CN104480190A - Method of isolating and screening antagonistic bacteria from rubber tree leaves - Google Patents
Method of isolating and screening antagonistic bacteria from rubber tree leaves Download PDFInfo
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- CN104480190A CN104480190A CN201410756455.6A CN201410756455A CN104480190A CN 104480190 A CN104480190 A CN 104480190A CN 201410756455 A CN201410756455 A CN 201410756455A CN 104480190 A CN104480190 A CN 104480190A
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Abstract
The invention relates to a method of isolating and screening antagonistic bacteria from rubber tree leaves. According to the method, bacteria are isolated from diseased leaves by a conventional sampling isolation method, and a strain of the antagonistic bacteria with an obvious inhibition action on the anthracnose pathogen RC178 of rubber trees is obtained by carrying out confront culture on the bacteria and test bacteria colletotrichum RC178 of the rubber trees. The isolated antagonistic bacteria are usually used as biocontrol bacteria and expected to be prepared into a microbial preparation, and therefore a basic preparation is provided for biocontrol of anthracnose of the rubber trees.
Description
Technical field
The invention belongs to biological control field of bacteria, specifically, relate to a kind of method of separation screening antagonistic bacterium from rubber tree blade.
Background technology
Anthracnose of rubber trees is the important disease having a strong impact on latex yield and quality during rubber tree produces, pathogenic bacteria is colletotrichum gloeosporioides Penz (C.gloeosporioides Penz.) and sharp spore anthrax-bacilus (C.acutatum Simmons), colletotrichum gloeosporioides Penz sharper spore anthrax-bacilus is more common, and sharp spore anthrax is China's rubber tree kainogenesis disease.Anthracnose of rubber trees is caused harm and is caused leaf tissue necrosis or come off in a large number, and cause a large amount of fallen leaves and the tender tip to return withered time serious, tree body is weak, shortens the time limit of tapping rubber, and postpones and opens the time of cutting, rubber ultimate production is significantly declined.
At present, a series of research is carried out to rubber anthrax both at home and abroad, mainly concentrated on plant disease epidemic and Chemicals aspect.Cui Changhua etc. (2006) adopt colony growth rate method to carry out the sensitivity testing of anthracnose of rubber trees bacterium to triazolone, Wocosin 50TK, m-tetrachlorophthalodinitrile, zineb, derosal 5 kinds of medicaments in indoor.Cai Zhiying etc. (2008) adopt growth rate method indoor measurement Yunnan Province rubber anthrax bacteria colletotrichum gloeosporioides Penz and sharp spore anthrax-bacilus to the susceptibility of prochloraz, derosal, thiophanate methyl, bromothalonil, m-tetrachlorophthalodinitrile and zinc manganese ethylenebisdithiocarbamate.Zheng Xiaolan (2008) adopts colony growth rate method to determine 18 kinds of rubber anthrax bacteria bacterial strains to the susceptibility of 4 kinds of conventional fungoid medicaments such as Bao Zhida.Wang Lanying etc. (2010) research reports the inhibit activities that 8 kinds of sterilant are sprouted rubber anthrax spores.But, in production for the control of rubber anthrax still based on chemical bactericide, and germ resistance, toxicity and problem of environmental pollution that chemical pesticide causes become increasingly conspicuous, and have become the major obstacle of restriction agricultural sustainable development.Therefore, urgently need to find and a kind ofly there is good, the nontoxic Biocontrol microorganism of environment compatibility, as the ideal substitute of chemical pesticide.
Summary of the invention
The object of this invention is to provide a kind of method of separation screening antagonistic bacterium from rubber tree blade, comprise the following steps:
(1) rubber infected leaves is gathered;
(2) soak blade with mercuric chloride, then use aseptic water washing;
(3) blade is placed on PDA substratum, after 28 DEG C of cultivation 48h, the thalline grown is forwarded on identical substratum, purifying after 28 DEG C of cultivation 48h; Meanwhile, adopted by the bacterium of gained after purifying in gradient dilution coating LB plate culture medium, after 28 DEG C of cultivation 48h, picking list bacterium colony is connected in corresponding medium slant;
(4) single bacterium colony 28 DEG C concussion is cultivated about 12 hours, draw and be placed on a small quantity on LB solid medium, smear evenly, 28 DEG C of cultivations; Then single bacterial strain is obtained by the process of infinite dilution method;
(5) from bacterial strain, filter out bacterium, be transferred to LB flat board, obtain single bacterium colony by spread plate method; With for trying bacterium rubber tree anthrax-bacilus RC178, opposite culture is carried out to the single strain obtained;
(6) by observing with or without inhibition zone, the antagonistic bacterium occurring inhibition zone is selected.
In a concrete embodiment, step (1) is: the rubber infected leaves gathering different sources, and clear water rinses; And clip size is that the vanelets of 5 × 5mm is for subsequent use.
In a concrete embodiment, step (2) is: the vanelets mercuric chloride of clip is soaked 30 seconds, then uses aseptic water washing 3 times, dries.
In a concrete embodiment, step (3) is: be placed with on PDA solid medium with sterilized tweezers by the vanelets of clip, every ware evenly puts 4 pieces, then is sealed by culture dish preservative film.Be inverted in constant incubator, 28 DEG C of cultivations.After 12h carries out observing 48h, the thalline grown is forwarded on identical substratum, after 28 DEG C of cultivation 48h, carries out purifying; Meanwhile, adopted by the bacterium of gained after purifying in gradient dilution coating LB plate culture medium, after 28 DEG C of cultivation 48h, picking list bacterium colony is connected in corresponding medium slant.
In a concrete embodiment, step (4) is: taken out by the slant tube be positioned in 8 ~ 10 DEG C of incubators.In aseptic operating platform, LB liquid medium is dispensed in Erlenmeyer flask, then chooses a small amount of bacterium in Erlenmeyer flask with inoculating needle, sealing.Be positioned over concussion on 28 DEG C of shaking tables to cultivate about 12 hours, take out.And then obtain single bacterial strain by the process of infinite dilution method.
In a concrete embodiment, PDA culture medium prescription is: potato 200g/L, glucose or sucrose 10g/L, agar 18g/L.
In a concrete embodiment, LB culture medium prescription is: Tryptones 10g/L, yeast extract powder 5g/L, NaCl 10g/L, pH=7; In above-mentioned formula, add agar form LB solid medium.
Compared with prior art, separating screening method of the present invention has following beneficial effect:
(1) the inventive method is easy to obtain new Antagonistic bacteria strains, and this bacterial strain has fairly obvious antagonistic action to rubber tree anthrax-bacilus RC178; It is commonly used for biocontrol microorganisms, is expected to make microbial preparation, for anthracnose of rubber trees biological control provides basis to prepare.
(2) Antagonistic bacteria strains using the inventive method to obtain significantly can reduce the pollution that chemical pesticide brings environment, meanwhile, can reach the object controlling disease and occur.
Brief description
Fig. 1 shows antagonistic bacterium and RC178 face-off figure.
Fig. 2 shows colonial morphology and the spore shape of antagonistic bacterium.
Embodiment
Further illustrate method of the present invention as embodiment below, will contribute to further understanding of the invention, protection scope of the present invention is not limited to the examples, and its protection domain is decided by claims.
Embodiment 1
From rubber tree blade, the method for separation screening antagonistic bacterium specifically comprises the following step:
1 sampled acquisition different areas rubber infected leaves, puts into plastics bag and record by the blade be collected.
2 material surface sterilizations are separated with antagonistic bacterium
2.1 clear water rinse the sick leaf clear water with clip and wash away the dust gravel on blade, dry.Being good for intersection clip size with scissors in disease is that the vanelets of 5 × 5mm is for subsequent use;
The minor illness leaf of clip is put into beaker by 2.2 sample sterilizations, soaks 30 seconds with mercuric chloride, should note constantly stirring minor illness leaf gently, then use aseptic water washing three times, dry in the process of soaking, for subsequent use;
2.3 bacteria distribution are cultivated and are placed with on PDA solid medium with sterilized tweezers by the vanelets of clip, and every ware evenly puts 4 pieces, then is sealed by culture dish preservative film.Be inverted in constant incubator, 28 DEG C of cultivations.After 12h carries out observing 48h, the thalline grown is forwarded on identical substratum, after 28 DEG C of cultivation 48h, carries out purifying; Meanwhile, adopted by the bacterium of gained after purifying in gradient dilution coating LB plate culture medium, after 28 DEG C of cultivation 48h, picking list bacterium colony is connected in corresponding medium slant;
The screening of 3 antagonistic bacteriums
The slant tube be positioned in 8 ~ 10 DEG C of incubators takes out by the purifying of 3.1 bacteriums, in aseptic operating platform, is dispensed into by LB liquid medium in Erlenmeyer flask, then chooses a small amount of bacterium in Erlenmeyer flask with inoculating needle, sealing.Be positioned over concussion on 28 DEG C of shaking tables to cultivate about 12 hours, take out.Then draw 200uL on LB solid medium with pipettor, paint daubs is smeared evenly, cultivates in 28 DEG C of incubators.And then obtain single bacterial strain by the process of infinite dilution method;
3.2 opposite culture screening Antagonistic bacteria strains filter out bacterium from each bacterial strain that sick leaf is separated, and are transferred to LB flat board, then obtain the strain of single bacterium colony 29 by spread plate method.The bacterium cake that cut-off footpath is 6mm bought by the aseptic punch tool of strains tested RC178, be placed in the middle of flat board that diameter is 90mm, the 29 strain bacterial strains through primary dcreening operation are inoculated in 4 corners around RC178 right-angled intersection respectively, and each bacterial strain 3 repetition, seals up culture dish with anti-pollution with preservative film.In addition, adopt dotted line facture and other face-off forms, carry out opposite culture.Only to connect pathogenic bacteria, do not connect isolated strains for contrast.Cultivate 4 ~ 7d in 28 DEG C, the bacterial isolates that observation is separated is to the bacteriostatic action of strains tested RC178;
Antagonistic bacterium to be identified is inoculated on LB flat board by 4 morphological features, cultivates 48h for 37 DEG C, observes colonial morphology and record result.As can be seen from Figure 1, antagonistic bacterium and anthrax-bacilus RC178 cultivate by various face-off mode all can there is obvious inhibition zone, and namely this inhibition zone 2d after connecing Antagonistic Fungi occurs, and this place pathogenic bacteria stops growing, Continuous Observation 10d, inhibition zone remains unchanged, and bacteriostasis rate all can reach 65.8%.
The dull and stereotyped upper 37 DEG C of Antagonistic Fungis to be identified cultivating 48h of picking LB carry out gramstaining, 1000 times of its thalline of light microscopic microscopic examination and gemma form, and record observations; Antagonistic Fungi in intense violet color, is gram-positive microorganism under microscopic visualization.Under 1000 times of light microscopics, the gemma of bacterium is corynebacterium, as shown in Fig. 2 right side.The bacterial strain of antagonistic bacterium is flats on LB flat board, and in faint yellow opaque, subcircular, surface purse up, edge is irregular, in wavy and splintery [12], as shown in Fig. 2 left side.
Strain gene group DNA is extracted for template, its 16S rDNA fragment of employing bacterial universal primers pcr amplification after 5DNA amplification and 16S rDNA gene amplification and sequential analysis utilize purifying.Get PCR primer carry out 1% agarose gel electrophoresis detect after, PCR primer is utilized DNA reclaim test kit reclaim, purifying.Then through the Guangzhou Branch order-checking of Shanghai Ying Jun Bioisystech Co., Ltd;
PCR the primer is UP1:5 '-TACGTGCCAGCAGCCGCGGTAATA-3 '; UP2:5 '-AGTAAGGAGGTGATCCAACCGCA-3 '.
PCR reaction system: bacterium liquid 1 μ L; DNTP 2.0 μ L; UP11.0 μ L; UP21.0 μ L; 10 × PCR Buffer2.5 μ L; Taq archaeal dna polymerase 0.2 μ L; Ultrapure water complements to 25 μ L.
PCR reaction conditions: 94 DEG C of 5min; 94 DEG C of 30Sec, 52 DEG C of 30Sec, 72 DEG C of 1min, 30 circulations; 72 DEG C of 10min, 4 DEG C of preservations.
PCR primer checks order after kits.
Subtilis (B.subtilis) is accredited as according to strain morphology and cultural characteristic and 16SrRNA genetic analysis.
And subtilis is as the Antagonistic Fungi of multiple pathogenic bacteria, it is the important biocontrol fungi of a class.Utilize biocontrol microorganisms to prepare microbial preparation to carry out controlling disease and significantly can reduce the pollution that chemical pesticide brings environment, meanwhile, the object controlling disease and occur can be reached.The control of anthracnose of rubber trees is also towards the future development of biological control.Isolating Antagonistic Fungi is the prerequisite preparing microbial preparation, for preparation has been made in the biological control better carrying out anthracnose of rubber trees.
Claims (5)
1. the method for separation screening antagonistic bacterium from rubber tree blade, comprises the following steps:
(1) rubber infected leaves is gathered;
(2) soak blade with mercuric chloride, then use aseptic water washing;
(3) blade is placed on PDA substratum, after 28 DEG C of cultivation 48h, the thalline grown is forwarded on identical substratum, purifying after 28 DEG C of cultivation 48h; Meanwhile, adopted by the bacterium of gained after purifying in gradient dilution coating LB substratum, after 28 DEG C of cultivation 48h, picking list bacterium colony is connected in corresponding medium slant;
(4) single bacterium colony 28 DEG C concussion is cultivated about 12 hours, draw and be placed on a small quantity on LB solid medium, smear evenly, 28 DEG C of cultivations; Then single bacterial strain is obtained by the process of infinite dilution method;
(5) from bacterial strain, filter out bacterium, be transferred to LB flat board, obtain single bacterium colony by spread plate method; With for trying bacterium rubber tree anthrax-bacilus RC178, opposite culture is carried out to the single strain obtained;
(6) by observing with or without inhibition zone, the antagonistic bacterium occurring inhibition zone is selected.
2. method according to claim 1, wherein said PDA culture medium prescription is: potato 200g/L, glucose or sucrose 10g/L, agar 18g/L.
3. method according to claim 1, wherein said LB culture medium prescription is: Tryptones 10g/L, yeast extract powder 5g/L, NaCl 10g/L, pH=7; In above-mentioned formula, add agar form LB solid medium.
4. method according to claim 1, the concussion of wherein said step (4) is cultivated and is: taken out by the slant tube be positioned in 8 ~ 10 DEG C of incubators, in aseptic operating platform, LB liquid medium is dispensed in Erlenmeyer flask, then a small amount of bacterium is chosen in Erlenmeyer flask with inoculating needle, sealing; Be positioned over concussion on 28 DEG C of shaking tables to cultivate about 12 hours, take out.
5. preparation method according to claim 1, the opposite culture of wherein said step (5) is: obtain single bacterium colony by spread plate method, the bacterium cake that cut-off footpath is 6mm bought by the aseptic punch tool of strains tested RC178, be placed in the middle of flat board that diameter is 90mm, the multiple bacterial strains through primary dcreening operation are being inoculated respectively around 4 corners of RC178 right-angled intersection, each bacterial strain 3 repetition, seals up culture dish with anti-pollution with preservative film; Adopt dotted line facture and other face-off forms, carry out opposite culture; Only to connect pathogenic bacteria, do not connect isolated strains for contrast.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105524871A (en) * | 2016-01-29 | 2016-04-27 | 江苏省中国科学院植物研究所 | Method for separating and screening out antagonistic bacterium from lycoris aurea bulb |
| CN105936879A (en) * | 2015-11-23 | 2016-09-14 | 中国热带农业科学院环境与植物保护研究所 | Bacillus subtilis K13, and culture method and application thereof |
| CN111607501A (en) * | 2020-06-05 | 2020-09-01 | 重庆工商大学 | Vegetable leaf microorganism collection system |
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| CN102643760A (en) * | 2011-02-18 | 2012-08-22 | 中国热带农业科学院环境与植物保护研究所 | Antagonistic bacterium capable of generating siderophore for controlling plant diseases |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102643760A (en) * | 2011-02-18 | 2012-08-22 | 中国热带农业科学院环境与植物保护研究所 | Antagonistic bacterium capable of generating siderophore for controlling plant diseases |
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| 贺春萍 等: "一株产铁载体橡胶树拮抗细菌的分离鉴定及耐药性分析", 《热带作物学报》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105936879A (en) * | 2015-11-23 | 2016-09-14 | 中国热带农业科学院环境与植物保护研究所 | Bacillus subtilis K13, and culture method and application thereof |
| CN105936879B (en) * | 2015-11-23 | 2019-09-13 | 中国热带农业科学院环境与植物保护研究所 | Bacillus subtilis K13 and its cultural method and application |
| CN105524871A (en) * | 2016-01-29 | 2016-04-27 | 江苏省中国科学院植物研究所 | Method for separating and screening out antagonistic bacterium from lycoris aurea bulb |
| CN111607501A (en) * | 2020-06-05 | 2020-09-01 | 重庆工商大学 | Vegetable leaf microorganism collection system |
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Application publication date: 20150401 |