CN103642727B - The bacillus amyloliquefaciens of one strain Antifungi and application thereof - Google Patents
The bacillus amyloliquefaciens of one strain Antifungi and application thereof Download PDFInfo
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Abstract
Do you the invention discloses a kind of bacillus amyloliquefaciens (Bacillus? amyloliquefaciens) KB, does is preserving number CGMCC? No.8179.Described bacillus amyloliquefaciens KB adopts rear brown rot germ to peach, rear ash arrhizus bacteria adopted by apple, rear ash arrhizus bacteria adopted by tomato and apple to adopt rear mould germ inhibited; Rear brown heart is adopted to peach, that rear gray mold adopted by apple, rear gray mold adopted by tomato, rear penicilliosis adopted by apple is inhibited.The most obvious to the restraining effect of Monilinia fructicola and apple ash arrhizus bacteria, antibacterial circle diameter is respectively 22mm and 20mm, reaches 100% to the inhibiting rate of peach brown rot spot and apple gray mold scab.
Description
Technical field
The bacillus amyloliquefaciens of one strain Antifungi and application thereof, belong to technical field of microbe application.
Background technology
Fungal diseases of plants brings massive losses to agriculture production, and after Fruit, fungal disease is the important disease of a class, causes a large amount of underproduction of fruit, has a strong impact on edibleness and the marketable value of fruit, and the income that result in orchard worker reduces.In developed country, the loss amount that after picking fruit, disease causes accounts for more than 15% of fruit total amount, and in developing country owing to lacking necessary Control Technology and equipment, the loss amount that after picking fruit, disease causes accounts for more than 30% of total amount.After picking fruit, the generation of disease has caused to the production of the especially high-end fruit and vegetable food of modern agricultural production and processing and has had a strong impact on.
The peach brown rot caused by Moniliniaspp. pathogenic fungi is all worldwide the first disease of peach at present, is also the first problem affecting peach industry development.According to statistics, in developed country as the U.S., Israel, Italy, France etc., adopt the 10%-30% that loss amount that rear brown heart causes just accounts for peach ultimate production; In developing country owing to lacking necessary Prevention Technique and relevant device, its loss amount reaches 30%-50%, even higher.Some area, the big area of peach brown rot seriously occurs, and almost causes the total crop failure of peach fruit.Therefore, the control of peach brown rot and control are seemed more urgent and important.China's peach garden area, more than 9,000,000 mu, is peach effective cultivated area first big country in the world; Producing peach per year more than 400,000 tons, is peach output first big country in the world.Beijing peach output occupies domestic last eight, only Pinggu County, beijing one ground, and peach cultivated area just reaches more than 220,000 mu (1.47 ten thousand hectares).Peach industry has become the pillar of the economy industry in Pinggu county, and some peaches and relevant converted products have exported to European Union, Japan and other countries.Since 2000, peach brown rot big area in Beijing increases, and increases the weight of year by year; The peach garden that wherein morbidity in 2005 is heavier, peach fruit is injured, and come off in a large number, have no the fruit of aobvious disease during harvesting, 2-3 days after harvesting, the fruit of more than 50% rots because brown heart infects, and this causes serious financial loss to orchard worker.Beijing plant protection unit peach brown rot investigation result display in 2012, each suburban county peach producing region, Beijing all has peach brown rot to occur, and the transport of peach after adopting and duration of storage sickness rate are all more than 30%.This greatly reduces edibleness and the marketable value of fruit, has had a strong impact on the development of Beijing peach industry.Therefore effective prevention and control of rear brown heart are adopted to peach and Economic development is especially closely connected with the income of orchard worker.
In current production to the control of peach brown rot still based on chemoprevention, but " 3R " negative impact brought thus is very serious.A large amount of use havocs of chemical pesticide ecotope, causes pesticide residue a large amount of in Peach fruits, affects people healthy.In northern peach producing region, before the harvesting of peach and after plucking, be equally also based on chemical pesticide to the means of prevention of peach brown rot, this not only affects the healthy of the people, also limit processing and the outlet of peach product.In China's Agenda 21 has been listed development biological pesticide and green food by China, and construction requirements Beijing of Beijing modern agriculture Science and Technology City and surrounding area crop pest realize green prevention and control, agricultural prods carries out green production.
Genus bacillus (Bacillusspp.) ubiquity in soil, it is one of superior microorganism population be present in soil and plant microecosystem, owing to producing endogenous spore, there is the ability of stronger opposing ambient pressure environment, can resist because of the injury that dry, thermal and uv radiation cause in survived environment, maintain its own existence ability unaffected.This biological property makes genus bacillus have very good application prospect, particularly in the biological products using active bacteria formulation, shows powerful vitality.Genus bacillus is of a great variety, enormous amount, can produce diversified physiologically active substance and meta-bolites, has prospects for commercial application widely.
Bacillus amyloliquefaciens is very wide in occurring in nature distribution, not yet has the report of the bacillus amyloliquefaciens preventing and treating peach brown rot at present.
Summary of the invention
The object of the present invention is to provide a kind of bacillus amyloliquefaciens (Bacillusamyloliquefaciens), bacterial strain KB, preserving number is CGMCCNo.8179.
Object of the present invention is also to provide a kind of pathogenic bacteria inhibitor, and its activeconstituents is above-mentioned bacillus amyloliquefaciens and/or its metabolite.
Object of the present invention is also to provide a kind of disease suppression agent, and its activeconstituents is above-mentioned bacillus amyloliquefaciens and/or its metabolite.
The bacterial strain number of bacillus amyloliquefaciens provided by the present invention (Bacillusamyloliquefaciens) is KB, and it is numbered CGMCCNo.8179 in registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center.
Shown in the described 16SrDNA sequence SEQIDNo.1 stating bacillus amyloliquefaciens (Bacillusamyloliquefaciens) KB.
The morphological feature of described bacillus amyloliquefaciens (Bacillusamyloliquefaciens) KB is: somatic cells is rod-short, directly or closely straight, and raw in gemma, column or ellipse, have mobility; LB substratum does not produce pigment, and bacterium colony is White-opalescent, flat or circular, matt, and along with incubation time increases, the thickening exsiccation of bacterium colony, edge is irregular, and bacterium colony has fold, becomes creamy.The physiological and biochemical property of described bacillus amyloliquefaciens (Bacillusamyloliquefaciens) KB is Gram-positive, form mycoderm, methyl red, nitrate reduction, acetyl methyl carbinol, indole reaction, catalase is positive, hydrolyzable gelatin, urea, Mierocrystalline cellulose, starch and Vitamin C2, oxydase reaction, decompose tyrosine negative, produce hydrogen sulfide, utilize Citrate trianion, do not utilize malonate, arginine, Methionin, ornithine decarboxylase reacting positive, phenylalanine deaminase reaction negative, glucose, sucrose produces sour aerogenesis, glucose can be utilized, maltose, sorbyl alcohol, rhamnosyl, synanthrin, semi-lactosi, inositol, wood sugar, seminose, N.F,USP MANNITOL, ribose, sucrose etc., do not utilize pectinose and sorbose.Described bacillus amyloliquefaciens (Bacillusamyloliquefaciens) KB is in containing 1%-7%(mass content) all can normal growth in the substratum of sodium-chlor, in containing 10%(mass content) can not grow in the substratum of sodium-chlor, all can normal growth in the substratum of pH4.5-9.0, under pH4.0, growth potential is weak, can not grow under pH9.5, at envrionment temperature is 4 DEG C, growth potential is weak, at 55 DEG C-80 DEG C, process 15min, still can normal growth.Described bacillus amyloliquefaciens (Bacillusamyloliquefaciens) KB produces addicted to iron element, cellulase, proteolytic enzyme and chitinase, and these are all the key biological and ecological methods to prevent plant disease, pests, and erosion indexs of biocontrol microorganisms.
The present invention also aims to provide above-mentioned bacillus amyloliquefaciens and/or its metabolite at suppression pathogenic bacteria, preparation pathogenic bacteria inhibitor, suppress disease, the application prepared in disease suppression agent.
Described pathogenic bacteria is following at least one:
A, peach adopt rear brown rot germ;
Rear ash arrhizus bacteria adopted by B, apple;
Rear ash arrhizus bacteria adopted by C, tomato;
Rear mould germ adopted by D, apple;
Described disease is following at least one:
A, peach adopt rear brown heart;
Rear gray mold adopted by B, apple;
Rear gray mold adopted by C, tomato;
Rear penicilliosis adopted by D, apple.
Wherein, described peach is adopted rear brown rot germ and can be Moniliniafructicola or Monilinialaxa or Moniliniafructigena; Rear ash arrhizus bacteria adopted by described apple is Botrytiscinerea; Rear ash arrhizus bacteria adopted by described tomato is Botrytiscinerea; Rear mould germ adopted by described apple is Penicilliumexpansum.
The metabolite of described bacillus amyloliquefaciens (Bacillusamyloliquefaciens) KB can obtain from the fermented liquid of described bacillus amyloliquefaciens (Bacillusamyloliquefaciens) KB.
The metabolite of described bacillus amyloliquefaciens (Bacillusamyloliquefaciens) KB specifically can be prepared as follows: cultivate described bacillus amyloliquefaciens (Bacillusamyloliquefaciens) KB in liquid medium within, and described bacillus amyloliquefaciens (Bacillusamyloliquefaciens) KB in removing liquid culture (fermented liquid) namely obtains the metabolite of described bacillus amyloliquefaciens.
Bacillus amyloliquefaciens (Bacillusamyloliquefaciens) KB in the present invention adopts rear brown rot germ to peach, rear ash arrhizus bacteria adopted by apple, rear ash arrhizus bacteria adopted by tomato and apple to adopt rear mould germ inhibited; Rear brown heart is adopted to peach, that rear gray mold adopted by apple, rear gray mold adopted by tomato, rear penicilliosis adopted by apple is inhibited.To in selected plant pathogenic fungi, the most obvious to the restraining effect of Monilinia fructicola and apple ash arrhizus bacteria, antibacterial circle diameter is respectively 22mm and 20mm, reaches 100% to the inhibiting rate of peach brown rot spot and apple gray mold scab.Illustrate that bacillus amyloliquefaciens (Bacillusamyloliquefaciens) KB has anti-microbial property that is stable, efficient, wide spectrum.
Biomaterial preservation explanation
Classification And Nomenclature: bacillus amyloliquefaciens (Bacillusamyloliquefaciens)
Strain number: KB
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism is called for short: CGMCC
Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Preservation date: on September 12nd, 2013
Register on the books numbering: CGMCCNo.8179. at preservation center
Accompanying drawing explanation
Fig. 1 .a) described bacillus amyloliquefaciens (Bacillusamyloliquefaciens) KB on PDA substratum to the inhibition of Monilinia fructicola Moniliniafructicola; B) described bacillus amyloliquefaciens (Bacillusamyloliquefaciens) KB is to the inhibition of apple gray mold Botrytiscinerea; C) described bacillus amyloliquefaciens (Bacillusamyloliquefaciens) KB is to the inhibition of graw mold of tomato Botrytiscinerea;
Fig. 2 .a) described solution amylase genus bacillus (Bacillusamyloliquefaciens) KB is to the inhibition of peach brown rot Lesion size; B) described solution amylase genus bacillus (Bacillusamyloliquefaciens) KB is to apple gray mold Lesion size inhibition; C) described solution amylase genus bacillus (Bacillusamyloliquefaciens) KB is to green mold of apple Lesion size inhibition; D) described solution amylase genus bacillus (Bacillusamyloliquefaciens) KB is to graw mold of tomato Lesion size inhibition.
Embodiment
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
The pathogenic bacteria of using in following embodiment, the public from field acquisition, also can obtain from Beijing City Agriculture and Forestry Institute, to repeat the application's experiment:
Botrytis cinerea---Botrytis cinerea (BotrytiscinereaPer.exFr.) (Li Xinghong etc. Beijing area botrytis cinerea is to the liquefaction resistance of phonetic mould amine. and plant protection .2012.38 (4): 141-143)
Monilinia fructicola---chain sclerotinia sclerotiorum (Moniliniafructicola (wint.) Rehm) (Wang Fei etc. the occurrence and control of peach brown rot. fruit tree flowers .2012 the 5th phase p58).
Rear ash arrhizus bacteria adopted by apple--Botrytis cinerea (BotrytiscinereaPer.exFr);
Rear mould germ adopted by apple--Penicillium patulum enzyme bacterium (PenicilliumexpansumLink, 1809)
Embodiment 1
The separation of bacillus amyloliquefaciens of the present invention (Bacillusamyloliquefaciens) KB and identification of strains
1, sample collecting
From having the organic orchard of north of China, gather peach and Kiwifruit.
2, the separation screening of bacterial strain and antagonism screening
Isolated strains from organic fruits, conventional gradients dilution spread is adopted to be separated, with LB, NA, KB and CM0002 totally 4 kinds of substratum cultivate respectively under 28 DEG C of conditions, and picking colony morphological differences is comparatively large and can the bacterium of pure culture, purification storage on LB substratum, and be that target pathogens carries out the primary dcreening operation of Antagonistic Fungi and repeatedly sieves again with Monilinia fructicola, finally obtain the bacterial isolates that a strain bacteriostatic activity is strong, name it to be KB.
3, the qualification of bacterial strain
Bacterial strain KB is carried out to the analysis of morphology, physio-biochemical characteristics and part conserved sequence.With reference to " soil microorganisms research principle and method " (Lin Xiangui. soil microorganisms study principle and method [M], Higher Education Publishing House, 2010) middle described method carries out the mensuration of Physiology and biochemistry and environmental resistance characteristic.
Result shows, the somatic cells of bacterial strain KB is rod-short at 400 times of opticmicroscope hypothallus cells, directly or closely straight, and raw in gemma, column or ellipse, have mobility; LB substratum does not produce pigment, and bacterium colony is White-opalescent, flat or circular, matt, and along with incubation time increases, the thickening exsiccation of bacterium colony, edge is irregular, and bacterium colony has fold, becomes creamy.The physiological and biochemical property of bacterial strain KB is Gram-positive, form mycoderm, methyl red, acetyl methyl carbinol, nitrate reduction, indole reaction, catalase, urine enzyme positive, hydrolyzable gelatin, Mierocrystalline cellulose, starch and Vitamin C2, oxydase reaction, decompose tyrosine negative, produce hydrogen sulfide, utilize Citrate trianion, do not utilize malonate, arginine, Methionin, ornithine decarboxylase reacting positive, phenylalanine deaminase reaction negative, glucose, sucrose produces sour aerogenesis, glucose can be utilized, maltose, sorbyl alcohol, rhamnosyl, synanthrin, semi-lactosi, inositol, wood sugar, seminose, N.F,USP MANNITOL, ribose, sucrose etc., do not utilize pectinose and sorbose.The environmental resistance of bacillus amyloliquefaciens (Bacillusamyloliquefaciens) KB shows as in containing 1%-7%(mass content) all can normal growth in the substratum of sodium-chlor, in containing 10%(mass content) can not grow in the substratum of sodium-chlor, all can normal growth in the substratum of pH4.5-9.0, under pH4.0, growth potential is weak, can not grow under pH9.5, at envrionment temperature is 4 DEG C, growth potential is weak, at 55 DEG C-80 DEG C, process 15min, still can normal growth.The 16SrDNA sequence of bacterial strain KB is as shown in SEQIDNo.1.
Embodiment 2
The mensuration of bacillus amyloliquefaciens of the present invention (Bacillusamyloliquefaciens) KB antimicrobial spectrum
For examination pathogenic bacteria:
Monilinia fructicola---chain sclerotinia sclerotiorum (Moniliniafructicola (wint.) Rehm);
Botrytis cinerea---Botrytis cinerea (BotrytiscinereaPer.exFr);
Rear ash arrhizus bacteria adopted by apple--Botrytis cinerea (BotrytiscinereaPer.exFr);
Rear mould germ adopted by apple--Penicillium patulum enzyme bacterium (PenicilliumexpansumLink, 1809)
Selecting that peach adopts rear brown heart germ, rear ash arrhizus bacteria adopted by apple, rear mould germ adopted by apple etc. is instruction fungi, adopts dull and stereotyped opposite culture method.
Concrete operations are as follows: bacillus amyloliquefaciens (Bacillusamyloliquefaciens) KB bacterial strain 28 DEG C of constant temperature culture 48h on LB substratum.Plant pathogenic fungi is 25 DEG C of constant temperature culture 3-4 days on PDA flat board, make from the aseptic stainless steel punch tool of colony edge diameter 0.7cm the agar block that carries disease germs; With aseptic inoculation pin picking pathogenic bacteria bacterium sheet, mycelia faces down and is inoculated in PDA plate center place, simultaneously apart from all about 3.00cm place, pathogenic fungi bacterium sheet both sides with aseptic inoculation ring line bacillus amyloliquefaciens (Bacillusamyloliquefaciens) KB, only to connect the flat board of pathogenic bacteria for contrast, each process repeats for three times, 25 DEG C of constant temperature culture 5-7 days, measure the antibacterial bandwidth between pathogenic fungi edge and the bacterium colony bandwidth center of bacillus amyloliquefaciens (Bacillusamyloliquefaciens) KB.
By as follows for artificial punching inoculation method concrete operations in sporocarp (adopt rear gray mold be described to prevent and treat apple):
Bacillus amyloliquefaciens (Bacillusamyloliquefaciens) KB bacterial strain 28 DEG C of constant temperature culture 48h on LB liquid nutrient medium, being adjusted to bacterial concentration with distilled water is 5 × 10
7cfu/ml.Apple ash arrhizus bacteria B.cinerea is 25 DEG C of constant temperature culture 5-7 days on PDA flat board, collect botrytis cinerea spore, make 5 × 10 with distilled water
4the spore suspension of cfu/ml; Ripe apple, after first using 0.5% hypochlorite disinfectant, with sterilized pipettor gun head, along apple waist jack, 3, hole pricked by each fruit, and each hole is 30mm × 30mm.Every hole inoculation 30ulKB bacterium liquid, room temperature is dried, and then inoculates the pathogenic bacteria spore suspension of 30ul.Finally, at the fruit of process is positioned over 25 DEG C, after 5-7 days, measure fruit rot spot size.Rotten spot is less than 10mm and is regarded as not falling ill.According to rotten scab account for total inoculation hole count number, calculate sickness rate, the scab number of not falling ill account for total inoculation hole count number, calculate scab inhibiting rate.
Experimental result is in table 1, bacillus amyloliquefaciens (Bacillusamyloliquefaciens) KB is in selected plant pathogenic fungi, the most obvious to the restraining effect of Monilinia fructicola and apple ash arrhizus bacteria, antibacterial circle diameter is respectively 22mm and 20mm, reaches 100% to the inhibiting rate of peach brown rot spot and apple gray mold scab.Illustrate that bacillus amyloliquefaciens (Bacillusamyloliquefaciens) KB has anti-microbial property that is stable, efficient, wide spectrum.
Table 1B.amyloliquefaciensKB is to the inhibition (PDA, 28 DEG C, 72h) of pathogenic bacteria
Wherein, described peach is adopted rear brown rot germ and can be Moniliniafructicola or Monilinialaxa or Moniliniafructigena; Rear ash arrhizus bacteria adopted by described apple is Botrytiscinerea; Rear ash arrhizus bacteria adopted by described tomato is Botrytiscinerea;
Rear mould germ adopted by described apple is Penicilliumexpansum.
Bacillus amyloliquefaciens (Bacillusamyloliquefaciens) KB all has good inhibition as shown in table 1 and Fig. 2 to the rotten scab that the 5 kind of plant pathogenic fungies for examination cause.
Embodiment 3
Under holding conditions, described bacillus amyloliquefaciens (Bacillusamyloliquefaciens) KB is to apple, the experiment of tomato post-harvest diseases prevention effect:
1., after apple-picking, through KB bacterium liquid, (bacterial concentration is respectively 10
6cfu/ml, 10
7cfu/ml, 10
8cfu/ml, 10
9cfu/ml) immersion treatment 30-60 second, under room temperature condition, air-dry, be then 1-2 DEG C in temperature, relative humidity is under 90-95% condition, dark storage.Preserve after 10 months, compared with control group, when bacterial concentration is 10
6its preventive effect of cfu/ml is 45%, when bacterial concentration reaches 10
7during cfu/ml, its preventive effect reaches 65%, when bacterial concentration reaches 10
8cfu/ml and 10
9when cfu/ml, its preventive effect is respectively 70% and 75%.
2., after tomato picking, through KB bacterium liquid, (bacterial concentration is respectively 10
6cfu/ml, 10
7cfu/ml, 10
8cfu/ml, 10
9cfu/ml) immersion treatment 30-60 second, under room temperature condition, air-dry, be then 20 DEG C in temperature, relative humidity is under 90-95% condition, dark storage.Preserve after 5 days, compared with control group, when bacterial concentration is 10
6its preventive effect of cfu/ml is 40%, when bacterial concentration reaches 10
7during cfu/ml, its preventive effect reaches 55%, when bacterial concentration reaches 10
8cfu/ml and 10
9when cfu/ml, its preventive effect is respectively 60% and 65%.
Claims (6)
1. a bacillus amyloliquefaciens (Bacillusamyloliquefaciens), bacterial strain KB, preserving number is CGMCCNo.8179.
2. a pathogenic bacteria inhibitor, its activeconstituents is bacillus amyloliquefaciens described in claim 1.
3. pathogenic bacteria inhibitor according to claim 2, is characterized in that, described pathogenic bacteria inhibitor is inhibited to following at least one pathogenic bacteria:
A, peach adopt rear brown rot germ;
Rear ash arrhizus bacteria adopted by B, apple;
Rear ash arrhizus bacteria adopted by C, tomato;
Rear mould germ adopted by D, apple.
4. a disease suppression agent, its activeconstituents is bacillus amyloliquefaciens described in claim 1.
5. disease suppression agent according to claim 4, is characterized in that, described disease is following at least one:
A, peach adopt rear brown heart;
Rear gray mold adopted by B, apple;
Rear gray mold adopted by C, tomato;
Rear penicilliosis adopted by D, apple.
6. following arbitrary application of bacillus amyloliquefaciens described in claim 1:
1) application in pathogenic bacteria is being suppressed;
2) application in preparation pathogenic bacteria inhibitor;
3) application in disease is being suppressed;
4) application in disease suppression agent is being prepared;
Described pathogenic bacteria is following at least one:
A, peach adopt rear brown rot germ;
Rear ash arrhizus bacteria adopted by B, apple;
Rear ash arrhizus bacteria adopted by C, tomato;
Rear mould germ adopted by D, apple;
Described disease is following at least one:
A, peach adopt rear brown heart;
Rear gray mold adopted by B, apple;
Rear gray mold adopted by C, tomato;
Rear penicilliosis adopted by D, apple.
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| CN106520607B (en) * | 2016-11-04 | 2019-09-24 | 北京市农林科学院 | Bacillus amyloliquefaciens and application thereof in inhibition of plasmopara viticola |
| CN107090419B (en) * | 2017-05-24 | 2020-04-14 | 浙江大学 | Bacillus amyloliquefaciens and application thereof |
| CN107338202B (en) * | 2017-06-27 | 2020-09-11 | 浙江理工大学 | Bacillus amyloliquefaciens with broad-spectrum pathogenic bacterium inhibiting function and application thereof |
| CN108034611A (en) * | 2018-01-12 | 2018-05-15 | 中国科学院成都生物研究所 | One bacillus amyloliquefaciens and its application |
| CN108118017B (en) * | 2018-02-09 | 2021-06-29 | 北京市农林科学院 | Deodorization microbial agent and preparation method and application thereof |
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