CN102329756B - Streptomyces albospinus strain BWL15-4 for preventing and treating banana vascular wilt and application thereof - Google Patents
Streptomyces albospinus strain BWL15-4 for preventing and treating banana vascular wilt and application thereof Download PDFInfo
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Abstract
The invention discloses a streptomyces albospinus strain BWL15-4 for preventing and treating a banana vascular wilt. The strain BWL15-4 is classified and named as streptomyces albospinus, and is preserved in the China General Microbiological Culture Collection Center on May 9, 2011; and the preservation number is CGMCC No. 4836. The strain has strong antagonistic capability to the pathogenic bacteria of the banana vascular wilt and can be used for preventing and treating the banana vascular wilt; the invention further discloses a microbial inoculum containing the strain, a preparation method and application thereof; and by using the microbial inoculum, not only can the banana vascular wilt be inhibited, but also the microbial inoculum has a certain growth promotion effect on a plant.
Description
Technical field
The present invention relates to a kind of bacterial strain and application thereof that prevents and treats banana blight, be specifically related to a kind of white thorn streptomyces strain BWL15-4 and application thereof that prevents and treats banana blight.
Background technology
The crushing soil-borne disease in a kind of banana production causing infects in banana blight Shi You Fusarium oxysporum Cuba specialized form (Fusarium oxysporum f.sp.cubense).This pathogenic bacteria has 4 physiological strains, what in China, distribute is No. 1 and No. 4 two physiological strains, to the harm of China banana industry maximum be No. 4 physiological strains, at present in Hainan, Guangdong, Yunnan, most of banana plantation, Fujian big area occurs, onset area is over 500,000 mu.Guangdong and Hainan banana blight serious place large-scale planting banana again of falling ill, has to replant other crop.
Banana blight bacteria is soil inhabitant, and it is invaded from the root of banana, along vascular bundle, up spreads and spreads, obstruction conduit, produce toxin, destroy the conveying function of fascicular moisture and nutrient, thereby make the over-ground part of banana plant can not get moisture and nutrient and turn to be yellow withered.The chlamydospore of pathogenic bacteria can survive more than 10 year in soil, is a kind of popular disease for many years.
Because Pathogen Causing Banana Fusarium Wilt is expanded and infected in vascular bundle, there is no so far highly effective Agro-chemicals control and high resistance to wilt good kind.Therefore, biological control is subject to extensive concern.The banana blight bio-control method of having reported or having applied for a patent, major part is to adopt genus bacillus as biocontrol strain, this class bacterial strain has many advantages as biocontrol microorganisms, but also there is a wretched insufficiency, be that bacterial strain itself is not strong to the antagonistic ability of pathogenic bacteria, thereby likely affect its biocontrol effect.
Summary of the invention
First object of the present invention is to provide a kind of white thorn streptomyces strain BWL15-4 that prevents and treats banana blight, because this bacterial strain itself is separated, from plant rhizosphere soil, be soil inhabitant, rhizospere competition is strong, biocontrol effect is good, especially strong to the antagonistic ability of Pathogen Causing Banana Fusarium Wilt.
Second object of the present invention is to provide the application of above-mentioned white thorn streptomyces strain BWL15-4 in control banana blight.
The 3rd object of the present invention is to provide a kind of preparation that contains above-mentioned white thorn streptomyces strain BWL15-4.
The 4th object of the present invention is to provide the preparation method of above-mentioned preparation, and this preparation method's technique is simple, and fermention medium is without valuable raw material, and cost is low, easily promotes.
Last object of the present invention is to provide the application of above-mentioned preparation in control banana blight.
First object of the present invention realizes by following technique means: a kind of white thorn streptomyces strain BWL 15-4 that prevents and treats banana blight, the Classification And Nomenclature of this bacterial strain BWL 15-4 is white thorn streptomycete (Streptomyces albospinus), on May 9th, 2011, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.4836.
The 16S rDNA portion gene sequence (1491bp) of this bacterial strain has been submitted the Genebank geneseq database of American National biotechnology information center (NCBI) to, and accession number is JN038327; The 16S rDNA nucleotide sequence of this bacterial strain BWL15-4 is as shown in SEQ ID NO:1.
Second technical scheme of the present invention is achieved by the following technical solution: the application of above-mentioned white thorn streptomyces strain BWL15-4 in control banana blight.
The 3rd technical scheme of the present invention is achieved by the following technical solution: a kind of preparation of preventing and treating banana blight, the liquid bacterial agent or the solid fungicide that contain above-mentioned white thorn streptomyces strain BWL15-4.
The 4th technical scheme of the present invention is achieved by the following technical solution: the preparation method of above-mentioned preparation, prepares liquid bacterial agent or solid fungicide by above-mentioned white thorn streptomyces strain BWL15-4 through actication of culture, liquid or solid fermentation procedure.
Wherein, in the preparation method of above-mentioned preparation, can also contain seed culture operation, be about to above-mentioned white thorn streptomyces strain BWL15-4 and prepare liquid bacterial agent or solid fungicide through actication of culture, seed culture, liquid or solid fermentation procedure.
Specifically, liquid bacterial agent in the present invention can obtain by following technical process: bacterial classification BWL15-4 activation (being that first order seed is cultivated) → spawn culture (is that secondary seed is cultivated, optional) → liquid fermenting (fermentor tank, sterile air, nutrient solution) → liquid bacterial agent.
Wherein liquid nutrient medium can be: glucose 1-3%, soybean cake powder 1-4%, Zulkovsky starch 1-4%, yeast extract paste 0.2%, NaCl 0.4%, CaCO
30.25%, defoamer 0.1-0.3%, by quality percentage composition.Below be only preferred substratum, in fact the present invention is to culture condition and not strict especially restriction of liquid nutrient medium, as long as this condition and liquid nutrient medium are applicable to the growth of bacterial strain.
Specifically, solid fungicide in the present invention can obtain by following technical process: bacterial classification BWL15-4 activation (being that first order seed is cultivated) → spawn culture (is that secondary seed is cultivated, optional) → solid fermentation (solid culture matrix) → fermentation cake → drying and crushing, mix in 1: 1 or other ratio with absorption carrier, mix rear suitable being dried, make solid fungicide.Wherein absorption carrier can be the variety carrier such as non-arable layer soil, kaolin.
The solid medium wherein adopting during solid fermentation is preferably and comprises rice 60-70%, analysis for soybean powder 5-15%, and wheat skin 10-20%, sandy soil 5%, by quality percentage composition.These are only the preferred substratum of the present invention, in fact the present invention is to the selection of culture condition and solid medium not strict especially restriction in fact, as long as this condition and solid medium are applicable to the growth of bacterial strain.
Last object of the present invention realizes by following technique means: the application of above-mentioned preparation in control banana blight.
Preparation in the present invention is in control during banana blight, liquid bacterial agent or solid fungicide and common fertilizer can be mixed to mix thoroughly in the ratio of 2: 100 and as base fertilizer, use afterwards, and each field planting is spreaded manuer in holes 2.5 kilograms, before banana planting, uses; In vegetative period after banana planting, can join liquid bacterial agent or solid fungicide in the conventional fertilizer of dissolving with dilution 5-10 ratio doubly, and banana plant is watered to fertilising.Or the liquid bacterial agent in the present invention control banana blight can be by liquid bacterial agent being diluted to 2 times or dilute as required rear liquid irrigating root or water and impose on plant base portion, or soak root before banana seedlings is transplanted.
Compared with prior art, tool of the present invention has the following advantages:
(1) the present invention is usingd and is stung in vain streptomycete BWL15-4 as antagonistic strain, strong to banana blight bacteria restraining effect, and banana blight is had to good prevention effect;
(2) preparation of the present invention has growth promoting function to banana;
(3) to sting in vain streptomycete bacterial strain BWL15-4 culture condition simple in the present invention, easily preserves, and is easy to suitability for industrialized production, has good development prospect;
(4) the agent preparation method technique of system control banana blight of the present invention is simple, and fermention medium is without valuable raw material, and cost is low, easily promotes.
Accompanying drawing explanation
Fig. 1 is the white stereoscan photograph that stings streptomycete BWL15-4 bacterial strain spore and spore chain that in embodiment 1, screening obtains, and spore surface has spinelet;
Fig. 2 is the fungistatic effect that the white thorn streptomycete BWL15-4 bacterial strain of preparation in embodiment 1 and the indoor flat plate face-off of banana wilt germina number-four biological strain are cultivated, and wherein, 1 is white thorn streptomycete BWL15-4 bacterial strain, and 2 is banana wilt germina number-four biological strain;
Fig. 3 is the fungistatic effect that the white thorn streptomycete BWL15-4 bacterial strain of preparation in embodiment 1 and the indoor flat plate face-off of banana wilt germina number-four biological strain are cultivated, and wherein, 1 is white thorn streptomycete BWL15-4 bacterial strain, and 2 is banana wilt germina number-four biological strain.
Separated and the evaluation of bacterial strain
Biocontrol microorganisms BWL15-4 in the present embodiment be from Altitudinal Gradient In Bawangling Nature Reserve, Hainan virgin forest soil screening and separating to a streptomycete, this bacterial strain has strong restraining effect to banana blight bacteria (Fusarium oxysporum f.sp.cubense), be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC on May 9th, 2011, preserving number is CGMCC No.4836, through identifying that called after stings streptomycete BWL 15-4 bacterial strain (Streptomyces albospinus BWL 15-4) in vain.
The separation screening process of bacterial strain BWL15-4 is as follows: will in Altitudinal Gradient In Bawangling Nature Reserve, Hainan virgin forest, carry out air-dry by herborization rhizosphere soil sample, with coating in screening culture medium after sterilized water dilution suitable multiple, screening culture medium is to add potassium bichromate, the PDA solid medium of Nalidixic Acid and cycloheximide, cultivate more than 5 days for 28 ℃, on flat board to be screened, grow after actinomycetes bacterium colony, on flat board, one or more indicator banana blight bacteria bacterium cakes are accessed in appropriate location again, when certain unwrapping wire bacterium colony has antagonistic action, indicator just can not be crossed this actinomycetes growth, present obvious antagonism band, picking has the actinomycetes of antagonistic action, line purifying three times, carry out flat board face-off experiment, select the wider actinomycetes of antagonism band as candidate strain, candidate strain is carried out to liquid fermenting, get fermented liquid 10mL filtration sterilization, with Oxford cup, carry out the production of antibacterial substance in dull and stereotyped face-off examination fermented liquid, the fermented liquid that produces antimicrobial substance carries out field efficacy screening again in the sick garden of banana blight, filter out thus the antagonistic strain BWL15-4 that field control effect is good.
The qualification process of bacterial strain BWL15-4 is as follows: first it is carried out to 16S rDNA sequential analysis, the 16S rDNA sequence of this bacterial strain is submitted to online in gene bank (http://www.ncbi.nlm.nih.gov/blast) and carries out Blast comparison, find that it stings streptomycete (Streptomyces albospinus) in same evolutionary branching with white, homology reaches more than 99%, the 16S rDNA Sequence gene order (1493bp) of this bacterial strain, submitted the Genebank geneseq database of American National biotechnology information center (NCBI) to, accession number is JN038327, its nucleotide sequence is as shown in SEQ ID NO:1.Then BWL15-4 bacterial strain is carried out to morphological feature, cultural characteristic and Analysis of The Physiological And Biochemical Properties and identify, its morphological specificity is by analysis: under scanning electron microscope, fibrillae of spores is 2-3 circle volution, and spore is oval, surface band spinelet (seeing Fig. 1); Its cultural characteristic is: on nutrient agar plate, there are abundant aerial hyphae and substrate mycelium, and gas silk canescence, base silk olive is yellow, without soluble pigment; Its physio-biochemical characteristics are: energy liquefy gelatin, a little less than Starch Hydrolysis, reduction nitrate, do not produce hydrogen sulfide and melanochrome, utilize urea, can utilize fructose, wood sugar, glycerine, lactose, saligenin, synanthrin, glucose, inositol, N.F,USP MANNITOL, semi-lactosi, maltose, dextrin, starch, sorbose, not utilize pectinose, sucrose, galactitol, on the culture medium that is sole carbon source at raffinose, seminose, growth is poor.Result shows: the morphological feature of this bacterial strain is identical with Bai Ci streptomycete with cultural characteristic, and the physio-biochemical characteristics overwhelming majority is identical with white thorn streptomycete.Therefore, the comprehensive comparison result of 16S rDNA sequence and the result of morphological specificity, cultural characteristic and Analysis of The Physiological And Biochemical Properties, by BWL15-4 identification of strains, be white thorn streptomycete, called after stings streptomycete BWL15-4 bacterial strain (Streptomyces albospinus BWL15-4) in vain.
This bacterial strain bacteriostatic action is strong, antibacterial circle diameter average out to 18mm in indoor flat plate face-off culture experiment, expand at repressed indicator banana wilt germina number-four biological strain mycelia tip, blackening, tenuigenin liquid leaks (seeing Fig. 2 and Fig. 3), illustrates that the meta-bolites of this bacterial strain is to pathogenic bacteria mycelia tool killing effect and very strong restraining effect.
Liquid bacterial agent and preparation method thereof
The liquid bacterial agent that contains above-mentioned white thorn streptomyces strain BWL15-4 that the present embodiment provides prepares by the following method:
(1) actication of culture, will sting in vain streptomyces strain BWL15-4 and be inoculated on solid medium, and substratum is nutrient agar;
(2) seed culture, is inoculated into liquid nutrient medium by activated spawn, fills a prescription to be: glucose 1%, and soybean cake powder 1%, yeast extract paste 0.2%, NaCl 0.3%, CaCO
30.1%, K
2hPO
40.05%; By quality percentage composition, all the other are water; PH value 7.0-7.2; At 28 ℃, triangular flask 160rpm shake flask fermentation is cultivated 72 hours, if be that after first order seed is cultivated, bacterial classification quantity is abundant can directly carry out liquid fermenting through actication of culture in step (1), and not need be that secondary seed is cultivated operation through seed culture again;
(3) liquid fermenting, adds seed liquor in liquid nutrient medium to by 6% (V/V), and filling a prescription is: glucose 2%, soybean cake powder 2%, Zulkovsky starch 2%, yeast extract paste 0.2%, NaCl 0.4%, CaCO
30.25%, defoamer 0.1%, and by quality percentage composition, all the other are water, what in the present embodiment, adopt is substratum preferably, other substratum also can, be not particularly limited, pH value 7.2,28 ℃ of tank temperature, tank pressure 0.07Mpa, stirring velocity 200rpm, oxygen-supply quantity > 60%, fermentation time approximately 7 days, obtains liquid fermenting preparation, for field control disease.
Embodiment 3
Solid fungicide and preparation method thereof
The solid fungicide that contains above-mentioned white thorn streptomyces strain BWL15-4 that the present embodiment provides prepares by the following method:
(1) actication of culture: will sting in vain streptomyces strain BWL15-4 and be inoculated on solid medium, substratum is nutrient agar;
(2) seed culture: activated spawn is inoculated into liquid nutrient medium, and filling a prescription is: glucose 1%, soybean cake powder 1%, yeast extract paste 0.2%, NaCl 0.3%, CaCO
30.1%, K
2hPO
40.05%, by quality percentage composition, all the other are water; PH value 7.0-7.2; At 28 ℃, 160 revs/min of shake flask fermentations of triangular flask are cultivated 72 hours, if be after first order seed is cultivated through actication of culture in step (1), bacterial classification quantity is abundant can directly carry out solid fermentation, and not need through seed culture be that secondary seed is cultivated operation again;
(3) solid culture: seed culture fluid is mixed with solid medium, solid culture based formulas is: rice 65%, analysis for soybean powder 15%, wheat skin 15%, sandy soil 5%, above by quality percentage composition, what in the present embodiment, adopt is substratum preferably, other substratum also can, be not particularly limited; 28 ℃ of-30 ℃ of incubation times are about 14 days, and now solid medium should fully be grown and large volume production spore;
(4) microbial inoculum preparation: the solid culture of fully fermentation and non-arable layer or kaolin are mixed to drying and crushing afterwards in 1: 1 or other ratio, make solid fungicide.
Embodiment 4
Liquid bacterial agent and solid fungicide are to banana blight field control effect:
The white thorn streptomycete BWL15-4 liquid bacterial agent of preparation in test microbial inoculum: embodiment 2, the white thorn streptomycete BWL15-4 solid fungicide of preparation in embodiment 3, clear water contrast.
Test site: the sick garden of China tropic Agriculture Academy Sciences tropic Biotechnology Research Institute's Haikou banana blight.
For trying banana variety: sense blight kind Brazil any of several broadleaf plants.
Application method:
Planting pit pre-treatment: water pouring planting pit with above-mentioned 2 times of diluent 1L of liquid bacterial agent containing white thorn streptomycete BWL15-4 bacterial strain, then cover thin soil; After solid fungicide mixes by 1: 1 with fertilizer, each planting pit is put with 2.5 kilograms, fully mixes thoroughly with soil in cave.
Banana seedlings is executed in advance microbial inoculum and is processed: transplant the day before yesterday, by 2 times of dilution immersion roots that contain white thorn streptomycete BWL15-4 strain liquid microbial inoculum for banana seedlings; Solid fungicide soaks root for banana seedlings after diluting 5 times.
Transplant: the banana seedlings of soaking after root is transplanted in the planting pit of processing.
Management: regularly apply fertilizer, liquid bacterial agent processing is monthly filled with roots once with 5 times of diluent 2L of liquid bacterial agent, solid fungicide is processed and is monthly used 5 times of solid fungicide to dilute liquid irrigating roots once, records plant disease index, sickness rate, and calculate prevention effect after 6 months.
Sickness rate %=morbidity strain (leaf) number * 100/ total strain of investigation (leaf) number
Disease index %=∑ (diseased plants at different levels (leaf) number * this disease level value)/investigate total strain (leaf) to count * highest value
Prevention effect %=(contrast disease index-processing disease index) * 100/ contrast disease index
Banana blight state of an illness investigation grade scale: 0 grade of outward appearance, without Visual symptoms, is dissected false cauline bundle tissue and had no browning, and 1 grade of outward appearance is without Visual symptoms, dissect false cauline bundle and organize browning, 2 grade blade yellows or vertical, 3 grades of false basal part of stem crackings, but diseased plant is not yet withered, 4 grades of diseased plants are withered.
Test-results:
Check plot starts to occur banana blight symptom for 40 days after banana seedlings field planting, after processing 150 days, the banana of white thorn streptomycete BWL15-4 strain liquid preparation or solid fungicide processing just has the slight yellow leaf symptom of indivedual plant base portion leaves performance, the prevention effect of the test that liquid bacterial agent is processed after 6 months is 84% (referring to table 1), prevention effect after 6 months of solid fungicide processing is 80% (referring to table 2), shows that banana blight is had to good prevention effect.
The prevention effect of the white thorn of table 1 streptomycete BWL15-4 liquid bacterial agent to banana blight
| Process | Sickness rate (%) | Disease index | Prevention effect (%) |
| CK | 90 | 62.5 | - |
| Liquid bacterial agent | 20 | 10 | 84 |
The prevention effect of the white thorn of table 2 streptomycete BWL15-4 solid fungicide to banana blight
| Process | Sickness rate (%) | Disease index | Prevention effect (%) |
| CK | 100 | 65.6 | - |
| Liquid bacterial agent | 23 | 13.1 | 80 |
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, be included in protection scope of the present invention.
Claims (2)
1. a white thorn streptomycete bacterial strain BWL15-4 who prevents and treats banana blight, is characterized in that: the Classification And Nomenclature of this bacterial strain BWL15-4 for white thorn streptomycete (
streptomyces albospinus), on May 9th, 2011, being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No. 4836.
2. the application of white thorn streptomycete bacterial strain BWL15-4 claimed in claim 1 in control banana blight.
3. a preparation of preventing and treating banana blight, is characterized in that: the liquid bacterial agent or the solid fungicide that contain white thorn streptomycete bacterial strain BWL15-4 claimed in claim 1.
4. the preparation method of preparation described in claim 3, is characterized in that: will sting in vain streptomycete bacterial strain BWL15-4 through preparing liquid bacterial agent or solid fungicide containing actication of culture, liquid or solid fermentation procedure.
5. the preparation method of preparation according to claim 4, is characterized in that: also comprise seed culture operation.
6. the preparation method of preparation according to claim 5, is characterized in that: the substratum adopting during liquid fermenting comprises glucose 1-3%, soybean cake powder 1-4%, Zulkovsky starch 1-4%, yeast extract paste 0.2%, NaCl 0.4%, CaCO
30.25%, defoamer 0.1-0.3%, by quality percentage composition, all the other are water.
7. the preparation method of preparation according to claim 5, is characterized in that: the solid medium adopting during solid fermentation comprises rice 60-70%, analysis for soybean powder 5-15%, and wheat skin 10-20%, sandy soil 5-10%, by quality percentage composition.
8. the application of preparation in control banana blight described in claim 3.
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| CN105400714A (en) * | 2015-10-29 | 2016-03-16 | 海南大学 | Streptomyces aureoverticillatus HN6 solid fermentation method |
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| CN107904196B (en) * | 2017-12-26 | 2020-04-07 | 中国热带农业科学院热带生物技术研究所 | Streptomyces yanshi and application thereof |
| CN108865934B (en) * | 2018-07-04 | 2022-06-28 | 中国热带农业科学院热带生物技术研究所 | Bacillus safensis HMD9204 and microbial inoculum and application thereof |
| CN113558058B (en) * | 2021-08-17 | 2022-04-26 | 中国科学院华南植物园 | Application of compound Xerucitririn A in preparation of medicines for preventing and treating banana vascular wilt pathogenic bacteria |
| CN115627248B (en) * | 2022-12-21 | 2023-04-28 | 中国热带农业科学院三亚研究院 | Novel streptomyces with broad-spectrum antifungal effect and microbial inoculum, preparation method and application thereof |
| CN115948301B (en) * | 2023-01-06 | 2023-10-24 | 中国热带农业科学院热带生物技术研究所 | Oligosaccharin composite microbial inoculant for preventing and controlling banana vascular wilt and application thereof |
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