CN102329755A - Nonabsorbent streptomyces ahygroscopicus strain BWL58 for preventing and treating banana vascular wilt and application thereof - Google Patents
Nonabsorbent streptomyces ahygroscopicus strain BWL58 for preventing and treating banana vascular wilt and application thereof Download PDFInfo
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Abstract
The invention discloses a nonabsorbent streptomyces strain BWL58 for preventing and treating a banana vascular wilt. The strain BWL58 is classified and named as nonabsorbent streptomyces ahygroscopicus, and is preserved in the China General Microbiological Culture Collection Center on May 9, 2011; the preservation number is CGMCC No. 4837; 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 not streptomyces hygroscopicus strain BWL58 and application thereof that prevents and treats banana blight.
Background technology
Banana blight is to infect the crushing soil-borne disease on a kind of banana production that causes by Fusarium oxysporum Cuba specialized form (Fusarium oxysporum f.sp.cubense).This pathogenic bacteria has 4 physiological strains; In China's distribution 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, the most of banana planting in Fujian district big area takes place, onset area is above 500,000 mu.The serious place of Guangdong and Hainan banana blight morbidity is the large-scale planting banana again, has to replant other crop.
Banana blight bacteria is a soil inhabitant, and it is invaded from the root of banana, up spreads along vascular bundle and spreads; Stop up 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 in soil more than 10 year, was a kind of disease of popular for many years.
Because the banana blight pathogenic bacteria expands and infects, still there are not highly effective chemical agent control and high resistance to wilt good kind so far in vascular bundle.Therefore, biological control receives 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 type bacterial strain has many advantages as biocontrol microorganisms; But have a wretched insufficiency, promptly bacterial strain itself is not strong to the antagonistic ability of pathogenic bacteria, thereby might influence its biocontrol effect yet.
Summary of the invention
First purpose of the present invention is to provide a kind of not streptomyces hygroscopicus strain BWL58 that prevents and treats banana blight; This bacterial strain is strong to the antagonistic ability of banana blight pathogenic bacteria, because bacterial strain itself separates from plant rhizosphere soil, is soil inhabitant; The rhizosphere colonization ability is strong, and biocontrol effect is good.
Second purpose of the present invention is to provide the application of above-mentioned not streptomyces hygroscopicus strain BWL58 in the control banana blight.
The 3rd purpose of the present invention is to provide a kind of preparation that contains above-mentioned not streptomyces hygroscopicus strain BWL58.
The 4th purpose of the present invention is to provide preparing such formulations, and this preparing method's technology is simple, and fermention medium need not valuable raw material, and cost is low, promotes easily.
Last purpose of the present invention is to provide the application of above-mentioned preparation in the control banana blight.
First purpose of the present invention realizes through following technique means: a kind of not streptomyces hygroscopicus strain BWL58 that prevents and treats banana blight; The classification called after of this bacterial strain BWL58 is streptomyces hygroscopicus (Streptomyces ahygroscopicus) not; Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on May 9th, 2011, deposit number is CGMCC No.4837.
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; Accession number is JN038326, and the 16S rDNA nucleotide sequence of this bacterial strain BWL58 is shown in SEQ ID NO:1.
Second technical scheme of the present invention realizes through following technical scheme: the application of above-mentioned not streptomyces hygroscopicus strain BWL58 in the control banana blight.
The 3rd technical scheme of the present invention realizes through following technical scheme: a kind of preparation of preventing and treating banana blight, contain liquid bacterial agent or the solid fungicide of above-mentioned bacterial strains BWL58.
The 4th technical scheme of the present invention realizes through following technical scheme: the preparation method of the preparation of above-mentioned control banana blight, with above-mentioned not streptomyces hygroscopicus strain BWL58 through contain actication of culture, the liquid or solid fermentation procedure prepares liquid bacterial agent or solid fungicide.
Wherein, Among the preparation method of the preparation of above-mentioned control banana blight; Can also contain the seed culture operation, be about to above-mentioned not streptomyces hygroscopicus strain BWL58 and prepare liquid bacterial agent or solid fungicide through containing actication of culture, seed culture, liquid or solid fermentation procedure.
Specifically; Liquid bacterial agent among the present invention can obtain through following technical process: bacterial classification BWL58 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.
The substratum that wherein adopts during liquid fermenting in the liquid nutrient medium can comprise glucose 1-3%, soybean cake powder 1-4%, Zulkovsky starch 1-4%, yeast extract paste 0.2%, NaCl 0.4%, CaCO
30.25%, skimmer 0.1-0.3%, by the quality percentage composition, all the other are water.More than be the preferred substratum of the present invention, in fact the present invention does not have special strict restriction to culture condition and liquid nutrient medium, as long as this condition and liquid nutrient medium are fit to the growth of bacterial strain.
Specifically; Solid fungicide among the present invention can obtain through following technical process: bacterial classification BWL58 activation (being that first order seed is cultivated) → spawn culture (is that secondary seed is cultivated; Optional) → solid culture matrix → fermentation cake (solid fermentation) → drying and crushing; Fermentation cake that obtains after the drying and crushing and absorption carrier were by 1: 1 or other mixed, and be suitably dry behind the mixing, processes solid fungicide.Wherein absorption carrier can be variety carrier such as kaolin, non-arable layer soil.
The solid medium that wherein adopts during solid fermentation is preferably and comprises rice 60-70%, analysis for soybean powder 5-15%, wheat skin 10-20%, sandy soil 5-10%, more than by the quality percentage composition.More than be the preferred solid medium of the present invention, in fact the present invention does not have special strict restriction in fact to the selection of culture condition and solid medium, as long as this condition and solid medium are fit to the growth of bacterial strain.
Last purpose of the present invention realizes through following technique means: the application of above-mentioned preparation in the control banana blight.
When the solid fungicide among the present invention is prevented and treated banana blight; Can mix liquid bacterial agent or solid fungicide and common fertilizer thoroughly back by 2: 100 (mass ratio) or other mixed uses as base fertilizer; Each field planting 2-3 kilogram of spreading manuer in holes is used before banana planting; Can join liquid bacterial agent or solid fungicide in the conventional fertilizer of dissolved with dilution 5-10 ratio doubly the vegetative period behind banana planting, and banana plant is watered fertilising.Perhaps the liquid bacterial agent among the present invention control banana blight can be through being diluted to fermented liquid 2 times or dilute the back liquid irrigating root as required or water and impose on the plant base portion, or before banana seedlings is transplanted, soak root.
Compared with prior art, the present invention has following advantage:
(1) the present invention with streptomyces hygroscopicus BWL58 not as antagonistic strain, strong to the banana blight bacteria restraining effect, banana blight is had good control effect;
(2) preparation of the present invention can stimulate the banana root growth and promote the banana growth of seedlings, and banana is had growth promoting function;
(3) not streptomyces hygroscopicus bacterial strain BWL58 culture condition of the present invention is simple, and easy storing is easy to suitability for industrialized production, and the excellent development application prospect is arranged;
(4) the agent preparation method technology of system control banana blight of the present invention is simple, and fermention medium need not valuable raw material, and cost is low, promotes easily.
Description of drawings
Fig. 1 is the spore of the not streptomyces hygroscopicus BWL58 bacterial strain that screening obtains among the embodiment 1 and the stereoscan photograph of spore chain, and spore surface has short thorn;
Fig. 2 is the fungistatic effect of the indoor flat plate face-off cultivation of not streptomyces hygroscopicus BWL58 bacterial strain and the banana blight bacteria of preparation among the embodiment 1; Banana wilt germina number-four biological strain receives after the inhibition of streptomyces hygroscopicus BWL58 bacterial strain not that the mycelia tip is expanded, blackening, tenuigenin liquid leak; 1 is streptomyces hygroscopicus BWL58 bacterial strain not, and 2 is banana wilt germina number-four biological strain.
Embodiment
Embodiment 1
The separation of bacterial strain and evaluation
Biocontrol microorganisms among the present invention be from the virgin forest soil of overlord mountain range, Hainan Island screening and separating to a streptomycete; This bacterial strain has had strong inhibitory effects to banana blight bacteria (Fusarium oxysporum f.sp.cubense); This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on May 9th, 2011; Preserving number is CGMCC No.4837, and the classification called after is streptomyces hygroscopicus (Streptomyces ahygroscopicus) not.
The separation screening process of bacterial strain BWL58 is following: from herborization rhizosphere soil the virgin forest of overlord mountain range, Hainan Island, then soil sample is carried out air-dry, with coating on the screening culture medium after the sterilized water dilution suitable multiple; Screening culture medium is to add the PDA solid medium of SRM 935a, Nalidixic Acid and cycloheximide, cultivates more than 5 days for 28 ℃, wait to screen grow the actinomycetes bacterium colony on the flat board after; One or more indicator banana blight bacteria bacterium cakes are inserted in the appropriate location on flat board again, and when certain unwrapping wire bacterium colony had antagonistic action, indicator just can not be crossed this actinomycetes growth; Present tangible antagonism band; Picking has the actinomycetes of antagonistic action, and line purifying three times carries out flat board face-off experiment; The actinomycetes of selecting antagonism band broad are as candidate strain; Candidate strain is carried out liquid fermenting, get fermented liquid 10mL filtration sterilization, carry out the production of antibacterial substance in the dull and stereotyped face-off examination fermented liquid with the Oxford cup; The fermented liquid that produces antimicrobial substance carries out the field efficacy screening again in the sick garden of banana blight, filter out the good antagonistic strain BWL58 of field control effect thus.
The qualification process of bacterial strain BWL58 is following: at first it is carried out 16S rDNA sequential analysis; Carry out the Blast comparison among the online gene of the being submitted to bank of 16S rDNA sequence (http://www.ncbi.nlm.nih.gov/blast) with this bacterial strain; Find that itself and streptomyces hygroscopicus (Streptomyces ahygroscopicus) not are in same evolution branch, homology reaches more than 99%.The 16S rDNA sequence 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 JN038326, and its 16S rDNA nucleotide sequence is shown in SEQ ID NO:1.Then the BWL58 bacterial strain is carried out morphology, cultural characteristic and physio-biochemical characteristics Analysis and Identification, through analyzing its morphological specificity be: fibrillae of spores is long volution under ESEM, the 2-6 circle, and spore is most of spherical to oval, and there is the spinelet (see figure 1) on the surface; Abundant aerial hyphae and substrate mycelium are arranged on nutrient agar plate, gas silk pearl, base silk olive is yellow; No soluble pigment, the ability liquefy gelatin, the starch hydrolysis is strong; Do not utilize Mierocrystalline cellulose, reduction nitrate salt does not produce hydrogen sulfide and melanochrome; Utilize urea; Can utilize fructose, rhamnosyl, seminose, glycerine, lactose, synanthrin, glucose, inositol, N.F,USP MANNITOL, SANMALT-S, dextrin, starch, sorbose, not utilize raffinose, wood sugar, saligenin, galactitol, be that growth is poor on the culture medium of sole carbon source at pectinose, sucrose, semi-lactosi.Morphological feature of this bacterial strain and cultural characteristic and streptomyces hygroscopicus is not identical, the physio-biochemical characteristics overwhelming majority is with streptomyces hygroscopicus is not identical.Therefore; The result that the comparison result of comprehensive 16S rDNA sequence and morphological specificity, cultural characteristic and physio-biochemical characteristics are analyzed; The BWL58 identification of strains is streptomyces hygroscopicus not, and called after is streptomyces hygroscopicus BWL58 bacterial strain (Streptomyces ahygroscopicus BWL58) not.
This bacterial strain bacteriostasis and rhizosphere colonization ability are strong; Can be at the rhizosphere colonization of banana; Antibacterial circle diameter average out to 18mm in flat board face-off experiment, blackening is expanded at indicator banana blight bacteria mycelia tip; Tenuigenin liquid leaks (seeing shown in Figure 2), explains that the meta-bolites of this bacterial strain has killing effect and very strong restraining effect to Cuba Fusarium oxysporum mycelia.
Embodiment 2
Liquid bacterial agent and preparation method thereof
The liquid bacterial agent that contains above-mentioned bacterial strains BWL58 that present embodiment provides prepares through following method:
(1) actication of culture is inoculated into bacterial strain BWL58 on the solid medium, and substratum is a nutrient agar;
(2) seed culture is inoculated into liquid nutrient medium with activated spawn, and by the quality percentage composition, filling a prescription is: glucose 2%, and soybean cake powder 1%, yeast extract paste 0.2%, NaCl 0.3%, CaCO
30.1%, K
2HPO
40.05%, all the other are water; PH value 7.0-7.2; Cultivated 72 hours at 160 rev/mins of shake flask fermentations of 28 ℃ of following triangular flasks, in fact abundant like the bacterial classification of preparation in the step (1), then can directly carry out liquid fermenting and get final product again through the seed culture operation;
(3) liquid fermenting adds seed liquor in the liquid nutrient medium to by 6% (volumn concentration), and filling a prescription is: glucose 2%, soybean cake powder 1%, Zulkovsky starch 2%, yeast extract paste 0.2%, NaCl 0.4%, CaCO
30.25%, skimmer 0.1%, and by the quality percentage composition, all the other are water, and what adopt in the present embodiment is preferable substratum; Other substratum also can, do not have special restriction, pH value 7.2, jar the temperature 28 ℃; Tank pressure 0.07Mpa, stirring velocity 200rpm, oxygen-supply quantity＞60%; About 7 days of fermentation time obtains the liquid fermenting preparation, supplies the field control disease to use.
Embodiment 3
Solid fungicide and preparation method thereof
The solid fungicide that contains above-mentioned bacterial strains BWL58 that present embodiment provides prepares through following method:
(1) actication of culture: bacterial strain BWL58 is inoculated on the solid medium, and substratum is a nutrient agar;
(2) seed culture: activated spawn is inoculated into liquid nutrient medium, and by the quality percentage composition, filling a prescription is: glucose 1%, and soybean cake powder 1%, soy peptone 0.2%, yeast extract paste 0.2%, NaCl 0.3%, CaCO
30.1%, K
2HPO
40.05%, all the other are water, adopt in the present embodiment for preferable substratum, other substratum also can, do not have special restriction; PH value 7.0-7.2; Cultivated 72 hours at 160 rev/mins of shake flask fermentations of 28 ℃ of following triangular flasks, in fact abundant like the bacterial classification of preparation in the step (1), then can directly carry out solid fermentation and get final product again through the seed culture operation;
(3) solid culture: seed culture fluid is mixed with solid medium, and by the quality percentage composition, the solid culture based formulas is: rice 65%; Analysis for soybean powder 5%, wheat skin 20%, sandy soil 10%; More than by the quality percentage composition, the proportioning of solid medium is preferred here, other proportioning also can; No particular requirement, 28 ℃ of-30 ℃ of incubation times are about 14 days, and this moment, solid medium should fully be grown and big volume production spore;
(4) fungicide preparation: the solid culture matrix drying and crushing that will fully ferment, promptly processed solid fungicide by 1: 1 behind the mixing with kaolin or non-arable layer soil.
Embodiment 4
Liquid bacterial agent and solid fungicide are to the banana blight field control effect:
The not streptomyces hygroscopicus strain BWL58 liquid bacterial agent of preparation among microbial inoculum: the embodiment 2 is used in test, the white thorn streptomycete BWL58 solid fungicide of preparation among the embodiment 3, clear water contrast;
Test place: the sick garden of China tropic Agriculture Academy Sciences tropic Biotechnology Research Institute's Haikou banana blight;
Supply the examination banana variety: sense blight kind Brazil any of several broadleaf plants.
Application method:
Planting pit pre-treatment: contain not with above-mentioned that 2 times of diluent 1L of liquid bacterial agent of streptomyces hygroscopicus strain BWL58 bacterial strain water the pouring planting pit, cover thin soil then; BWL58 bacterial strain solid fungicide is with after fertilizer mixed by 1: 1, and each planting pit is put and used the 2-3 kilogram, fully mixes thoroughly with soil in the cave.
Banana seedlings is executed microbial inoculum in advance and handled: transplant previous day, 2 times of diluents of streptomyces hygroscopicus BWL58 strain liquid microbial inoculum soak root with containing not with banana seedlings; Be used for banana seedlings after solid fungicide dilutes 5 times and soak root.
Transplant: the banana seedlings that will soak behind the root is transplanted in the planting pit of handling.
Management: fertilising regularly, liquid bacterial agent are handled every month 5 times of diluent 2L with liquid bacterial agent and are irritated roots once, and solid fungicide is handled every month with 5 times of dilution liquid irrigating roots of solid fungicide once, record plant disease index, sickness rate after 6 months, and calculate control effect.
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) number * superlative degree value
Control 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 does not have visible symptom, dissects false cauline bundle and organizes and do not see browning, and 1 grade of outward appearance does not have visible symptom; 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 withered as yet, and 4 grades of diseased plants are withered.
Test-results:
The check plot began to occur the banana blight symptom in 40 days after the banana seedlings field planting;, the banana that streptomyces hygroscopicus BWL58 bacterial strain fermentation liquor is not handled or solid preparation is handled all do not have plant performance blight symptom after handling 120 days; Control effect after 6 months; The BWL58 bacterial strain fermentation liquor is treated to 82% (seeing table 1 for details); BWL58 bacterial strain solid preparation is treated to 79.5% (seeing table 2 for details), shows that the BWL58 bacterial strain is that fermented liquid or solid preparation all have good control effect to banana blight.
Table 1 not streptomyces hygroscopicus BWL58 bacterial strain fermentation liquor to the control effect investigation statistics table of banana blight
| Handle | Sickness rate % | Disease index % | Preventive effect % |
| Clear water contrast (CK) | 100％ | 66.5 | - |
| The BWL58 liquid bacterial agent | 15％ | 12 | 82 |
Table 2 not streptomyces hygroscopicus BWL58 bacterial strain solid preparation to the control effect investigation statistics table of banana blight
| Handle | Sickness rate % | Disease index % | Preventive effect % |
| Clear water contrast (CK) | 100％ | 68.5 | - |
| The BWL58 liquid bacterial agent | 22％ | 14.1 | 79.5 |
The foregoing description is a preferred implementation of the present invention; But embodiment of the present invention is not restricted to the described embodiments; Other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; All should be the substitute mode of equivalence, be included in protection scope of the present invention.
Claims (8)
1. not streptomyces hygroscopicus strain BWL58 who prevents and treats banana blight; It is characterized in that: the classification called after of this bacterial strain BWL58 is streptomyces hygroscopicus (Streptomyces ahygroscopicus) not; Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on May 9th, 2011, deposit number is CGMCC No.4837.
2. the application of the described bacterial strain BWL58 of claim 1 in the 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 the described not streptomyces hygroscopicus of claim 1 strain BWL58.
4. the preparation method of the said preparation of claim 3 is characterized in that: with the described not streptomyces hygroscopicus of claim 1 strain BWL58 through contain actication of culture, the liquid or solid fermentation procedure prepares liquid bacterial agent or solid fungicide.
5. according to the preparation method of the said preparation of claim 4, it is characterized in that: also comprise the seed culture operation.
6. according to the preparation method of the said preparation of claim 5, it is characterized in that: the substratum that adopts during liquid fermenting comprises glucose 1-3%, soybean cake powder 1-4%, Zulkovsky starch 1-4%, yeast extract paste 0.2%, NaCl0.4%, CaCO
30.25%, skimmer 0.1-0.3%, by the quality percentage composition, all the other are water.
7. according to the preparation method of the said preparation of claim 5, it is characterized in that: the solid medium that adopts during solid fermentation comprises rice 60-70%, analysis for soybean powder 5-15%, wheat skin 10-20%, sandy soil 5-10%, more than by the quality percentage composition.
8. the application of the described preparation of claim 3 in the control banana blight.
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| CN104894018A (en) * | 2015-05-27 | 2015-09-09 | 中国热带农业科学院热带生物技术研究所 | Luna Lin Haresii streptomycete and application thereof |
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| CN104894018A (en) * | 2015-05-27 | 2015-09-09 | 中国热带农业科学院热带生物技术研究所 | Luna Lin Haresii streptomycete and application thereof |
| CN104894018B (en) * | 2015-05-27 | 2018-09-25 | 中国热带农业科学院热带生物技术研究所 | A kind of Lu Nalinrui streptomycetes and its application |
| CN105400714A (en) * | 2015-10-29 | 2016-03-16 | 海南大学 | Streptomyces aureoverticillatus HN6 solid fermentation method |
| CN107904196A (en) * | 2017-12-26 | 2018-04-13 | 中国热带农业科学院热带生物技术研究所 | A kind of Yan Shi streptomycetes and its application |
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