CN103555616B - One bacillus amyloliquefaciens and the application of liquid preparation in control walnut root rot thereof - Google Patents

One bacillus amyloliquefaciens and the application of liquid preparation in control walnut root rot thereof Download PDF

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CN103555616B
CN103555616B CN201310493899.0A CN201310493899A CN103555616B CN 103555616 B CN103555616 B CN 103555616B CN 201310493899 A CN201310493899 A CN 201310493899A CN 103555616 B CN103555616 B CN 103555616B
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bacillus amyloliquefaciens
liquid
root rot
walnut
root
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CN103555616A (en
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朱天辉
朱涵明月
李姝江
张静
刘洋
韩珊
谯天敏
张丽娜
郑磊
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Sichuan Agricultural University
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Sichuan Agricultural University
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Abstract

The invention discloses a bacillus amyloliquefaciens and the application of liquid preparation in control walnut root rot thereof, the Classification And Nomenclature of this bacterial strain is bacillus amyloliquefaciens, Bacillus? amyloliquefaciens, complete called after bacillus amyloliquefaciens Ba4, being deposited in No. 1, North Star West Road, Chaoyang District, city of BeiJing, China institute No. 3 China Committee for Culture Collection of Microorganisms's common micro-organisms centers on September 12nd, 2013, does is deposit number CGMCC? No.8177.Bacillus amyloliquefaciens Ba4 provided by the invention has special efficacy to walnut root rot, and has resistance, and Storage is good, and high temperature resistance, low temperature, resistance to drying, be suitable for merchandized handling, and use once can permanently effective, to continue control sick, significantly reduce costs.

Description

One bacillus amyloliquefaciens and the application of liquid preparation in control walnut root rot thereof
Technical field
The present invention relates to field of ecology, in particular a bacillus amyloliquefaciens and the application of liquid preparation in control walnut root rot thereof.
Background technology
Walnut has important economic worth and Ecology Action, has very consequence in China's forestry development and improvement of the ecological environment, particularly has positive effect for raising Agricultural Economy of The Mountainous Regions level.Walnut root rot (Fusariumsolani) is injured based on Seedling Stage, and the nursery stock the lighter that is injured affects growth, and severe one causes mortality, becomes a large obstacle of nursery work.Susceptible seedling, main manifestations is that root blackening is rotted, and overground part yellow leaf, leaf margin blackening, mortality ratio is very high.The large tree that grows up also can fall ill cultivation area, present Sichuan, is a potential threat to Walnut Industry.Main employing chemical prevention and tending technique at present, and bio-control method research not yet relates to.Though chemical prevention is a kind of effectively preventing means, there is many practical problemss such as pathogenic bacteria resistance, environment and food safety, poisoning etc., some chemical bactericides strictly limit use at many developed countries and regions.And be more and more subject to the favor of people with eco-friendly biological pesticide, be the important component part of the future of agriculture Sustainable development.
Therefore, prior art existing defects, needs to improve.
Summary of the invention
Technical problem to be solved by this invention is the defect of preventing and treating walnut root rot for prior art, provides a bacillus amyloliquefaciens and the application of liquid preparation in control walnut root rot thereof.
Technical scheme of the present invention is as follows:
One bacillus amyloliquefaciens, its Classification And Nomenclature is bacillus amyloliquefaciens, Bacillusamyloliquefaciens, be deposited in No. 1, North Star West Road, Chaoyang District, city of BeiJing, China institute No. 3 China Committee for Culture Collection of Microorganisms's common micro-organisms centers on 09 12nd, 2013, deposit number is CGMCCNo.8177.
The making method of the liquid preparation of described bacillus amyloliquefaciens, comprises the following steps:
(1) primary inclined plane seed: ordinary method makes beef extract-peptone slant medium, after inoculating described bacillus amyloliquefaciens, cultivates, makes first order seed;
(2) secondary liquid seed: nutrition meat nutrient solution is after autoclaving, under logical oxygen controls, the bottling of 300mL triangular flask ratio is contained in 100mL liquid, then described bacillus amyloliquefaciens inclined-plane seed is inoculated under aseptic conditions, every bottle graft kind 2 inclined-plane seeds, shaking culture, makes bacillus amyloliquefaciens liquid seeds;
(3) liquid fermenting: nutrition meat nutrient solution is after autoclaving, the bottling of 500mL triangular flask ratio is contained in 200mL liquid under logical oxygen controls, the bacillus amyloliquefaciens liquid seeds that inoculation step (2) is obtained under aseptic conditions, 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, it is characterized in that, 100-300 times of extent of dilution of the described bacillus amyloliquefaciens liquid formulation volume ratio of walnut forest land of not falling ill fills with root along root system breadth, every strain 100 milliliters, annual spring uses once, and root rot can be prevented to occur; Morbidity walnut fills with root and neighbouring soil pouring filling than 50-200 times of extent of dilution along root system breadth by described bacillus amyloliquefaciens liquid formulation volume, and every strain 200 milliliters, spring, summer respectively once, can imitate treatment root rot.
The present invention has filtered out the specific new strains 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 drying, be suitable for merchandized handling, and use once can permanently effective, to continue control sick, 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
Gather healthy Walnut Roots in walnut root rot spot, Chaotian District, Sichuan Guangyuan city, take back laboratory and be separated.The sample aseptic water washing adopted back is clean, take 1.0g root tissue, with the alcohol disinfecting 30s of 70%, after aseptic washing 3 times, again with after the NaClO sterilization 3min of 4%, aseptic washing 3 times, draws last washings 100 μ l and smears NA flat board (extractum carnis 3g, peptone 10g, sodium-chlor 5g, agar powder 15-20g, distilled water 1000ml, pH7.0,121 DEG C of autoclaving 30min after mixing packing), 28 DEG C 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, puts into aseptic mortar, add quartz sand and the 10ml sterilized water of sterilizing, fully grind, leave standstill 30min, dilution 10 -1, 10 -2, 10 -3, 10 -4, 10 -5totally 5 gradients, draw 100 μ l10 respectively -3, 10 -4, 10 -5these 3 gradient lapping liquids are coated with NA flat board (extractum carnis 3g respectively, peptone 10g, sodium-chlor 5g, agar powder 15-20g, distilled water 1000ml, pH7.0,121 DEG C of autoclaving 30min after mixing packing), each process repeats for 3 times, 28 DEG C of dark culturing 48h, choose the most uniform gradient of growth, carry out plate count.
According to the size of bacterium colony, color, whether projection, 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, and NA inclined-plane of transferring after purifying is for subsequent use in 4 DEG C of preservations.Obtain 10 endogenetic bacterias altogether.
2, endogenetic bacteria is to the dull and stereotyped antagonistic action of walnut root rot germ
Dull and stereotyped face-off method: at walnut root rot germ (Fusariumsolani) the bacterium cake (diameter 5mm) of culture dish central authorities inoculation 7d, 3cm place is about from this germ cake, the endophyte cultivating 2d is picked respectively with transfering loop, at each standardized bar fine rule in the symmetrical both sides of germ, 28 DEG C of cultivations, only to inoculate the flat board of germ for contrast, in time contrasting and cover with ware, measure the colony growth diameter of germ, often process repetition 3 times.Be calculated as follows colony growth inhibiting rate:
Colony growth inhibiting rate=(contrast bacterium colony clean growth diameter-clean growth diameter of process bacterium colony)/clean growth diameter × 100% of contrast bacterium colony
Table 1 is the antagonistic action of endogenetic bacteria to walnut root rot germ (Fusariumsolani), 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 (7 days) of walnut root rot germ
Note: * otherness compares: be that difference is remarkable containing same letter, otherwise be remarkable.
3, the Identification of Species of interior raw Antagonistic Fungi
Through morphological observation, be bacillus amyloliquefaciens in conjunction with physiological and biochemical index (table 2) and this endophyte of molecular biology identification Ba4.
NA plate culture medium has been cultivated single bacteria colony white of the Ba4 bacterial strain of 2d, and 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 prepares
Nutrition meat nutrient solution (extractum carnis 7.0g, peptone 10g, NaCl5g, water 1000mL) after autoclaving, be contained in 300mL triangular flask ratio (logical oxygen controls) bottling in 100mL liquid, at sterile state inoculation Ba4 inclined-plane seed, every bottle graft kind 2 inclined-plane seeds, temperature 26-28 DEG C, initial pH value 7.0,120r/min shaking culture 36h, makes Ba4 microbial inoculum.
(2) cause of disease inoculation and process
Choosing biennial basin walnut seedling is experimental subjects, PDA inclined-plane walnut pine root fungus (Fusariumsolani) conidium (concentration 3 × 10 5) inoculate root system after one week, adopt root-pouring method to divide different concns to carry out root irrigation to Juglans with above-mentioned microbial inoculum, every basin 200mL, and compare with sterilized water, repeat 3 times, statistics walnut incidence.Often process Juglans 50 strain.
Table 3 is Ba4 endogenetic bacteria prevention effect after pot experiment 30d, can find out, after the dilution of Ba4 endogenetic bacteria fermentation liquor, show different prevention effect, Be very effective below stoste to 200 times, can substantially control disease and not occur by particularly less than 100 times.
The prevention effect of table 3Ba4 endogenetic bacteria after pot experiment 30d
5, field control example
5.1, Ba4 liquid preparation makes
(1) primary inclined plane seed: ordinary method makes beef extract-peptone slant medium (extractum carnis 7.0g, peptone 10g, NaCl5g, agar 17g, water 1000mL), cultivates after inoculation Ba4 and makes first order seed in 24-36 hour at 26-28 DEG C.
(2) secondary liquid seed: nutrition meat nutrient solution (extractum carnis 7.0g, peptone 10g, NaCl5g, Ramulus Juglandis steeping fluid 1000mL) after autoclaving, be contained in 300mL triangular flask ratio (logical oxygen controls) bottling in 100mL liquid, at sterile state inoculation Ba4 inclined-plane seed, every bottle graft kind 2 inclined-plane seeds, temperature 26-28 DEG C, 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, be contained in 500mL triangular flask ratio (logical oxygen controls) bottling in 200mL liquid, at sterile state inoculation Ba4 liquid seeds, inoculum size 10%(volume ratio), temperature 26-28 DEG C, 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 therapy effect 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 obtained filtrate is Walnut juice steeping fluid.
5.2, field prevention and treatment
(1) preventive treatment: the above-mentioned liquid preparation 100-300 of walnut forest land that do not fall ill doubly (volume ratio) extent of dilution fills with root along root system breadth, and every strain 100 milliliters, annual spring uses once, and root rot can be prevented to occur.The results are shown in Table 4.
(2) Prevention technology: morbidity walnut presses 50-200, and doubly (volume ratio) extent of dilution is along root system breadth filling root and neighbouring soil pouring filling, and every strain 200 milliliters, spring, summer respectively once, can be imitated treatment root rot, the results are shown in Table 5.
Table 4Ba4 endogenetic bacteria field preventive effect: walnut forest land of not falling ill (life in 1 year)
Note: * otherness compares: be that difference is remarkable containing same letter, otherwise be remarkable.
Table 5Ba4 endogenetic bacteria Field treatment effect: morbidity walnut forest land
Note: * otherness compares: be that difference is remarkable containing same letter, 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 improve and convert the protection domain that all should belong to claims of the present invention.

Claims (3)

1. the application of bacillus amyloliquefaciens liquid preparation in control walnut root rot, it is characterized in that, the deposit number of bacillus amyloliquefaciens is CGMCCNo.8177; The making method of bacillus amyloliquefaciens liquid preparation comprises the following steps:
(1) primary inclined plane seed: ordinary method makes beef extract-peptone slant medium, after inoculating described bacillus amyloliquefaciens, cultivates, makes first order seed;
(2) secondary liquid seed: nutrition meat nutrient solution is after autoclaving, under logical oxygen controls, the bottling of 300mL triangular flask ratio is contained in 100mL liquid, then described bacillus amyloliquefaciens inclined-plane seed is inoculated under aseptic conditions, every bottle graft kind 2 inclined-plane seeds, shaking culture, makes bacillus amyloliquefaciens liquid seeds;
(3) liquid fermenting: nutrition meat nutrient solution is after autoclaving, the bottling of 500mL triangular flask ratio is contained in 200mL liquid under logical oxygen controls, the bacillus amyloliquefaciens liquid seeds that inoculation step (2) is obtained under aseptic conditions, inoculum size is 10% of total liquid volume, shaking culture, make bacillus amyloliquefaciens liquid preparation, then preserve;
Application method is: 100-300 times of extent of dilution of the described bacillus amyloliquefaciens liquid formulation volume ratio of walnut forest land of not falling ill fills with root along root system breadth, every strain 100 milliliters, and annual spring uses once, and root rot can be prevented to occur; Morbidity walnut fills with root and neighbouring soil pouring filling than 50-200 times of extent of dilution along root system breadth by described bacillus amyloliquefaciens liquid formulation volume, and every strain 200 milliliters, spring, summer respectively once, can imitate treatment root rot.
2. application according to claim 1, 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.
3. application according to claim 1, is characterized in that, the composition of nutrition meat nutrient solution is extractum carnis 7.0g, peptone 10g, NaCl5g, Ramulus Juglandis steeping fluid 1000mL.
CN201310493899.0A 2013-10-21 2013-10-21 One bacillus amyloliquefaciens and the application of liquid preparation in control walnut root rot thereof Expired - Fee Related CN103555616B (en)

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CN104593284B (en) * 2014-09-06 2017-10-10 山西农业大学 One plant of Endophyte of walnut and its 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
CN110192486B (en) * 2019-04-24 2021-03-30 新疆林科院经济林研究所 Method for preventing and treating virus diseases of jujube trees
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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CN102839142B (en) * 2012-09-14 2017-02-08 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
CN103555618B (en) * 2013-10-21 2015-05-06 四川农业大学 Bacillus amyloliquefaciens and application of liquid preparations thereof in treatment of cenangium ferruginosum

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