CN103642727A - Bacillus amyloliquefaciens inhibiting fungi and application thereof - Google Patents
Bacillus amyloliquefaciens inhibiting fungi and application thereof Download PDFInfo
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Abstract
The invention discloses Bacillus amyloliquefaciens KB with the collection number of CGMCC No. 8179. The Bacillus amyloliquefaciens KB has an inhibiting effect on peach postharvest monilinia fructicola, apple postharvest botrytis cinerea, tomato postharvest botrytis cinerea and apple postharvest penicillium notatum, as well as the inhibiting effect on peach postharvest brown rot, apple postharvest gray mold, tomato postharvest gray mold and apple postharvest blue mold. The inhibiting effect on the peach monilinia fructicola and the apple botrytis cinerea is most obvious, the diameters of inhibition zones are 22mm and 20mm respectively, and the inhibition rate against peach brown rot spots and apple gray mold spots achieves 100%.
Description
Technical field
The bacillus amyloliquefaciens of Antifungi and an 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, has caused a large amount of underproduction of fruit, has a strong impact on edibleness and the marketable value of fruit, has caused orchard worker's income to reduce.In developed country, the loss amount that after picking fruit, disease causes accounts for the more than 15% of fruit total amount, and in developing country, owing to lacking necessary prevention and control technology and equipment, the loss amount that after picking fruit, disease causes accounts for the 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 to have a strong impact on.
The peach brown rot being caused by Monilinia spp. pathogenic fungi is all worldwide the first disease of peach at present, is also the first problem that affects 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 peach really to be had no harvest.Therefore, the control of peach brown rot and control are seemed to more urgent and important.China's peach garden area surpasses 9,000,000 mu, is the effective cultivated area of peach the first big country in the world; Producing peach per year and surpass 400,000 tons, is peach output the first big country in the world.Beijing peach output occupies domestic last eight, Pinggu County, beijing one ground only, 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 heavier peach garden of morbidity in 2005 wherein, peach is really injured, and comes off in a large number, has no the fruit of aobvious disease during harvesting, 2-3 days after harvesting, more than 50% fruit rots because brown heart infects, and this causes serious financial loss to orchard worker.Within 2012, Beijing plant protection unit peach brown rot investigation result shows, each suburban county peach producing region, Beijing all has peach brown rot to occur, and the transportation of peach after adopting and duration of storage sickness rate are all more than 30%.Edibleness and marketable value that this greatly reduces fruit, had a strong impact on the development of Beijing peach industry.Therefore effective prevention and control and the Economic development of peach being adopted to rear brown heart are especially closely connected with orchard worker's income.
In producing at present, the control of peach brown rot still be take to chemoprevention as main, but " 3R " negative impact bringing is thus very serious.A large amount of use havocs of chemical pesticide ecotope, cause pesticide residue a large amount of in peach fruit, affect people healthy.In northern peach producing region, before the harvesting of peach and after plucking, to the means of prevention of peach brown rot, be equally also to take chemical pesticide as main, this not only affects the healthy of the people, has also limited processing and the outlet of peach product.In < < China Agenda 21 > > has listed development biological pesticide and green food in 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 is carried out green production.
Genus bacillus (Bacillus spp.) ubiquity in soil, to be present in one of superior microorganism population in soil and plant microecosystem, owing to producing endogenous spore, the ability with stronger opposing ambient pressure environment, can resist the injury causing because of dry, thermal and uv radiation 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 that use active bacteria formulation, shows powerful vitality.Genus bacillus is of a great variety, and enormous amount can produce diversified physiologically active substance and meta-bolites, has prospects for commercial application widely.
Bacillus amyloliquefaciens distributes very wide at occurring in nature, not yet have the report of the bacillus amyloliquefaciens of the peach brown rot of preventing and treating at present.
Summary of the invention
The object of the present invention is to provide a kind of bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacterial strain KB, preserving number is CGMCC No.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 (Bacillus amyloliquefaciens) is KB, and registering on the books of Qi China Committee for Culture Collection of Microorganisms common micro-organisms center is numbered CGMCC No.8179.
Shown in the described 16S rDNA sequence SEQ ID No.1 that states bacillus amyloliquefaciens (Bacillus amyloliquefaciens) KB.
The morphological feature of described bacillus amyloliquefaciens (Bacillus amyloliquefaciens) KB is: somatic cells is rod-short, straight or closely straight, and raw in gemma, column or ellipse, have mobility; On LB substratum, do not produce pigment, bacterium colony is White-opalescent, flat or circular, tarnish, and along with incubation time increases, bacterium colony thickening becomes dry, and edge is irregular, has fold on bacterium colony, becomes creamy.The physiological and biochemical property of described bacillus amyloliquefaciens (Bacillus amyloliquefaciens) 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, can utilize glucose, 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 (Bacillus amyloliquefaciens) 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 a little less than growth potential, under pH9.5, can not grow, in envrionment temperature, be at 4 ℃ a little less than growth potential, at 55 ℃-80 ℃, process 15min, still can normal growth.Described bacillus amyloliquefaciens (Bacillus amyloliquefaciens) KB produces and has a liking for 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 suppressing pathogenic bacteria, preparation pathogenic bacteria inhibitor, suppress disease, preparing the application in disease suppression agent.
Described pathogenic bacteria be following at least one:
A, peach are adopted rear brown rot germ;
B, apple are adopted rear ash arrhizus bacteria;
C, tomato are adopted rear ash arrhizus bacteria;
D, apple are adopted rear mould germ;
Described disease be following at least one:
A, peach are adopted rear brown heart;
B, apple are adopted rear gray mold;
C, tomato are adopted rear gray mold;
D, apple are adopted rear penicilliosis.
Wherein, described peach is adopted rear brown rot germ and can be Monilinia fructicola or Monilinia laxa or Monilinia fructigena; It is Botrytis cinerea that described apple is adopted rear ash arrhizus bacteria; It is Botrytis cinerea that described tomato is adopted rear ash arrhizus bacteria; It is Penicillium expansum that described apple is adopted rear mould germ.
The metabolite of described bacillus amyloliquefaciens (Bacillus amyloliquefaciens) KB can obtain from the fermented liquid of described bacillus amyloliquefaciens (Bacillus amyloliquefaciens) KB.
The metabolite of described bacillus amyloliquefaciens (Bacillus amyloliquefaciens) KB specifically can be prepared as follows: in liquid medium within, cultivate described bacillus amyloliquefaciens (Bacillus amyloliquefaciens) KB, described bacillus amyloliquefaciens (Bacillus amyloliquefaciens) KB removing in liquid culture (fermented liquid) obtains the metabolite of described bacillus amyloliquefaciens.
Bacillus amyloliquefaciens in the present invention (Bacillus amyloliquefaciens) KB adopts rear brown rot germ, apple to peach and adopts rear ash arrhizus bacteria, tomato and adopt rear ash arrhizus bacteria and apple to adopt rear mould germ inhibited; Peach is adopted to rear brown heart, apple, and to adopt that rear gray mold, tomato are adopted rear gray mold, apple is adopted rear penicilliosis inhibited.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, and the inhibiting rate of peach brown rot spot and apple gray mold scab is reached to 100%.Illustrate that bacillus amyloliquefaciens (Bacillus amyloliquefaciens) KB has anti-microbial property stable, efficient, wide spectrum.
Biomaterial preservation explanation
Classification And Nomenclature: bacillus amyloliquefaciens (Bacillus amyloliquefaciens)
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
The preservation center numbering of registering on the books: CGMCC No.8179.
Accompanying drawing explanation
Fig. 1 .a) described bacillus amyloliquefaciens (Bacillus amyloliquefaciens) KB inhibition to Monilinia fructicola Monilinia fructicola on PDA substratum; B) inhibition of described bacillus amyloliquefaciens (Bacillus amyloliquefaciens) KB to apple gray mold Botrytis cinerea; C) inhibition of described bacillus amyloliquefaciens (Bacillus amyloliquefaciens) KB to graw mold of tomato Botrytis cinerea;
Fig. 2 .a) inhibition of described solution amylase genus bacillus (Bacillus amyloliquefaciens) KB to peach brown rot Lesion size; B) described solution amylase genus bacillus (Bacillus amyloliquefaciens) KB is to apple gray mold Lesion size inhibition; C) described solution amylase genus bacillus (Bacillus amyloliquefaciens) KB is to green mold of apple Lesion size inhibition; D) described solution amylase genus bacillus (Bacillus amyloliquefaciens) KB is to graw mold of tomato Lesion size inhibition.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
The pathogenic bacteria of using in following embodiment, the public can also can obtain from Beijing City Agriculture and Forestry Institute from field acquisition, to repeat the application's experiment:
Botrytis cinerea---Botrytis cinerea (Botrytis cinerea Per.ex Fr.) (Li Xinghong etc. Beijing area botrytis cinerea detects the resistance of phonetic mould amine. plant protection .2012.38 (4): 141-143)
Monilinia fructicola---chain sclerotinia sclerotiorum (Monilinia fructicola (wint.) Rehm) (Wang Fei etc. the generation of peach brown rot and control. fruit tree flowers .2012 the 5th phase p58).
Apple is adopted rear ash arrhizus bacteria--Botrytis cinerea (Botrytis cinerea Per.ex Fr);
Apple is adopted rear mould germ--Penicillium patulum enzyme bacterium (Penicillium expansum Link, 1809)
Embodiment 1
Separation and the identification of strains of bacillus amyloliquefaciens of the present invention (Bacillus amyloliquefaciens) KB
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, adopt conventional gradient dilution coating separated, with LB, NA, KB and CM0002 totally 4 kinds of substratum cultivate respectively under 28 ℃ of conditions, large and the bacterium that can pure culture of picking colony morphological differences, purification storage on LB substratum, and take Monilinia fructicola as target pathogens carries out the primary dcreening operation of Antagonistic Fungi and repeatedly sieves again, and finally obtain the strong bacterial isolates of a strain bacteriostatic activity, naming it is KB.
3, the evaluation 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 is studied principle and method [M], Higher Education Publishing House, 2010) in, 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, straight or closely straight, and raw in gemma, column or ellipse, have mobility; On LB substratum, do not produce pigment, bacterium colony is White-opalescent, flat or circular, tarnish, and along with incubation time increases, bacterium colony thickening becomes dry, and edge is irregular, has fold on bacterium colony, 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, can utilize glucose, 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 a little less than growth potential, under pH9.5, can not grow, in envrionment temperature, be at 4 ℃ a little less than growth potential, at 55 ℃-80 ℃, process 15min, still can normal growth.The 16S rDNA sequence of bacterial strain KB is as shown in SEQ ID No.1.
Embodiment 2
The mensuration of bacillus amyloliquefaciens of the present invention (Bacillus amyloliquefaciens) KB antimicrobial spectrum
For trying pathogenic bacteria:
Monilinia fructicola---chain sclerotinia sclerotiorum (Monilinia fructicola (wint.) Rehm);
Botrytis cinerea---Botrytis cinerea (Botrytis cinerea Per.ex Fr);
Apple is adopted rear ash arrhizus bacteria--Botrytis cinerea (Botrytis cinerea Per.ex Fr);
Apple is adopted rear mould germ--Penicillium patulum enzyme bacterium (Penicillium expansum Link, 1809)
Select peach to adopt rear brown heart germ, apple and adopt rear ash arrhizus bacteria, apple to adopt rear mould germ etc. be indication fungi, adopt dull and stereotyped face-off culture method.
Concrete operations are as follows: bacillus amyloliquefaciens (Bacillus amyloliquefaciens) KB bacterial strain 28 ℃ of constant temperature culture 48h on LB substratum.Plant pathogenic fungi is 25 ℃ of constant temperature culture 3-4 days on PDA flat board, from colony edge, with the aseptic stainless steel punch tool of diameter 0.7cm, make the agar block that carries disease germs; With aseptic inoculation pin picking pathogenic bacteria bacterium sheet, mycelia faces down and is inoculated in the dull and stereotyped center of PDA, simultaneously at equal aseptic inoculation ring bacillus amyloliquefaciens (Bacillus amyloliquefaciens) KB that rules for approximately 3.00 cm places apart from pathogenic fungi bacterium sheet both sides, the flat board that only connects pathogenic bacteria of take is contrast, each is processed three times and repeats, 25 ℃ of constant temperature culture 5-7 days, measure the antibacterial bandwidth between pathogenic fungi edge and the bacterium colony bandwidth center of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) KB.
Artificial punching inoculation method concrete operations in sporocarp (take prevent and treat apple adopt rear gray mold describe as example) are as follows:
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) KB bacterial strain 28 ℃ of constant temperature culture 48h on LB liquid nutrient medium, with distilled water, being adjusted to bacterial concentration is 5 * 10
7cfu/ml.Apple ash arrhizus bacteria B.cinerea is 25 ℃ of constant temperature culture 5-7 days on PDA flat board, collect botrytis cinerea spore, with distilled water, make 5 * 10
4the spore suspension of cfu/ml; Ripe apple, first with after 0.5% clorox sterilization, with sterilized pipettor gun head, along apple waist jack, each fruit is pricked 3, hole, and each hole is 30mm * 30mm.Every hole inoculation 30ul KB bacterium liquid, room temperature is dried, and then the pathogenic bacteria spore suspension of inoculation 30ul.Finally, the fruit of processing is positioned at 25 ℃, after 5-7 days, measures fruit rot spot size.Rotten spot is less than 10mm and is regarded as not falling ill.According to the scab that rots account for total inoculation hole count number, calculate sickness rate, not the scab number of morbidity account for total inoculation hole count number, calculate scab inhibiting rate.
Experimental result is in Table 1, bacillus amyloliquefaciens (Bacillus amyloliquefaciens) KB is in selected plant pathogenic fungi, restraining effect to Monilinia fructicola and apple ash arrhizus bacteria is the most obvious, antibacterial circle diameter is respectively 22mm and 20mm, and the inhibiting rate of peach brown rot spot and apple gray mold scab is reached to 100%.Illustrate that bacillus amyloliquefaciens (Bacillus amyloliquefaciens) KB has anti-microbial property stable, efficient, wide spectrum.
Table 1B.amyloliquefaciens KB to the inhibition of pathogenic bacteria (PDA, 28 ℃, 72h)
Wherein, described peach is adopted rear brown rot germ and can be Monilinia fructicola or Monilinia laxa or Monilinia fructigena; It is Botrytis cinerea that described apple is adopted rear ash arrhizus bacteria; It is Botrytis cinerea that described tomato is adopted rear ash arrhizus bacteria;
It is Penicillium expansum. that described apple is adopted rear mould germ
The rotten scab that bacillus amyloliquefaciens (Bacillus amyloliquefaciens) KB causes the 5 kind of plant pathogenic fungies for examination all has good inhibition as shown in table 1 and Fig. 2.
Embodiment 3
Under holding conditions, described bacillus amyloliquefaciens (Bacillus amyloliquefaciens) KB tests apple, tomato post-harvest diseases prevention effect:
1. after apple-picking, through KB bacterium liquid, (bacterial concentration is respectively 106cfu/ml, 107cfu/ml, 108cfu/ml, 109cfu/ml) immersion treatment 30-60 second, under room temperature condition, air-dry, then in temperature, be 1-2 ℃, relative humidity is under 90-95% condition, dark storage.Preserve after 10 months, compare with control group, when bacterial concentration is that its preventive effect of 106cfu/ml is 45%, when bacterial concentration reaches 107cfu/ml, its preventive effect reaches 65%, and when bacterial concentration reaches 108cfu/ml and 109cfu/ml, its preventive effect is respectively 70% and 75%.
2. after tomato picking, through KB bacterium liquid, (bacterial concentration is respectively 106cfu/ml, 107cfu/ml, 108cfu/ml, 109cfu/ml) immersion treatment 30-60 second, under room temperature condition, air-dry, then in temperature, be 20 ℃, relative humidity is under 90-95% condition, dark storage.Preserve after 5 days, compare with control group, when bacterial concentration is that its preventive effect of 106cfu/ml is 40%, when bacterial concentration reaches 107cfu/ml, its preventive effect reaches 55%, and when bacterial concentration reaches 108cfu/ml and 109cfu/ml, its preventive effect is respectively 60% and 65%.
Claims (7)
1. a bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacterial strain KB, preserving number is CGMCC No.8179.
2. a pathogenic bacteria inhibitor, its activeconstituents is the metabolite of bacillus amyloliquefaciens described in bacillus amyloliquefaciens and/or claim 1 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 are adopted rear brown rot germ;
B, apple are adopted rear ash arrhizus bacteria;
C, tomato are adopted rear ash arrhizus bacteria;
D, apple are adopted rear mould germ.
4. a disease suppression agent, its activeconstituents is the metabolite of bacillus amyloliquefaciens described in bacillus amyloliquefaciens and/or claim 1 described in claim 1.
5. disease suppression agent according to claim 4, is characterized in that, described disease be following at least one:
A, peach are adopted rear brown heart;
B, apple are adopted rear gray mold;
C, tomato are adopted rear gray mold;
D, apple are adopted rear penicilliosis.
6. following arbitrary application of the metabolite of bacillus amyloliquefaciens described in bacillus amyloliquefaciens and/or claim 1 described in claim 1:
1) application in suppressing pathogenic bacteria;
2) application in preparation pathogenic bacteria inhibitor;
3) application in suppressing disease;
4) application in preparing disease suppression agent.
7. application according to claim 6, is characterized in that, described pathogenic bacteria be following at least one:
A, peach are adopted rear brown rot germ;
B, apple are adopted rear ash arrhizus bacteria;
C, tomato are adopted rear ash arrhizus bacteria;
D, apple are adopted rear mould germ;
Described disease be following at least one:
A, peach are adopted rear brown heart;
B, apple are adopted rear gray mold;
C, tomato are adopted rear gray mold;
D, apple are adopted rear penicilliosis.
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| CN104593297A (en) * | 2014-12-31 | 2015-05-06 | 广东森度生态农业科技有限公司 | Microbial strain with acid-producing function and organic fertilizer using microbial strain |
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