CN104726384A - Bacillus subtilis for prohibiting rhizoctonia solani of potatoes - Google Patents
Bacillus subtilis for prohibiting rhizoctonia solani of potatoes Download PDFInfo
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- CN104726384A CN104726384A CN201510188051.6A CN201510188051A CN104726384A CN 104726384 A CN104726384 A CN 104726384A CN 201510188051 A CN201510188051 A CN 201510188051A CN 104726384 A CN104726384 A CN 104726384A
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- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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Abstract
The invention provides bacillus subtilis for prohibiting rhizoctonia solani of potatoes and lays the foundation for biological control of rhizoctonia solani of potatoes. The bacillus subtilis for prohibiting rhizoctonia solani of the potatoes is bacillus subtilis PB12, filed in China General Microbiological Culture Collection Center by the CGMCC No. 10603 in March 11, 2015. The bacillus subtilis PB12 has high capability in prohibiting growth of hypha of rhizoctonia solani of the potatoes. Over culturing days, prohibiting effect is more remarkable.
Description
Technical field
The present invention relates to a bacillus subtilis.
Background technology
Rhizoctonia is the fungi that a class is widespread in nature, in global farming, non-anthropogenic soil, all have distribution, and many rhizoctonias are easy to be separated from infected plant and soil obtain.The host range of rhizoctonia is extremely wide, can infect 43 section more than 200 kind of plant.Rhizoctonia is a kind of Soil-born plant pathogenic fungi, easily causes the rotten kind of plant, seedling stage dampings off.The saprophytic competitive power of rhizoctonia in soil is strong, and the survival time is long, is considered to one of soil-borne plant pathogen of most destructive force.Rhizoctonia is hung contracting near mycelia branch origination point, forms barrier film near branching-point, and mycelia often at right angles or acute angle branch.Biological control mainly utilizes the antagonistic action between microorganism, selects the growth to the Antimicrobial pathogenic bacteria that farm crop do not work the mischief.Bacterium, actinomycetes, fungi development prospect in biocontrol of plant disease is good, and the research of biocontrol fungicide and exploitation have important realistic meaning.Microbial pesticide is a class biological pesticide most widely used, with fastest developing speed, the most promising at present, is one of key breakthrough mouth solving China's current agricultural environmental problem.
Heilongjiang Province is the township of potato planting, and potato planting area constantly expands in recent years, and Development of Potato Industry is swift and violent.But because production of seed stock person provides the idea of anosis potato seed not strong, often non-potato seed is done potato seed application with kind of person, kind is introduced and is ignored quarantine in allocation and transportation process and checks on; The planting area production of seed stock area even had, does not note crop rotation; Also have in planting process to the prophylactico-therapeutic measures of disease implement half-hearted, various reasons causes potato disease to be on the rise.Potato miliary damping-off is exactly one of them, and the morbidity of this disease is general, and sickness rate reaches more than 5%, and hazard rating is also increasing the weight of increasingly, not only makes output reduce by 20%, and has a strong impact on Potato Quality, become a large obstacle of Development of Potato Industry.
Pathogenic bacteria dry thread Pyrenomycetes is the pathogen of a large amount of farm crop in a kind of whole world and wild host, its sclerotium is survived the winter on stem tuber or in soil, or the plant residue of mycelium in soil is survived the winter, Second Year spring, when temperature, humidity condition are applicable to, sclerotial germination invades potato young shoot, seedling, particularly have invade during wound more faster.Root, subterraneous stem, stolon, stem tuber can be invaded again in the season of growth.The sclerotium that new stem tuber is formed, or the sclerotium of surviving the winter again in soil, next year runs into adapt circumstance condition, can infect again.In a word, this disease is the disease that potato seed combines with soil-borne in spite of illness, therefore, makes troubles to control.
Summary of the invention
The invention provides a bacillus subtilis, can inhibition of potato miliary damping-off germ, for the biological control of potato miliary damping-off lays the foundation.
The subtilis of inhibition of potato silk core germ of the present invention is subtilis (Bacillus subtilis) PB12, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date is on March 11st, 2015, and deposit number is CGMCC No.10603.
Subtilis PB12 Gram-positive of the present invention, shaft-like, two ends are blunt flat, and most thalline series connection bunchiness, in irregularly shaped.Produce gemma, cultivate 24h spore forming rate for 30 DEG C and reach more than 80%, gemma end is raw, and oval, do not expand, be cultured to 48h, gemma all comes off.Bacterium colony on NYDA solid medium is creamy white, fold, and edge is irregular, and bacterium colony central protuberance is carinate, easily provokes.
Subtilis PB12 of the present invention can grow at 50 DEG C, and pH5.7 can grow, and 7%NaCl can grow, and does not grow under anaerobic condition.Glucose fermentation produces acid not aerogenesis, catalase reacting positive, and Starch Hydrolysis is positive, VP test is positive, and utilizes Citrate trianion to react and is positive, and gelatine liquefication reaction is positive, litmus milk reduction peptonizes reaction and is positive, and nitrate reaction is positive, and clark and Lubsreaction is positive.
Subtilis PB12 of the present invention can grow on NYD substratum, and optimum growth temperature is 30 DEG C, and optimal pH is 7.2 ~ 7.5, rotating speed 150r/min.NYD substratum is made up of beef extract 8g, yeast extract paste 5g, glucose 10g, agar 15g and distilled water 1000mL.
Subtilis PB12 of the present invention is by 16S rDNA sequence alignment analysis, and have high homology with bacillus, homology is more than 98%.By determining that subtilis PB12 belongs to bacillus (Bacillus Cohn) in conjunction with morphological features, growth conditions, Physiology and biochemistry qualification result, be subtilis (Bacillus subtilis).
Bacteriostatic test plate and culturing bottle bacteriostatic experiment show, under solid culture and liquid culture condi, subtilis PB12 of the present invention all has the ability of stronger inhibition of potato miliary damping-off germ mycelial growth, and along with the increase of cultivated days, fungistatic effect is more obvious.
Subtilis PB12 of the present invention, belong to bacillus (Bacillus Cohn), be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date is on March 11st, 2015, and deposit number is CGMCC No.10603.
Accompanying drawing explanation
The antagonistic action of subtilis PB12 when Fig. 1 is solid culture; The antagonistic action of subtilis PB12 when Fig. 2 is liquid culture.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: the subtilis of present embodiment inhibition of potato silk core germ is subtilis PB12, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date is on March 11st, 2015, and deposit number is CGMCCNo.10603.
Present embodiment subtilis PB12 Gram-positive, shaft-like, two ends are blunt flat, and most thalline series connection bunchiness, in irregularly shaped.Produce gemma, cultivate 24h spore forming rate for 30 DEG C and reach more than 80%, gemma end is raw, and oval, do not expand, be cultured to 48h, gemma all comes off.Bacterium colony on NYDA solid medium is creamy white, fold, and edge is irregular, and bacterium colony central protuberance is carinate, easily provokes.
Present embodiment subtilis PB12 can grow at 50 DEG C, and pH5.7 can grow, and 7%NaCl can grow, and does not grow under anaerobic condition.Glucose fermentation produces acid not aerogenesis, catalase reacting positive, and Starch Hydrolysis is positive, VP test is positive, and utilizes Citrate trianion to react and is positive, and gelatine liquefication reaction is positive, litmus milk reduction peptonizes reaction and is positive, and nitrate reaction is positive, and clark and Lubsreaction is positive.
Present embodiment subtilis PB12 can grow on NYD substratum, and optimum growth temperature is 30 DEG C, and optimal pH is 7.2 ~ 7.5, rotating speed 150r/min.NYD substratum is made up of beef extract 8g, yeast extract paste 5g, glucose 10g, agar 15g and distilled water 1000mL.
Present embodiment subtilis PB12 is celebrated in the soil in susceptible region between the potato fields of flat village to be separated by Bai Kui town, Hulan District and obtains.Separation method carries out according to the following steps: one, with julienne potatoes pyrenomycetes for target, get susceptible regional soil 10 grams between potato fields, be put in 100mL and be with in the triangular flask of granulated glass sphere and sterilized water, jolt 20 minutes, soil sample and water are fully mixed, by cell dispersal.Two, gradient dilution method is utilized to be diluted to 10
3, 10
4, 10
5three gradients, carry out gradient dilution by the concentrated solution of this batch and coat on NYDA substratum, and be placed in 32 DEG C of incubators and cultivate 18h, the bacterial strain selecting different colonial morphology carries out label and separation and Culture; Isolated single bacterium colony access is equipped with in the test tube of NYD substratum (5mL), and in 32 DEG C, 150r/min cultivates 18h.Strong, that antimicrobial spectrum the is wide biocontrol strain of antagonistic action is selected to carry out purifying, preservation.Be the subtilis PB12 of present embodiment.
Carry out Molecular Identification to the subtilis PB12 that separation screening obtains, carry out according to the following steps: the STb gene extracting bacterial strain, adopting the 16S rDNA universal primer of bacterium, is that template carries out pcr amplification with genomic dna.Then utilize glue to reclaim test kit and reclaim purified pcr product; Afterwards, carry out cloning, transforming, screening positive clone daughter colony, checks order after enlarged culturing.16S rDNA sequence in sequencing result and GenBank carries out sequence analysis, and have high homology with bacillus (Bacillus Cohn), homology is more than 98%.By determining that subtilis PB12 belongs to bacillus (Bacillus Cohn) in conjunction with morphological features, growth conditions, Physiology and biochemistry qualification result, be subtilis (Bacillus subtilis).
For the fungistatic effect of checking present embodiment subtilis PB12, carry out following experiment:
The antagonistic action of julienne potatoes core germ biocontrol strain:
(1) early-stage preparations
By subtilis PB12 picking list colony inoculation in the 5mL test tube that NYD substratum is housed, in 30 DEG C of 150r/min vibrators, cultivate 24h.Be inoculated in be equipped with in the 50mL triangular flask of NYD substratum with the inoculum size of 2% again, in 30 DEG C of 150r/min vibrators, cultivate 24h stand-by.
Potato miliary damping-off germ (Rhizoctonia solani K ü hn) is connected to PDA flat board to cultivate as in 28 DEG C of incubators, treats that mycelia covers with culture dish.Described potato miliary damping-off germ (Rhizoctonia solani K ü hn) is provided by Plant De-toxin Nursery Stock Inst., Heilongjiang Prov. Academy of Agriculture Sc.
(2) antagonistic action under solid culture condition
With potato miliary damping-off germ for target object, the antagonistic action of research subtilis PB12.First subtilis PB12 is carried out gradient dilution (10 times, 100 times), on PDA culture medium flat plate, then evenly fill each dilution bacterium liquid 100 μ L (comprising bacterium stoste, 10 times of diluents, 100 times of diluents).Dry rear every flat board and access the target pathogens bacterium cake that diameter is 8mm respectively, if be not coated with subtilis PB12 bacterium liquid for contrast, repeat 3 times, in 28 DEG C of cultivations.Every day observes fungal growth situation, measures inhibition zone size.
Bacteriostasis rate (%)=(contrast hyphal diameter-process hyphal diameter)/contrast hyphal diameter × 100%.
(3) antagonistic action under liquid culture condi
With potato miliary damping-off germ for target object, the antagonistic action of research subtilis PB12.First subtilis PB12 is carried out gradient dilution (10 times, 100 times), then in the 50ml culturing bottle that PDA liquid nutrient medium is housed, add each dilution bacterium liquid 1mL (comprising bacterium stoste, 10 times of diluents, 100 times of diluents) respectively with the inoculum size of 2%.In every bottle, access the target pathogens bacterium cake that diameter is 8mm more respectively, if what do not inoculate subtilis PB12 bacterium liquid is contrast, repeats 3 times, in the cultivations of 28 DEG C of constant-temperature tables.Every day observes fungal growth situation, when control group fungal growth situation comparatively experimental group clearly time stop cultivating, mycelia is filtered, and claims its dry weight.
Bacteriostasis rate (%)=(contrast mycelia weight-process mycelia weight)/contrast mycelia weight × 100%.
Experimental result is as follows:
(1) antagonistic action under solid culture condition
By bacteriostatic test plate, result shows under solid culture condition, the growth of the suppression silk core germ mycelia that PB12 can be stronger, and along with the increase of cultivated days, fungistatic effect more obvious (as Fig. 1 and table 1).
The antagonistic effect of subtilis PB12 under table 1 solid culture condition
Note: in table, data are the mean value repeated for 3 times.
As shown in figure 1 and table 1, along with the increase of cultivated days, mycelia grows control group gradually, when cultivation covers with whole flat board to control group when the 3rd day, and now experimental group each silk core germ mycelium growth vigor is obviously weaker than control group, and almost no longer grow in the cultivation middle and later periods.Simultaneously along with dilution increase, fungistatic effect declines to some extent.
(2) antagonistic action under liquid culture condi
By culturing bottle bacteriostatic experiment, result shows under liquid culture condi, and PB12 has the ability of stronger suppression silk core germ mycelial growth equally, and along with the increase of cultivated days, fungistatic effect more obvious (as Fig. 2 and table 2).
The antagonistic effect of silk core germ biocontrol strain under table 2 liquid culture condi
| Bacterial strain | Dry weight (mg) | Bacteriostasis rate/% |
| PB12 stoste | 5.0 | 98.67 |
| PB1210 times of diluent | 5.7 | 98.49 |
| PB12100 times of diluent | 6.0 | 98.41 |
| CK | 376.3 | - |
Note: in table, data are the mean value repeated for 3 times.
As shown in 2 and table 2, along with the increase of cultivated days, the ramp of control group mycelia becomes agglomerate, when cultivation is to stopping when the 4th day cultivating, is carried out by each group of mycelia filtering, dry, weighing.Can find out that control group silk core germ mycelia agglomerate is obviously greater than each experimental group silk core germ mycelia agglomerate.Simultaneously along with dilution increase, fungistatic effect has downtrending.
Claims (1)
1. the subtilis of a strain inhibition of potato miliary damping-off germ, it is characterized in that this bacterial strain is subtilis (Bacillus subtilis) PB12, be deposited 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, preservation date is on March 11st, 2015, and deposit number is CGMCC No.10603.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201510188051.6A CN104726384A (en) | 2015-04-20 | 2015-04-20 | Bacillus subtilis for prohibiting rhizoctonia solani of potatoes |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105296381A (en) * | 2015-08-31 | 2016-02-03 | 哈尔滨师范大学 | Bacillus subtilis CYY-25 and application thereof |
| CN105505836A (en) * | 2016-01-27 | 2016-04-20 | 黑龙江省科学院微生物研究所 | Bacillus subtilis and methods for preparing and using bacillus subtilis microbial agent for controlling potato scab |
| CN105746579A (en) * | 2016-02-02 | 2016-07-13 | 安建慧 | Application of bacillus subtilis HMB19198 preparation in control of rhizoctonia silani |
| CN115044494A (en) * | 2022-04-27 | 2022-09-13 | 内蒙古自治区农牧业科学院 | Bacillus subtilis WZ10 and application thereof |
| CN115161247A (en) * | 2022-08-22 | 2022-10-11 | 河南大学 | Bacillus subtilis 201015 and application thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105296381A (en) * | 2015-08-31 | 2016-02-03 | 哈尔滨师范大学 | Bacillus subtilis CYY-25 and application thereof |
| CN105296381B (en) * | 2015-08-31 | 2018-11-30 | 哈尔滨师范大学 | One bacillus subtilis CYY-25 and its application |
| CN105505836A (en) * | 2016-01-27 | 2016-04-20 | 黑龙江省科学院微生物研究所 | Bacillus subtilis and methods for preparing and using bacillus subtilis microbial agent for controlling potato scab |
| CN105746579A (en) * | 2016-02-02 | 2016-07-13 | 安建慧 | Application of bacillus subtilis HMB19198 preparation in control of rhizoctonia silani |
| CN115044494A (en) * | 2022-04-27 | 2022-09-13 | 内蒙古自治区农牧业科学院 | Bacillus subtilis WZ10 and application thereof |
| CN115044494B (en) * | 2022-04-27 | 2023-06-23 | 内蒙古自治区农牧业科学院 | Bacillus subtilis WZ10 and application thereof |
| CN115161247A (en) * | 2022-08-22 | 2022-10-11 | 河南大学 | Bacillus subtilis 201015 and application thereof |
| CN115161247B (en) * | 2022-08-22 | 2023-07-28 | 河南大学 | Bacillus subtilis 201015 and application thereof |
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