4 physiological strains of banana blight and other banana common disease kind such as the microscler leaf spot fungi of banana, Glorosprium musarum Cookeet Mass, the big greyness germ of banana, the mould leaf spot fungi of mango chain lattice spore, lichee anthrax-bacilus, mango anthrax-bacilus are all had stronger antagonistic action.
The methylotrophy type Bacillus strain 4-L-16 that second technical problem to be solved by this invention is to provide above-mentioned preventing and controlling banana fusarium wilt has the purposes in the medicine of preventing and controlling banana fusarium wilt effect in preparation.
The 3rd technical problem to be solved by this invention is to provide a kind of preparation of preventing and controlling banana fusarium wilt; Said preparation contains the methylotrophy type Bacillus strain 4-L-16 of above-mentioned preventing and controlling banana fusarium wilt; Said preparation has better antagonistic action to multiple pathogenic bacterias such as No. 1, No. 4 physiological strains of banana blight and other banana common disease kind such as the microscler leaf spot fungi of banana, Glorosprium musarum Cookeet Mass, the big greyness germ of banana, the mould leaf spot fungi of mango chain lattice spore, lichee anthrax-bacilus, mango anthrax-bacilus, also plant strain growth is had growth-promoting functions simultaneously.
The technical problem that the present invention will solve at last is the preparation method who is to provide above-mentioned preventing and controlling banana fusarium wilt preparation.
First technical problem to be solved of the present invention realizes through following technical scheme: a kind of methylotrophy type Bacillus strain 4-L-16 of preventing and controlling banana fusarium wilt; The preservation name of this bacterial strain is called methylotrophy type genus bacillus 4-L-16; Classification called after Bacillus methylotrophicus4-L-16; Depositary institution is Chinese typical culture collection center, and preservation date is on June 26th, 2012, and preserving number is CCTCC NO:M2012248.
The methylotrophy type Bacillus strain 4-L-16 of the preventing and controlling banana fusarium wilt among the present invention adopts dull and stereotyped dilution coating method to separate the mixed fermentation liquid from culture medium cake fertilizer, brown sugar and water; And through dull and stereotyped face-off culture method screening acquisition; Wherein cake fertilizer is preferably one or more in peanut cake, rapeseed cake and the soya-bean cake, and concrete separation screening process can be consulted the description in the hereinafter specific embodiment.
Second technical problem of the present invention realizes through following technical scheme: the methylotrophy type Bacillus strain 4-L-16 of above-mentioned preventing and controlling banana fusarium wilt has the purposes in the medicine of preventing and controlling banana fusarium wilt effect in preparation.
This medicine is not only to the dull and stereotyped antagonistic action of No. 1, No. 4 physiological strain of banana blight bacteria; Multiple pathogenic bacterias such as the microscler leaf spot fungi of banana, Glorosprium musarum Cookeet Mass, the big greyness germ of banana, the mould leaf spot fungi of mango chain lattice spore, lichee anthrax-bacilus, mango anthrax-bacilus also had an antagonistic action.
The 3rd technical problem of the present invention realizes through following technical scheme: a kind of preparation of preventing and controlling banana fusarium wilt, contain the methylotrophy type Bacillus strain 4-L-16 of above-mentioned preventing and controlling banana fusarium wilt.
As a kind of improvement of the present invention: the preparation of preventing and controlling banana fusarium wilt of the present invention, also contain culture medium cake fertilizer, brown sugar and water, wherein the gross weight of cake fertilizer and brown sugar and the gross weight of water are than being 1:4, the C/N ratio of culture medium is 15 ~ 25:1.
Described cake fertilizer is one or more in peanut cake, soya-bean cake and rapeseed cake preferably.
Last technical problem of the present invention realizes through following technical scheme: the preparation method of above-mentioned preventing and controlling banana fusarium wilt preparation contains following steps:
(1) with the brown sugar brown sugar water that gets soluble in water; Cake fertilizer is placed in the ventilative property wrapping body and will place the intravital cake fertilizer of ventilative property packing to place brown sugar water or directly cake fertilizer is dissolved in brown sugar water, and regulating the pH value is 6 ~ 8, places the shady and cool ventilation place; Sealing is preserved, and must contain the brown sugar water solution of cake fertilizer;
The methylotrophy type Bacillus strain 4-L-16 of brown sugar water solution and the preventing and controlling banana fusarium wilt described in the claim 1 that (2) will contain cake fertilizer was 25 ~ 30 ℃ of condition bottom fermentations 7 ~ 15 days; And stir the maintenance ventilation frequently to accomplishing fermenting process; Get red tan solution, filter and promptly get the resisting banana vascular wilt fermented liquid that contains multiple mikrobe.
The volume ratio of the methylotrophy type Bacillus strain 4-L-16 of brown sugar water solution that contains cake fertilizer described in the step of the present invention (2) and preventing and controlling banana fusarium wilt is preferably 8 ~ 12:1.
Compared with prior art, the present invention has following advantage:
(1) the present invention utilizes raw materials such as culture medium cake fertilizer, brown sugar and water according to the direct liquid fermenting of optimization of C first; Through the mikrobe that produces in the fermented liquid is separated, screening obtains a strain all has better antagonistic effect to No. 1, No. 4 physiological strains of banana blight and the former bacterium of other several diseases methylotrophy type genus bacillus 4-L-16;
(2) because bacterium source produces in biological fermentation, be easy to field planting breeding, the growth of can effectively suppress banana blight bacteria No. 1, No. 4 physiological strains and other banana common disease kind, environmental safety is good, and is significant to the banana blight prevention and control;
(3) the methylotrophy type genus bacillus 4-L-16 among the present invention is processed microbial preparation with other organic matter; No. 1, No. 4 physiological strains of banana blight and the former bacterium of other several diseases are had better antagonistic effect, also plant strain growth is had growth-promoting functions simultaneously;
(4) the bacterial strain 4-L-16 strain culturing condition of the preventing and controlling banana fusarium wilt among the present invention simultaneously is simple, and reproduction speed is fast, is easy to preserve, and is easy to suitability for industrialized production, has the excellent development application prospect.
Description of drawings
Fig. 1 be among the embodiment 2 the 4-L-16 bacterial strain to the antagonistic effect of No. 1 physiological strain of Fusarium oxysporum;
Fig. 2 be among the embodiment 2 the 4-L-16 bacterial strain to the antagonistic effect of No. 4 physiological strains of Fusarium oxysporum;
Fig. 3 is that the 4-L-16 bacterial strain is to the fungistatic effect figure of the microscler leaf spot fungi of banana, the mould leaf spot fungi of mango chain lattice spore, the big gray leaf spot bacterium of banana, lichee anthrax-bacilus, mango anthrax-bacilus and banana anthrax-bacilus among the embodiment 2, and wherein A represents the microscler leaf spot fungi of banana; B represents the mould leaf spot fungi of mango chain lattice spore; C represents the big gray leaf spot bacterium of banana; D represents the lichee anthrax-bacilus; E represents the mango anthrax-bacilus; F represents the banana anthrax-bacilus;
Fig. 4 contains the prevention and control design sketch of methylotrophy type genus bacillus 4-L-16 microbiobacterial agent to dwarf banana blight in seedling stage among the embodiment 3;
Fig. 5 contains the prevention and control design sketch of methylotrophy type genus bacillus 4-L-16 microbiobacterial agent to Brazilian any of several broadleaf plants blight in seedling stage among the embodiment 4.
Embodiment
Embodiment 1
The source of bacterial strain, separation and evaluation
This bacterial strain obtains by separating in rapeseed cake, brown sugar and the water by fermentation liquid; Concrete preparation of fermentation liquid method is following: brown sugar 155g is dissolved in the 1000mL water; Rapeseed cake 95g is dissolved in the brown sugar water, and the pH value of regulating brown sugar water is 7.0, places shady and cool ventilative place; Sealing is preserved, and must contain the brown sugar water solution of cake fertilizer; The brown sugar water solution that will contain cake fertilizer places the 5L volumetric flask, and at 180rpm, under 30 ℃ of conditions, concussion was cultivated 7 days, crosses and filters the fermented liquid that the red tan solution agent is accomplished for fermentation.
In the fermented liquid antimicrobial short of money separate, primary dcreening operation
Adopt dull and stereotyped dilution coating method, extent of dilution (10
-1～10
-3), adopt PDA substratum separating to fungi; The LB substratum separates bacterium; The Gause I substratum separates actinomycetic, and adding the quality percentage composition before separating is that 3% SRM 935a suppresses bacterium, fungi growth.Draw the dilution solution 100 μ L of above-mentioned difference respectively with liquid-transfering gun, drip on culture medium flat plate, evenly with the coating of L type glass rod; Dry slightly; Be inverted petridish, in 25～28 ℃ of incubators, cultivated 3 days, according to the color of the bacterium colony that grows on the flat board, form, size etc.; Different fungies, bacterium and actinomycetic single bacterium colony are chosen to the new substratum purifying cultivate, bacterium, actinomycetes are used the PDA culture medium culturing with LB culture medium culturing, fungi.Adopt dull and stereotyped face-off culture method primary dcreening operation antagonistic strain, primary dcreening operation obtains 28 strain antagonism bacterium altogether, with bacterial classification freeze-drying-20 ℃ preservation, is used for further screening.
2. antimicrobial short of money sieves again in the fermented liquid
Adopt dull and stereotyped face-off culture method, detect antagonism bacterium behind the primary dcreening operation respectively the bacteriostatic activities of No. 1 physiological strain of Fusarium oxysporum and No. 4 physiological strains, with the antibacterial bandwidth (cm) between antagonistic strain and the pathogenic bacteria as the strong and weak index of antagonistic action.
Antagonistic strain to be measured cultivated 72 hours in 28 ℃ of incubators after; Insert the FOC1/FOC4 mycelia piece of 5mm in potato glucose culture medium flat plate central authorities; Apart from 2.5cm place around the FOC1/FOC4 mycelia piece, inoculate antagonism bacterium 5 μ L to be measured, place 28 ℃ of incubators to cultivate; Only to inoculate pathogenic bacteria is contrast, when treating that contrast has just been covered with petridish.Antibacterial bandwidth (cm) between measurement antagonistic strain and the pathogenic bacteria is as the strong and weak index of antagonistic action.
Final screening obtains FOC1 and FOC4 pathogenic bacteria are all had the bacterial strain 4-L-16 of better antagonistic effect.
3. antimicrobial short of money is identified
The main biological characteristics of methylotrophy type genus bacillus 4-L-16 is: this bacterium is a gram-positive microorganism, and culture condition is the LB substratum, 37 ℃, and pH7.0.Detect the 16sRNA sequence; And with carrying out the BLAST comparison on the online GENBANK of being submitted to of sequence; Find that its similarity bacterium in Bacillus amyloliquefaciens subsp.Plantarum and two kinds of bacterium of Bacillus methylotrophicus reaches 99.859%; Identify finally through Physiology and biochemistry and confirm that this bacterium is Bacillus methylotrophicus, its 16sRNA sequence can be consulted shown in the sequence table SEQ ID NO:1.
Table 1 bacterial strain 4-L-16 phenotype and physio-biochemical characteristics-enzyme is lived, carbon assimilation
Annotate :+: positive reaction;-: negative reaction; W: weak positive reaction
Table 2 bacterial strain 4-L-16 physio-biochemical characteristics-carbon assimilation
Annotate :+: positive reaction;-: negative reaction; W: weak positive reaction
With this bacterial strain after on June 26th, 2012 sending to Chinese typical culture collection center and carry out preservation; Preservation name through this bacterial strain of isolation identification 4-L-16 is called methylotrophy type genus bacillus 4-L-16; Classification called after Bacillus methylotrophicus4-L-16; Depositary institution is Chinese typical culture collection center, and preservation date is on June 26th, 2012, and preserving number is CCTCC NO:M2012248.
2.1 methylotrophy type genus bacillus 4-L-16 is to the dull and stereotyped antagonistic effect of No. 1 microspecies FOC1 of banana blight bacteria
With methylotrophy type genus bacillus 4-L-16 activation on the LB substratum; Place 37 ℃ of incubators to cultivate after 72 hours; At the FOC1 mycelia piece of the access 5mm of potato glucose PDA culture medium flat plate central authorities,, inoculate methylotrophy type genus bacillus 4-L-16 nutrient solution 5 μ L apart from FOC1 mycelia piece 2.5cm place; With the flat board that does not meet methylotrophy type genus bacillus 4-L-16 serves as that contrast places 28 ℃ of incubators to cultivate 5 ~ 7 days; Measuring antibacterial bandwidth is 9mm, and No. 1 microspecies of banana blight bacteria are had obvious restraining effect, sees table 3 and Fig. 1.
2.2 methylotrophy type genus bacillus 4-L-16 is to the dull and stereotyped antagonistic effect of No. 4 microspecies FOC4 of banana blight bacteria
With methylotrophy type genus bacillus 4-L-16 activation on the LB substratum; Place 37 ℃ of incubators to cultivate after 72 hours, insert the FOC4 mycelia piece of 5mm, apart from FOC4 mycelia piece 2.5cm place in potato glucose PDA culture medium flat plate central authorities; Inoculation methylotrophy type genus bacillus 4-L-16 nutrient solution 5 μ L; Flat board not meet methylotrophy type genus bacillus 4-L-16 is contrast, places 28 ℃ of incubators to cultivate 5 ~ 7 days, and measuring antibacterial bandwidth is 12mm; No. 4 microspecies of banana blight bacteria are had obvious restraining effect, see table 3 and Fig. 2.
Table 3 methylotrophy type genus bacillus 4-L-16 is to the restraining effect of No. 1, No. 4 physiological strain of banana blight
Annotate :-: the unrestraint effect; +: antibacterial band diameter 1～4mm; ++: antibacterial band diameter 4～8mm; The antibacterial band diameter＞8mm of +++:.
2.3 methylotrophy type genus bacillus 4-L-16 is to the antagonistic action of multiple pathogenic bacteria
Adopt dull and stereotyped face-off method; To the antagonistic effect of 6 kinds of pathogenic bacterias such as the microscler leaf spot fungi of banana, the mould leaf spot fungi of mango chain lattice spore, Glorosprium musarum Cookeet Mass, the big greyness germ of banana, lichee anthrax-bacilus, mango anthrax-bacilus, these six kinds of pathogenic bacterias provide by Chinese Academy of Tropical Agricultural Sciences's resource environment and the Xie Yixian researcher of plant protection institute.With methylotrophy type genus bacillus 4-L-16 activation on the LB substratum; Place 37 ℃ of incubators to cultivate after 72 hours, insert the target bacterium mycelia piece of 5mm, apart from target bacterium mycelia piece 2.5cm place in potato glucose PDA culture medium flat plate central authorities; Inoculation methylotrophy type genus bacillus 4-L-16 nutrient solution 5 μ L; Flat board not meet methylotrophy type genus bacillus 4-L-16 is contrast, places 28 ℃ of incubators to cultivate 5 ~ 7 days, detects and measures antibacterial bandwidth.Like table 4 and shown in Figure 3.
Table 4 methylotrophy type genus bacillus 4-L-16 is to the restraining effect of multiple pathogenic bacteria
Annotate :-: the unrestraint effect; +: antibacterial band diameter 1～4mm; ++: antibacterial band diameter 4～8mm; The antibacterial band diameter＞8mm of +++:.
Embodiment 3 contains the biotechnological formulation of methylotrophy type genus bacillus 4-L-16
At first the methylotrophy type genus bacillus 4-L-16 bacterial strain for preparing among the embodiment 1 is carried out activation on the LB substratum, place 37 ℃ of incubators to cultivate after 72 hours, draw thalline 5 ~ 10 μ L on the LB substratum with liquid-transfering gun; Placing 100mL quality percentage composition is 10% brown sugar water solution, and shaking culture, culture temperature are 30 ℃; Shaking speed is 180rpm, and incubation time is 3 ~ 5 days, promptly obtains containing the seed liquor of methylotrophy type genus bacillus 4-L-16 bacterial strain; Then with the ratio of this seed liquor with 1:10; Join in the mixed solution of rapeseed cake and brown sugar, the preparation ratio of this mixed solution is: according to the C/N and the nitrogen content of rapeseed cake, and the carbon content of brown sugar; The C/N that adopts ordinary method to regulate material in the mixed solution is 15/1, and adds an amount of water.
With fermentation 1000mL microbial inoculum and to make rapeseed cake with the gross weight of brown sugar gross weight/water be 1/4 to be example, need adding seed liquor 100mL, rapeseed cake 141g, brown sugar 109g adds water 900mL.It is that 30 ℃, shaking speed are shaking culture 7 ~ 15 days under the 180rpm condition that the fermented liquid for preparing is placed temperature; Under the natural condition,, adopt the good tun of ventilation type, place the shady and cool ventilation place according to above ratio if need bulk fermentation also passable, direct fermentation, but to guarantee sooner or later respectively to stir 5 ~ 10min, and guarantee air flow, ferment got final product in 15 days; Adopt large-scale fermentation to irritate when fermenting, above temperature and stirring velocity, ferment get final product in 7 days, obtained the red tan solution filtration after the fermentation completion and promptly got and contain a large amount of methylotrophy type genus bacillus 4-L-16 bacterial strains and other multiple beneficial mikrobes get microbiobacterial agent.
Embodiment 4 contains the biotechnological formulation of methylotrophy type genus bacillus 4-L-16
At first the methylotrophy type genus bacillus 4-L-16 bacterial strain for preparing among the embodiment 1 is carried out activation on the LB substratum, place 37 ℃ of incubators to cultivate after 72 hours, draw thalline 5 ~ 10 μ L on the LB substratum with liquid-transfering gun; Placing 100mL quality percentage composition is 10% brown sugar water solution, and shaking culture, culture temperature are 30 ℃; Shaking speed is 180rpm, and incubation time is 3 ~ 5 days, promptly obtains containing the seed liquor of methylotrophy type genus bacillus 4-L-16 bacterial strain; Then with the ratio of this seed liquor with 1:10; Join in the mixed solution of peanut cake and brown sugar, the preparation ratio of this mixed solution is: according to the C/N and the nitrogen content of peanut cake, and the carbon content of brown sugar; The C/N that adopts ordinary method to regulate material in the mixed solution is 20/1, and adds an amount of water.
With fermentation 1000mL microbial inoculum and to make peanut cake with the gross weight of brown sugar gross weight/water be 1/4 to be example, need adding seed liquor 80mL, peanut cake 88g, brown sugar 162g adds water 900mL.It is that 30 ℃, shaking speed are shaking culture 7 days under the 180rpm condition that the fermented liquid for preparing is placed temperature; Under the natural condition,, adopt the good tun of ventilation type, place the shady and cool ventilation place according to above ratio if need bulk fermentation also passable, direct fermentation, but to guarantee sooner or later respectively to stir 5 ~ 10min, and guarantee air flow, ferment got final product in 15 days; Adopt large-scale fermentation to irritate when having ready conditions, above temperature and stirring velocity, ferment get final product in 7 days, obtained the red tan solution filtration after the fermentation completion and promptly got and contain a large amount of methylotrophy type genus bacillus 4-L-16 bacterial strains and other multiple beneficial mikrobes get microbiobacterial agent.
Embodiment 5 contains the biotechnological formulation of methylotrophy type genus bacillus 4-L-16
At first the methylotrophy type genus bacillus 4-L-16 bacterial strain for preparing among the embodiment 1 is carried out activation on the LB substratum, place 37 ℃ of incubators to cultivate after 72 hours, draw thalline 5 ~ 10 μ L on the LB substratum with liquid-transfering gun; Placing 100mL quality percentage composition is 10% brown sugar water solution, and shaking culture, culture temperature are 30 ℃; Shaking speed is 180rpm, and incubation time is 3 ~ 5 days, promptly obtains containing the seed liquor of methylotrophy type genus bacillus 4-L-16 bacterial strain; Then with the ratio of this seed liquor with 1:10; Join in the mixed solution of peanut cake and brown sugar, the preparation ratio of this mixed solution is: according to the C/N and the nitrogen content of soya-bean cake, and the carbon content of brown sugar; The C/N that adopts ordinary method to regulate material in the mixed solution is 25/1, and adds an amount of water.
With fermentation 1000mL microbial inoculum and to make soya-bean cake with the gross weight of brown sugar gross weight/water be 1/4 to be example, need adding seed liquor 120mL, soya-bean cake 62g, brown sugar 188g adds water 900mL.It is that 30 ℃, shaking speed are shaking culture 7 days under the 180rpm condition that the fermented liquid for preparing is placed temperature; Under the natural condition,, adopt the good tun of ventilation type, place the shady and cool ventilation place according to above ratio if need bulk fermentation also passable, direct fermentation, but to guarantee sooner or later respectively to stir 5 ~ 10min, and guarantee air flow, ferment got final product in 15 days; Adopt large-scale fermentation to irritate when having ready conditions, above temperature and stirring velocity, ferment get final product in 7 days, obtained the red tan solution filtration after the fermentation completion and promptly got and contain a large amount of methylotrophy type genus bacillus 4-L-16 bacterial strains and other multiple beneficial mikrobes get microbiobacterial agent.
Embodiment 6 contains the prevention and control effect of methylotrophy type genus bacillus 4-L-16 microbiobacterial agent to dwarf banana blight in seedling stage
Test place: China tropic Agriculture Academy Sciences tropic Biotechnology Research Institute's warmhouse booth.
The test kind: No. 1 dwarf banana of bronze, available from academy of agricultural sciences, Guangxi group training factory, about height of seedling 20cm, robust growth, five leaves wholeheartedly, no disease and pest.
Pathogenic bacteria: No. 1 physiological strain Fusarium of Fusarium oxysporum Cuba specialized form oxysopoyum f.sp.cubence race 1 (FOC1) provided by Chinese Academy of Tropical Agricultural Sciences's resource environment and the Xie Yixian researcher of plant protection institute.FOC1 is seeded on the PDA substratum, and 25 ℃ of following constant temperature culture l0d wash spore that to be made into concentration in the sterilized water be 1 * 10
5Individual/the mL spore suspension, supply inoculation to use.
Supply examination soil: latosol, pick up from the Haikou City Longhua District.
Microbial preparation: embodiment 3 fermentation makes contains methylotrophy type genus bacillus 4-L-16 microbiobacterial agent
Test is handled: (1) CK: do not execute pathogenic bacteria, do not execute microbial inoculum; (2) pathogenic bacteria contrast: execute pathogenic bacteria, do not execute microbial inoculum; (3) microbial inoculum is handled: execute pathogenic bacteria, execute microbial inoculum.Each handles 20 strain any of several broadleaf plants seedlings.
TP: choose the consistent banana seedling of big or small growing way, seedling is hindered root, being immersed in concentration then is 5 * 10
630min in individual/mL spore suspension, CK is immersed in the equivalent clear water.Soak in the plastic tub of the paramount 10cm of back seedling replanting, diameter 12cm, apply microbial inoculum (diluting 50 times), every basin 100mL by different treatment; CK and pathogenic bacteria contrast apply the equivalent clear water, every fermented liquid and clear water that repeated to apply equivalent at a distance from 7 days again, duration of test; It is all consistent that each handles potted plant control measures; Observe incidence that each is handled every day, the sickness rate of treating the pathogenic bacteria contrast reaches 80% when above, disease index of each processing of investigation, measures plant height and calculates relative control effect.
Test-results: through tracing observation; Any of several broadleaf plants seedling begins morbidity after transplanting 10 days, when waiting to transplant 22 days, any of several broadleaf plants seedling sickness rate of pathogenic bacteria control treatment reaches 85%; Each disease index of handling of investigation; Pathogenic bacteria contrast disease index is 26.42, and the disease index that microbial inoculum is handled is 12.85, and relative control effect is 51.36%.Average plant height was 29.27 after microbial inoculum was handled simultaneously, exceeded 8.05cm than the pathogenic bacteria contrast, showed that this microbial inoculum has growth-promoting functions to plant strain growth.Like Fig. 4, shown in the table 5.
Table 5 contains the prevention and control effect of methylotrophy type genus bacillus 4-L-16 microbiobacterial agent to dwarf banana blight in seedling stage
Embodiment 7 contains the prevention and control effect of methylotrophy type genus bacillus 4-L-16 microbiobacterial agent to Brazilian any of several broadleaf plants blight in seedling stage
Test place: China tropic Agriculture Academy Sciences tropic Biotechnology Research Institute's warmhouse booth.
The test kind: Brazilian any of several broadleaf plants, available from the tropical AS of China group training center, about height of seedling 15cm, robust growth, 3 leaves wholeheartedly, no disease and pest.
Pathogenic bacteria: Fusarium oxysporum Cuba specialized form No. 4 physiological strain Fusarium oxysopoyum f.sp.cubence race4 (FOC4) is provided by professor Jiang Zide of Agricultural University Of South China.FOC4 is seeded on the PDA substratum, and 25 ℃ of following constant temperature culture l0d wash spore that to be made into concentration in the sterilized water be 5 * 10
6Individual/the mL spore suspension, supply inoculation to use.
Supply examination soil: latosol, pick up from the Haikou City Longhua District.
What fermentation made among microbial preparation: the embodiment 3 contains methylotrophy type genus bacillus 4-L-16 microbiobacterial agent
Test is handled: (1) CK: do not execute pathogenic bacteria, do not execute microbial inoculum; (2) pathogenic bacteria contrast: execute pathogenic bacteria, do not execute microbial inoculum; (3) microbial inoculum is handled: execute pathogenic bacteria, execute microbial inoculum.Each handles 20 strain any of several broadleaf plants seedlings.
TP: choose the consistent banana seedling of big or small growing way, seedling is hindered root, being immersed in concentration then is 5 * 10
630min in individual/mL spore suspension, CK is immersed in the equivalent clear water.Soak in the plastic tub of the paramount 10cm of back seedling replanting, diameter 12cm, apply microbial inoculum (diluting 50 times), every basin 100mL by different treatment; CK and pathogenic bacteria contrast apply the equivalent clear water, every fermented liquid and clear water that repeated to apply equivalent at a distance from 7 days again, duration of test; It is all consistent that each handles potted plant control measures, observes each incidence of handling every day, treats that the sickness rate of pathogenic bacteria contrast reaches at 80% o'clock; Each disease index of handling of investigation is measured plant height and is calculated relative control effect.
Test-results: through tracing observation; Any of several broadleaf plants seedling begins morbidity after transplanting 14 days, when waiting to transplant 30 days, any of several broadleaf plants seedling sickness rate of pathogenic bacteria control treatment reaches 90%; Each disease index of handling of investigation; Pathogenic bacteria contrast disease index is 34.28, and the disease index that microbial inoculum is handled is 11.43, and relative control effect is 66.56%.The average plant height of plant is 20.67cm after the processing of plant height microbial inoculum is respectively handled in investigation simultaneously, and the average plant height of pathogenic bacteria contrast is 15.46cm, and growth has certain growth-promoting functions to banana to show this microbial inoculum; Shown in Fig. 5 and table 6.
Table 6 contains the prevention and control effect of methylotrophy type genus bacillus 4-L-16 microbiobacterial agent to banana blight in seedling stage
The foregoing description is a preferred implementation of the present invention; But embodiment of the present invention is not restricted to the described embodiments; Other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; All should be the substitute mode of equivalence, be included in protection scope of the present invention.
Claims (7)
1. the methylotrophy type Bacillus strain 4-L-16 of a preventing and controlling banana fusarium wilt; It is characterized in that: the preservation name of this bacterial strain is called methylotrophy type genus bacillus 4-L-16; Classification called after Bacillus methylotrophicus4-L-16; Depositary institution is Chinese typical culture collection center, and preservation date is on June 26th, 2012, and preserving number is CCTCC NO:M2012248.
2. the methylotrophy type Bacillus strain 4-L-16 of the described preventing and controlling banana fusarium wilt of claim 1 has the purposes in the medicine of preventing and controlling banana fusarium wilt effect in preparation.
3. the preparation of a preventing and controlling banana fusarium wilt is characterized in that: the methylotrophy type Bacillus strain 4-L-16 that contains the preventing and controlling banana fusarium wilt described in the claim 1.
4. the preparation of preventing and controlling banana fusarium wilt according to claim 3 is characterized in that: also contain culture medium cake fertilizer, brown sugar and water, wherein the gross weight of cake fertilizer and brown sugar and the gross weight of water are than being 1:4, and the C/N ratio of culture medium is 15 ~ 25:1.
5. the preparation of preventing and controlling banana fusarium wilt according to claim 4 is characterized in that: described cake fertilizer be in rapeseed cake, peanut cake and the soya-bean cake one or more.
6. the preparation method of the preparation of claim 4 or 5 described preventing and controlling banana fusarium wilts is characterized in that: contain following steps:
(1) with the brown sugar brown sugar water that gets soluble in water; Cake fertilizer is placed in the ventilative property wrapping body and will place the intravital cake fertilizer of ventilative property packing to place brown sugar water or directly cake fertilizer is dissolved in brown sugar water, and regulating the pH value is 6 ~ 8, places the shady and cool ventilation place; Sealing is preserved, and must contain the brown sugar water solution of cake fertilizer;
The methylotrophy type Bacillus strain 4-L-16 of brown sugar water solution and preventing and controlling banana fusarium wilt that (2) will contain cake fertilizer was 25 ~ 30 ℃ of condition bottom fermentations 7 ~ 15 days; And stir the maintenance ventilation frequently to accomplishing fermenting process; Get red tan solution, filter and promptly get the resisting banana vascular wilt fermented liquid that contains multiple mikrobe.
7. the preparation method of the preparation of preventing and controlling banana fusarium wilt according to claim 6 is characterized in that: the volume ratio of methylotrophy type Bacillus strain 4-L-16 that contains brown sugar water solution and the preventing and controlling banana fusarium wilt of cake fertilizer described in the step (2) is 8 ~ 12:1.
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