CN103555616A - Bacillus amyloliquefaciens and application of liquid preparations thereof in prevention and control of fusarium solani - Google Patents
Bacillus amyloliquefaciens and application of liquid preparations thereof in prevention and control of fusarium solani Download PDFInfo
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Abstract
The invention discloses bacillus amyloliquefaciens and application of liquid preparations thereof in prevention and control of fusarium solani. The strain has a classification name of bacillus amyloliquefaciens, the complete name of the strain is bacillus amyloliquefaciens Ba4, and the strain is collected in China General Microbiological Culture Collection Center in 3# No.1 courtyard on Beichen west road in Chaoyang district in Beijing, China on 12, September in 2013 and has a collection number of CGMCC No.8177. The provided bacillus amyloliquefaciens Ba4 has a special effect on fusarium solani, has high stress resistance, high keeping property, high high/low-temperature resistance and high drying resistance and is suitable for commercial production. Moreover, when the bacillus amyloliquefaciens Ba4 is applied once, the disease can be effectively and continuously controlled for a long time, and the cost is greatly reduced.
Description
Technical field
The present invention relates to field of ecology, an in particular bacillus amyloliquefaciens and liquid preparation thereof the application in control walnut root rot.
Background technology
Walnut has in important economic worth and Ecology Action ， China's forestry development and improvement of the ecological environment and has very consequence, particularly for improving Agricultural Economy of The Mountainous Regions level, has positive effect.Walnut root rot (Fusarium solani) be take Seedling Stage and is injured as main, and the nursery stock the lighter that is injured affects growth, and severe one causes mortality, becomes a large obstacle of the work of growing seedlings.Susceptible seedling, main manifestations is that root blackening is rotted, overground part yellow leaf, leaf margin blackening, mortality ratio is very high.The adult large tree in cultivation area, Sichuan also can fall ill now, to Walnut Industry, is a potential threat.Mainly adopt at present chemical prevention and forest culture and management technology, and bio-control method research not yet relates to.Though chemical prevention is a kind of effectively preventing means, has many practical problemss such as pathogenic bacteria resistance, environment and food safety, poisoning, some chemical bactericides strictly limit use in many developed countries and area.And be more and more subject to people's favor with eco-friendly biological pesticide, be the important component part of the future of agriculture Sustainable development.
Therefore, there is defect in prior art, needs to improve.
Summary of the invention
Technical problem to be solved by this invention is the defect to the control of walnut root rot for prior art, and the application in control walnut root rot of a bacillus amyloliquefaciens and liquid preparation thereof is provided.
Technical scheme of the present invention is as follows:
One bacillus amyloliquefaciens, its Classification And Nomenclature is bacillus amyloliquefaciens, Bacillus amyloliquefaciens, in on 09 12nd, 2013, be deposited in No. 3 China Committee for Culture Collection of Microorganisms's common micro-organisms centers of No. 1, North Star West Road, Chaoyang District, BeiJing, China city institute, deposit number is CGMCC No.8177.
The making method of the liquid preparation of described bacillus amyloliquefaciens, comprises the following steps:
(1) primary inclined plane seed: ordinary method is made beef extract-peptone slant medium, inoculates after described bacillus amyloliquefaciens, cultivates, and makes first order seed;
(2) secondary liquid seeds: nutrition meat nutrient solution is after autoclaving, under logical oxygen is controlled, in 100mL liquid, be contained in the bottling of 300mL triangular flask ratio, then under sterile state, inoculate described bacillus amyloliquefaciens inclined-plane seed, 2 inclined-plane seeds of every bottle graft kind, shaking culture, makes bacillus amyloliquefaciens liquid seeds;
(3) liquid fermenting: nutrition meat nutrient solution is after autoclaving, under controlling, logical oxygen is contained in the bottling of 500mL triangular flask ratio in 200mL liquid, the bacillus amyloliquefaciens liquid seeds that inoculation step under sterile state (2) makes, inoculum size is 10% of total liquid volume, shaking culture, make bacillus amyloliquefaciens liquid preparation, then preserve.
The making method of the liquid preparation of described bacillus amyloliquefaciens, in step (1), the composition of beef extract-peptone slant medium is extractum carnis 7.0g, peptone 10g, NaCl5g, agar 17g, water 1000mL.
The making method of the liquid preparation of described bacillus amyloliquefaciens, the composition of its nutrition meat nutrient solution is extractum carnis 7.0g, peptone 10g, NaCl5g, Ramulus Juglandis steeping fluid 1000mL.
Described making method obtains liquid preparation.
The application of described liquid preparation in control walnut root rot, the walnut forest land of not falling ill is filled with root with 100-300 times of extent of dilution of described bacillus amyloliquefaciens liquid preparation volume ratio along root system breadth, 100 milliliters of every strains, use once annual spring, can prevent root rot to occur; The walnut of falling ill is filled with root and near soil pouring filling by 50-200 times of extent of dilution of described bacillus amyloliquefaciens liquid preparation volume ratio along root system breadth, 200 milliliters of every strains, and spring, summer respectively once, can be imitated treatment root rot.
The present invention has filtered out the specific new bacterial strain Ba4 of walnut root rot, and it has the brood gemma of strong stress resistance, and Storage is good, high temperature resistance, low temperature, resistance to dry, be suitable for commercialization and produce, and use once can be permanently effective, can continue to control disease, significantly reduce costs.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
1, the separation of endogenetic bacteria, purifying and preservation
In walnut root rot spot, Chaotian District, Sichuan Guangyuan city, gather healthy Walnut Roots, take back laboratory separation.The sample of adopting is back clean with aseptic water washing, takes 1.0g root tissue, the alcohol disinfecting 30s with 70%, after aseptic washing 3 times, then with after 4% NaClO sterilization 3min, aseptic washing 3 times, draw last washings 100 μ l and smear NA flat board (extractum carnis 3g, peptone 10g, sodium-chlor 5g, agar powder 15-20g, distilled water 1000ml, pH7.0, mix 121 ℃ of autoclaving 30min after packing), 28 ℃ of dark culturing 24h, for the contrast of surface sterilization, check that whether surface sterilization is thorough.
The root tissue of surface sterilization is shredded, put into aseptic mortar, add quartz sand and the 10ml sterilized water of sterilizing, fully grind, standing 30min, dilutes 10
-1, 10
-2, 10
-3, 10
-4, 10
-5totally 5 gradients, draw respectively 100 μ l10
-3, 10
-4, 10
-5these 3 gradient lapping liquids are coated with respectively NA flat board (extractum carnis 3g, peptone 10g, sodium-chlor 5g, agar powder 15-20g, distilled water 1000ml, pH7.0, mix 121 ℃ of autoclaving 30min after packing), each is processed 3 times and repeats, 28 ℃ of dark culturing 48h, choose growth gradient the most uniformly, carry out plate count.
According to the size of bacterium colony, color, projection whether, edge feature, smooth surface whether, the difference of the aspect such as transparency, picking list bacterium colony is at the flat lining out purifying of NA, the NA inclined-plane of transferring after purifying is standby in 4 ℃ of preservations.Obtain altogether 10 endogenetic bacterias.
2, the dull and stereotyped antagonistic action of endogenetic bacteria to walnut root rot germ
Dull and stereotyped face-off method: at walnut root rot germ (Fusarium solani) the bacterium cake (diameter 5mm) of the inoculation 7d of culture dish central authorities, from the about 3cm of this germ cake place, with transfering loop, pick the endophyte of cultivating 2d respectively, at each standardized fine rule of the symmetrical both sides of germ, 28 ℃ of cultivations, the flat board of only inoculating germ of take is contrast, when ware is covered with in contrast, measures the colony growth diameter of germ, and every processing repeats 3 times.Be calculated as follows colony growth inhibiting rate:
Colony growth inhibiting rate=(the clean growth diameter of contrast bacterium colony clean growth diameter-processing bacterium colony)/clean growth diameter * 100% of contrast bacterium colony
Table 1 is the antagonistic action of endogenetic bacteria to walnut root rot germ (Fusarium solani), and result shows: after 7d, have 4 kinds of endogenetic bacteria colony growth inhibiting rates to reach more than 61.54%, wherein Ba4 bacterial strain inhibiting rate reaches 100%.
Table 1 endogenetic bacteria is to the antagonistic action of walnut root rot germ (7 days)
Note: the comparison of * otherness: containing same letter, be that difference is not remarkable, otherwise be remarkable.
3, the Identification of Species of interior raw Antagonistic Fungi
Through morphological observation, in conjunction with physiological and biochemical index (table 2) and this endophyte of molecular biology identification Ba4, be bacillus amyloliquefaciens.
Single bacteria colony white of having cultivated the Ba4 bacterial strain of 2d on NA plate culture medium, rounded, edge is irregular, and surface drying has fold, inviscid, opaque, G+.
Table 2 bacterial strain Ba4 physiological and biochemical index
4, pot experiment
(1) Ba4 biocontrol microorganisms is prepared
Nutrition meat nutrient solution (extractum carnis 7.0g, peptone 10g, NaCl5g, water 1000mL) after autoclaving, in 100mL liquid, be contained in 300mL triangular flask ratio (logical oxygen is controlled) bottling, at sterile state inoculation Ba4 inclined-plane seed, 2 inclined-plane seeds of every bottle graft kind, temperature 26-28 ℃, initial pH value 7.0,120r/min shaking culture 36h, makes Ba4 microbial inoculum.
(2) cause of disease inoculation and processing
Choosing biennial basin walnut seedling is experimental subjects, PDA inclined-plane walnut pine root fungus (Fusarium solani) conidium (concentration 3 * 10
5) inoculate root system after one week, with above-mentioned microbial inoculum, adopt root-pouring method to divide different concns to carry out root irrigation to Juglans, every basin 200mL, and compare with sterilized water, repeat statistics walnut incidence 3 times.Every processing Juglans 50 strains.
Table 3 is Ba4 endogenetic bacteria prevention effect after pot experiment 30d, can find out, Ba4 endogenetic bacteria fermented liquid is after dilution, show different prevention effect, stoste to 200 times following effect is remarkable, particularly, below 100 times, can substantially control disease and not occur.
The prevention effect of table 3Ba4 endogenetic bacteria after pot experiment 30d
5, field control example
5.1, Ba4 liquid preparation is made
(1) primary inclined plane seed: ordinary method is made beef extract-peptone slant medium (extractum carnis 7.0g, peptone 10g, NaCl5g, agar 17g, water 1000mL) is cultivated and within 24-36 hour, is made first order seed after inoculation Ba4 at 26-28 ℃.
(2) secondary liquid seeds: nutrition meat nutrient solution (extractum carnis 7.0g, peptone 10g, NaCl5g, Ramulus Juglandis steeping fluid 1000mL) after autoclaving, in 100mL liquid, be contained in 300mL triangular flask ratio (logical oxygen is controlled) bottling, at sterile state inoculation Ba4 inclined-plane seed, 2 inclined-plane seeds of every bottle graft kind, temperature 26-28 ℃, initial pH value 7.0,120r/min shaking culture 36h, makes Ba4 liquid seeds.
(3) liquid fermenting: nutrition meat nutrient solution (extractum carnis 7.0g, peptone 10g, NaCl5g, Ramulus Juglandis steeping fluid 1000mL) after autoclaving, in 200mL liquid, be contained in 500mL triangular flask ratio (logical oxygen is controlled) bottling, at sterile state inoculation Ba4 liquid seeds, inoculum size 10%(volume ratio), temperature 26-28 ℃, initial pH value 7.0,120/min shaking culture 72h, makes Ba4 liquid preparation.
(4) preparation is preserved: bottled preparation normal temperature preserves half a year or low temperature (4 degree) does not affect prevention and result for the treatment of in 1 year half.
(5) preparation of the Walnut juice steeping fluid in step (2), (3): 10g Walnut juice is put into 1000ml water and boiled 15 minutes, filters, and the filtrate making is Walnut juice steeping fluid.
5.2, field prevention and treatment
(1) preventive treatment: doubly (volume ratio) extent of dilution is along root system breadth filling root with above-mentioned liquid preparation 100-300 for the walnut forest land of not falling ill, and 100 milliliters of every strains, use once every year spring, can prevent root rot to occur.The results are shown in Table 4.
(2) control processing: the walnut of falling ill is pressed 50-200 times of (volume ratio) extent of dilution and drenched filling along root system breadth filling root and near soil, 200 milliliters of every strains, spring, summer respectively once, can be imitated treatment root rot, the results are shown in Table 5.
Table 4Ba4 endogenetic bacteria field preventive effect: the walnut forest land of not falling ill (life in 1 year)
Note: the comparison of * otherness: containing same letter, be that difference is not remarkable, otherwise be remarkable.
Table 5Ba4 endogenetic bacteria Field treatment effect: morbidity walnut forest land
Note: the comparison of * otherness: containing same letter, be that difference is not remarkable, otherwise be remarkable.
Should be understood that, for those of ordinary skills, can be improved according to the above description or convert, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.
Claims (6)
1. a bacillus amyloliquefaciens, is characterized in that, deposit number is CGMCC No.8177.
2. the making method of the liquid preparation of bacillus amyloliquefaciens according to claim 1, is characterized in that, comprises the following steps:
(1) primary inclined plane seed: ordinary method is made beef extract-peptone slant medium, inoculates after described bacillus amyloliquefaciens, cultivates, and makes first order seed;
(2) secondary liquid seeds: nutrition meat nutrient solution is after autoclaving, under logical oxygen is controlled, in 100mL liquid, be contained in the bottling of 300mL triangular flask ratio, then under sterile state, inoculate described bacillus amyloliquefaciens inclined-plane seed, 2 inclined-plane seeds of every bottle graft kind, shaking culture, makes bacillus amyloliquefaciens liquid seeds;
(3) liquid fermenting: nutrition meat nutrient solution is after autoclaving, under controlling, logical oxygen is contained in the bottling of 500mL triangular flask ratio in 200mL liquid, the bacillus amyloliquefaciens liquid seeds that inoculation step under sterile state (2) makes, inoculum size is 10% of total liquid volume, shaking culture, make bacillus amyloliquefaciens liquid preparation, then preserve.
3. the making method of the liquid preparation of bacillus amyloliquefaciens according to claim 2, is characterized in that, in step (1), the composition of beef extract-peptone slant medium is extractum carnis 7.0g, peptone 10g, NaCl5g, agar 17g, water 1000mL.
4. the making method of the liquid preparation of bacillus amyloliquefaciens according to claim 2, is characterized in that, the composition of nutrition meat nutrient solution is extractum carnis 7.0g, peptone 10g, NaCl5g, Ramulus Juglandis steeping fluid 1000mL.
5. the liquid preparation obtaining according to the arbitrary described making method of claim 2-4.
6. the application of liquid preparation according to claim 5 in control walnut root rot, it is characterized in that, the walnut forest land of not falling ill is filled with root with 100-300 times of extent of dilution of described bacillus amyloliquefaciens liquid preparation volume ratio along root system breadth, 100 milliliters of every strains, use once annual spring, can prevent root rot to occur; The walnut of falling ill is filled with root and near soil pouring filling by 50-200 times of extent of dilution of described bacillus amyloliquefaciens liquid preparation volume ratio along root system breadth, 200 milliliters of every strains, and spring, summer respectively once, can be imitated treatment root rot.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104593284A (en) * | 2014-09-06 | 2015-05-06 | 山西农业大学 | Walnut endophyte and use thereof |
| CN107760640A (en) * | 2017-11-24 | 2018-03-06 | 四川农业大学 | A kind of Methylotrophic bacillus B18 and its liquid preparation preparation and application |
| CN110192486A (en) * | 2019-04-24 | 2019-09-03 | 新疆林科院经济林研究所 | A method of for preventing and treating jujube tree virosis |
| CN111139199A (en) * | 2020-01-13 | 2020-05-12 | 中化农业（临沂）研发中心有限公司 | Bacillus amyloliquefaciens CGMCC No.17843 and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102839142A (en) * | 2012-09-14 | 2012-12-26 | 杨凌绿都生物科技有限公司 | Bacillus amyloliquefaciens (Ba 168) and fermenting culture method and application of bacillus amyloliquefaciens |
| CN102864100A (en) * | 2012-08-16 | 2013-01-09 | 北京雷力农用化学有限公司 | Bacillus amyloliquefaciens and application thereof |
| CN103555618A (en) * | 2013-10-21 | 2014-02-05 | 四川农业大学 | Bacillus amyloliquefaciens and application of liquid preparations thereof in treatment of cenangium ferruginosum |
-
2013
- 2013-10-21 CN CN201310493899.0A patent/CN103555616B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102864100A (en) * | 2012-08-16 | 2013-01-09 | 北京雷力农用化学有限公司 | Bacillus amyloliquefaciens and application thereof |
| CN102839142A (en) * | 2012-09-14 | 2012-12-26 | 杨凌绿都生物科技有限公司 | Bacillus amyloliquefaciens (Ba 168) and fermenting culture method and application of bacillus amyloliquefaciens |
| CN103555618A (en) * | 2013-10-21 | 2014-02-05 | 四川农业大学 | Bacillus amyloliquefaciens and application of liquid preparations thereof in treatment of cenangium ferruginosum |
Non-Patent Citations (1)
| Title |
|---|
| 郝建安 等: "解淀粉芽孢杆菌NK10.BAhjaWT抑真菌作用的研究", 《微生物学通报》, vol. 35, no. 6, 20 June 2008 (2008-06-20) * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104593284A (en) * | 2014-09-06 | 2015-05-06 | 山西农业大学 | Walnut endophyte and use thereof |
| CN104593284B (en) * | 2014-09-06 | 2017-10-10 | 山西农业大学 | One plant of Endophyte of walnut and its application |
| CN107760640A (en) * | 2017-11-24 | 2018-03-06 | 四川农业大学 | A kind of Methylotrophic bacillus B18 and its liquid preparation preparation and application |
| CN107760640B (en) * | 2017-11-24 | 2020-06-09 | 四川农业大学 | Bacillus methylotrophicus B18 and preparation and application of liquid preparation thereof |
| CN110192486A (en) * | 2019-04-24 | 2019-09-03 | 新疆林科院经济林研究所 | A method of for preventing and treating jujube tree virosis |
| CN111139199A (en) * | 2020-01-13 | 2020-05-12 | 中化农业（临沂）研发中心有限公司 | Bacillus amyloliquefaciens CGMCC No.17843 and application thereof |
| CN111139199B (en) * | 2020-01-13 | 2021-08-31 | 中化农业（临沂）研发中心有限公司 | Bacillus amyloliquefaciens CGMCC No.17843 and application thereof |
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