CN105255795A - Cytospora chrysosperma biocontrol bacterium - Google Patents
Cytospora chrysosperma biocontrol bacterium Download PDFInfo
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Abstract
The invention relates to a cytospora chrysosperma biocontrol bacterium capable of effectively preventing and treating cytospora chrysosperma. The cytospora chrysosperma biocontrol bacterium is streptomyces sioyaensis F58, and is preserved in the China general microbiological culture collection center, and the preservation number is CGMCC No.11469. The width of a living bacterium inhibition zone in a cytospora chrysosperma treadmill standoff test of the streptomyces sioyaensis F58 ranges from 30.2 mm to 35.5 mm, the inhibition zone diameter measured by a fermentation broth cylinder plate method ranges from 50.7-56.4 mm, and an artificial inoculation test indicates that bioactive substances in fermentation broth of the cytospora chrysosperma biocontrol bacterium achieve a remarkable prevention and treatment effect on cytospora chrysosperma, the protection effect rate is 88.7%, and the treatment effect rate is 73.4%.
Description
Technical field
The present invention relates to a strain Poplar Diseases biocontrol microorganisms.
Background technology
Willow bark rot is one of important disease of willow, and this disease worldwide generally distributes, extensively occurs.The pathogenic bacteria perfect stage of willow bark rot is dirty black skin shell bacterium (ValsasordidaNit.), and the imperfect stage is golden yellow shell capsule spore bacterium (Cytosporachrysosperma).At present, the control of this disease is mainly based on chemical prevention and breeding resistant variety, but above-mentioned two kinds of prevention and controls all exist obvious drawback.There is the problems such as resistance is degenerated, resistance is low in the kind that disease-resistant variety seed selection is cultivated; And chemical prevention easily causes environmental pollution and destroys the problems such as the eubiosis.
Fungus treatment utilizes antagonistic microbe controlling plant diseases to be avoid the effective solution of the problems referred to above, and the method belongs to and is regulated to main sustainable control techniques with the ecosystem, has advantage free from environmental pollution, not destroy the eubiosis.But at present can the biocontrol microorganisms of antagonism Cytospora chrysosperma few, the microbiotic for the control of willow bark rot is also extremely rare, and majority still rests on the laboratory screening stage.
Summary of the invention
The invention provides a strain willow bark rot biocontrol microorganisms, can effectively prevent and treat willow bark rot.
Willow bark rot biocontrol microorganisms of the present invention is salt room streptomycete (Streptomycessioyaensis) F58, is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCCNo.11469.
Salt room streptomycete (Streptomycessioyaensis) F58 antimicrobial spectrum is wide, to Cytospora chrysosperma (Vasalsordida), dothiorella gregaria bacterium (Botryosphaeriadothidea), Alternaria mali (Alternariaalternataf.sp.mali), Melon fusarium Wilt (Fusariumoxysporiumf.sp.melonis), fusarium moniliforme (Fusariummoniliforme), Rhizoctonia solani Kuhn (Rhizoctoniasolani), Pyricularia oryzae (Magnaporthegrisea), Colletotrichum truncatum (Colletotrichumtruncatum), Cochliobolus sativus (Bipolarissorokiniana), fusarium graminearum (Fusariumgraminearum), Tobacco target spot bacterium (Thanatephoruscucumeris), Exserohilum turcicum (Exserohilumturcicum), southern corn leaf blight (Bipolarismaydis) all has bacteriostatic activity.
Salt room streptomycete (Streptomycessioyaensis) F58 reaches 30.2 ~ 35.5mm to the antibacterial bandwidth of viable bacteria in the dull and stereotyped dual test of Cytospora chrysosperma, fermented liquid cylinder plate method measures antibacterial circle diameter and reaches 50.7 ~ 56.4mm, artificial inoculation controlling experiment shows that in its fermented liquid, bio-active substance confrontation willow bark rot has significant prevention effect, provide protection and therapeutic action reach 88.7% and 73.4% respectively, its prevention effect difference is not remarkable compared with the upper chemical agent preventing and treating willow bark rot of production, and possess low toxicity, noresidue, with eco-friendly advantage.
Salt room streptomycete (Streptomycessioyaensis) F58 can make gelatine liquefication; Milk can not be made to solidify and peptonize; Do not utilize decomposition of cellulose; Hydrolyzed starch is positive; Catalase, urase, Esterase reaction are the positive; Do not produce H
2s; M.R. negative, V.P. tests the positive; Nitrate reduction reaction negative; Pectinose, wood sugar, fructose, starch, lactose, glucose, maltose can be utilized; N.F,USP MANNITOL, raffinose, rhamnosyl can not be utilized.Salt room streptomycete (Streptomycessioyaensis) F58 can utilize extractum carnis, peptone, yeast powder, (NH4)
2sO
4and KNO
3five kinds of nitrogenous sources; Comparatively strong to the tolerance of NaCl, can grow containing on the substratum of 5%NaCl; Have stronger tolerance to temperature, can grow at 15 ~ 37 DEG C, 28 DEG C is optimum growth temperature; Good to the tolerance of alkali, all can grow in the scope of pH5 ~ 12, and pH5 ~ 7 are the optimal pH of growth.
Salt room streptomycete (Streptomycessioyaensis) F58 riotous growth on Gause I substratum, aerial hyphae is luxuriant, 28 DEG C cultivate 1 ~ 2d bacterium colony smooth, generate without spore, 3d starts adularescent spore and grows from colony edge, starts to gradually become greyish white to snow weasel ash after 4d.Basis of microscopic observation, substrate mycelium, without horizontal film, is not ruptured; Electron microscopic observation result shows (as shown in Figures 1 to 3): the spacious or tight spiral of fibrillae of spores pine, often irregular, accidental short and loose-screw shape, spore column; By microbial morphology and physiological and biochemical index qualification, think that willow bark rot biocontrol microorganisms of the present invention belongs to streptomyces.Salt room streptomycete (Streptomycessioyaensis) F58 is through 16SrDNA sequence alignment analysis, the highest with the 16SrDNA homology of known actinomycetic streptomycete Streptomycessioyaensis (gene accession number KF475886), reach 99%.
Salt room streptomycete (Streptomycessioyaensis) F58 is salt room streptomycete, belongs to streptomyces (Streptomyces); Be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number is CGMCCNo.11469, and preservation date is on September 30th, 2015.
Accompanying drawing explanation
Fig. 1 is spore chain and the spore electron microscopic observation figure of salt room streptomycete (Streptomycessioyaensis) F58.
Fig. 2 is spore chain and the spore electron microscopic observation figure of salt room streptomycete (Streptomycessioyaensis) F58.
Fig. 3 is spore chain and the spore electron microscopic observation figure of salt room streptomycete (Streptomycessioyaensis) F58.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: present embodiment willow bark rot biocontrol microorganisms is salt room streptomycete (Streptomycessioyaensis) F58, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCCNo.11469.
Salt room streptomycete (Streptomycessioyaensis) F58 picks up from Shuangliao City of Jilin Province experimental forest, by pedotheque sterilized water dilution 10
3doubly, utilize Gause I substratum to cultivate under 28 DEG C of conditions, adopt conventional plating dilutions culture method to be separated and obtain.Salt room streptomycete (Streptomycessioyaensis) F58 after separation and purification proceed to Gause I substratum cultivate under 28 DEG C of conditions, expand numerous.
Salt room streptomycete before is all separated from ocean mud, and salt room streptomycete (Streptomycessioyaensis) F58 of the present invention the first is separated the salt room streptomycete obtained from soil.Salt room streptomycete (Streptomycessioyaensis) F58 of the present invention and salt room streptomycete type strain difference to some extent on utilization of carbon source, salt room streptomycete (Streptomycessioyaensis) F58 can not utilize raffinose and rhamnosyl, but can L-arabinose be utilized, D-Fructose, W-Gum; 16SrDNA comparison result is not also 100% homology simultaneously, therefore judges that salt room streptomycete (Streptomycessioyaensis) F58 should be salt room streptomycete new variant.
Embodiment 1
In salt room streptomycete (Streptomycessioyaensis) F58 fermention medium, the concentration of groundnut meal is 2.5% (w/v), the concentration of starch is 5.0% (w/v), the concentration of yeast powder is 0.08% (w/v), the concentration of glucose is 0.02% (w/v), (NH
4)
2sO
4concentration be 0.08% (w/v), CaCO
3concentration be 0.32% (w/v), the concentration of NaCl is 0.2% (w/v), fermention medium pH value is 7.0 ~ 7.2.7d is cultivated in fermention medium 150r/min concussion under 28 DEG C of constant temperatures of inoculation salt room streptomycete (Streptomycessioyaensis) F58, then the centrifugal 15min of 6000r/min, crosses bacterial filter removing thalline and obtains salt room streptomycete (Streptomycessioyaensis) F58 fermented supernatant fluid.
Salt room streptomycete (Streptomycessioyaensis) F58 fermented liquid is to willow bark rot controlling experiment
Willow bark rot pathogen infection: inoculate by the raw yellow fast poplar cuttage seeding of 1a, by Cytospora chrysosperma after PDA substratum 26 DEG C of constant temperature culture 5 ~ 7d, bacterium cake is bought for subsequent use with 7mm punch tool, scratch to willow cadre with blade, wound about size 5mm, is attached to wound by former for willow bark rot bacterium bacterium cake, the moisturizing of poplar leaf juice is dipped in absorbent cotton, often process 15 strains, repeat, after moisturizing 7d, remove bacterium cake for 3 times.
The antibacterial contrast of fermented liquid: 5d, 10d, 15d after bacterium cake are removed in 15d, 10d, 5d and inoculation process before inoculation, respectively by F58 fermented supernatant fluid 5 times of diluents, 100 times of diluents of 50 times of diluents of 50% Tuzet, 100 times of diluents of 40% Asomat, 70% thiophanate methyl and clear water contrast uniform application are in the willow cadre infected by Cytospora chrysosperma, nursery stock incidence is observed in timing, calculates prevention effect.
Disease index grade scale, with reference to Jilin Province's provincial standard " willow bark rot process for comprehensively treating code " [17]
I grade (typical value 0)---anosis;
II grade (typical value 1)---scab lateral length accounts for less than 1/4 of trunk girth;
III level (typical value 2)---scab lateral length accounts for 1/4 ~ 2/4 of trunk girth;
IV grade (typical value 3)---scab lateral length accounts for 2/4 ~ 3/4 of trunk girth;
V level (typical value 4)---more than 3/4 ~ trees that scab lateral length accounts for trunk girth are dying or dead.
Experimental result: in removing bacterium cake 15d " Invest, Then Investigate " sickness rate and disease index (as shown in table 1), contrast nursery stock sickness rate and disease index are distributed as 82.2% and 49.4; Before the inoculation of T1 (preventing and treating with salt room streptomycete F58) pathogenic bacteria, the nursery stock sickness rate of coating process is 15.5%, disease index is 5.57, sickness rate and the disease index of coating process after pathogenic bacteria inoculation are 35.6% and 13.4 respectively, and provide protection and therapeutic action are distributed as 88.7% and 73.4%; The provide protection that T2 (100 times of diluent controls with 40% Asomat) and T4 (100 times of diluent controls with 70% thiophanate methyl) processes is 89.8, and therapeutic action is distributed as 74.4% and 79.3%.Experiment finds that contrast nursery stock scab within several weeks afterwards expands gradually, and at scab place, visible significantly pycnidium, has individually conidium angle to occur; The susceptible plant scab of T1 ~ T4 does not almost continue to expand, and scab place has no pycnidium.Experimental result shows, salt room streptomycete (Streptomycessioyaensis) F58 fermented liquid has significant restraining effect to Cytospora chrysosperma, even if willow infects Cytospora chrysosperma, also has good result for the treatment of; Although its provide protection is a little less than T2 and T4, higher than T3 (the 50 times of diluents control with 50% Tuzet), difference is not remarkable; Therapeutic action and T2 ~ T4 difference not remarkable.
Table 1
In table, data are mean number ± standard deviation.Show through the inspection of DuncanShi new multipole difference method remarkable at P<0.01 and P<0.05 level difference, lower same with large and small lowercase alphabet different after column data.
Salt room streptomycete (Streptomycessioyaensis) F58 bacteriostatic test
Streptomycete (Streptomycessioyaensis) F58 dull and stereotyped dual test in salt room is to Tobacco target spot bacterium, Cytospora chrysosperma and the effect of dothiorella gregaria bacterium the strongest, visible obvious antibacterial band, antibacterial band width average reaches more than 30mm, to Alternaria mali, rice blast fungus and Rhizoctonia solani Kuhn have very strong restraining effect, antibacterial bandwidth is respectively 27.6mm, 25.6mm, 18.3mm, to fusarium graminearum, Cochliobolus sativus, Melon fusarium Wilt, fusarium moniliforme, Colletotrichum truncatum, Exserohilum turcicum is relative with southern corn leaf blight restraining effect more weak, but antibacterial more than bandwidth 6.3mm.
Cylinder plate method measures the bacteriostatic activity of salt room streptomycete (Streptomycessioyaensis) F58 and fermented liquid thereof: all kinds of pathogenic bacteria is made mixed bacterium flat board, cylinder plate method is adopted to measure the bacteriostatic activity of fermented liquid, fermented liquid dilutes 5 times and uses application of sample amount to be 100 μ l, often processes 3 times and repeats.The bacteriostatic activity of salt room streptomycete (Streptomycessioyaensis) F58 and fermented liquid thereof is as shown in table 2.
Table 2
Experiment proves that the fermented liquid of salt room streptomycete (Streptomycessioyaensis) F58 still maintains good bacteriostatic activity, extremely strong antagonistic action is had to fusarium graminearum, Tobacco target spot bacterium, Cytospora chrysosperma, dothiorella gregaria, bacteriostatic diameter reaches more than 50mm, and all having obvious restraining effect to all for examination pathogenic bacteria, salt room streptomycete (Streptomycessioyaensis) F58 antimicrobial spectrum is wide.
Embodiment 2
Salt room streptomycete (Streptomycessioyaensis) F58 is inoculated in respectively 10 kinds of different substratum, cultivate 36 ~ 48h for 28 DEG C and start have bacterium colony to occur, after 72 ~ 96h, spore starts generation.On different substratum, the form of lawn, the color of sorus, the color of matrix mycelia, the soluble pigment of generation, growing state all have difference, the method provided according to " streptomycete identification handbook " and color version carry out observing, comparison, result is as shown in table 3, and salt room streptomycete (Streptomycessioyaensis) F58 is produced soluble pigment on most substratum; Aerial hyphae becomes snow weasel grey from canescence or freshwater mussel meat is white, substrate mycelium is milky white to snow weasel ash.
Table 3
Note: "-": not grow, "+": more growths are more vigorous
Salt room streptomycete (Streptomycessioyaensis) F58 fermented liquid controlling experiment A
Employing is smeared and two kinds of different insecticide-applying way of spraying are prevented and treated Poplar Plantation, and test-results is as shown in table 4.
Table 4
Test-results shows, the mode prevention effect of manually smearing salt room streptomycete (Streptomycessioyaensis) F58 fermented liquid is better than spraying process, but the Method compare manually smeared is time-consuming, and labour cost is also higher.Advise falling ill heavier plot to previous year, before next onset peak period, adopt this method.No matter be manually smear or spray along with the increase of extension rate between process three concentration, prevention effect decreases, but between three concentration process, difference is not remarkable.
Salt room streptomycete (Streptomycessioyaensis) F58 fermented liquid controlling experiment B
Coexist the mycocide one that salt room streptomycete (Streptomycessioyaensis) F58 fermented liquid and control bark rot are commonly used Shuangliao City of Jilin Province test prevention effect, and test-results is as shown in table 5.
Table 5
Test-results shows, after using salt room streptomycete (Streptomycessioyaensis) F58 fermented liquid, on trunk, established scab starts dry and hard, good prevention effect is had to willow bark rot, 7d after 2nd dispenser, the preventive effect of salt room streptomycete (Streptomycessioyaensis) F58 fermented liquid with contrast medicament---70% first holder wettable powder, 800 times of diluents are suitable; Slightly lower than 50% Tuzet, 50 times of diluents and 40% asomate, 100 times of diluents.Fermented liquid nontoxic residue-free of the present invention.
Salt room streptomycete (Streptomycessioyaensis) F58 fermented liquid controlling experiment C
With cutter, scab is struck off, be coated with dry with Tuzet and salt room streptomycete (Streptomycessioyaensis) F58 fermented liquid.Experimental result is as shown in table 6.
Table 6
After result shows to use salt room streptomycete (Streptomycessioyaensis) F58 fermented liquid, on trunk, established scab starts dry and hard; And having good prevention effect to willow bark rot, 7d after the 2nd dispenser: the preventive effect of salt room streptomycete (Streptomycessioyaensis) F58 fermented liquid 5 times, 10 times, 20 times diluents is 45.68 ~ 61.79%; After 3rd dispenser, 10d preventive effect is 48.95 ~ 64.35%, and preventive effect significantly improves; The results of analysis of variance shows: the preventive effect of salt room streptomycete (Streptomycessioyaensis) F58 fermented liquid is lower by about 25% than contrast medicament 50% Tuzet wettable powder 800 times of liquid, but this fermented liquid and environmental friendliness, nontoxic residue-free.
Claims (1)
1. a strain willow bark rot biocontrol microorganisms, it is salt room streptomycete (Streptomycessioyaensis) F58, is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCCNo.11469.
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| CN201810736154.5A CN108795825B (en) | 2015-12-01 | 2015-12-01 | One plant of poplar bark rot biocontrol microorganisms is inhibiting the application in pathogenic activity |
| CN201810736186.5A CN108795826B (en) | 2015-12-01 | 2015-12-01 | Application of one plant of poplar bark rot biocontrol microorganisms in control of plant disease |
| CN201810735604.9A CN108935520B (en) | 2015-12-01 | 2015-12-01 | A kind of biological prevention and control agent, preparation method and its application method |
| CN201510859507.7A CN105255795B (en) | 2015-12-01 | 2015-12-01 | One plant of willow bark rot biocontrol microorganisms |
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| CN201810736186.5A Division CN108795826B (en) | 2015-12-01 | 2015-12-01 | Application of one plant of poplar bark rot biocontrol microorganisms in control of plant disease |
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| CN109452293A (en) * | 2018-09-19 | 2019-03-12 | 河南农业大学 | A kind of plant source compound formulation and preparation and spraying method for preventing and treating east area of the Liao River, coextensive with eastern and southern Liaoning Province flue-cured tobacco target spot |
| CN110699304A (en) * | 2019-11-27 | 2020-01-17 | 吉林省林业科学研究院 | Biocontrol streptomyces |
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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 |
| CN112931534B (en) * | 2020-03-28 | 2021-10-29 | 吉林省林业科学研究院 | Application of streptomycete in biological control |
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| CN109452293A (en) * | 2018-09-19 | 2019-03-12 | 河南农业大学 | A kind of plant source compound formulation and preparation and spraying method for preventing and treating east area of the Liao River, coextensive with eastern and southern Liaoning Province flue-cured tobacco target spot |
| CN110699304A (en) * | 2019-11-27 | 2020-01-17 | 吉林省林业科学研究院 | Biocontrol streptomyces |
| CN110699304B (en) * | 2019-11-27 | 2021-04-27 | 吉林省林业科学研究院 | Biocontrol streptomyces |
| 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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| CN108795826B (en) | 2019-08-09 |
| CN108795825A (en) | 2018-11-13 |
| CN108935520A (en) | 2018-12-07 |
| CN105255795B (en) | 2018-08-28 |
| CN108795825B (en) | 2019-08-09 |
| CN108935520B (en) | 2019-08-09 |
| CN108795826A (en) | 2018-11-13 |
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