CN108795825A - Application of one plant of willow bark rot biocontrol microorganisms in inhibiting pathogenic activity - Google Patents
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Abstract
The present invention relates to application of one plant of willow bark rot biocontrol microorganisms in inhibiting pathogenic activity, willow bark rot biocontrol microorganisms are sioyaensis (Streptomyces sioyaensis) F58, it is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.11469；The pathogenic bacteria are plant pathogen.The present invention has many advantages, such as that good antimicrobial effect, antimicrobial spectrum are wide, environmentally friendly.
Description
The present invention is divisional application, and former Chinese Patent Application No. is：201510859507.7 the applying date is：2015 12
Month 1 day, patent name was when application：One plant of willow bark rot biocontrol microorganisms.
Technical field
The invention belongs to biotechnologies more particularly to one plant of willow bark rot biocontrol microorganisms in inhibiting pathogenic activity
Application.
Background technology
Willow bark rot is one of important disease of willow, the disease worldwide generally distribution, extensively generation.Poplar
The pathogen perfect stage for setting bark rot is dirty black skin shell bacterium (Valsa sordida N it.), and the imperfect stage is golden yellow shell
Capsule spore bacterium (Cytospora chrysosperma).Currently, the prevention of the disease is mainly with chemical prevention and breeding resistant variety
It is main, but there is apparent drawback in above two control method.There are resistances to degenerate for the kind that disease-resistant variety selection and breeding are cultivated, anti-
The problems such as property is low；And chemical prevention easily causes the problems such as environmental pollution and destruction ecological balance.
Fungus treatment is to avoid the effective solution of the above problem, and be somebody's turn to do using antagonistic microbe controlling plant diseases
Method belong to the ecosystem regulate and control based on sustainable control technology, have it is free from environmental pollution, do not destroy the excellent of the ecological balance
Point.But at present can antagonism Cytospora chrysosperma biocontrol microorganisms it is fewer and fewer, the antibiotic also pole for the prevention of willow bark rot
To be rare, majority remains in the laboratory screening stage.
Invention content
In view of the problems of prior art, the present invention provides one plant of willow bark rot biocontrol microorganisms and pathogenic bacteria is being inhibited to live
Application in property, has many advantages, such as that good antimicrobial effect, antimicrobial spectrum are wide and environmentally friendly.
The technical solution that the present invention solves above-mentioned technical problem is as follows：
The present invention provides application of one plant of willow bark rot biocontrol microorganisms in inhibiting pathogenic activity.
Willow bark rot biocontrol microorganisms of the present invention are sioyaensis (Streptomyces sioyaensis) F58,
It is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.11469.
The pathogenic bacteria are plant pathogen.
Preferably, the pathogenic bacteria are selected from Cytospora chrysosperma (Vasal sordida), dothiorella gregaria bacterium
(Botryosphaeria dothidea), Alternaria mali (Alternaria alternata f.sp.mali), sweet tea
Cucurbit wilt bacterium (Fusarium oxysporium f.sp.melonis), fusarium moniliforme (Fusarium
Moniliforme), Rhizoctonia solani Kuhn (Rhizoctonia solani), Pyricularia oryzae (Magnaporthe grisea), big
Beans anthrax bacteria (Colletotrichum truncatum), Cochliobolus sativus (Bipolaris sorokiniana), wheat
Gibberellic hypha (Fusarium graminearum), Tobacco target spot bacterium (Thanatephorus cucumeris), the big spot of corn
One or more of germ (Exserohilum turcicum), southern corn leaf blight (Bipolaris maydis).
Sioyaensis (Streptomyces sioyaensis) F58 has many advantages, such as that antimicrobial spectrum is wide, for above-mentioned cause
Germ has bacteriostatic activity.
The present invention provides one plant of willow bark rot biocontrol microorganisms, can effectively prevent willow bark rot.
Sioyaensis (Streptomyces sioyaensis) F58 is in the tablet dual test of Cytospora chrysosperma
The antibacterial bandwidth of viable bacteria is up to 30.2~35.5mm, and zymotic fluid cylinder plate method measures antibacterial circle diameter and reaches 50.7~56.4mm, manually
Inoculation controlling experiment shows that biological active matter confrontation willow bark rot has significant control effect, protective effect in its zymotic fluid
With therapeutic effect respectively up to 88.7% and 73.4%, its control effect compared with the chemical agent for preventing willow bark rot in production
Difference is not notable, and has low toxicity, noresidue and environmental-friendly advantage.
Sioyaensis (Streptomyces sioyaensis) F58 can make gelatin liquefaction；It cannot make milk solidification and peptone
Change；Do not utilize decomposition of cellulose；Hydrolysis starch is positive；Catalase, urase, Esterase reaction are the positive；It does not generate
H2S；M.R. negative, V.P. experiments are positive；Nitrate reduction reaction negative；Can utilize arabinose, xylose, fructose,
Starch, lactose, glucose, maltose；Mannitol, raffinose, rhamnose cannot be utilized.Sioyaensis (Streptomyces
Sioyaensis) F58 can utilize beef extract, peptone, yeast powder, (NH4)2SO4And KNO3Five kinds of nitrogen sources；To the resistance to of NaC l
It is stronger by property, it can be grown on the culture medium of the l containing 5%NaC；There is stronger tolerance to temperature, can be given birth at 15~37 DEG C
Long, 28 DEG C are optimum growth temperature；It is good to the tolerance of alkali, it can be grown in the range of pH5-12, and pH5~7 are made a living
Long optimal pH.
Sioyaensis (Streptomyces sioyaensis) F58 riotous growth on Gause I culture medium, gas life
Mycelia is luxuriant, and 28 DEG C of culture 1~2d bacterium colonies are smooth, are generated without spore, and 3d begins with white spore and grown from colony edge, 4d
After start to gradually become greyish white to snow weasel ash.Microscopically observation, substrate mycelium are not broken without horizontal film；Electronic Speculum observes result table
It is bright (as shown in Figures 1 to 3)：Fibrillae of spores pine is spacious or tight spiral, often irregularly, accidental short and loose-screw shape, sporophore
Shape；It is identified by microbial morphology and physiological and biochemical index, it is believed that willow bark rot biocontrol microorganisms of the present invention belong to streptomyces.Salt
Room streptomycete (Streptomyces sioyaensis) F58 is through 16SrDNA sequence alignment analysis, the strepto- with known actinomyces
The 16S rDNA homology highests of bacterium Streptomyces sioyaensis (gene accession number KF475886), reach 99%.
Sioyaensis (Streptomyces sioyaensis) F58 is sioyaensis, belongs to streptomyces
(Streptomyces)；It is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation
Location is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, deposit number CGMCC
No.11469, the deposit date is Septembers in 2015 30 days.
Description of the drawings
Fig. 1 is the spore chain and spore Electronic Speculum observation chart of sioyaensis (Streptomyces sioyaensis) F58.
Fig. 2 is the spore chain and spore Electronic Speculum observation chart of sioyaensis (Streptomyces sioyaensis) F58.
Fig. 3 is the spore chain and spore Electronic Speculum observation chart of sioyaensis (Streptomyces sioyaensis) F58.
Specific implementation mode
The principle and features of the present invention will be described below with reference to the accompanying drawings, and the given examples are served only to explain the present invention, and
It is non-to be used to limit the scope of the present invention.
Specific implementation mode one：Present embodiment willow bark rot biocontrol microorganisms are sioyaensis (Streptomyces
Sioyaensis) F58, is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is
CGMCC No.11469。
Sioyaensis (Streptomyces sioyaensis) F58 picks up from Shuangliao City of Jilin Province experimental forest, by soil
Sample dilutes 10 with sterile water3Times, it is cultivated under the conditions of 28 DEG C using Gause I culture medium, is trained using conventional plating dilutions
The method separation of supporting obtains.Sioyaensis (Streptomyces sioyaensis) F58 after isolating and purifying is transferred to Gause I training
Support base cultivated under the conditions of 28 DEG C, expand it is numerous.
Sioyaensis before is isolated from ocean mud, and sioyaensis (Streptomyces of the present invention
Sioyaensis) F58 is the first sioyaensis isolated from soil.Sioyaensis of the present invention
(Streptomyces sioyaensis) F58 and sioyaensis the type strain difference on utilization of carbon source, salt room strepto-
Bacterium (Streptomyces sioyaensis) F58 cannot utilize gossypose and rhamnose, but can utilize L-arabinose, D-
Fructose, cornstarch；16SrDNA comparison results are not also 100% homologous simultaneously, therefore judge sioyaensis
(Streptomyces sioyaensis) F58 should be sioyaensis new variant.
Embodiment 1
Groundnut meal is a concentration of in sioyaensis (Streptomyces sioyaensis) F58 fermentation mediums
2.5% (w/v), a concentration of 5.0% (w/v) of starch, a concentration of 0.08% (w/v) of yeast powder, glucose it is a concentration of
0.02% (w/v), (NH4)2SO4A concentration of 0.08% (w/v), CaCO3A concentration of 0.32% (w/v), NaC l concentration
For 0.2% (w/v), fermentation medium pH value is 7.0~7.2.It is inoculated with sioyaensis (Streptomyces sioyaensis)
The fermentation medium of F58 150r/mi n shake culture 7d under 28 DEG C of constant temperatures, then 6000r/mi n centrifuge 15mi n,
It crosses bacterial filter and removes thalline acquisition sioyaensis (Streptomyces sioyaensis) F58 fermented supernatant fluids.
Sioyaensis (Streptomyces sioyaensis) F58 zymotic fluids are to willow bark rot controlling experiment
Willow bark rot pathogen infection：The inoculation yellow fast poplar cuttage seeding of 1a lifes, by Cytospora chrysosperma in PDA culture medium
After 26 DEG C of 5~7d of constant temperature incubation, it is spare to beat bacteria cake with 7mm card punch, gives willow cadre to scratch with blade, wound size 5mm is left
Willow bark rot opportunistic pathogen bacteria cake is attached to wound, poplar leaf juice moisturizing is dipped in absorbent cotton by the right side, often handles 15 plants, 3 weights
It is multiple, remove bacteria cake after moisturizing 7d.
The antibacterial comparison of zymotic fluid：5d, 10d, 15d after bacteria cake are removed in 15d, 10d, 5d and inoculation processing before inoculation, respectively
By 5 times of dilutions of F58 fermented supernatant fluids, 50 times of dilutions of 50% Tuzet, 100 times of dilutions, 70% of 40% asomate
100 times of dilutions and the clear water control of thiophanate methyl are uniformly applied to the willow cadre infected by Cytospora chrysosperma, timing
Nursery stock incidence is observed, control effect is calculated.
Disease index grade scale, with reference to Jilin Province's provincial standard《Willow bark rot process for comprehensively treating regulation》
I grade (typical value 0) --- it is disease-free；
II grade (typical value 1) --- scab lateral length accounts for 1/4 or less trunk perimeter；
III level (typical value 2) --- scab lateral length accounts for the 1/4~2/4 of trunk perimeter；
IV grade (typical value 3) --- scab lateral length accounts for the 2/4~3/4 of trunk perimeter；
3/4 or more~trees that V grades (typical values 4) --- scab lateral length accounts for trunk perimeter are dying or dead.
Experimental result：Investigate incidence and disease index (as shown in table 1) after removing bacteria cake 15d, control nursery stock morbidity
Rate and disease index are distributed as 82.2% and 49.4；Coating processing before T1 (with sioyaensis F58 preventions) pathogen inoculation
Nursery stock incidence is 15.5%, and disease index is 5.57, the incidence and disease index that coating is handled after pathogen inoculation
It is 35.6% and 13.4 respectively, protective effect and therapeutic effect are distributed as 88.7% and 73.4%；T2 is (with 40% asomate
The prevention of 100 times of dilutions) and the protective effects that are handled (with the prevention of 100 times of dilutions of 70% thiophanate methyl) of T4 be
89.8, therapeutic effect is distributed as 74.4% and 79.3%.Experiment finds that control nursery stock scab within several weeks later is gradually expanded,
The visible apparent pycnidia at scab has individually the appearance of conidium angle；Plant scab susceptible T1~T4 does not almost have
Have and continue to expand, pycnidia is had no at scab.The experimental results showed that sioyaensis (Streptomyces
Sioyaensis) F58 zymotic fluids have significant inhibiting effect to Cytospora chrysosperma, even if willow has infected willow bark rot
Bacterium also has preferable therapeutic effect；Although its protective effect be slightly below T2 and T4, higher than T3 it is (dilute with 50 times of 50% Tuzet
Release liquid prevention), but difference is not notable；Therapeutic effect and T2~T4 differences is not notable.
Table 1
Data are average ± standard deviation in table.Show with different large and small lowercase alphabets after column data newly multiple through DuncanShi
Very poor method is examined in P<0.01 and P<0.05 level difference is notable, similarly hereinafter.
Sioyaensis (Streptomyces sioyaensis) F58 bacteriostatic tests
Sioyaensis (Streptomyces sioyaensis) F58 tablets dual test is to Tobacco target spot bacterium, willow
Bark rot bacterium and the effect of dothiorella gregaria bacterium are most strong, it is seen that apparent antibacterial band, antibacterial band mean breadth reaches 30mm or more, right
Alternaria mali, rice blast fungus and Rhizoctonia solani Kuhn are respectively with very strong inhibiting effect, antibacterial bandwidth
27.6mm, 25.6mm, 18.3mm, to fusarium graminearum, Cochliobolus sativus, Melon fusarium Wilt, fusarium moniliforme, big
Beans anthrax bacteria, Exserohilum turcicum and southern corn leaf blight inhibiting effect are relatively weaker, but antibacterial bandwidth 6.3mm or more.
Cylinder plate method measures the bacteriostatic activity of sioyaensis (Streptomyces sioyaensis) F58 and its zymotic fluid：
Mixed bacterium tablet is made in all kinds of pathogens, the bacteriostatic activity of zymotic fluid is measured using cylinder plate method, zymotic fluid dilutes 5 times using sample-adding
Amount is 100 μ l, often handles 3 repetitions.The suppression of sioyaensis (Streptomyces sioyaensis) F58 and its zymotic fluid
Bacterium activity is as shown in table 2.
Table 2
| Strains tested | Antibacterial band (mm) | Bacteriostatic diameter (mm) |
| Cytospora chrysosperma | 32.3±2.55Bb | 56.4±0.70Aa |
| Dothiorella gregaria bacterium | 30.2±1.41BCb | 42.7±0.98Dc |
| Alternaria mali | 27.6±1.60CDc | 35.5±0.60Ee |
| Melon fusarium Wilt | 15.7±1.34EFe | 22.8±0.87Ff |
| Fusarium moniliforme | 11.3±1.04GHf | 21.3±0.62Ffg |
| Rhizoctonia solani Kuhn | 18.3±0.78Ed | 50.3±2.09Bb |
| Rice blast fungus | 25.6±1.27Dc | 22.7±1.22Ff |
| Colletotrichum truncatum | 18.0±1.38Ed | 47.3±1.05Cc |
| Cochliobolus sativus | 8.7±0.34HIg | 18.5±1.153Gh |
| Fusarium graminearum | 6.3±0.78Ih | 56.8±1.27Aa |
| Tobacco target spot bacterium | 35.5±1.70Aa | 50.8±1.17Bb |
| Exserohilum turcicum | 13.2±0.98FGf | 20.5±0.85FGg |
| Southern corn leaf blight | 11.5±0.85GHf | 23.2±1.13Ff |
Experiments have shown that the zymotic fluid of sioyaensis (Streptomyces sioyaensis) F58 still maintains preferably
Bacteriostatic activity has extremely strong antagonism to fusarium graminearum, Tobacco target spot bacterium, Cytospora chrysosperma, dothiorella gregaria,
Bacteriostatic diameter reaches 50mm or more, and has obvious inhibiting effect, sioyaensis for examination pathogen to all
(Streptomyces sioyaensis) F58 antimicrobial spectrums are wide.
Embodiment 2
Sioyaensis (Streptomyces sioyaensis) F58 is inoculated in 10 kinds of different culture mediums respectively, 28
DEG C 36~48h of culture begins with bacterium colony appearance, and spore starts to generate after 72~96h.The form of lawn, spore on different culture mediums
The color of sub- heap, the color of matrix mycelia, the soluble pigment of generation, growing state are different, according to《Strepto- dientification of bacteria hand
Volume》The method and color version of offer are observed, are compared, and the results are shown in Table 3, by sioyaensis (Streptomyces
Sioyaensis) F58 generates soluble pigment on most culture mediums；Aerial hyphae becomes snow weasel grey or freshwater mussel meat from canescence
In vain, substrate mycelium is milky white to snow weasel ash.
Table 3
| Culture medium | Aerial mycelium | Substrate mycelium | Soluble pigment | Luxuriant degree |
| Gause I agar | Freshwater mussel meat is white | It is milky white | It is yellowish pink | +++ |
| Glucose asparagine | It is greyish white | VI6-3’ | Realgar | +++ |
| Sucrose Cha Shi | It is greyish white | It is greyish white | Pale orange | + |
| Kirschner synthesizes No.1 agar | Snow weasel ash | Snow weasel ash | Wither leaf palm fibre | ++ |
| Potato dextrose agar | II5-1’ | III6-1’ | Gold leaf is yellow | +++ |
| Yeast extract malt extract agar | It is greyish white | It is milky white | It is apricot yellow | +++ |
| Starch ammonium agar | III5-1’ | Sallow | +++ | |
| Wa Shi nutrient agars | It is greyish white | It is greyish white | It is yellowish pink | + |
| Shellfish Knight | It is greyish white | Snow weasel ash | I7-7’ | ++ |
| Emol pine | It is greyish white | It is milky white | Marigold is yellow | +++ |
Note："-":It does not grow, "+":More growths are more vigorous
Sioyaensis (Streptomyces sioyaensis) F58 zymotic fluid controlling experiments A
Using smearing and spraying, two kinds of different insecticide-applying way prevent Poplar Plantation, test result such as 4 institute of table
Show.
Table 4
Test result shows manually to smear the side of sioyaensis (Streptomyces sioyaensis) F58 zymotic fluids
Formula control effect is better than spraying treatment, but the mode manually smeared is relatively time-consuming, and labour cost is also higher.It is recommended that upper one year
The heavier plot of degree morbidity, before next onset peak period, using the method.It is either artificial to smear still spraying treatment three
As the increase of extension rate, control effect decrease between a concentration, but difference is not notable between three concentration processing.
Sioyaensis (Streptomyces sioyaensis) F58 zymotic fluid controlling experiments B
Sioyaensis (Streptomyces sioyaensis) F58 zymotic fluids and prevention bark rot are commonly killed very
Microbial inoculum tests control effect in Shuangliao City of Jilin Province together, and test result is as shown in table 5.
Table 5
Test result shows using after sioyaensis (Streptomyces sioyaensis) F58 zymotic fluids, trunk
Above established scab starts dry and hard, has good control effect, 7d after the 2nd dispenser, sioyaensis to willow bark rot
The preventive effect and comparison medicament of (Streptomyces sioyaensis) F58 zymotic fluids --- 70% 800 times of first support wettable powder
Dilution is suitable；100 times of dilutions of 50 times of dilutions of slightly less than 50% Tuzet and 40% asomate.Zymotic fluid of the present invention is nontoxic
Noresidue.
Sioyaensis (Streptomyces sioyaensis) F58 zymotic fluid controlling experiments C
Scab is struck off with knife, with Tuzet and sioyaensis (Streptomyces sioyaensis) F58 zymotic fluids
Tu Gan.Experimental result is as shown in table 6.
Table 6
The result shows that after using sioyaensis (Streptomyces s i oyaens i s) F58 zymotic fluids, on trunk
Established scab starts dry and hard；And there are good control effect, 7d after the 2nd dispenser to willow bark rot：Salt room strepto-
The preventive effect of bacterium (Streptomyces sioyaensis) 5 times, 10 times, 20 times dilutions of F58 zymotic fluids be 45.68~
61.79%；10d preventive effects are 48.95~64.35% after 3rd dispenser, and preventive effect significantly improves；The results of analysis of variance shows：Salt room
The preventive effect of streptomycete (Streptomyces sioyaensis) F58 zymotic fluids is than 50% Tuzet wettable powder of comparison medicament
800 times of liquid low 25% or so, but the zymotic fluid and environmental-friendly, nontoxic residue-free.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all the present invention spirit and
Within principle, any modification, equivalent replacement, improvement and so on should all be included in the protection scope of the present invention.
Claims (2)
1. application of one plant of willow bark rot biocontrol microorganisms in inhibiting pathogenic activity, which is characterized in that the willow bark rot
Biocontrol microorganisms are sioyaensis (Streptomyces sioyaensis) F58, are deposited in Chinese microorganism strain preservation management committee
Member's meeting common micro-organisms center, deposit number are CGMCC No.11469；The pathogenic bacteria are plant pathogen.
2. application according to claim 1, which is characterized in that the pathogenic bacteria are selected from Cytospora chrysosperma (Vasal
Sordida), dothiorella gregaria bacterium (Botryosphaeria dothidea), Alternaria mali (Alternaria
Alternata f.sp.mali), Melon fusarium Wilt (Fusarium oxysporium f.sp.melonis), rice bakanae disease
Germ (Fusarium moniliforme), Rhizoctonia solani Kuhn (Rhizoctonia solani), Pyricularia oryzae
(Magnaporthe grisea), Colletotrichum truncatum (Colletotrichum truncatum), Cochliobolus sativus
(Bipolaris sorokiniana), fusarium graminearum (Fusarium graminearum), Tobacco target spot bacterium
(Thanatephorus cucumeris), Exserohilum turcicum (Exserohilum turcicum), southern corn leaf blight
One or more of (Bipolaris maydis).
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| CN113025537A (en) * | 2020-03-28 | 2021-06-25 | 吉林省林业科学研究院 | Fermentation method of streptomycete |
| CN113061551A (en) * | 2019-11-27 | 2021-07-02 | 吉林省林业科学研究院 | Application of biocontrol streptomyces in preventing and treating plant disease pathogenic bacteria |
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| CN110218671A (en) * | 2019-05-29 | 2019-09-10 | 江苏师范大学 | One plant of production water streptomycete and its application in biological control |
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| AGBESSI S等: "Antagonistic properties of two recombinant strains of Streptomyces melanosporofaciens obtained by intraspecific protoplast fusion", 《APPLIED MICROBIOLOGY AND BIOTECHNOLOGY》 * |
| 任嘉红等: "杨树腐烂病拮抗细菌的筛选及其定殖研究", 《西部林业科学》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113061551A (en) * | 2019-11-27 | 2021-07-02 | 吉林省林业科学研究院 | Application of biocontrol streptomyces in preventing and treating plant disease pathogenic bacteria |
| CN113025537A (en) * | 2020-03-28 | 2021-06-25 | 吉林省林业科学研究院 | Fermentation method of streptomycete |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108795825B (en) | 2019-08-09 |
| CN108795826B (en) | 2019-08-09 |
| CN108795826A (en) | 2018-11-13 |
| CN108935520B (en) | 2019-08-09 |
| CN108935520A (en) | 2018-12-07 |
| CN105255795A (en) | 2016-01-20 |
| CN105255795B (en) | 2018-08-28 |
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