CN108795825B - One plant of poplar bark rot biocontrol microorganisms is inhibiting the application in pathogenic activity - Google Patents
One plant of poplar bark rot biocontrol microorganisms is inhibiting the application in pathogenic activity Download PDFInfo
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Abstract
The present invention relates to one plant of poplar bark rot biocontrol microorganisms to inhibit the application in pathogenic activity, poplar 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, former Chinese Patent Application No. are as follows: 201510859507.7, the applying date are as follows: 2015 12
Month 1 day, patent name when application are as follows: one plant of poplar bark rot biocontrol microorganisms.
Technical field
The invention belongs to field of biotechnology more particularly to one plant of poplar bark rot biocontrol microorganisms in inhibiting pathogenic activity
Application.
Background technique
Poplar bark rot is one of important disease of poplar, 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 and treatment of the disease is mainly with chemical prevention and breeding resistant variety
It is main, but there is obvious drawback in above two control method.There are resistances to degenerate for the kind that disease-resistant variety breeding is 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 the effective solution for avoiding the above problem using antagonistic microbe controlling plant diseases, and should
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 and treatment of poplar bark rot
To be rare, majority remains in the laboratory screening stage.
Summary of the invention
In view of the problems of prior art, the present invention provides one plant of poplar bark rot biocontrol microorganisms and is inhibiting pathogenic bacteria living
Application in property, has many advantages, such as that good antimicrobial effect, antimicrobial spectrum are wide and environmentally friendly.
The technical scheme to solve the above technical problems is that
The present invention provides one plant of poplar bark rot biocontrol microorganisms and is inhibiting the application in pathogenic activity.
Poplar 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 poplar bark rot biocontrol microorganisms, can effectively prevent poplar bark rot.
Sioyaensis (Streptomyces sioyaensis) F58 is in the plate dual test of Cytospora chrysosperma
The antibacterial bandwidth of viable bacteria reaches 50.7~56.4mm up to 30.2~35.5mm, fermentation liquid cylinder plate method measurement antibacterial circle diameter, manually
Inoculation controlling experiment shows that biological active matter confrontation poplar bark rot has significant control efficiency, protective effect in its fermentation liquid
With therapeutic effect respectively up to 88.7% and 73.4%, its control efficiency compared with the chemical agent for preventing and treating poplar bark rot in production
Difference is not significant, 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. test are positive；Nitrate reduction reaction negative；Can use 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 are raw
Mycelia is luxuriant, and 28 DEG C of culture 1~2d bacterium colonies are smooth, generate without spore, and 3d begins with white spore and grows 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
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 poplar bark rot biocontrol microorganisms of the present invention belong to streptomyces.Salt
Strepto- of room streptomycete (Streptomyces sioyaensis) F58 through 16SrDNA sequence alignment analysis, with known actinomyces
The 16S rDNA homology highest of bacterium Streptomyces sioyaensis (gene accession number KF475886), reaches 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 on September 30th, 2015.
Detailed description of the invention
Fig. 1 is the spore chain and spore Electronic Speculum observation figure of sioyaensis (Streptomyces sioyaensis) F58.
Fig. 2 is the spore chain and spore Electronic Speculum observation figure of sioyaensis (Streptomyces sioyaensis) F58.
Fig. 3 is the spore chain and spore Electronic Speculum observation figure of sioyaensis (Streptomyces sioyaensis) F58.
Specific embodiment
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 invention.
Specific embodiment 1: present embodiment poplar 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, 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 result is not also 100% homologous simultaneously, therefore judges sioyaensis
(Streptomyces sioyaensis) F58 should be sioyaensis new variant.
Embodiment 1
The concentration of groundnut meal is in sioyaensis (Streptomyces sioyaensis) F58 fermentation medium
2.5% (w/v), starch concentration be 5.0% (w/v), the concentration of yeast powder is 0.08% (w/v), the concentration of glucose is
0.02% (w/v), (NH4)2SO4Concentration be 0.08% (w/v), CaCO3Concentration be 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 is centrifuged 15mi n,
It crosses bacterial filter and removes thallus acquisition sioyaensis (Streptomyces sioyaensis) F58 fermented supernatant fluid.
Sioyaensis (Streptomyces sioyaensis) F58 fermentation liquid is to poplar bark rot controlling experiment
Poplar bark rot pathogen infection: the inoculation raw Huang Kuaiyang cuttage seeding of 1a, by Cytospora chrysosperma in PDA culture medium
After 26 DEG C of 5~7d of constant temperature incubation, it is spare that bacteria cake is beaten with 7mm punch, gives poplar cadre to scratch with blade, wound size 5mm is left
Poplar bark rot opportunistic pathogen bacteria cake is attached to wound by the right side, dips in poplar leaf juice moisturizing, every 15 plants of processing, 3 weights with absorbent cotton
It is multiple, bacteria cake is removed after moisturizing 7d.
The antibacterial comparison of fermentation liquid: 5d, 10d, 15d after bacteria cake are removed in 15d, 10d, 5d and inoculation processing before being inoculated with, respectively
By 5 times of dilutions of F58 fermented supernatant fluid, 100 times of dilutions, 70% of 50 times of dilutions of 50% Tuzet, 40% asomate
100 times of dilutions and the clear water control of thiophanate methyl are uniformly applied to the poplar cadre infected by Cytospora chrysosperma, timing
Nursery stock incidence is observed, control efficiency is calculated.
Disease index grade scale, with reference to Jilin Province's provincial standard " poplar 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；
V grades (typical value 4) --- 3/4 or more~trees that 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；T1 (prevents and treats) coating processing before pathogen is inoculated with sioyaensis F58
Nursery stock disease incidence is 15.5%, and disease index is 5.57, the disease incidence and disease index that coating is handled after pathogen inoculation
It is respectively 35.6% and 13.4, protective effect and therapeutic effect are distributed as 88.7% and 73.4%；T2 is (with 40% asomate
The prevention and treatment of 100 times of dilutions) and the protective effect that handles of T4 (being prevented and treated with 100 times of dilutions of 70% thiophanate methyl) be
89.8, therapeutic effect is distributed as 74.4% and 79.3%.Experiment discovery control nursery stock scab within several weeks later is gradually expanded,
The visible apparent pycnidia at scab has individually the appearance of conidium angle；T1~T4 susceptible plant scab does not almost have
Have and continue to expand, pycnidia is had no at scab.The experimental results showed that sioyaensis (Streptomyces
Sioyaensis) F58 fermentation liquid has significant inhibiting effect to Cytospora chrysosperma, even if poplar has infected poplar bark rot
Bacterium also has preferable therapeutic effect；Although its protective effect be slightly below T2 and T4, be higher than T3 it is (dilute with 50 times of 50% Tuzet
Release liquid prevention and treatment), but difference is not significant；Therapeutic effect and T2~T4 difference is not significant.
Table 1
Data are average ± standard deviation in table.Show with large and small lowercase alphabets different after column data newly multiple through DuncanShi
Very poor method inspection is significant in P < 0.01 and the level difference of P < 0.05, similarly hereinafter.
Sioyaensis (Streptomyces sioyaensis) F58 bacteriostatic test
Sioyaensis (Streptomyces sioyaensis) F58 plate dual test is to Tobacco target spot bacterium, poplar
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 have very strong inhibiting effect, and antibacterial bandwidth is respectively
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 fermentation liquid:
Mixed bacterium plate is made in all kinds of pathogens, using the bacteriostatic activity of cylinder plate method measurement fermentation liquid, fermentation liquid dilutes 5 times using sample-adding
Amount is 100 μ l, 3 repetitions of every processing.The suppression of sioyaensis (Streptomyces sioyaensis) F58 and its fermentation liquid
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 fermentation liquid 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 spectrum is 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 " provide method and color version observed, compared, 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
White, 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 agar | 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: "-": do not grow, "+": more growths are more vigorous
Sioyaensis (Streptomyces sioyaensis) F58 fermentation liquid controlling experiment A
Poplar Plantation is prevented and treated using smearing and spraying two kinds of different insecticide-applying way, test result such as 4 institute of table
Show.
Table 4
Test result shows the side for manually smearing sioyaensis (Streptomyces sioyaensis) F58 fermentation liquid
Formula control efficiency 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
With the increase of extension rate between a concentration, control efficiency decreases, but difference is not significant between three concentration processing.
Sioyaensis (Streptomyces sioyaensis) F58 fermentation liquid controlling experiment B
Sioyaensis (Streptomyces sioyaensis) F58 fermentation liquid and prevention and treatment bark rot are commonly killed very
Microbial inoculum tests control efficiency in Shuangliao City, Jilin Province together, and test result is as shown in table 5.
Table 5
Test result shows using after sioyaensis (Streptomyces sioyaensis) F58 fermentation liquid, trunk
Above established scab starts dry and hard, has good control efficiency, 7d after the 2nd application, sioyaensis to poplar bark rot
The preventive effect and comparison medicament of (Streptomyces sioyaensis) F58 fermentation liquid --- 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.Fermentation liquid of the present invention is nontoxic
Noresidue.
Sioyaensis (Streptomyces sioyaensis) F58 fermentation liquid controlling experiment C
Scab is struck off with knife, with Tuzet and sioyaensis (Streptomyces sioyaensis) F58 fermentation liquid
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 fermentation liquid, on trunk
Established scab starts dry and hard；And there are good control efficiency, 7d after the 2nd application: salt room strepto- to poplar bark rot
The preventive effect of bacterium (Streptomyces sioyaensis) 5 times, 10 times, 20 times dilutions of F58 fermentation liquid be 45.68~
61.79%；10d preventive effect is 48.95~64.35% after 3rd application, and preventive effect significantly improves；The results of analysis of variance shows: salt room
The preventive effect of streptomycete (Streptomyces sioyaensis) F58 fermentation liquid is than 50% Tuzet wettable powder of comparison medicament
800 times of liquid low 25% or so, but the fermentation liquid 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 in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (1)
1. one plant of poplar bark rot biocontrol microorganisms is inhibiting the application in pathogenic activity, which is characterized in that the poplar bark rot
Biocontrol microorganisms be sioyaensis (Streptomyces sioyaensis) F58, it is 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 selected from Alternaria mali
(Alternaria alternata f.sp.mali), Melon fusarium Wilt (Fusarium oxysporium
f.sp.melonis), Tobacco target spot bacterium (Thanatephorus cucumerisOne or more of).
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